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Applications of Polymer, Composite, and Coating Materials

Nanostructured Thermo-responsive Surfaces Engineered via Stable Immobilization of Smart Nanogels with Assistance of Polydopamine Lei Zhang, Zhuang Liu, Lu-Yue Liu, Jun-Li Pan, Feng Luo, Chao Yang, Rui Xie, Xiao-Jie Ju, Wei Wang, and Liang-Yin Chu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b20395 • Publication Date (Web): 26 Nov 2018 Downloaded from http://pubs.acs.org on November 29, 2018

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ACS Applied Materials & Interfaces

Nanostructured

Thermo-responsive

Surfaces

Engineered via Stable Immobilization of Smart Nanogels with Assistance of Polydopamine Lei Zhang†, Zhuang Liu†,‡ *, Lu-Yue Liu†, Jun-Li Pan§, Feng Luo†, Chao Yang†, Rui Xie†, ‡, XiaoJie Ju†, ‡, Wei Wang†, ‡, and Liang-Yin Chu†, ‡, *

† School ‡ State

of Chemical Engineering, Sichuan University, Chengdu, Sichuan 610065, P. R. China.

Key Laboratory of Polymer Materials Engineering, Sichuan University, Chengdu, Sichuan

610065, P. R. China. §

West China School of Preclinical and Forensic Medicine, Sichuan University, Sichuan 610064,

China.

ACS Paragon Plus Environment

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ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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ABSTRACT Thermo-responsive surfaces featured with nanostructures have found wide potential applications in biological and chemical fields. Herein, we report nanostructured thermo-responsive surfaces engineered via stable immobilization of thermo-responsive nanogels with assistance of polydopamine.

The results show that the thin layer of polydopamine on the poly(N-

isopropylacrylamide) (PNIPAM) nanogels nearly does not affect the thermo-responsive property of the nanogels. The stability of thermo-responsive nanogels on the substrate surfaces immobilized under different pH conditions of dopamine solutions are quatitively studied by fluid shearing experiments inside capillaries, and the characterization results show that the strong interaction forces between the polydopamine layer on the substrate surfaces and the thermo-responsive nanogels are heavily depended on the oxidation state of dopamine molecules. With the proposed strategy, thermo-responsive nanostructured surfaces immobilized with PNIPAM nanogels on twodimensional and three-dimensional substrate surfaces are generated to respectively achieve smart cell culture plates and smart gating membranes, which demonstrate versatile applications of the nanostructured thermo-responsive surfaces.

KEYWORDS Nanostructured surfaces; Responsive nanogels; Surface modification; Smart cell culture plates; Smart gating membranes


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1. INTRODUCTION Responsive surfaces, which could render novel chemicophysical functions to materials,1,2 are of great interests for the development of smart gating membranes, self-cleaning and antifouling surfaces, sensor devices, micro- and nanofluidic devices, and so on.3-6 Thereinto, the thermoresponsive surfaces have shown great importance in numerous applications, including tunable cell culture platforms,7-10 controlled release,11,12 biomimetics,13-15 advanced membranes for separation,5,16-18 and “on/off” switches for chemical reactions.19,20 Engineering nanostructures for thermo-responsive surfaces could improve the interfacial properties such as specific surface area, wettability and hardness, due to the binary collaboration of nano-size effect and thermosensitivity.21-23 Up to now, two main strategies have been developed to fabricate nanostructured thermoresponsive surfaces, in which one is a two-step strategy and the other is a one-step strategy. The two-step strategy is creating nanostructured architectures on the substrate surfaces in advance by techniques of nanoparticle-depositing, wet etching, laser cutting, etc.21,23 And then, thermoresponsive polymer brushes are covalently connected to the nanostructure surfaces by either “grafting-to” or “grafting-from” method.5,24 For examples, Liu et al. have prepared the siliconnanopillars via photolithography with wet etching approach, and then poly(N-isopropylacrylamide) (PNIPAM) brushes are grafted on silicon-nanopillars by surface-initiated atom-transfer radical polymerization (ATRP).24 Such thermo-responsive nanostructured surfaces could capture and release of targeted cancer cells by hydrophobic interactions and topographic interactions. To achieve the thermo-responsive functionality of the surface, the surface modifications involve skilled micromaching technology and chemical processes are requisite, which are torturous and complicated. The other strategy is a one-step approach, which is directly depositing thermo-


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responsive nanogels on the substrate surfaces via chemical or physical processes.25-28

