Engineering of Stable Cross-Linked Multilayers Based on Thermo-Responsive PNIPAM-Grafted-Chitosan/Heparin to Tailor Their Physiochemical Properties and Biocompatibility

The thermo-responsive poly(N-isopropylacrylamide) (PNIPAM) is ubiquitously applied in controlled drug release and tissue engineering. However, the lack of bioactivity of PNIPAM restricts its use in cell-containing systems being a thermo-responsive adhesive substratum with no regulating effect on cell growth and differentiation. In this study, integrating PNIPAM with chitosan into PNIPAM-grafted-chitosan (PNIPAM-Chi) allows a layer-by-layer assembly with bioactive heparin to fabricate PNIPAM-modified polyelectrolyte multilayers (PNIPAM-PEMs). Grafting PNIPAM chains of either 2 (LMW) or 10 kDa (HMW) on the chitosan backbone influences the cloud point (CP) temperature in the range from 31 to 33 °C. PNIPAM-Chi with either a higher molecular weight or a higher degree of substitution of PNIPAM chains exhibiting a significant increase in diameter above CP as ensured by dynamic light scattering is selected to fabricate PEM with heparin as a polyanion at pH 4. Little difference of layer growth is detected between the chosen PNIPAM-Chi used as polycations by surface plasmon resonance, while multilayers formed with HMW-0.02 are more hydrated and show striking swelling-and-shrinking abilities when studied with quartz crystal microbalance with dissipation monitoring. Subsequently, the multilayers are covalently cross-linked using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide to strengthen the stability of the systems under physiological conditions. Ellipsometry results confirm the layer integrity after exposure to the physiological buffer at pH 7.4 compared to those without cross-linking. Moreover, significantly higher adhesion and more spreading of C3H10T1/2 multipotent embryonic mouse fibroblasts on cross-linked PEMs, particularly with heparin terminal layers, are observed owing to the bioactivity of heparin. The slightly more hydrophobic surfaces of cross-linked PNIPAM-PEMs at 37 °C also increase cell attachment and growth. Thus, layer-by-layer constructed PNIPAM-PEM with cross-linking represents an interesting cell culture system that can be potentially employed for thermally uploading and controlled release of growth factors that further promotes tissue regeneration.