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Advanced Artificial Extracellular Matrices using Amphiphilic NanogelCrosslinked Thin Films to Anchor Adhesion Proteins and Cytokines Yoshihide Hashimoto, Sada-atsu Mukai, Shin-ichi Sawada, Yoshihiro Sasaki, and Kazunari Akiyoshi ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00485 • Publication Date (Web): 09 Feb 2016 Downloaded from http://pubs.acs.org on February 23, 2016

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ACS Biomaterials Science & Engineering

Advanced Artificial Extracellular Matrices using Amphiphilic Nanogel-Crosslinked Thin Films to Anchor Adhesion Proteins and Cytokines

Yoshihide Hashimoto†, Sada-atsu Mukai†, ‡, Shin-ichi Sawada, Yoshihiro Sasaki, and Kazunari Akiyoshi* Department of Polymer Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto 615-8510, Japan Japan Science and Technology Agency (JST), The Exploratory Research for Advanced Technology (ERATO), Katsura Int’tech Center, Katsura, Nishikyo-ku, Kyoto 615-8530, Japan

†

These authors contributed equally to this work.

* Correspondence and requests for materials should be addressed to K.A. Email: [email protected]

ACS Paragon Plus Environment

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ABSTRACT

A novel type of nanogel-crosslinked (NanoClik) film composed of acryloyl-modified cholesterol-bearing pullulan nanogels with pentaerythritol tetra(mercaptoethyl) polyoxyethylene as a crosslinker is created through the Michael addition coupled with solvent evaporation. Tensile testing and atomic force microscopy shows that the elastic property of the NanoClik films can be controlled by changing the crosslinker concentration. The NanoClik films strongly absorb proteins after simple immersion in solutions of functional proteins, including the hormone insulin, cytokine bone morphogenetic protein-2 (BMP-2), and vitronectin. The amphiphilic nanogels in the films induce this absorption by acting as anchoring and loading proteins. Mouse embryo fibroblast cells adhere to and proliferate on the NanoClik films anchoring vitronectin, while NanoClik films loaded with BMP-2 strongly increase the differentiation of human mesenchymal stem cells into osteoblasts. These results suggest that the NanoClik films act as novel artificial extracellular matrix that enable the reservation of various biological proteins to the nanogels.

Keywords: self-assembled nanogels, nanogel tectonics, bio-inspired artificial extracellular matrix, chaperone-like activity, tissue engineering

Introduction Developing smart materials to control cellular functions and behaviors is a key challenge in the fields of regenerative medicine and tissue engineering. Biological cells generally communicate with the extracellular environment and surrounding cells and tissues via signaling molecules, namely, biological mediators, which control cell function and behavior, including


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cellular responses and fate.1-2 Growth factors in particular play a critical role by regulating cell behavior including migration, proliferation, differentiation, and matrix synthesis. Thus, growth factors are a key regulator of cellular function for regenerative medicine applications, such as the treatment of injured tissue.3-4 However, water-soluble growth factors are rapidly deactivated by enzymes and are sensitive to chemical and physical degradation,5 which results in their extremely short half-life, on the order of several minutes, in vivo.6-10 In addition, a long-term large-dose administration is needed to achieve sufficient therapeutic effects, and distribution control is also necessary to avoid undesirable side effects caused by the diversity of bioactivity throughout the body.11 To address these issues, various spatially and temporally controlled delivery systems for signaling molecules, including growth factors, have been reported. In biological systems, extracellular matrix (ECM) plays a crucial role in managing and presenting growth factors for cells. For instance, for growth factors that directly bind to ECM, the ECM acts as a reservoir that can readily supply growth factor and as a barrier to protect them from degradation.12 In addition, ECM provides cell adhesion sites, allowing for an increased local concentration of growth factors in proximity to their specific receptors embedded in the membranes of attached cells. Further, the physical properties, such as stiffness and surface morphology, among others, of the ECM also affect cell behaviors.13-16 Thus, controlling the physical properties of the ECM, as well as its interactions with growth factors, is of crucial importance. A variety of artificial ECMs (aECMs), both naturally derived and chemically synthesized, have been reported.17 The properties of the aECM surfaces can be modified by coating proteins, which provide cell adhesion sites. Naturally derived hydrogels aECMs have frequently been used to provide cellular microenvironments.18 Such hydrogels act as a reservoir


