Fabrication Of Thermoresponsive Polymer Film And Its Manipulation For Cell Adhesion And Detachment

Biomaterial science is an important interdisciplinary subject derived frommaterial science and life science.Biomaterials or biomedical materials couldsubstitute the function of a part of tissues or organics through a safe,reliable,biocompatible way.The Thermoresponsive poly(N-isopropylacrylamide) (PNIPAAm)has a lower critical solution termperature (LCST) at about 32℃in pure water.It isuseful for cell recovery without enzymatic digestion.Cells can adhere,spread,andproliferate on the surface grafted with PNIPAAm at 37℃,but can be spontaneouslydetached by only lowering the culture temperature to below the LCST of PNIPAAm.Here,we fabricated thermoresponsive cell culture surfaces using surface-initiatedatom transfer radical polymerization (SI-ATRP) of NIPAAm on the silicon substratesand then studied the manipulation of the surface on cell adhesion and detachment.Firstly,3-(aminopropyl)triethoxysilane (APS) was immobilized on the siliconsurface to introduce-NH₂ groups on the surface.Then the substrate was treated withATRP initiator,bromoisobutyryl bromide,to give rise to the Si-Br surface.TheSI-ATRP of NIPAAm on the Si-Br surface was carried out using a reaction mixture ofNIPAAm,CuBr,PMDETA (1,1,4,7,7-pentamethyldiethylenetriamine),H₂O/MeOH.After polymerization,the PNIPAAm grafted surfaces were characterized by atomicforce spectroscopy (AFM),ellipsometry,X-ray photoelectron spectroscopy (XPS),contact angle,quartz crystal microbalance (QCM),respectively.The experimentalresults showed that PNIPAAm chains had been successfully grafted on the siliconsurfaces,the chains of PNIPAAm grew linearly from the silicon substrates,and thePNIPAAm surfaces showed temperature sensibility.At 37℃,cells adhere,proliferate on the PNIPAAm surfaces,while lowering the culture temperature to theLCST of PNIPAAm,cells detached spontaneously from the surfaces.To study the effect of the thickness of PNIPAAm grafted on the silicon surface oncell adhesion and detachment,the thickness gradient surface has been prepared usinga combinatorial method:a micropump was used to control the polymerization time ofdifferent positions on the silicon substrate.The results showed that the surface became more hydrophilic with the increase of the thickness of PNIPAAm.When the thicknessof PNIPAAm was higher than 45nm,cells could not adhere on the surface.Lowerthan 20nm,the cells could not detach from the surface as lowering culturetemperature to 20℃.So the suitable thickness for cell adhesion and thermoresponsivedetachment of PNIPAAm on the silicon surface was about 20-45nm.Introducing poly(ethylene glycol) (PEG) molecules into PNIPAAm chains couldaccelerate the hydration of PNIPAAm chains.So a layer of PEG was firstlyimmobilized on the silicon surface by the ATRP of poly(ethylene glycol) methylmethacrylate (PEGMA),then PNIPAAm chains were formed from the PEG surfaceusing P(PEGMA) as macromolecular initiators.Thus block copolymer brushes ofP(PEGMA)-b-PNIPAAm were achieved.Combined with the injecting method,threetypes of gradient surfaces with different gradient direction were fabricated on thesilicon substrates.Cell culture results showed that the introduction of PEG moleculesmade the PNIPAAm chains hydrate quickly during the process of lowering the culturetemperature,thus the cells could detach quickly from the PNIPAAm surfaces.To improve cell adhesion on the PNIPAAm grafted surface,Arg-Gly-Asp (RGD)peptide was gradiently immobilized on the thermoresponsive surface.Firstly,PNIPAAm brushes were grafted on the silicon surface by the SI-ATRP of NIPAAm,then polyacrylic acid (PAA) chains grew from the PNIPAAm surface through theATRP of sodium acrylate.Finally,the RGD peptide was covalently grafted on thesurface by the functional group reaction of carboxyl group and amino group.Theeffect of PAA grafting thickness and RGD grafting quantity on cell adhesion anddetachment of thermoresponsive surface was studied.With the increase of PAAthickness,grafting quantity of RGD increased and so the cells adhered on the surface.But when the PAA thickness increased to some content,the negative effect ofcarboxyl groups on cell adhesion appeared,because the surface became morehydrophilic and thereby the number of cells adhered on the surface decreased.