Poly(N-isopropylacrylamide) based thin microgel films for use in cell culture applications

Abstract

Poly(N-isopropylacrylamide) (PNIPAm) is widely used to fabricate cell sheet surfaces for cell culturing, however copolymer and interpenetrated polymer networks based on PNIPAm have been rarely explored in the context of tissue engineering. Many complex and expensive techniques have been employed to produce PNIPAm-based films for cell culturing. Among them, spin coating has demonstrated to be a rapid fabrication process of thin layers with high reproducibility and uniformity. In this study, we introduce an innovative approach to produce anchored smart thin films both thermo- and electro-responsive, with the aim to integrate them in electronic devices and better control or mimic different environments for cells in vitro. Thin films were obtained by spin coating of colloidal solutions made by PNIPAm and PAAc nanogels. Anchoring the films to the substrates was obtained through heat treatment in the presence of dithiol molecules. From analyses carried out with AFM and XPS, the final samples exhibited a flat morphology and high stability to water washing. Viability tests with cells were finally carried out to demonstrate that this approach may represent a promising route to integrate those hydrogels films in electronic platforms for cell culture applications.

Figure 1

Schematic representation of the functionalization of P(NIPAm-co-AAc) and IPN microgels with –ene reactive groups. The P(NIPAM-co-AA) and IPN nanogels are is ene-functionalized by allylamine at room temperature in the presence of EDC/NHS couple. Then, allylamine is added to the for the formation of the amide bonds onto the carboxylic acid. The reaction is allowed to proceed for 48 h.

Figure 2

ATR FT-IR spectra of the NIPAm based microgels before and after the amidation reaction with allylamine. Inset: a zoom of the peak at 1725 cm⁻¹, to show the carboxylic acid moiety of AAc. The spectra are normalized with respect to the adsorption band at 1645 cm⁻¹, due to the PNIPAm network.

Figure 3

1H-NMR spectra of P(NIPAm-co-AAc) (A) and P(NIPAm-co-AAc)-ENE (B) microgels in D₂O. The chemical structure represents the P(NIPAm-co-AAc)-ene microgel. Insets in (B) right, a zoom of the peak at 3.07 ppm due to the vinyl moiety; left, a zoom of the peak at 5.5 ppm. The spectra are normalized with respect to the peak at 3.82 ppm, due to the isopropyl group of the PNIPAm network.

Figure 4

Scheme of the reaction carried out to functionalize the glass coverslips with vynil groups. Organic residues can be removed by washing the substrates in piranha solution (a,b), and then silanized with TMSPMA (c).

Figure 5

Contact angle (CA) measurements for a glass coverslip not treated (control), after piranha treatment and after TMSPMA silanization.

Figure 6

Preparation scheme of anchored hydrogel films: ene-functionalized colloidal suspensions with the addition of DTT (a); spin coating on glass coverslips functionalized with TMSPMA (b); thiol-ene click reactions activated in oven at 120 °C (c); washing in water at room temperature and final drying (d).

Figure 7

Top row: AFM images of pristine PNIPAm, IPN-ene and P(NIPAm-co-AAc)-ene films (a–c, respectively). Bottom row: AFM images after thermal treatment and washing of PNIPAm, IPN-ene and P(NIPAm-co-AAc)-ene films (d–f, respectively).

Figure 8

AFM images and height profiles along the edges of the cuts for PNIPAm (a), P(NIPAm-co-AAc)-ene (b) and IPN-ene (c) films.

Figure 9

High resolution XPS spectra: N1s (a), O1s (b) and Si2p (c) peaks obtained for glass treated with NaOH, pristine and washed PNIPAm films.

Figure 10

XPS spectra of P(NIPAm-co-AAc)-ene (a). In (b) Si2p spectra of glass treated with TMSPMA and of a P(NIPAm-co-AAc)-ene film before and after washing. In (c) a bar graph with the elemental composition of P(NIPAm-co-AAc) films grafted and rinsed. The measurements were taken in different areas of the same sample.

Figure 11

XPS spectra of IPN-ene (a). In (b) Si2p spectra of glass treated with TMSPMA and of an IPN-ene film before and after washing. In (c) a bar graph with the elemental composition of IPN-ene films grafted and rinsed. The measurements were taken in different areas of the same sample.

Figure 12

Cell proliferation and spreading after three different incubation intervals: 5, 24, 72 h. The number of cells is plotted in (a), the cell spread area in (b). Data reported are mean values ± standard error, *p < 0.5, ***p < 0.001.

Figure 13

Live/dead assay images for cell viability of cells grown on pristine glass, glass/TMSPMA, PNIPAm, P(NIPAm-co-AAc)-ene and IPN-ene (named IPN). In (a–e) we show fluorescent live/dead assay images representing cell viability. In (f) a statistical analysis of a number of cells dead and the number of cells alive is reported. All data represent the mean values ± standard error, *p < 0.5. Red is for dead cells and green for living cells. The initial seeding density was 20000 cells/cm² and the incubation time 24 h. Red scale bar: 50 µm.