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for relatively large molecules, including growth factors, allowing controlled diffusion of the molecules trapped within their polymer network.19 Dry gel films (e.g., collagen-based films) have also been used as aECM.20-21 These films are advantageous for their stability and ease of handling due to their shape retention. In addition, various bioactive molecules can be loaded into the films by layer-by-layer deposition, for example.22-23 Electrospun nanofibrous films with high specific surface area and structural toughness have also been used as scaffolds for regenerative medicine.24-26 Our approach for constructing aECMs is to use amphiphilic nanogels that are prepared through self-assembly of hydrophobized polysaccharides. We proposed the concept of “nanogel tectonics”, which involves the construction of functional hierarchical gels and bio-interfaces using polymerizable nanogels and crosslinkers.27-28 This polymerizable cholesterol-bearing pullulan (CHP) nanogel-crosslinked (NanoClik) gel allows protein encapsulation and release. The gel, was developed from CHP nanogels without chemical modification of the target molecules,29-30 such as cytokine bone morphogenetic protein-2 (BMP-2), which is useful for bone tissue engineering.31 Recently, NanoClik porous gels and NanoClik microspheres were also reported based on similar nanogel tectonic methods.28, 32 However, the strength, flexibility, and stability of existing NanoClik gels have been insufficient to withstand biomechanical stress and storage. Here, we report a novel type of NanoClik dry thin film created by solvent evaporation of NanoClik hydrogels obtained by the reaction of acryloyl group-modified cholesterol-bearing pullulan (CHPOA), as the polymerizable nanogel, and pentaerythritol tetra (mercaptoethyl) polyoxyethylene (PEGSH) as the crosslinker.33 The resulting NanoClik films are transparent and flexible shape-retaining films whose elasticity can easily be controlled by changing the


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crosslinker concentration. The characterization of these films and their function as an aECM are reported in the present study.

Experimental Section Materials: CHP, in which pullulan (Mw: 1×105 g mol−1) was substituted with 1.2 cholesterol moieties per 100 anhydrous glucoside units and PEGSH (Mw: 1×104 g mol−1) was purchased from NOF corporation (Tokyo, Japan). 2-Acryloyloxyethyl isocyanate (AOI) was purchased from Showa Denko K. K (Tokyo, Japan). Di-n-butyltin (IV) dilaurate (DBTDL; 95%), Rhodamine B isothiocyanate, and FITC-labelled recombinant human insulin were purchased from Sigma-Aldrich Co., LLC (St. Louis, MO, USA). Dimethylsulfoxide (DMSO, Super Dehydrated grade) was purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Fetal bovine serum (FBS) was purchased from Life Technologies Corporation (Carlsbad, CA, USA). All other regents were obtained from commercial sources and were used without further purification.

Synthesis of CHPOA: CHPOA was synthesized as previously described.34 Briefly, CHP (2.5 g) was dissolved in 100 mL of super dehydrated DMSO, and then DBTDL (10 mM) and AOI (30 mM) were added. Next, the mixture was stirred at 45 °C for 24 h and dropped into excess ether/ethanol. The precipitate was dissolved in DMSO, dialyzed with milli-Q water, and lyophilized. The degrees of substitution of the acryloyl groups were determined by 1H NMR (400MHz, Bruker Biospin, Avance 400, DMSO-d6/D2O: 9/1 (v/v), 25 °C), δ: 0.64 (s, 3H, cholesterol 18-H), 0.80–2.40 (m, cholesterol H), 2.95–4.00 (glucose, 2-H, 3-H, 4-H, 5-H, and 6H), 4.69 (1H, glucose 1-H (1→6)), 5.03 (2H, glucose, 1-H (1→4)), and 5.96–6.37 (3H,


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CH2=CH-). The hydrodynamic diameter of the CHPOA nanogels was measured by dynamic light scattering (DynaPro NanoStar; Wyatt Technology Corporation, Santa Barbara, CA, USA).

