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Osmosis-Driven Protein Distribution Optimization via a Single-Vessel Process
Protein-loaded microspheres prepared via the water-in-oil-in-water (w/o/w) emulsion method are porous with poor protein distribution due to the inner water phase leaving the microspheres following polymer precipitation, resulting in the generation of cavities and large pores. Attempts to prevent this by osmosis-driven escape of the inner water phase were unsuccessful, as a polymer precipitation front formed instantly resulting in hollow microspheres. A method to tightly control the events during microsphere formation was developed. Polymer precipitation was controlled by saturating the outer water phase in the organic solvent and isolating the secondary emulsification step from the environment to prevent organic solvent extraction prior to osmosis-driven water escape. Moreover, a single-vessel microsphere preparation method was developed to eliminate product loss and contamination resulting from multiple vessel transfers.
Microspheres were prepared in a single vessel, from polymer dissolution to final product storage (fig. 1). BSA-FITC, 25 kDa PLGA 50:50 and methylene chloride (DCM) were used. 0.1% w/v PVA in water was used as the outer water phase, with or without 1% w/v NaCl. The microspheres were characterized by SEM, confocal microscopy, drug loading, encapsulation efficiency, and burst release (24h) at 37oC.
SEM and confocal microscopy revealed non-porous microspheres with homogenous distribution of protein throughout the polymer matrix when osmosis was applied, as opposed to microspheres where no osmosis was applied (Figure 2). Applying osmosis without isolating the system in a closed vial resulted in hollow microspheres. Moreover, while the application of osmosis in the preparation process did not alter significantly the drug loading or the encapsulation efficiency, the 24 h burst release decreased dramatically as shown in Figure 3.
Our results confirmed our hypothesis that isolating the system in a closed vial during the second emulsification step in combination with application of osmosis prevents early DCM extraction and polymer precipitation, resulting in non-porous microspheres with homogenous protein distribution. Protein-loaded microspheres prepared via this method showed low burst release, a significant property for controlled release microsphere systems. This work showcases the importance of excipients during microsphere preparation, as the careful choice of excipients significantly affects product quality and performance.
Opalescence and Liquid-Liquid Phase Separation in a Protein Solution
To study the effect of concentration, temperature, pH, ionic strength and buffer species on opalescence and liquid-liquid phase separation (LLPS) in a protein solution
Turbidity of a protein solution was measured as percent transmittance using UV-Vis spectrophotometer at different solution conditions. LLPS of the protein was confirmed by equilibrium studies and by visually observing LLPS under microscope.
Structural changes in protein before and after phase separation were studied using Circular Dichroism. Temperature ramp studies were conducted to determine the phase separation temperature (Tcloud) at different solution conditions. Tcloud (temperature where transmittance is 70%) was correlated to protein-protein interactions measured in dilute solutions using DLS (kD). Opalescence measurements, Tcloud measurements and DLS studies were also performed in the presence of different salt ions at pH 6.5 and 15 mM ionic strength.
For the protein molecule studied maximum opalescence is observed near the pI of the molecule and at low ionic strengths. From equilibrium studies, it was observed that protein concentration remains constant in two phases (protein-rich and protein-poor) irrespective of the initial protein concentrations. At low ionic strength and close to the pI of the molecule, Tcloud values were higher indicating a relatively less stable solution, which shifted to lower temperature values at pH away from pI. At relatively higher ionic strength, Tcloud decreased at a pH closer to pI of the molecule and increased away from pI. There was discrepancy in the magnitude of the change in attractive interactions measured by DLS and shift in Tcloud with change in solution conditions.
This study shows that phase separation is an equilibrium/thermodynamic process; protein does not undergo structural changes on phase separation. Strong attractive interactions are observed in systems exhibiting LLPS as indicated by shifts to higher Tcloud and large negative kD. Tcloud measurements can be utilized as a potential screening tool to study the effect of excipients on opalescence and phase separation in early stages of protein formulation development.
A Detailed Mechanistic Study on Adjuvants and Optimizing Antigenicity of Particulate Cancer
Sperm protein 17 (Sp17) is a cancer/testis antigen which is expressed aberrantly in several cancers like prostate, ovarian cancer, hepatocellular carcinoma and multiple myeloma. Its restricted expression in normal tissues and aberrant expression in cancers makes it an attractive target for cancer immunotherapy. Enhancing delivery of Sp17 may significantly improve clinical outcome by eliciting a specific and sustained anti-tumor response. The main goals of this project were 1) to formulate Sp17 microparticles (MP) and investigate its efficacy in vitro. 2) Conduct a detailed mechanistic study on adjuvants that may
augment anti-tumor efficacy of Sp17 MP. 3) To test combination of two adjuvants in conjunction with Sp17 MP for synergetic effect
Recombinant Sp17 was expressed in M15 cells, isolated and purified using the Ni-NTA fast start kit (Qiagen). Sp17 and adjuvants were encapsulated separately in MP using the Buchi B-290 spray drier. Particle size, zeta potential and SEM imaging was performed on microparticles. SDS-PAGE was performed to confirm the stability of Sp17 in MP. Release of Sp17 from MP was performed in gastric and intestinal pH conditions. Eight adjuvant MP were screened on DC 2.4 cells by studying several innate and adaptive immune markers like nitric oxide, CD40, CD80, CD86, CD54 and MHC-II.