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The

nanostructured thermo-responsive surfaces constructed with nanogels as building units could exhibit significant deformation effect on surface topography when compared with the abovementioned polymer brushes. The responsive nanogels could be physically immobilized on the surfaces via either Van der Waals forces or electrostatic interactions.25-27 However, the adhesion interactions are usually weak especially upon long storage under water or changes of environmental conditions such as temperature, ions and pH. To stably immobilize the thermo-responsive nanogels on substrate surfaces, specific reactions are needed to creat chemical bonds connecting with nanogels, for which the substrates are usually needed to be pre-treated.28 Recently, Serpe et al. have reported a method utilizing polydopamine to coat surfaces with PNIPAM-based microgels.6 The results showed that microgels could be coated on the various kinds of surfaces. However, the effect of polydopamine on the thermo-responsive property of PNIPAM microgels still remians unknown, and the thermo-responsive performances of the microgel-immobilized surfaces are not investigated systematically yet. Moreover, the stability and corresponding mechanism of the immobilization of microgels still remains unclear.6 Therefore, to solve these remained problems, it is still highly desired to further study the nanostructured thermo-responsive surfaces engineered via stable immobilization of smart nanogels with assistance of polydopamine. Herein, we engineer thermo-responsive nanostructured surfaces with PNIPAM nanogels (PNGs) with assistance of a polymerized dopamine (PDA) in aqueous solutions with different pH values (Figure 1). The dopamine molecules contain catechol and amine functional groups,29 which can be spontaneously polymerized into adhesive polydopamine (PDA).30 The pH condition could significantly affect the self-polymerization of dopamine, which would result in diverse stabilities of PNGs-immobilized surfaces (Figure 1a and b). The resultant PDA shows chemical versatility and strong adhesivity, which can be used to coat the PNGs on most of organic and inorganic


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surfaces6. The PDA firmly coats on the PNGs due to high-strength irreversible covalent bond formation between the o-quinone groups of PDA and the polymeric networks of PNGs.31,32 PNIPAM is a well-known thermo-responsive polymer, which can change the conformation between a swollen/hydrophilic state and a shrunken/hydrophobic state when the environmental temperature is changed across the volume phase transition temperature (VPTT) around 32 °C, and vice versa.33,34 In this study, the effect of the PDA layer on PNIPAM nanogels on the thermoresponsive property of the nanogels is investigated, and the stability of thermo-responsive nanogels on the substrate surfaces immobilized under different pH conditions of dopamine solutions are quatitively studied by fluid shearing experiments inside capillaries. To demonstrate versatile applications of nanostructured thermo-responsive surfaces fabricated with the proposed strategy, we create thermo-responsive nanostructured surfaces on two-dimensional (2D) flat substrates (Figure 1c) and three-dimensional (3D) porous substrates (Figure 1e) to respectively achieve smart cell culture plates with responsive cell adhesion property (Figure 1d) and smart gating membranes with responsive gating property (Figure 1f), and systematically investigate the thermo-responsive performances of the microgel-immobilized surfaces.

2. EXPERIMENTAL SECTION 2.1. Synthesis of Thermo-Responsive Nanogels PNIPAM nanogels (PNGs) were prepared by precipitation polymerization.19,35 Typically, 2.26 g monomer N-isopropylacrylamide (NIPAM), 0.154 g crosslinker N,N´-methylenebisacrylamide (BIS), 0.08 g initiator potassium persulfate (KPS) were dissolved in 300 mL deionized water that has been bubbled with nitrogen for 20 min. Then, the reaction proceeded under stirring for 4 h at 70 oC using a thermo-stated water bath. Immediately after the reaction, the round-bottom flask


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containing solution was placed in an ice bath to terminate further polymerization. Next, the prepared PNGs were purified by repeated centrifugations. Finally, the nanogels were freeze-dried for further use.

