Abstract
BackgroundThe purpose of this study is to modulate the transfection efficiency of adenovirus (Ad) on the cornea by the covalent attachment of chitosan on adenoviral capsids via a thioether linkage between chitosan modified with 2-iminothiolane and Ad cross-linked with N-[γ-maleimidobutyryloxy]succinimide ester (GMBS).Methodology/Principal FindingsModified Ad was obtained by reaction with the heterobifunctional crosslinking reagent, GMBS, producing maleimide-modified Ad (Ad-GMBS). Then, the chitosan-SH was conjugated to Ad-GMBS via a thioether bond at different ratios of Ad to GMBS to chitosan-SH. The sizes and zeta potentials of unmodified Ad and chitosan-modified Ads were measured, and the morphologies of the virus particles were observed under transmission electron microscope. Primary cultures of bovine corneal epithelial cells were transfected with Ads and chitosan-modified Ads in the absence or presence of anti-adenovirus antibodies. Chitosan modification did not significantly change the particle size of Ad, but the surface charge of Ad increased significantly from −24.3 mV to nearly neutral. Furthermore, primary cultures of bovine corneal epithelial cells were transfected with Ad or chitosan-modified Ad in the absence or presence of anti-Ad antibodies. The transfection efficiency was attenuated gradually with increasing amounts of GMBS. However, incorporation of chitosan partly restored transfection activity and rendered the modified antibody resistant to antibody neutralization.Conclusions/SignificanceChitosan can provide a platform for chemical modification of Ad, which offers potential for further in vivo applications.
Highlights
Gene delivery to the three major cell types of the cornea using viral and non-viral methods has been demonstrated in vivo and in vitro [1]
Characterization of chitosan-modified Ad In the present study, the covalent attachment of chitosan on Ad was achieved by first modifying chitosan with 2-iminothiolane and cross-linking Ad to GMBS, and subsequently conjugating thiolated chitosan to the GMBS-modified Ad by thioether formation
When compared to the average size of unmodified Ads, dynamic light scattering analysis showed that the average size of chitosan-modified Ads gradually increased with increasing amounts of GMBS or/and chitosan
Summary
Gene delivery to the three major cell types of the cornea using viral and non-viral methods has been demonstrated in vivo and in vitro [1]. Recombinant adenovirus (Ad) has been extensively evaluated in gene delivery to the cornea in many studies [2,3,4,5,6,7,8] This method can achieve a high level of transgene expression, but immune responses may be raised against Ad proteins, thereby limiting further administration of the virus. Seymour et al used a multivalent hydrophilic polymer based on poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA) to modify Ad vectors [19,20,21,22]. These modified vectors show extended plasma circulation time, reduced toxicity, and evasion of neutralizing antibodies. The purpose of this study is to modulate the transfection efficiency of adenovirus (Ad) on the cornea by the covalent attachment of chitosan on adenoviral capsids via a thioether linkage between chitosan modified with 2iminothiolane and Ad cross-linked with N-[c-maleimidobutyryloxy]succinimide ester (GMBS)
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