Self-Assembled Two-Dimensional Thermoresponsive Microgel Arrays

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Self-Assembled Two-Dimensional Thermoresponsive Microgel Arrays for Cell Growth/Detachment Control Yongqing Xia,† Xinlong He,† Meiwen Cao,† Xiaojuan Wang,† Yawei Sun,† Hua He,† Hai Xu,*,† and Jian Ren Lu*,‡ †

State Key Laboratory of Heavy Oil Processing and Centre for Bioengineering and Biotechnology, China University of Petroleum (East China), Qingdao, 266555, China ‡ Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Schuster Building, Oxford Road, Manchester, M13 9PL, U.K. S Supporting Information *

ABSTRACT: Monodisperse poly(N-isopropylacrylamide-styrene) (PNIPAAmSt) microgels with different St/NIPAAm ratios have been synthesized via a one-step surfactant-free emulsion polymerization process. The resulting microgel dispersions were used to fabricate 2D arrays on the surface of silicon wafers/glass coverslips through dip coating. The thermal responsiveness of the PNIPAAmSt microgel arrays was examined by spectroscopic ellipsometry and the results unraveled that the thermoresponsive behavior of the arrays was highly consistent with the microgels dispersed in the bulk, showing high dependence on the content of styrene. The structure of the films varied from nonclose-packed 2D arrays to close-packed 2D arrays, depending on both properties of the microgels and array fabrication conditions. When the weight ratio of styrene was below 40%, the microgel arrays demonstrated effective control for cell growth and detachment across their volume phase transition temperatures (around 28 °C). The extent of swelling of the microgels was the key factor to determine whether the cells could detach from the film easily. For the rather close-packed 2D arrays prepared by the same kind of PNIPAAmSt microgels, the gaps between microgel particles showed no obvious effect on the rate of cell detachment.

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INTRODUCTION Submicron colloids are capable of self-assembling into two- or three-dimensional (2D or 3D) periodic arrays. These ordered arrays are useful materials for optical devices,1,2 biosensors,3 and biomedical applications.4 In most cases, hard particles such as silicon, polystyrene, and poly(methyl methacrylate) particles, are used to form periodic arrays due to their easy availability.5,6 However, rather few studies have been made for the surface self-assembly of soft microgel particles.7,8 Poly(N-isopropylacrylamide)-carrying (PNIPAAm) microgels are thermoresponsive particles that exhibit a volume phase transition temperature (VPTT) in water with a sharp phase transition point in the physiologically relevant range around 30 °C. The synthesis, characterization, and applications of the PNIPAAm microgels have been extensively studied over the past few years.9−12 Compared with hard particles, it is difficult to fabricate ordered 2D arrays from soft PNIPAAm © 2014 American Chemical Society

microgels. In most cases, self-assembly of 2D microgel arrays suffers from 3D spoiling and even if this issue is kept under control, the 2D ordering may become lost upon drying. Nevertheless, formation of 2D microgel arrays has already been explored for different applications.13−16 Surface micropatterning offers a convenient approach for exploring fundamental cell-biomaterial interactions, in particular, allowing the effect of some fundamental features such as surface patterning on cell adhesion, proliferation, migration and differentiation to be examined.17−25 However, to the best of our knowledge, few studies explored how micropatterned surfaces of thermoresponsive microgels could be utilized to control cell detachment or cell sheet harvesting. Most studies on the Received: July 22, 2014 Revised: September 18, 2014 Published: October 13, 2014 4021

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000 Da) for 3 days against water with frequent water change until the dialysate conductivity was close to that of pure water (around 1 μS/cm). For all synthesis processes of microgel particles, the MBA was kept constant at 0.041 g. The total weight of NIPAAm and styrene monomers was kept constant at 4.0 g, but the ratio was varied according to the composition designated. The resultant microgels were referred to as PS25, PS40, PS55, and PS70, meaning that the fed styrene in the total monomers was 25 wt % (26.5 mol %), 40 wt % (42.0 mol %), 55 wt % (57.1 mol %) and 70 wt % (71.7 mol %), respectively. The actual percentage of polystyrene in the corresponding microgel after purification was about 24.5 ± 0.4 wt % (26.1 ± 0.3 mol %), 37.7 ± 0.2 wt % (41.1 ± 0.2 mol %), 67.4 ± 0.4 wt % (69.2 ± 0.3 mol %), and 79.4 ± 0.3 wt % (80.8 ± 0.4 mol %), respectively, as determined from elemental analysis (Thermo, Flash EA1112 series). Four replicate runs were produced for each sample with furnace temperature at 900 °C. Microgel Characterization in Bulk Phase. The hydrodynamic diameters and thermoresponsive behaviors of the PNIPAAmSt microgels were characterized by dynamic light scattering (DLS, Zetasizer Nano instrument from Malvern Instruments Ltd., with the detector positioned at the scattering angle of 173°) in the temperature range of 20−50 °C. Each microgel dispersion (0.01 wt % microgel in water) was heated steadily and the microgel size determined every 2 °C by letting the microgel dispersion equilibrate at each temperature for 10 min. At each temperature, five consecutive runs were performed and each run composed of 15 individual radius measurements using a 10 s integration time. Transmission electron microscopy (TEM, JEM-2100UHR, JEOL) and cryogenic temperature TEM (cryo-TEM, JEM1400, JEOL) were also used to characterize the structures of PS25 microgels. 2.3. Preparation and Characterization of PNIPAAmSt Microgel Films. Film Preparation. Surface self-assembly of the PNIPAAmSt microgels on silicon wafers or glass slides was achieved via a dip-coating process. To obtain different interparticulate distances within the microgel monolayers, the concentrations of the microgel dispersions in water were varied in the range of 0.1−1.0 wt % and the withdrawing speeds were varied in the range of 1−10 μm/s, respectively. After dip coating, the silicon wafers or glass slides were annealed in an oven set to 120 °C for 2 h. Film Characterization. Scanning electron microscopy (SEM, S-4800, Hitachi) was used to help examine the resulting microgel films, with sample surfaces coated with a thin Au layer to increase the contrast and quality of the images. The AFM images of the dried PNIPAAmSt microgel films coated on silicon wafers were acquired from a Nanoscope IVa system (Digital Instruments, Santa Barbara, CA, U.S.) in the tapping mode under laboratory conditions. Images of scan size of 10 μm were obtained simultaneously at a scan rate of 1 Hz with a matrix of 512 × 512 data points along the xy plane. AFM images of the PNIPAAmSt microgel films in water environment were acquired by contact mode at room temperature (about 20 °C). The thermoresponsive changes of the microgel films were determined using a variable angle spectroscopic ellipsometry (Jobin-Yvon UVISEL). Ellipsometry is a technique that has been widely used to determine the thickness (L) and the refractive index (n) of an adsorbed or deposited thin film on an optically flat surface. The principle of the technique is described elsewhere.31,32 In our work, the L and n values for the microgel