2.2. Immobilizing Stability Tests Glass capillaries (length 5 cm, inner diameter 400 μm) were immobilized with PNGs on the inner surfaces by injecting the freshly prepared dispersion containing PNGs (5 mg/mL) and DA∙HCl (2 mg/mL) at pH=8.5 into the tube for 12 h with a flow rate of 100 μL/h. The resulted glass capillary is referred as pH 8.5-DA. For contrast groups, dispersions containing PNGs (5 mg/ mL) without DA at pH=8.5, or with DA (2 mg/ mL) at pH=1, or without DA at pH=1 were continuously inject into the capillaries for 12 h under a flow rate of 100 μL/h, respectively. The resulted glass capillaries were referred as pH 8.5-no DA, pH 1-DA and pH 1-no DA, respectively. Subsequently, the above-mentioned PNGs-immobilized glass capillaries were rinsed with water at flow rates of 1000 μL/h, 2000 μL/h, 3000 μL/h and 5000 μL/h at 25 oC, respectively. After each rinsing, the morphological characteristics of inner surfaces of the PNGs-immobilized glass capillaries were carefully characterized by scanning electron microscope (SEM) to check the status of PNGs. The PNGs numbers on the inner surfaces of capillaries within the same areas were counted from at least five different SEM images to calculate the average values. Due to the laminar flow in the capillaries, the fluid shear stress (τ) in cylindrical capillaries with inner diameter of 400 μm obeys to the Newtonian shearing law, which can be described as equation (1)36:

 

du dr


(1)

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where, τ stands for fluid shear stress (N∙m-2), du/dr for the velocity gradient (s-1), and μ for the viscosity of liquid (Pa∙s). Thus, the boundary fluid shear stress (τ0) at the inner surface of capillary can be derived from equation (1) with r = R, where R is the inner radius of capillary (m).

2.3. Immobilizing of PNGs on the Surfaces of Different Substrates The substrates of glass, PDMS, PMMA, Al and stainless steel were ultrasonically cleaned in water for 10 min and ethanol for 1 min respectively. The PC porous membranes were soaked in water in advance. The commercial cell culture plates were used as received. In a typical procedure, PNGs were dispersed in Tris buffer solution (15 mM, pH=8.5) with nanogel concentration of 5 mg/mL, and the concentration of DA∙HCl was 2 mg/mL in the Tris buffer solution. Then, the substrates were immersed in the above-mentioned dispersion in a shaker for 12 h at room temperature. The immobilizing cycle was repeatedly carried out for the PC membranes to get multiple immobilizings. For the cell culture plates, the wells were added with dispersions (1 mL for each well) and placed in a shaking water bath for 12 h. Then, the substrates were rinsed with deionized water and ultrasonically washed for 1 min to remove the unstably immobilized PNGs.

The solution

containing PNGs immobilized with polymerized dopamine (PDA) was centrifuged for four times to obtain the PDA-immobilized PNGs (PNGs@PDA).

2.4. Characterization of Components and Morphology The hydrodynamic diameters and Zeta potential values of nanogels were measured by dynamic light scattering (DLS, Zetasizer Nano-ZEN3690, Malvern) at different temperatures with scattering angle of 90°. Transmission electron microscopy (TEM) was carried out on a Tecnai G2 F20 STWIN (FEI) transmission electron microscope operated at an acceleration voltage of 200 kV. All of the samples of PNGs and PNGs@PDA with a concentration of 0.1 mg/mL were ultrasonically


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dispersed before TEM characterization. The compositions of the nanogels were analyzed by an Xray Photoelectron Spectroscopy (XPS, XSAM800, KRATOS) instrument using Al Kα (1486.6 eV) as radiation source. The take-off angle of the photoelectron was set at 70° for XPS measurements. The water contact angles (CA) on flat substrates at 25 °C and 45 °C were characterized by a contact angle measurement instrument (DSA25, Krüss GmbH) to study the thermo-responsive wettability of PNGs-immobilized surfaces. The UV spectra of DA solutions stored at pH=1 and pH=8.5 were recorded using a UV-vis spectrophotometer (UV-1700, Shimadzu). A SEM (JSM-5900LV, JEOL) was employed to observe the microstructures of PNGs immobilized on the substrates. The underwater morphology of PNGs-immobilized PC membranes and the elastic modulus of PNGsimmobilized surfaces were in-situ characterized using Atomic Force Microscopy (AFM) (MultiMode 8, Bruker) equipped with liquid cell for temperature controlling at peak force tapping mode.

2.5. Cell Culture and Release Testing for PNGs-immobilized Cell Culture Plates L929 mouse fibroblast cells provided by cell library of Chinese Academy of Sciences were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% bi-antibiotics of penicillin/streptomycin at 37 oC and 5% CO2.26 After incubation on blank culture plate and PNGs-immobilized culture plate for 24 h at 37 oC, the cells were observed by optical microscope (IX73-U, Olympus) and imaged, then the culture plates were cooled down to 25 oC. The same cell areas were continuously monitored during the cooling process. The cells were re-seeded on the PNGs-immobilized culture plate for successive culture and then the controlled cell culture/release behavior was studied in the same way. The cells on the PNGsimmobilized culture plate at 37 oC and those at 25 oC were immediately freeze-dried for observing their morphologies at different temperatures by SEM.


