DDL Career Development Grant

Lucy Goodacre - PhD Researcher in Pharmaceutical Science at King’s College London and Nanopharm Ltd, an Aptar Pharma Company.

Lucy previously studied for an MPharm at UCL School of Pharmacy, University College London (2018-2022). She was notably awarded a visiting scholarship to the University of North Carolina (UNC) Eshelman School of Pharmacy, to complete her Master’s project in pharmacokinetic modelling of caffeine in neonatal hypoxic ischaemic encephalopathy (September - December 2021).

Under the primary supervision of Professor Ben Forbes, Lucy’s PhD project focusses on advanced experimental and computational methods to optimise nasal drug delivery. More specifically, Lucy aims to develop a biologically-relevant suite of nasal mucus simulants, to be used to study mucus-drug particle interaction in the nasal cavity and guide the design of drug formulations for effective intranasal administration.

Lucy is also on the committee board for the New Researcher Network (NRN) at DDL, where she encourages like-minded PhD students and early career scientists to engage in discussion and improve their connections within the field of inhalation/respiratory science.

Spray-Drying Sumatriptan Succinate Powders to Evaluate the Effect of Mucus in Nasal Deposition Studies

Incorporating nasal mucus simulants as coatings into nasal casts (e.g. Aeronose® or Alberta Idealised Nasal Inlet, AINI®) offers a promising approach for studying the effect of the mucosal surface on deposition in the nasal cavity. By utilising these mucus-like coatings, we aim to observe their impact on regional deposition patterns and drug recovery from the casts. This allows the study of physical or chemical interactions between the mucus and depositing drug particles.

As part of the investigation, we wish to explore the hypothesis that the effect of mucus simulant-coating on deposition of powder formulations may differ from liquid formulations when delivered into the nasal cavity. For example, a biologically-relevant coating agent, such as a nasal mucus simulant, may reduce particle bounce for powder formulations. Consequently, spray drying (SD) an API powder will be necessary to produce a particle size distribution within the optimal nasal delivery range of 20-150 µm. Sumatriptan succinate, which is used for the treatment of migraines, has been selected as the API of choice due to its availability in both liquid and powder formulations, making it suitable for testing dripping and particle bounce hypotheses. The commercial product Onzestra® Xsail will serve as the benchmark due to its established in vivo performance. The availability of clinical data will enable analysis via a physiologically-based pharmacokinetic (PBPK) model to provide a mechanistic understanding of regional deposition impact on nasal pharmacokinetics. This proposal seeks funding for the development of bespoke SD sumatriptan powders (via academic collaboration with a partner laboratory), which will enable the hypothesis to be tested.

Aims:

Phase 1
To develop SD formulations of sumatriptan succinate for studies to probe the effect of nasal cast mucus-lining on regional deposition.

Phase 2
To formulating a suite of SD sumatriptan powders containing mucoadhesive excipients to evaluate the effect on nasal cast deposition, and other mucus-drug interactions (based on a wider suite of mucus simulants).

I am currently a second-year PhD student at King’s College London supervised by Professor Ben Forbes and Dr Cecile Dreiss. My PhD project focusses specifically on advancing experimental and computational methods to optimise nasal drug delivery.

I plan to use the career development award to enhance my PhD work by collaborating with the University of Parma (Dr Fabio Sonvico & Dr Eride Quanta). Currently, my PhD focuses on the design and manufacture of nasal mucus simulants. I have demonstrated the feasibility of producing mucus simulants by preparing prototypes and characterising these through bulk- and micro-rheological techniques and adhesion studies (Goodacre et al, 'Use of Rheology and Mucoadhesivity to Guide the Development of Nasal Mucus Simulants', Drug Delivery to the Lungs, 2023). This suite of characterisation techniques provided an insight into the simulants’ performance and suitability for evaluating nasal formulations. The next steps will be to evaluate the application of these mucus simulants in studies of nasal deposition, drug dissolution and transport.

Experimental Design:

Phase 1 of the proposed Career Development Award project will focus specifically on the use of nasal casts coated with a thermosensitive polymer containing mucins. Both liquid and powder formulations of sumatriptan succinate will be tested for nasal deposition and drug recovery. Studies will evaluate the effects of dripping for liquids and particle bounce for powders. Producing bespoke spray-dried powders is crucial to study the effects of particle size distribution, which cannot be manipulated with commercial powder products (which are also prohibitively expensive).

Phase 2 will involve production of SD powders containing mucoadhesive excipients to study a broader range of particle-mucus interactions. This phase will use a suite of mucus simulants with different attributes to assess the interactions of formulations with mucus simulants in nasal casts, dissolution and permeability experiments.

Methods:

1) Setting up spray dryer equipment - optimising solid content, feed rate, atomisation
2) Characterisation of SD powder and quality control - particle size distribution, moisture content, morphology, API stability

Scientific Value:

The availability of powders is a crucial enabler of the proposed project. Producing bespoke powder formulations will allow multiple hypotheses to be tested, including the significance of mucus lining on in vitro deposition and whether this has differential effects for powders and liquid formulations. Understanding mucus-drug particle interactions within the nasal cavity can lead to the development of more effective nasal drug delivery systems, ultimately improving patient outcomes. Therefore, this research has the potential to make a significant contribution to the field.

Value for Career Development:

There is currently no spray drying equipment at King’s College London; this collaborative project will provide an invaluable opportunity to obtain vital test material for my project, enhance my research skills and expand my global network. Through this project, I will gain hands-on experience with spray drying techniques, which is highly relevant for my field of pharmaceutical science. As part of the DDL New Researcher Network committee, it would also allow me to strengthen my networks and align with my ambitions of improving connectivity within PhD students and early researchers.

Joana Pinto da Silva is a PhD candidate in Biomedical Sciences at the University of Algarve, Portugal.
Her research, conducted within the Drug Delivery Lab, focuses on developing innovative dry powder formulations for pulmonary delivery, specifically aiming to create an inhalable platform for immunization against respiratory infections.

Prior to her doctoral studies, Joana earned her MSc in Biomedical Sciences from the University of Algarve in 2020. In 2021, she was awarded a PhD scholarship from the Foundation for Science and Technology in Portugal.

Under the expert guidance of Dr. Ana Grenha, director of the Drug Delivery Lab, Joana explores various carriers and excipients to develop inhalable therapeutics. The research conducted in the Drug Delivery Lab focuses on pulmonary drug delivery strategies and targeted therapies for respiratory conditions.

So far, Joana’s work has resulted in one provisional patent application. She has presented her research through four oral presentations and seven posters at various scientific conferences. From 2021 to
2023, she has participated annually in the Drug Delivery to the Lungs Conference. In 2023 DDL, she delivered a podium presentation entitled “Repurposing bacterial lysates: could inhalation be a possibility in respiratory infection management?”. In 2024, she was awarded Best Poster for her work on “Pulmonary delivery of bacterial lysates mediated by locust bean gum microparticles” at the Pulmonary Drug Delivery Workshop.

In vivo assessment of bacterial lysate-loaded locust bean gum microparticles aimed at respiratory disease prevention upon inhalation

Background and Significance

Respiratory infections continue to pose significant health challenges globally, with diseases such as Covid-19, pneumonia, and COPD-related infections causing substantial morbidity and mortality [1]. Although bacterial lysates have been used as immunomodulators for preventing respiratory tract infections due to their ability to stimulate the immune system, their clinical significance remains uncertain [2]. Contradictory trial results and the European Medicines Agency's (EMA) recent recommendations limit their usage primarily to cases of recurrent respiratory infections [3]. To overcome the posed limitations of lack of efficacy in respiratory infections, we propose the development of an innovative inhalable formulation of bacterial lysates to provide enhanced protection and improve the therapeutic outcomes.

Bacterial lysates (BL) consist of extracts of pathogenic bacteria, such as S. aureus, S. pyogenes, and K. pneumoniae, which are responsible for various respiratory infections [2]. Currently, they are predominantly administered orally; however, their low efficacy in inducing mucosal immunity limits their protective effect against lung infections. An alternative approach involves delivering the antigens directly to the lungs via inhalation, allowing for targeted interaction with mucosa-associated lymphoid tissues (MALT) in the bronchial zone (BALT), thereby enhancing local immune responses [4].
Our proposed solution leverages locust bean gum (LBG), a galactomannan known for its high affinity towards macrophages, as an antigen carrier for the inhalable formulation [5,6]. By combining BL with LBG, we developed an innovative approach for the inhalation of BL, with the intention to elicit an immune response in the site of entry of pathogens.

Output summary

The results obtained thus far indicate that BL were successfully microencapsulated by spray-drying using LBG as the matrix material, achieving an association efficiency of 81%. MP exhibited suitable morphology, and those with an LBG:BL ratio of 10:0.2 (w/w) demonstrated optimal aerodynamic properties for lung deposition (MMAD of 4.6 µm and FPF of 29%). Serum agglutination and immunochromatography assays confirmed the presence of native antigens in BL-loaded MP formulations, indicating that spray-drying did not compromise antigen integrity. In vitro release studies showed gradual BL release, reaching approximately 80% within 6 h. Mucoadhesive properties of LBG-based MP were demonstrated, potentially prolonging lung residence time and, potentially enhancing antigen uptake by antigen-presenting cells (APCs), improving the immune response. Furthermore, the formulated inhalable MP demonstrated no adverse effects on respiratory cell viability, suggesting a favorable safety profile. This career development proposal is particularly dedicated to the last phase of the project, which is expected to comprise the in vivo proof of concept.

Work performed

Thus far, extensive work has been performed and presented in several conferences including DDL, with a poster presentation at the DDL2022 [7], and an oral presentation in last year’s DDL [8]. Those communications showcased the development and optimization of inhalable LBG-based microparticles loaded with BL. MP characterization studies evaluated aerodynamic properties for lung deposition, as well as MP morphology and mucoadhesive properties. Furthermore, the performed analysis included evaluation of antigen integrity after processing and antigen release profile.
Current studies are evaluating MP safety and antigen presenting cell targeting. This includes analysing the cytocompatibility of MP with respiratory cell lines A549, Calu-3 and THP-1, as well as in vitro uptake of MP by antigen presenting cell models such as differentiated THP-1 cells (dTHP-1). The results already obtained provide a positive outlook and indicate the potential success of the application. However, to provide a proof-of-concept , in vivo studies are now required.

Proposal goal

Evaluate in vivo protective efficacy by using an animal model to compare the elicited immune response (mucosal and systemic) and the real protection against respiratory challenge following inhaled vs. oral immunization with BL-loaded LBG MP.

Detailed Description of Research Design

Wistar rats will be used in this study, performed in collaboration with Dr. Manuela Gaspar from iMed.ULisboa and following ethical guidelines. Based on power analyses derived from comparable studies, seven groups consisting of six rats per group will participate in the experiment [9, 10]. The studies will follow a rigorous experimental design, including multiple study groups:
Group 1 (Test - Inhalation): Rats in this group will receive a first dose of BL-loaded LBG MP by inhalation using a nose-only inhalation device optimized for the assay [6] on day 0 and a booster dose on day 14.
Group 2 (Control - Naïve): These rats will serve as naïve controls and will not undergo any treatment.
Group 3 (Positive Control - Oral): Rats in this group will receive commercially available BL orally (by gavage) on days 0 and 14, serving as a reference standard for comparative purposes.
Group 4 (Control – LBG MP): Rats in this group will receive LBG MP formulated without BL on days 0 and 14, to understand the effects caused by the empty MP.
Immune Response Evaluation:
At day 28, all rats will be euthanized using CO2 inhalation. Serum will be collected to determine the levels of IgG1 and IgG2a against Streptococcus pyogenes M protein using ELISA tests employing previously isolated antigens. Bronchoalveolar lavage (BAL) procedures will also be conducted to assess the lung mucosal immune response. Specific IgA concentrations will be measured in BAL fluids, while cell counts for macrophages, neutrophils, and eosinophils will be determined.
Challenge Experiment:
Groups 5 (Test - Inhalation + Challenge) and 6 (Positive Control - Oral + Challenge) will be challenged with a lethal dose of Streptococcus pyogenes on day 28 [11], after receiving the vaccine doses in days 0 and 14 as for groups 1 and 3. Rat survival rates will be recorded and compared to observe any protective effects conferred by the pre-existing immunity developed through either inhalation or oral administration. Group 7 (Control - Infected but Not Immunized) will serve as a negative control, with death expected within two days post infection.
One week after the challenge, surviving rats from Groups 5 and 6 will be sacrificed, and lung tissue samples will be taken for histopathology examination and bacterial counts in lung homogenates [12]. Throughout the study, animal weights will be recorded weekly, and signs of pain, distress, or morbidity will be closely monitored.

Research team and facilities

The proposed studies will be conducted at iMed.ULisboa (Faculty of Pharmacy, University of Lisbon) in collaboration with Dr. Manuela Gaspar, who has certification from Direção Geral de Alimentação e Veterinária (DGAV), and facilities for conducting animal experimentation studies according to European regulations. This collaborative environment will foster the applicant’s development as a scientist and provide essential networking opportunities.

Expected Outcomes and Impact

This project aims to demonstrate the feasibility and advantages of a novel inhaled bacterial lysate immunization strategy. Success will support further pre-clinical studies and offers potential for translation into improved prevention for respiratory infections. The research findings are to be disseminated through presentations at scientific conferences and meetings. Emphasis will be placed on international meetings relevant to pulmonary drug delivery and vaccine development. Specifically, we intend to present the obtained results at the Drug Delivery to the Lungs conference.

Applicant Career Development

This project provides interdisciplinary training in formulation development, biological evaluation, and immunology, enhancing the applicant's skills as a pharmaceutical scientist and vaccine development researcher. Throughout the course of the in vivo study, the applicant will acquire diverse skillsets spanning other aspects of the development of a pharmaceutical formulation not addressed so far, including animal experimentation, immunology, and histopathology, with the corresponding data analysis and scientific communication. Particularly, the understanding of immunology will expand exponentially with the analyzes of systemic and mucosal immune responses, performance of bronchoalveolar lavage procedures and ELISA assays, and dissect cellular responses. Moreover, the applicant will hone crucial skills in ethical animal handling, model selection, immunization protocol execution (both inhalation and oral methods), challenge experiments, and operating nose-only inhalation device.
Upon completion of this project, the applicant will emerge as a versatile pharmaceutical scientist, uniquely positioned to excel in various roles related to vaccine development and mucosal immunology. Some possible career trajectories would include becoming a researcher in respiratory drug delivery, working in academia or industry on the development of novel inhalation therapies. There would be opportunities to become a formulation scientist, specializing in the creation and optimization of inhalable formulations for a variety of respiratory conditions. An additional path would be exploring respiratory therapy research, independently investigating mechanisms of the respiratory system, and designing innovative inhalation-based treatment strategies. With the acquisition of some particular skills in the field of immunization, the applicant might also seek opportunities in the development of inhaled vaccines.

References:

[1] Forum of International Respiratory Societies. The global impact of respiratory disease. Third Edition. European Respiratory Society, 2021. Accessed 22 September, 2021. firsnet.org/images/publications/FIRS_Master_09202021.pdf
[2] S.C. Kearney, M. Dziekiewicz, W. Feleszko, Immunoregulatory and immunostimulatory responses of bacterial lysates in respiratory infections and asthma, Annals of Allergy, Asthma & Immunology, 114 (2015) 364-369.
[3] EMA, Bacterial lysate medicines for respiratory conditions to be used only for prevention of recurrent infections, in, European Medicines Agency, 2019.
[4] M. Hellfritzsch, R. Scherließ, Mucosal vaccination via the respiratory tract, Pharmaceutics, 11 (2019) 375.
[5] S. Rodrigues, A. Alves, J. Cavaco, J. Pontes, F. Guerreiro, A. Rosa da Costa, F. Buttini, A. Grenha, Dual antibiotherapy of tuberculosis mediated by inhalable locust bean gum microparticles, International Journal of Pharmaceutics, 529 (2017) 433-441.
[6] A. Grenha, A.D. Alves, F. Guerreiro, J. Pinho, S. Simões, A.J. Almeida, M.M. Gaspar, Inhalable locust bean gum microparticles co-associating isoniazid and rifabutin: Therapeutic assessment in a murine model of tuberculosis infection, European Journal of Pharmaceutics and Biopharmaceutics, 147 (2020) 38-44.
[7] J. Pinto-da-Silva, J. Cruz, A. Grenha, Locust bean gum microparticles as carriers for lung delivery of bacterial lysates, Drug Delivery to the Lungs Conference 2022, Edinburgh, Scotland, 2022, pp. 544-547.
[8] J. Pinto-da-Silva, M. Berzosa, A. Delgado-López, C. Gamazo, A. Grenha, Repurposing bacterial lysates: an inhalation approach for respiratory infection management, Drug Delivery to the Lungs Conference 2023, Edinburgh, Scotland, 2023, pp. 106-109.
[9] S. Vyas, S. Dhoble, V. Ghodake, V. Patravale, Xyloglucan based mucosal nanovaccine for immunological protection against brucellosis developed by supercritical fluid technology, International Journal of Pharmaceutics: X, 2 (2020) 100053.
[10] E. Darzi Eslam, S. Darvish Alipour Astaneh, I. Rasooli, S. Nazarian, A. Jahangiri, Passive immunization with chitosan-loaded biofilm-associated protein against Acinetobacter baumannii murine infection model, Gene Reports, 20 (2020) 100708.
[11] S. Roberts, J.R. Scott, L.K. Husmann, C.A. Zurawski, Murine models of Streptococcus pyogenes infection, Current protocols in microbiology, Chapter 9 (2006) Unit 9D.5.
[12] D. Sanchez-Guzman, P. Le Guen, B. Villeret, N. Sola, R. Le Borgne, A. Guyard, A. Kemmel, B. Crestani, J.-M. Sallenave, I. Garcia-Verdugo, Silver nanoparticle-adjuvanted vaccine protects against lethal influenza infection through inducing BALT and IgA-mediated mucosal immunity, Biomaterials, 217 (2019) 119308.

Ho Wan (Howard) Chan is a final-year PhD Candidate in the Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, the University of Hong Kong (HKU). He obtained his BPharm degree (with First Class Honours) from HKU in 2020 and registered as a pharmacist in Hong Kong in 2021. He is now pursuing a PhD under the supervision of Associate Professors Prof. Aviva Chow and Prof. George Leung, with the financial support of the Hong Kong PhD Fellowship Scheme and the HKU Presidential PhD Scholarship.

Howard's research interest is in developing orally/nasally inhalable nanoagglomerate formulations for pulmonary/nose-to-brain drug delivery. He has published ten peer-reviewed articles to date, including two original research articles in the International Journal of Pharmaceutics and a review article in AAPS PharmSciTech under the special issue "Advances in Drug Delivery by Inhalation - Official Collection from AAPS Inhalation & Nasal Community (INC)" as first author. He has received several accolades during his PhD studies, including the prestigious Sir Edward Youde Memorial Fellowship for Research Postgraduate Research Students 2023/24 (awarded only to three full-time research postgraduate students in Hong Kong annually), the HKU Research Postgraduate Student Innovation Award 2022/23, and a Silver Medal at the 49th International Exhibition of Inventions Geneva. Patent applications in the US and China have been filed for his invention of a continuous manufacturing platform for inhalable nanoagglomerate powders.

In vitro NSCLC organoid efficacy screening platform for inhaled chemotherapeutics

Lung cancer is the deadliest and 2nd most common cancer type worldwide, accounting for 1.8 million deaths annually. Non-small cell lung cancer (NSCLC) constitutes 85% of lung cancer cases and displays a poor response to chemotherapy. Novel treatment strategies are urgently required to improve NSCLC treatment efficacy and survival rates.

Effective chemotherapy requires efficient delivery to the primary lung tumor. Yet, conventional intravenous infusion can only deliver 5 – 10% of the administered drug dose to the lungs, with even lower penetration into primary tumors. Direct aerosol inhalation enables localized pulmonary chemotherapy accumulation and reduces systemic adverse effects by reducing off-target distribution. However, clinical translation of inhaled chemotherapeutics remains sluggish due to insufficient preclinical models that appropriately predict treatment efficacy. Sub-confluent 2D monolayers or 3D spheroids commonly used for in vitro drug testing do not fully represent human tumor architecture and function, while animal models can be challenging to construct and pose significant animal ethics concerns. Organoids have garnered considerable interest in disease modeling and drug screening as they preserve tumor morphology and genetics after long-term culture while being relatively easy to construct. They are also considered valuable alternatives to animal studies for seeking regulatory approval with growing public sentiment against animal research. However, organoids cultured using standard protocols do not mimic direct contact of the lung epithelia with gas, a key feature of respiratory tract structure and function.
The proposed project aims to develop an NSCLC organoid efficacy screening platform for inhaled chemotherapeutics using inhalable paclitaxel nanoagglomerate dry powder (PTX-NADP; Chan et al., Int J Pharm, 2024;653:123877) as the model formulation. Organoid lines will be established from surgically resected NSCLC tissues by Matrigel-embedded 3D culture and used to generate 3D air-liquid interface (ALI) cultures by seeding organoids onto collagen gel-containing Transwell® inserts to mimic the direct contact of the lung epithelium with the gas surface. Upon successful ALI culture, organoids will be treated with different doses of PTX solution by direct PTX inoculation, nebulized PTX/redispersed PTX-NADP using an Aerogen Solo® nebulizer or aerosolized PTX-NADP by dispersing PTX-NADP using a syringe enclosed by a cone to minimize environmental aerosol contamination. The organoids’ growth curve will be monitored by brightfield microscopy, and staining will be conducted for confocal laser scanning microscopy and flow cytometry evaluation of live/dead cells. Western blots will be performed to probe the molecular antitumor mechanism. If successful, the organoid platform will be extended to evaluate other novel inhaled NSCLC treatments, such as targeted therapies and immunotherapies, ultimately expediting their clinical translation.

My interest in respiratory pharmaceutical research stemmed during my pre-registration pharmacy internship in the early wave of COVID-19. I encountered first-hand the unmet medical needs of patients with various respiratory conditions, including COVID-19, lung cancer, and chronic obstructive pulmonary disease. I was highly fascinated by the application of nanotechnologies in medicine when taking my undergraduate drug delivery courses and my final-year research project during my BPharm studies at the University of Hong Kong (HKU). My interests in inhaled drug delivery and nanotechnology have motivated me to continue my research journey as a PhD student at my alma mater. My PhD research focuses on developing nanoagglomerate dry powder formulations for advanced pulmonary drug delivery. The ongoing research work has already led to the successful development of inhalable remdesivir and paclitaxel nanoagglomerate dry powders with promising therapeutic potential, with two first-author original research articles published in the International Journal of Pharmaceutics and two related first-author review articles published in AAPS PharmSciTech and Journal of Pharmaceutical Sciences.

Despite considerable efforts to develop inhalable nanotherapeutics, very few inhalable nanomedicine products have been approved by regulatory authorities, indicating a translational gap between research and pharmaceutical development. Challenges in clinical translation of inhalable nanomedicines, as discussed in our recently published review article (Chan et al., AAPS PharmSciTech, 2023;24(4):98), include manufacturing scale-up and process control and articulation of preclinical data and clinical consideration. To tackle the challenge of manufacturing scale-up, my PhD research work has led to the successful development of an integrated continuous manufacturing platform for inhalable nanoagglomerate dry powders, which eliminates batch-to-batch variations and provides easier process control and greater scalability (Chan et al., Int J Pharm, 2023;644:123303). Several accolades were won based on this invention, including the HKU Research Postgraduate Student Innovation Award and a Silver Medal at the recent 49th International Exhibition of Inventions Geneva.

This proposed project aims to address the difficulty in articulating preclinical data with clinical considerations for inhalable (nano)chemotherapeutics. We have previously demonstrated the superior in vitro antitumor efficacy of inhalable paclitaxel nanoagglomerate dry powders (PTX-NADP) compared to unformulated paclitaxel in 2D A549 lung adenocarcinoma cell monolayers (Chan et al., Int J Pharm, 2024;653:123877), and studies are ongoing to demonstrate their in vivo efficacy in orthotopic lung cancer models. However, significant challenges are encountered before the successful development of these models due to the surgical skills required for orthotopic tumor inoculation and endotracheal intubation for intratracheal drug administration. These procedures may also cause unintentional pain or discomfort to animals, which poses concerns in animal ethics. Indeed, the use of animals for pharmaceutical research has been an active topic of debate. With the US FDA recently announcing that animal studies are no longer a prerequisite for premarket applications, this ignites my strong research interest in developing a novel analytical method evaluating in vitro performance for inhaled chemotherapeutics with a close correlation to clinical efficacy. To this end, an NSCLC organoid efficacy screening platform for inhaled chemotherapeutics is proposed in my research proposal.

