Synthesis of Thermoresponsive Chitosan- graft-Poly(N-isopropylacrylamide) Hybrid Copolymer and Its Complexation with DNA

Abstract

A hybrid synthetic-natural, thermoresponsive graft copolymer composed of poly(N-isopropyl acrylamide) (PNIPAM) side chains, prepared via RAFT polymerization, and a chitosan (Chit) polysaccharide backbone, was synthesized via radical addition-fragmentation reactions using the "grafting to" technique, in aqueous solution. ATR-FTIR, TGA, polyelectrolyte titrations and ¹H NMR spectroscopy were employed in order to validate the Chit-g-PNIPAM copolymer chemical structure. Additionally, ¹H NMR spectra and back conductometric titration were utilized to quantify the content of grafted PNIPAM side chains. The resulting graft copolymer contains dual functionality, namely both pH responsive free amino groups, with electrostatic complexation/coordination properties, and thermoresponsive PNIPAM side chains. Particle size measurements via dynamic light scattering (DLS) were used to study the thermoresponsive behavior of the Chit-g-PNIPAM copolymer. Thermal properties examined by TGA showed that, by the grafting modification with PNIPAM, the Chit structure became more thermally stable. The lower critical solution temperature (LCST) of the copolymer solution was determined by DLS measurements at 25-45 °C. Furthermore, dynamic and electrophoretic light scattering measurements demonstrated that the Chit-g-PNIPAM thermoresponsive copolymer is suitable of binding DNA molecules and forms nanosized polyplexes at different amino to phosphate groups ratios, with potential application as gene delivery systems.

Keywords: DNA polyplexes; RAFT polymerization; chitosan; gene delivery systems; grafting; poly(N-isopropylacrylamide); thermal response.

Scheme 1

Schematical representation of (A) PNIPAM synthesis by RAFT polymerization and (B) proposed mechanism of the PNIPAM “grafting to” the Chit chains: (1) KPS extracts an H atom from the Chit amino group, generating a stable salt (potassium bisulfate) and Chit amino and bisulfate radicals; (2) the bisulfate radical attacks the PNIPAM chains at the trithiocarbonate active end group, generating a PNIPAM macroradical; (3) the PNIPAM macroradical reacts with Chit NH radical forming Chit-g-PNIPAM copolymer.

Figure 1

ATR-FTIR spectra of PNIPAM, Chit and Chit-g-PNIPAM copolymer.

Figure 2

Validation of Chit-g-PNIPAM structure via ¹H NMR. The spectra of PNIPAM, Chit and Chit-g-PNIPAM are shown in comparison.

Figure 3

TGA curves of Chit, Chit-g-PNIPAM and PNIPAM polymers.

Figure 4

Back conductometric titration curves of (a) Chit and Chit-g-PNIPAM, and (b) PNIPAM solutions.

Figure 5

The influence of temperature on the: (a) scattered light intensity, (b) particle size, (c) polydispersity index and (d) size distribution of Chit-g-PNIPAM (polymer concentration: 1.2 mg/mL, pH 6.5, number of experiment replications = 3).

Figure 6

DLS and ELS results for the Chit-g-PNIPAM/DNA complexes were (a) the scattered intensity values, (b) the zeta potential values, (c) the size distributions of the hydrodynamic radii, R_h, and (d) the R_h values of the peaks of the size distributions (the different symbols: triangle, inverted triangle, equilateral triangle, and diamond correspond to the different peaks according to size), depending on the N/P ratio.

Figure 7

DLS results for representative Chit-g-PNIPAM and DNA complexes at N/P = 1 and 4 were (a) the scattered intensity values, (b) the R_h values of the peaks of the corresponding size distributions, and (c,d) the size distributions of the hydrodynamic radii, R_h, are shown as a function of temperature.

Figure 8

Schematic representation of the complexation process between the Chit-g-PNIPAM copolymer and the DNA chains, at two N/P ratios and their response to the increase of temperature (components are not drawn exactly in scale).