thermoresponsive cell detachment have so far utilized linear PNIPAAm polymeric chain brushes to fabricate different micropatterned surfaces.26−28 In our previous work, we have found that CaCO3 mineralized poly(N-isopropylacrylamide-acrylic acid) microgels can form ordered 2D arrays, and NIH 3T3 cells can grow and then detach from the arrays via temperature stimuli.29 Inspired by the results, polystyrene, a hydrophobic polymer with good biocompatibility was introduced into the PNIPAAm to form a new hybrid PNIPAAmSt microgel.30 We then demonstrated that the copolymerization of styrene could also mediate the adhesion and detachment behaviors of NIH 3T3 cells on the PNIPAAmSt microgel films by temperature stimuli.30 The incorporation of styrene can not only alter the amphiphilicity of the microgels, but also tune their physicochemical nature, directly impacting their self-assembling ability and cytocompatibility. Thus, in this work, we have focused on studying the influence of styrene content on the self-assembly of the microgels and the effect of corresponding assembled 2D arrays on the efficiency of cell growth and detachment. The results showed that while cells could grow well on all microgel arrays formed with a wide range of styrene content, those containing a high content of styrene did not allow easy cell detachment due to the lack of obvious thermoresponsive changes upon cooling. For PNIPAAmSt microgels containing less than 40 wt % of styrene, 3T3 fibroblast cells could grow well and detach over 90% from their arrays, indicating the significant role played by the thermoresponsive swelling. Furthermore, for the microgel films fabricated in this study, the gaps between the microgels showed no obvious effect on the rate of cell detachment.

2. MATERIALS AND METHODS 2.1. Materials. All the chemicals were obtained from SigmaAldrich. N-isopropylacrylamide (NIPAAm) was purified by recrystallization from a toluene/hexane mixture (1:3) and dried in vacuum. Styrene (St) was purified by distillation under reduced pressure, ammonium persulfate (APS) was purified by recrystallization from water. N,N-methylene bis(acrylamide) (MBA) was used as received. All water used in this experiment was processed by Milli-Q system (Milli-Q Advantage A10 Water System Production Unit). Silicon wafer cuts (15 mm × 20 mm, of ⟨111⟩ orientation and bearing a native surface oxide layer of 1−2 nm, from Compart Technology UK) and glass coverslips (20 mm × 20 mm, from Sail Brand, China) were immersed into piranha solution (H2O2:H2SO4 = 1:3 by volume) at 90 °C for 1 h, followed by abundantly rinsing with the processed ultrapure water from the Milli-Q system. Cell culture plates (6-well plates, from Corning) were use as received. 2.2. Preparation and Characterization of PNIPAAmSt Microgels. Microgel Preparation. PNIPAAmSt microgels were prepared by surfactant-free precipitation polymerization. After dissolving appropriate amounts of NIPAAm, MBA and styrene monomers in 190 mL of water, the reaction mixture was transferred to a four-necked round-bottom flask equipped with a condenser and a nitrogen inlet, and then heated to 70 °C under a gentle stream of nitrogen. After 1 h, 0.120 g of an initiator (APS) was dissolved in 10 mL of water (oxygen free) and added to the flask to initiate polymerization. The reaction was continued for 4 h while keeping the reaction in a nitrogen environment by continuous N2 purging. Following the synthesis the microgels were purified by dialysis (cutoff 11 4022