As the proposed work involves clinical specimens of NSCLC tissues, we will collaborate with Prof. David CL Lam, Clinical Associate Professor and Chief of the Respiratory Medicine Division in the Department of Medicine, School of Clinical Medicine, HKUMed and Honorary Consultant in the Department of Medicine in Queen Mary and HKU-Shenzhen Hospitals. Prof. Lam is a respiratory physician with a research interest in translational research in lung cancer and has extensive experience in establishing new lung cancer organoid models in his laboratory. He and his team’s expert insights into the design of the proposed 3D air-liquid interface-cultured organoid models will bring synergy to the project’s success in providing novel clinically relevant models for inhaled chemotherapeutic treatment screening.

The award of the DDL Career Development Grant will allow me to gain exposure to the field of organoid research, which I had not been exposed to in my current studies and are currently not available in my department and inspire me to integrate organoid and organ-on-a-chip technologies in my future research. Organoid antitumor performance results from the proposed project will complement my ongoing in vivo efficacy studies on PTX-NADP to further enrich the quality of the manuscript in preparation for publication in prestigious academic journals. At the same time, through dissemination of the research outcomes of the proposed project at the DDL Conference and other oncology or drug delivery conferences, I aim to polish my presentation skills and initiate further collaborations with other members of the scientific community to enhance translational research of inhaled chemotherapeutics further. I will also explore establishing commercial opportunities upon the successful development of the NSCLC organoid platform, such as inhaled chemotherapeutic screening services or platform licensing via opening a start-up. In the long run, the Grant will assist me in establishing an independent and comprehensive research portfolio in inhaled drug delivery from formulation, manufacturing, and physicochemical characterization to preclinical evaluation, such that I can continue to pursue my initiatives to improve the quality of life of patients suffering from respiratory conditions and allow them to “Inhale the future.”

Dr. Ashlee Brunaugh is an Assistant Professor in the University of Michigan, Department of Pharmaceutical Sciences. Dr. Brunaugh’s lab at the University of Michigan works on the development of inhaled and systemic drug delivery systems for improved treatment of respiratory diseases. Her major research interests center on the development of new approaches to understand how drugs interact with delivery systems and biological interfaces present in the respiratory tract. These findings are then utilized by her lab in several ways, including 1) the development of new particle engineering approaches and delivery systems to achieve fine-tuned control over macro- and micro- drug distribution patterns and drug release profiles to maximize target engagement; 2) identification of product and human factors which impact performance of orally inhaled and nasal drug products (OINDP) to facilitate generic OINDP development; and 3) formulation approaches to reduce cost and enhance stability of protein therapeutics for respiratory diseases. Dr. Brunaugh also has prior experience in entrepreneurship, having co-founded a company focused on the commercialization of repurposed drugs for respiratory infections (CloXero Therapeutics), and worked several years as a practicing pharmacist in a variety of patient care settings. These combined experiences enhance the clinical translatability of her research approach.

Development of an image-analysis based platform to monitor dissolution and mucosal diffusion of inhaled drug particles

There exists no standardized assay to assess dissolution and mucosal diffusion for inhaled drug products. The major goal of this project is to optimize and validate an image-analysis based approach to monitor the dissolution rate of drug particles on a substrate. The system is designed to be coupled with existing flow-through cell apparatus designs (e.g., DissolvIt) to provide a surface-level view of particle dissolution following deposition. The novelty of our approach is that dissolution and mucosal diffusion will be monitored simultaneously by two methods: 1) the particle diameter shrinkage rate, as assessed by image analysis; and 2) appearance rate of the drug in the flow-through media. We hypothesize discrepancies between the two methods (i.e., a low mass balance) will indicate the potential for mucus to limit the bioavailability of the drug product.

We will first optimize our image analysis algorithm to enhance its ability to discriminate between critical quality attributes of inhaled drug particles. We will utilize a library of drug particles with distinct physicochemical properties. Powders will be dispersed in a single layer onto the surface of a substrate. This will then be placed in the flow through cell unit (containing simulated lung lining fluid) under microscope, and a video will be recorded for two hours. The recording will be separated into individual frames, with an automated script performing the subsequent processing and analysis. Briefly, each frame will be converted into a binary image, undergo edge detection, water-shedding, and contouring to delineate individual particles, and the Feret's diameter will be measured for each detected particle. The difference in size values representing a particular interval of time would then be used to calculate the rate of dissolution across that time interval. We hypothesize that for particles of a similar size distribution, early-stage dissolution phenomenon will vary as a function of particle surface attributes. We will compare our results against those obtained using an alternative aerosol dissolution assay, such as the modified NGI cup.

We will next determine the ability of our assay to capture the impact of mucus-drug interactions on dissolution and diffusion rate. We will perform a preliminary analysis to determine which members of our library exhibit bulk molecular interactions with airway mucus using an equilibrium dialysis assay. Library members at either extreme will be subjected to the dissolution method described above, with the addition of human sputum as the dissolution medium. We will concurrently observe appearance of the drugs in the flow through perfusate using LC/MS. We hypothesize that discrepancies in mass balance between the two methods will be correlated to the extent of mucus-drug binding identified in the equilibrium dialysis assay, and this discrepancy will be greater for poorly water soluble drugs. One year is requested for the proposed work.

I am currently a tenure-track Assistant Professor at the University of Michigan (located in Ann Arbor, MI, USA). My lab was established in November 2021. My PhD training was completed under the direction Dr. Hugh Smyth (University of Texas at Austin), and my research career has been primarily focused on the development of inhaled drug products for the treatment of respiratory infections. In addition to my academic training, I have also worked as a practicing pharmacist in a variety of patient-care settings following obtainment of my PharmD in 2016, and following my PhD, I helped establish two pharmaceutical start-ups (Via Therapeutics and CloXero Therapeutics). These experiences have provided me a pragmatic approach to my current research endeavors, and I strive to develop inhaled drug products that can quickly move forward from the preclinical stage to the clinical stages.

In my current role, I propose to expand my Ph.D. and post-graduate research through the creation of a world-class respiratory drug delivery laboratory at the University of Michigan College of Pharmacy (the first of its kind at this institute). A current major research interest of my group is to model the interactions between drugs and the lung environment and understanding how these interactions impact drug distribution in the lungs and drug efficacy. We then utilize these findings in the development of cost-effective drug products to 1) resolve and prevent occurrence of opportunistic infections in chronic disease states and 2) reduce the hyperinflammatory response noted in acute and chronic respiratory disease. These studies are also important in establishing a pathway for generic inhaled drug product development, which will overall improve patient access to these lifesaving technologies and reduce the economic burden for healthcare.

Development of an improved aerosol dissolution assay would greatly facilitate the achievement of my research vision, as such work is quite preliminary in nature and not supported through typical funding mechanisms available in the United States through National Institutes of Health. For this reason, in spite of its potential utility, it is unlikely to be supported by traditional extramural funding routes. However, I believe the development of an image-analysis based approach to inhaled drug dissolution monitoring, particularly one which uses equipment already available in many aerosol research labs, could have far-reaching impact on the DDL community in addition to solidifying my position as an independent researcher.

Based upon my prior training and experiences, as well as the support provided through my institute, I am well-equipped to oversee all aspects of this proposed work and successful complete all milestones within the one-year timeline. This career development award will provide an important foundation for the continued development of my laboratory and will enable me to make a substantial impact on the field of inhaled drug delivery.

Mr. Sagar R. Pardeshi is an Assistant Professor in the Department of Pharmaceutics at St. John Institute of Pharmacy & Research (SJIPR), Palghar, Maharashtra, India. He earned his M. Pharm in Pharmaceutics and is currently pursuing his Ph.D. under the supervision of Dr. Jitendra Naik, Senior Professor at the University Institute of Chemical Technology, Jalgaon. His research focuses on dry powder inhalation formulations and ophthalmic drug delivery systems.
Sagar's current research project involves the development of a Combined Microfluidic and Drying Process for Continuous Fabrication of Mannosylated Dry Powder Inhalation Formulations for the effective treatment of tuberculosis. This novel drug delivery system aims to enhance drug deposition directly into the lungs, specifically targeting alveolar macrophages.

He has authored numerous research articles related to pulmonary drug delivery, published in journals such as the International Journal of Biological Macromolecules and Drying Technology. Sagar has contributed to over 30 publications in renowned journals published by leading publishers such as ACS, Elsevier, Springer, Taylor & Francis, and Wiley. His Google Scholar profile reflects 480 citations, with an H-index of 13 and an i10-index of 16. Sagar has also guided postgraduate students on projects related to novel drug delivery systems and biomaterials for tissue engineering. He actively reviews for several scientific journals and is a registered pharmacist with the Maharashtra State Pharmacy Council. Additionally, he is a life member of the Indian Pharmaceutical Association and the Indian Society of Technical Education.

Combined Microfluidic and Drying Process for Continuous Fabrication of Mannosylated Dry Powder Inhalation Formulation for Effective Therapy of Tuberculosis

Tuberculosis, affecting one-third of the world's population, is a deadly and highly contagious disease caused by Mycobacterium tuberculosis (M. TB), an intracellular pathogen. While oral administration is a common route for anti-TB drug delivery, issues such as limited drug availability, short biological half-life, and enzymatic barriers hinder their efficacy. Thus, enhancing TB treatment through drug delivery strategies is imperative. Direct lung delivery presents a promising alternative to address these limitations.

This study aims to develop Mannose-anchored mucoadhesive dry powder inhalation formulations (DPIs) containing anti-tubercular (Anti-TB) drugs. Leveraging the affinity of the mannose receptor for M. tuberculosis, the mannosylated formulation targets alveolar macrophages for effective TB therapy. Traditional methods of creating respirable drug particles involve crystallization followed by milling, necessitating further processing for stability. Our research innovatively utilizes a microfluidic approach combined with thin film freezing (TFF) technology to prepare mannose-anchored fine drug particles. By bypassing the need for additional homogenization or stabilization, this approach offers enhanced formulation stability. We believe that the TFF technology will enable many of these medications to be formed into the convenient, direct-to-lung dry powder inhaler, allowing drug delivery directly to the target site for the first time.

The TFF technology produces particles that are ideal for DPI delivery. The process produces a "Brittle Matrix Particle," which has a low bulk density, a large surface area, and an amorphous morphology, enabling the particles to supersaturate once they come into contact with the target, such as lung tissue. According to lab experiments, the aerodynamic attributes of the particles allow for up to 75% of the drug to be deposited in the deep lung. The TFF process entails dissolving therapeutics in a solvent, which may include agents meant to enhance dispersion and avert clumping, as well as excipients to enhance adhesion to the target site.

Using a T or Y junction microfluidic reactor feeding directly into the freezing chamber, particulate suspension is obtained, followed by lyophilization. When combined with lactose and dispersed in an inhaler, the resulting DPIs exhibit fine particles with an aerodynamic diameter of 1-5 µm, ensuring good aerosol efficiency. We hypothesize that the low-density brittle matrix obtained can be aerosolized by inhaler shear force, eliminating the need for additional additives in DPI formulations. Furthermore, mannosylated drug delivery vehicles may enhance macrophage uptake, leading to improved activity and reduced side effects, thereby offering a more efficient treatment strategy for tuberculosis.

My research on dry powder inhalations for pulmonary drug delivery systems has significantly broadened my personal and professional horizons as a pharmaceutical solid-state and formulation scientist. Throughout my career, I have actively pursued opportunities to explore various pathways, with a particular interest in respiratory medication delivery research. Currently, I am pursuing my Ph.D. (submitted Ph.D thesis) under the guidance of Dr. Jitendra Naik at the Research Group of the University Institute of Chemical Technology (KBC North Maharashtra University). My research focuses on the formulation and development of dry powder inhalations and ophthalmic formulations.

Recently, I took on the role of Assistant Professor at St. John Institute of Pharmacy and Research, Palghar, where my responsibilities include academic lecturing, student mentoring, and overall class management to provide undergraduate and postgraduate students with a deeper understanding of research methodologies.

I have authored two research articles in the area of respiratory research: "Mannose-anchored N,N,N-trimethyl chitosan nanoparticles for pulmonary administration of etofylline," published in the International Journal of Biological Macromolecules by Elsevier, and "Preparation and characterization of sustained-release pirfenidone-loaded microparticles for pulmonary drug delivery: Spray drying approach," published in Drying Technology by Taylor & Francis. Additionally, I have contributed to several research papers, review articles, and book chapters in the field of microreactor and drying technology. In total, I have published more than 30 research papers, reviews, and book chapters in reputable journals like ACS, Elsevier, Springer, Taylor & Francis, and Wiley, focusing on pulmonary and ophthalmic formulations. My Google Scholar citations are 384, with an H-index of 12 and an i10-index of 15.

The next stage of my research aims to assess the suitability of a combined microfluidic and drying process for the continuous fabrication of mannosylated mucoadhesive dry powder inhalation formulations targeting macrophages for effective tuberculosis therapy. Utilizing mannose-conjugated mucoadhesive carriers such as chitosan and N,N,N-trimethyl chitosan, I will load anti-TB active pharmaceutical ingredients (APIs) into the carrier using microreactor technology in conjunction with thin-film freezing (TFF) to evaluate their impact on dry powder inhalation (DPI) performance. This study will employ APIs like rifampicin and rifabutin to assess the functionality and drug delivery properties of TFF-assisted dry powder inhalations and measure output and aerosol properties. Manufacturing techniques based on microreactors and TFF can address persistent issues in inhalation drug delivery due to engineered particle shapes. Although still in its early stages, recent advancements have enhanced the utility and applicability of these techniques. However, access to appropriate manufacturing technology for complex designs in high resolution remains a significant challenge.

Financial and technical support is crucial for the development of this research. A DDL Career Development Grant would mark a significant turning point in my interdisciplinary career as a scientist specializing in lung drug delivery. My long-term objective is to compile a comprehensive scientific report detailing the preliminary investigations of intricate dry powder inhalations. I envision continued research in academic and institutional settings, as this topic remains substantially unexplored. My ultimate goal is to contribute to the advancement of this emerging field of study and present my research to an international audience of aerosol scientists. The research outcomes will be published in reputable peer-reviewed journals. If successful, this proposed research will establish a method for consistently producing inhalable particles of drugs at a larger scale, demonstrating sufficient storage stability and performance efficiency.

This work will enhance our understanding of API processing techniques in DPI-based formulations, particularly for drugs with poor aqueous solubility. It may also enable the production of drug-polymer mixtures suitable for pulmonary delivery, with the potential for technology transfer from lab to industry. Pulmonary drug delivery systems have always intrigued me, and I am eager to delve deeper into this field as a formulation scientist. This research will also provide valuable insights into dry powder inhaler systems.

Mr. Maan Singh is a Ph.D. research scholar working under the supervision of Dr. Dinesh Kumar in the Pharmaceutical Solid-State Research Laboratory (PSSR) at the Indian Institute of Technology, Banaras Hindu University, Varanasi, India. He is an expert in crystal habit modification of the APIs without changing their internal structure utilizing ultrasonication. He worked for two years (2022 to 2024) as a junior research fellow (JRF) on a project entitled “Crystal Engineering to Improve its Pharmaceutical Properties”. He has one granted German patent and has published four review articles and two research articles in reputed peer-reviewed international journals. Mr. Maan Singh also has experience in the Intellectual Property domain at IQVIA, India, from September 2020 to March 2022. He is an expert in thorough secondary searches for patents on over 30 countries' patent websites.
During his undergraduate program, he developed a keen interest in pursuing further research in the pharmaceutical sciences and cleared the national level examination Graduate Pharmacy Aptitude Test (GPAT) in the year 2018. Through this examination, he secured a scholarship award from the All India Council for Technical Education (AICTE), India, for pursuing his Master’s degree. Further, he completed his Master of Pharmacy (2-year postgraduate program) from Babasaheb Bhimrao Ambedkar University, Lucknow, Uttar Pradesh, India. He joined Sun Pharmaceutical Industries Limited (R&D-II), Haryana, India as an intern to complete his Master of Pharmacy thesis project entitled “Optimization and Evaluation of Pharmaceutical Supramolecular System of Itraconazole utilizing co-crystals”.

Explore the applicability of the sonocrystallization technique to produce DPI formulations for the treatment of fungal infection in the lungs

Lung infection is a major health issue worldwide and occurs when a virus, bacteria, or fungus gets into the lungs and causes inflammation. Invasive pulmonary aspergillosis (IPA) is a major lung infection caused by a fungus called Aspergillus and is now the leading cause of death due to invasive fungal infections worldwide.

Presently, anti-fungal drugs (terbinafine, amphotericin-b, itraconazole, etc.) are used to treat IPA. Sometimes these drugs are ineffective in treating fungal infections and need surgery to remove the fungus ball from the lungs which is painful and costly. Current strategies such as oral or parenteral delivery of these drugs are inefficient as they require very high doses to achieve optimal drug concentration at the site of infection. Itraconazole is widely used as an anti-fungal drug for the treatment of IPA administered through oral route, but due to its poor aqueous solubility, a high dose is required.

Dry powder inhaler (DPI) through the inhalational route could be a better option for a direct delivery of itraconazole to treat the IPA. The surface area of drug particles in DPI is increased due to reduced particle size (1-5µm) which increases solubility and ultimately reduces drug dose. Through this route, the drug can be inhaled directly into the lungs where the drug start showing its action. Generally, particles with good flowability are desired for delivery of drugs to the lungs. Itraconazole possesses poor flowability due to needle-shape crystals. This hinders the direct delivery of itraconazole to the lungs in the form of DPI. Till now, several approaches have been explored to obtain particles with acceptable physicochemical properties such as micronization, spray drying, spherical crystallization, etc. Micronization causes electrostatic charge development on the particle surface which causes poor flowability. Spray drying results in poor yield & doesn’t always produce fully dried particles and requires additional processing to dry it completely. Spherical crystallization is not a well-established process and might result in the breakdown of spherical agglomerates during storage and transportation.

Sono-crystallization is an ultrasound-based technique for modifying crystal habit & obtaining desired crystal size distribution. Ultra-sound energy enhances the nucleation rate and, therefore, produces smaller crystals with uniform size distribution. Narrow crystal size distribution, modified crystal habit, and reduced particle size results in improvement in the aqueous solubility & flow properties. The project outcome shall give a piece of detailed information on the applicability of sonocrystallization to make DPI for the treatment of invasive pulmonary aspergillosis infection. I believe that this research work provides valuable insights about the applicability of sonocrystallization in producing DPI formulations. If successful, we will explore the scalability of sonocrystallization to produce DPI on a large scale.

As a research scholar in the pharmaceutical sciences, I always tried to develop cost-effective novel formulations to treat fungal infections at low doses. My interest was developed in new formulations during B. Pharm (four-year undergraduate program, 2014-2018). During this program, I learned the basis of drug delivery and development related to different dosage forms and their route of administration. My curiosity about designing the novel formulation experimentally developed during the M. Pharma (two-year post-graduate program, 2018-2020). I worked for two years on a project entitled “Optimization and Evaluation of Pharmaceutical Supramolecular System of Itraconazole Utilizing Co-Crystals” at R&D II, Sun Pharmaceutical Industries Limited, Haryana, India. I explore the utility of cocrystallization to enhance the solubility and dissolution rate of itraconazole. The solubility of itraconazole cocrystal is improved by approximately 123% compared to plain itraconazole API. Itraconazole cocrystal showed a 4-times higher initial dissolution rate at a 15-minute time point and later gave a similar dissolution profile compared to the marketed formulation. This project motivated me to go deeper to explore the research gap in the field of pharmaceutical sciences.

I got the opportunity to work as a junior research fellow (JRF) on the Science and Engineering Research Board (SERB) sponsored project entitled “Crystal Engineering of Dapagliflozin to Improves its Pharmaceutical Properties” under the supervision of Dr. Dinesh Kumar in the Department of Pharmaceutical Engineering and Technology, Indian Institute of Technology, Banaras Hindu University, India. In this project, we were able to modify the crystal habit without changing its internal structure by using the sonication to improve the solid-state properties.

Currently, I am exploring the applicability of sonocrystallization in controlling nucleation rate and crystal growth, improving the flowability and dissolution rate of the APIs, etc. These experiences have reinforced my interest in pharmaceutical research to produce DPI formulations for the treatment of fungal infections in the lungs with the help of sonication. I believe that the sonocrystallization technique can be helpful for improving physicochemical properties of the APIs and enable its delivery in the form of DPI to the lungs. I am hoping that this research work with the help of the DDL Career Development Grant will take me one step forward to boost my research capabilities and motivate me to go deeper to explore the possibilities of producing the DPI on a large scale.

Taye Tolu Mekonnen is a postdoctoral research associate at the University of Sydney, specializing in imaging technologies for diagnosis and treatment. He earned his PhD in Biomedical Engineering from Macquarie University in 2020, where he developed a pioneering imaging technology known as multichannel optical coherence tomography (MC-OCT). This patented device has significant applications in high-speed imaging of upper airway dynamics and non-destructive industrial testing. He was a recipient of the international Macquarie University Research Training Program (iMQRTP) scholarship for his doctoral studies.

After completing his PhD, Dr. Mekonnen began his academic career in Ethiopia as a lecturer at Jimma University. He then moved to the University of Houston as a postdoctoral researcher, where he significantly contributed to the development of optical elastography methods, including a novel form of reverberant optical coherence elastography (OCE), to characterize tissue biomechanical properties. Since January 2023, he has been advancing his research at the University of Sydney, focusing on developing efficient imaging technologies aimed at reducing the cost of inhalers through faster and more effective screening methods.

With a strong foundation in computer engineering (MSc, Addis Ababa University) and electrical engineering (BSc, Bahir Dar University), Dr. Mekonnen is dedicated to enhancing biomedical imaging techniques and their applications in healthcare. His current research interests include advancing and implementing high-resolution optical coherence tomography and optical coherence elastography for drug delivery to the lungs.

Development of an optofluidic system to characterize lower airway fluid dynamics

Understanding the intricate dynamics of respiratory airflow and the transport mechanisms of inhaled aerosols in the airways is crucial for advancing respiratory health. This includes evaluating respiratory risks associated with inhaled airborne aerosols and optimizing inhalation therapy for systemic and topical drug delivery. Extensive research has focused on extra-thoracic fluid dynamics to enhance drug delivery efficacy and comprehend the fate of inhaled aerosols. For instance, the complex geometry and relatively high flow rate in the upper airways leads to flow separation, turbulence, and recirculation zones, which in turn affects the deposition patterns of particles in this region. Particle deposition on airway walls in this region predominantly results from inertia impaction. Conversely, lower airway particle transport and deposition are mainly influenced by gravitational and Brownian motion forces. The slow resident respiratory airflow in regions such as the bronchioles play significant role in determining the fate of microparticles, leading to distinct aerosol transport mechanism and deposition patterns.
Characterization of the fluid dynamics in the narrow lower airway has been mostly limited to computational fluid dynamics (CFD). While CFD is powerful for simulating and understanding fluid flow in various regions of the narrow lower airways, modelling particle dynamics and its interactions with airflow, as well as deposition mechanisms can complicate the simulation. The emergence of microfluidic channel offers an opportunity to assess fluid dynamics in this region in vitro, potentially serving as validation tools for CFD and offer insights into the characteristics of in vivo airflow in these regions.
This study aims to introduce a unique approach for characterizing fluid dynamics in microchannels that mimic lower airway regions using optical coherence tomography (OCT). Unlike high-speed cameras and other microscopic imaging techniques which are limited to focal plane imaging, OCT provides a visualization of microchannel cross-sections, enabling a real-time characterization of flow dynamics. Its high temporal and spatial resolution imaging capacity is vital for accurately quantifying dynamic particle behavior, including tracking its motion and deposition dynamics. We have recently demonstrated the capabilities of OCT in imaging sub-surface microstructures of dry powder depositions and two-phase detection at high resolution (< 5 µm) and substantial penetration depth (~2.5 mm) for the first time. Integrating microscale imaging with microfluidic channels holds promise in providing unparalleled insights into the airflow dynamics in narrow airways. The core aims of this study are to: • develop an optofluidic system integrating microfluidic system with our existing OCT system; • examine the dynamic characteristics of aerosols including particle flow patterns, tracking its trajectories, and analysing retention time and deposition behaviour.

As a postdoctoral research associate at the University of Sydney, I have been devoted to advancing optical imaging techniques in the fields of biomedical and respiratory science. In my recent research efforts, I have led the development and optimization of a high-resolution OCT technique designed specifically for analysing the dynamic behaviour of inhalable pharmaceutical powders. Our research, which focuses on characterizing the deposition and dissolution properties of aerosols, with a specific emphasis on applications in inhalation drug delivery, has been provisionally patented.