Synthesis of acryloyl group-modified CHP-Rh (CHPOA-Rh): rhodamine-labeled CHP (CHPRh) was synthesized as previously described.34 Briefly, CHP (3.0 g) was dissolved in 150 ml of dehydrated DMSO containing DBTDL (20 mM). Separately, rhodamine B isothiocyanate (4 mM) was dissolved in dehydrated DMSO and stirred for 30 min at room temperature. The two solutions were combined and stirred at 45°C for 24 h and added to excess ether/ethanol. The precipitates were dissolved in DMSO, dialyzed with DMSO for 1 day and milli-Q water for 7 days, and then lyophilized. The degree of substitution of rhodamine groups was assessed by ultraviolet (UV) measurement at 558 nm using a UV/visual spectrophotometer (V-660, Jasco, Tokyo, Japan). CHP-Rh contained 0.3 rhodamine groups per 100 anhydrous glucoside units. Acryloyl group-modified CHP-Rh was synthesized in the same manner as CHPOA.

Preparation of the NanoClik Films: The NanoClik films were prepared by combining a Michael addition between the acryloyl groups of the CHPOA and the thiol group of the PEGSH with solvent evaporation. Briefly, CHPOA was dissolved in milli-Q water at a concentration of 3%. Separately, PEGSH was dissolved in milli-Q water and then added into the CHPOA solution. The molar ratios of acryloyl to thiol groups were 100:1, 100:2, or 100:10. The mixtures were placed in polystyrene dishes and incubated at 40 °C for at least 24 h to create the diskshaped NanoClik films.


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Film Thickness and Swelling Ratio: The film thickness was measured using a micrometer with 2-µm accuracy (MDQ-30M; Mitutoyo Corporation, Kawasaki, Japan). The values are expressed as the mean ± standard deviation (n=6). The swelling ratio of the NanoClik films was determined by immersion in water. Each specimen was weighed dry (Wd), and then immersed in 5 mL of water at 25 °C. After 24 h, the specimens were reweighed (Wh) after removing excess fluid by blotting on filter paper. The swelling ratio was calculated using the following equation: Swelling ratio (%) =

Wh − Wd × 100 Wd

(1)

The values are expressed as the mean ± standard deviation (n=5).

Tensile Testing: The mechanical properties of the NanoClik films were measured using a dynamic shear rheometer (MCR302; Anton Paar GmbH, Graz, Austria) equipped with a tensile testing module and Peltier temperature controller. All specimens were cut into dumbbell shapes (12 mm long and 2 mm wide), and the thickness was measured using a micrometer before tensile testing. A 50 N load cell was employed, and the specimens were loaded at a rate of 10 mm/min at 40 °C. Four specimens from each group were each separately tested.

Contact angle measurement: Contact angle of NanoClik films under dry and wet conditions was determined by a half angle method from the side-viewed shape of an ultrapure water droplet on the film. In the case of dry film, 3 µl of ultrapure water was dropped on the dry NanoClik film surface, and the shape was immediately observed from the side. In the case of wet film, the NanoClik film was swollen in ultrapure water, and excess water on the top of the swollen film was removed with a filter paper. Before the film had been completely dried, 3 µl of ultrapure water was dropped on the film surface, and the shape was immediately observed from the side.


