Glycosylation improves α-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic)acid microspheres

Abstract

Enhancing protein stability upon encapsulation and release from polymers is a key issue in sustained release applications. In addition, optimum drug dispersion in the polymer particles is critical for achieving release profiles with low unwanted initial “burst” release. Herein, we address both issues by formulating the model enzyme α-chymotrypsin (α-CT) as nanoparticles to improve drug dispersion and by covalently modifying it with glycans to afford improved stability during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres. α-CT was chemically modified with activated lactose (500 Da) to achieve molar ratios of 4.5 and 7.1 lactose-to-protein. The bioconjugates were co-lyophilized with methyl-β-cyclodextrin followed by suspension in ethyl acetate to afford nanoparticles. Nanoparticle formation did not significantly impact protein stability; less than 5% of the protein was aggregated and the residual activity remained above 90% for all formulations. Using a solid-in-oil-in-water (s/o/w) methodology developed in our laboratory for nanoparticles, we obtained a maximum encapsulation efficiency of 61%. Glycosylation completely prevented otherwise substantial protein aggregation and activity loss during encapsulation of the non-modified enzyme. Moreover, in vitro protein release was improved for glycosylated formulations. These results highlight the potential of chemical glycosylation to improve the stability of pharmaceutical proteins in sustained release applications.

Bone biomaterials and interactions with stem cells

“…Synthetic biodegradable polymers include poly(lactic acid) (PLA), 134 poly(glycolic acid) (PGA), 135 poly(lactic-co-glycolic acid) (PLGA), 136 poly(vinyl alcohol) (PVA), 137 PCL, 138 and polyoxyethylene (POE). They greatly differ from each other in terms of structure and properties.…”

Glycosylated Nanoparticles for Cancer-Targeted Drug Delivery

“…Additionally, the directional organization of molecules on the nanoparticle periphery can help by increasing the electrophile affinity to target molecules due to the high surface area to volume ratio of NPs (62,63). Therefore, the optimum drug dispersion and homogeneity in a nanoparticle system and the linkage to cargo molecules should be well controlled and reproducible to obtain the desired therapeutic effect (64).…”

“…Regarding size, reports on organic and inorganic NPs indicate that glycosylation increases the size and molecular weight of NPs (14,64,65). Additionally, glycoconjugates exhibit a neutralization of zeta potential without significant alterations in colloidal stability (34,66).…”

Milling solid proteins to enhance activity after melt-encapsulation

“…There is also the potential for increased hydrogen bonding between the sugars on the surface of GOx and carboxylic acid units formed during PLGA degradation, resulting in increased retention of GOx within the PLGA cylinder. A previous study has demonstrated that synthetic glycosylation of a protein with a long sugar that was encapsulated within PLGA hindered the release of the protein, potentially due to similar mechanisms (Flores-Fernández and Griebenow, 2012). The slightly lower release of 50 Hz milled GOx was likely due to the localization of more GOx in one area, resulting in larger clusters of protein due to hydrogen bonding and further diminishment of release.…”