During my doctoral studies at Macquarie University, I pioneered the development of a novel multichannel optical coherence tomography (MC-OCT) system, a technology which enables the reconstruction of dynamic upper airway geometries during physiological respiration. Subsequently, as a postdoctoral researcher at the University of Houston, I introduced a non-invasive opto-acoustic technique to facilitate a comprehensive assessment of biomechanical properties in soft materials. Building upon these achievements, my current research focus lies in advancing high-resolution optical techniques for evaluating aerosol mechanics relevant to inhalation drug delivery systems. Through this work, I aim to contribute to the development of more effective and targeted drug delivery methods, ultimately improving treatment outcomes.
I am committed to advancing the field of respiratory science through innovative optical techniques. Within this goal, I am exclusively focused on research, specifically dedicated to the development of innovative optical techniques that advance the imaging capabilities of pharmaceutical aerosols. For the next few years, I am committed to establishing myself as a recognized researcher in both Australian and international academic communities. My aim is to build a strong publication record consisting of high-quality and impactful research that is relevant to practitioners both within and outside academia. Securing the DDL Career Development Grant would be of immense value in facilitating this goal, as it would provide me with the necessary resources and dedicated time to further enhance my research output.
In addition to developing my research profile, my short-term goal is to transition into an academic position where I can lead a research group dedicated to advancing optical techniques in the field of inhaled drug delivery. Academic position would provide me with the opportunity to contribute as educator, mentor, and researcher, making significant contributions to scientific knowledge and clinical practice in this domain.
T

To achieve my career aspirations, I recognize the importance of continuous learning, skill development, and networking. I plan to pursue additional training opportunities, such as workshops and courses, to enhance my expertise in areas like grant writing, project management, and teaching pedagogy. I will actively seek out collaborations with leading researchers and industry partners to broaden my research scope and impact. Throughout my PhD and beyond, I have had the privilege to work with and learn from distinguished professors across various fields, including biomedical engineering, fluid mechanics, optical imaging and pharmaceutics. The invaluable training I received from these esteemed mentors has equipped me with the necessary knowledge and skills to conduct impactful research, and they continue to be close and reliable mentors. Recognizing the value of collaboration, I seek to leverage my global network to foster additional partnerships beyond my current mentors at the University of Sydney.

Cheng Ma is currently a 4th-year Ph.D. student in the Department of Pharmacology and Pharmacy at the University of Hong Kong and a visiting scientist in the School of Pharmacy at University College London. Cheng obtained his bachelor's degree and master’s degree (supervisor: Dr. Chuanbin Wu) from Sun Yat-sen University, China. After graduation, he joined Dr. Jenny Lam’s team in the Department of Pharmacology and Pharmacy at the University of Hong Kong as a Ph.D. student. He is an awardee of the HKU Presidential Ph.D. Scholarship. His current research project focuses on peptide-based nucleic acid delivery systems for pulmonary delivery. He aims to develop safe, robust, and effective RNA delivery platforms for the treatment of lung diseases in clinics. He has presented his research on DDL2023 and CRS2024 and published five peer-reviewed articles as the first/co-first author in journals such as Drug Delivery and Translational Research and Powder Technology.

Development of an inhalable mRNA delivery system with high mucus penetration ability for cystic fibrosis

The mRNA therapy emerges as a promising approach for treating cystic fibrosis (CF), leveraging its capacity to produce functional cystic fibrosis transmembrane conductance regulator (CFTR) protein. PEG12KL4 is a cationic peptide-based carrier, capable of binding to mRNA through electrostatic interactions, demonstrating remarkable in vivo transfection efficiency in the lung. (1) However, CF has a highly concentrated mucus layer, composed of a high content of macromolecules such as mucin, globular proteins, and DNA fragments. (2) Upon inhalation, PEG12KL4/mRNA complexes face obstacles in traversing this dense, viscoelastic CF mucus layer due to interactions like steric hindrance, hydrophobic interactions, and electrostatic binding, which decreases the delivery efficacy of PEG12KL4/mRNA complexes to lung epithelial cells. To overcome these barriers, an efficient formulation capable of facilitating the penetration of PEG12KL4/mRNA through the mucus layer is needed. This can be achieved by reducing the positive surface charge of the complexes to avoid their electrostatic interaction with the negatively charged mucin protein. Moreover, enhancing water influx into the airways with osmotic pressure regulators can dilute the mucus and improve the mucus penetration of complexes. Additionally, the co-administration of mucus-clearing agents will help reduce the viscosity of mucus by dissociating the disulfide bond within mucus.
This project aims to develop an inhalable formulation of PEG12KL4/mRNA designed to significantly enhance mucus penetration and transfection efficiency in the CF model. The influence of excipients such as buffer salts, nonionic surfactants, sugar, and mucus-clearing agents on the mucus penetration ability will be evaluated. Commercial artificial pulmonary mucus and artificial CF medium will be employed. The penetration of complexes loading fluorescence-labeled mRNA in mucus will be evaluated using nanoparticle tracking analysis (NTA). Additionally, the in vitro transfection efficiency of the formulation will be tested on lung epithelial cells in the presence of artificial mucus. An air-liquid interface model based on Calu-3 cells will also be employed to evaluate the mucus penetration and transfection ability within the mucus secreted by Calu-3 cells.
References
1. Qiu et al. Journal of Controlled Release 314 (2019): 102-115.
2. Turuvekere Vittala Murthy et al. Advanced Drug Delivery Reviews (2024): 115305.

My research interest in developing pulmonary drug delivery systems began during my Master degree under the supervision of Dr. Chuanbin Wu at Sun Yat-sen University. During that time, I learned about the great clinical therapeutic value of the pulmonary delivery system, which inspired my curiosity and enthusiasm for developing inhaled formulations. Upon completing my Master degree, I had the privilege of joining Dr. Jenny Lam's laboratory at the University of Hong Kong as a Ph.D. student. Since then, I have delved into the promising field of mRNA therapy, captivated by its potential to treat lung disease by expressing specific proteins in targeted tissues. Throughout my Ph.D., my focus was on pioneering a novel peptide-based gene delivery system for the pulmonary delivery of mRNA and siRNA.
Inhaled gene therapy is a promising strategy to cure lung disease, especially for those lacking druggable intracellular molecular targets. However, challenging barriers are hindering the clinical pulmonary application of gene therapy, such as lung deposition of particles, mucus trap, and cell membrane obstruction, and there are urgent needs for systematic studies in these fields. My research goal is to bridge the gap between novel gene therapy strategies and their clinical application for pulmonary diseases.
During my PhD study, I had the opportunity to exchange to UCL School of Pharmacy as a visiting scientist, which equipped me with the experience of various advanced techniques such as nanoparticle tracking analysis and mass photometry. These techniques allowed me to perform an investigation and have a deep understanding of the interaction between particles and lung mucus at the particle level.
My current research ambition is to tackle these mucus penetration challenges by employing several promising excipients. Support from the Drug Delivery to the Lungs (DDL) and the DDL career development grant would be instrumental in advancing this project. The grant would enable me to acquire fluorescence-labeled mRNA to track and evaluate the movement of complexes in the artificial mucus, facilitating the development of a novel peptide-based formulation for mRNA pulmonary delivery. Moreover, it would help establish robust methods to assess the mucus penetration capabilities of our formulations, providing valuable insights for addressing similar challenges in other systems.

Dr Shyamal Das is an Associate Professor and Associate Dean of Research at the School of Pharmacy, University of Otago, New Zealand. Prior to this, he was a research fellow for five years at Monash Institute of Pharmaceutical Sciences from where he completed his PhD. He is also the current President (2023–2024) of the Otago Medical School Research Society and elected President (2024-2025) of the NZ Chapter of the Controlled Release Society.

Dr Das leads the research on dry powder inhalers (DPI) in New Zealand. He focuses on high dose DPIs for lung infections such as tuberculosis (TB), and is leading a Phase I Clinical study for TB treatment. He is recognized leader of powdersurface energy characterization. His current research also includes understanding dissolution in the lungs, microRNA and cannabinoids delivery. Dr Das secured research funding of $2.3M, published >100 peer reviewed publications, most of these are in top quartile journals, and contributed to a further 120 abstracts and presentations. He has been an invited speaker at 25 conferences and seminars including the prestigious RDD Europe, the Inhalation Asia Conference 2015, a keynote speech at an international conference. In addition to mentoring 8 Postdoctoral/Assistant Research Fellows, he is or has been the supervisor of 8 PhD, 1 Masters, 18 Honours, and 83 other research students. His PhD graduates secured positions in prestigious universities and industries. Theses of two students were enlisted as Exceptional Theses in the Division of Health Sciences.
Dr Das is an excellent teacher, and he received multiple awards for teaching from the University of Otago including the ‘Top Five Teachers 2016’, ‘Premier Lecturer 2017 for Health Science Division’, the School of Pharmacy (SOP)’s ‘Excellence in Teaching Award 2016’ and ‘The Engagement Award 2021’and ‘Supervisor of the Year 2017 for the Health Science Division’. He is a UK Council of Graduate Education Recognized Research Supervisor (2022). He was also awarded School’s ‘Emerging Researcher Award 2016’ and the ‘Research Excellence Award 2019’ in the senior category, and the ‘Best Research Paper of the Year Award’ in 2019, 2020 and 2021 from Green Cross Health Pharmacy Awards. He has developed and led BPharm(Hons) Programme and a number of papers.

Dr Das serve on the Editorial Board of International Journal of Pharmaceutics (IJP) and Pharmaceutics, and acted as Editor of two special issues of IJP. He is an Associate Editor of Frontiers of Drug Delivery’s Respiratory Drug Delivery section, and a member of DDL Scientific Committee since 2015. Dr Das was a session chair of conferences, including DDL in and Inhalation Asia conference. He organized a symposium at the APSA Conference in 2021 and a workshop on inhaler therapy at the New Zealand International Science Festival (NZISF) in 2021 and 2023. Dr Das is a sought-after grant reviewer internationally and a Reviewer and Judge for international conferences including DDL, Inhalation Asia and APSA. He has examined 23 PhD and Masters theses and peer reviewed 115 manuscripts for high-impact journals. He was declared a KiwiBank Local Hero 2021 as a part of New Zealanders of the Year competition.

Respiratory drug delivery platform in New Zealand

Respiratory diseases are a major health issue in New Zealand. Although expertise on aetiology, disease mechanisms, infectious agents, drug development, clinical and epidemiological research are spread all over the country, there is no shared platform for these researchers. Moreover, research on formulation for inhaled delivery is only done in the ‘Das laboratory’ in the University of Otago. The goal of this project is to bring lung scientists of the country together to share their knowledge and interact among themselves and globally for greater impact. This platform will support and provide mentorship for academics and postgraduate students through: a) setting up a website to help with outreach efforts. b) organizing workshops, symposiums or conferences; d) learning from opinion leaders of respiratory drug delivery.

The Respiratory Drug Delivery is unique in New Zealand and complementary to the research conducted by many organizations. My laboratory initiated and is now continuing research on dry powder inhaler. My goal is to create a respiratory research platform to bring together expertise currently dispersed across the University of Otago to facilitate a programme bid in respiratory disease to enable translation/commercialisation. The overarching goal is to create a platform for the research and translation of respiratory medicines. This platform will integrate entrepreneurs for translation of research with policy makers, to become a focal point for research translation, communicating with key stakeholders and entrepreneurs in respiratory diseases. This platform will expand national research collaborations to enable: access to translational funding; strengthened cross-disciplinary research; identification of critical expertise and strong external linkages to industry and the community. Thus, this platform is aiming to foster, develop and promote research into respiratory diseases in New Zealand.

Ziru Xu is currently a third-year PhD student at King’s college London. Her supervisors are Professor Ben Forbes, Dr. Simon Pitchford and Dr Magda Swedrowska. She graduated from the Joint College of Queen’s University of Belfast (QUB) and China Medical University (CMU) with First Class Honours in Pharmaceutical Science.
She has lab experiences in different countries. During the undergraduate, she contributed to several projects related to Traditional Chinese Medicine, Metabolic Syndrome, and clinical medication. In 2018, She took part in an international exchange program held by International Federation Medical Students’ Association and visited Federal University of Parana, Brazil. Ziru was also involved in the QUB research project, Comparative study of conventional polymer processing and 3D-printing methods for the manufacture of drug-eluting devices.

Generally, she is interested in lung inflammation and pulmonary delivery of biological drugs. Her PhD project focuses on the development of inhalable platelet-based therapy for acute lung injury. It starts from producing platelet-based bioproducts and explores their therapeutical effects on lungs. How to delivery bioproducts to lungs effectively is also an important part of her project.

Develop Respirable Powder of Human Platelet Extract by Freeze Dry/ Spray Freeze Dry

Platelet-related products have already been applied in skin and skeleton as a form of regenerative medicine, amplifying the natural growth factors to heal tissues. To apply platelet growth factor directly on lung, we developed sonicated platelet extract that showed the potential to repair lung epithelial barrier damage on cells. It could form a nice aerosol to reach the deep lung by mesh nebulizer. This autologous biologic in liquid form is far from large-scale application as it requires qualified staff for blood manipulation and cold storage conditions. Making the liquid biomolecular-based drugs into powder is a common way to prolong their shelf life. Especially for platelet-related products, drying into solid state means a start to transfer clinic-based therapy to a ready-to-used commercial products. Among different drying techniques, a proper choice is significant for the quality attributes of the final product.
Freeze drying (FD) is the most common way to remove solvent by sublimation and desorption, but this method is time-consuming, expensive, and creates flaky particles. Another renowned method is spray drying (SD), which transforms aqueous samples into dried particulate forms by atomizing the feed solution into a hot drying medium. However, it is not suitable for heat-sensitive drugs. A combination of FD and SD is called spray freeze dry (SFD). There are three steps in the drying process. It starts from dispersion of bulk liquid solution into droplets, which is called atomization. The next freezing step is solidification of droplets by direct contact with the cold fluid. In the end, solidified droplets experience sublimation at a very low temperature and pressure. The products of SFD have some favorable physical properties, such as spherical structure, high specific surface area, and low density.
Dry powder inhalers (DPI) are activated by the patient’s airflow and don’t need hand-breath coordination. They also have some other advantages such as compactness, portability, and rapid delivery time.
In this project, we are going to find the best formulation to make human platelet extract into respirable powder by freeze drying or spray freeze drying. Developing aqueous platelet extract to solid form is the first step to commercialize platelet-related products and standard their production and doses. Administration by dry powder inhalers means these biologics could be daily used by patients themselves. It saves time and costs for patients as they don’t need venepuncture with the help of qualified staff at the point of care. Off-the-shelf products also make sure timely use in case of emergency and benefit the patients who have blood diseases. It would be a milestone for the application of platelet-related products and a new way to maintain lung homeostasis.
Aim
1 Make human platelet extract into bioactive and respirable powder of by freeze dry or spray freeze dry
2 Find the best dry powder formulation for pulmonary delivery

1 General statement
Since platelet therapies have been well studied and applied on skeleton and skin, we tried to develop a respirable human platelet product sample for lung diseases last two years. As the first defense line in the lungs, the epithelium can be damaged by infections, inflammation, toxic compounds, and trauma. It is critical to recover a healthy epithelial barrier after lung injury. Platelets produce many different types of growth factors which promote cell growth and tissue regeneration. We investigated the regenerative role of platelet extract on lung alveolar epithelial cells and the potential for development into an inhaled medicine. To gain the extract, platelets were subjected to ultrasound sonication to release their cell contents. Cell proliferation assays using A549 cells showed that sonicated platelet extract enhanced alveolar epithelial cell growth in a time- and concentration- dependent manner. When nebulized with an Aerogen Pro® mesh nebulizer, the extract produced an aerosol with a fine particle fraction below 5 μm of ca. 50%. In conclusion, sonicated platelet extract was effective at promoting alveolar epithelial cell growth and can be nebulized to form a bioactive respirable aerosol. However, aqueous platelet-related products all face some disadvantages, like clinic-based production, poor stability, and high storage requirement. We bring the idea developing new formulations to make this bioproduct into powder. This is a revolution for the administration of platelet-related products. This project will be a collaboration between Professor Ben Forbes at King's College London and Dr. Jenny Lam at University of London.
2 Experiment Design
2.1 Preparation
2.1.1 Prepare sonicated platelet extract from fresh human blood
Sonication is a rapid and efficient method to break platelet membrane and release platelet content to gain platelet extract. To minimize the variation among individuals, two parameters will be controlled in this study. One is to dilute the platelet density to the same level before disrupting platelets and after sonication, platelet extract will be diluted to a certain protein level for the following experiments.
2.1.2 Make powder by freeze drying or spray freeze drying
Pressure and temperature are the key parameters to control during freeze-drying. Besides stabilizing excipients may be needed to protect the biological activity of protein from several stresses both during freezing and drying. These encompass a wide variety of compounds including sugars, polyols, polymers, surfactants, and amino acids.
Atomization is the primary stage of spray freeze drying. There are many choices of nozzles, like two-fluid, three- fluid four-fluid and ultrasonic nozzles, which results in final samples of different properties. The process also determines determining the particle size distribution of the sprayed droplets by adjusting feed viscosity, atomization energy, the feed flow rate and surface tension. Similar with SD, a combination of excipients is usually applied to the protein formulation in SFD. Common excipients used in dry powder formulations include Polyols (such as mannitol), sugars (such as lactose and trehalose) and surfactants (such as polysorbates 20 and 80).
2.2 Evaluation
2.2.1 Physical Properties
2.2.1.1 Scanning Electron Microscopy (SEM)
A scanning electron microscope will be used to obtain the morphology of SFD microparticles. Morphology of the SFD-based particles is a function of variables such as the drying phase, spraying, freezing phases, excipients, and solid content. Image analysis of SEM pictures could derive the size distribution of SFD powder.
2.2.1.2 Differential Scanning Calorimetry (DSC)
The thermal behavior and crystallization tendency of the powder formulations will be studied by differential scanning calorimetry (DSC). The Tg and its onset as well as the energy of crystallization above Tg can be determined.
2.2.1.3 Thermogravimetric Analysis (TGA)
The water content of the powder formulations will be determined by thermogravimetric analysis (TGA). It is a method of thermal analysis in which the mass of a sample is measured over time as the temperature changes.
2.2.1.4 Production Yield
The powder will be restored to liquid form after drying. Then the total protein level will be tested by BCA assay. Compared with the starting protein concentration in the original liquid sample, it could tell the loss of proteins caused by the drying process.
2.2.2 Bioactivity
To assess the protein stability and understand in vitro bioactivity of platelet extract after. drying, the powder would be dissolved in PBS first and diluted with cell medium to a certain protein level before applying on cells.
2.2.3 Aerosol performance
A next generation impactor will be used to evaluate the aerosolisation efficacy of the powder formulations. The deposition profile was defined by the following parameters: recovered dose (RD), emitted dose (ED), emit- ted fraction (EF), fine particle dose (FPD), fine particle frac- tion (FPF), mass median aerodynamic diameter (MMAD), and geometric standard deviation (GSD).
3 Gain
3.1 Access techniques that are not available in King’s lab and more skills
The common techniques to make powdery samples includes freeze dry, spray dry and spray freeze dry. There is just one freeze dryer in King’s College London. Though it is the most widely used drying technology for biomolecules, it still has a lot of disadvantages such as the high processing costs, long cycle times, batch-to-batch heterogeneity, phase separation propensity, and lack of continuous operation. Emerging as a viable alternative, spray freeze drying combines the atomization feature of spray drying with the vacuum drying feature of freeze drying. Dr. Jenny Lam has special interests in the use of particle engineering methods to produce inhaled dry powder formulations for the treatment of respiratory diseases. Buchi B-290 spray dryer and B-90 nano spray dryer are available in her lab, which can convert liquids to dry powders in a gentle, continuous, and scalable drying process. Besides the choices of dryers, several related parameters could decide the properties of final powdery products, such as feed flow rate, atomization energy and excipient types. In the freeze-dried/ spray-freeze-dried biopharmaceuticals area, several key papers are published Dr. Jenny Lam’s teams. With her help, I could gain more knowledge in a short time and carry out experiments much more smoothly.
This cooperation is not only academic communication but also a chance to learn how different research groups work and how they run their lab. The ability of arranging a team or a lab is also important as an independent researcher.
3.2 Enable publication in better scientific journals and prospects of future scientific appointments in academia or industry
So far, my experiments are completed in order of preparing the bioproduct, testing its bioeffect by in-vitro study and exploring the nebulized aerosol performance. To develop a mature biotherapy and make it into a commercial product further, it is essential to simplify and standard the production, control the costs and improve the patience compliance. These goals can be achieved by exploring an ideal formulation. My work will contribute to realize commercial production rather than being limited in biotherapy development. This is a good chance to think about research from industry side from which I could learn how to transfer my research results to the final product that can bring real benefits to patients and create economic value. It also means a rich structure of a publication. The topics will cover from bioproduct study, novel drug delivery and formulation development. Then we could target the journals whose influence impactors are much higher.

Jorge Alves-Silva is a Post-doctoral fellow at the Institute for Clinical and Biomedical Research, at the Faculty of Medicine of the University of Coimbra (UC) and an Invited Assistant Professor at the Coimbra Health School (ESTeSC-IPC). He holds a PhD in Pharmaceutical Sciences, specialty Pharmacognosy and Phytochemistry, from the UC and has focused his research on the preventive/therapeutic potential of plant volatile extracts and metabolites. Over the years, Jorge has demonstrated the bioactive potential of these compounds namely as antifungal, anti-inflammatory, and anti-ageing agents. He is now interested in the clinical translation of this knowledge and aims to develop non-invasive therapeutic approaches, particularly inhalatory strategies. Indeed, despite the undeniable potential of these compounds, several drawbacks limit their clinical translation, such as volatility, lipophilicity and oxidation, and consequently frequent administrations are required. Therefore, strategies that improve their stability, lung retention and sustained release are needed. Moreover, inhalatory modes of administration are more amenable for patients, contributing to higher adherence and consequent increased efficacy. This breakthrough approach consolidates Jorge Alves-Silva research interests and will pave the way for the development of effective inhalatory preventive/therapeutic strategies for lung diseases.

ALVEOLUS - Inhaled Plant Volatiles for Lung Diseases

Chronic respiratory diseases present high mortality and disability rates, particularly in high income countries, where the presence of associated risk factors is highly prevalent. Among respiratory diseases, aspergillosis and cystic fibrosis have been in the spotlight due to increasing resistance and lack of effective treatments, respectively. Indeed, Aspergillus fumigatus, one of the causative agents for aspergillosis, was included in the recent WHO fungal priority pathogens list, highlighting its relevance and urgent need for mitigation. In addition, the same list reports very high mortality rates in immunocompromised patients with invasive aspergillosis caused by azole-resistant strains. Furthermore, aspergillosis is often prevented resorting to prophylaxis that also contributes to the emergence of resistant strains. Importantly, respiratory diseases are often associated with an inflammatory response that often leads to other complications such as increased mucus production and tissue remodeling. For example, in cystic fibrosis, the mucus clogs the airways and traps germs leading to infections, inflammation, respiratory failure, and other complications. Validating the role of inflammation in lung diseases, it has been shown that oral anti-inflammatory drugs delay cystic fibrosis. Nevertheless, anti-inflammatory drugs may present toxic effects and their use is limited in some patients with other underlying pathologies, such as asthma. Therefore, the development of less toxic and more amenable anti-inflammatory agents is paramount. Aromatic and medicinal plants stand out as a potential source of new anti-inflammatory drugs with many traditional uses supporting these effects. Moreover, their widely known antimicrobial effects, confirmed by their widespread use in cleaning agents, is also relevant in the context of lung infections.

Over the years our research group has validated the anti-inflammatory and antimicrobial potential of many plant volatiles and pointed out possible mechanisms of action. Despite the undeniable potential, these compounds present several drawbacks that continue to limit their clinical translation, such as volatility, lipophilicity and oxidation. Therefore, to achieve therapeutic efficacy, frequent administrations are required which jeopardizes patients’ adherence to therapy. In addition, due to their liquid form and high volatility, conventional techniques used to increase lung deposition, such as the addition of carrier systems, cannot be efficiently applied. To overcome these limitations the present proposal aims to develop an innovative inhalatory strategy to improve their stability, lung retention and sustained release, by resorting to solid matrix-based lipid nanoparticles. Overall, the ALVEOLUS proposal will boost the clinical translation of plant volatiles by paving the way for the development of effective inhalatory preventive and therapeutic strategies for lung diseases.

Scientific research is one of my main passions, with natural products drawing my attention since early days. In the last years, I have directed my research to the potential of these products, namely plant extracts and their volatile compounds as health promoting agents. This field of research has a direct impact on population welfare and can improve their quality of life. In this context, disruptive and impactful ideas are a must in order to fill knowledge gaps and truly impact patient’s health.
The foundation for my career plan was established during my PhD in Pharmaceutical Sciences, concluded in 2022 at the University of Coimbra (UC). During my PhD studies I had the opportunity to develop a breakthrough study that pointed out a cardioprotective potential for a volatile natural compound (doi: 10.1016/j.phrs.2022.106151). In addition, I collaborated in several projects showing the potential of essential oils and isolated volatile compounds as anti-inflammatory and antimicrobial agents (doi: 10.3389/fphar.2019.00446; 10.1016/j.jep.2018.06.025; 10.1016/j.indcrop.2018.01.024; 10.1016/j.jep.2019.112120). These studies demonstrated that volatile extracts (essential oils) obtained from aromatic and medicinal plants present strong effects against several pathogenic fungi, including Aspergillus spp. highly relevant in lung diseases. Furthermore, we demonstrated that several volatile extracts present a strong anti-inflammatory effect by modulating the canonical NF-κB signaling pathway. More recently, I also collaborated in studies showing the anti-aging potential of essential oils (doi: 10.3390/nu15081930; 10.3390/antibiotics12010179), which is highly relevant in the context of lung diseases, due to the high susceptibility of this organ to senescence and “inflammageing”. At this stage of my career, I would like to establish an independent research path and I´m truly interested in developing non-invasive routes of administration for plant volatiles, being the inhalation route one of my main goals. Indeed, lung diseases such as cystic fibrosis, aspergillosis or chronic obstructive pulmonary disease greatly impact patient’s quality of life and present very high mortality and disability rates. Plant extracts and compounds could be an excellent preventive/therapeutic strategy due to their widely reported anti-inflammatory and antimicrobial potential that could alleviate the inflammatory response and infection progress in these pathologies, thus improving patient’s quality of life.

