Development of Covalent Chitosan-Polyethylenimine Derivatives as Gene Delivery Vehicle: Synthesis, Characterization, and Evaluation.

Laura Nicolle,Jens Casper,Tomaž Einfalt,Pascal Detampel,Beat Thöny,Sandrine Gerber-Lemaire,Hiu Man Grisch-Chan,Céline M. A. Journot,Jörg Huwyler,Melanie Willimann

doi:10.3390/ijms22083828

Abstract

There is an increasing interest in cationic polymers as important constituents of non-viral gene delivery vectors. In the present study, we developed a versatile synthetic route for the production of covalent polymeric conjugates consisting of water-soluble depolymerized chitosan (dCS; MW 6–9 kDa) and low molecular weight polyethylenimine (PEI; 2.5 kDa linear, 1.8 kDa branched). dCS-PEI derivatives were evaluated based on their physicochemical properties, including purity, covalent bonding, solubility in aqueous media, ability for DNA condensation, and colloidal stability of the resulting polyplexes. They were complexed with non-integrating DNA vectors coding for reporter genes by simple admixing and assessed in vitro using liver-derived HuH-7 cells for their transfection efficiency and cytotoxicity. Using a rational screening cascade, a lead compound was selected (dCS-Suc-LPEI-14) displaying the best balance of biocompatibility, cytotoxicity, and transfection efficiency. Scale-up and in vivo evaluation in wild-type mice allowed for a direct comparison with a commercially available non-viral delivery vector (in vivo-jetPEI). Hepatic expression of the reporter gene luciferase resulted in liver-specific bioluminescence, upon intrabiliary infusion of the chitosan-based polyplexes, which exceeded the signal of the in vivo jetPEI reference formulation by a factor of 10. We conclude that the novel chitosan-derivative dCS-Suc-LPEI-14 shows promise and potential as an efficient polymeric conjugate for non-viral in vivo gene therapy.

Highlights

Gene therapy has emerged in the last decades as a powerful strategy for the treatment of acquired and inherited disorders
Conjugation to BPEI was initially attempted via direct activation of depolymerized chitosan (dCS) primary amines in the presence of 1,1 -carbonyldiimidazole (CDI) or N,N’-disuccinimidyl carbonate (DSC), followed by condensation with the BPEI units and formation of urea as covalent linkage (Table S1, Scheme S1)
A succinyl (Suc) spacer was introduced for further functionalization via carboxylic acid functionality (Figure 2b)

Summary

Introduction

Gene therapy has emerged in the last decades as a powerful strategy for the treatment of acquired and inherited disorders. Various diseases could benefit from it, including cancer and viral infections, cardiovascular diseases, and inherited Mendelian disorders [1,2] This approach relies on the modification of gene expression by gene replacement, i.e., transfer of exogenous nucleic acids as a therapeutic gene copy, or precise genome editing [3]. Due to their polyanionic nature and their sensitivity to endonucleases, nucleic acids should preferably be delivered to target cells by means of nanocarriers, called delivery vectors. The interest in synthetic non-viral vectors for gene delivery as a potentially safer alternative has steadily increased over the past years

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