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.