Synthesis and Evaluation of a Thermoresponsive Degradable Chitosan-Grafted PNIPAAm Hydrogel as a "Smart" Gene Delivery System

Abstract

Thermoresponsive hydrogels demonstrate tremendous potential as sustained drug delivery systems. However, progress has been limited as formulation of a stable biodegradable thermosensitive hydrogel remains a significant challenge. In this study, free radical polymerization was exploited to formulate a biodegradable thermosensitive hydrogel characterized by sustained drug release. Highly deacetylated chitosan and N-isopropylacrylamide with distinctive physical properties were employed to achieve a stable, hydrogel network at body temperature. The percentage of chitosan was altered within the copolymer formulations and the subsequent physical properties were characterized using ¹H-NMR, FTIR, and TGA. Viscoelastic, swelling, and degradation properties were also interrogated. The thermoresponsive hydrogels were loaded with RALA/pEGFP-N1 nanoparticles and release was examined. There was sustained release of nanoparticles over three weeks and, more importantly, the nucleic acid cargo remained functional and this was confirmed by successful transfection of the NCTC-929 fibroblast cell line. This tailored thermoresponsive hydrogel offers an option for sustained delivery of macromolecules over a prolonged considerable period.

Keywords: drug delivery; hydrogel; hydroxyapatite; nanoparticles; thermoresponsive.

Figure 1

(a) Schematic of Chitosan-grafted-Poly(NIPAAm) synthesis. (b) Crosslinking of Cs-g-PNIPAAm with genipin (GE).

Figure 2

Verification of Cs-g-PNIPAAm composition via (a) ¹H-NMR spectra of NIPAAm monomer, PNIPAAm, Chitosan in 1% acetic acid, and Cs-g-PNIPAAm copolymer; (b) comparison of Cs in acetic acid and Cs-g-PNIPAAm copolymer. ¹H-NMR confirmed presence of PNIPAAm and chitosan in the structure. Lack of peaks between 5.5–6.5 ppm demonstrates no monomers in the copolymer. (c) The ζ-potential of Cs and Cs-g-PNIPAAm with 10%, 20%, and 30% Cs in relation to PNIPAAm after reconstitution in different media (DDW, Tris-EDTA (TE) buffer, PBS).

Figure 3

(a) FTIR confirming presence of the individual component in the copolymer, (b) grafting of the copolymers analyzed via TGA: Representative degradation profiles of hydrogel with varied Cs% showed two transition phases (typical of pure Cs and PNIPAAm). Mass remaining at 600 °C was attributed to Cs component and the mass % of Cs component from TGA corresponded to Cs utilized during hydrogel synthesis. Insert shows grafting efficiency and Cs context calculated with weight remaining at 600 °C.

Figure 4

Rheological characterization of Cs-g-PNIPAAm: (a) Tan δ of gel as a function of temperature achieved using ramp temperature analysis, where the tan δ value shifted from a value > 1 to a value < 1. Dashed line represents equilibrium state of the gel when liquid solution and solid gel phases were equal (G′ = G″). (b) Hydrogel stability assessed via time sweep at 37 °C over a 20 min period. (c) Viscosity of gels in liquid state at 20 °C. (d) Dynamic temperature sweep of Cs-g-PNIPAAm copolymers in PBS, DDW, and Tris-EDTA (TE) buffer demonstrating ability to modulate mechanical properties while maintaining consistent sol-gel transition point. (e) Shear storage modulus (G’) and shear loss modulus (G”) values extracted from the dynamic temperature sweep.

Figure 5

(a) Swelling behavior of Cs-g-PNIPAAm hydrogels in different media (PBS, DDW, and Tris-EDTA (TE) buffer). (b) Viscosity of the hydrogels determined in a flow mode at 20 °C. (c) Force required to inject 200 µL hydrogel through 25-G needle and viscosity of copolymers measured via rheological measurement in flow mode.

Figure 6

SEM of hydrogel at ×400 (above) and ×10,000 (below): (a) Cs-g-PNIPAAm hydrogel, (b) Cs-g-PNIPAAm hydrogel with pure HA and, (c) Cs-g-PNIPAAm hydrogel with RALA-HA NPs.

Figure 7

(a) Cell viability analyzed via MTS assay for NCTC L929 fibroblasts co-cultured for 24 h with the hydrogel degradation products for 10%, 10% crosslinked with GE, and 30% Cs in relation to NIPAAm. The dashed line delineates 70% viability, below which is deemed cytotoxic, as designated by the ISO 10993–5 [35]. (b) Degradation profile of Cs-g-PNIPAAm in DDW over eight weeks. (c) Release profiles of RALA/pEGFP-N1 NPs) from 5% w/v 10% Cs and 30% Cs-g-PNIPAAm hydrogels in DDW at 37 °C. Values represent mean ±SEM (n = 3).

Figure 8

Assessment of DNA transfection efficiency of NCTC-929 cells after incubation with RALA/GFP NPs released from Cs-g-PNIPAAm hydrogels at 24 h time point. (a) Flow cytometry analysis showed varied transfection efficiency of RALA/GFP NPs released from the hydrogel formulations. (b) Cells transfection efficiency was visualized with the presence of green fluorescent protein expressed by the NCTC-929 cells for (1) fresh NPs, (2) NPs released from 10% Cs-g-PNIPAAm in DDW, (3) NPs released from 30% Cs-g-PNIPAAm in DDW, (4) NPs released from 10% Cs-g-PNIPAAm in Tris-EDTA (TE) buffer, and (5) NPs released from 10% Cs-g-PNIPAAm in DDW crosslinked with 1% GE.

Figure 9

(a) Mechanism of primary radical initiation with APS and TEMED. (b) Proposed scheme of radial polymerization. Secondary initiation of Cs by TEMED radical, leading to chain reaction propagation with NIPAAm and terminated with a hydrogen atom shift by radical scavenger species (TEMED or bisulfate) to liberate C-2 directed graft of Cs-PNIPAAm copolymer.