A Spontaneous In Situ Thiol-Ene Crosslinking Hydrogel with Thermo-Responsive Mechanical Properties

Abstract

The thermo-responsive behavior of Poly(N-isopropylacrylamide) makes it an ideal candidate to easily embed cells and allows the polymer mixture to be injected. However, P(NiPAAm) hydrogels possess minor mechanical properties. To increase the mechanical properties, a covalent bond is introduced into the P(NIPAAm) network through a biocompatible thiol-ene click-reaction by mixing two polymer solutions. Co-polymers with variable thiol or acrylate groups to thermo-responsive co-monomer ratios, ranging from 1% to 10%, were synthesized. Precise control of the crosslink density allowed customization of the hydrogel's mechanical properties to match different tissue stiffness levels. Increasing the temperature of the hydrogel above its transition temperature of 31 °C induced the formation of additional physical interactions. These additional interactions both further increased the stiffness of the material and impacted its relaxation behavior. The developed optimized hydrogels reach stiffnesses more than ten times higher compared to the state of the art using similar polymers. Furthermore, when adding cells to the precursor polymer solutions, homogeneous thermo-responsive hydrogels with good cell viability were created upon mixing. In future work, the influence of the mechanical micro-environment on the cell's behavior can be studied in vitro in a continuous manner by changing the incubation temperature.

Keywords: Michael-type addition; N-isopropylacrylamide; hydrogel.

Figure 1

Graphical representation of covalent crosslink formation by a Michael-type, click-reaction between a thiol and an acrylate functionality. Different feed ratios of functional monomers resulted in a different crosslink density of the hydrogel network, allowing customization of the hydrogel’s mechanical properties to match different tissue stiffness levels.

Scheme 1

On top, the synthetic route to synthesize P(NiPAAm-co-Cys). Below: the synthetic route to synthesize P(NiPAAm-co-HEAacr).

Figure 2

(a) The maximum solubility of polymer solutions was plotted in function of feed ratio of functional monomer. (b) Results obtained from DSC measurement for P(NiPAAm-co-Cys) with different feed ratio. (c) Results obtained from DSC measurement for P(NiPAAm-co-HEAacr) with different feed ratio. T_cp = cloud point temperature, the onset of an increase in the heat flow. T_max = maximum temperature, the temperature at the maximum heat flow. Error bars: standard deviation.

Figure 3

(a) Gel fraction of hydrogels in function of feed percentage of functional monomer. * Hydr1% is too unstable to obtain accurate results. (b) Swelling degree (SD) of hydrogels consisting of polymers with different feed ratio. Hydr1% (25 wt%) in black, Hydr2% (22.5 wt%) in purple, Hydr3% (20 wt%) in green and Hydr4% (10 wt%) in blue. Error bars: standard deviation.

Figure 4

X-axis represents hydrogels consisting of polymers with a different incorporation ratio and dissolved at maximum solubility between brackets. Points depicted in blue are measured at 4 °C, red points at 45 °C. (a,b) Storage modulus (squares) and loss modulus (triangles). (c) Loss factor. (d) Complex modulus. Error bars: standard deviation. All measurements were performed at a frequency of 1Hz and 1% strain. This data is summarized in Table S6.

Figure 5

Comparison of hydrogel described in this manuscript (Hydr3%) to the state of the art (Bearat et al. and Wang et al. [36,37]) at 20 °C in blue and at 37 °C in red.

Figure 6

(a) Frequency sweep of Hydr3% at 4 °C. (b) Frequency sweep of Hydr3% at 45 °C. (c) Strain sweep of Hydr3% at 4 °C. (d) Strain sweep of Hydr3% at 45 °C.

Figure 7

Complex modulus of (a) Hydr2% and (b) Hydr3% in function of temperature. All measurements were performed at a frequency of 1Hz and a 1% strain.

Figure 8

Normalized complex modulus as a function of time shows the degradation over time. The complex modulus was normalized by the complex modulus measured at the start.

Figure 9

Results for the compression-relaxation experiments. Compression moduli are depicted as squares, relaxation moduli as triangles. Blue = 4 °C and red = 45 °C. The following data was summarized in Table S7.

Figure 10

Cell viability assessed after 1, 3 and 7 days of incubation in cell culture medium at 37 °C and 5% CO₂ humidified atmosphere. The living cells were visualized in green using Calcein AM (488/515) and the dead cells were visualized red using BOBO-3 Iodide (570/602) and indicated with white arrows.