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AFM Force Measurement: The elasticity of hydrated NanoClik films was measured using a NanoWizard Ultra Speed AFM (JPK Instruments AG, Berlin, Germany). The specimens were loaded with rectangular silicon nitride cantilevers (OMCL-RC800PSA; Olympus Corp., Tokyo, Japan) that were 100 µm long, 40 µm wide, and 800 nm thick, with a resonant frequency of 71 kHz and spring constant of 0.76 N m−1. AFM images were collected using Quantitative Imaging mode (QI-mode, JPK), working in a liquid environment. For force mapping, a sequence of forcedistance curves was collected over different 30-µm × 30-µm areas of the NanoClik films. The collected force spectra were analyzed using JPK data processing software. A baseline was first subtracted from the non-contact z-range of the force–displacement data, and the effect of cantilever bending was also subtracted, resulting in the force–deformation (F-δ) data.

Protein Trapping and Release: The NanoClik films (φ: 8 mm) were immersed in 1 mL of FITC-insulin (80 µg mL−1; Sigma-Aldrich) in PBS pH 7.4 (GIBCO, Life Technologies) at 25 °C. Next, 5 µL of supernatant from each sample was collected at specific times and the fluorescence intensity was measured at an excitation wavelength of 495 nm and an emission wavelength of 519 nm using a fluorescent spectrometer (FP-8500; JASCO, Tokyo, Japan) equipped with a onedrop measurement unit (SAF-851; JASCO). The encapsulation efficiency was estimated based on the decrease in fluorescent intensity of the FITC-insulin bathing solution. The release of FITC-insulin from the NanoClik films in the presence of serum was also assessed. The NanoClik film (φ: 8 mm) loaded with FITC-insulin was immersed in 1 mL of PBS supplemented with 10% FBS at 37 °C. Then, 5-µL samples of the supernatant were collected at specific times and their


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fluorescence intensity was measured as described above. These experiments were repeated four times and the results are expressed as the mean ± standard deviation.

Degradation of the NanoClik Films: Rhodamine-labeled NanoClik films (φ: 8 mm) were immersed in 1 mL of PBS (pH 7.4) supplemented with 10% FBS at 37 °C. Then, 5-µL samples of the supernatant were collected at specific times. After sampling, 5 µL of fresh PBS supplemented with 10% FBS was immediately added to maintain a constant volume. The cumulative release of nanogels from the NanoClik film was estimated based on fluorescent spectrometry of the supernatant samples at an excitation wavelength of 550 nm and an emission wavelength of 580 nm. The experiments were repeated four times, and the results are expressed as the mean ± standard deviation.

Confocal Laser Scanning Microscopy: CLSM of the rhodamine-labeled NanoClik film was performed using a multiphoton microscope (LSM780; Carl Zeiss, Oberkochen, Germany) equipped with a Coherent Chameleon Vision S laser source and a GaAsP detector. Images of the rhodamine-labeled NanoClik films were obtained at a magnification of 64× with an excitation wavelength of 561 nm.

Cell Culture on the NanoClik Films: The mixtures of CHPOA nanogel and PEGSH were placed into 4-well glass bottom dishes and the Michael addition and solvent evaporation were performed to create the NanoClik film surfaces. The molar ratio of acryloyl to thiol groups was 100:0 (nanogel cast film), 100:1, 100:2, and 100:10. Each specimen was treated with 300 µl of re combinant human vitronectin (Vn) solution (20 µg/ml, Sigma-Aldrich) for 24 h at 37 °C and


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washed with PBS for 3 h to remove excess vitronectin prior to cell seeding. Mouse embryonic fibroblasts (NIH3T3, clone 5611; JCRB cell bank, Osaka, Japan) were seeded onto each of the NanoClik films at a density of 1×104 cells cm−2 and maintained in Dulbecco’s Modified Eagle Medium (DMEM; GIBCO, Life Technologies) supplemented with 1% antibiotics in a humidified atmosphere of 95% CO2 at 37 °C during 48 h.