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2.6. Cytotoxicity of the Nanogels and Nanogel-immobilized Surfaces The viabilities of cells incubated with nanoparticles of PNGs and PNGs@PDA, and those grown on PNGs-immobilized culture plates were analyzed by CCK-8 array.37,38 100 μL of complete medium containing L929 mouse fibroblast cells were added into a 96-well plate at a density of 1×104 cells/well. After overnight incubation, 100 μL of PBS solution containing concentrated nanoparticles was added to each well to achieve a final concentration of 0.0001, 0.001, 0.01 and 0.1 mg/mL. The control culture plate was added with 100 μL PBS solution without PNGs. The 96-well plate was placed in incubator (MCO-18AIC，SANYO) at 37 oC and 5% CO2 for 24 h. During this period, the solutions in wells were replaced with fresh medium twice to remove the PNGs completely, and then incubated for an additional 72 h in medium without PNGs. After the incubation, the medium was removed and 10 μL CCK-8 reagent was added in each well and incubated for 2 h. The absorbance was measured at 450 nm using microplate reader (Multiskan FC, Thermo Fisher), and the cell viability was calculated as the ratio of the absorbance of the tested samples to the control wells. The cell viability of PNGs-immobilized culture plate was studied in the same way as that for PNGs. Briefly, L929 mouse fibroblast cells with a density of 1×104 cells/well in 100 μL of complete medium were seeded into a blank 96-well plate and a PNGsimmobilized 96-well plate, then the plates were placed in incubator at 37 oC and 5% CO2 for 24 h, washed twice with fresh medium, and then incubated for an additional 72 h. After incubation, the medium was removed and 10 μL CCK-8 reagents were added in each well and cell viability was analyzed by microplate reader. In order to ensure the accuracy of the experimental data, the absorbance ratio of ten wells were measured and averaged for each test group.

2.7. Thermo-Responsive Gating Property Testing for PNGs-immobilized Porous Membranes


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The trans-membrane water fluxes (J) at 45 oC and 25 oC were recorded for investigation of thermoresponsive gating properties of PNGs-immobilized PC porous membranes.

The water flux

experiments were performed in a dead-end filtration apparatus under a constant static pressure of 6 cm water-column.39 The diameter of effective trans-membrane area was 37 mm, and the operation temperature was controlled by a thermostatic water bath at alternating temperatures of 25 oC and 45 oC for three cycles. At least five times of measurements were carried out to obtain an average value of the water flux. The PC porous membranes were immobilized with PNGs twice for studying the responsive rate of the gating function. The temperature of experimental water was reversibly and rapidly changed between 50 oC and 20 oC, and the water flux was recorded instantly.

3. RESULTS AND DISCUSSION 3.1. Thermo-responsive property of PDA-coated PNGs PDA-coated PNGs, which are referred as PNGs@PDA, are prepared by incubating the PNGs in DA solution at alkaline conditions (Tris-buffer, pH 8.5) for 12 h. The color of solution containing PNGs is lacte (Figure 2a, Insert), while that containing PNGs@PDA is black gray (Figure 2b, Insert), which shows the same color after freeze drying (Supplementary Figure S1). Figures 2a and 2b show the transmission electron microscopy (TEM) images of PNGs and PNGs@PDA, in which the PDA surface layer is confirmed to be well-formed on the outer networks of PNGs@PDA nanogels after modification (Figure 2b). Moreover, due to the coating of PDA surface layer, a 4.23 % rise of nitrogen in the PNGs@PDA compared with that of PNGs is confirmed by XPS spectra (Figure 2c). After coating with PDA layer, the hydrodynamic radius of PNGs@PDA is increased to ~300 nm at 20 oC comparing with that of ~280 nm of PNGs at the same temperature. Importantly, the PNGs@PDA nanogels still shows significant thermo-responsive volume phase transition


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around 32 oC (Figure 2d), which confirms that the PDA coating does not change the thermoresponsive characteristics of PNIPAM nanogels.

