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

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.

Chitosan Functionalization: Covalent and Non-Covalent Interactions and Their Characterization

“…With the exception of methacrylic anhydride, all reported anhydrides for CS functionalization are cyclic, which allow further functionalization with amino-based systems after ring-opening ( Figure 4 ). This strategy was frequently reported for the conjugation to small molecules and polymers such as branched and linear polyethylenimines (B/LPEI), leading to gene delivery vehicles [ 39 , 41 ].…”

“…Following this procedure, the conjugation to dodecyl amine led to CS-based self-assembled micelles for gene delivery [ 89 ]. Alternatively, CS-PEI copolymers were further conjugated to PEG derivatives through O-functionalization to evaluate the shielding effect of PEG chains on the targeted gene delivery vehicles [ 41 ].…”

“…The very low solubility of some commercial CS batches in aqueous solutions is an obstacle to their characterizations by NMR analysis before and after derivatization. Nevertheless, with the advances of high field NMR spectrometers, detailed characterization reports were provided for CS–small molecule conjugates and CS-based copolymers [ 36 , 37 , 39 , 40 , 41 , 48 , 70 ].…”

“…We believe that 1 H NMR analysis is not sufficient for the characterization of covalent systems, and we suggest the implementation of DOSY NMR in the list of routine analyses for such derivatives. We illustrated the relevance of 2D DOSY NMR in our recent publication describing the functionalization and characterization of CS with small molecule and polymers [ 41 ].…”

Application of Non-Viral Vectors in Drug Delivery and Gene Therapy

“…Chitosan (CS) is one of the most abundant biopolymers derived from natural chitin that commonly exists in the exoskeletons of arthropods, crustacean shells, insects, and fungal cell walls [ 93 ]. CS can be degraded by internal enzymes, which makes chitosan have good biocompatibility [ 94 , 95 ]. Like other cationic polymers, chitosan is linked to nucleic acids by electrostatic interaction [ 96 , 97 ].…”