Sp17 MP had an average particle size of 3.59 ± 0.5μm and zeta potential of +9.36mV. Encapsulation efficiency of Sp17 was found to be 78%. SEM images confirmed particles were irregular in shaped with surface indentations. SDS-PAGE confirmed the presence of Sp17 encapsulated in its native form. Cumulative release of Sp17 was approximately 15% in simulated murine gastric and intestinal pH conditions. Nitric oxide release was significantly (p<0.05) higher in adjuvant treated groups compared to Sp17 MP. CD 40 and CD 80 expression was elevated in R848, MPL and MF59 treated groups (p<0.05)
compared to Sp17 MP. Combination of R848 and Alum, R848 and MF59 and R848 and P4 showed enhanced expression of CD80. CD40 elevation was highest in MPL and R848 combination.
Sp17 MP in combination with R848, MPL and MF59 MP significantly improve innate and adaptive immune response to cancer antigens.
In Vitro and In Vivo Studies on Transdermal Particulate HPV Vaccine
Human Papillomavirus (HPV) vaccines are recommended by the World Health Organization for cervical cancer control programs world-wide. However, the cost of these vaccines and requirements for administration are significant barriers for vaccination in developing countries. Microparticulate vaccines have the potential to alleviate these problems. The purpose of this study is to develop an HPV16 microparticulate vaccine for transdermal administration and evaluate its efficacy in both in-vitro and in-vivo studies.
HPV 16 virus-like particles (VLPs) were produced in human embryonic kidney cells 293TT. VLPs were incorporated into a cellulosic polymer matrix and formulated into microparticles using a Buchi B-290 spray dryer in a single step. VLP encapsulation was determined using transmission electron microscopy (TEM) and Western blot analysis. For in–vitro study, antigen-presenting cells (APCs) were exposed to vaccine and characterized for cell-surface expression (CD40, CD80/86 and MHC II). For in-vivo study, AdminPatch® transdermal administration of VLPs as microparticles was compared to VLPs in solution. Female BALB/c mice (n=6 for each group) received 4 doses. Blood samples were collected and antibodies were detected with a direct HPV16 VLP IgG ELISA. Spleen and lymph node pools were prepared at week 40 to analyze memory T and B cells using flow cytometry.
The microparticle yield after spraying was 90% w/w, with average size 3.5+ 0.6 μm and average zeta potential -19.7 + 0.3 mV. VLP encapsulation efficiency was 85% based on Western blot detection of HPV16 L1 protein. APCs expressed significantly higher CD40, CD80/86 and MHCII in the particulate vaccine group compared to the solution group. HPV 16 antibodies were detected more frequently in the microparticle group (3 of 6 mice by week 7 and 6 of 6 mice by week 12) than in the solution group (1 of 6 mice by week 12). Spleen and lymph node CD4+, CD27, CD62L and CD45R cell populations were significantly higher (p<0.02) for the microparticle cohort compared to the solution group.
Transdermal administration of HPV VLP as microparticulate vaccine is more immunogenic than HPV VLP in solution.
Phase Separation and Component Crystallization in Freezing Segment of Protein and Amino
Many freeze-dried protein formulations contain glass-forming stabilizing excipients (e.g., trehalose) that protect proteins from dehydration-induced irreversible conformation changes and chemical changes during storage. Some amino acid excipients also form glass-state solids upon lyophilization. The purpose of this study was to elucidate miscibility of proteins and amino acid excipients in frozen solutions and its effect on their crystallization.
Aliquots of frozen solutions containing a model protein (e.g., recombinant human albumin) and amino acids were applied for heating thermal analysis from -70°C to obtain glass transition temperatures of maximally freeze-concentrated solutes Tgô€€€OEand solute crystallization peaks. Some frozen solutions were annealed at elevated temperatures (e.g., -10°C) before their second scan from -70°C.
Some amino acid excipients (e.g., L-valine, glycine) showed high propensity to crystallize during the freezing process. Other excipients freeze-concentrated into narrow non-ice regions between ice crystals remained amorphous (e.g., sodium Lglutamate, L-arginine hydrochloride) or crystallized (e.g., L-histidine hydrochloride) upon the annealing. Frozen solutions containing the protein and amorphous excipients showed single or double Tgô€€€OE transitions that indicate their varied miscibility depending on the combinations and concentration ratios. Many protein-rich frozen solutions showed single Tgô€€€OE transitions in the first heating scans and after their annealing, indicating maintenance of the amorphous concentrated solute mixture. Frozen solutions containing rHA and higher mass ratio of L-Arg HCl showed double Tgô€€€OE transitions. The transition temperature profiles suggested separation of the non-crystalline solutes into the solute-mixture and excipient phases. Frozen solutions containing rHA and higher mass ratio of L-His HCl showed the amorphous/amorphous phase separation and following crystallization of the excipient.
The phase separation should allow nucleation of amino acid crystals in the excipient-dominant concentrated phase. Information on the solute mixing state should be valuable for appropriate use of the amino acid excipients either as a crystalline bulking agent or an amorphous stabilizer in freeze-dried formulations.
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