Despite their huge potential, the translation of plant volatiles to a clinical setting is very challenging due to their high volatility, lipophilicity, and proneness to oxidation, which decreases their stability and bioavailability. Importantly, these compounds are highly suitable for inhalation, but some features such as their small size limits their use due to poor lung retention, being easily and readily removed upon exhalation. Built upon my previous work and skills acquired during my academic studies, including motivation and resilience to achieve the proposed goals, I designed the current proposal that gathers my main research interests and reflects the scientific path I intend to build.

The ALVEOLUS proposal includes several tasks that spawn from the development of inhalable particles in a dry powder to the in vivo validation of the therapeutic potential of this inhalatory system. The DDL Career Development Grant from the Aerosol Society would be of utmost importance to carry out the first task, namely the development of the solid lipid nanoparticles (SLN) containing the volatile compound(s) of interest. In this stage, relevant parameters for nanoparticle formulation need to be optimized such as particle size, polydispersity coefficient and zeta potential assessed by dynamic light scattering, Fourier transform infrared spectroscopy and differential scanning calorimetry (methodologies and equipment available at my institution). The encapsulation and loading efficiency will also be determined to assess the capacity of the SLN to incorporate the compound(s). In vitro release of the compound(s) will be determined using the dialysis bag method. Particle morphology will be assessed by transmission electron microscopy. Stability assays of the compound-containing SLN in solution for short- and long-term storage will be assessed by monitoring particle size and zeta potential. The following tasks aim to improve mucoadhesion and lung retention, by adsorbing SLNs to a mucoadhesive compound. A dry powder will be obtained by submitting the SLNs to spray-drying. Alternatively, bulking systems will be used to increase particle size, thus improving the aerodynamic properties of the SLNs, which allows for a deep lung deposition and increased retention. Particle size, density, flow behavior and surface characteristics of the obtained powders will be assessed according to the European Medicines Agency guidelines (EMEA/CHMP/QWP/49313/2005 Corr). Lung distribution of the powder will be disclosed using an Andersen Cascade Impactor, according to the European Pharmacopoeia. The final stages of this proposal resort to a pre-clinical validation using animal models of lung diseases, such as cystic fibrosis, chronic obstructive pulmonary disease, thus paving the way for the clinical translation of these compounds. The results obtained during the DDL Career Development Grant will be of the utmost importance, since they will leverage my applications to other funding opportunities, in order to fully accomplish the present proposal.

ALVEOLUS project brings novelty to my previous studies as it resorts to pharmaceutical technology to develop advanced drug delivery systems and is a step forward in the clinical translation of plant volatiles. The proposal also enriches my knowledge on drug formulation and routes of administration, namely the inhalatory one supporting my scientific maturity and will enable me to leverage of my own research line, thus consolidating an independent research career.
Having in mind that multidisciplinary teams are the cornerstone for a successful proposal, scientific collaborations will be key to bring to the project the skills and experience I lack. This proposal counts on previously established collaborations with researchers of the Faculty of Pharmacy and Chemistry Department of the UC and clinicians from Coimbra Hospital and University Centre. Moreover, new collaborations have been established namely with the Drug Sciences Department of University of Porto. Clinical collaborations are quite relevant, and I aim to reinforce them to bring clinical relevance and the patient need perspective to future projects. I also intend to collaborate with the pharmaceutical industry to further optimize the delivery support device, resorting to the wide network of industrial sponsors of the DDL Conference. These collaborations are critical as they will facilitate the clinical translation of the drug delivery system developed during the project. In addition, the direct collaboration with the pharmaceutical industry, will grant me a product-focused approach, which will provide the necessary skills to create a drug delivery system that could generate value and reach the market, thus improving patient’s quality of life. Furthermore, being awarded the DDL Career Development Grant, will open up my network by allowing the contact with several members of the DDL committee that have extensive experience in several fields of research relevant for my career plan.
Furthermore, I believe that mentorship, leadership, and communicational skills are an essential part of the skillset required for a successful independent research career. Therefore, this research project will allow me to establish my research line, which will allow me to mentor master and PhD students, thus contributing to their growth as resilient and motivated scientists. In addition, I plan to participate in several dissemination initiatives, both for academic, such as attending the DDL Conference 2023, Global Conference on Pharmaceutics and Novel Drug Delivery Systems or International Summit on Pharmaceutics and Drug Delivery Systems, and non-academic audiences.

Finally, the line of research I intend to create, based on the current proposal, is highly relevant to my institution as a specialized and trained researcher in inhalable encapsulated nanoparticles will be available. This skillset is currently missing at the institution and could greatly contribute to the research in disease-related strategic areas. Indeed, this research project will allow to install a workflow that can be adapted and applied to different research areas

Amritha G. Nambiar is perusing Ph.D. in pharmaceutical sciences from IIT BHU, Varanasi in pharmaceutical solid-state research particularly focusing on API development. She holds post graduate degree in pharmacy (Pharmaceutics) from IIT BHU, Varanasi and undergraduate degree in pharmacy from Priyadarshini J.L. College of Pharmacy, Nagpur (RTMNU). She is an undergraduate degree university gold medalist and awarded with few meritorious achievements in academics. She had published few review articles on continuous manufacturing of amorphous solid dispersions, cocrystals, and API-coprocessing. She is also playing the role teaching assistant in different subjects like pharmaceutical technology, pharmacokinetics, biology, hospital and clinical pharmacy.

Exploring the utility of co-processing techniques to produce DPI formulations

Lung diseases and infections such as asthma and chronic pulmonary disease (COPD), tuberculosis, etc. are chronic and serious conditions. Inhalation-based therapies can be considered as an effective approach for treating the mentioned diseases. The direct delivery of drugs to the lungs ensures its prompt onset of action and better treatment efficacy. Dry powder-based inhalation of drug with carrier particles such as lactose constitutes an effective pulmonary drug delivery system. For a drug to be administered in dry powder inhaler form, the aerodynamic diameter of the particles must be in the range 1-5µm. Usually, milling or spray drying is carried out to obtain micron size-range particles of drugs. Milling causes static charge generation thereby making the powder very cohesive. The flowability of powders also tends to reduce, thus, compromising the accuracy of dose delivery. Also, milled powder is irregular in shape and contain regions of both crystalline and amorphous forms. This makes the particle behaviour prediction highly challenging. Spray drying on the other hand, shows a very high variability in the powder yield (20-70%). Also, spray drying can induce amorphization of the particles leading to production of thermodynamically unstable powder with increased susceptibility towards moisture sorption, agglomeration, and recrystallization (phase conversion).
The coprecipitation technique is commonly used to prepare amorphous solid dispersion of poorly water-soluble drugs. Herein, a suitable API and polymer mixture is coprecipitated (by anti-solvent addition or solvent evaporation). There is a possibility of controlling the particle size of the final solid-dispersion obtained by employing high shear mixers. This way, the particle properties of coprecipitated amorphous solid dispersion (cPASD) can be optimized for DPI applications. Use of high shear mixers in producing micron sized particles is well known. The process can be suitably scaled up for continuous production by using in-line homogenizers. The density of the produced particles can also be effectively controlled by washing the obtained powders with suitable solvents. Also, there can be experiments wherein the API-polymer mixture are co-precipitated in the presence of lactose or other common carriers and explore its applicability for pulmonary delivery. Through this experiment, I intend to explore the applicability of co-precipitated powder for pulmonary delivery using Rifabutin as a model API.

The Research on dry powder inhalations for pulmonary drug delivery system has substantially broadened my personal and professional horizons as a Pharmaceutical solid state and formulation scientist. I have looked for opportunity to explore several pathways throughout my career. I was especially interested in the research on respiratory medication delivery. I am pursuing Ph.D. at Dr. Dinesh Kumar’s Research group at Indian Institute of Technology (Banaras Hindu University) which has a focus on respiratory drug delivery. Formulation and development of dry powder inhalation has been the focus of my dissertation research. My other responsibilities include academic lecturing, student monitoring, and overall class management for a pharmaceutics lab in order to provide undergraduate students a better understanding of research.
I have published a review article in the prestigious journal of AAPS SciTech with title “Continuous Manufacturing and Molecular Modeling of Pharmaceutical Amorphous Solid Dispersions”.
The subsequent stage of my research is determining the coprecipitation of API by forming amorphous solid dispersions. I will use several powder blends for amorphous solid dispersions with various active pharmaceutical ingredients (APIs) diverse sizes to examine their impact on DPI performance. This study uses rifabutin to assess the functionality and drug delivery properties of amorphous solid dispersions for dry powder inhalations and to measure the output and aerosol properties. The manufacturing based on coprecipitation techniques can address persistent problems in inhalation drug delivery because of the engineered particle shapes. Even though the technology is still in its infancy, recent developments have increased its usefulness and applicability. Access to appropriate manufacturing technology for complex designs in high resolution, however, continues to be a significant constraint. Support on both a financial and technical level is essential to the development of this research. This DDL Career Development Grant would be a crucial turning point in my interdisciplinary career as a scientist specializing in drug delivery to the lungs. My long-term objective is to create a thorough scientific report that details the preliminary investigations of intricate co-precipitation and dry powder inhalation. I believe that further research will continue in academic and institutional contexts because this topic is still substantially unexplored. My personal goal is to contribute to the growth of this new area of study, and I'm hoping that it will enable me to present my research to an international audience of aerosol scientists. The research outcomes shall be published in reputed peer-reviewed journals. If successful, through the proposed research work, we shall develop a method to consistently produce inhalable co-precipitated particles of (drug) at a larger scale showing sufficient storage stability and performance efficiency. Through this work, we shall be able to better understand the utility of API co-processing technique in production of DPI-based formulations. The technique can be adopted in producing DPI-based formulations of drugs exhibiting poor aqueous solubility. There is a possibility of producing co-precipitated mixtures of drug and polymer suitable for pulmonary delivery. We strongly intend to transfer the technology from the lab to industry. The pulmonary drug delivery systems have always intrigued me and I wish to explore the field as a formulation scientist. This research shall also provide me valuable insights of dry powder inhaler systems.

Stefania Glieca is currently a PhD student in Professor Francesca Buttini's group at the University of Parma, Italy. The aim of her PhD is the development of dry inhalation powders containing biological products.

In 2021 she obtained a Master's Degree in Pharmaceutical Chemistry and Technology at the University of Parma. The master thesis was focused on the development of a dry inhalation powder containing calcium phosphate coated liposomes with Cyclosporine A for the prevention of lung transplantation rejection and for the treatment of COVID-19.

She started her PhD in “Drug Sciences” in 2022. During her PhD she worked on the formulation of a dry powder formulation for the peripheral lungs delivery of a small anti-SARS-CoV-2 protein that acts as a decoy on the ACE2 receptor.
Her research is currently focused on the formulation of dry powders for inhalation containing Lactobacilli for the modulation and the restoration of the healthy lung microbiota for cystic fibrosis or other lungs infections.

She was awarded for the Best Poster entitled “Formulation Approach of Miniprotein Decoy Dry Powder Inhaler for SARS-CoV-2 Infection Inhibition” in 2022 and for the Best Poster entitled “Development of Inhalation Powders Containing Lactobacilli with Antimicrobial Activity Against Pseudomonas aeruginosa” in 2023 at the Pulmonary Drug Delivery Workshop.

Development of Lactic Acid Bacteria Inhalation Powders for the Pathogens Infection Containment

Chronic pulmonary diseases are characterized by a dysbiosis, meaning a variation in abundance and diversity of the microbiota, the colonizing microorganisms of the lung. In the case of cystic fibrosis (CF), the abundance of mucus on the lung epithelial cells and the loss of the microbiota's defence capacity leads to the development of chronic infections by pathogens, such as P. aeruginosa. The widespread use of systemic and inhaled antibiotics for the treatment of infections has led to the development of multidrug resistances, responsible for patients’ morbidity and mortality.
To date, the only attempts that have been made to restore the pulmonary microbiota concern the administration of oral probiotics, the effect of which is mediated by the gut-lung axis.
An innovative therapeutic approach could be to deliver probiotics to the lungs, in particular to the bronchi, where they can interact with the resident microflora. After their adhesion to the lung epithelium, probiotics could both interfere competitively with pathogenic bacteria and promote the proliferation of resident bacteria endowed with anti-inflammatory and antimicrobial properties. The outcome could be increased compared to the oral administration of the same strains.
The aim of this project is to develop an inhalation formulation as a dry powder containing probiotics that has antimicrobial activity.
Being multidisciplinary research, in addition to the Prof. Buttini’s team, of which I am part, within the Advanced Drug Delivery Research Lab (ADDRes Lab) of the University of Parma, a broad collaboration of partners has been created including:
• Microbiology research group, Food and Drug Department, University of Parma (Prof. Benedetta Bottari)
• Preclinical studies research group, Food and Drug Department, University of Parma (Prof. Simona Bertoni)
• Risk Analysis and Genomic Epidemiology Unit, Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia-Romagna (Dr. Erika Scaltriti)

The group has already managed to obtain by spray drying three dry powder inhalers containing probiotics, Lpb. plantarum, Lcb. rhamnosus or L. acidophilus. The manufacturing has been optimized to maintain the vitality of Lactobacilli and preliminary in vitro studies have demonstrated the ability of these powders to inhibit the growth of P. aeruginosa.
The project now focuses on further investigating the stability of the formulation both in terms of respirability and in terms of vitality and ability of probiotics to adhere to lung cells in different temperature conditions (25°C and 4°C).
Furthermore, prebiotics will be included in the optimized formulation, in order to increase the vitality and growth capacity of bacteria

I approached the world of inhalation for the first time during my master's thesis, in January 2021, joining the team of Prof. Buttini at University of Parma. The project undertaken in those six months was based on the development of microparticles for pulmonary administration starting from liposomes loaded with biotechnological drugs. After this period, I was interested in continuing the research I had the opportunity to remain in the group with a one-year research fellowship. This year allowed me to deepen many aspects of lung administration, studying the formulation of dry powder inhalers but also the characterization of pMDIs and nebulizers.
COVID-19 pandemic, because of the numerous complications in its management, brought attention to the revaluation of administration of proteins or vaccines by inhalation route. Hence, my work last year focused on the development of a formulation containing a protein that acts as a decoy against the SARS-CoV-2 spike protein, preventing it from entering the host cell. The formulation, in the form of dry inhalation powder, allowed a direct deposition to the lung, overcoming the problem of the limited stability of the protein in liquid form and the alteration it can undergo during its nebulization.
In November 2022 I became a Ph.D. student, and my project is based on the development of drug delivery platforms for the administration of biologics to the lung.
In addition to research, part of my Ph.D. regards the supervision of undergraduate Pharmacy students as a tutor during the galenic laboratory lessons.

Currently, our research work is focused on the development of an inhaled powder containing probiotics as an adjunctive treatment to the traditional one for patients affected by cystic fibrosis. The powder has already been optimized using a quality by design approach and has proved capable of having excellent aerodynamic characteristics and maintaining the viability of the bacteria included in the formulation. Moreover, in vitro studies were conducted to prove the formulation safety on human pulmonary cells, the adhesion capacity of the bacteria and their ability to prevent the growth of P. aeruginosa strains with different virulence.

The next step is to conduct an extensive study on the stability of the formulation in order to evaluate which are the best storage conditions of the formulation. In this study, it will be evaluated not only the ability of the powder to maintain good respirability, but also to test the ability of probiotics to remain viable and maintain their ability to adhere to lung cells when stored at different storage temperature conditions.
The data collected up to now have highlighted, especially for L. acidophilus, the poor ability to resist to the spray drying process, leading to an excessive decrease in viability and in the ability to adapt to the culture medium and start growing once the powder has been resuspended. For this reason, a further investigation will be conducted on the possible beneficial effect on the introduction of prebiotic substances, such as inulin or maltodextrin. In fact, the introduction of prebiotics could allow not only to increase the growth of the probiotics included in the formulation but also help to strengthen the pulmonary microbiota. Information related to probiotics post-production viability, ability to adhere, safety on lung cells will be collected.

The DDL career development award would provide me support to:
- Investigate the stability of the optimized probiotics inhalation powders after storage in controlled temperature conditions (i.e. 25°C and 4°C). The formulations will be characterized in terms of:
o Respirability: the aerodynamic particle size distribution will be assessed using a Next Generation Impactor. The aerodynamic parameters will be obtained by quantified the bacteria deposited in each stage of the impactor.
o Viability and vitality: a LIVE/DEAD BacLight Kit will be employed to count the ratio between the live and total cells in the powder (viability). A BacTrac Kit will be employed to obtain the parameters which describe the growth capacity of the bacteria.
o Adhesion capacity: the bacteria in the powders will be tested for their capacity to adhere to the Calu-3 and A549 pulmonary cell lines. Powder will be suspended in PBS and deposited on the cells and after 2h it will be calculated the percentage of the adherent bacteria to the deposited.
- Transferring the drug delivery platform to evaluate the inclusion of prebiotics, such as inulin, in the formulation with the aim of increasing the viability and the growth capacity of the bacteria. The tests that will be conducted are reported above. In addition, a safety study will be conducted as well on Calu-3 and A549 cells.

Alison Lansley is a Principal Lecturer in Pharmaceutics at the University of Brighton (UoB). She obtained her degree in Pharmacy and PhD in mucociliary clearance from the UoB and then undertook post-doctoral positions at UCLA (cellular control of ciliary activity) and King’s College London (the use of airway cell lines as drug absorption models of the lung) where she then took up a lectureship. Following a career break, she was awarded a Daphne Jackson Fellowship which she undertook at the University of Sussex before accepting her current position. Her research interests include the use of in vivo-reflective in vitro models to study the effect of mucus on drug/particle deposition behaviour and absorption in the respiratory tract, factors affecting mucociliary clearance and inhalation toxicity.
She has previously led the MPharm programme at the UoB and is a Senior Fellow of the Higher Education Academy, a Fellow of the Academy of Pharmaceutical Sciences (APS) and a member of the APS Biopharmaceutics Focus Group and the Nasal Research Focus Group. She teaches biopharmaceutics and formulation science to undergraduate and postgraduate students and has supervised PhD students and postdoctoral researchers with funding from research councils, charities, and industry. She has published papers and book chapters on pulmonary and nasal drug delivery.

The use of ciliary activity to determine the local toxicity of inhaled pharmaceuticals

The airway epithelium is the first tissue to encounter inhaled chemicals, pollutants and applied pharmaceuticals, so in vitro airway epithelial models have been considered as potential replacements for regulatory acute inhalation toxicity tests. Regulatory assessment of acute inhalation toxicity relies on in vivo testing. However, there is increasing interest in developing new approach methodologies (NAMs) to identify toxic effects with a view to the eventual replacement of animal tests.

The lining of the airways has evolved to provide a barrier to the entry of external substances. This is contributed to by mucociliary clearance (MCC) which is the movement of secreted mucus by hair-like cilia which constantly beat beneath the mucous layer. Inhaled particles and droplets tend to deposit on the mucus and are removed from the airways in a conveyor belt-like manner. MCC is an advantage when removing noxious substances such as inhaled pollutants but can be undesirable when the retention of pharmaceuticals is required for local activity e.g., delivery via the nose and lung to treat allergic rhinitis, asthma or COPD or absorption e.g., the systemic delivery of poorly absorbed drugs such as desmopressin and other biologics. However, substances toxic to MCC are also problematic as they make the individual more prone to respiratory infections. Therefore, measurement of ciliary beat frequency (CBF) is a useful endpoint in assessing the local toxicity of inhaled pharmaceuticals.

In order to study CBF without using animal tissue, primary cultures from human bronchial epithelial cells are required. Organotypic 3-D in vitro airway epithelial models are available commercially e.g. Mucilair , EpiAirway that are well characterised regarding their pseudostratified structure, barrier properties, cilia beating, etc. However, these are prohibitively expensive for routine use in acadaemia. Therefore, primary cultures from human bronchial epithelial cells (commercially supplied) will be cultured in-house on transparent permeable supports (Transwell Clear) at an air-liquid interface to form ciliated epithelia. This is technically demanding and, while the applicant has initiated this, further work is required to obtain fully differentiated cultures expressing beating cilia. Without this element, further work cannot proceed.

Once fully differentiated cultures have been achieved, CBF will be measured in the absence and presence of various drugs/excipients (surfactants, lipids, preservatives)/pollutants (ultrafine particles of AgCl, Fe(OH)3, polystyrene), or after the cells have been cultured in the presence of certain pharmaceuticals/pollutants (as above).

Being able to measure CBF will complement a suite of other techniques such as the measurement of mucus secretion, mucociliary clearance, airway epithelial permeability, cytotoxicity and therefore provide a strong technical base for future grant applications related to inhaled drug delivery or air pollution

This proposal has an element of career development and an element of service development.

Career development
I do not consider myself to be a typical ‘career development’ applicant. I have been a successful researcher with a record of external funding and a reputation in the field of nasal and pulmonary drug delivery (PubMed search: “Lansley AB” and “Batts AH” Publications (Scopus): 39; H-Index score (Scopus): 17). I also co-write a book chapter on Intranasal Drug Delivery in one of the the leading pharmaceutics textbook used by industrialists and academics (Aulton’s Pharmaceutics. The Design and Manufacture of Medicines. 6th edition. Eds. Aulton, M.E. and Taylor, K. Churchill Livingstone Elsevier, London).

However, while undertaking significant teaching-focused administrative roles e.g., MPharm Course leader (600 students; ~40 staff) my research lost its momentum (three PhD students completed but there was insufficient time to apply for further funding and write all the papers). This was recognised by my employer, the University of Brighton, and in 2020-21 I was given a sabbatical from teaching to bring about a step change in my research.

During my sabbatical, I wrote four papers and recommissioned the ciliary beat frequency (CBF) -image analysis equipment that is to be used in the proposed project. The CBF-image analysis apparatus has previously been used to study the effect of endothelin and various nasal formulations on CBF (Pawsey et al. Pulm Pharmacol Ther 2011: 24: 602-9: Ayoub PhD Thesis 2015). It was updated in 2015 at a total cost of £12,205 (£8,510 for a high speed digital charged-coupled device (CCD) camera, image analysis software image analysis system and £3,695 for 37oC incubation chamber). As part of its recent re-commissioning, software has been written in-house for data acquisition and image analysis. Preliminary data has shown that the system works as expected.

Unfortunately, my sabbatical coincided with the COVID-19 pandemic, and I was unable to achieve all that I had hoped due to lockdown. In particular, I was unable to undertake training with a local company to learn how to culture human bronchial epithelial cells such that they will express beating cilia. The company has generously shared its standard operating procedures with me and offered support. Therefore, part of the DDL Career Development Grant will be spent on developing these organotypic 3-D in vitro airway epithelial models. Commercially obtained human bronchial epithelial cells (already purchased) will be cultured on Transwell Clear polyester inserts using Promocell medium. Both myself and a technician at the University of Brighton will work on gaining these skills which will then be taught to any research students/fellows that may subsequently use the CBF equipment.

Once beating cilia are expressed, the membrane bearing the cells will be mounted in a custom-built perfusion chamber. Ciliary activity is temperature-dependent, so the chamber will be placed on the temperature-controlled stage of the microscope and maintained within a temperature-controlled chamber at 37oC. The cilia will be observed using phase contrast microscopy at x40 magnification using a microscope mounted on an anti-vibration table while the tissue is perfused with warmed (37oC) medium at a controlled flow rate in the absence and presence of pharmaceuticals/pollutants, or after having been cultured in the presence of certain pharmaceuticals/pollutants (UFPs) (see project summary). Images of ciliary activity will be detected with a CCD camera (340 fps; resolution 2048 x 1088) and images will be recorded and analysed using the programs developed in-house.

Data obtained will be published and form the basis of future funding applications.

Collaboration
I am a member of an international, multidisciplinary consortium of researchers (both industrial e.g., Nemura, Proveris, Aptar and academic e.g., KCL, Universities of Kiel, Brussels, Parma). Our aim is to raise the awareness of nasal drug delivery and obtain funding for this type of research (Nasal Research Focus Group (NRFG)). My expertise in ciliary activity is unique to this group and having a means of measuring CBF would enhance our success in funding applications. Successful applications will permit the future development of research students and fellows in this area.

