The synergistic effects of stimuli-responsive polymers with nano- structured surfaces: wettability and protein adsorption

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<ul><li><p>The synergistic effects of stimuli-responsive polymers with nano- structuredsurfaces: wettability and protein adsorption{</p><p>Qian Yu,ab Xin Li,a Yanxia Zhang,ab Lin Yuan,*a Tieliang Zhaoab and Hong Chen*a</p><p>Received 25th May 2011, Accepted 27th May 2011</p><p>DOI: 10.1039/c1ra00201e</p><p>Surface modification with stimuli-responsive polymers leads to switchable wettability and</p><p>bioadhesion that varies in response to environmental stimuli. The introduction of nanoscale structure</p><p>onto surfaces also results in changes to the surface properties. However, the synergistic effects of</p><p>stimuli-responsive polymers with nanoscale structures are unclear. In this work, two typical stimuli-</p><p>responsive polymers, thermo-responsive poly(N-isopropylacrylamide) (poly(NIPAAm)) and pH-</p><p>responsive poly(methacrylic acid) (poly(MAA)), were grafted from initiator-immobilized silicon</p><p>nanowire arrays (SiNWAs) with nanoscale topography via surface-initiated atom transfer radical</p><p>polymerization. Because of the synergistic effects of the stimuli-responsive conformation transition of</p><p>polymer chains and the nano-effects of three-dimensional nanostructured SiNWAs, these new</p><p>platforms possess several unique properties. Compared with their corresponding modified flat silicon</p><p>surfaces, the introduction of nanoscale roughness enhanced the thermo-responsive wettability of</p><p>SiNWAs-poly(NIPAAm) but weakened the pH-responsive wettability of SiNWAs-poly(MAA).</p><p>More importantly, these surfaces exhibited special protein-adsorption behavior. The SiNWAs-</p><p>poly(NIPAAm) surface showed good non-specific protein resistance regardless of temperature,</p><p>suggesting a weakened thermo-responsivity to protein adsorption. The SiNWAs-poly(MAA) surface</p><p>showed obvious enhancement of pH-dependent protein adsorption behavior.</p><p>Introduction</p><p>In recent years, nanomaterials have been widely used for various</p><p>biomedical and biotechnology applications, including biosensors,</p><p>drug delivery, and diagnostics.1,2 Understanding and controlling</p><p>the interactions of nanomaterials with biological molecules, such</p><p>as proteins is not only of great theoretical interest but also of</p><p>crucial practical importance. Properties inherent to the nano-</p><p>material, such as size, curvature and surface chemistry strongly</p><p>influence the quality, secondary structure and activity of adsorbed/</p><p>conjugated proteins, all of which influence biofunctionality.35 On</p><p>the other hand, conjugation of nanomaterials with appropriate</p><p>proteins offers potential opportunities for biomolecule delivery,</p><p>targeted therapy and the detection of small molecules.69</p><p>Silicon nanowires are widely used as a typical one-dimensional</p><p>nanomaterial for field-effect transistors,10 biosensors11,12 and</p><p>thermoelectric materials.13,14 Silicon nanowire arrays (SiNWAs)</p><p>have attracted increased attention because of their novel</p><p>electronic properties and surface activity; they are good</p><p>candidate substrates for surface-enhanced Raman scattering15</p><p>and solar cells.16 In recent years, some groups have investigated</p><p>the biocompatibility of SiNWAs and broadened their applica-</p><p>tion to the biomedical and biomaterial fields.1721 It was found</p><p>that the nano-topography of SiNWAs enhanced the interaction</p><p>between cells and surfaces and therefore benefits cell adhe-</p><p>sion.22,23 With proper modification, SiNWAs acquired unique</p><p>wettability that enabled resistance to the adhesion of platelets24</p><p>and bacteria.25 Moreover, SiNWAs exhibited efficient tumor cell</p><p>capture19 and controlled drug release20 because of their three-</p><p>dimensional (3D) nanostructure. However, to the best of our</p><p>knowledge, there are few reports concentrating on protein</p><p>adsorption on SiNWAs.26</p><p>The interfaces that control protein adsorption and