Immunostaining: Cells were washed twice with PBS and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. After two more washes, the cells were permeabilized with 0.2% TritonX-100 in PBS for 5 min and blocked with PBS containing 5% goat serum for 30 min. Next, the specimens were incubated with anti-vinculin antibody (1:400 dilution, Clone hVIN-1, Sigma-Aldrich) overnight at 4 °C, followed by incubation with Alexa Fluor 568conjugated goat anti-mouse immunoglobulin G antibody (1:400 dilution, Life Technologies), Alexa Fluor 488-conjugated Phalloidin (0.2 µM, Life Technologies), and 2 mg mL−1 42,6diamidino-2-phenylindole dihydrochloride (DAPI, Dojindo Laboratories, Kumamoto, Japan). Finally, the specimens were washed twice with PBS and imaged by laser scanning confocal microscope (LSM780, Carl Zeiss). The cell numbers and areas were quantified using Image J software (NIH) according to pixel area (n=5).

Cell Viability: Cell viability was assessed following established methods.35 The NIH3T3 cells were cultured in DMEM supplemented with 10% FBS, penicillin (100 units mL−1; Life Technologies), and streptomycin (100 µg mL−1; Life Technologies). The cells were seeded into 96-well plates at a density of 1.5×104 cells cm−2. After incubation for 24 h at 37 °C, the culture medium was replaced by 100 µL of CHPOA nanogel that was dissolved in culture media (1–5


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mg mL−1), and the cells were incubated for another 24 h at 37 °C in 5% CO2. The cell viability was then determined using a modified MTT dye reduction assay using WST-8 2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (Cell counting kit-8, Dojindo Laboratories) according to the manufacturer’s instructions.

Human Mesenchymal Stem Cell Differentiation on the NanoClik Films: hMSCs (UCB408E6E7TERT-33), which were immortalized by transformation with HPV-E6, HPV-E7, and human Telomere Reverse Transcriptase, were purchased from the JCRB cell bank. Cells were cultured on three substrates: the Vn/NanoClik film, BMP2/Vn/NanoClik film, and TCPS. Cells were seeded onto each substrate at a density of 5×104 cells cm−2, maintained in mesenchymal stem cell basal medium (Plusoid-M; GlycoTechnica Ltd., Yokohama, Japan), and cultured for 2 days. After the cells reached confluence, the basal medium was replaced with osteogenic differentiation medium consisting of Plusoid-M supplemented with 200 ng mL−1 of recombinant human BMP-2 (HumanZyme Inc., Chicago, IL, USA) and cultured for 3 days. ALP staining and the ALP assay were performed to assess the osteogenic differentiation of the hMSCs. For ALP staining, the cells were fixed with citrate buffer containing 45% acetone and 10% methanol for 10 min at room temperature. The cells were treated with 5-bromo-4-chloro-3indolyl phosphate and nitro blue tetrazolium chloride solution for 30 min at 37 °C, rinsed three times with PBS, and observed using a fluorescence microscope equipped with a digital camera (BIOREVO BZ-9000; Keyence, Tokyo, Japan). For the ALP assay, the cells were lysed with 1% Nonidet P-40 and half of the volume was set aside for the protein assay. A 0.2 M Tris-HCl buffer (pH 9.5) containing 12.5 mM p-nitro-phenyl phosphate and 1 mM MgCl2 was added to the cell lysates and incubated for 30 min at 37 °C. The ALP activity was determined by the absorbance at


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405 nm using an EnSpire Multimode plate reader (PerkinElmer, Waltham, MA, USA). The values were expressed as nmol of p-nitrophenol produced per 30 min per mg of protein. The total protein contents were determined using Pierce BCA protein assay kits (Thermo Fisher Scientific Inc., Waltham, MA, USA).

Statistical Analysis: All results are expressed as the mean ± standard deviation. After assessing data set variances, Student's t-tests or one-way ANOVAs were used to detect significant differences among the experimental groups. P-values less than 0.05 were considered statistically significant.

