Controllable Stabilization of Poly(N-isopropylacrylamide)-Based

Jun 20, 2012 - ... Diana María Escobar-García , Christian Grandfils , Bernardino Isaac ... Shengtong Sun , Li-Bo Mao , Zhouyue Lei , Shu-Hong Yu , H...
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Controllable Stabilization of Poly(N-isopropylacrylamide)-Based Microgel Films through Biomimetic Mineralization of Calcium Carbonate Yongqing Xia,† Yanfeng Gu,† Xuan Zhou,† Hai Xu,*,† Xiubo Zhao,‡,§ Mohammed Yaseen,§ and Jian Ren Lu*,§ †

Centre for Bioengineering and Biotechnology, 66 Changjiang West Road, Qingdao Economic Development Zone, Qingdao 266555, China ‡ Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield, S1 3JD, United Kingdom § Biological Physics Laboratory, School of Physics and Astronomy, University of Manchester, Schuster Building, Oxford Road, Manchester, M13 9PL, United Kingdom S Supporting Information *

ABSTRACT: Two types of thermoresponsive microgels, poly(N-isopropylacrylamide) (PNIPAM) microgels and poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAMAC) microgels were synthesized and used as templates for the mineralization of amorphous calcium carbonate (ACC) by diffusion of CO2 vapor under ambient conditions. Thermosensitive PNIPAM/CaCO3 hybrid macroscopic hydrogels and micrometer-sized PNIPAMAC/CaCO3 hybrid microgels were controllably obtained and different mineralization mechanistic processes were proposed. The impact of the loaded CaCO3 on the size, morphology, stability, and thermosensitivity of the microgels was also analyzed. PNIPAM/CaCO3 hybrid macrogels had a slight decrease in thermoresponsive phase transition temperature, while PNIPAMAC/CaCO3 hybrid microgels showed a clear increase in phase transition temperature. The difference reflected different amount and location of ACC in the gel network, causing different interactions with polymer chains. The PNIPAMAC/CaCO3 microgels formed stable monolayer films on bare silica wafers and glass coverslips upon drying. The microgel films could facilitate the attachment and growth of 3T3 fibroblast cells and their subsequent detachment upon temperature drop from 37 °C to the ambient condition around 20 °C, thus, offering a convenient procedure for cell harvesting.

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INTRODUCTION Natural biogenic hybrid materials are often composed of soft organic and hard inorganic components that have distinctive mechanical and biological properties far superior to their synthetic counterparts. Inspired by natural biogenic minerals, many researchers have attempted to mimic natural biomineralization through exploring mechanistic processes underlying organic−inorganic hybridization.1−3 Calcium carbonate is one of the most abundant biogenic minerals found in mollusc shells. It has three anhydrous crystalline polymorphs, that is, vaterite (the least thermodynamically stable), aragonite, and calcite (the most stable). In the last two decades, numerous additives and substrates, such as inorganic ions,4 organic molecules,5 selfassembled monolayers,6 and polymers7−10 have been used to synthesize CaCO3 crystals and control their morphologies and polymorphs. Unlike the well-documented crystalline polymorphs, amorphous calcium carbonate (ACC) has often been overlooked for its instability and higher solubility. However, recent investigations have suggested that ACC plays many key roles in the early stage of biomineralization,11−14 particularly with regards to the transformation to aragonite and calcite and the mediating effects of organic species such as proteins.15−20 © 2012 American Chemical Society

Poly(N-isopropylacrylamide) (PNIPAM) is a typical thermoresponsive polymer and bears the basic chemical structure in close resemblance to proteins and peptides. It exhibits a lower critical solution temperature (LCST) in aqueous environment. Below the LCST around 32 °C, the polymer is hydrophilic and water-soluble. Above it, it is in a collapsed, hydrophobic state. For this reason PNIPAM and its copolymers are frequently used as key constituents for fabricating thermoresponsive materials in many applications, including drug release,21−23 bioseparation,24,25 and cell culture engineering.26−28 Microgels are cross-linked latex particles which swell in a specific solvent. Compared with other additives or substrates, microgels as templates to mineralize CaCO3 provide several advantages, including uniform size, extremely large surface area, and huge flexibility for surface functionalization. Most importantly, microgels can be used as confined micro- and nanoenvironment to grow CaCO3 inside the particles, a process similar to biomineralization in single cell organisms. Recently, Received: April 6, 2012 Revised: June 6, 2012 Published: June 20, 2012 2299