desorption</p><p>through the modulation of environmental stimuli are important</p><p>for many biomedical and biotechnological applications, including</p><p>controlled drug delivery, separation science and biosensors.2731</p><p>Persistent efforts have been made towards the design of such smart</p><p>interfaces, and many reports concern surface modification with</p><p>stimuli-responsive or smart polymers.3235 poly(N-isopropyla-</p><p>crylamide) (poly(NIPAAm)) is the best known thermo-responsive</p><p>polymer,36 and numerous reports indicate that poly(NIPAAm)-</p><p>modified surfaces can switch their wettability, topography and</p><p>bioadhesion in response to environmental temperature in the</p><p>aCollege of Chemistry, Chemical Engineering and Materials Science,Soochow University, Suzhou, 215123, China. E-mail:;; Fax: +86-512-65880827; Tel: +86-512-65880827bSchool of Materials Science and Engineering, Wuhan University ofTechnology, Wuhan, 430070, China{ Electronic Supplementary Information (ESI) available: Detailedprocedures of preparation and optimization of SiNWAs, contact angleimages of oil drop and bubble in PBS, and HSA and lysozyme adsorptionon SiNWAs and SiNWAs-poly(NIPAAm) surfaces. See DOI:10.1039/c1ra00201e/</p><p>RSC Advances Dynamic Article Links</p><p>Cite this: RSC Advances, 2011, 1, 262269</p><p> PAPER</p><p>262 | RSC Adv., 2011, 1, 262269 This journal is The Royal Society of Chemistry 2011</p><p>Publ</p><p>ished</p><p> on </p><p>02 A</p><p>ugus</p><p>t 201</p><p>1. D</p><p>ownl</p><p>oade</p><p>d on</p><p> 10/</p><p>11/2</p><p>013 </p><p>19:5</p><p>4:33</p><p>. View Article Online / Journal Homepage / Table of Contents for this issue</p></li><li><p>vicinity of its lower critical solution temperature (LCST).3740 This</p><p>novel property has attracted considerable attention and can be</p><p>applied to the fabrication of surfaces with switchable wettability,41</p><p>the separation of proteins and other biomolecules,42,43 and the</p><p>regulation of the adhesion/detachment of cells,44 platelets45 and</p><p>bacteria.46 Poly(methacrylic acid) (poly(MAA)) is another type of</p><p>smart polymer that exhibits an extended/collapsed conformational</p><p>transition as pH changes; its swelling behavior has been extensively</p><p>studied.4751 When grafted onto a solid surface, this transition</p><p>results in pH-responsive changes in surface wettability and charge,</p><p>which in turn influences protein-surface interactions because of the</p><p>change in hydrophobic and electrostatic interactions.52</p><p>Because previous reports have shown that the stimuli-respon-</p><p>sivity of surface wettability can be greatly enhanced by nanoscale</p><p>roughness,41,53,54 it is natural to explore whether protein adsorp-</p><p>tion can also be enhanced when nanoscale topography is</p><p>introduced on these smart surfaces. However, the influence of</p><p>nanoscale structure on the interactions of smart surfaces with</p><p>biomolecules or cells is not consistent. Chen et al. found that the</p><p>introduction of nanoscale topography onto poly(NIPAAm)</p><p>modified SiNWAs surfaces resulted in the disappearance of</p><p>thermo-responsive platelet adhesion.24 In contrast, our recent</p><p>research indicates that the poly(MAA)-modified SiNWAs</p><p>surfaces exhibit significantly pH-responsive lysozyme adsorption</p><p>as compared with poly(MAA)-modified flat silicon surfaces.26</p><p>These results suggest that it is important to investigate the</p><p>synergistic effects of both stimuli-responsive polymers and</p><p>nanoscale structures on smart surface properties (wettability</p><p>and bioadhesion), which are not only of great theoretical interest</p><p>but also of crucial importance for designing and fabricating</p><p>novel devices in biomaterial and biomedical applications.</p><p>In the present study, two nanoscale surfaces of SiNWAs</p><p>modified with typical smart polymers, namely thermo-responsive</p><p>poly(NIPAAm) and pH-responsive poly(MAA) were prepared via</p><p>surface-initiated atom-transfer radical polymerization (SI-ATRP).