Formulation of PEG-ylated L-asparaginase loaded poly (lactide-co-glycolide) nanoparticles: influence of Pegylation on enzyme loading, activity and in vitro release.

The present paper describes the advantage of PEG-ylation of L-asparaginase before encapsulation over its incorporation in the native form. During encapsulation a considerable amount of native protein undergoes denaturation and forms insoluble aggregates. In an effort to overcome this problem, L-asparaginase was PEG-ylated before subjecting it to the harsh conditions as encountered during double emulsion solvent evaporation technique. L-asparaginase was conjugated with succinimidyl succinate derivative of polyethylene glycol (SS-PEG, MW 5000) followed by characterization of the formed conjugate using size exclusion-HPLC and SDS PAGE. The PEG-ylated L-asparaginase consisted of different isomers from mono to multi PEG-ylated depending upon the number of Lysine residues (14 in case of L-asparaginase) with about 5% as native protein. The specific activity as retained after PEG-ylation was 62.84 +/- 8.2% and further about 82.7% of activity was recovered from the particles. Imitated studies with the native protein confirmed the enhanced stability of the conjugated protein when exposed to the organic solvent and sonication and showed comparatively less encapsulation efficiency due to increased hydrophilicity. Release profiles for native as well as conjugated proteins consisted of sustained release of about 66.66% and 44.45% in 28 days, respectively. The decrease in the release can be attributed to the increase in the molecular weight of the conjugated protein. The study finally proved that PEG-ylation protected the enzyme and prevented it from denaturation during encapsulation.

Controlled release of bioactive recombinant human growth hormone from PLGA microparticles.

Controlled release formulation of recombinant human growth hormone (r-hGH) was achieved using poly lactide-co-glycolide (PLGA) polymer. Denaturation of r-hGH by dichloromethane during primary emulsification step of particle preparation was minimized by using human serum albumin whereas inclusion of sucrose and sodium bicarbonate helped in reducing protein denaturation during lyophilization and polymer particle degradation. Encapsulation efficiency of r-hGH entrapped in PLGA particles (size approximately 30 microm) was around 45% with protein load 20 microg of r-hGH/mg of polymer particles. Porous particles showed quick release of r-hGH in comparison to non-porous particles in vitro. More than 10 ng/mL of bioactive r-hGH was found in the serum of the experimental animals observed for a 30-day period after a single intramuscular injection of the polymeric formulation. Incorporation of optimal stabilizers is thus essential for the development of a stable, month long controlled release of polymer particle based r-hGH formulation.