3.2. Stability of Immobilized Nanogels on Surfaces The pH condition could significantly affect the self-polymerization of dopamine,40 which would result in diverse stabilities of PNGs-immobilized surfaces. To investigate the self-polymerization of DA under different pH conditions, two pieces of glass substrates are immersed into aqueous solutions containing DA at pH 1 and pH 8.5 for 12 h, respectively (Figure 3a). The DA molecules possess few oxidation at pH=1, but are strongly oxidized at pH=8.5 according to the specific peaks of catechol at ~280 nm and quinone complex at ~403 nm of UV-vis spectra (Figure 3b).41 The O1s spectra of glass substrates shows that the content of quinone C=O is 26.27 % on surface in case of pH=8.5, which indicates the intensely oxidative self-polyerization of DA. However, few C=O is found at the situation of pH=1 (Figure 3c). The large amount of quinone C=O in PDA layer would exactly strengthen the interaction to the outer interface of PNGs, while the phenolic hydroxyl groups are preferred to connect with inorganic surfaces (e.g., Fe3O4, SiO2) via reversible coordination bonds (Figure 3d).31,40 Therefore, the strong interactions between oxidized DA and substrate surfaces would be the main reason for the stable immobilization of PNGs. The stability of the PNGs immobilization with or without assistance of polydopamine are studied by flow shearing tests, in which the shearing force acts on the immobilized PNGs on the inner surface of glass capillary (Figure 4a and Supplementary Figure S2). The SEM images in Figures 4b and 4c show the results of flow shearing tests on the inner walls of PNGS-immobilized capillaries prepared at pH 1-DA and pH 8.5-DA respectively. The PNGs immobilizated on the surface at pH 1-DA is unstable when flow shearing stress is higher than 0.05 N/m2 (Figure 4d). As expected, the PNGs immobilizated at pH 8.5-DA shows strong adhesion force, and the nanogels


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are firmly immobilized on surface even the boundary shear stress (τ0) reaches to 0.2 N/m2 (Figure 4c and 4d). Without the assistance of DA at pH=1, the PNGs also could be immobilizated on the inner surface of capillaries beacuse of the electrostatic interactions. The PNGs in solutions at varied pH values of 1 or 8.5 are all negatively charged with zeta potential of ~25 mV (Figure 4e), while the glass surfaces are positively charged in solution at pH 1 and negatively charged at pH 8.5.41 However, after the flow-shearing tests, nearly no PNGs are left on the inner surfaces of capillaries if the PNGs are immobilized without the assistance of polydopamine at both pH=1 amd pH=8.5 (Supplementary Figure S3 and Figure 3d).

The results confrim that the PNGs

immobilizated at pH=8.5 with the assistance of polydopamine is so stable although repulsive force is existing between the interfaces, which further demonstrates that it is the oxidation of DA molecules rather than the electrostatic interaction enabling the stable immobilization of PNGs on substrates. The surfaces of various flat substrates such as glass, polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), aluminum (Al) and stainless steel are immobilized with PNGs@PDA nanogels to study the thermo-responsive properties (Supplementary Figure S4). The water wettability of the PNGs-immobilized surfaces are studied by contact angles (CA) to comfirm the thermo-responsive change of hydrophilicity and hydrophobicity. For the uncoated substrates, all the water CA values of substrate surfaces decrease with increasing the temperature from 25 oC to 45 oC, due to the temperature-caused reduction of the liquid surface tension at the liquid/air interface (Figure 5a and 5c). After immobilied with PNGs@PDA nanogels, all the water CA of PNGs-immobilized surfaces increase as temperature increasing from 25 oC to 45 oC (Figure 5b and 5c). For instance, the water CA of PNGs-immobilized glass increases 12.5o from 77.3 ± 2o to 90.8 ± 2o when temperature changes from 25 oC to 45 oC (Figure 5b and c). The results show that all the PNGs-immobilized surfaces exhibit significant thermo-responsive performances.


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3.3. Thermo-Responsive Cell Culture Plates Fabricated by Immobilizing PNGs on Commercial Plates PNGs are immobilized on the surfaces of commercial cell culture plates to achieve thermocontrolled culture and release of cells. For commercial tissue culture plate, L929 mouse fibroblasts strongly adhere on the surface after 24-hour culture at 37 oC, and cannot be detached by simply lowering the temperature even with strong water rinsing (Supplementary Figure S5).

