The lack of bioactivity of poly(N-isopropylacrylamide) (PNIPAM) restricts the use in cell-containing systems to the support as thermoresponsive adhesive substratum with no effect on cell differentiation. In this study, layer-by-layer assembly of polyelectrolytes, allows the combination of PNIPAM-grafted-chitosan (PNIPAM-Chi) with bioactive heparin (Hep) to fabricate PNIPAM−modified polyelectrolyte multilayers (PNIPAM−PEM). Owing to lower critical solution temperature of PNIPAM at around 32 °C, the physiochemical and biological properties of PNIPAM−PEM can be thermally modulated.
The thermorepsonsive behaviors of PNIPAM-Chi with different sizes and degree of substitution (DS) are ensured by dynamic laser scattering. The PNIPAM-PEMs are formed from selected PNIPAM-Chi derivatives as polycation and Hep as polyanion at pH 4. Subsequently, chemical cross-linking is introduced to stabilize the PEM. The temperature effects on wetting properties roughness and stiffness of the PNIPAM−PEMs with either PNIPAM-Chi− or Hep− terminal layer are investigated by different analytical methods. In addition, the stability of the PEMs is tested by rinsing with PBS, pH 7.4 and DMEM. Furthermore, the association of adhesive protein vitronectin with PNIPAM-PEMs at 20 and 37 °C is studied for their biological function. The application of PNIPAM-PEMs as cell culture substrate is finally examined using multipotent mouse stem cells in the presence of vitronectin.
PNIPAM-Chi with either higher DS or size showing strong thermoresponsiveness across LCST are selected to from multilayer with heparin. Ellipsometry results confirm that irreversible cross-linking retains the layer integrity after exposure to physiological buffer at pH 7.4 compared to those without cross-linking. Upon temperature change, PNIPAM-PEMs particularly Hep as terminal layer not only exhibit smoother and more hydrophobic surface at 37 °C than at 20 °C but also are stiffer than those PEMs without PNIPAM. This might also take account of higher retention of vitronectin on Hep− terminated at 37 °C. Finally, with the aid of pre-adsorbed vitronectin, cell adhesion and spreading are improved on both PNIPAM-Chi− and Hep− terminated layers.
In conclusion, the amide bonds make an irreversible bond formation between PNIPAM-Chi and Hep to enhance stability of PEM. In addition, the PNIAPM immobilized on the surface affects the wetting and mechanical properties of the surfaces. Therefore, the PNIPAM-PEMs with cross-linking provides greater stability and biological function as a cell culture system to potentially promotes tissue regeneration.</div>