A thorough mechanistic understanding of the phenomena of protein adsorption and desorption with respect to the role of the nature of the surface involved (such as hydrophobic or hydrophilic) and conditions of the microenvironment (such as pH and ionic strength), is highly desirable to design polymer-based systems for protein drugs, including human growth hormone (r-hGH). Previous experiments have shown that adsorption of proteins at hydrophobic polymer surfaces results in unfolding of the protein molecule, loss of structure, and a possible loss of activity too. Biocompatible surface coatings (such as polyethylene glycols, dextrans, and proteins) have been shown to prevent and/or control the nonspecific adsorption of proteins. Among these, the potential of protein-coated surfaces as drug delivery platforms for protein drugs is largely unexplored as yet. A layer of serum proteins immediately covers the surface of any particle-based system administered in vivo and determines the identity of that system henceforth. This layer of surface-bound proteins also known as the “corona” can be made up of a diverse range of proteins (depending on the type of bodily fluid it encounters) The utilization of protein corona as a platform for delivering an active molecule such as a delicate protein (like hGH or insulin) is an exciting and nascent area of research in colloidal drug delivery systems.
The current project has been able to study and understand the interactions of rhGH (a model protein drug) with protein-coated colloidal surfaces. The project involved development and characterization of protein-coated surfaces (magnetic & non-magnetic) by covalent attachment of model proteins on the surfaces of polystyrene particles by means of covalent spacers of increasing length and different chemical natures. Three model proteins were chosen to create these surface coatings; different functional groups on the surfaces of these model proteins and particles were used for creating these covalent linkages. The protein-coated surfaces thus created were characterized for their thickness of the surface coating & surface charge (photon correlation spectroscopy) and amount of protein immobilized (particle-based micro-BCA assay). The interaction of r-hGH with these protein-coated surfaces under different conditions of pH and ionic strength were explored using radioactive-binding assays. The effect of the type of protein immobilized on the surface, type of linkage used in immobilization, pH and ionic strength of the microenvironment on the amount, strength, thermodynamics and kinetics of the r-hGH adsorption/desorption process were all studied.
The model proteins were observed to completely cover the nanoparticle surfaces. It was found that the nanoparticle-protein conjugates were stable at values of pH and ionic strength that were used in the study. The interaction of r-hGH with the protein-coated surfaces was due to interplay of electrostatic and hydrophobic interactions. Hydrophobic interaction forces governed adsorption & desorption of r-hGH when the pI of the surface bound protein and r-hGH were observed to be close. When the pKa pH of the surface bound protein and r-hGH were far apart (on the pH scale), the adsorption & desorption of r-hGH was governed by electrostatic attraction. The interaction of r-hGH with the protein-coated surfaces was found to be of moderate strength. Desorption of r-hGH was found to be higher in presence of 1% BSA solutions at higher temperatures (reversible interactions). It was needed to develop a particle-based assay for determining the extent of surface bound protein. This assay would serve invaluable for particles ≥ 20nm, which do not easily submit to traditional methods of separation. Several different analytical approaches were tried to develop this assay.
In summary it was observed that the behavior of protein-particle conjugates (in response to environmental factors such as pH and ionic strength) is mainly directed by the properties of the surface-bound protein. Such protein-coated surfaces may interact favorably with another protein such as r-hGH and have the potential to act as drug delivery platforms for protein drugs. Such interactions were observed to be of moderate strength, reversible and influenced by factors of the microenvironment such as pH and ionic strength.
|Commitee:||Bruist, Michael, Jannalgadda, Kamal, Motheram, Rajeshwar|
|School:||University of the Sciences in Philadelphia|
|School Location:||United States -- Pennsylvania|
|Source:||DAI-B 77/04(E), Dissertation Abstracts International|
|Keywords:||Bio configuration, Nanoparticles, Protein adsorption, Protein-protein interactions, Proteins|
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