</p><p>We investigated the synergistic effects of stimuli-responsive</p><p>properties and nanoscale roughness on the surface wettability</p><p>and protein adsorption. It is interesting that the introduction of</p><p>nanostructure results in some novel properties, which may be</p><p>favorable for biomaterial and biomedical applications. The</p><p>poly(NIPAAm)-modified SiNWAs exhibit high resistance to</p><p>non-specific proteins regardless of temperature, whereas the</p><p>poly(MAA)-modified SiNWAs show an extremely high capacity</p><p>for binding protein at low pH. Most of the adsorbed protein can be</p><p>released by simply increasing the pH.</p><p>Experimental section</p><p>Materials</p><p>N-Isopropylacrylamide (NIPAAm, Acros, 99%) was recrystal-</p><p>lized from toluene/hexane solution (50%, v/v) and dried under</p><p>vacuum prior to use. tert-Butyl methacrylate (tBMA, Aldrich,</p><p>98%) was passed through a column of activated basic alumina</p><p>prior to use. Copper(I) bromide (CuBr, Fluka) and Copper(II)</p><p>bromide (CuBr2, Aldrich) were recrystallized before use.</p><p>3-Aminopropyltriethoxysilane (APTES, Aldrich), bromoisobu-</p><p>tyryl bromide (BIBB, Fluka) and 1,1,4,7,7-pentamethyldiethyle-</p><p>netriamine (PMDETA, Aldrich) were used as received. All other</p><p>solvents were purchased from Shanghai Chemical Reagent Co.</p><p>and purified according to standard methods before use. Silicon</p><p>wafers were purchased from Guangzhou Semiconductor</p><p>Materials (Guangzhou, China). The as-received silicon wafers</p><p>were cut into square chips about 0.5 cm 6 0.5 cm. Deionizedwater was purified by a Millipore water purification system to</p><p>give a minimum resistivity of 18.2 MV?cm and used in all</p><p>experiments. Fibrinogen (MW = 341 kDa) was purchased from</p><p>Calbiochem (La Jolla, CA).</p><p>Preparation of polymer-grafted silicon nanowire arrays</p><p>The SiNWAs were prepared by chemical etching of crystalline</p><p>silicon in HF/AgNO3 aqueous solution (the detailed procedure is</p><p>given in ESI{). Then, the initiator was immobilized on theSiNWAs following the procedures reported previously.40 SI-</p><p>ATRP grafting of NIPAAm was carried out in a glovebox</p><p>purged with argon. NIPAAm (6.25 g, 55.23 mmol), PMDETA</p><p>(0.7 mL, 3.35 mmol) and CuBr (0.16 g, 1.12 mmol) were</p><p>dissolved in a 1 : 1 mixture of methanol and water (25 mL). The</p><p>reaction solution was sonicated for 2 min and then added to a</p><p>glass vessel, into which the initiator-functionalized SiNWAs were</p><p>also placed. The polymerization was carried out at room</p><p>temperature for 2 h. The obtained SiNWAs-poly(NIPAAm)</p><p>surfaces were rinsed with deionized water and dried under an</p><p>argon flow. SI-ATRP grafting of tBMA was carried out as</p><p>follows. tBMA (4 mL, 25 mmol), CuBr2 (3.3 mg, 0.015 mmol),</p><p>CuBr (7.15 mg, 0.05 mmol), PMDETA (0.031 mL, 0.15 mmol)</p><p>and 3 mL acetone were mixed in a flask. The heterogeneous</p><p>reaction solution was degassed with three freezepumpthaw</p><p>cycles and then stirred at 60 uC for 20 min until it became clearand homogeneous. The solution was then transferred to a</p><p>Schlenk flask containing the initiator-functionalized SiNWAs via</p><p>syringe. The polymerization was allowed to proceed at 60 uC for4 h. The obtained SiNWAs-poly(tBMA) surfaces were rinsed</p><p>thoroughly with acetone, dried under an argon flow, and placed</p><p>in a flask containing a mixture of 1,4-dioxane (20 mL) and</p><p>concentrated HCl (37%, 3 mL). The reaction was carried out at</p><p>80 uC for 2 h to hydrolyze poly(tBMA) to poly(MAA). Theresulting surfaces were thoroughly rinsed with acetone and</p><p>ethanol and dried under an argon flow. For comparison,</p><p>polymer-grafted smooth silicon surfaces (Si-poly(NIPAAm)</p><p>and Si-poly(MAA)) were also prepared following the same</p><p>procedures mentioned above.</p><p>Surface characterization</p><p>The chemical composition of the modified silicon surfaces was</p><p>determined with an ESCALAB MK II X-ray photoelectron</p><p>spectrometer (XPS) (VG Scientific Ltd.). All XPS data were</p><p>analyzed using XPS Peak 4.1 software. The thicknesses of the</p><p>polymer grafts on the smooth silicon substrate were measured by</p><p>an M-88 spectroscopic ellipsometer (J. A. Woollam Co., Inc.).