Results and Discussion Preparation and Characterization of the NanoClik Films: CHPOA was synthesized as previously described.27-28 The degree of substitution of the acryloyl group was estimated to be 26 per 100 glucose units using 1H NMR (Supporting Information, Figure S1). The hydrodynamic diameter of the CHPOA nanogel was approximately 40 nm (Figure 1A, B). The NanoClik films were prepared by reacting the acryloyl groups of the CHPOA with the thiol group of the PEGSH upon solvent evaporation, as illustrated in Figure 2A. The effect of the molar ratio of acryloyl to thiol on the crosslinking was assessed to show the differences in the NanoClik film characteristics. The transparency of the NanoClik films is an important property for tissue engineering and cell culture applications because it allows observation of the healing process and cellular behavior. Transparent NanoClik films were created at low crosslinker concentrations of 0.066, 0.13 and 0.67% (acryloyl:thiol molar ratios of 100:1, 100:2, and 100:10) (Figure 2B–D), but not at higher crosslinker concentrations of 7.9%.


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(acryloyl:thiol molar ratio of 1:1). This difference can be attributed to partial gelation prior to the constitution of a thin film. In addition, the flexibility of the resulting NanoClik films increased with increasing crosslinker concentration. An adequate range of crosslinker (PEGSH) concentrations were tested to produce flexible and transparent NanoClik films. The thickness of each film was in the range of 16 to 24 µm, and there were statistical differences in thickness among the different crosslinker concentrations (P < 0.05), as shown in Table 1. The film thickness can easily be controlled within the range from a few micrometer to several hundreds of micrometers by changing the volume of the solution. The elasticity of biomaterials is another important property for tissue engineering and cell culture applications.16, 36

Elasticity depends on the crosslinking density, which can be controlled by the crosslinker

concentration. Therefore, adjusting the crosslinker concentration imbues the various types of NanoClik films with widely varying elastic properties. Typical stress–strain (S-S) curves for the nanogel cast film (without crosslinker) and three types of the NanoClik films are shown in Figure 3A. During the initial phase, the stress rapidly increased in proportion to the strain. The inset graph shows the linear response region (up to ~1%) of the S-S curves obtained from each NanoClik film. Tensile testing showed that NanoClik-2 and NanoClik-10 exhibited typical yield behavior with increasing stress during tension, but the nanogel cast film and NanoClik-1 did not. In general, chemical crosslinking is considered to be stronger than physical crosslinking, while the elastic properties of hydrogels with a high crosslinking density are much higher than that of hydrogels with low crosslinking density.37 Surprisingly, increasing the crosslinker concentration resulted in a drastic decrease in the Young’s modulus (E), yield strength (σyield), and ultimate tensile strength (σtensile), while the failure strain (εfailure) tended to markedly increase. This phenomenon may be attributed to the high mobility of the polyethylene glycol (PEG) chains.38


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For a more quantitative analysis, we summarized the dependence of E, σyield, σtensile, and εfailure on crosslinker concentration (Figure 3B–E). E, σyield, and σtensile show an exponential decay, while εfailure rises exponentially, with increasing crosslinker concentration. Thus, these results suggest that the NanoClik films created with high crosslinker concentration are much more flexible than those created with low crosslinker concentration, and indicate the possibility of controlling the flexibility of the NanoClik films. Further characterization of the transparent NanoClik films was performed using a swelling test and water content analysis (Table 1). The NanoClik films had high water absorbability and reached equilibrium swelling within a few minutes. The equilibrium swelling ratio decreased with increasing crosslinker concentration, but the water content after reaching equilibrium swelling remained nearly the same. The surface morphology of the NanoClik films created with all crosslinker concentrations was extremely smooth without any pores, and there were no apparent morphological differences among them, as assessed by scanning electron microscopy. The nanogel structure of re-swollen NanoClik films was also observed by stimulated emission depletion (STED)39 microscopy (Supporting Information, Figure S2). The water contact angle was measured to characterize the surface hydrophilicity and wetting properties. The static contact angles of the NanoClik films in dry and wet conditions are shown in Figure 4A. The contact angles of 80°–100° of the NanoClik films in the dry condition showed that the surfaces were relatively hydrophobic, possibly because of the cholesteryl groups of the CHP nanogel. Increasing the PEGSH content decreased the contact angles, suggesting that the fraction of PEG chains at the surface had increased. In the wet condition, all NanoClik film contact angles were close to 48°–57°, independent of the crosslinker concentration. The polysaccharide chains should be located at the surface of the films in the wet condition.