By attending national and international meetings such as DDL and RDD and presenting the project results, I would be able to enhance my research profile, and chances of collaboration. This would enable me to build wider and stronger networks with other academics and industrialists which would enhance future funding application. Part of the DDL Career Development Grant would be spent on networking.

Service Development
There are relatively few academic laboratories in the UK able to undertake studies of ciliary activity. In addition, there is increasing interest in developing new approach methodologies (NAMs) for acute inhalation toxicity testing of chemicals and pharmaceuticals (drugs, excipients, nanoparticles). Measurement of CBF, alongside a suite of other techniques such as the measurement of mucus secretion, barrier function e.g., transepithelial electrical resistance, cytotoxicity (MTT and/or LDH assays), cytokine release, etc., would provide a better understanding of the toxicological impact of inhaled substances.

I would be open to collaborations with academic and industrial partners.

Dr. Michael Chow earned his BPharm(Hons) degree from the University of Hong Kong. After becoming a registered pharmacist, he commenced his PhD studies at the same institution in 2013, under the supervision of Dr. Jenny Lam and Dr. Judith Mak. His research project focused on the development of inhaled siRNA formulations for pulmonary delivery.

Upon completing his PhD, Dr. Chow worked as a postdoctoral research associate in the Advanced Drug Delivery Group at the School of Pharmacy, The University of Sydney, Australia, under the leadership of Prof. Hak-Kim Chan. During this time, his research expanded to include the study of the pharmacokinetics and pharmacodynamics of inhaled bacteriophage formulations in both healthy and diseased murine models.
Currently, Dr. Chow serves as a postdoctoral research fellow at the School of Pharmacy, University College London. His primary research interests involve the design and in vitro and in vivo characterization of inhalable formulations of biopharmaceuticals, with a particular emphasis on powder formulations prepared using spray drying and spray-freeze drying techniques. His long-term research goal is to facilitate the clinical translation of inhaled biologics that address unmet medical needs and enhance pharmaceutical care for patients.

Dr. Chow has published over 30 peer-reviewed articles and two book chapters dedicated to the field of pulmonary drug delivery for respiratory diseases. Additionally, he actively contributes to the scientific community by serving as a peer reviewer for various journals in his field and as a special issue editor, alongside Dr. Philip Kwok, for the themed issue titled 'Inhaled Treatment of Respiratory Infections' in the journal Pharmaceutics.

Strategies to enhance solid state stability of inhalable antibodies powders through particle engineering with spray-freeze drying

Antibodies have wide therapeutic potential for respiratory diseases, such as lung infections, asthma, and cancers. Currently, they have been administered parenterally, which is invasive and has suboptimal lung distribution with higher risks of systemic toxicities. The lack of inhalable antibodies highlights an unmet medical need. By pulmonary administration, antibodies can accumulate in the lungs for enhanced and localised therapeutic effects. It also permits antibodies to be formulated and directly administered as solid formulations for improved stability.

This study aims to prepare highly inhalable, stable powders of IgG using spray-freeze drying (SFD). SFD is viable alternative to spray drying (SD) in preparing inhalable powders of biologics. It uses different particle formation mechanisms to SD and does not expose samples to elevated temperatures. SFD powders often have a larger geometric size that better evade macrophage phagocytosis, but with small aerodynamic diameters due to their high porosity. It is hypothesised that particle properties can be engineered primarily through the choice and compositions of excipients and the lyophilisation conditions. Formulation design and optimisation is achieved using Design of Experiment approach alongside our experience in formulation strategies for inhaled biologics. Generally-regarded-as-safe excipients including sugar (e.g. trehalose), polyols (e.g. cyclodextrins and inositol), sugar alcohols, amino acids, and fatty acids (e.g. sodium stearate) will be explored for their capacity to confer solid state stabilities of antibodies and particle dispersibility. Solid properties, aerosol performance and bioactivity of the powder formulations will be characterised. Stability over short- (< 2 weeks) and medium- (4-8 weeks) duration will be evaluated, emphasising on the effects of exposure to elevated humidity on protein conformation and powder aerosol performance. In vitro toxicity of excipient systems and bioactivity of formulated antibodies will be studied. The expected deliverables of the project are identification of promising candidate formulation suitable for the pulmonary delivery of antibodies. The pool of potential excipients for inhaled medicine, especially for solid formulations, will be expanded. Insights on the mechanisms of particle formation in SFD and enhanced understanding on solid state stabilisation of proteins would be obtained, and formulation strategies to improve particle dispersion of SFD powders shall be informed. The project outcomes shall guide future studies that includes in vivo safety and efficacy studies of candidate formulations in healthy and diseased animals. Feasibility of expanding the candidate formulations to other antibody formats (e.g., Fab, nanobodies) or therapeutic proteins and vaccines will be studied. Also, strategic combinations with small molecular drugs that have potential dual functionalities as active and particle property modifiers will be explored.

I developed my interest in pharmaceutics during my BPharm undergraduate, as its interdisciplinary nature aligned with my favourite subjects of science and mathematics. Besides, pharmaceutics substantially involves translational research that strives to extend scientific advancement in laboratory to the hands of patients in real life that improve their health. With basic science research, we gain precious knowledge that expand our horizon to the unknown world, but at times these advancements could first appear remote and irrelevant to the wider public. My motivation to bridge this gap has driven me to take a more pragmatic perspective towards research. I believe that translational research can enrich new scientific findings with meanings that benefit people with quality-of-life improvement, even though they might be less glamorous and eye-catching. Pharmaceutics is also a research discipline where pharmacists can fully utilise their knowledge from the drug development pipeline to their clinical use and appreciate the intricate interactions in between.

Under the supervision of Dr. Jenny Lam, Dr. Judith Mak, and Dr. Philip Kwok, my research project during PhD candidature on developing a powder formulation of therapeutic siRNA has become the first realisation of my research aspiration. During the post-doctoral training in University of Sydney, I expanded my research area to the pulmonary delivery of bacteriophage as an alternative or combined therapy to antibiotics for the treatment resistant pulmonary infections, leading to the publication of two pioneer studies on the pharmacokinetics and pharmacodynamics of inhaled bacteriophage. These experiences have reinforced my interest in pharmaceutical research of inhaled biologics. My upcoming goal is to continue my research in the field, including the proposed project. This proposed project on inhaled antibodies if funded will support my research career in multiple ways through further polishing my research, analytical, problem-solving, and presentation skills, and offering me with opportunities to new publications and funding. All of which form a foundation for me to transit to an independent research scientist. As I continue to grow and establish, I would like to get involved through collaboration in conducting clinical studies, which I believe is an essential component in translational research.
In the long run, my present research ambition lies in making solid contributions to advancing pharmaceutical development on inhaled biologics to fill unmet medical needs and bring actual improvements to the pharmaceutical care of patients, which can be fulfilled through participation or coordination in the development of a pharmaceutical product, making significant breakthroughs or discoveries that expedite product development, or proposing solutions that overcome major development challenges.

My other career aspiration is in education, and I want to pass on my knowledge and experience to the future young generation. I strongly believe in the mutual growth of teachers and students through teaching and learning in education. Not only is teaching an efficient way to consolidate concepts and knowledge, but by empowering students I can also reflect on myself and learn from them. With respect to this, I am eager to take on the challenge of lecturing and supervising students, sharing with them my knowledge and experience in the same way as how I was educated and inspired by my teachers.

Tanu is currently in her final year pursuing a Ph.D. in the Department of Pharmaceutics at the University of Connecticut, USA. Her academic journey began with a Bachelor's and Master's degree in Pharmacy from the University of Delhi, India. Her Ph.D. research focuses on assessing the influence of process parameters on the performance of dry powder inhalers. She employs both experimental and computational tools in her work, all with the overall objective of optimizing vacuum drum-filling parameters to enhance product performance. This project is the result of a collaborative partnership between the University of Connecticut and Experic in New Jersey. In addition to her primary research, Tanu is also involved in other exciting projects, including the development of a Computational Fluid Dynamics (CFD) model for inkjet-based 3D printing of pharmaceutical tablets and the creation of a Discrete Element Method (DEM) model for the continuous twin-screw wet granulation process."

Impact of Process Parameters on Dry Powder Inhaler Performance

Dry powder inhalers (DPI) are becoming increasingly popular due to growing interest in pulmonary drug delivery. The DPI performance is the net result of formulation properties and a series of processes carried out during the manufacturing process such as drug micronization, blending, and capsule filling. The DPI formulations are usually developed by considering formulation properties, but the impact of unit operations such as capsule filling is often the least explored. Therefore, this proposal has been designed with the goal to evaluate the impact of powder-filling conditions on DPI performance. The outcomes from this project will also be used to develop a machine learning Artificial Neural Network (ANN) model to predict the product performance. This project is a collaboration between Dr. Justin Lacombe’s group at Experic, USA, and the University of Connecticut, USA.
DPI formulation usually consists of micronized drug, coarse carrier, and additional components such as fine lactose. DPI manufacturing usually begins with API micronization to generate particles with an aerodynamic diameter of 1-5μm. However, this small size of API particles results in poor flow properties making the capsule filling a challenge. In this study, we will evaluate a formulation consisting of micronized Albuterol sulfate and a lactose carrier to study the impact of low-dose capsule filling conditions on DPI performance. The API will be micronized using an Air Jet mill and will be analyzed for amorphous content and surface energy. The formulation will be then blended using a high-shear mixer at different blending conditions (blending time and speed) and the blended formulation will be then filled in HPMC capsules using Harro Hofliger vacuum drum filler. The vacuum drum filler involves a rotating dosing drum below a hopper where the powder is continuously stirred, and powder is pulled into the drum bore by vacuum and dispensed into the capsule with positive pressure. The amount of powder dispensed is influenced by multiple factors such as vacuum pressure, drum bore size, dispense pressure, stirrer type, stirrer speed, and powder properties which consequently affect the product performance. After that, the filled capsules will be evaluated for their aerosol performance using NGI.
The ANN model will be developed using the experimental data where formulation properties (bulk density, tap density, and particle size distribution) and process parameters (vacuum, stirrer type, and stirrer speed) will be used as the input features to predict the filling performance (capsule fill weight and fill weight variability) and product performance (FPF and MMAD). The outcomes from this study will be useful in identifying and optimizing the critical process parameters during low-dose vacuum drum filling to achieve optimum product performance. The outcomes from this project will be published in renowned journals and will also be presented at various conferences such as DDL, AICHE, and AAPS.

I have always been drawn to the power of healthcare sciences to heal and improve human lives. This led me to pursue my career in a field which has the potential to revolutionize the way we treat diseases and make a real difference in the world by improving the quality of life for millions of people. I completed my bachelor’s degree in pharmacy from the Delhi Institute of Pharmaceutical Sciences and Research (DIPSAR), India, where I got an opportunity to get exposed to the pharmaceutical industry. As my course progressed, my fascination with pharmaceuticals, especially pharmaceutics, only grew stronger. During my coursework, I was granted the opportunity to intern in the drug delivery and pharmacological sciences department at the top medical college in India, All India Institute of Medical Sciences (AIIMS). At AIIMS, I worked with research scientists, professors, and doctors to learn about their work with ophthalmic formulations. I spent most of my time preparing ophthalmic formulations for use in surgeries and I got to see firsthand how these formulations could help to restore sight and improve quality of life. This work was incredibly impactful, as it helped to improve the lives of countless people and further grew my interest in pharmaceutical research. Therefore, I decided to continue my education in pharmacy by enrolling in a Master of Pharmacy program at the same university where I completed my bachelor’s degree. While pursuing my master’s degree, my passion narrowed down to exploring and designing ways to improve pharmaceutical drug product development, which motivated me to design my thesis in a similar domain. During this period, I also got the opportunity to work as a pharmaceutical sciences intern at Vyome Biosciences, where I worked with a team of esteemed research scientists to gain more exposure to the industry. Subsequently, I decided to dive deeper into my areas of interest and enrolled in a PhD program in the pharmaceutical department at the University of Connecticut. My initial research work was primarily focused on developing the computational models for pharmaceutical processes such as three-dimensional binder jet printing of pharmaceutical tablets and powder blending followed by experimental validation of the developed models. Shortly after that, I got an exciting opportunity to work with Dr. Justin Lacombe as a pharmaceutical development and engineering intern at Experic in New Jersey, where I learnt about the industrial aspects of formulation development and pharmaceutical manufacturing. Most of my work at Experic focused on the optimization of low dose powder filling process for Dry Powder Inhalers (DPI) and the manufacturing of inhalation formulations. I learned that research and innovation should go hand in hand with ease of manufacturing, packaging, and scalability, and because of that, I decided to design my doctoral thesis with an overall goal to bridge the gap between Dry Powder Inhaler manufacturing and final product performance. My doctoral thesis involves understanding the impact of process parameters such as formulation blending, and the DPI capsule filling on the final product performance with an overall goal to optimize the dry powder inhaler performance. Later, we will also develop a machine learning Artificial Neural Network (ANN) model to predict product performance. The DPI formulation development is mainly focused on material attributes to improve the product aerosol performance. However, the impact of process parameters on DPI performance is often less investigated.
The Drug Delivery to the Lung (DDL) career development grant will be instrumental in helping us achieve our project goals by providing the necessary funding for raw materials and equipment. With the help of this funding, we will be able to perform required performance analysis such as inverse gas chromatography, scanning electron microscopy (SEM), high-performance liquid chromatography (HPLC), and next generation impactor (NGI) analysis.
DDL grant will also play a vital role in funding my travel to various conferences such as DDL and American Association for Pharmaceutical Scientists Pharm Sci. (AAPS) where I will be able to present our work and provide an impetus to my professional growth in the inhalation sciences field. Being an international student, participating in conferences will be helpful in expanding my professional network and opening opportunities for research collaborations. The outcome of this project will serve as a vital source of information to understand the role of process parameters on DPI performance and consequently improve the manufacturing process.

Dr. Alberto Baldelli completed his Ph.D. in Particle and Surface Engineering at the University of Alberta in 2016. After that, he joined the University of British Columbia as a postdoctoral fellow in the Department of Mechanical Engineering and as a Research Fellow in the Faculty of Food and Land Systems. His research interests are particle formation of proteins, characterization of carbonaceous nanoparticles, encapsulation of bioactive compounds, pharmaceutical and food bioprocessing, and superhydrophobicity. Currently, he works at Hovione where he can apply his knowledge in spray drying in the design of dry powders for the nasal and lung deliveries.

Development of a prophylactic drug against airborne viruses

The recent pandemic emphasizes the importance of studies on developing methods for preventing airborne viruses from entering or infecting a host. Identified and known airborne viruses are varicella (also called chickenpox), mumps, measles, tuberculosis, diphtheria, meningitis, and Respiratory syncytial (RSV). Some types of influenza, rhinoviruses, hantaviruses, and SARSs, currently lack invalidated and long-term vaccines. Recognized receptors for influenza A, rhinoviruses, and parainfluenza are ACE2, ICAM1, and sialic acid receptors. These combinations are not the only ones; it is still unknown which receptors show the highest adhesion forces with one or more types of airborne viruses. Rhinoviruses can be subdivided into 74 HRV-A, 25 HRV-B, and 6 HRV-C. Depending on the groups, the receptor needed for cell entry might differ.
Similarly, Influenza A (IAV) shows several subtypes of HA-NA combinations, H1N1 and H3N2 viruses circulating among humans. These types seem to IAVs appear to bind to N-linked glycans on proteins especially, but these are not unconditionally essential for host cell entry in vitro. Specific sialoglycan chains may influence the functional receptors for the cell entry of IAV; however, a precise identification of which chain is activating the entry of IAV the most is unclear. Regardless, the sialoside-rich mucus layer certainly turns into a decoy for IAV. Moreover, angiotensin-converting enzyme 2 (ACE2) is proven to be the key in the SARS-CoV-2 infection process due to the virus-receptor affinity. SARS-CoV-2 contains the receptor-binding domain (RBD), which directly binds to the peptidase domain (PD) of angiotensin-converting enzyme 2 (ACE2). The ACE2 structural transmembrane domain anchors its extracellular domain to the plasma membrane and enters the host cell for SARS-CoV-2.
Furthermore, the cellular serine protease TMPRSS2 is employed by SARS-CoV-2 for S-protein priming. The activation of the spike protein through proteolytic cleavage by a host protease near the junction between its S1 and S2 domains generates the binding to the receptor. The newly liberated S2 domain N-terminus into the cell membrane produces a viral and cellular membranes fusion. The above-mentioned airborne viruses are examples of viruses without a proper cure, treatment, or vaccine.
The affinity of recombinant receptors and airborne viruses has already been explored. The idea of using recombinant receptors as a possible treatment has been published in a few research studies. The use of the affinity rhACE2 and SARS-CoV-2 in the development of prophylactic drugs has been addressed in a previous publication, achieved as a collaboration of some members of this proposal. However, the application of this idea to a variety of receptors-airborne viruses is new and could potentially develop an innovative type of drug. Moreover, the optimization achieved through the engineering of dry powder of the drug could increase the drug efficiency.

Dr Baldelli has shown breakthrough results in the areas of inhalable drugs, environmental aerosols and coatings, theory of particle formation, and food bioprocessing. As inhalable drugs, via SD, he produced alternatives to antibiotics, to which humans are developing resistance, for defeating biofilms in the upper respiratory tract. As environmental aerosols and coatings, he correlated the disordered carbon content in soot and the diameter of primary particles composing soot aggregates disrupting previous assumptions in computational models for the understanding of how to prevent the formation of soot from combustion sources. Moreover, he produced an affordable superhydrophobic, icephobic, and electrically conductive coating for avoiding ice formation on wings, aircraft, or antennas in cold regions. As theoretical methods for controlling particle formation, he released the main references in the area by discovering the time to saturation, nucleation, and crystal growth in the evaporation of salt droplets. This allowed for reducing the cohesion forces between inhalable microparticles and, thus, increasing the efficiency of inhalable drugs. As food bioprocessing, he created dispersible dairy powder and mucoadhesive encapsulated iron for food fortification. The last-mentioned powder is now inserted in bread and rice in India and is expected to reduce the number of people affected by anaemia. These outcomes generated an h-index of 17 and an i10-index of 24, with a total citation of 746 in journals with an average impact factor 6.7 and with total funding achieved of 2.2 million CAD. Collaborations in 6 continents and 13 countries have been established producing 5 patents, 44 publications, 14 invited talks, and 14 conference oral presentations.
With the above-mentioned proposal, Dr. Baldelli would be able to strengthen his knowledge and experience in the pharmaceutical bioprocessing area. As a result, Dr. Baldelli could be able to achieve his ultimate goal to become an Assistant Professor in Engineering in the near future.

Dan Hardy, founder of Microsol, undertook his PhD in aerosol science at the University of Bristol. He specialised in understanding droplet microphysics and microparticle formation with experimental and computation methods. Dan developed a novel technique for studying freefalling microdroplets and co-developed a new software package for the accurate simulation of aerosol droplet evaporation. In 2020 Dan was awarded the Aerosol Society Doctoral Student Award.

In line with completing his PhD, Dan founded Microsol. Microsol provides advanced formulation optimisation tools for respiratory drug delivery, has been backed by funding from Innovate UK, the University of Bristol, and the University of Hertfordshire, and has progressed from start-up to revenue generation in its first 12 months.

Dan is also the assistant course manager EPSRC CDT in Aerosol Science, which provides world-class training to the next generation of aerosol scientists. The CDT is a collaborative organisation, consisting of seven academic institutions and over 60 industrial partner organisations. Training and research span all areas and application of aerosol science.

In addition to advancing Microsol’s research, covering aerosol hygroscopic response in physiological conditions and regional deposition, Dan continues to pursue research interests in particle formation processes.

Validation of coupled aerosol kinetics and regional deposition model using an NGI

Project Summary
Understanding regional deposition is essential for optimising the dose delivery of inhaled medicines. Currently, there are a range of laboratory and computational tools available to scientists for measuring and predicting the dynamics of aerosol plumes during inhalation. Some tools, such as computational fluid dynamics simulations provide detailed representations of the air flow within the lungs during inhalation. Other tools, such as Next Generation Impactors (NGIs) provide experimental validation of regional deposition through sequential filtration stages. Despite the variety of tools available there is still significant challenge in understanding the interplay between aerosol dynamics in physiological conditions and deposition.

The challenges faced are multifaceted: current state-of-the-art simulations are limited in their ability to represent the hygroscopic response of aerosol plumes or accurately predict phase change - either solidification of evaporating droplets or dissolution of airborne particles, NGI studies are advancing, but it is difficult to consistently reproduce physiological conditions such as high relative humidity (RH) and temperature. In studies where measurements are performed at more accessible conditions, the impact of aerosol dynamics is not captured.

Microsol is developing a software tool for the prediction of regional deposition, based on experimentally validated models of droplet behaviour at the single-particle scale. Crucially, this software will account for the unique physicochemical properties of any formulation - particularly the hygroscopicity. Furthermore, it will be applicable to the simulation of NGI studies. This enables accelerated formulation development, in addition to establishing the model's validity in humans. The optimisation of formulation properties may be achieved using simulations to predictively assess regional deposition and then, by modifying the formulation, tuning the deposition behaviour.

This project will be focussed on the simulations of NGI deposition, specifically on the benchmarking of Microsols software against experimental data. The core model has been developed to a proof of concept level and now needs validating.

A test formulation will be chosen based on advice from Microsol’s advisory board. The time-evolution of a typical size distribution of aerosol as it passes through an NGI and the corresponding deposition behaviour will be simulated - this may be performed for a range of environmental conditions. In parallel, NGI experiments using the same candidate formulation and conditions will be performed. This will require external laboratory access and will be the main allocation of grant funds. Discussions are currently underway with academic and industry contacts to find a suitable project partner to perform these measurements. Finally, a data analysis phase will provide a comparison of results, assessment of the model and a route to improvements where needed.

Having approached aerosol science from a physical chemistry background and having performed research focused on fundamental aerosol phenomena, I have found many exciting applications for the science I have worked on within the field of inhaled medicines.

My interest in research has shaped my career to-date. My first experience was a year in industry at the Engineering and Physical Sciences Research Council, providing me with an understanding of the impact the high quality research can have, both at the societal level and for the individual. I pursued my interest in physical science, acquiring an MSci in Chemical Physics at the University of Bristol.

Subsequently I undertook a PhD with Professor Jonathan Reid at the University of Bristol in Aerosol Science, specifically, focussing on particle formation through droplet drying. Throughout my PhD, I learned of the many challenges and opportunities for innovation within the pharmaceutical industry and respiratory drug delivery. I developed novel measurement techniques for observing droplet evaporation and crystallisation at the single-particle scale, enabling the relationships between droplet properties, environmental conditions, and particle morphologies to be probed. I also co-developed a software package for the simulation of single droplet heat and mass transfer kinetics in a cross-institution collaboration.

In the late stages of my PhD, I noticed a need within industry for measurement capability and advanced computational tools such as those I had experience with. I therefore joined the QTEC QUEST research commercialisation business incubator and, six months later, founded Microsol to service this need. Since being founded, Microsol has been awarded over £70k from organisations including Innovate UK, the University of Bristol, and the University of Hertfordshire. This has enabled the commercialisation of the Electrodynamic Balance technology (single particle analysis technique), the development of my commercial skills, and the growth of the business.

Having recently completed my PhD I am the assistant course manager at the EPSRC Centre for Doctoral Training in Aerosol Science alongside my role as a founder/CEO at Microsol. This role enables me to share my knowledge and experience with new PhD students and engage with cutting-edge, interdisciplinary research from multiple world-class universities. Through my role at Microsol I am continuing to pursue my research interests and translate advanced technologies to address industry needs.

The current priority for Microsol is the advancement of predictive computational tools to accelerate formulation research and development. This may be achieved by coupling accurate simulations of aerosol dynamics and regional deposition simulations. Currently, industry faces significant challenges, such as the transition away from MDI propellants with high climate impact, challenges within particle engineering for dry powder inhalers, and the emergence and rapidly growing interest in respiratory and nasal delivery of biologics. Digitalisation of formulation research and development will accelerate industry in addressing these challenges. By assessing formulation performance in-silico across a wide range of formulation candidates and modifying formulation properties, top candidates formulations may be identified prior to major laboratory studies. Thus, laboratory resources may be devoted to candidates with the highest chance of ideal performance. My aim within this field is to leverage the tools I have experience with for analysing aerosol processes at the single-particle scale through computational augmentation to address the formulation challenges faced by industry today.

Funding from the DDL Career Development Grant would enable me to perform the study outlined, upskilling myself, gaining access to equipment that I would not otherwise have, and continuing to advance the field of respiratory drug delivery. Furthermore, it would expand my hands-on experience to the tools used by industry as the majority of my work to-date has been performed with custom made instrumentation in academia. This opportunity will enable the advancement of my research career as I seek to translate fundamental research into valuable tools of industrial applications. I founded Microsol as a vehicle to help me enact this vision. This funding opportunity will enable me to continue to develop the company, advancing my own expertise and commercial skills and widening the impact of the research I perform. Additionally, this project will help me widen my network, interacting with leading researchers from both academia and industry both during the project and the presentation of subsequent results.

