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.