By

immobilizing PNGs on the surface of commercial cell culture plate, the nanostructured surface becomes hydrophobic and stiff at 37 oC (above VPTT) (Figure 6a-c), which are beneficial to cell culture and adhesion.42,43 The average elastic modulus of shrunken PNGs is ca. 110 MPa (Figure 6c). Therefore, the spindle-liked fibroblasts spread well on the nanostructured surface immobilized with PNGs at 37 oC (Figure 6d1 and Inset). After cooling to 25 oC, the PNGs-immobilized surface becomes hydrophilic and ultrasoft (Figure 6b), and the average elastic modulus becomes 0.235 MPa (Figure 6c). Thus, the cells turn to sphere shapes within 30 min (Figure 6d2 and Inset). Subsequently, most of the cultured cells could be removed by rinsing with water (Figure 6d3). Moreover, as shown in Figure 6d4-d6, the second adhesion/detachment cycle of fibroblasts can be also triggered by temperature changes on the same washed PNGs-immobilized plate due to the superior stability of the PNGs immobilizing (Supplementary Figure S6), where the PNGsimmobilized surfaces still show high cell removal rate over than 95 % in successive culture cycles (Figure 7a). To reveal the suitability of the thermo-responsive cell culture plates fabricated by our strategy for further biological applications, the cytotoxicity of PNGs, PNGs@PDA and PNGsimmobilized substrate are studied. The cell viabilities are over 100 % tested by Cell Counting Kit-8 (CCK-8) array (Figure 7b), which indicate the materials in our strategy have good


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biocompatibility. The results confirm that thermo-responsive nanostructured surface with superior stability can be applied for cell culture with high cell-detaching efficiency and satisfactory biocompatibility.

3.4. Thermo-Responsive Gating Membranes Fabricated by Immobilizing PNGs on Porous Substrates To demonstrate the immobilization of PNGs on porous surfaces with the proposed strategy, thermo-responsive gating membranes are fabricated by immobilizing PNGs on commercial polycarbonate (PC) porous membranes with straight pores of 1.2 μm in diameter. SEM images show the PNGs are evenly immobilized not only on the outer surfaces but also on the inner surfaces of pores (Figure 8a,b). With increasing the immobilizing run, the number of immobilized PNGs increases (Figure 8a,b).

The surface morphologies of PC gating membrane fabricated by

immobilizing PNGs twice, which are studied by in-situ AFM at peak force tapping mode in water at 30 oC (below VPTT) and 40 oC (above VPTT), are shown in Figure 8c,d. At a temperature lower than VPTT, the PNGs are in the swollen state, leading to decreased pore size (Figure 8c). At a temperature higher than VPTT, the pore size increases due to the shrunken conformation of PNGs (Figure 8d). The height profiles show the dimension of nanogels decreases while the pore dimension increases upon increasing temperature (Figure 8e,f).

The PNGs-immobilized

membranes exhibit excellent thermo-responsive gating property and reversibility (Figure 9a,b). As shown in Figure 9c, for the blank PC membrane, the water flux (J) is 364 kg h-1 m-2 at 25 oC under a constant static pressure of 6 cm water-column, which slightly increases with increasing the temperature due to the thermo-induced decrease of viscosity. By contrast, the water flux of PNGsimmobilized membranes at 25 oC is as low as 19 kg h-1 m-2 under the same condition of driving


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force (Figure 9c). With increasing the temperature over the VPTT, the immobilized PNGs significantly shrink; as a result, the water flux across the PNGs-immobilized membrane increases remarkably. For instance, the water flux of the membrane immobilized twice with PNGs is 428 kg h-1 m-2 at 45 oC under the same condition of driving force, and the gating coefficient (R) that defined as the ratio of water flux across the membrane at 45 oC to that at 25 oC is as large as 23 (Supplementary Figure S7). The thermo-responsive gating behavior is reversible (Figure 9c,d,e), indicating the superior stability of the immobilizing. The responsive rate of the gating by immobilized PNGs is super-fast, and the trans-membrane water flux can change within 10 s by altering the temperature (Figure 9d,e) and vice versa. After being immersed in water for three months, the PNGs are still stably immobilized on the pore surface of the membrane (Supplementary Figure S8). This demonstration confirms that thermo-responsive gating membranes can be effectively achieved by immobilizing PNGs nanogels on the porous substrates with assistance of polydopamine, which provides a facile and efficient route for fabricating smart gating membranes.