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of PNIPAM microgels, it was noted that the microgel beads selfassembled into macroscopic hydrogels after 48 h. A small sample of these hybrid hydrogels was freeze-dried without any treatment. Another small sample was immersed in water for seconds and then freeze-dried. The rest of the hybrid stock was diluted with water and shaken vigorously to disperse them back into hybrid microgels (referred to as PNIPAM/CaCO3 hybrid microgels). These samples were used for different experiments. Microgel Characterization. The hydrodynamic diameters and thermoresponsive behavior of the mineralized and nonmineralized 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 20−40 °C. The dispersed microgels in water were allowed to equilibrate thermally for 10 min before measurements were taken at each temperature. A graphite furnace high resolution atomic absorption spectrometer (contra AA 700, Analytik Jena AG) was used for the determination of calcium content in the hybrid particles. Transmission electron microscopy (TEM, JEM-2100UHR, JEOL) was used to characterize the synthesized microgels before and after mineralization. Specimens for TEM imaging were taken from diluted dispersions, deposited on a 400 mesh carbon-coated copper grid, and dried under the infrared lamp before being observed. Scanning electron microscopy (SEM, S-4800, Hitachi) was also used to help examine microgel’s morphological structures. Microgel dispersions were diluted with water, dropped onto cleaned glass slides, and dried at room temperature. Samples were coated with a thin Au layer to increase the contrast and quality of the images. Atomic force microscopy (AFM, Nanoscope IVa MultiMode AFM, Digital Instruments, Santa Barbara, CA) was used to undertake images in tapping mode. Samples were prepared by dropping 10 μL of microgel dispersions onto freshly cleaved mica surfaces. After 10 s, the mica surfaces were dried gently with nitrogen gas. Microgel Film Preparation and Characterization. As substrates, either bare silicon wafers or microscopic glass coverslips were used. The glass coverslips and silicon wafers were cleaned with a 3:7 mixture of H2O2 (30%) and H2SO4 (98%) for 30 min, followed by abundant rinsing with water. Then 0.25 wt % microgel suspension was dropped on the bare substrates and left to dry. The substrates with dried microgels were immersed in purified water for at least 12 h while replacing the water every 3 h before tapping mode AFM was employed to characterize the morphology of dried microgel particles remaining on the bare silicon substrates after rinsing treatments. In the case of formation of microgel films, film stability was also determined using a variable angle spectroscopic ellipsometry (Jobin-Yvon UVISEL). The thickness of the native oxide layer on clean silicon wafer was first determined prior to PNIPAMAC/CaCO3 microgel coating. The measurement processes were performed following the protocols reported previously.29 Following film formation and annealing in water for 12 h, its dry film thickness (dried under ambient vacuum oven) was first measured, followed by measuring its thickness in pure water daily while the water was changed daily. On the fifth day, the film was dried again with its dry thickness measured again to assess film stability. The experimental data were analyzed using DeltaPsi I software provided by Jobin-Yvon. Cell Culture, Growth, and Detachment Monitoring. The glass coverslips coated with a particulate monolayer film of PNIPAMAC/ CaCO3 microgels were sterilized for 2 h by UV and then transferred into six-well tissue culture plates for subsequent use. NIH 3T3 cells (20000 per well) were planted uniformly on differently coated coverslips placed in plate wells and cultivated in DMEM medium containing 10% FBS, 2 mM L-glutamine, and 1% penicillin/ streptomycin at 37 °C and 5% CO2. All the experiments were undertaken in triplicate, and fresh and warm culture medium (preheated to 37 °C to avoid any possible cell detachment due to temperature drop) was used to replace the old medium every other day. At days 1, 2, and 4, the cells were observed by a phase contrast inverted microscope (Leica, DMI3000, Germany). In addition to assessing cell growth by microscopic visualization of cell appearance and counting numbers, standard MTT assays were also undertaken to

PNIPAM-based materials have been used as templates to mineralize CaCO3.29,30 Kuang et al. have used poly(Nisopropylacrylamide-co-(4-vinylpyridine)) microgels as templates to prepare submicrometer-sized inorganic−organic composite particles with unusual shapes (disk-like, cell-like, and hemispheroidal shapes),29 Nassif et al. have used particles that are composed of poly(diethylaminoethyl methacrylate)-bpoly(N-isopropyl-acrylamide)-b-poly(methacrylic acid) as templates to prepare homogeneous aragonite crystals with structural complexity.30 Those studies have focused mainly on the formation of hybrid particles and their dynamics with good control of the growth of CaCO3 crystals. However, the potential applications of these hybrid beads or particles have been little explored. In this study, we report the copolymerization of PNIPAM microgels with different monomers and their subsequent use as templates to prepare CaCO3/microgel hybrid materials, with the focus on investigating the influence of different comonomers used during microgel polymerization on mineralization process and hybrid material properties. We also explore the potential application of these hybrid materials in cell culture and tissue engineering.

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EXPERIMENTAL SECTION

Materials. All the chemicals were obtained from Sigma-Aldrich. NIsopropyl acrylamide (NIPAM) was purified by recrystallization from a toluene/hexane mixture (1:3) and dried in vacuum. Acrylic acid (AC) was purified by distillation under reduced pressure, ammonium persulfate (APS) was purified by recrystallization from water. N, NMethylenebisacrylamide (MBA), calcium chloride (CaCl2), and ammonium carbonate ((NH4)2CO3) were used as received. All water used in this experiment was processed by Milli-Q system (MilliQ Advantage A10 Water System Production Unit). Preparation of PNIPAM-Based Microgels. PNIPAM microgels were prepared by surfactant-free precipitation polymerization. Typically, 1.132 g of NIPAM (10 mmol) and 0.031 g of the crosslinking agent MBA (0.2 mmol) were dissolved in 95 mL of water. The reaction mixture was then 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, 0.060 g of an initiator (APS) was dissolved in 5 mL of (oxygen free) water and added to the flask to initiate polymerization. The reaction was continued for 4.5 h in a nitrogen environment by continuous purging. Following synthesis the PNIPAM microgels were purified by four successive centrifugations at 40000 g for 30 min (HANILSUPRA22K preparative ultracentrifuge), each followed by decantation and redispersion in water. In contrast, the poly(N-isopropylacrylamideco-acrylic acid) microgels, referred to as PNIPAMAC microgels, were prepared and purified as in the case of PNIPAM microgels, except that the monomers were 0.792 g of NIPAM (7 mmol) and 0.216 g of AC (3 mmol). The amount of functional group −COOH was determined using conductometric titration. The results showed that there was 1.60 mmol of −COOH groups per gram of PNIPAMAC microgels, indicating the incorporation of 54.4% of the added AC monomers to the microgels. Mineralization of CaCO3 Using PNIPAM-Based Microgels as Templates. Gas diffusion method was used to perform the mineralization of CaCO3 in the presence of microgels. Briefly, in glass beakers, the microgels were well dispersed in 10 mM CaCl2 aqueous solution. The concentration of microgels was kept at 1 wt %. The beakers were then put in a closed desiccator where fresh (NH4)2CO3 was used as the CO2 vapor source. All mineralization experiments were carried out at the ambient temperature of 20 ± 2 °C for 48 h. The mineralized PNIPAMAC microgels, referred to as PNIPAMAC/CaCO3 hybrid microgels, were then purified by four successive centrifugations at 40000 g for 30 min, each followed by decantation and redispersion in water. In the case of the mineralization 2300

