A Thermoresponsive Chitosan−NIPAAm/Vinyl Laurate Copolymer Vector for Gene Transfection

Abstract

A carboxyl-terminated N-isopropylacrylamide/vinyl laurate (VL) copolymer was prepared and coupled with chitosan (molecular weight = 2000) to produce a chitosan-NIPAAm/VL copolymer (PNVLCS) vector. The aqueous solution of PNVLCS displayed an obvious thermoresponsive behavior with a lower critical solution temperature (LCST) about 26 degrees C. The transmission electron microscopy (TEM) showed that the size of PNVLCS/DNA complexes varied with charge ratios (+/-), and the smaller nanoparticles were formed at higher charge ratios. DLS revealed that the size of complex particles was dependent on temperature. The results of temperature-variable circular dichroism (CD), UV, and electrophoresis retardation indicated that at lower charge ratios, DNA in the complexes assume a B conformation, whereas increasing charge ratios caused B --> C type conformation transformation; the dissociation-formation of PNVLCS/DNA complexes could be tuned by varying temperature: at 37 degrees C, the collapse of PNIPAAm in PNVLCS was favorable for the formation of compact complexes, shielding more DNA from exposure; at 20 degrees C, the hydrated and extended PNIPAAm chains facilitated the unpacking of DNA from PNVLCS, increasing the exposure of DNA. PNVLCS was used to transfer plasmid-encoding beta-galactosidase into C2C12 cells. The level of gene expression could be controlled by varying incubation temperature. The transfection efficiency of PNVLCS was well improved by temporarily reducing culture temperature to 20 degrees C, whereas naked DNA and Lipofectamine 2000 did not demonstrate the characteristics of thermoresponsive gene transfection.

The potential of chitosan for pulmonary drug delivery

“…Increased transfection efficiency was also achieved by coupling chitosan to a carboxyl-terminated Nisopropylacrylamide/vinyl laurate, derived from N-isopropylacrylamide which is a well known temperature-sensitive polymer (33). Finally, the same effect on transfection was described as a result of the interaction of chitosan with surfaces through specific ligands which were previously immobilised on the molecule, such as galactose, transferrin, folate and mannose, which interact with specific cell receptors (33).…”

Chitosan-DNA/siRNA Nanoparticles for Gene Therapy

“…It is actually a contradiction between the stability and dissociation ability of complexes. A temperature-sensitive modification of poly(N-isopropylacrylamide) (PNIPAAm) can control the dissociation of PNVLCS (N-isopropylacrylamide/vinyl laurate copolymer with chitosan) complexes with DNA by a temporary reduction in the culture temperature to 20℃ (Sun et al, 2005) The cytoplasm, a mesh-like network of microfilaments and microtubules, will limit the diffusion of complexes or DNA about 500-1000-fold. Adenovirus particles naturally bind to dynein and are actively transported towards the nuclear pore complexes once they are inside the cytoplasm.…”

Medical Polymer-Based Gene Therapy

“…Many research studies have been conducted for enhancement of transfection efficiency, such as pHsensitive modification , Jones, et al, 2003, Kim, et al, 2003, Wong, et al, 2006, temperature-sensitive modification , Dang, et al, 2006, Sun, et al, 2005, specific target ligand modification (Hashimoto, et al, 2006, Mansouri, et al, 2006, Wu & Wu, 1998 and so on. Among the chemical modifications of chitosan, PEI grafted chitosan showed some benefit due to high transfection efficiency.…”