To summarise, funding from the DDL Career Development Grant would enable me to broaden my technical skills, increase my research impact, and advance the field of drug delivery to the lung through the development of a validated tool for the scientific community.

I obtained my Bachelor of Respiratory Therapy in 2013 at Chang Gung University in Taiwan. After that, I passed the Professional and Technical Exams for Respiratory Therapists. In 2015, I received my Master's degree in Occupational Medicine and Industrial Hygiene from National Taiwan University (NTU) under the supervision of Professor Chih-Chieh Chen. I became a research assistant in the same group until 2017. I then undertook my PhD studies on the development of dry powder aerosol formulations under the supervision of Professor Hak-Kim Chan and Dr. Philip Chi Lip Kwok at the School of Pharmacy, The University of Sydney, and graduated in 2021. Subsequent to my PhD, I held a post-doctoral fellow position at the School of Pharmacy, National Taiwan University (2021-2022). In 2022, I was appointed as a Assistant Professor in the School of Pharmacy at the National Taiwan University, a position I still hold.

My research focuses on pulmonary drug delivery, which is closely tied to four major areas, including (1) the engineering and physicochemical characterization of pharmaceutical aerosol formulations, (2) the development of inhaled drug devices, (3) the enhancement of in vitro-in vivo correlations for orally inhaled drugs, and (4) the evaluation of performance of aerosol delivery devices for optimization in inhaled drug delivery during respiratory support. I have evaluated and characterized various aerosol formulations and delivery systems used in the treatment of pulmonary and systemic diseases in vitro.

Standardizing off-label use of intravenous amikacin for nebulization

Hospital-acquired pneumonia (HAP) and ventilator-associated pneumonia (VAP) are two of the most serious nosocomial infections associated with high morbidity and mortality. Multidrug-resistant pathogens have compounded the problem, as effective antibiotic options are becoming limited. Amikacin, an aminoglycoside antibiotic, is effective against both Gram-negative bacilli and Gram-positive cocci, and resistance has remained relatively low and stable over years of use. The intravenous administration of amikacin carries a potential risk of systemic toxicity, such as nephrotoxicity and ototoxicity.

Aerosol inhalation of amikacin provides an alternative that delivers high concentrations of the drug directly to the lungs, leading to rapid attainment of the minimum inhibitory concentration in the infected area and reducing toxicity and adverse effects caused by systemic exposure of amikacin. Currently, there is only one commercialized amikacin product for oral inhalation use, which is not yet available in Taiwan and is indicated only for the treatment of lung infections caused by Mycobacterium avium complex. As a result, alternative ways to administer amikacin through inhalation as adjunctive therapy for treating VAP and HAP are urgently needed.

The off-label use of an intravenous amikacin solution given by nebulization has shown promising results in terms of achieving improved clinical outcomes while lowering amikacin-related toxicity. However, nurses or respiratory therapists often dilute the amikacin solution with water or saline using arbitrary dilution factors and select nebulizers arbitrarily. This arbitrary preparation can lead to inappropriate physicochemical and pharmacological properties of the amikacin solution when given off-label by nebulization.

To date, there are no investigations exploring the physicochemical characteristics and aerosol performance of intravenous amikacin solution when administered off-label by nebulization for inhalation. Thus, the objective of this project is to establish recommendations for off-label usage of amikacin solution in clinical practice by conducting in vitro examinations of various solution properties and aerosol performance in various breathing models.

As a first-year Assistant Professor at the National Taiwan University School of Pharmacy (NTUSP), my primary goal is to contribute to the field of pulmonary drug delivery. I aim to achieve this by focusing on four key career development goals:

Establishing a research program that addresses the unmet needs in pulmonary drug delivery: To achieve this goal, I plan to develop novel drug delivery technologies and evaluate their efficacy in both in vitro and in vivo studies. I have recently established my own laboratory at NTUSP and bought a spray dryer (BUCHI S300) for my lab. Moreover, I will develop a research plan that focuses on innovative and feasible approaches to developing drug delivery technologies. This research plan will be aligned with the strategic priorities of my institution and will address key challenges in the field of pulmonary drug delivery.

Securing funding to support my research program: To support my research program, I will need to secure funding from various sources, including government agencies, private foundations, and industry sponsors. I plan to achieve this goal by developing a strong research proposal, building collaborations with other researchers, and leveraging my existing network of contacts. I am now collaborating with my previous supervisor, Prof. Hak-Kim Chan (School of Pharmacy, The University of Sydney, Australia), Senior Lecturer, Dr. Darson Li (Department of Mechanical and Manufacturing Engineering, University of New South Wales, Australia), and Dr. Ronan Mac Loughlin (Director R&D, Science and Emerging Technologies in Aerogen). In addition, my research has been recently funded by the National Science and Technology Council (Taiwan) and the pharmaceutical industry.

Mentoring students: As an Assistant Professor, I have an opportunity to mentor the next generation of researchers in my field. I plan to achieve this goal by recruiting and supervising students and postdoctoral fellows, providing them with the necessary resources and guidance to develop their research skills, and fostering a supportive research environment. This year, I have recruited 4 undergraduate students with 1 master’s student.

Building a national and international reputation as an expert in pulmonary drug delivery: To build my reputation as an expert in this field, I plan to disseminate my research findings through publications, presentations, and collaborations with other researchers in my field. I will focus on publishing my research in high-impact journals. I have published 17 papers, of which one of them was published in the top 2% of journals in its research field. (Advanced Drug Delivery Reviews; five-year impact factor is as high as 21.65). Additionally, by joining international conferences, I can enhance the visibility of my research. Thus, I am planning to join the DDL 2023 conference in person this year.

To achieve these career development goals, I will take the following steps:

‧Develop a research plan that addresses the unmet needs in pulmonary drug delivery.
‧Seek funding opportunities from government agencies, private foundations, and industry sponsors.
‧Recruit and supervise students and postdoctoral fellows to assist me in my research program.
‧Publish my research in high-impact journals and present my research at national and international conferences.
‧Engage in professional development activities, such as attending workshops and conferences, to enhance my research and teaching skills.

Basanth Babu Eedara is currently a Research Assistant Professor in Prof. Heidi M. Mansour’s lab at the Florida International University (FIU) Center for Translational Science in Port Saint Lucie, Florida. As a former Postdoctoral Research Associate (Jul 2020-Feb 2022 at The University of Arizona, Tucson) and current Research Assistant Professor at FIU in Prof. Mansour’s lab, his research focuses on the development and evaluation (i.e., physicochemical, and biological) of novel inhalable drug formulations for treating lung conditions.

Dr. Eedara completed his PhD in the Pharmaceutical Sciences under the supervision of Dr. Shyamal C. Das and Prof. Ian G. Tucker at the University of Otago in Dunedin, New Zealand. Prior to this, he worked as an Assistant Professor (Feb 2012- Dec 2014) in the Department of Pharmaceutics at St. Peter’s Institute of Pharmaceutical Sciences, Telangana, India. His long-term career goals involve becoming an independent researcher and developing formulations to deliver drugs in a targeted manner to prolong and localize in the lungs. He is also interested in understanding the dissolution behavior of respirable size particles which can be useful to establish differences between formulations and to estimate the dissolution behavior in the lungs. Techniques used for development and evaluation of inhalable dry powder formulations include drying techniques (i.e., spray drying and freeze drying), in vitro and in vivo aerosolization studies, solid state characterization methods, surface characterization techniques, particle size analysis, electron microscopy, in vitro dissolution and drug release studies, in vitro acellular, in vitro cellular, and in vivo animal studies.

Nanocomposite Dry Powder Particles for Inhalation Delivery of Combination Anti-Cancer Drugs

Lung cancer is a leading cancer killer worldwide and the American Cancer Society estimates about 130,180 deaths from lung cancer in 2022. The lack of effective first-line chemotherapeutics, the existence of resistant tumors, and the non-optimal route of administration contribute to poor prognosis and high mortality in lung cancer. The addition of localized delivery of anti-cancer drugs via pulmonary route to the systemic delivery could allow a therapeutic intensification due to a loco-regional diffusion of the drug close to the tumor, while fighting invasive and diffuse cancerous cells. Literature reports suggest inhalation delivery of a combination of docetaxel with a nonsteroidal anti-inflammatory drug, celecoxib showed anticancer activity through cell proliferation inhibition and induction of apoptosis, along with reduced gastrointestinal toxicity. This study aims to develop highly aerosolizable, stable nanocomposite dry powder particles loaded with docetaxel and celecoxib for inhalation delivery. We hypothesize that the nanocomposite dry powder particles disintegrate in the lung lining fluid upon deposition and produce nanoparticles, which will escape alveolar macrophage uptake and release the drug in controlled manner for prolonged periods of time. Nanocomposite dry powder particles will be produced from nanoparticle suspension using a binder (leucine or trileucine) by spray drying approach. The produced powders will be characterized for solid state nature, in vitro aerosolization behavior, in vitro drug release and stability at accelerated conditions. Our lab has demonstrated expertise in producing dry powder formulations of anti-cancer drugs using spray drying technique and has all facilities to characterize the formulations. Following the success of this study, I plan to evaluate formulation tolerability of a most promising dry powder formulation in a small group of Sprague Dawley rats (male, 280-350 g, 3-4 weeks) intratracheally using a dry powder insufflator, Penn-Century Dry Powder Insufflator -Model DP-4R (Penn Century Inc., Wyndmoor, PA, USA) and in vivo studies using an animal model of lung cancer in future.

Career Goal(s):
My goal is to become an independent academic investigator in the field of pharmaceutical sciences and a leader in pulmonary drug delivery research with an aim to deliver drugs in a targeted manner to treat the localized lung diseases. With this interest, I did a Ph.D. on various strategies to improve the residence time of the inhaled drug particles in the lungs. As a Postdoctoral Research Associate (Jul 2020-Feb 2022 at the University of Arizona) and Research Assistant Professor, my current position since Feb 2022 in Prof. Heidi M. Mansour’s lab, my current research focuses on the development and evaluation (physicochemical and biological) of novel inhalable drug formulations for treating lung conditions. Continuing my progress towards my career goal will require additional training in improving my research skills, writing grant applications, and collaborations with multidisciplinary research teams.
This project will allow me to improve my research skills in engineering nano-composite dry powder particles with better dispersibility. The extensive training in research methods and co-supervision skills that I received while earning a Ph.D. and postdoctoral training has provided the necessary foundation for me to be able to lead the proposed research project and advance my development as an independent investigator. The success of this project will build my confidence in developing my own research ideas and securing more grants particularly from the National Institutes of Health (NIH) to reach my career goal as an academic investigator. The presentation of this work at inhalation research conferences such as Drug Delivery to the Lungs, Respiratory Drug Delivery and International Society for Aerosols in Medicine (ISAM) would expand my scientific network and collaborations with multidisciplinary research teams.

Project Background:
Lung cancer is the leading cause of cancer death worldwide. The American Cancer Society estimates 1 in 16 people will be diagnosed with lung cancer in their lifetime.1 Combination drug therapies are being explored, as the synergistic action of multiple drugs could potentially lead to better therapeutic efficacy and reduced opportunity for the development of drug resistance by the cancer cells.2 However, the systemic routes of administration are limited by off-target toxicity and poor pulmonary selectivity of the drug combinations used, leading to low therapeutic efficacy.3
Several programs are currently investigating pulmonary delivery of anti-cancer drugs via inhalation.4 Pulmonary delivery of drugs has several advantages over conventional treatment, including a) being non-invasive, b) circumventing first pass metabolism and systemic toxicity, c) reducing the frequency of administration, and d) higher local drug concentrations. Among several pulmonary drug delivery systems, biodegradable nanoparticles have demonstrated several advantages in terms of protecting the active ingredient from degradation and releasing the drug in controlled manner for prolonged periods of time.5 Although few attempts have been made to deliver anticancer agents using nanoparticles and liposomes via inhalation route, the major limitations of these systems are instability during nebulization, biodegradability, drug leakage and associated drug adverse side effects.6,7 Other issue with nanoparticle pulmonary delivery is that their size is not suitable for deep lung deposition.
In my proposed study, I aim to develop nanocomposite dry powder particles loaded with two anti-cancer drugs, docetaxel and celecoxib which have synergistic action for treatment of lung cancer.8-10 Nanocomposite particles consist of drug-loaded nanoparticles and excipients. Nanoparticles are combined with the binder to render micron-sized particles of 3 μm size, which is the most effective dimension to deposit deep into lungs. After deposition in the surface fluid layer of alveoli, the complex is designed to liberate primary drug-loaded nanoparticles.11 Having deposited beyond the mucociliary escalator, the drug-loaded nanoparticles are sized to avoid recognition of alveolar macrophages and delivered near the lung epithelium for uptake and prolonged lung residence.

Nivedita Shetty works for the Small Molecule Pharmaceutical Sciences Department of Genentech, Inc., in South San Francisco, CA responsible for formulation development of inhaled and oral drugs to support phase I clinical studies.

Prior to joining Genentech, she completed her PhD in Pharmaceutics at Purdue University with a focus in particle engineering for inhalation. During her PhD she was awarded the Outstanding Student Research Award from National Institute for Pharmaceutical Technology & Education (NIPTE); Excipient Graduate Student Scholarship by International Pharmaceutical Excipients Council (IPEC); McKeehan Graduate Fellowship and Ronald W. Dollens Graduate Scholarship from Purdue University. She holds undergraduate Pharmacy degree from Bombay College of Pharmacy, India and Master’s degrees in Pharmaceutics from Northeastern University, Boston.

She served as co-chair for New Devices and Emerging Therapies networking group at International Society for Aerosol in Medicine (ISAM) and current secretary of Inhalation and Nasal Community of American Association of Pharmaceutical Scientists (AAPS). She serves as an expert reviewer on numerous journals including AAPS PharmSciTech, Molecular Pharmaceutics and Pharmaceutics (MDPI).

Her current research interests are particle engineering for high drug load inhaled formulation and mechanistic understanding of the role of material properties on aerosol performance.

For locally acting dry powder inhaler formulations deposition of drug particles in the lung followed by its dissolution in lung mucosal fluid is very complex. In recent years, inhaled drug molecules seem to be poorly soluble and high doses of drugs need to be delivered to the lungs to exert local therapeutic effect. Such scenarios where the drug dose is high and solubility is low could result in dose number >>1 thereby resulting in deposition of insoluble particles in the lung which could have toxicity implications.

Our current research aims to work with one such poorly soluble model internal compound to better understand the difference in in-vitro deposition and dissolution performance between jet milled and spray dried high dose formulation for inhalation. Jet milling is a well-established cost-effective technology for inhaled formulation development. Spray drying has also been used for producing inhaled formulations but could be more cost and time consuming. Jet milled particles are crystalline whereas solution-based spray dried particles tend to be amorphous. We aim to evaluate and compare the in-vitro deposition and dissolution advantage of spray dried to jet milled formulations for inhalation. We will also characterize the formulations for its flow and solid-state property.

The outcome of this research aims to help early formulation feasibility decisions for high dose drugs with poor solubility. As per the Inhalation Ad Hoc Advisory Panel for the USP Performance Tests of Inhalation Dosage Forms dissolution testing is not a requirement for inhaled products. Through this research we might be able to provide a scenario where in-vitro dissolution of inhaled DP is critical during early formulation screening (high target lung dose with poor solubility).

1. Through this research I hope to get a better understanding on the importance of in-vitro dissolution testing for inhaled drug products.
2. Internally, we do not have the capability to run analytical testing’s such as NGI and dissolution. Hoping to collaborate with a university to execute these analytical testings
and mentor a PhD student in the process.
3. Looking to get a Publication upon completion of this project.

Ahmad is a Pharmacist and a PhD student in the group of Prof. Sally-Ann Cryan at the Royal College of Surgeons in Ireland (RCSI) – School of Pharmacy and biomolecular sciences. In 2019, he received the Strategic Academic Recruitment (StAR) PhD scholarship from the Royal College of Surgeons in Ireland. His PhD work focuses on developing inhalable All Trans Retinoic Acid-loaded nanoparticles as host directed immunotherapy for tuberculosis in collaboration with St. James’ hospital (Trinity College Dublin), Imperial College London and Aerogen Ltd.

He completed his Erasmus Mundus Joint Master’s degree in Nanomedicine for drug delivery (NANOMED EMJMD) jointly from four universities; University of Paris and University of Angers in France, University of Pavia in Italy and University of Patras in Greece, funded by Erasmus+ scholarship. He did his Master’s thesis at Queen’s university Belfast in United Kingdom focusing on engineering targeted nanomedicines for advanced prostate cancer. He obtained his BSc in Pharmacy from the University of Jordan in 2016.

Ending Tuberculosis (TB) by 2030 was listed in the UN sustainable development goals as TB kills more people globally than any other bacterial infection. 5.8 million people were newly diagnosed with TB in 2020. In the same year 1.3 million HIV negative and 214,000 HIV positive people died from TB.
Current treatment regimens are based on lengthy oral and parenteral dosage regimens with medications associated with many adverse effects which leads to poor patient adherence and the significant rise of multi drug resistance TB strains (MDR-TB). So, there is unmet clinical need to develop new treatment strategies to overcome MDR-TB and improve the treatment dosing regimen. An adjunctive host-directed therapy (HDT) could address these issues. HDTs act on the host instead of the bacteria, by boosting the host immune response, to augment the beneficial features of fighting the bacteria and reduce tissue damage. Administration of TB drugs locally via the pulmonary route to achieve high concentrations at the site of infection is important to enhance efficacy, target drugs towards the alveolar macrophages which are the niche for Mycobacterium tuberculosis (Mtb) and avoid systematic side effects, thereby providing better prognosis for TB patients, and reducing the incidence rate of MDR-TB.

One of the emerging HDTs for TB from our lab is All trans retinoic acid (ATRA) which is the active metabolite of vitamin A, currently a FDA approved drug for acne and acute promylocytic leukemia (Bahlool AZ et al, Curr Res Immunol, 2022). Drug repurposing of licensed medications such as ATRA is considered a streamlined approach for market access and reduces the industrial development costs and length of time to reach the patient. We have developed a formulation for targeted, host-directed TB treatment, using ATRA-loaded nanoparticles (NPs) that are suitable for nebulization. These nanoparticles have proven their in vitro efficacy against H37Ra Mtb and their safety in an in vitro airway epithelial cell model. This formulation has been successfully integrated with Aerogen Solo® vibrating mesh nebulizer (VMN) and the generated aerosol has favourable droplet size and aerodynamic properties for deep lung deposition (drafted manuscript). The nanoparticles have been successfully scaled up using the Ignite Nanoassembler® microfluidics system to enable more efficient clinical and commercial translation.

The project is a collaboration between academia, industry and clinical teams led by Prof. Sally-Ann Cryan at RCSI, the TB immunology group led by Prof. Joseph Keane and Dr. Mary O’Sullivan at St. James’ Hospital, Dublin and Dr. Ronan MacLoughlin at Aerogen Ltd, Galway. The project is now focusing on testing this inhalable TB therapy using state-of-the art animal models and performing advanced aerosol characterization to support the effective and timely clinical translation offering hope to millions of patients worldwide who are infected with TB annually.

I look back a few years and feel very grateful for choosing this path, getting interested in science at a young age and craving to understand what is behind natural phenomena around me, sparking my curiosity and fuelled with love for science. Undertaking a PhD is never easy and throwing a pandemic on the top of this would make it more difficult. However, this made me more resilient and passionate to pursue my project focusing on respiratory diseases and respiratory drug delivery. The COVID-19 pandemic is significantly impacting Tuberculosis (TB) case-finding, management and access to treatment, leading to a drop in diagnosis and an increase in deaths compared with previous years. The pandemic has renewed the focus on the benefits of pulmonary drug delivery and drug repurposing as part of the global efforts to treat and tackle COVID-19. My ultimate goal is to build a strong academic healthcare-focused career and experience in the field of inhaled nanomedicines and therapeutic aerosol bioengineering for infectious diseases.

I completed my Pharmacy degree in 2016 with a research project focused on developing a silver nanoparticles-hydrogen peroxide based system to treat multi drug resistant infections (PLoS One. 2019. PMID: 31393906). In 2017, I got an Erasmus+ scholarship to do my Master’s degree. I was accepted into Erasmus Mundus Joint Master’s degree in Nanomedicine for drug delivery (NANOMED EMJMD) which is a mobility joint program between four universities; University of Paris Cite, University of Angers in France, University of Pavia in Italy and University of Patras in Greece. This diverse scientific and multicultural travel experience has provided me with many scientific and life skills that helped me grow in the academic field, learning to work with multiple research groups within multiple labs with different cultures and a unique multi-cultural travel experience that sharpened my personality and flexibility. During my Masters, I did a three months’ internship in MiNT research lab in Angers, working on a pre-clinical evaluation of a nanomedicine formulation for ovarian cancer in collaboration with a start-up company and Institute Curie. At the end of my second year, I completed my Master’s thesis at Queen’s University Belfast, UK, working on engineering targeted nanomedicines for advanced prostate cancer.

In 2019, I was accepted into the StAR (Strategic academic recruitment) PhD program at the Royal College of Surgeons in Ireland (RCSI), and this is when destiny took me into a whole new level. I was able in the first year to do 3 lab rotations on 3 different projects on tissue engineering, cancer bioinformatics and respiratory drug delivery. Then, I chose to develop and defend my PhD proposal on developing inhalable host directed immunotherapy for tuberculosis. During my PhD, I am developing an adjunctive inhaled ATRA-Loaded polymeric nanoparticles as host directed immunotherapy for TB to overcome the issue of multidrug resistance and improving the treatment regimen.

We have developed ATRA loaded poly-lactic-co-glycolic acid (PLGA) loaded nanoparticles at RCSI and proved their safety and the efficacy in an in vitro infection model of TB at the Trinity Translational Medicine Institute, St. James’ Hospital (Dublin). In collaboration with Aerogen, I have successfully integrated these ATRA-nanoparticles with the Aerogen Solo® vibrating mesh nebulizer and characterized the aerodynamic characteristics of the aerosol using laser diffraction and cascade impaction and assessed the delivered dose in simulated adult breathing pattern using a breathing simulator (drafted paper). Besides research, academic teaching and student supervision is part of my academic PhD life at RCSI, I’m enjoying supervising undergraduate Pharmacy students in their research projects and delivering practical pharmaceutics lab sessions.

We are currently moving on with this project to in vivo studies, in collaboration with Dr. Brian Robertson at School of Medicine at Imperial College London. Dr. Robertson’s lab has established a murine infection model of tuberculosis and we are hoping to test our formulation in vivo in this model to determine the in vivo efficacy and safety of our targeted delivery approach for ATRA as an adjunct treatment for Tuberculosis. During this part of my PhD, I will learn about the Mycobacterium tuberculosis murine infection model, and the delivery of the ATRA-nanoparticles to the lungs. We will monitor the bacterial burden and pathology in the lungs to assess efficacy of treatments compared to untreated controls and collect lung and Bronchoalveolar lavage (BALs) samples for further analysis of the effect on the immune cell population and cytokine profile. Lung tissue samples will be examined by a veterinary pathologist to assess the safety of the formulation. This part of my project will provide in vivo data to complement and extend my current studies and give me experience of working with specialized TB animal models, biological samples and working with class 3 biosafety animal facilities. In addition, the generated nanoparticle-loaded aerosols will be further characterized in simulated breathing adult and paediatric patterns with manikin head models for more clinically relevant characterization with our collaborators in Aerogen. This will allow me to gain a new set of skills and techniques that are not available for me to learn at my university and will allow me to bring it back to my research group, bridging the gap of experience that we need for the project.

In order to achieve my goal, a broad range of interdisciplinary knowledge and hands on experience will be required and the progress of this work is highly dependent on financial and technical support. The DDL career development grant would provide me with the support to strengthen the interdisciplinary collaboration between two different institutions in two different countries and enable me to expand my professional network. This grant if funded will support;
• My development of additional technical skills in the pre-clinical in vivo assessment of inhaled medicines and advanced aerosol characterization methods
• The pre-clinical assessment of our novel nanomedicine targeting TB in order to generate comprehensive data on the pre-clinical efficacy and safety of ATRA-nanoparticles with a view to applying for further funding to support clinical development and/or commercialisation with our clinical and industrial collaborators
• The presentation of project outcomes at international drug delivery, aerosol, and TB related conferences including DDL conference, Controlled Release Society (CRS), UK-Ireland Controlled Release Society local chapter (UKICRS), European Foundation of Clinical Nanomedicine (CLINAM) and Keystone TB, to gain valuable presentation and communication skills and experience with peers worldwide
• This grant will also enable me to engage with a group of outstanding infectious disease researchers in Imperial College London and thereby expand my professional network. It will give me the opportunity to get the experience of collaborating internationally across campuses and communicating my science to a scientific audience outside my field.