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Figure 1. TEM micrographs to show the core−shell structures of PNIPAM (a) and PNIPAMAC (c) microgels and the AFM images of the ordered arrays formed from PNIPAM (b) and PNIPAMAC (d) microgel dispersions dried on mica; (e, f) cross-section analysis performed as shown in (b) and (d), respectively.

Figure 2. Temperature dependency of the hydrodynamic diameters for (a) PNIPAM microgels and their corresponding CaCO3 hybrid microgels, and (b) PNIPAMAC microgels and their corresponding CaCO3 hybrid microgels at pH 7.4. The data shown represent measurements with increasing temperature, close to those measured from decreasing temperature (not shown for clarity) within experimental errors. help assess cell viability at days 1, 2, and 4 using bare glass coverslips as control. To aid thermoresponsive cell detachment, the well plates were left on the lab bench at 20−22 °C for cooling and the cells were monitored in situ. Cell numbers and their shapes (rounded versus spread) were counted at the same observation field to indicate the detachment at the early stage. The results were averaged from five representative view fields.

low degree of cross-linking (2%), these PNIPAM-based microgels are mechanically soft and may collapse in an anisotropic fashion when dried on support surface. On the basis of TEM imaging, the PNIPAMAC microgels were larger than the PNIPAM microgels due to the copolymerization of the hydrophilic monomer acrylic acid. Latex particles and silica spheres can pack densely into hexagonal lattices.33,34 Yoshida and co-workers35 prepared 2D monolayer films of PNIPAM microgel beads via doubletemplate polymerization. Kawaguchi and co-workers36,37 prepared films of PNIPAM particles which, upon air-drying, resulted in highly ordered 2D colloidal crystals with a controllable distance. The balance between capillary attraction and steric repulsion was regarded as the driving force for regulating the distance between the particles. In our work, both PNIPAM and PNIPAMAC microgels could be well distributed on freshly cleaved mica, but the latter tended to become more orderly distributed only on small areas of typical 10 × 10 μm, with representative AFM images shown in Figure 1b,d. It thus appears that there is sufficient repulsive interaction between the PNIPAM microgel particles to keep them apart and that the

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RESULTS AND DISCUSSION Preparation of Thermoresponsive Core−Shell PNIPAM-Based Microgels. In the present work, two types of thermoresponsive microgels, PNIPAM and PNIPAMAC, were prepared using the recently reported precipitation polymerization offering narrow size distribution.31 Acrylic acid (AC) was used in the case of PNIPAMAC microgels to introduce carboxylate groups, thus, providing active nucleation sites for calcium minerals. Figure 1a,c shows the TEM images of the two types of PNIPAM-based microgels. Both display core−shell structures in which each dark spherical area represents the dense cross-linked core, and the light corona around each dark core represents the loose cross-linked shell.32 Because of the 2301

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Figure 3. (a) Optical photograph of the PNIPAM/CaCO3 hybrid hydrogel taken from mother liquor; (b) a low-magnification SEM image of the surface of freeze-dried hybrid hydrogel without any treatment, showing that most part of the surface is smooth, with some rectangle caves formed by expelled calcite crystals (arrows indicating the areas); (c) the surface of the hybrid hydrogel treated by water immersion (inset shows high magnification image); (d) high-magnification SEM image of dispersed PNIPAM/CaCO3 hybrid microgels with many CaCO3 nanoparticles surrounding microgel beads.

bottom of a beaker (Figure 3a). This phenomenon could be repeated in our experimental microgel concentration range from 0.01 to 1.0 wt %, with the thickness of hydrogels decreasing with decreasing concentration. After freeze-drying without any treatment, most of the surface area of the hydrogel disk is smooth, with some pores or cavities visible, as indicated by arrows in Figure 3b. From the size and shape on the deposited surface (Figure S1), we can deduce that these cavities were occupied by calcite crystals that had formed in dispersion and then entrapped by the hydrogel. Subsequent removal of these crystals from the surface created these pores or holes. After reimmersed in water for seconds, the smooth surface layer became disintegrated as a result of detachment of some of the microgel particles. It, thus, becomes clearly visible that the disk is comprised of many compact microgel particles (Figure 3c). When immersed in water for hours, the hydrogel swelled to the full extent, but its overall 3D structure still remained. The mechanical property of the swollen hydrogel film was very weak and could easily be disassembled into microgel particles upon vigorous shaking and rinsing. Further SEM imaging revealed that the hybrid microgels were surrounded by many CaCO3 nanoparticles (about 100 nm) with irregular shape (Figure 3d). EDS (energy dispersive X-ray spectroscopy) analysis (Figure S2) revealed the existence of Ca, and HRTEM observation revealed little sign of obvious crystal lattices from these hybrid particles. Thus, CaCO3 in the hybrid microgels must exist in the form of amorphous calcium carbonate (ACC), consistent with our XRD analysis. Previous studies have reported the use of preformed inorganic materials such as silica particles and calcium carbonate crystals to prepare hybrid hydrogels from poly(N-

