Synthesis and Characterization of Injectable Sulfonate-Containing Hydrogels

Abstract

Sulfonate-containing hydrogels are of particular interest because of their tunable mechanical and swelling properties, as well as their biological effects. Polysulfonate copolymers were synthesized by reacting 2-acrylamido-2-methylpropanesulfonic acid (AMPS), acrylamide (AM), and acrylic acid (AA). We found that the incorporation rate of sulfonate-containing monomer and the molecular weight of the copolymer were significantly enhanced by increasing the ionic strength of the solution. We introduced thiol groups by modifying the pendant carboxylates or copolymerizing along with a disulfide-containing monomer. The thiol-containing copolymers were reacted with a 4-arm acrylamide-terminated poly(ethylene glycol) via a thiol-ene click reaction, which was mediated by a photoinitiator, a redox initiator, or a base-catalyzed Michael-Addition. We were able to tailor the storage modulus (33-1800 Pa) and swelling capacity (1-91 wt %) of the hydrogel by varying the concentration of the copolymers. We determined that the injectable sulfonate-containing hydrogels were biocompatible up to 20 mg/mL, as observed by an electric cell-substrate impedance sensing (ECIS) technique, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay using three different cell lines: human retinal pigment epithelial cells (ARPE-19), fibroblasts (NIH 3T3), and Chinese hamster ovary cells (CHO).

Figure 1

(a) Copolymerization of AM and AMPS in an aqueous solution. (b) Characteristic ¹H NMR spectrum of the reaction mixture. This sample was taken at 25 min of polymerization with a molar feed ratio of 50:50 AMPS/AM.

Figure 2

Conversions of AMPS and AM at 25 min of polymerization under different conditions showed that the presence of NaCl enhanced the reactivities of both monomers and reduced the difference between their reactivities.

Figure 3

Addition of NaCl and AM increased the molecular weight of copolymers.

Figure 4

Synthesis of a thiol- and sulfonate-containing copolymer by copolymerization of AM, AMPS, and BAC in DMF, followed by reduction of the disulfide bonds using DTT.

Figure 5

Thiolation of copoly(AM-co-AMPS-co-AA) via carbodiimide and triazine methods.

Figure 6

Structures of the components of sulfonate-containing hydrogel: the thiol- and sulfonate-containing copolymer and the 4-arm PEG-acrylamide.

Figure 7

Hydrogels with a higher concentration of sulfonate-containing copoly(AM-co-AMPS-co-BAC) (AM/AMPS/BAC = 7.5:2:0.5) swelled more and took a longer time to equilibrate.

Figure 8

Sulfonate-containing hydrogel with increasing concentrations of copolymer (10–40 mg/mL) showed increasing storage moduli.

Figure 9

Resistance measurements of (A) ARPE-19, (B) NIH 3T3, and (C) CHO cells plated at 20000 cells/well and exposed to sulfonate-containing hydrogel at 15 and 20 mg/mL showed excellent recovery and a minor lag phase. Unlike ARPE-19 cells, NIH 3T3, and CHO cells require almost 50 h to reach confluency; therefore, the hydrogel addition points for NIH 3T3 and CHO cells were different from that for ARPE-19 cells. Because media change is not possible following the addition of the hydrogels, a slight decrease in cell attachment was observed. Overall, the presence of hydrogels did not show any toxic effect on the cell types tested. Values are expressed in mean ± SEM, with each condition tested (n = 4).

Figure 10

Overall, the viability of ARPE-19, NIH 3T3, and CHO cells exposed to sulfonate-containing hydrogels at 15 and 20 mg/mL for 96 h were greater than 80%. CHO cells were the least affected. Each column represents the average of four wells. Error bars represent the standard error as established by the standard deviation divided by the square root of the well number (n = 4).

Figure 11

Inverted light microscopy images of (A) ARPE-19, (B) NIH 3T3, and (C) CHO cells exposed for 72 h to sulfonate-containing hydrogel at different concentrations showed similar morphology and cell number compared to control cells. The images of confluent cell monolayers were obtained using an Olympus CKX41 inverted microscope with ×10 magnification. The visualization window is 60 × 60 μm; the scale bars correspond to 15 μm.