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layers were derived from the (ψ, Δ) spectra using a Cauchy fitting model. In this fitting procedure, only L and n were treated as fitting parameters dependently. The measurement processes were performed following the protocols as previously reported.33 In brief, each measurement was first carried out in air and then in water using a specially designed liquid cell, with the oxide layer of the silicon wafer being first measured. Its thickness and composition were assumed to remain unchanged subsequently. The liquid cell facilitating the measurements at the solid/water interface had a pair of fused quartz plates (0.2 mm thick) as windows and the incoming and exiting beams were aligned perpendicular to the windows. Changes in film thickness and refractive index as a function of temperature were recorded, and the microgel film was allowed to equilibrate at each temperature for ∼1.5 h prior to each measurement. The process was recycled and repeated to examine the extent of film reversibility in its expansion and contraction. The experimental data were analyzed using DeltaPsiII software provided by JobinYvon. Surface wettability of the microgel films was determined by a static contact angle measurement instrument (DSA100; KRÜ SS, Hamburg, Germany). Water droplets were placed onto the surfaces at 25 or 38 °C to examine the wettability using a sessile drop method. Data were averaged from five separate experiments for each surface and shown with their standard deviations. 2.4. Cell Culture. The glass coverslips dip coated with PNIPAAmSt microgel films were sterilized for 2 h by UV light and then transferred into 6-well tissue culture plates for subsequent use. NIH 3T3 cells (20000 per well) were seeded uniformly on PNIPAAmSt microgel dip coated coverslips and cultivated in DMEM medium containing 10% FBS at 37 °C and 5% CO2. After being cultured for 48 h, 200 μL of 5 mg/mL MTT solution in PBS was added to each well and incubated at 37 °C for 4 h. After removal of the medium carefully, 1 mL of DMSO was added to each well to dissolve the formazan crystals for 10 min, and then 100 μL of the DMSO solution transferred to 96 well plate. The absorbance at 490 nm was determined using a microplate autoreader (Molecular Devices, M2e). Each condition was assayed in duplicate in three different experiments. For cell detachment, 4 °C fresh DMEM was added to replace the old culture medium rapidly, and then the plate was left under ambient temperature ready for microscopic monitoring of the detachment process. It took about 2 min for this process to complete. The cell detachment process at a fixed spot was monitored using a phase contrast inverted microscope (Leica, DMI3000, Germany) at ambient temperature (about 20 °C), and detached cells were counted at the same field, with the starting time being defined as 2 min after the start of the culture medium replacement.

improve the bioadhesiveness of the PNIPAAm copolymer films.38,39 Two recent studies have demonstrated the development of PNIPAAm microgel films aiming at controlling cell adhesion and detachment.40,41 Polystyrene is a typical hydrophobic polymer widely used in cell culturing. In this work, styrene was introduced via copolymerization to tune the properties of the microgels prepared, including their amphiphilicity and physiochemical properties. In our work, we have demonstrated that PNIPAAm microgels are too soft to self-assemble into wellordered 2D arrays. Such microgel films could only enable some 55% cells to detach (Figure SI1, Supporting Information). Thus, the broad aim of the work reported here was to examine how the amount of styrene incorporated affects the thermoresponsiveness and self-assembling ability of the newly synthesized microgels, and the subsequent impact on cell growth and detachment from the microgel films formed. 3.1. Characterization of PNIPAAmSt Microgels in Bulk Phase. DLS experiments were first used to characterize the PNIPAAmSt microgels with respect to their size distribution and swelling performance. Figure 1 shows the swelling curves of

Figure 1. Temperature dependence of microgel diameters in aqueous phase with different styrene contents as determined by DLS, showing that the thermoresponsive swelling of the microgels diminishes with increasing styrene content. The numbers in the names of the PS microgels denote the percentages of polystyrene in the starting composition of the synthesis.

PNIPAAmSt microgels with different styrene contents by presenting their diameter changes. The diameters are the peak values taken from each size distribution measured by DLS. Generally, they show an increasing trend with decreasing temperature, indicating the swelling of the PNIPAAmSt microgels. With increasing the percentage of styrene, the swelling capacity of the microgels decreases, especially for PS70, where no significant shrinkage of the microgels was observable with increasing temperature. Thus, as the content of styrene increased, the microgel particles behaved more and more like polystyrene hard spheres. The TEM and cyro-TEM images of PS25 microgels showed no obvious core−shell structure from the one-pot polymerization procedure (Figure SI2) . Thus, these microgel particles must have a broadly uniform composition and spherical structure. For thermoresponsive microgels, their VPTT were around 28 °C, lower than pure PNIPAAm microgels. The differences may arise from the copolymerization of the hydrophobic monomers. The results as shown in Figure 1 indicate that the PNIPAAmSt microgels as synthesized offer a wide range of

3. RESULTS AND DISCUSSION Thermoresponsive film surfaces prepared by surface grafting of linear PNIPAAm polymers have been used by Okano et al. and others for harvesting different cells and cell sheets.34,35 However, these PNIPAAm polymers may not be optimal for cell attachment and growth due to their rather hydrophilic nature. Alternative methods have been developed to improve the performance of the PNIPAAm coatings.36,37 It was reported that the bioadhesiveness of the PNIPAAm films could be improved by copolymerizing with a more hydrophobic monomer. For example, Selezneva and Rollason reported the successful copolymerization with N-tert-butylacrylamide to 4023

