In situ forming poly(ethylene glycol)-based hydrogels via thiol-maleimide Michael-type addition

Abstract

The incorporation of cells and sensitive compounds can be better facilitated without the presence of UV or other energy sources that are common in the formation of biomedical hydrogels such as poly(ethylene glycol) hydrogels. The formation of hydrogels by the step-growth polymerization of maleimide- and thiol-terminated poly(ethylene glycol) macromers via Michael-type addition is described. The effects of macromer concentration, pH, temperature, and the presence of biomolecule gelatin on gel formation were investigated. Reaction kinetics between maleimide and thiol functional groups were found to be rapid. Molecular weight increase over time was characterized via gel permeation chromatography during step-growth polymerization. Swelling and degradation results showed incorporating gelatin enhanced swelling and accelerated degradation. Increasing gelatin content resulted in the decreased storage modulus (G'). The in vitro release kinetics of fluorescein isothiocyanate (FITC)-labeled dextran from the resulting matrices demonstrated the potential in the development of novel in situ gel-forming drug delivery systems. Moreover, the resulting networks were minimally adhesive to primary human monocytes, fibroblasts, and keratinocytes thus providing an ideal platform for further biofunctionalizations to direct specific biological response.

FIGURE 1

Synthetic scheme of PEG dithiol. Adapted from Ref .

FIGURE 2

Synthetic scheme of poly(ethylene glycol) hydrogel via macromeric thiol-maleimide addition.

FIGURE 3

PEG terminal maleimide conversion over time in THF via thiol-maleimide addition.

FIGURE 4

GPC Chromatograms of PEG polymers via thiol-maleimide addition obtained at 20 h, 2 d, and 1 month (A) and degraded PEG SH-Mal hydrogels (B).

FIGURE 5

Equilibrium weight swelling ratio of PEG hydrogels and sIPNs via thiol-maleimide addition and photopolymerization over time. (n=3)

FIGURE 6

Storage modulus (G’) and loss modulus (G”) at 25 °C as a function of frequency for non-swollen PEG SH-Mal hydrogels (A), swollen PEG SH-Mal hydrogels (B), non-swollen PEG SH-Mal sIPNs (C), swollen PEG SH-Mal sIPNs (D).

FIGURE 7

In vitro release kinetics of FD-40 from PEG SH-Mal hydrogel and PEG SH-Mal sIPN with 10% gelatin at 37 °C.

FIGURE 8

Optical micrographs of adherent monocytes on PEG SH-Mal hydrogel (A, B), photopolymerized PEG hydrogel (C, D), and TCPS (E, F) at 24 h (A, C, E) and 7 d (B, D, F) (20× magnification).

FIGURE 9

Adherent cell density on TCPS, photopolymerized PEG hydrogel and PEG SH-Mal hydrogel. Three cell types were included, primary human monocyte (A), keratinocyte (B), and fibroblast (C). Cells were observed at 10× magnification. Five images per sample were taken at random fields of view. †, p < 0.05 vs. the cell adhesion density on TCPS at the same time point; ‡, p < 0.05 vs. the cell adhesion density on photopolymerized PEG hydrogel at the same time point. All data presented as average ± standard deviation (n=3).

FIGURE 10

Microscopic images of adherent fibroblasts and keratinocytes on TCPS surface in the presence of PEG SH-Mal hydrogels at 24, 48 and 96 h (20× Magnification). (A) Fibroblast optical microscopy images (left-hand side) and fluorescent microscopy images (right-hand side, green, live stained cells; red, dead stained cells); (B) Keratinocyte optical microscopy images (left-hand side) and fluorescent microscopy images (right-hand side, green, live stained cells; red, dead stained cells).

FIGURE 11

Normalized cell viability of primary human monocytes, fibroblasts, and keratinocytes treated with hydrogel extracts on 24 and 48 h. Control: cells treated with serum-depleted medium only. (n=6)