Positively charged cholesterol–recombinant human gelatins foster the cellular uptake of proteins and murine immune reactions

Pallavi A Kadengodlu,^1,3 Takehisa Hebishima,² Shin-Nosuke Takeshima,² Mika Ito,¹ Mingzhe Liu,¹ Hiroshi Abe,¹ Yoko Aida,² Toshiro Aigaki,³ Yoshihiro Ito<sup>1,3

1</sup>Nano Medical Engineering Laboratory, ²Viral Infectious Diseases Unit, RIKEN Advance Science Institute, Wako, Saitama, Japan; ³Graduate School of Biological Science, Tokyo Metropolitan University, Tokyo, Japan

Purpose: Recombinant human gelatins with defined molecular weights were modified with cholesterol to make them amphiphilic in nature. We investigated the feasibility of these modified human gelatins acting as a carrier of antigenic proteins for inducing cellular immunity. The aim of this study was to synthesize novel and effective compounds for vaccine delivery in vivo.
Methods: Two types of cholesterol-modified gelatin micelles, anionic cholesterol-modified gelatin (aCMG) and cationic-cholesterol modified gelatin (cCMG), were synthesized using different cholesterol derivatives such as the cholesterol-isocyanate (Ch-I) for aCMG and amino-modified cholesterol for cCMG. One was anionic and the other cationic, and therefore they differed in terms of their zeta potential. The aCMG and cCMG were characterized for their size, zeta potential, and in their ability to form micelles. Cytotoxicity was also evaluated. The modified human gelatins were then investigated as a carrier of antigenic proteins for inducing cellular immunity both in vitro in DC 2.4 cells, a murine dendritic cell line, as well as in vivo. The mechanism of entry of the polymeric micelles into the cells was also evaluated.
Results: It was found that only cCMG successfully complexed with the model antigenic protein, fluorescein-isothiocyanate ovalbumin (OVA) and efficiently delivered and processed proteins in DC 2.4 cells. It was hypothesized that cCMG enter the cells predominantly by a caveolae-mediated pathway that required tyrosine kinase receptors on the cell surface. Animal testing using mice showed that the cationic cholesterol-modified gelatin complexed with OVA produced significantly high antibody titers against OVA: 2580-fold higher than in mice immunized with free OVA.
Conclusion: Conclusively, cCMG has shown to be very effective in stimulating an immune response due to its high efficiency, stability, and negligible cytotoxicity.

Keywords: recombinant human gelatin, cholesterol, micelle, protein delivery, caveolae pathway, receptor-mediated endocytosis