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swelling in response to the ratio of NIPAAm to St. Under the swollen state around 20 °C, the swollen diameter for the microgels with a nominal 25 wt % styrene (PS25) is peaked around 630 nm, compared to that of 380 nm obtained for the microgels with a nominal 70 wt % styrene (PS70), showing a drastic decrease of the swollen size with increasing styrene content. In contrast, microgel sizes in the dehydrated state around 38 °C show a smaller but clearly opposite trend of changes. PS70 microgels have the dehydrated diameters close to 370 nm, as compared to those around 300 nm for PS25 microgels, showing that as the styrene content increasing, the dehydrated microgel sizes increase. Thus, as the styrene content becomes high, the diameters do not change much over the same temperature range. This feature is further demonstrated in Figure SI3. 3.2. Fabrication of 2D Arrays of PNIPAAmSt Microgel Films. Microgels can be deposited on substrate surfaces such as glass, plastics, gold, and silicon oxide by a variety of methods including dip coating and spin coating. Similar to the practice often adopted in the control of hard spheres, microgels could also be assembled into ordered arrays on substrate surfaces. In most cases, self-assembly onto a substrate surface leads to the formation of close-packed microgels, but subsequent drying often reveals nonclose packed arraying with varying gaps between surface confined microgel particles.42−45 Under these conditions, the diameters of the microgels can be deduced by the center-to-center distances (Dctc) of the neighboring microgels. For example, from the cationic poly(styrene-co-Nisopropylacrylamide) core−shell microgel particles prepared, Lu et al. found that the microgels formed 2D ordered arrays upon assembly on a highly negatively charged mica surface46 and that the distances between the microgel particles after drying were larger than the hydrodynamic diameters measured by DLS. Kawaguchi et al. studied their 2D arrays fabricated using PNIPAAm-carrying hairy particles by air-drying and found that the interparticle distances were affected by the graft chain length of the hairy particles attached.47 In both of these cases, the authors proposed that when the microgel particles approached the substrate surface and became adsorbed, their positions were fixed on the substrate. Thus, interparticulate center-to-center distances were influenced by charge repulsion and steric protrusion, resulting in values greater than the hydrodynamic diameters as measured in bulk phase. Because the microgels were soft, they became deformed upon contact with substrate and filled some of the gaps between them. We have previously shown that the lateral dimensions of surface confined soft PNIPAAm microgels (dried under an infrared lamp before observation) were greater than their diameters measured by DLS in bulk phase at 38 °C,29 also consistent with the deformations as suggested above. Scheme 1 shows a schematic outline of the dynamic interfacial process, starting with the formation of an initial wet microgel film onto the substrate surface upon withdrawing the substrate from the microgel dispersion (Step 1). Depending on softness and hydration, microgels become flattened on the substrate surface (Step 2), showing apparently greater lateral dimensions (DAdb) than the sizes (DDLS) as measured in bulk phase by DLS. The surface confined microgel particles tend to shrink upon drying to different extent, but their focal centers do not tend to move, thus leaving rather uniform gaps between them (Step 3). The drying process forces the soft particles to become further confined, forming a nonclosed packed arraying. As indicated in Scheme 1, the center-to-center distance (Dctc) does not change

Scheme 1. Schematic Illustration of Mechanistic Progress of Self-Assembly of Soft Microgels onto Substrate during Water Evaporation

much, but the value is mostly larger than the respective microgel diameter in swollen state in bulk phase. Thus, at temperatures below VPTT, Dctc is affected by properties of the microgels including surface charges and softness. This explains why in almost all PNIPAAm microgel self-assembly cases, the Dctc values were dictated by the initial surface 2D packing and subsequently remained rather constant.7,8,13,14,43,45,46,48 Within this limiting length range, the interparticulate gaps upon surface assembly can vary, depending on the hardness, surface characteristics, and extent of hydration of the microgels. Figure 2a−d shows AFM topographic images of the PS25 microgels and their corresponding cross-section profiling (e−h) in the dry and swollen states. To make sure the footprint of an individual microgel could be determined clearly, samples a and b were prepared by spin coating 0.5 wt % microgel dispersion at 1000 rpm, then dried at 120 °C for 2 h. The glass transition temperature (Tg) of polystyrene is about 80−110 °C.49 When PNIPAAmSt microgels were heated up to 120 °C, they became softened, and the flexibility of the polymer chains could well help strengthen their interactions with the substrate, resulting in strong surface immobilization. Immersion of the dry sample surface (Figure 2a) into water (Figure 2b) led to a fast increase in the height of the microgels from 160 nm (Figure 2e) to 232 nm (Figure 2f), showing fast swelling. The swelling of the microgels also occurred in the lateral direction, increasing the footprint diameters from less than 500 nm for the dry microgels (Figure 2e) to more than 900 nm for the swollen ones (Figure 2f). When dip coating was performed using 1 wt % microgel dispersion with a withdrawing speed of 5 μm/s, highly ordered 2D arrays could also be formed (Figure 2c). Figure 2d shows the AFM imaging of the 2D film formed at the swollen state and at ambient temperature. The cross-section profiling (Figure 2h) shows a reduced relative vertical distance about 120−160 nm, but the vertical distance to the bottom surface is about 200−220 nm, similar to the height measured in Figure 2f. This observation suggests that the space was filled between neighboring microgels, that is, the adjacent microgels were interpenetrated. More importantly, after immersion into liquid phase, the surface particle number density was reduced compared to that in air. Two main factors contributed to the loss of the microgels. First, because the swelling increased the 4024