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Interactions Between the NanoClik Films and Proteins: To demonstrate the potential of the NanoClik films as in vitro biomimetic reservoirs for protein delivery, we investigated the interactions between the NanoClik films and FITC-insulin as a model protein. Concerning the nature of CHP nanogel-protein (insulin) interaction, we had previously discussed in detail.40-41 Hydrophobic associated domain by cholesterol groups of CHP was important to the interaction with proteins. After immersion in a solution of FITC-insulin, the amount of FITC-insulin trapped in the NanoClik film gradually increased over time, reaching a plateau within 24 h. This implies that the CHP nanogels effectively trapped proteins mainly through hydrophobic interactions (Figure 5A). However, the maximum loading amount decreased with increasing crosslinker concentration, indicating that the encapsulation of FITCinsulin within the CHP nanogels was inhibited by the PEG chains. In fact, PEG hydrogels are very resistant to protein adsorption and cell adhesion.42 In addition, we visualized the distribution of FITC-insulin within the NanoClik-1 using confocal laser scanning microscopy (CLSM). The CLSM images demonstrated that the FITC-insulin interacted with the NanoClik film in a homogeneous manner (Figure 5B). Notably, this complex was remarkably stable, as the FITCinsulin remained in the NanoClik film without spontaneous release for at least 1 month in phosphate-buffered saline (PBS; pH 7.4) at 4 °C. The release profiles of FITC-insulin sequestered within the NanoClik films into PBS supplemented with 10% fetal bovine serum were estimated (Figure 5C). No burst release of FITC-insulin from the NanoClik films was observed, and the release profiles could be controlled by changing the crosslinker concentration. The degradation of the NanoClik films and the releases of nanogels were also investigated in the same conditions using rhodamine-labeled CHP nanogels (Figure 4B). The nanogels were


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gradually released from the NanoClik films through progressive hydrolysis of the ester bond. The release behaviors nearly coincided with the release of FITC-insulin. These results suggest that the FITC-insulin was released from the film as a protein-nanogel complex upon degradation of the chemically crosslinked moiety, an ester bond.

Cell Culture on the NanoClik Films: We investigated the cell adhesion and spreading patterns of mouse embryo fibroblasts (NIH3T3) cells on the NanoClik films. On the nanogel cast film, the cells adhered, but did not spread out, retaining a round shape (Figure 6A). In contrast, the cells aggregated and did not adhere to any of the NanoClik films, regardless of the PEGSH concentration (Supporting Information, Figure S3A–C). This result suggests that the NanoClik films inhibit nonspecific cell adhesion, likely because of the PEG crosslinker components. The CHP nanogels, components of the NanoClik films, strongly interact with proteins, mainly through hydrophobic interactions.40-41 The CHP nanogel domains on the surfaces of the films should act as anchoring sites for ECM adhesion proteins such as vitronectin and members of the integrin family (αvβ1, αvβ3, αvβ5, αIIbβ3) via the RGD motif.43 Therefore, we investigated the adhesion of cells onto each NanoClik film in the presence of vitronectin. Vitronectin binds to cell adhesion proteins such as integrin on cell membrane. As a result, cells can attach to the film and form stress fiber, in other words, cells can growth properly and take a spreading form. The NanoClik films complexed with vitronectin allowed cells to adhere and spread out (Figure 6B–D), implying that the nanogels within the NanoClik films interacted with the vitronectin, promoting cell adhesion. As a control for comparison, cells were also cultured on pullulan-crosslinked (PClik) films treated with vitronectin (Supporting Information, Figure S3D–F). No cell adhesion occurred on the PClik