presence of additional electrostatic repulsion arising from the incorporation of acrylic acid monomers in PNIPAMAC microgels does not offer any clear advantage in forming the orderly surface microgel particle arrays. The heights of the dried PNIPAM and PNIPAMAC microgels were about 56 and 37 nm, respectively (Figure 1e,f). The greater heights in the case of PNIPAM microgels implied greater mechanical strength and less deformation in contact with the surface and upon drying. Figure 2 shows DLS measurements of variations of the hydrodynamic diameters of the dispersed PNIPAM and PNIPAMAC microgels in bulk water as a function of temperature. Both microgels show similar thermoresponsive behavior, having a rather steady size change over 25−33 °C. At low temperature, the microgels were at a swollen state and a wide particle size distribution appeared with irregular surface features. However, the microgels shrank to hard spheres when temperature was above 33 °C. The deswelling process was associated with the expulsion of hydrated water, accompanied by increase in hydrophobic interaction and formation of new hydrogen bonding network. The decreased hydrodynamic diameters were also associated with narrower size distribution observed. It was found that this thermoresponsive phase transition was fully reversible and the microgels remained stable at higher temperatures, that is, no particle aggregation occurred. Both PNIPAM and PNIPAMAC microgels remained stable even after addition of CaCl2 (10 mM) at the ambient temperature, showing good gel phase stability. Mineralization of CaCO3 within PNIPAM Microgel Dispersion. Figure 3 shows the macro- and micromorphologies of the mineralized PNIPAM microgels. After 48 h, the PNIPAM microgels formed a macroscopic hydrogel disk on the 2302

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Figure 4. (a) TEM micrograph of the dispersed PNIPAM/CaCO3 hybrid microgels with core−shell structure; (b) AFM image of ordered arrays formed by PNIPAM/CaCO3 hybrid microgels dried on mica; (c) TEM micrograph of the dispersed PNIPAMAC/CaCO3 hybrid microgels with reduced core−shell structure; (d) AFM image of ordered arrays formed by PNIPAMAC/CaCO3 hybrid microgels dried on mica.

Scheme 1. Schematic Representation to Show the Dynamic Biomineralization Processa

a

PNIPAM microgels are initially well dispersed in solution (A), but upon CO2 diffusion, CaCO3 nanoparticles form on the surface of PNIPAM microgel particles and in solution (B). The hierarchal assembly of PNIPAM microgel particles stimulated by the CaCO3 nanoparticles causes the sequential integration of the hybrid PNIPAM microgels into a single 3D gel entity (C). Depending on system conditions, it typically takes some 20 h for microgel particles to grow from state (B) to (C).

vinylcaprolactam) systems.38,39 The results in our work have demonstrated that ACC can also initiate the PNIPAM microgels to further assemble into 3D hydrogels. In the present system the amide groups in the polymers have weak interactions with CaCO3 nanoparticles. Consequently, the microgels only have a weak influence on calcification, leading to the physical entrapment of the CaCO3 nanoparticles. Thus, affinity of microgels to CaCO3 nanoparticles provides a driving force in aggregation and hydrogel formation. Because ACC has a high solubility in water,40 the assembly and disassembly of the hydrogel network can be manipulated by controlling ACC solubility as demonstrated above. At 33 °C, the hybrid hydrogels formed showed little thermosensitivity in its mother liquor as they were already in the compact state. When cooled down to the ambient temperature, the hydrogel became swollen and thus showed large thermosensitivity. The thermosensitivity of PNIPAM/

CaCO3 hybrid microgels is thus similar to that of the PNIPAM microgels, except incorporation of CaCO3 makes the effective hydrodynamic diameters larger. The hydrodynamic diameters of the PNIPAM microgels are around 220 nm at 35 °C, and those of the PNIPAM/CaCO3 hybrid microgels increase to 270 nm (Figure 2), clearly showing the effect of loading of CaCO3 into PNIPAM microgels. The hybrid microgels still hold the core−shell structure, evident from changing packing densities between the core and shell regions (Figure 4a). The phase transition temperature of hybrid microgels does not vary much, both around 25−33 °C, indicating the weak impact of CaCO3 loading. The amount of CaCO3 associated in the hydrogels was in fact low, only about 0.6%, calculated from atomic absorption spectroscopy. TEM revealed that CaCO3 was mainly distributed within the shell region. Further AFM imaging unraveled that the PNIPAM/CaCO3 hybrid particles assembled into rather ordered arrays and that the heights of PNIPAM/ 2303

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Figure 5. SEM images of (a) PNIPAMAC microgels and (b) PNIPAMAC/CaCO3 hybrid microgels dried on a glass substrate. The insets in both cases show the corresponding microgels at higher magnifications.

Figure 6. Microscopic image (a), SEM (b) image, and (c) XRD pattern of calcite crystals deposited on the glass slide in PNIPAMAC/CaCO3 microgel dispersion.