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Figure 2. AFM topographic images and cross section profiling of PS25 microgels coated on silica wafer in the dry and swollen states: spin coated from the microgel concentration of 0.5 wt % at dry state (a,e) and swollen state (b,f); ordered arrays of the same microgels prepared by dip coating at the microgel concentration of 1.0 wt % (the withdrawing speed at 5 μm/s) at dry state (c,g) and at swollen state (d,h).

volume of the microgels, the space occupation will force some microgels detach from surface. Second, the AFM measurements in contact mode could also remove some of the microgels.50 In fact, with the increase of the cycle of scanning, more microgels were lost from the observation area (Figure SI4a-d). To quantify the extent of particle loss, we counted the microgel particles within an area of 2800 μm2 from the PS25 microgel film using AFM imaging over an immersion period (in water) of 6 days (analyzed by ImageJ software). This study revealed about 10% loss of the surface immobilized microgel particles over the period (Figure SI4e). To advance this understanding, we have further investigated the concentration dependent surface assembly of the microgels and characterized the surface film morphological structures using SEM. Figure 3 shows typical SEM images of the 2D arrays formed by dip-coating PS25 microgel dispersions onto silicon wafer, showing a clear trend of increase of surface packing density with concentration over the range of 0.25−1.0 wt % studied. When the withdrawing speed was reduced to 1 μm/s, 2D close-packed arrays were obtained. As withdrawing speed increased toward 5 μm/s, the dried microgels formed

Figure 3. SEM images of 2D microgel arrays formed by dip-coating aqueous dispersions of PS25 microgels under different microgel concentrations (0.25, 0.5, 1 wt %) and different withdrawing speeds (1, 5, 10 μm/s).

4025

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Figure 4. SEM images of 2D microgel arrays formed by dip-coating 1.0 wt % aqueous dispersions of PNIPAAmSt: (A) PS25, (B) PS40, (C) PS55, and (D) PS70 at a withdrawing speed of 5 μm/s and (a) PS25; (b) PS40; (c) PS55 and (d) PS70 at a withdrawing speed of 10 μm/s. All the scale bars were at 20 μm and those placed in the insets were 2 μm for A and a and 5 μm for the rest of the insets.

Figure 5. Changes in the thickness and refractive index (readings at λ = 600 nm) of the microgel films prepared from (a) PS25; (b) PS40; (c) PS55 and (d) PS70 measured as a function of temperature over 20−40 °C. The films were prepared by dip-coating 1.0 wt % microgel dispersions at a withdrawing speed of 5 μm/s. The results show a trend of changes of microgel diameters with increasing temperature and content of styrene, consistent with the changes measured from bulk microgel dispersions by DLS as shown in Figure 1.

contents. Figure 4 shows the surface morphological features of the microgel films prepared from PS25 to PS70 under the two withdrawing speeds. As evident from the images shown in Figure 4, when the withdrawing speed is 5 μm/s, PS25 and PS40 formed microgel monolayers, with no evidence of any building up into the 3D morphologies (Figure 4A,B). However, PS55 and PS70 showed some evident building up of 3D morphologies (Figure 4C,D). There is thus a clear trend of increase in the packing density with increasing styrene content. Also, as the styrene content increases, the microgel particles tend to form close-packed and well-ordered arrays in small area. PS55 and PS70 behaved like pure PS hard particles in surface packing performance.48,51,52 At the withdrawing speed of 10 μm/s, monolayers formed from all four types of microgels. While PS25 and PS40 formed ordered nonclose-packed arrays with Dctc values of 585 ± 250 and 447 ± 40 nm, respectively, PS55 and PS70 formed rather disordered arrays, showing a deteriorating influence with increasing the styrene content.

ordered but nonclose-packed arrays, and the center-to-center distance (Dctc) was about 380 ± 50 nm. When the withdrawing speed increased to 10 μm/s, surface microgel packing further reduced, with a larger center-to-center distance of about 585 ± 273 nm. As the center-to-center distance increased, the microgels within the film became less ordered. In fact, for films formed at the withdrawing speed larger than 10 μm/s, we could not obtain well-ordered microgel arrays in areas larger than 1 cm2 any longer. AFM imaging was also used to determine Dctc changes, and the values were consistent with those obtained from SEM (Figure SI5). Thus, the surface packing density and ordering of the microgel particles can be tuned by concentration and withdrawing speed in the dip-coating process. As the combination of the microgel concentration of 1 wt % and withdrawing speed of 5 and 10 μm/s produced rather well packed 2D PS25 monolayer films, we have chosen these 2D microgel film forming conditions to assess film properties formed by the other microgel particles with different styrene 4026

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Figure 6. (a) Changes of the PS25 microgel film thickness when placed under water at 20 °C, heated to 37 °C, cooled to 20 °C, again heated to 37 °C, and then cooled to 20 °C to complete the two full thermal cycles; (b) Lack of changes in film thickness (and their matching refractive indices) measured from the PS25 microgel layer immersed in water at ambient temperature (20 °C) for 6 days indicated high stability of the microgel film formed.