As an international student, I’m hoping to be able to bring the new skillset that I learn during my training back to my home country to do my own research and train new generations in my country on respiratory drug delivery research which is a field that we lack skilled expertise in. I believe that this grant will not only benefit me personally, but will indirectly benefit many people globally with the transferable skills that I will gain and teach to peers and younger generations.

This award will help the clinical translation of my project which could potentially save millions of patients worldwide who are infected with TB annually. It will allow me to build strong transferable interdisciplinary knowledge, skills, techniques and professional experience which will help me bridge to a strong career path in the field of respiratory drug delivery and help me to grow as a leader in the field.

Jana Schembera is a pharmacist by training and a PhD student in the group of Prof. Regina Scherließ at Kiel University. The aim of her doctoral thesis is the development of an inhalable dry powder platform for mRNA vaccines.

During her pharmacy studies she gained first insights in the pharmaceutical industry as an intern within the inhalation characterisation team in the Pharmaceutical Sciences Department of AstraZeneca in Gothenburg.

In 2018 she completed her studies of pharmacy at Kiel University and spent one half of her practical year at Boehringer Ingelheim in Biberach an der Riß. At Boehringer Ingelheim she joined a group handling late-stage drug product development to gain further industrial experience. After being registered as a pharmacist she came back to Kiel University in 2020 to start her PhD in the field of inhalation.

Apart from her research project she is responsible for the practical teaching of pharmaceutics to pharmacy students and continuing her training to become a specialised pharmacist in pharmaceutics and analytics.

The route of administration has tremendous impact on the efficacy of pharmaceutical products such as vaccines. As many viral infections enter the body through the respiratory tract, the aim of my doctoral thesis is the development of an inhalable dry powder platform for mRNA vaccines. The delivery of vaccines to the respiratory tract aims directly at the main entry point of respiratory pathogens and the mucosal immune system.
Delivery of mRNA into eukaryotic cells requires the use of transfection reagents such as lipids forming lipoplexes with the mRNA. The lipid carrier not only enhances transfection efficiency but protects the mRNA against hydrolysis and RNases as well. To focus on the formulation in the solid state we chose two well-established lipids for lipoplex formulation in an equal weight ratio: the cationic lipid DOTAP and the neutral lipid DOPE.
In order to determine the most effective lipoplex composition, I investigated the efficiency of transfecting Calu-3 cells with various weight ratios of mRNA and the equal mixture of the two lipids. As a model system, I used reporter gene mRNA coding for firefly luciferase. A luminescence assay measures the abundance of this enzyme. This assay system also verifies that the mRNA is still intact and translatable to a functional protein.
After determining the best working weight ratio, I investigated the compatibility of excipients commonly used in inhalation with mRNA-lipoplexes during transfection and their particle characteristics. These excipients are going to form the matrix surrounding the mRNA-lipoplexes in inhalable particles. As my next steps, I am going to spray dry the mRNA-lipoplexes and excipients in a solution together to reach a particle size less than 5 µm. The spray drying process is a critical step because the formulation and process parameters have to ensure the integrity of mRNA and lipids to preserve a proper transfection efficiency.
One of the experts in the field of mRNA formulation for the respiratory tract is Jenny Lam, the DDL2020 Emerging Scientist Award Winner. Jenny Lam and her group were among the first reporting dry powder delivery of mRNA to the respiratory tract to induce transfection. Thus, I plan a scientific stay in her lab for two weeks to get a deeper insight and hands-on experience especially in the quantitative analysis of mRNA in complex matrices. Due to the expertise of Jenny Lam’s group utilising polymers as vesicles, I hope to find a way of adapting their methods to my lipid-vesicles and transferring them to Kiel. Apart from this, I aim to obtain new perspectives for the evaluation of my own procedures, to develop innovative methods, and to gain insights into in vivo assessment.
The overall aim of this project is the development of a readily adaptable dry powder platform for pulmonal mRNA vaccination. To fulfil this challenging task, expertise for tailored drug delivery and know-how in molecular biology need to be synergised.

Early on in my studies, I discovered an interest in Pharmaceutics and started to work as a student assistant within the group of Prof. Regina Scherließ at Kiel University. A key aspect of the group’s research is the development and characterisation of inhalable formulations, especially dry powder formulations. During my time as a student assistant, I was able to gain an impression of the complexity and challenges of respiratory drug administration, as well as to grasp its tremendous potential. During an internship, I had the opportunity to gain insight in the work of the inhalation characterisation team in the Pharmaceutical Sciences Department of AstraZeneca in Gothenburg. Here I got to know the industrial point of view on formulations meant for inhalation and their aspects concerning pharmaceutical activity in pre-clinical development and at the start of clinical trials.
Subsequently, after finishing my studies I spent one half of my practical year at the main German research site of Boehringer Ingelheim in Biberach an der Riß where I joined a group handling late-stage drug product development. In this group, I worked on my own project within which I established a newly arrived instrument for particle size measurements in order to support the analytics of late-stage drug products. My time at Boehringer Ingelheim and AstraZeneca did not only give me a very valuable peek into the world of industrial research, but it also emphasised how many facets along the way critically influence the efficacy and quality of the finished formulation.
Now, I am a registered pharmacist and a doctoral candidate in the group of Prof. Regina Scherließ at Kiel University. During my past two years, I was not only doing research but also spending half of my time teaching students in the second and fourth year of their pharmacy studies. In the second year students learn about the common formulations, which have to be prepared in a public pharmacy, and the fourth year is all about formulations that are produced in industry. Watching the students enhance their independent thinking and scientific competences is one of the best parts of my teaching duties. Teaching future pharmacists comes with great responsibility. For example, some day in the pharmacy, they may need to prepare capsules meant for children. Apart from this I am currently introducing a master student into the world of cell culture and providing advice on experiment design.
Whenever I see the students develop their skills, I think about how I can develop myself further. This comprises enhancing my teaching skills and especially becoming a better researcher as well as making progress with my research project. I am convinced visiting a foreign lab and seeing their way to approach research questions and address challenges is going to benefit both my personal and research progress.
The goal of my research project is the development of an inhalable dry powder formulation for mRNA vaccination. Already at the beginning of my time as a doctoral candidate, I realised that the industrial insights are often not suitable for formulation development in academia.
Apart from this I am the first and currently only researcher working with mRNA in my department. Hence, I started from scratch and established handling procedures and analytics of mRNA in our lab. After facing many challenges working out the basics and gaining experience in performing complex assays, I began with the actual research and formulation development.
Now, with Covid-19 not interfering to such extends anymore, I would greatly benefit from visiting another academic lab that is highly experienced in handling mRNA.
This opportunity would allow me to learn innovative techniques and reflect as well as improve my own methods. It would also show me aspects I might have missed. In addition, this new point of view would give me the chance to enhance my creativity of implementing procedures, and my abilities of overcoming challenges and solving problems. As my stay will only last two weeks, I will also have to put my time management and team working skills to the test.
The visit would not only add to my scientific expertise, but it would also give me the chance of networking and getting in touch with scientists working in my field on an international level. I aim to stay in touch with the group and hopefully a joint project can be approached together in the future. This is an opportunity to be recognised by the international community, which will enhance the progress of my project and career.
As I love to travel and get to know people, I was happy to attend my first face-to-face conference, the PBP World Meeting in Rotterdam in March this year, where I presented a poster with the title “Transfection of Calu-3 cells with mRNA/DOTAP:DOPE-lipoplexes: influence of weight ratio and forming media”. After that experience, I am thrilled for my first face-to-face Drug Delivery to the Lungs conference in the end of this year and looking forward to presenting the next steps of my work.
Since the spirit of scientific exchange is something I never want to miss, I plan to proceed with formulation development for inhalation in either academia or the industry after finishing my doctoral thesis in 2024.
Succinctly, the experiences of the stay in Jenny Lam’s lab are going to enhance my chances of a confident scientific carrier, bring me a huge step closer to the inhalable dry powder platform for mRNA vaccines, and provide me with a data set I would like to publish in a manuscript and present at the DDL 2023.

Simon Bock is a Pharmacist by training and PhD student at the Department of Pharmaceutics and Biopharmaceutics at Kiel University, Germany. His research focuses on DPI formulation development through implementation of Additive Manufacturing technologies in pharmaceutical processes. His PhD project comprises the evaluation of complex dispersing aids and unconventional carrier geometries in dry powder inhalation. Prior to his academic research, he completed his studies of pharmacy at the Technical University of Braunschweig, Germany, in 2016. After graduation, he worked for several local pharmacies in Hamburg and the Frankfurt area, Germany. In addition, he gained first experiences in the pharmaceutical industry working in the quality management unit of Sanofi Germany. Since April 2018, he has been employed as a research associate in the working group of Prof. Regina Scherließ at the Institute of Pharmacy in Kiel, Germany. Besides research on particle engineering of DPI formulations he has been responsible for the practical academic teaching to undergraduates. To present his scientific findings he was previously selected for giving several live talks including the NordicPOP conferences in Oslo in 2019 and Copenhagen in 2020.

My PhD project entitled Application of additive manufacturing in the development of DPI formulations has two objectives: On the one hand, the application of additive manufacturing (AM) processes to fabricate complex, uniform carrier geometries. On the other hand, the development and production of accessories with intricate details to improve dispersion of dry powder formulations. For such formulations, API particles are oftentimes blended with coarse carrier particles. The production process of the standard carrier, irregularly shaped lactose particles, implies a high degree of inter-particle variability, which can potentially lead to dose-to-dose variations. Despite the advantages of processability and dosability, perfect spheres may be similarly unsuitable as carriers with regards drug loading capacity, surface properties, fluidisability, etc. It remains unclear which specific carrier geometry and size is most suitable. The production of non-spherical and uniform particles on a microscale to systematically assess the influence of geometry and size has not been technically possible in the past.
The aim of the project is thus to systematically assess the influence of both characteristics by high-precision fabrication of uniform particles. The focus is on how such techniques could contribute to a better understanding of fundamental processes that occur during inhalation of DPI formulations. Small-scale manufacturing and processing enable the production and evaluation of innovative interactive blends with tailored API capacity and delivery performance. As a hypothetical result, it might be feasible in the future to make active substances available for inhalation therapy that hitherto could only be administered to the lungs in insufficient or uncontrolled doses by specifically altering the carriers.
In addition to the production of customised carriers as such, my project includes the investigation of relatively large dispersing aids as free-levitating accessories in DPI devices. It remains unclear to what extend such accessories can influence the dispersion of powder formulations within an inhaler. Since the deagglomeration of formulation components is influenced by particle-particle interactions, dispersing aids could provide additional kinetic energy to overcome particle adhesion forces. This might result in a better aerosolisation and thus higher dispersing efficiency. The magnitude and intensity of energy input might be related to the behaviour of the object in the fluid within the device, which is influenced by its’ morphology and size. Furthermore, surface structure and roughness of such aids are considered to influence the particle interactions and thus the dispersing capacity. Only state-of-the-art manufacturing techniques, which are challenging and costly, can guarantee accurate control of such properties. Hence, using appropriate fabrication techniques allow a better statement of the actual impact of various types of dispersing aids.

As a pharmacist by training and a formulation scientist in the current position, the combination of classic pharmaceutics with AM techniques offers a broad widening of my personal and professional horizon.
The urge to wander off the beaten track has always been part of my professional lifetime. In 2016, I finished my studies of pharmacy at Technische Universität Braunschweig, Germany, with the 2nd state examination. During my studies I spent some time at the University of Agder, Norway, with a non-pharmaceutical curriculum. Furthermore, voluntary internships at Boehringer Ingelheim and Bayer Healthcare allowed me to gain insights in the pharmaceutical industry. After my 2nd state examination, I spent the following practical year in a pharmacy and at the pharmaceutical company Sanofi. Having been licensed as a pharmacist in 2017, I worked in the Frankfurt area in various pharmacies but also on the development of a smartphone app for pharmaceutical professionals. With work experience from pharmacies, the pharmaceutical industry and other ventures, the research related to respiratory drug delivery intrigued me the most. It stroke me, that this particular branch of medicine and pharmaceutics, respectively, is still relatively small and that there could be a lot to explore. In addition, I have found that my personal interest lies mainly in research and development. Therefore, I applied as a PhD student in the research group of Prof. Regina Scherließ at the Department of Pharmaceutics and Biopharmaceutics at Kiel University, whose research activities already covered this topic for many years.
Since April 2018, academic teaching and student supervision is an essential part of my current work at Kiel University. The content covered in lectures and practical courses comprises a wide range of topics related to pharmaceutics and biopharmaceutics: from solid, liquid and sterile dosage forms to inhalatives and transdermal application systems etc. To explain the need of established but also of innovative dosage forms is a crucial part of the lectures. In addition to the teaching as such, for the time being I am responsible for the overall organisation and scheduling of an extensive lab class on pharmaceutics. It involves the transformation from analogue to digital teaching, which became necessary due to the current pandemic. This digitalisation of academia offers novel ways of communication and collaboration. As an additional effect, the potential of computational tools for research and development in all aspects of pharmaceutics gets increasingly into focus.
On top of the daily routine of academic teaching, to provide insights into research to undergraduates means a lot to me. In 2019, I initiated and was responsible for an internship in the Department of Pharmaceutics and Biopharmaceutics for a student from the United States of America. With the student being a chemical engineer, the internship was characterized by a lively exchange of different scientific perspectives from both parties. This period strengthened my conviction, that interdisciplinarity is always beneficial in research. I am convinced, that to wander off the beaten tracks can generally be worth it.
From the very beginning of my doctorate, I have been working on particle engineering of dry powder inhalation carriers in interactive blends. Initially, I worked with basic operations such as carrier coating to investigate the effect of surface modification on the aerodynamic performance. In this work package, model MCC-carriers with relatively consistent sphericity and narrow particle size distribution were covered with several amino acids in a fluidised bed coating process. Subsequently, the effect of both the particle size and the coating of the carrier on emitted dose and fine particle fraction of API were investigated. The diversity and magnitude of variability in production and processing of carriers has hindered a systematic study of isolated, individual parameters ever since. In the framework of this work package, the question arose whether novel technologies could provide new investigatory concepts. As a result of attending the NordicPOP conference in January 2019, at which I was accepted to give a live talk on my research, an opportunity for a scientific collaboration with the institute of pharmacy in Oslo, Norway, came up. Specifically, a temporary stay in Prof. Ingunn Tho's research group was arranged. The aim of this visit was to work out how 3D printing (3DP) could generally be useful for the development of orally inhaled and nasal drug products.
During that two-week stay in March 2019, I was kindly introduced to the principles of AM with the help of Eric Kissi, Postdoctoral Research Fellow at the University of Oslo. Hands-on experiments with standard equipment supplemented my theoretical knowledge about 3DP. With two undergraduates, who accompanied me for one week, plenty of tools and prototypes were manufactured to acquire advanced knowledge. From digital design, slicing, preparation of filament or resin to printing and post-processing, the variability of 3DP workflows is unimaginably high. Therefore, and in view of the following work package, I decided to focus on refining a handful of prototypes. These prototypes were then used as accessories in DPI formulations. Particularly, several relatively large prints with well-defined shape were inserted into the main chamber of a Twister® device. The aim of this insertion was to proof, if such free-levitating structures provide additional dispersing effects. Proof of concept on whether these prints could be used as dispersing aids was first mentioned in an abstract submitted for the DDL 2019 conference. This concept and its preliminary results matured not only into the central theme of my doctoral research but also into a submission for a European patent.
The task now is to further evaluate the optimal geometry and size of a dispersing aid. Based on the prototypes previously used, a handful of geometries with intricate details and much higher complexity were selected for three-dimensional printing. Once these objects will each be fabricated in different sizes with highest resolution, the influence of the resulting products on the DPI performance can be investigated. For this purpose, the aids will be inserted into an inhaler device together with interactive powder blends or soft pellet formulations, using different APIs in each case. In the course of an aerodynamic assessment, the effect on the dispersion performance can then be analysed.
The considerable degrees of freedom of AM in terms of particle morphology and manufacturing allow new routes to tackle long-standing challenges. This technology is still considered to be in its infancy. However, with the progress made in the recent past, the applicability and accessibility are steadily increasing. At this point in time, the first use of additively manufactured, extraordinarily complex DPI carriers in a size of a few micrometres is within reach. Nevertheless, sound decision-making for the allocation of finances, software, equipment, and personnel is imperative for further progress in this area.
Due to the need to fabricate objects with intricate details in high resolution, access to suitable manufacturing technologies is a major bottleneck. Therefore, the progress of the current work package is heavily dependent on financial and technical support. In the long term, numerical simulations complemented with formulation-specific parameters and coupled with deep-learning algorithms could yield a continuous optimisation of both carrier and dispersing aid designs. With technologies capable of producing such geometries, access to an in-depth study of particle characteristics and behaviour appears attainable in the future.
It took two years of theoretic, hands-on and collaborative experience in 3DP to reach the current state. Technology for manufacturing of complex designs down to a few microns is now available. With the knowledge acquired in the past, the DDL Career Development Grant would open a new chapter of interdisciplinarity in my personal and professional career as a scientist. Financial support could help to give general insights how AM processes can potentially be applied in respiratory sciences. My personal goal is to elaborate explanations of fundamental mechanisms in DPI formulations and to raise new scientific questions in the intersect of formulation development and AM. This grant would provide access to state-of-the-art printing technologies, enable collection and validation of data and accelerate the research process.
My intention is to publish a comprehensive scientific paper outlining initial explorations of the potential capabilities of complex dispersing aids and unconventional carrier geometries in dry powder inhalation. Since the use of the corresponding fabrication technologies is still unexplored in many respects, it can be expected that research in this area will continue in both academic and institutional settings. Contributing to the expansion of this emerging field of research is my personal goal. I would be delighted if this work would eventually culminate in a presentation to an international auditorium of aerosol scientists. The Career Development Grant could substantially support this project that would mark a cornerstone of interdisciplinarity in my personal career in the field of drug delivery to the lungs

Snezana Radivojev is currently working on her PhD thesis in a cooperation between RCPE GmbH (Research Center Pharmaceutical Engineering) and the Medical University of Graz in Austria, under the supervision of Dr. Eleonore Fröhlich. Her PhD research is focused on the establishment of in-vitro-in-vivo correlations through the development of new and/or improved in-vitro and in-silico methodologies, following the rational understanding of orally inhaled product’s biopharmaceutics. Additionally, she investigates the development and characterization of new orally inhaled products. She acquired her BSc diploma in Chemistry, from University of Novi Sad in Serbia. In 2017, she earned Dipl. -Ing degree in Chemical and Pharmaceutical Engineering from Graz University of Technology in 2017 with the master thesis ‘‘Characterization of potential new dry powder inhaler formulations’’ under the supervision of Prof. Dr. Johannes Khinast (Institute of Process and Particle Engineering) and Dr. Sarah Zellnitz. Following graduation, she started her carrier in RCPE as a scientist and join their Area II-Advanced Products and Delivery, supervised by Dr. Amrit Paudel and Dr. Simone Pival-Marko. Aside from her research, she is interested in actively promoting networking opportunities between PhD students coming from different backgrounds of knowledge and universities in the field of inhalation. During DDL2019, together with Dr. Magda Swedrowska (King’s College London) and Marie Hellfritzsch (Kiel University), she founded the New Researcher Network (NRN) as a mean to facilitate cooperation between young researchers and support them throughout their scientific journey

The proposed research project is a part of the work essential to finalizing my doctoral thesis. The topic of my thesis is focusing on the optimization and improvement of the current state of the art in-vitro systems which would result in a trustworthy set of data. These are subsequently used to improve the in-vivo predictions by application of in-silico models. My personal development in the inhalation field started early in my master thesis. During this work, I gained an insight into different manufacturing processes, DPI performance and challenges that arise when formulating drugs intended for inhalation use. Thus, a further step was to tackle the open research questions of inhalation biopharmaceutics. This field is still emerging and represents a wide space for gathering the lacking scientific knowledge. My thesis is done at Medical University in Graz, coupled with Research Center Pharmaceutical Engineering and two industrial partners. The work within this framework displayed to me the relevance of investigating the questions interesting to academia but, in the same time, possessing the potential industrial application. The first part of my work was focused on the investigation of how mishandling of DPIs could potentially impact the predicted pharmacokinetic parameters. This study showed how in-vitro studies can be successfully coupled with the physiologically based pharmacokinetic modelling as a risk-assessment approach for the stability of DPI formulations (Radivojev et al., Jou. of Drug Deliv. and Sci. 2019). This topic was further investigated with the support of professor Forbes and SimInhale COST Action. The opportunity to work with the groups that have different specialties so early in my career helped me to acquire unique perspectives and enriched my laboratory and research skills. The second phase of my PhD was focused on the investigation of different SLFs and their application (Radivojev et al. Int. Jou. of Pharm. 2019) as well as how different components present can impact the solubility of inhaled drugs and their predicted in-vivo performance (Radivojev et al, Int. Jou. of Pharm. 2021, submitted). It tackles the challenges widely discussed in the inhalation field such as the relevance of using certain SLFs (e.g. simple systems such as phosphate buffer or addition of surfactants) or apparatus design. The proposed research project is a final step in my work that can help in elucidating whether the use of more complex in-vitro apparatus is necessary or simple approaches can be considered. Furthermore, it will contribute towards an overall better understanding of inhaled biopharmaceutics.
Early on in my career as a researcher in inhalation, I was fortunate to realize how important collaborations and open discussions are in the world of science. I became passionate in helping early carrier researchers, such as myself, in facilitating exchanges of research ideas and challenges as well as the work in joint projects. With the help of Drug Delivery to the Lungs conference committee and my colleagues, New Researcher Network has been founded. We aim that this group provides continuous support of early carrier researches, for both professional and personal development, and to enrich contributions to the aerosol research. The DDL grant would be significant support in my career development as well as the possibility to advance collaboration between three groups i.e. King’s College London, Medical University and Research Center Pharmaceutical Engineering in Graz. Professor Ben Forbes has a vast knowledge in the inhalation field and is a recognized expert in the field of inhalation biopharmaceutics. His supervision and experience gained in his research group will be a great opportunity to widen the horizons of my knowledge. The proposed cooperation falls within the umbrella of the carrier pathway I would like to follow and promotes international cooperation as well as interdisciplinary research by combining different methodologies.

Aneesh Thakur is currently an Assistant Professor at University of Copenhagen, Department of Pharmacy, where he is working in the research group of Vaccine Design and Delivery. Prior to this, he was an Assistant Professor in the Department of Veterinary Microbiology at CSK Himachal Pradesh Agricultural University (2013-2016). He carried out postdoctoral research positions at University of Copenhagen (2016-2020) and Technical University of Denmark (2012-2013). He was a research scientist in the Tuberculosis Aerosol Challenge Facility at International Centre for Genetic Engineering and Biotechnology (2007-2009). He obtained his undergraduate (DVM) and master degree (MVSc) in Veterinary Microbiology and Immunology from CSK Himachal Pradesh Agricultural University (2007) and his PhD in Immunology from Technical University of Denmark (2013). His research is highly multidisciplinary and involves (i) the development of next-generation nanoparticle-based formulation strategies to enhance efficacy of subunit and nucleic acid-based vaccines and therapies and (ii) strategies for controlled modulation of pulmonary T cell immunity using adjuvants. He investigates topics at the interface of immunology, drug delivery, and nanotechnology to pursue novel vaccine designs for translational applications against infectious diseases and cancer.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) is one of the top 10 causes of death worldwide, despite decades-long use of the Bacillus Calmette–Guérin (BCG) vaccine. Hence, a new vaccine is urgently needed to control this TB pandemic. TB typically affects the lungs, and the transmission of Mtb occurs primarily through inhalation of droplets containing bacilli from infected individuals. As the respiratory tract is the natural route of Mtb infection, mucosal immunization in the airways with highly purified recombinant Mtb antigen(s) is a very promising strategy for the development of novel TB vaccines. We have made a major breakthrough by identifying the molecular components and a formulation strategy for a thermostable, dry powder-based inhalable TB subunit vaccine candidate, H56/CAF01. The overall goal of this project is to design a vaccine and a vaccination strategy that together induces strong H56-specific humoral and cellular immunity in the lungs and systemically, which is independent of cold-chain. We hypothesize that a vaccine, which elicits strong anti-TB immunity in the lungs, can induce rapid clearance of Mtb-infected cells very early at the site of infection and hence enhance protection. However, vaccine delivery in lungs is associated with many challenges such as: (1) design of inhalable, dry powder-based vaccine formulations suitable for inhalation, (2) the specific aerodynamic size requirement of particles for deep lung deposition (1.5-4 µm), and (3) lack of devices for preclinical administration of powders. Here, we aim to target the respiratory mucosa for immunization using PreciseInhale® equipment to induce strong immune responses at mucosal sites and systemically. We hypothesize that by using a combination of dry powder technology, prime-boost mucosal immunization, and precise dosing of aerosols, we will: (1) enhance delivery of the vaccine to the deep lungs and draining lymph nodes that will facilitate antigen uptake by dendritic cells, (2) generate and maintain local and systemic memory T cells and antibodies, and (3) eliminate the need for expensive cold-chain. To test our hypothesis, we will use a heterologous prime/boost immunization regimen consisting of parenteral prime vaccination with BCG, followed by boosting with dry powder-based CAF01-adjuvanted H56 in the airways. Our recent data show that mucosal boost immunization in the lungs robustly boost parenterally primed H56-specific CD4+ Th1/Th17 and IgA responses. We have also identified spray drying conditions and stabilizing excipients, which are optimal for manufacturing of the dry powder-based vaccine. In this project, we will identify the most efficient dry powder vaccine formulation and mucosal immunization regimen for enhancing vaccine-mediated protection against TB. A thermostable and self-administrable TB vaccine that can be mass distributed, e.g., to the developing world might in the long-term have a tremendous impact on global health.

