Dissertation/Thesis Abstract

Effect of surface modification of biodegradable poly(lactic-co-glycolic acid) microspheres on surface-protein interaction
by Park, Alice H., Ph.D., University of the Sciences in Philadelphia, 2015, 201; 10169467
Abstract (Summary)

The current project involves synthesizing surface modified poly(lactic-co-glycolic acid) (PLGA) microspheres which are coated with non-therapeutic proteins as colloidal vehicles for the delivery of therapeutic protein drugs. Since the plasma circulation time of human growth hormone (hGH) is relatively short, a biodegradable carrier that can interact with hGH reversibly was designed to extend the plasma circulating time and enhance patient compliance eventually. By modifying the surface of microspheres, the loading amount of non-therapeutic proteins onto the surfaces will be increased, which will result in better efficiency as a drug delivery system. Also, use of protein-protein interaction to load therapeutic proteins onto microspheres can decrease the degree of denaturation of the therapeutic protein.

Biodegradable PLGA microspheres were prepared using double emulsion method, and their physical properties were characterized. Size and surface charge were analyzed using dynamic light scattering (DLS) techniques, and density of carboxyl groups on the surface of the microspheres was determined using titration. The surface of PLGA microspheres was modified by incorporating poly(ethylene-co-maleic acid) (PEMA) as a surfactant in order to enhance capacity for covalently bound surface ligands as PEMA contains high number of carboxyl groups. Then, PLGA microspheres were functionalized with bovine serum albumin (BSA) and bovine β-casein (CAS), and protein attachment was verified by micro-BCA assay. Lastly, the interaction of nontherapeutic protein-coated PLGA microspheres with recombinant human growth hormone (r-hGH) was investigated using isotope dilution method with radioisotope of 125I-labeled r-hGH, and the effect of pH and ionic strength of the solvent medium on the protein-protein interaction was studied.

The microspheres were spherical with smooth surfaces, and the average size of the PLGA/PEMA microspheres was near 1000nm with the negatively charged surface in the range of -50 mV to -80 mV. The prepared microspheres were stored with 1% or 5% of sucrose as a cryoprotectant by lyophilization. The density of carboxyl groups was high compared to the one of polystyrene particles. BSA and β-casein were successfully conjugated onto the surface of PLGA/PEMA microsphere. The average amounts of BSA and CAS conjugated were 0.1μg and 0.25μg per cm2 of microspheres, respectively. Also, the size of protein conjugated microspheres was not significantly different from the one before conjugation. The binding study showed that as the concentration of r-hGH increased, the amount of r-hGH adsorbed onto the microspheres increased as well. However, it did not reach a saturation point in the concentration range of 0.0125- 0.75mg/ml. The adsorption process was found to be dependent upon the pH and ionic strength. Hydrophobic interaction governed the adsorption process of r-hGH when the pH (pH 5.3) was near the pI of participating proteins, and electrostatic interaction governed the adsorption when the pH (pH 4.0 or 7.2) was away from the pI of participating proteins. From desorption studies, it was observed that the adsorption of r-hGH onto the surfaces of PLGA and protein-coated PLGA was reversible at all pH and ionic strengths studied. However, the desorption results of r-hGH from protein-coated PLGA microspheres was inconclusive due to severe variations, except at pH 4.0. In general, BSA-coated and CAS-coated microspheres showed similar percentages of desorption. The highest desorption was observed at pH 4.0, and the lowest desorption was observed at pH 7.2.

In summary, PLGA/PEMA microspheres were successfully prepared using double emulsion method, and BSA and β-casein were conjugated onto the surface. It was observed that the interaction between protein-coated surface and protein in solution was complex and influenced by several processes, such as electrostatic interaction, hydrophobic interaction, hydrogen bond formation, and structural changes of proteins.

Indexing (document details)
Advisor: Gupta, Pardeep K.
Commitee:
School: University of the Sciences in Philadelphia
Department: Pharmaceutics
School Location: United States -- Pennsylvania
Source: DAI-B 78/02(E), Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Pharmacy sciences
Keywords: Adsorption, Conjugation, Desorption, Plcta microshperes, Protein-protein interaction, Recombinant human growth hormone (r-hgh)
Publication Number: 10169467
ISBN: 978-1-369-23660-6
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