Chemical Modification of Adenoviral Capsid with Chitosan

The purpose of this study is to covalently modify adenoviral capsid with chitosan, a cationic and natural polymer. To further expand applicability of chitosan, we have modified the amine group of chitosan with 2-iminothiolane to introduce thiol groups and obtained a 46.9％ yield. Adenoviruses (Ads) were reacted with N-[γ-maleimidobutyryloxy]succinimide ester (GMBS), a heterobifuctional crosslinker, and maleimide-modified Ads (MalN-Ads) were obtained. Then, the chitosan-SH was reacted with MalN-Ads via thioether at different ratios of Ads to GMBS to chitosan-SH. The sizes and the zeta potentials of unmodified Ads and chitosan-modified Ads were measured, and morphology of the virus was observed under transmission electron microscope (TEM). Primary culture of bovine corneal epithelial cells was transduced with either adenoviruses or chitosan-modified Ads in the absence or presence of anti-adenovirus antibodies. Furthermore, we incorporated targeting ligands, such as epidermal growth factor (EGF) and RGD, on chitosan to further modify adenovirus.
Analysis of particle sizes showed modification with chitosan did not obviously affect the size of Ads, but zeta potential analysis revealed that the surface charge of Ads significantly changed form -24.3 mV to nearly neutral charge. The results of TEM also showed chitosan-modified Ads had a rough and irregular appearance. Depending on the increasing amounts of GMBS, the transduction efficiency was attenuated gradually. However, incorporation of chitosan could restore a part of transduction activity. Chitosan-modified Ads were resistant to antibody neutralization at a low antibody concentration, but could not work at a high concentration. We also speculate that ligand-linked chitosan-modified Ads could transducer cells via alternative pathway.
In conclusion, chitosan can provide a great platform of chemical modification on adenoviruses. It permits incorporation of a range of targeting molecules, but also of other biological effectors. Hence, chitosan-modified Ads are potential for further in vivo studies.