CaCO3 hybrid microgels became more easily assembled into an ordered array when dried on mica than PNIPAM/CaCO3 hybrid microgels (Figure 4d). These features are similar to the structural advances observed from CaCO3-loaded PNIPAM copolymers containing 4-vinylpyridine microgels.29,41 With time, the PNIPAMAC/CaCO3 hybrid microgels tended to transform from initial plano-convex-like to disk-like, although their overall heights did not change much, showing structural deformation due to interactions with surface and under drying. After undergoing mineralization, the extent of swelling of PNIPAMAC microgels is greater than that of PNIPAM microgels. This together with the greater flexibility led to the greater flattened diameters of PNIPAMAC/CaCO3 microgels when adsorbed on the surface of mica, consistent with the swollen state of the PNIPAM/CaCO3 microgels. It is worth noting that native PNIPAMAC microgels become stuck to each other and form clusters due to the partial fusion of poorly cross-linked shells when dried on a support glass coverslip (Figure 5a). For the PNIPAMAC/CaCO3 hybrid microgels, the feature was reduced (Figure 5b). The insets in both cases show the corresponding microgels at higher magnifications. It is clearly visible that the outer surfaces of the PNIPAMAC/CaCO3 hybrid microgels appear to be spiky or hairy, preventing them from becoming fused. Thus, the hybrid particles remained well-separated spheres after drying. DLS data showed that the PNIPAMAC/CaCO3 particles also remain thermoresponsive, but the extent of deswelling is remarkably smaller than PNIPAMAC microgels at the same pH (8.50), showing that binding of CaCO3 particles reduced the extent of thermoresponsive size changes. The hydrodynamic diameters of the PNIPAMAC/CaCO 3 hybrid particles increased to 450 nm at 35 °C from some 250 nm measured from PNIPAMAC microgels (Figure 2b), showing a huge

CaCO3 hybrid particles remained unchanged due to the low CaCO3 incorporation (Figure 4b). The stages involved in the PNIPAM/CaCO3 hydrogel formation are depicted in Scheme 1. When CO2 diffuses into the PNIPAM microgels, CaCO3 nanoparticles formed both in the solution and in the pores of microgels. The CaCO3 nanoparticles formed on the surface of the microgels or in aqueous phase can be absorbed by neighboring microgels, resulting in bridging to physically cross-link the microgels and form small flocculates. As the reaction process goes on, more flocculates are formed, which then further aggregate and eventually integrate into a whole hydrogel entity. Under the conditions studied in this work, gel formation occurs within 20 h. It is useful to note that system setup and diffusion control have drastic influence on the dynamic process of microgel particle flocculation and gel formation. Mineralization of CaCO3 within PNIPAMAC Microgel Dispersion. When CO2 diffused into PNIPAMAC microgels (1 wt %), the microgel dispersion became translucent, gelated in the first 5 h and then turned into a suspension after 48 h. The pH value increased to 9.1 from its initial 3.0, accompanying a small amount of the bulk precipitation of CaCO3 particles in the liquid phase. When the gelated dispersion was subject to freeze-drying, the surface of the dried phase was found to be extremely smooth, showing little morphological features from SEM. AFM was subsequently used to trace smaller surface structures (Figure S3), with clear microgel connections being unraveled from the joining up of many nanoparticles. After being mineralized for 48 h, the shell of the precursor microgel surface became less obvious as observed by TEM (Figure 4c), indicating the penetration of CaCO3 nanoparticles into the PNIPAMAC microgels. AFM imaging also showed that the PNIPAMAC/ 2304

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Scheme 2. Schematic Representation of the Mineralization Process of PNIPAMAC Microgels upon CO2 Diffusiona

a

At the beginning, PNIPAMAC microgels are well dispersed in solution (A), but upon CO2 diffusion with NH3, the PNIPAMAC microgels swell and glue to each other accompanied with CaCO3 nanoparticles formed on the surface of PNIPAMAC microgel particles, resulting in the increase of dispersion viscosity (B). As the reaction progresses, the PNIPAMAC microgels entrap more CaCO3 nanoparticles and become more compact, resulting in the formation of a segregated suspension (C).

Figure 7. AFM images of PNIPAM-based microgels deposited on glass substrate after immersing in water using 0.25 wt % particle suspensions at pH 7.4: (a) PNIPAM, (b) PNIPAM/CaCO3, (c) PNIPAMAC, and (d) PNIPAMAC/CaCO3. The images were taken under the same conditions for direct comparison.

structural effect to the outer surface region of the hybrid hydrogel particles as a result of CaCO3 incorporation. Figure 2b also shows that the phase transition for the hybrid particles shifted to lower temperature compared to the PNIPAMAC microgel, clearly showing the influence of incorporation of CaCO3 in the microgel structure. Thus, when the polymer chains near the core were fixed up, a lower temperature was needed to transform the mobile polymer chains on the surface into a collapsed conformation. X-ray diffraction patterns of the PNIPAMAC/CaCO3 hybrid particles identified the formation of amorphous CaCO3 (ACC). Measurements from atomic absorption spectroscopy revealed that the amount of ACC entrapped in the hybrid microgels was about 1%.