4. CONCLUSION In summary, the nanostructured thermo-responsive surfaces can be engineered by stably immobilize PNGs nanogels on 2D or 3D substrate surfaces with assistance of polydopamine. In the proposed strategy, the PDA layer on the PNGs does not affect the thermo-responsive property of PNGs, and the superior stability of the PNGs immobilized on substrates is resulted from the strong interactions formed between oxidized DA and substrate surfaces. With the proposed strategy, nanostructured thermo-responsive surfaces on 2D and 3D substrate surfaces have been demonstrated to respectively achieve responsive cell culture plates and smart gating membranes. Because the immobilized PNGs exhibit significant thermo-responsive phase transition between the


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swollen, hydrophilic and soft state and the shrunken, hydrophobic and stiff state upon environmental temperature change across the VPTT, the PNGs-immobilized cell culture plates exhibit excellent cell culture/release performances, and the PNGs-immobilized porous membranes exhibit reversible and rapid gating performances. The proposed strategy exhibits versatile utility to generate universal responsive nanostructured surfaces with superior stability for myriad applications by introducing different kinds of responsive functional nanogels, and may open up new fields of applications for responsive nanogel-immobilized surfaces.5,44

ASSOCIATED CONTENT Supporting Information Digital photographs of freeze-dried samples of blank PNGs and PNGs coated with PDA for 12 h; Schematic illustration of the apparatus for generating shearing force caused by fluid flow on the immobilized PNGs on the inner surface of glass capillary; SEM images of the inner surfaces of PNGs-immobilized capillaries prepared without assistance of DA after rinsing with flow rate ranging from 0 to 5000 μL/h; SEM images of PNGs immobilizing on different substrates; Microscopy images of L929 mouse fibroblast cells on commercial cell plates at 37 oC, 25 oC, and after strong rinsing with PBS buffer at 25 oC; SEM image of the surface of thermo-responsive cell plate after second use; Effect of PNGs immobilizing runs on the gating coefficient (R) of asprepared thermo-responsive gating membranes; SEM image of cross-section view of a membrane that fabricated by immobilizing PNGs thrice and has been immersed in water for three months before observation. This material is available free of charge via the Internet at http://pubs.acs.org.


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AUTHOR INFORMATION Corresponding Authors *Address correspondence to [email protected] (Z.L.) or [email protected] (L.Y.C.). Author Contributions L.Y.C., Z.L. and L.Z. conceived and designed the study. L.Z. performed the experiments. All authors discussed the results and contributed to the data interpretation. L.Y.C., Z.L., and L.Z. wrote the manuscript and all authors commented on the manuscript.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT The authors gratefully acknowledge support from the National Natural Science Foundation of China (91434202, 21506127), the Program for Changjiang Scholars and Innovative Research Team in University (IRT15R48) and State Key Laboratory of Polymer Materials Engineering (sklpme2014-1-01).

ABBREVIATIONS DA, dopamine; PDA, polydopamine; PNIPAM, poly(N-isopropylacrylamide); VPTT, volume phase transition tempearature; PNGs, PNIPAM nanogels; TEM, transmission electron microscopy;


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XPS, X-ray photoelectron spectroscopy; AFM, atomic force microscope; SEM, scanning electron microscope. DLS, dynamic light scattering; CA, contact angles.


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FIGURES

Figure 1. Schematic illustration of the nanostructured thermo-responsive surfaces engineered via stable immobilization of smart nanogels with assistance of polydopamine. a) Immobilization under acid condition of pH 1. b) Immobilization under alkaline condition of pH 8.5. c,d) Thermoresponsive cell culture plate fabricated by immobilizing thermo-responsive nanogels on commercial plate for controlled culture and release of cells. e,f) Thermo-responsive gating membranes fabricated by immobilizing thermo-responsive nanogels on porous substrates.


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Figure 2. Characterization of PNGs and PNGs@PDA. a,b) TEM images of PNGs (a) and PNGs@PDA (b) and corresponding digital photographs of particle solutions (inserted). The scale bars are 500 nm. c) XPS spectra of PNGs and PNGs@PDA. d) Temperature-dependent hydrodynamic radii of PNGs and PNGs@PDA in water.