almost constant at 0.3 from 22 to 37 °C. Changes in refractive index with temperature matched thickness variations well, showing a clear trend of tending to the behavior of pure polystyrene particles with increasing styrene content. 3.4. Film Swelling against Thermal Cycles and Stability. Film stability against heating and cooling was tested by monitoring changes in the thickness of the PS25 microgel film prepared by dip coating at the withdrawing speed of 5 μm/ s. The SE measurements were made at the ambient temperature of 20 °C and cell culturing temperature of 37 °C. Before the microgel film was measured, the precise thickness of the native oxide layer on the freshly cleaned silica wafer surface was first determined (15 ± 2 Å). Figure 6a shows the thicknesses measured from the PS25 film that underwent two temperature cycles. During the first cycle, the hydrated PS25 film was found to be 310 ± 30 nm at 20 °C, but upon heating to 37 °C the dehydrated thickness dropped to 195 nm ±20 nm, with the refractive index also increased as expected. The drastic shrinkage with temperature confirms that the PS microgel film on the surface swells and collapses across VPTT, similar to the bulk PS25 microgel particles. However, a slight increase in thickness was observed during the second cycle (∼330 nm at 20 °C and ∼205 nm at 37 °C). This may arise from the sufficient time for the swelling and deswelling to reach equilibration. After 24 h, the thickness at 20 °C remained at 330 nm again. The results suggested no loss in the microgel film during the thermal cycles. The refractive index remained at about 1.345 and 1.365 (λ = 600 nm) for 20 and 37 °C, respectively, and showed no obvious change with the thermal cycles. The stability of the PS25 microgel film was further assessed by monitoring the variation in their thicknesses during 6 days of immersion in water at ambient temperature. As shown in Figure 6b, little variation in the thickness of the microgel film was observed, suggesting its good stability. These assessments indicated that the PS25 microgel film was stable and that no desorption of microgel particles from the surface occurred. We have previously shown that the surface zeta potential of the PS25 microgels in water was about −8.4 mV.30 As the bare silicon oxide surface also bears weak negative charges, the formation of stable PS25 microgel films on the same charged silicon wafer means that the electrostatic interaction alone does not dictate microgel film stability. Other interactions such as chain entanglement and hydrophilic affinity associated with

3.3. Thermal Film Responsiveness. Upon contact with the substrate, soft microgels are easily flattened. Furthermore, they can display swelling−deswelling behavior under surface confinement, resembling what is shown from their respective bulk microgels. SE was used to determine whether the deposited microgel films had thermal responsiveness via temperature stimuli in water, thereby unraveling the conditions under which the microgel layers swell and deswell. A simplified planar slab model was used to fit the measured SE data. A clear advantage in this work was that the film thickness under dry or swollen state was well above 50 nm, offering sufficient structural sensitivity to decouple the film thickness and density. This enabled us to derive reliable information on the extent of water mixing into the film under different film and solution conditions. Figure 5 shows the curves of film thickness plotted against temperature, with the corresponding refractive index also shown. These profiles are very similar to the bulk swelling curves from the corresponding microgels (Figure 1), showing that the surface deposited microgels could mimic the thermoresponsive changes of the bulk dispersions. Specifically, the largest thermoresponsive variation in film thickness and refractive index over the temperature range studied was observed from the PS25 microgel film. As styrene content increased, the thermoresponsive change diminished. Interestingly, however, film thicknesses under the hydrated state were all below 270 ± 30 nm and were significantly lower than the corresponding hydrated diameters for the matching microgels in the bulk, showing clear deformation upon surface deposition. Furthermore, increase in the styrene content led to the reduction in the extent of hydration and deformation reduced and the difference between the hydrated layer thicknesses and the matching bulk microgel diameters also reduced. As the temperature went above VPTT, the layer thicknesses were even closer to their respective dehydrated bulk microgel diameters and displayed a trend of increase of thickness with styrene content, consistent with the changes in bulk microgel diameter. The extent of water inside the film could be estimated from its measured refractive index. For PS25 and PS40, the water volume fraction changed from 0.9 at 22 °C to about 0.8 at 37 °C. The outcome meant that, even in its collapsed state, the microgel still retained a high content of water. But for PS55 and PS70, the indices have no significant changes with increasing temperature. For PS70, the water volume fraction remained 4027

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hydrogen bonding between the acrylamide groups and the substrate seemed to play a dominant role. 3.5. Wettability of the Microgel Films. Changes in the wettability of the PNIPAAm coatings control their surface responses to proteins expressed by cells. Water contact angle is an important parameter and is widely used to help assess surface physiochemical properties and their mediation to cell adhesion and proliferation. Table 1 shows water contact angle Table 1. Water Contact Angle Changes Measured from the Microgel Films against Temperature As Estimated by the Sessile-Drop Method θH2O microgel films PS25-5 PS25-10 PS40-5 PS40-10 PS55-5 PS55-10 PS70-5 PS70-10

25 °C 25.3 30.0 24.7 28.1 40.2 37.3 55.2 57.6

± ± ± ± ± ± ± ±

2.8 0.8 1.8 5.3 3.5 3.9 2.7 1.4

38 °C 55.8 58.1 51.0 50.3 49.9 49.6 65.5 57.9

± ± ± ± ± ± ± ±

Figure 7. Phase contrast microscopic images of NIH 3T3 cells 48 h after incubation at 37 °C (left) and 60 min after cooling the culture plate to 20 °C (right). The microgel films were prepared by drawing each glass slide from 1.0 wt % microgel dispersion with the speed of 5 μm/s. The scale bar marked was 100 μm. The results show that, although increase in the content of styrene had no adverse effect on cell proliferation and growth, it inhibited the detachment of the cells due to the lack of thermoresponsive changes of the film properties.