In these microgel systems, calcium carbonate crystals in the order of micrometers in size were usually obtained on a glass slide, resulting from solution mineralization. XRD revealed the formation of calcite crystals (Figure S1). Under different microgels but otherwise the same conditions, the morphologies of these deposited calcite crystals were very different. In the PNIPAM microgel dispersion, the calcite crystals were rhombohedra with six well-expressed {104} faces (Figure S1). However, in the PNIPAMAC microgel dispersion, most of the crystals looked like pine nuts (Figure 6a,b) even though XRD (Figure 6c) revealed that these crystals were also calcite. The difference may arise from the strong interaction between carboxylic acid groups and CaCO3 in the PNIPAMAC system. 2305

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Figure 8. Phase contrast inverted microscopy images of NIH-3T3 mouse fibroblast cells 48 h after incubation at 37 °C (a) and 30 min after staying at room temperature, 20−22 °C (b). The figures to the right show that more than 90% of the cells adhered at 37 °C (a). At the ambient temperature, approximately the same percentage of cell assumed a spherical shape and detached from the surface (b). More than 90% of the cells were removed from the surface by gentle rinsing with cell medium at room temperature (e); (c) and (d) enlarged area circled in (a) and (b), respectively; for (a), (b), and (e), bar = 200 μm; for (c) and (d), bar = 50 μm.

Although the exact mode of mediation requires further research, the general effect imposed by organic compounds is broadly similar to the features unraveled from amino acids and proteins in our previous studies.5,18−20 Compared with the PNIPAM microgels, the carboxylates at the periphery of the PNIPAMAC microgels have a strong interaction with the Ca2+ ions.42 The molecular recognition may well exist between the PNIPAMAC microgels and the surface of the calcite formed. Thus, the calcite grows through a distinct anisotropic process by which the PNIPAMAC microgels can act on crystal selectivity. Scheme 2 outlines the mineralization process of the PNIPAMAC microgels. The process is pH sensitive due to the acrylic acid monomer present within the copolymer, with dissociation pKa around 4.3. The dispersion had a low pH value around 3.0 before CO2 diffusion and the deionization of carboxylic groups led to an increase in the strength of hydrogen bonds. The PNIPAMAC microgels become shrunk and transparency decreased at a low pH. When NH3 vapor is slowly diffused into the dispersion accompanying CO2, the pH becomes increased. At pH > 4.3, the carboxylic groups are increasingly ionized, the PNIPAMAC microgels become swollen and transparency increases. The liquid appears viscous and gelation starts to occur due to the fusion of the microgels. At the same time, the carboxylate groups generated provide active nucleation sites for calcium mineralization.46,47 CaCO3

particles spontaneously form both in the porous shells and in bulk solution. As the reaction progresses, the density of the hybrid microgels increases due to the greater amount of CaCO3 nanoparticles formed and the hybrid microgels grow bigger in size and become more compact, resulting in the formation of segregated suspension associated with the reduction of dispersion viscosity. Applications of the Mineralized Microgels. Schmidt et al. have recently formed homogeneous and close-packed PNIPAM microgel films on PEI-precoated glass coverslips, which were successfully used for cell growth and removal through thermoresponsive switching.43 Hydrogel bead attachment avoids challenging covalent coupling procedures as often developed for generating surfaces switchable to cell adhesion.26 In this work, we found that the PNIPAMAC/CaCO3 microgels could also form smooth and close-packed monolayer films over an adequate range of concentration and surface conditions (Figure S4). We have thus deposited PNIPAMAC/CaCO3 microgels on glass coverslips, followed by thorough water evaporation. The coated surfaces were subsequently immersed in water for a few hours. The AFM images taken from different PNIPAM films after water swelling and rinsing are shown in Figure 7. Before immersing in water, all surfaces were covered with microgel particles. After immersing in water, however, the surface morphology and packing density look different, showing 2306

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contributed to the film stability, essential for the observed healthy cell growth and detachment. Nonenzymatic cell detachment avoids damages to cells as a result of chemical cleavages to extracellular matrix (ECM) proteins. Thermoresponsive cell detachment at the end of adherent cell culturing is particularly attractive because this process avoids ECM damages that could otherwise undermine their differentiation.

the different strengths of confinement to the substrate surface. The PNIPAM and PNIPAMAC microgels could not form wellpacked films due to the lack of surface binding force, though the PNIPAM microgels appear to perform better, consistent with the observation reported.44 In contrast, PNIPAM/CaCO3 microgels could not adhere on the substrate at all under the experimental conditions. However, deposition of the PNIPAMAC/CaCO3 microgels led to the formation of a highly stable and smooth film. Immersion in water in this case helps remove particles loosely attached to the outer surface but did not cause any detachment of particles in direct contact with the substrate. The stability clearly arose from the combined effect of the introduction of acrylic monomers and CaCO3 mineralization. Subsequent ellipsometric measurements were undertaken to monitor film stability over a period of 5 days. The results showed no measurable changes, suggesting that such film would remain stable over the typical period of cell culture. To test the feasibility of the films formed from PNIPAMAC/ CaCO3 microgels for cell culture, NIH 3T3 cells were seeded on the microgel films in cell culture medium and incubated at 37 °C. At day 2, the cells adhered efficiently and spread well, indicating that the microgel film surfaces were adherent at the physiological temperature (Figure 8a). This behavior is comparable to cells seeded on surface-grafted PNIPAM brushes and hydrogels.26,45 After cooling down to room temperature, nearly 90% cells assumed spherical shape within 15 min (Figures 8b and S6). At this stage, the cells could be effectively detached from the surface and removed by gentle rinsing, confirming that the PNIPAMAC/CaCO3 microgel-coated surface can be efficiently switched from cell-adherent to cellrepellent by convenient temperature stimuli (Figure 8e). MTT assays were used to assess cell viability at days 1, 2, and 4. The experiments were done to compare cells grown on the thermoresponsive microgel films with bare glass coverslips as control. The results indicate that cell growth on the PNIPAMAC/CaCO3 microgel films is equivalent to that on the bare glass coverslips, showing no toxicity to inhibit cell growth (Figure S5). In contrast, PNIPAM and PNIPAMAC microgel surfaces were improper for this purpose due to their lack of sufficient film stability, resulting in detachment of some of the microgels from the substrate. However, on areas where PNIPAM/ PNIPAMAC microgel films remained, cells could still grow and detach, though the effectiveness for thermoresponsive detachment was not as good as on the PNIPAMAC/CaCO3 microgelcoated films (Figure S7). In addition, cell detachment at ambient temperature was not observed on the bare coverslips (Figure S8). After the cells were detached from the film surface, the microgel film remained mostly intact, but minor detachment of the PNIPAMAC/CaCO3 microgel particles could still be observed (Figure S9). The general cell responses are in broad agreement with the physiochemical properties provided by the close-packed thermosensitive microgel particle films, which undergo a sharp phase transition by temperature changes. Under normal culture conditions at 37 °C, the microgel particles are relatively hydrophobic and cells attach, spread, and proliferate, similar to typical tissue culture surfaces. However, when temperature is reduced below the microgel’s LCST of 32 °C, the film becomes hydrophilic and swells, forcing the cells to detach spontaneously without the need for enzymatic or mechanical treatments. The crucial role played by the mineralization process in the case of PNIPAMAC/CaCO3 microgel particles

