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Vibhuti Agrahari, Mary Joseph, Sulabh Patel, Ravi Vaishya, Ashim K Mitra; Development, optimization and evaluation of biological activity of tailor-made pentablock copolymer based composite formulation for sustained delivery of protein therapeutics. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5028. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
The objective of this work is to develop, optimize and evaluate pentablock copolymer (PBC) based formulation which can carry large doses, retains biological activity during formulation development, storage and/or release and provide sustained release protein delivery system for the treatment of posterior segment ocular diseases.
Various ratios and molecular weights of each block were selected for synthesis and optimization of biodegradable and biocompatible PB copolymers for the preparation of nanoparticles (NPs) and thermosensitive gel. PBC were characterized for their molecular weight and purity by NMR, GPC. FTIR and XRD analysis were utilized to determine functional group and crystallinity, respectively. PBC NPs were successfully prepared with double emulsion solvent evaporation method. NPs were characterized for particle size, poly dispersity, entrapment efficiency and drug loading. In vitro release studies of protein from NPs alone and composite formulation were performed in PBS 7.4 at 370C. CD spectroscopy, ELISA, cell proliferation and cell invasion techniques were employed to evaluate conformation and binding affinity of released proteins. In order to investigate release mechanisms, release data were fitted to various kinetic models.
NMR, GPC, FTIR and XRD analyses of PBC provided complete characterization of the polymers. Composite formulations demonstrated zero-order release with none or negligible burst release. Optimization of various formulation parameters contributed to increase in %EE from ~25% to ~79% and %DL from ~5% to ~17%. A sustained in vitro drug release for more than three months was observed for proteins. CD spectroscopy confirmed retention of structural conformation of proteins. Biological activity of released bevacizumab was confirmed by in vitro cell culture. Retention of enzymatic activity of lysozyme and catalase in release samples were also confirmed. Based on the R2 value, best fit model was identified.
An improvement in drug loading through formulation optimization allowed higher amount of proteins in a smaller injection volume and delivery for extended duration. It is anticipated that these polymers can serve as a platform for ocular delivery of therapeutic proteins, for the treatment of posterior segment chronic indications such as AMD, DR and DME.
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