3.5 6.0 3.1 4.3 3.1 5.1 2.6 2.6

The postfixes 5 and 10 mean that the films were prepared with the withdrawing speeds of 5 μm/s and 10 μm/s in the dip coating, respectively. a

Figure 8 shows a set of phase contrast images of an area of NIH 3T3 cells in real-time on the PS25 film from 2 to 60 min after the cold fresh cell culture medium was added. The observation was made at an ambient temperature of about 20 °C. At first, most cells adopted a spindle shape with the formation of lamellipodia. For a typical cell, the initial length was measured to be 68.2 μm at 2 min and 20 °C. After 10 min under the ambient temperature, the length was significantly reduced to 32.8 μm. After 15 min, the cell became more spherical due to the active retraction of the cell body and the length was reduced to 29.2 μm. After 20 min, most cells turned to a round morphology as they had lost their anchoring points. It should be noted that in this work, many individual NIH 3T3 cells failed to detach completely from the microgel films because of the absence of intracellular interactions as observed in cell sheets, but they could detach from the surface by gently rinsing with the culture medium. Thus, the rounded cells were referred to as detached cells in the work. The morphological changes of the cells on the microgel films prepared with withdrawing speed of 10 μm/s via temperature stimuli are similar to those obtained from surfaces coated at 5 μm/s. While cell detachment on the films of PS25 and PS40 prepared with withdrawing speed of 10 μm/s was found to be similar to what was observed from those coated at the withdrawing speed of 5 μm/s, many cells grown on these film surfaces also became rounded, showing good detachment. As explained previously, increase in withdrawing speed led to the reduced microgel surface coverage, but the results together show that differences in the gaps between the surface confined microgels did not produce any pronounced difference on cell detachment under the conditions studied. To make a more systematic assessment of the effect of gaps within microgel films on cell detachment, we have quantified the percentage of cells detached on each of the microgel films studied against detachment time. Figure 9 shows cell detachment kinetics from 2 to 60 min at ambient temperature from all four types of microgel films coated. To make data directly comparable, we observed the same surface area during the detachment process. When observed after cold treatment, many cells retracted and became round already. It can be seen

data obtained from each surface coated with different microgels and withdrawing speeds using the sessile-drop method at 25 and 38 °C. PS25 and PS40 microgel films showed a marked difference in contact angle at temperatures above and below VPTT, with no obvious difference between the withdrawing speed of 5 and 10 μm/s. No obvious difference in surface wettability was found between PS55 and PS70 microgel films, indicating that as the styrene content was high, little change could be induced in surface hydrophobicity/hydrophilicity. As will be discussed next, the different thermoresponsive changes as revealed from the contact angle measurements could well impact cell responses across VPTT. 3.6. Cell Growth and Detachment. The viability of cells grown on the PNIPAAmSt microgel films was compared with that grown on the bare glass coverslips (control). The microgel films were again dip coated with a withdrawing speed of 5 μm/ s, with the microgel concentration fixed at 1 wt %. MTT assay was used to assess the cell viability after 2 days of growth at 37 °C. The MTT results revealed that similar absorbance from NIH 3T3 cells grown on both the PNIPAAmSt microgel films and the positive control (Figure SI6). The results thus showed no difference in their viability, meaning that the range of variation in styrene or PNIPAAm content caused little effect on cell attachment and growth. Cell detachment was also tested after incubation at 37 °C for 48 h. The NIH 3T3 cells were cold treated and observed at ambient temperature for 60 min, the adhesion and detachment of cells grown on the microgel films after cold treatment is shown in Figure 7. The results indicate that only the microgel films prepared from PS25 and PS40 were effective to trigger cell detachment when temperature fell below the VPTT points. For the PS55 microgel film, about half cells still remained adhered on the surface after 60 min. For the PS70 microgel film, few cells became even round after the observation period, consistent with it being lack of thermosensitivity. 4028

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Figure 8. Time lapse microscopic imaging (scale bar 100 μm in the top row) of NIH 3T3 cells detaching from a microgel surface prepared by dip coating 1.0 wt % PS25 microgel dispersion at the withdrawing speed of 5 μm/s. Images in the bottom row were taken from the corresponding circled areas in the top row. They together show a progressive shrinking and detaching process of the cell marked from the coated substrate over the time scale of 1 h.

