Synthesis and Characterization of 30Kc19-based Protein Nanoparticles for the Application to Drug Delivery

Abstract

30Kc19 protein is a member of the 30K protein family from silkworm, having molecular weights of around 30 kDa. 30Kc19 protein is the most abundant among 30K proteins in the hemolymph. In previous studies, 30K proteins exhibited anti-apoptotic effect in various cells by gene expression or addition of 30K proteins in recombinant form produced from Escherichia coli. 30Kc19 also enhanced productivity and glycosylation by expression of a 30Kc19 gene or supplementation with a recombinant 30Kc19 protein. Additionally, 30Kc19 exhibited enzyme-stabilizing and cell-penetrating abilities in vitro. In this study, it was hypothesized that supplemented 30Kc19 penetrated into the cell and enhanced the stability of the cellular enzyme, and investigated this using in vitro and cellular assessments. The activity of isolated mitochondrial complex I / III was enhanced with 30Kc19 in dose-dependent manner while initial reaction rate was unchanged, suggesting that 30Kc19 enhanced enzyme stability rather than specific activity. For intracellular enzyme activity assessment, mitochondrial complex II activity in HeLa cells increased more than 50% with 30Kc19. The enhanced mitochondrial complex activity increased mitochondrial membrane potential and ATP production in HeLa cells with 30Kc19, by over 50%. Then cell penetrating and enzyme stabilizing effect of 30Kc19 was exploited to efficient drug delivery. 30Kc19 and HSA were used as building block of protein nanoparticles to exploit both beneficial effect of 30Kc19 protein stability of HSA nanoparticles. 30Kc19-HSA nanoparticles were successfully prepared using the desolvation method, with uniform spherical morphology and stable dispersion. 30Kc19-HSA nanoparticles showed negligible toxicity when treated to cells, and 30Kc19-HSA nanoparticles also exhibited increase in cellular uptake compared with HSA nanoparticles. Because stable 30Kc19-HSA nanoparticles were successfully synthesized and characterized, nanoparticles loaded with model enzyme cargo to investigated effect of 30Kc19 on cargo enzyme. 30Kc19-HSA nanoparticles loaded with β-galactosidase were uniformly spherical in shape, dispersed evenly in phosphate buffered saline and cell culture media, and released β-galactosidase in a sustained manner. The 30Kc19-HSA nanoparticles had negligible toxicity to animal cells and exhibited enhanced cellular uptake and intracellular stability of β-galactosidase in HeLa and HEK293 cells when compared with those of HSA nanoparticles. These results suggest that 30Kc19-HSA protein nanoparticles could be used as a versatile tool for drug delivery to various cells. Next, 30kc19-HSA nanoparticles were used to deliver actual therapeutic protein to cells. Fabry disease is a genetic lysosomal storage disease caused by deficiency of α-galactosidase, the enzyme that degrades neutral glycosphingolipids transported to lysosomes. Enzyme replacement therapy (ERT) using recombinant α-galactosidase is the only treatment available for Fabry disease. Because enhancing cellular delivery and enzyme stability is a challenge of ERT using α-galactosidase to maximize treatment efficacy, 30Kc19-HSA protein nanoparticles were used to enhance delivery and intracellular α-galactosidase stability. The 30Kc19-HSA nanoparticles had a uniform spherical shape and were well-dispersed. The 30Kc19-HSA nanoparticles had negligible toxicity to human cells. The nanoparticles exhibited enhanced cellular uptake and intracellular stability of the delivered α-galactosidase in human foreskin fibroblasts. Additionally, the nanoparticles enhanced globotriaosylceramide degradation in fibroblasts from a patient with Fabry disease. It is expected that 30Kc19-HSA protein nanoparticles will be used as an effective tool for efficient delivery and enhanced stability of drugs.