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CONCLUSIONS Two types of poly(N-isopropylacrylamide)-based microgels were successfully developed and explored as templates for calcium carbonate mineralization in this work. The morphologies of the hybrid materials could be controlled by modulating the structure of the comonomers. Either macroscopic hydrogels or individual hybrid microgels could be obtained by using PNIPAM or PNIPAMAC microgels as templates. Interactions between CaCO3 nanoparticles and microgels were used to prepare different morphological hybrid materials. Both PNIPAM and PNIPAMAC microgels with and without CaCO3 showed broadly similar thermoresponsive switches in hydration/dehydration, but incorporation of comomoners such as acrylic acid and different amounts of ACC nanoparticles provided the hybrid microgels different properties for technological exploitations. Specifically, PNIPAMAC/CaCO3 microgels formed stable close-packed films on glass coverslips that facilitated good cell growth at 37 °C, followed by efficient thermoresponsive detachment at an ambient temperature. Thermoresponsive cell detachment can avoid ECM damages in contrast to enzymatic cleavages at the end of cell culturing. Thus, thermoresponsive microgel films offer a convenient route for generating switchable surfaces for cell adhesion and detachment.

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ASSOCIATED CONTENT

S Supporting Information *

Detailed experiment procedures and supplementary results. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86-532-86981569 (H.X.); +44-161-3063926 (J.R.L.). E-mail: [email protected] (H.X.); [email protected] (J.R.L.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support from the Natural Science Foundation of China (#20804057), Natural Science Fund of Shandong Province (#Q2008B03), Research Fund for the Doctoral Program of Higher Education (#200804251527), and the U.K. Physical Sciences and Engineering Research Council (EPSRC; EP/D064945/1, EP/ F062966/1).

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REFERENCES

(1) Dorozhkin, S. V.; Epple, M. Angew. Chem., Int. Ed. 2002, 41, 3130−3146. (2) Stupp, S. I.; Braun, P. V. Science 1997, 277, 1242−1248. (3) Sanchez, C.; Arribart, H.; Guille, M. M. G. Nat. Mater. 2005, 4, 277−288.

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(39) Wehnert, F.; Pich, A. Macromol. Rapid Commun. 2006, 27, 1865−1872. (40) Loos, W.; Verbrugghe, S.; Goethals, E. J.; Du Prez, F. E.; Bakeeva, I. V.; Zubov, V. P. Macromol. Chem. Phys. 2003, 204, 98−103. (41) Zhang, G.; Wang, D. Y.; Gu, Z. Z.; Hartmann, J.; Mohwald, H. Chem. Mater. 2005, 17, 5268−5274. (42) Perkin, K. K.; Turner, J. L.; Wooley, K. L.; Mann, S. Nano Lett. 2005, 5, 1457−1461. (43) Schmidt, S.; Hellweg, T.; von Klitzing, R. Langmuir 2008, 24, 12595−12602. (44) Schmidt, S.; Zeiser, M.; Hellweg, T.; Duschl, C.; Fery, A.; Mohwald, H. Adv. Funct. Mater. 2010, 20, 3235−3243. (45) Kumar, A.; Srivastava, A.; Galaev, I. Y.; Mattiasson, B. Prog. Polym. Sci. 2007, 32, 1205−1237. (46) Grassmann, O.; Lobmann, P. Biomaterials 2004, 25, 277−282. (47) Filmon, R.; Grizon, F.; Basle, M. F.; Chappaard, D. Biomaterials 2002, 23, 3053−3059.