Figure 9. NIH 3T3 cell detachment kinetics on different microgel films prepared with the withdrawing speed of (a) 5 μm/s and (b) 10 μm/s. The results show that as styrene content in the microgels increases, the extent of cell detachment (measured by percentage of cell spreading) slows down drastically. With increasing withdrawing speed in the microgel film coating, the gaps between microgel particles within the film increased. The results also indicate little impact in the gap size on cell detachment.

associated with the initial cold treatment for cells (t > 2 min). The fitted parameters were given in the Supporting Information (Table SI1). The results showed that more than 90% cells could detach from PS25 and PS40, and the detachment rate constant k for the cell detachment on PS25-5 and PS40-5 are 0.168 and 0.068 min−1, respectively, showing almost twice faster detachment rate from the PS25-5 surface. It is also intriguing that for the same microgels, the kinetic coefficients k for PS25-5 and PS25-10 and those for PS40-5 and PS40-10 are similar, respectively. This again confirms that the effects of the gap distance between the microgel particles on cell detachment are insignificant within experimental errors, and the extent of swelling plays a key role in cell detachment dynamics. Against the surface patterning work reported by Kumashiro et al.,55 the cells detached quicker from our microgel assembled PS25 and PS40 films than observed from the micropatterned PNIPAAm surface, and the difference could be attributed to the microgel film surface patterning, which could be more easily hydrated after cold treatment than flat grafted linear PNIPAAm film surface. Although Tsai et al. and Schmidt et al. have showed that PNIPPAm microgels could also be used to control cell growth and detachment,40,41 these authors did not assess the dynamic cell release processes. In the course of our own work

that the percentage of the spread cells decreased from 100 to almost 0 for PS25 and PS40. The detachment process clearly slowed down after the first 30 min. As explained already, the spread cells could clearly detach from the PS25 and PS40 microgel films by temperature stimuli. For PS55 and PS70, however, approximately 50% and 90% spread cells could not detach from the microgel films over the same screening period. In cell detachment by temperature-stimuli studies, individual cells or cell sheets would spontaneously detach from the film with extracellular membrane molecules, which are mainly adhesive proteins at the interface of cells/cell sheet and film; this means that cells/cell sheet detachment could be promoted by the detachment of proteins.53,54 Kumashiro et al. proposed that because the exponential Langmuir model is usually used to evaluate protein desorption,55 a single exponential decay model could also be used to calculate the rate of cell detachment in our study: Y = Yo + A exp(−k(t − 2))

(1)

where Y is the percentage of spread cells in the detachment process, Y0 is the final percentage of spread cells remaining, t is detachment time (min), k is the detachment rate constant, A is the constant of detachment, and 2 denotes the nominal time 4029

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results together indicate that the swelling ratio of the microgel films is the key factor influencing cell detachment dynamics.

using pure PNIPPAm microgel formed 2D arrays, we found that only 40−50% spread cells could detach from the pure PNIPAAm microgel films. These PNIPPAm microgel films were therefore less effective for cell harvesting. Thus, incorporation of some 20−40 wt % styrene in the case of PS25 and PS40 offers the most efficient cell detachment in terms of the dynamic detachment rate and the percentage of cells detached. However, as more styrene was introduced, the thermoresponsive response weakened and the fraction of the detached cells became fast reduced. To confirm the stability of the microgel films in the process of cell detachment, SEM was used to reveal the morphologies of PS25 and PS70 microgel films. It could be seen that, although microgel particles did become lost in some small areas after cell detachment, the majority of the film area could still hold its integrity (Figure 10).

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ASSOCIATED CONTENT

S Supporting Information *

Supplementary results on the characterization of PNIPAAm and PNIPAAmSt microgel films and cell responses. This material is available free of charge via the Internet at http:// pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +86-532-86981569; e-mail address: [email protected]. *Tel: +44-161-3063926; e-mail address: [email protected]. uk. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank the Natural Science Foundation of China (NSFC, #20804057), the Fundamental Research Funds for the Central Universities (#14CX02121A), UK Engineering and Physical Sciences Research Council (EPSRC EP/F062966/1) for funding support. We also thank the Royal Society (London) for support through a Sino-British Fellowship and an International Partnership Programme.

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Figure 10. SEM images of PNIPAAmSt microgel films after cell detachment, indicating that the microgel arrays pronominally remained on the surfaces. (A,B) PS25 film prepared by dip coating 1.0 wt % PS25 microgel dispersion at the withdrawing speed of 5 μm/s and 10 μm/s, respectively; (C,D) PS70 film prepared by dip coating 1.0 wt % PS75 microgel dispersion at the withdrawing speed of 5 μm/s and 10 μm/s, respectively.

4. CONCLUSIONS A convenient approach to prepare 2D arrays of thermoresponsive microgel films has been developed. By copolymerization with styrene, a series of microgels with different swelling capacities was synthesized. All PNIPAAmSt microgels as reported in this work could form rather well-ordered 2D patterns under appropriate coating conditions. However, only 2D films prepared from microgels with the content of styrene around 20−40 wt % showed obvious thermoresponsive changes while films containing over 40% styrene behaved more like hard polystyrene particles. Cells could grow well on all microgel films, but those containing high styrene content did not allow easy cell detachment due to the lack of thermoresponsive changes upon cooling. For PS25 and PS40 microgel films, 3T3 fibroblast cells could grow well and detach over 90% from the films. Furthermore, the cells detached faster from PS25 films than PS40 films, consistent with the different extent of thermoresponsive changes. It was also found that gaps manipulated from withdrawing speeds of 5 and 10 μm/s did not cause any major influence on cell detachment rates. The 4030

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