</p><p>The nanostructures and surface morphology of pristine and</p><p>modified SiNWAs were observed using scanning electron</p><p>microscopy (SEM, S-4800, Japan).</p><p>Contact angle measurements</p><p>Contact angle (CA) measurements were performed using an SL-</p><p>200C optical contact angle meter (Solon Information Technology</p><p>This journal is The Royal Society of Chemistry 2011 RSC Adv., 2011, 1, 262269 | 263</p><p>Publ</p><p>ished</p><p> on </p><p>02 A</p><p>ugus</p><p>t 201</p><p>1. D</p><p>ownl</p><p>oade</p><p>d on</p><p> 10/</p><p>11/2</p><p>013 </p><p>19:5</p><p>4:33</p><p>. </p><p>View Article Online</p></li><li><p>Co., Ltd.) with a heating element placed on the sample stage to</p><p>control the temperature of the sample surfaces. The static water</p><p>contact angles were measured using the sessile drop method in the</p><p>dry state. For poly(MAA)-grafted surfaces, the samples were first</p><p>immersed in phosphate buffered saline (PBS) at a specified pH for</p><p>30 min and dried immediately in an argon flow. The pH of the PBS</p><p>was pre-adjusted by adding aqueous NaOH or HCl solution until</p><p>the desired values were reached. To further investigate the</p><p>wettability of the surfaces in the wet state, the oil (dichloro-</p><p>methane, CH2Cl2) contact angles and captive bubble contact</p><p>angles in PBS under different temperatures (for poly(NIPAAm)-</p><p>grafted surfaces) or at different pH values (for poly(MAA)-grafted</p><p>surfaces) were also measured.</p><p>Protein adsorption</p><p>Fibrinogen adsorption from PBS solution (1 mg mL21) was</p><p>determined by radiolabeling with 125I using a Wizard 3991480</p><p>Automatic Gamma Counter (Perkin-Elmer Life Sciences).40 PBS</p><p>at different pH levels was used to prepare protein solutions.</p><p>Adsorption was allowed to proceed for 3 h under static</p><p>conditions. For PNIPAAm-grafted surfaces, the adsorption</p><p>temperatures were chosen at either room temperature (23 uC)or body temperature (37 uC) at pH 7.4; for PMAA-graftedsurfaces, the pH of protein solutions were 4 or 9 at 23 uC.Because it is very difficult to calculate the absolute surface area</p><p>of SiNWAs, the amount of protein adsorption is expressed as</p><p>mg/disc (for one disc, the apparent surface area is 0.5 cm2).</p><p>Results and discussion</p><p>Characterization of SiNWAs modified with polymers</p><p>The general process for the formation of poly(NIPAAm) or</p><p>poly(MAA) brushes on SiNWAs (SiNWAs-poly(NIPAAm) or</p><p>SiNWAs-poly(MAA)) is illustrated in Scheme 1. First, the</p><p>SiNWAs were prepared by a chemical etching method, as</p><p>previously reported.55 The length and diameter of the resulting</p><p>SiNWAs depended on the etching time; homogeneous SiNWAs</p><p>were prepared with an optimized etching time (Fig. S1, ESI{).The initiator was immobilized, followed by SI-ATRP of</p><p>NIPAAm or tBMA using these surfaces as substrates. The</p><p>poly(tBMA) chains were further hydrolyzed in acidic solution</p><p>to obtain the corresponding poly(MAA) chains. For com-</p><p>parison, polymers grafted onto smooth silicon surfaces</p><p>(Si-poly(NIPAAm) and Si-poly(MAA)) were also prepared</p><p>following the same procedures. The resulting poly(NIPAAm)-</p><p>and poly(MAA)-grafted layers had a thickness of y39.2 nm andy18.5 nm, respectively, as determined by ellipsometry. Thesevalues are consistent with the values reported elsewhere.40,56</p><p>Changes in the chemical composition of SiNWAs after the</p><p>grafting of the polymers were determined from XPS data. The</p><p>survey spectra of the poly(NIPAAm)- and poly(MAA)-grafted</p><p>surfaces are shown in Fig. 1, and the corresponding chemical</p><p>composition of these surfaces is summarized in Table 1. The</p><p>experimental atomic ratios for the resulting surfaces were close to</p><p>the theoretical values, suggesting a successful modification process.</p><p>The nanostructures and surface morphology of unmodified and</p><p>modified SiNWAs were observed using SEM. The top vi...</p></li></ul>


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