My current postdoctoral research work in the lab of Prof. Camilla Foged at University of Copenhagen focuses on image-guided design of a thermostable and self-administrable subunit vaccine against TB in humans. I have developed a novel immunization strategy and novel dual-isotope (111In/67Ga) radiolabeling of the candidate TB subunit vaccine, H56/CAF01 and characterized the immunogenicity and SPECT/CT-based biodistribution of the vaccine following pulmonary delivery in mice (Front. Immunol., 2018). I also engineered a novel gadoteridol-loaded liposomal CAF01 formulation using a quality-by-design (QbD) approach for studying pulmonary delivery of this vaccine in vivo by contrast-enhanced magnetic resonance imaging (MRI). My results show that QbD-optimized CAF01-gadoteridol formulation is safe and stable in vivo and enhances MRI signal by 1.5 fold and changes T1 relaxation by ~ 30% in mice lungs (Mol. Pharm., 2019). I have analyzed the uptake of H56/CAF01 vaccine by pulmonary antigen presenting cell (APC) populations and trafficking to regional lymph nodes in combination with Mass Spectrometry (MS) imaging (Front. Immunol., in press). The results show that the prime and mucosal boost immunization of the H56/CAF01 vaccine elicits a distinct innate myeloid response and activation of APCs (Front. Immunol., in press). The MS imaging data show that the H56/CAF01 vaccine is safe following pulmonary delivery as there is no upregulation of phospholipid lysophosphatidylcholine (16:0) and sphingolipid ceramide, which are known biomarkers of inflammation and local tissue damage (Front. Immunol., in press). I have also made considerable progress in my efforts to design a dry powder H56/CAF01 vaccine formulation. Using spray drying, we have previously produced dry powder CAF01 with preserved adjuvanticity (J. Control Release, 2013) and I have recently shown that spray-dried, reconstituted H56/CAF01 induces Th1/Th17 responses in mice (Vaccine, 2018). Using a QbD approach, I have now formulated dry powder CAF01 in the presence of trehalose and dextran as the stabilizing excipients and observed optimal aerosol performance (flyability and aerosol yield) of the 2 µm sized powders by using the PreciseInhale® equipment (unpublished data). Presently, I am working to optimize these dry powder vaccine formulations and to study their aerosol performance, exposure, and in vivo pulmonary delivery through PreciseInhale® system. In a side project in the lab, I standardized lipopolysaccharide-induced mice model of inflammation. We generated proof-of-concept that lipidoid-polymer-hybrid nanoparticles (LPNs) loaded with siRNA targeted against the pro-inflammatory cytokine tumor necrosis factor (TNF-α) can reduce experimental inflammation (unpublished data). Using a QbD approach, we identified the factors of importance for spray drying of TNF-α siRNA-loaded LPNs for inhalation (Pharm. Res., 2019). We found that the dispersed micro-embedded LPNs had preserved physicochemical characteristics as well as in vitro siRNA release profile and gene silencing, as compared to the non-spray-dried LPNs (Pharm. Res., 2019). We are continuing our efforts to evaluate the aerosol performance of these powders in PreciseInhale® with the ultimate goal to demonstrate the siRNA-mediated gene silencing of dry powder formulations in animal models of chronic obstructive pulmonary disease.

My motivation for applying to the DDL Career Development Grant proposal is to make use of the experience developed in the area of drug delivery to the lungs especially the delivery of vaccines. I aim to use this grant for optimizing pulmonary delivery of the dry powder-based H56/CAF01 vaccine using dry powder technology, prime-boost immunization, and powder administration with PreciseInhale® system. Specifically, my objectives are:
1. To optimize high-quality aerosol generation from H56/CAF01 dry powder and evaluate their detailed aerosol characteristics (particle size, flyability, density, shape, solid form), which are important before in vivo exposure.
2. To optimize the in vivo exposure of dry powder vaccine formulations in mice using the PreciseInhale® system and to evaluate the immunogenicity and safety of H56 in combination with the CAF01 adjuvant using homologous parenteral and mucosal prime‐boost immunization strategies.
3. To conduct an aerosol challenge study with Mtb in mice to measure the protective efficacy and safety of the dry powder vaccine after priming with BCG, using the best immunization strategy and adjuvant combination identified under objective 2.

I trust that the support from DDL Career Development Grant will allow me to generate a novel data on the in vivo immunogenicity and protective efficacy of the dry powder H56/CAF01 vaccine using the PreciseInhale® equipment. I expect to present this data at one of the DDL conferences. This interdisciplinary project at the interface between basic and applied research has exciting translational potential. If the results are promising, the optimized vaccine formulation will be scaled up, and preclinical toxicology and stability studies will be performed. Finally, the optimized dry powder H56/CAF01 formulation and immunization strategy, as determined by the iterative studies in animal models, will be tested in a non-human primate Mtb infection model. These studies will provide crucial pre-clinical data for translating the dry powder H56/CAF01 vaccine forward into human clinical trials as an improved and cold-chain independent TB vaccine candidate.

Following this project grant, I aim to not only expand my current research but also to broaden my scientific horizons in the field of drug delivery to the lungs in general and dry powder vaccines in particular. As described, I have experience in producing dry powder-based subunit vaccine and siRNA formulations. However, we have to date been limited in our preclinical studies by the lack of a suitable device for powder administration to mice. In this project proposal, the application of PreciseInhale® system will allow us the precision dosing of mice lungs with H56/CAF01 vaccine and evaluation of the dry powder-based vaccine immunogenicity and efficacy. These studies can be translated for testing the prophylactic and therapeutic efficacy of novel dry powder-based subunit vaccine and siRNA formulations for other respiratory infections, inflammatory conditions, and cancer. For example, using the PreciseInhale® system in the near future, I plan to evaluate the in vivo therapeutic effect of dry powder TNF-α siRNA-loaded LPNs against chronic obstructive pulmonary disease (COPD). The ability to evaluate the therapeutic effect of dry powder-based biologics will potentially benefit our research group in vaccine design and delivery at the University of Copenhagen.

Finally, through sharing my research results in one of the DDL conferences, I expect to get feedback for my results so as to improve my research. It will also give me an excellent opportunity to expand my scientific network in the field of drug delivery to the lungs and possibilities of future research collaborations and knowledge transfer. Therefore, I would highly appreciate the support under the DDL Career Development Grant for my personal and professional development.

Davide D’Angelo is currently a PhD student at the University of Parma. He completes his bachelor degree in 2017 in biotechnology at the University of Perugia, Italy, with a thesis on the methylation level of A.thaliana. In 2017 he moved to Parma, to complete his studies with a master's degree in Medical, Veterinary and Pharmaceutical Biotechnologies at the University of Parma, graduating with honours in 2019 with a thesis on the formulation of a pulmonary dry powder containing a vaccine against HPV. From 2019 he starts his PhD in the “Biopharmaceutics-Pharmacokinetics” program in the laboratory of pharmaceutical technology of Food and Drug Department at the University of Parma, working under the supervision of Professors Francesca Buttini and Fabio Sonvico on a project entitled “Particle engineering applied for the development of dry powders for inhalation containing biotechnological drugs”. His research is actually focused on the development of an inhalable powder containing cyclosporine for the prevention of pulmonary rejection after transplantation and potentially for the containment of the "cytokine storm" caused by Sars-CoV-2 infection.

While for inhaled products a great deal of effort has been dedicated in the last few years to improve the methods of characterization to go beyond the “simple” aerosol characterization requested by pharmacopoeias and guidelines, this has not been the case for nasal products.
However, when nasal delivery is suggested for vaccination, for systemic delivery of peptides and proteins or for nose-to-brain delivery, it appears clear that the evaluation of spray pattern and geometry characteristics and of the eventual respirable fraction with aerodynamic diameter below 5 µm, do not provide sufficient insight into the biopharmaceutical characteristics of the formulation under investigation. Nasal cavity regional deposition and formulation dissolution/permeation appear much more relevant indicator if the nasal product under development will perform in following in vivo and clinical studies.

Around these topics, in addition to the Advanced Drug Delivery Research Lab (ADDRes Lab) of the University of Parma, a broad collaboration of academic partners has been created including:
• School of Cancer and Pharmaceutical Sciences, King’s College London, UK (Prof. Ben Forbes).
• Department of Pharmaceutics and Biopharmaceutics, Kiel University, Germany (Prof. Regina Scherließ)
• Faculté de Pharmacie, Université Libre de Bruxelles, Belgium (Prof. Jonathan Goole)

The first part of the project consists in a joint effort from the various collaborating group to characterize with advanced methods that go beyond the pharmacopoeial tests a reference commercial nasal product, i.e. mometasone furoate nasal spray suspension. Each group will receive few nasal products from the same production batch of the same supplier and will apply their own in-house approach developed over the last few years.
In the case of UniPR group, we will perform the spray deposition in an anatomically correct, transparent, silicone human nose educational model (Koken, Japan). The evaluation of the deposition pattern in different region of interest (lower, middle and upper nasal cavity). At the same time the product will be tested with the Next Generation Impactor using a 3D printed expansion chamber designed to allow the collection of the nasal product droplets on Snapwell insert in view of dissolution experiments.

The second phase of the project consists in the organization of a one-and-a-half-day symposium to be held in Parma (tentative date 17-18 May 2021), that will see the participation of the groups that took part in the study. The event will focus not only on the presentation of the data collected by the different groups but also on a roundtable of discussion about the different techniques and methods employed with a critical appraisal of merits and limitations. This project and the concluding Symposium will contribute to establish and strengthen a network of collaboration on the field.

Project Title:
Advanced nasal product characterization: focus on deposition and dissolution

Description and Project Goals:
The main purpose of this work is to perform and compare some of the most recent nasal product characterization approaches in terms of deposition and dissolution. In detail the purpose of the study is to use all available cast models within the collaboration group to assess nasal deposition of the same commercial product utilising the in-house assessment strategy of each individual lab.

A second purpose is consisting in the organization of a one-and-a-half-day symposium to be held in Parma where data from the network will be presented along with details of the cast model and experimental details. This will strength the collaboration among the network members, disseminate best practices and the harmonization among these product characterization procedures.

Background

There is a wide interest in nasal administration of drugs due to peculiar characteristics of this site, for many years this route of administration has been limited to local delivery and later the potential of nasal administration for systemic delivery, mucosal vaccination and nose-to-brain delivery has been considered. Nevertheless, factors that influence drug deposition in the nasal cavity are less studied because this cavity is somewhat inaccessible, and delivery of aerosols into this site is a complex process that depends on many parameters including formulation characteristics, the device used, and the way the patient handles this device and the anatomical complexity of the nose (1).
During the optimisation of products for nasal delivery, the delivered dose, the spray pattern and geometry and droplet size distribution are indicated by pharmacopoeias and guidelines as the critical features to be assessed in a nasal delivery system. However, while this appears sufficient for local acting nasal products, it appears not adequate for nasal products designed for systemic, vaccine or nose-to-brain delivery. Indeed, there are still no recommendations or guidelines in terms of standard approaches to evaluate nasal deposition and/or dissolution profile of the formulation (2). No specific restrictions or recommendations are made in terms of nasal cast coating, flow profile or flow rate, insertion angle or insertion depth, manual vs. automated actuation and sample collection from cast. However, when results are reported, the experimental description should contain all these aspects.

In this project, each lab from the collaboration group will use its in-house cast and routine methods for the assessment of nasal deposition of a reference suspension spray (commercially available mometasone suspension spray 50 µg/shot). If applicable, the testing should be performed without and with nasal inspiration and application should be done in both nostrils at the same time with equal number of actuations.

Dissolution rates of nasal products are determined by the physicochemical properties of the formulation, the dosage form, and potentially the region in which the drug is deposited. A major problem with nasal administration is the rapid clearance of the formulation from the cavity due to mucociliary transport which correlates strongly with the duration of the drug action, its efficacy and can also jeopardize patient compliance by involving frequent dosing. More viscous liquid formulation or the administration of powder can increase the residence time of the drug on the nasal mucosa (3). However, the impact of these formulation choices on drug dissolution and availability are often not fully understood. Thus, also in this case an effort has to be produced towards the definition of an optimal dissolution methodology for nasal products that would involve the collection of the drug formulation as emitted from the device, deposition on a surface representative of nasal environment and dissolution at the liquid interface allowing a sufficient resolution to discriminate among different formulation compositions.

Expected results:
- The results shall show the variety of cast models deposition and dissolution methods with their capabilities (and shortcomings) which will allow cast critical evaluation for specific research questions during the roundtable at the concluding symposium.
- Comparison of single laboratory assessments will show how comparable/dissimilar are general conclusions being drawn from the different cast models (such as nasal vs. post-nasal fraction and regional deposition) and dissolution methods (different dissolution rates according to experimental setups).
- The comparison among the single laboratory assessment protocols will allow defining parameters which need or can be harmonized across labs in view of the proposal of a guideline for advanced nasal products testing.

Research Plan
Duration: 4 months.
Location: University of Parma, Italy.
Assessment of mometasone nasal spray deposition pattern
Deposition pattern of mometasone suspension spray will be assessed using a colour-based method consisting of uniformly coat the inner surface of a silicone human nose model (Koken Co, Japan) with Sar-Gel®, a water-indicating paste which changes from transparent to purple on contact with water.
Qualitative and quantitative estimation of the drug deposition will be obtained taking pictures of the nasal cast after aerosolization, with a digital camera. Koken nasal cast is constructed in two separable structures that could allow the quantification of nasal deposition selective washing of specific region of interest (e.g. lower, middle and higher nasal cavity section) with solvent mixture capable of dissolve the drug. The amount of mometasone in each part will be evaluated by HPLC.
The present of the mucus and mucociliary clearance, which are key parameters for nasal drug delivery, cannot be evaluated in such artificial models. However, this study will include a step of coating the nasal cast surface that could be performed by either nebulization or simple application of mucin dispersed in simulated nasal fluid to mimic nasal environment.
The emitted formulation per each actuation will be quantified by measuring the weight difference of the device before and after each actuation and administration-related variables will be specified when results will be reported.

Mometasone formulation collection and dissolution method
For nasal administration, cascade impactor coupled with expansion chambers are recommended by the Guidance for Industry “Nasal Spray and Inhalation Solution, Suspension, and Spray Drug Products - Chemistry, Manufacturing, and Controls Documentation” for the estimation of the deposition in the respiratory tract. The deposition in the expansion chamber represents the nasal fraction, while particles that reached the impactor represent the respirable fraction.
In this study, a 2 L modified expansion chamber and a next generation impactor, NGI will be employed (4). The modified expansion chamber designed by 3D-printing is comprised of a lower part that presents the connection to the impactor and an inlet hole to trigger the nasal device at 30° from the axis, and the upper half that allows the incorporation of three Snapwell cell culture inserts located opposite to the inlet hole. The impactor will be connected to a pump at a flow rate of 15 L/min.

This method will allow at the same time to: i) characterize the aerosol performance in terms of fraction above and below 5µm; ii) quantify the fraction collected in the whole expansion chamber and iii) collect the formulation deposited on the Snapwell insets for dissolution/permeation experiments.

In the case of the dissolution/permeation experiments will be performed in two experimental setups.
In the first case, dissolution experiments will be performed using Snapwell inserts will be non-coated or coated with a mucin solution in simulated nasal fluid (SNF). Inserts will be placed in a plate with wells filled with SNF. Samples will be collected at predetermined time intervals and replaced with the same volume of fresh buffer to evaluate the amount of drug dissolved.

In the case of permeation experiments, Snapwells will be coated with RPMI2650 human nasal cells. These cells when cultivated at air-liquid interface conditions form a pseudo-monolayer, express tight junctions and secrete mucus on their apical surface, thus providing an interesting model of the nasal epithelium. RPMI2650 will be cultivated on Snapwells for 14 days and then three cell inserts will be fitted into the modified expansion chamber before the experiment. After the actuation of the nasal device in the expansion chamber adapted on the NGI, the Snapwells will be removed from the modified chamber and placed in 6-well plates containing the fresh pre-warmed SNF as described before, for drug permeation assessment. Samples will be collected from the basal chamber at set time intervals. Subsequently, cells will be scraped from the insert membrane and lysed in order to quantify the amount of mometasone inside the cells by HPLC.
At the end of the experiments, the NGI will be disassembled and each impactor stage will be washed with a suitable solvent solution and samples will be analysed by HPLC.

Bibliography:

1. Kundoor V. and Dalby R. (2011) Pharm Res. 28(8):1895-1904.
2. Salade L. et al. (2019). Int J Pharm. 561:47-65.
3. Tiozzo Fasiolo L. et al. (2018). Eur J Pharm Sci, 113 2-1.7
4. Pozzoli M. et al. (2016) Eur J Pharm Biopharm, 107:223-233.

George Herbert is a final year PhD student in Professor Stephen J. Archibald’s research group based in the Department of Biomedical Sciences at the University of Hull. He completed an integrated masters (MChem Chemistry with Molecular Medicine) in the group before progressing to his PhD in 2018. George’s research aims to develop and validate pulmonary drug delivery systems using fluorine-18 radiolabelling and positron emission tomography technology.

Based in the Positron Emission Tomography Research Centre (PETRC), the groups work focuses at the interface of chemistry and biology, developing new PET radiotracers for the diagnosis and treatment of disease (oncology, cardiovascular and infection). Research performed in the PETRC is progressed from concept to pre-clinical in vivo assessment. The new Medical Imaging Research Centre (MIRC) based at Castle Hull hospital is a custom-built facility that will allow leading-edge research to translated through clinical development.

Following the success of the work supported by the DDL Career Development Grant, George was able to secure a job as a Lead Radiochemist based at GE Healthcare. Starting in the autumn of 2021, he is in the final stages of completing his research and hopes to submit his thesis by December 2021.

Compared to the conventional oral and intravenous routes, pulmonary drug delivery offers the opportunity for superior drug concentrations in the lung for rapid onset of action in addition to improved patient compliance. However, the development and validation of effective inhaled medications is often challenging. Patients with respiratory disease innately suffer from compromised pulmonary systems and therefore high drug output from minimum patient effort is a desirable quality. Nebulisers, DPI’s and pMDI’s are among the most commonly employed devices however suffer various drawbacks including variable output efficiencies and device-formulation complications. Vapes present as alternative drug delivery devices which could address some of these issues, yet remain vastly underexplored.

Non-invasive medical imaging can serve as an invaluable tool to aid the drug development process. Positron emission tomography (PET) is a highly sensitive and quantitative medical imaging modality which has been previously successfully employed to validate novel drug delivery systems. Organic radioisotopes can be covalently attached to drug molecules in place of their native, non-radioactive isotopes creating structurally identical imaging probes. Fluorine-18 (18F) benefits from high positron yields and low positron energies, promoting sensitive measurements. PET can be employed in pre-clinical proof-of-concept in vitro studies and more complex in vivo studies focussing on pulmonary penetration, deposition and clearance. Results from these studies can be used to optimise performance by making informed changes to the device or formulation.

This project aims to assess the output and drug delivery potential of vape devices using 18F labelled drug molecules. In order to quantify the output and aerosol parameters of the device, the vape liquid was first labelled with a radioactive tag. An organic molecule was designed and synthesised to ensure desirable miscibility, volatility and stability. This molecule was labelled with 18F in high yields and included into vape liquid with high efficiency. Preliminary results from a short 2 second vape indicate a 20x improved output efficiency compared to jet nebulisers. Following device assessment, various drugs will be radiolabelled with 18F and assessed for their vaporisation stability and output characteristics. One such drug is fluticasone propionate, a corticosteroid used to treat asthma and COPD, which possesses a native fluorine atom and hence favours 18F radiolabelling. Variable flow rate pumps will be used alongside valves operating on timers to ensure accurate control over the device puff volume. These can also be used to replicate the forced inhalation volume and flow rates of different disease states. Initially these formulations will be assessed in vitro using impactors to determine the MMAD, GSD, FPF and ED. Following the success of these studies, the devices will be evaluated in vivo using animal models.

In order to fulfil the aims of this project and successfully evaluate the drug delivery potential of vape devices, a broad range of interdisciplinary knowledge and skills will be required. My research experiences to-date provide me with a strong foundation in the relevant disciplines necessary to begin working towards addressing the research aims.

A Master’s degree in medicinal chemistry has educated me in the core disciplines of chemistry with a focus on synthetic organic chemistry for drug development. Theory and practical skills developed during this course will be useful for the synthesis of radiolabelled drug molecule that will be required to validate the vape devices. As a second year PhD student, my first year was spent acquiring proficiencies in new skills including organic radiochemistry, in vitro biological assays and in vivo PET analysis of novel tracers. In addition, my first year has taught me how to conduct research effectively, from planning experiments to interpreting the data and ultimately executing on-task problem solving. I am a motivated, hard-working and ambitious individual, and I believe my positive can-do attitude will allow me to overcome the challenges associated with fundamental scientific research allowing for time efficient progress.

Due to the diverse nature of the project, a small research team must be assembled to meet the demand of specialist knowledge. Working as part of a broad and dynamic team would allow me to further strengthen my communication skills, both verbal and written, which are essential for successful research collaboration and career progression. Furthermore, working closely alongside members of this research team provides the opportunity to develop new expertise in areas that will compliment my current knowledgebase. This will allow me to mature into a well-rounded scientist equipped with a desirable range of knowledge for career growth.

Drug development has remained a passion of mine since early in my undergraduate degree and my research experience to-date has taught me that molecular imaging can be a valuable asset in this process. The discovery of new molecular entities is a notoriously slow, expensive and often without success. Imaging modalities, such as PET, can increase the success rate of this process by evaluating the in vivo performance of new drug candidates before initiating costly and time-consuming clinical trials. In the short-term, following the completion of my PhD, I would like to further expand my organic radiochemistry toolbox, specifically using carbon-11 to radiolabel drug-like molecules. Compared to fluorine, carbon is much more abundant in pharmacological molecules and therefore would provide the opportunity for a larger library of drug candidates to be radiolabelled. In the long-term, I hope to apply my knowledge in organic radiochemistry to aid the development process of new drug candidates for a range of applications. This will speed up the bench-to-bedside translation of new molecular entities which could have a significant positive impact in disease management; I feel this would be particularly beneficial in pulmonary drug development which is inherently complex.

In order to achieve my career goals, I will need to possess a range of specialist knowledge and skills. This research experience presents the opportunity to build on existing proficiencies and will also stimulate the development of new expertise/ skills that will be invaluable for career progression. My synthetic knowledge and skills will be further expanded by the synthesis of clinically established molecular structures and their derivatisation for radiolabelling. Furthermore, I aim to develop an expertise in fluorine-18 radiochemistry and its numerous analytical and QC techniques. As aforementioned, the development of drugs for pulmonary administration presents with additional complications compared to the conventional intravenous and oral routes. This project will give me a unique insight into the tools required to validate drug delivery devices and provides me with a highly bespoke collection of skills for the evaluation radiolabelled aerosols. This will include the assembly, calibration and operation of impactors and the translation of drug formulations to pre-clinical animal models for in vivo evaluation. Specifically, this element of the project will provide me with the opportunity to become well-practised in animal handling and common in vivo and ex vivo experiments. This understanding will allow me to bridge the interdisciplinary gap between chemistry and biology and streamline the translation of new drug formulations to pre-clinical assessment throughout my career. Furthermore, I will develop the ability to efficiently assess risks and implement measures to mitigate the likelihood of these occurring. This is a highly transferrable and valuable asset for the progression of a safe scientific career.

More broadly, the research experience will allow me to assemble core skills that are intrinsic in successful project development. This ranges from conceptualisation by identifying a gap in the research through experimental design, data analysis and ultimately communication of the results. The hands-on experience will also encourage me to fine-tune my creative and resourceful problem-solving ability. Other core research skills, including time management and teamwork, will also be refined through this experience.

At the projects conclusion, I will have the opportunity to present the results from this research to a broad audience. This experience will allow me to network and become known in scientific community which is essential for career progression.

Collectively, my existing knowledge, experiences to-date and support of a specialist research team provide me with a strong foundation to generate results in an achievable timeframe. As the project progresses, new skills and applied knowledge will be acquired in addition to the further concretion of existing expertise. Ultimately this experience should allow me to develop into an independent and confident researcher endorsed with a range of expert scientific knowledge and valuable personal qualities.