(4) Han, Y. J.; Wysocki, L. M.; Thanawala, M. S.; Siegrist, T.; Aizenberg, J. Angew. Chem., Int. Ed. 2005, 44, 2386−2390. (5) Wu, C. M.; Wang, X. Q.; Zhao, K.; Cao, M. W.; Xu, H.; Xia, D. H.; Lu, J. R. Cryst. Growth Des. 2011, 11, 3153−3162. (6) Han, Y. J.; Aizenberg, J. Angew. Chem., Int. Ed. 2003, 42, 3668− 3670. (7) Belcher, A. M.; Wu, X. H.; Christensen, R. J.; Hansma, P. K.; Stucky, G. D.; Morse, D. E. Nature 1996, 381, 56−58. (8) Meldrum, F. C.; Colfen, H. Chem. Rev. 2008, 108, 4332−4432. (9) Sommerdijk, N. A.; de With, G. Chem. Rev. 2008, 108, 4499− 4550. (10) Gao, Y. X.; Yu, S. H.; Cong, H.; Jiang, J.; Xu, A. W.; Dong, W. F.; Colfen, H. J. Phys. Chem. B 2006, 110, 6432−6436. (11) Pouget, E. M.; Bomans, P. H.; Goos, J. A.; Frederik, P. M.; de With, G.; Sommerdijk, N. A. Science 2009, 323, 1455−1458. (12) Volkmer, D.; Harms, M.; Gower, L.; Ziegler, A. Angew. Chem., Int. Ed. 2005, 44, 639−644. (13) Han, J. T.; Xu, X. R.; Kim, D. H.; Cho, K. Adv. Funct. Mater. 2005, 15, 475−480. (14) Xu, G. F.; Yao, N.; Aksay, I. A.; Groves, J. T. J. Am. Chem. Soc. 1998, 120, 11977−11985. (15) Lakshminarayanan, R.; Loh, X. J.; Gayathri, S.; Sindhu, S.; Banerjee, Y.; Kini, R. M.; Valiyaveettil, S. Biomacromolecules 2006, 7, 3202−3209. (16) Aizenberg, J.; Lambert, G.; Weiner, S.; Addadi, L. J. Am. Chem. Soc. 2002, 124, 32−39. (17) Addadi, L.; Raz, S.; Weiner, S. Adv. Mater. 2003, 15, 959−970. (18) Wang, X. Q.; Sun, H. L.; Xia, Y. Q.; Chen, C. X.; Xu, H.; Shan, H. H.; Lu, J. R. J. Colloid Interface Sci. 2009, 332, 96−103. (19) Wang, X.; Kong, R.; Pan, X.; Xu, H.; Xia, D.; Shan, H.; Lu, J. R. J. Phys. Chem. B 2009, 113, 8975−8982. (20) Wang, X. Q.; Wu, C. M.; Tao, K.; Zhao, K.; Wang, J. Q.; Xu, H.; Xia, D. H.; Shan, H. H.; Lu, J. R. J. Phys. Chem. B 2010, 114, 5301− 5308. (21) Yoshida, R.; Sakai, K.; Okano, T.; Sakurai, Y. Adv. Drug Delivery Rev. 1993, 11, 85−108. (22) Coughlan, D. C.; Quilty, F. P.; Corrigan, O. I. J. Controlled Release 2004, 98, 97−114. (23) Na, K.; Park, J. H.; Kim, S. W.; Sun, B. K.; Woo, D. G.; Chung, H. M.; Park, K. H. Biomaterials 2006, 27, 5951−5957. (24) Kawaguchi, H.; Fujimoto, K. Bioseparation 1998, 7, 253−258. (25) Tan, I.; Zarafshani, Z.; Lutz, J. F.; Titirici, M. M. Appl. Mater. Interface 2009, 1, 1869−1872. (26) Yang, L.; Pan, F.; Zhao, X. B.; Yaseen, M.; Padia, F.; Coffey, P.; Freund, A.; Yang, L. Y.; Liu, T. Q.; Ma, X. H.; Lu, J. R. Langmuir 2010, 26, 17304−17314. (27) Zhao, Z. Q.; Chen, Z. B.; Zhao, X. B.; Pan, F.; Cai, M. H.; Wang, T.; Zhang, H. G.; Lu, J. R.; Lei, M. J. Biomed. Sci. 2011, 18, DOI: 10.1186/1423-0127-18-37. (28) Yang, J.; Yamato, M.; Shimizu, T.; Sekine, H.; Ohashi, K.; Kanzaki, M.; Ohki, T.; Nishida, K.; Okano, T. Biomaterials 2007, 28, 5033−5043. (29) Kuang, M.; Wang, D. Y.; Gao, M. Y.; Hartmarm, J.; Mohwald, H. Chem. Mater. 2005, 17, 656−660. (30) Nassif, N.; Gehrke, N.; Pinna, N.; Shirshova, N.; Tauer, K.; Antonietti, M.; Colfen, H. Angew. Chem., Int. Ed. 2005, 44, 6004− 6009. (31) David, G.; Simionescu, B. C.; Albertsson, A. C. Biomacromolecules 2008, 9, 1678−1683. (32) Varga, I.; Gilanyi, T.; Meszaros, R.; Filipcsei, G.; Zrinyi, M. J. Phys. Chem. B 2001, 105, 9071−9076. (33) Lopez, C. Adv. Mater. 2003, 15, 1679−1704. (34) Xia, Y. N.; Gates, B.; Yin, Y. D.; Lu, Y. Adv. Mater. 2000, 12, 693−713. (35) Sakai, T.; Takeoka, Y.; Seki, T.; Yoshida, R. Langmuir 2007, 23, 8651−8654. (36) Tsuji, S.; Kawaguchi, H. Langmuir 2005, 21, 2434−2437. (37) Tsuji, S.; Kawaguchi, H. Langmuir 2005, 21, 8439−8442. (38) Brecevic, L.; Nielsen, A. E. J. Cryst. Growth 1989, 98, 504−510. 2308

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