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Figure 3. Immobilizaiton mechanism. a) Schematic illustration of the modification of glass substrates by immersing in DA solution at pH=1 (a1) and pH=8.5 (a2) for 12 h. b) UV-Vis spectra of aqueous DA solutions after storage at pH=1 for 12 h and at pH=8.5 for 12 h. The insets are the digital photographs of DA solutions with different pH values after 12 h. c) High-resolution of O1s spectra of blank glass substrate prepared by immersing in pure water for 12 h (c1), PDA-coated glass substrate prepared by immersing in 2 mg/mL DA solution at pH=1 for 12 h (c2), and PDAcoated glass substrate prepared by immersing in 2 mg/mL DA solution at pH=8.5 for 12 h (c3). d) Schematic illustration of the functional groups on surface of glass substrates after modified in DA solution at pH=8.5 (d1) and pH=1 (d2), in which the glass surface modified at pH=8.5 shows much more o-quinone and primary amines, indicating the strong oxidation of DA at pH=8.5.


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Figure 4. Immobilizaiton stability of PNGs. a) Schematic illustration of the shearing force caused by fluid flow acting on the immobilized PNGs. b,c) SEM images of the inner surfaces of the PNGsimmobilized capillaries prepared at pH 1-DA (b) and pH 8.5-DA (c) after flow-shearing test under different flow rates. d) Statistic number of PNGs on the inner surfaces of capillaries after rinsing with water at different flow rates. e) Zeta potential values of PNGs under different pH and DA conditions.


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Figure 5. a,b) Contact angle measurements at 25 oC and 45 oC for different substrates before (a) and after (b) immobilizing PNGs. c) The differences between contact angles on the virgin and PNGs-immobilized surfaces when temperature changing from 25 oC to 45 oC.


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Figure 6. Thermo-responsive cell culture plates fabricated by immobilizing PNGs on commercial plates. a,b) Elastic modulus maps of PNGs-immobilized surface at 37 oC (a) and 25 oC (b) obtained by in-situ measurement in Derjaguin-Müller-Toporov (DMT) mode. c) The elastic modulus of the PNGs at 37 oC along the section line (L-1) in (a) and that of the PNGs at 25 oC along the section line (L-2) in (b), in which L-1 and L-2 are located in the same position. d) Microscopic images of L929 mouse fibroblast cells on the same PNGs-immobilized cell plate in first-run culturing (d1d3) and second-run culturing (d4-d6), in which the inserted SEM images in (d1) and (d2) show the cells’ spreading shape at 37 oC (d1) and spherical shape at 25 oC (d2) on the thermo-responsive cell culture plates. The scale bars are 500 nm in (a,b), 200 μm in (d), and 10 μm in the inserted SEM images in (d1,d2).


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Figure 7. a) Quantitative characterization of the adhesion and release of cells on the commercial and PNGs-immobilized cell culture plates at different temperatures, which indicates that more than 90% of the cells can detach from the PNGs-immobilized surface after first and second incubation by gentle rinsing at 25 oC, instead, few cell can be removed from control cell plate surface even by strong rinsing. b) Cell viability of L929 mouse fibroblast cells after 72 h culture with blank PNGs and PNGs@PDA at different concentrations, and PNGs-immobilized substrate.


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Figure 8. Thermo-responsive gating membranes fabricated by immobilizing PNGs on porous substrates. a,b) SEM images of surface (a) and cross-section (b) views of blank PC membrane (a0, b0) and PNGs-immobilized PC membranes by immobilizing PNGs once (a1, b1), twice (a2, b2) and thrice (a3, b3). c,d) AFM images of PNGs-immobilized PC membranes (fabricated by immobilizing PNGs twice) at 30 oC (c) and 40 oC (d) in pure water, in which AFM 3D images of (c2) and (d2) are locally enlarged from (c1) and (d1) respectively. e) The height profiles across the PNGs immobilized on the membrane surface at 30 oC (marked as 30-1 in (c1)) and 40 oC (marked as 40-1 in (d1)). f) The height profiles across the membrane pores immobilized with PNGs at 30 oC (marked as 30-2 in (c1)) and 40 oC (marked as 40-2 in (d1)).


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Figure 9. Test for the thermo-responsive ability of the gating membrane immobilized with PNGs. a) Schematic illustration of the testing apparatus. b,c) The instantaneous liquid height in graduated tube is recorded by camera immediately after adding with pure water at temperature of 20 oC (b) and 50 oC (c). The water is dyed by methylene blue for eye catching. c) Water flux (J) values of the membranes with immobilizing runs of PNGs from 0 to 3 at 25 oC and 45 oC under static pressure of 6 cm water column. d,e) The rapid change of water flux across the PNGs-immobilized PC membrane (fabricated by immobilizing PNGs twice) when the temperature is changed from 50 oC to 20 oC (d) and reversely changed from 20 oC to 50 oC (e).


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