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films, despite the vitronectin treatment. To visualize the actin fibers and focal adhesions, the cells were simultaneously stained with Alexa Fluor 488-conjugated phalloidin and anti-vinculin antibody.44 Interestingly, the CLSM images demonstrated that the cells cultured on NanoClik-10 film showed prominent actin filaments, whereas most of the cells cultured on NanoClik-1 and NanoClik-2 were spread out with a partially cobblestone-like appearance and lacked mature stress fibers (Figure 7F–H). The number of adherent cells and the area of cell spreading were quantified using image processing software (ImageJ, NIH, Bethesda, MD, USA). Both the number of adherent cells and the cell area significantly increased with increasing crosslinker concentration (Figure 6I–L). Cell adhesion and actin organization depend strongly on the properties of the substrate, especially the matrix stiffness.45 Many cells cultured on soft substrates with low elasticity fail to elongate and form stress fibers, whereas cells cultured on hard substrates with high elasticity tend to form stress fibers and focal adhesions.45-46 Therefore, we investigated the local stiffness of each NanoClik film using force mapping via atomic force microscopy (AFM) (Figure 7). The local stiffness increased with increasing crosslinker concentration. Thus, the difference in cellular behaviors on the NanoClik films is likely caused by the differences in elasticity of the NanoClik films in their hydrated condition. Comparing the result to that of the tensile test, the dependency of measured stiffness on crosslinker (PEGSH) concentration was opposite. In the case of the tensile test, the film was entirely stretched on the dry condition. With the increase of the PEG contents of the films, the failure strains increased. This is probably due to the lower Tg and higher mobility of PEG compared with that of polysaccharide, pullulan. On the other hands, in the AFM measurement, we could obtain information of nanometer scale elasticity under the hydrated condition. The experimental conditions (dry or wet) and the scale of observation (macro or nanoscale) are completely


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different between the tensile test and the AFM measurement. In the AFM measurement, measured Young’s modulus corresponds to the mobility of network of nanoscale in the surface of the films. The increase of the contents of PEG derivative as crosslinker resulted in the increase of the density of crosslinking points and the increase of Young’s modulus.

Differentiation of Human Mesenchymal Stem Cells on the NanoClik Films: BMPs promote the differentiation of mesenchymal stem cells (MSCs) toward the osteogenic lineage and are also widely used for bone tissue engineering.47 However, their clinical application has been limited by their short half-life, rapid local clearance, and adverse side effect.48 Here, we aimed to demonstrate the efficacy of the NanoClik film as a growth factor reservoir for delivering osteoinductive biomolecular cues to human MSCs in a controlled manner. Alkaline phosphatase (ALP) activity is a well-known early marker of osteogenesis. Therefore, the osteogenic differentiation of hMSCs cultured on tissue culture polystyrene (TCPS), NanoClik-10, and NanoClik-10 preloaded with a small amount of BMP-2 was assessed by ALP staining (Figure 8). All of the culture conditions supplemented with exogenous BMP-2 in the basal media appeared to result in an increased number of ALP-positive cells compared with those without exogenous BMP-2 in the basal media (Figure 8A–F). Quantification of the ALP activity, which demonstrated the enzymatic quantity per mg of protein, was consistent with the ALP staining (Figure 8G). The presence of exogenous BMP-2 in the basal media had little effect on the ALP activity of the cells on the TCPS and NanoClik-10. It is notable that the ALP activity of the BMP-2/NanoClik-10 group without exogenous BMP-2 in the basal media was approximately twice as high as any of the other groups. Moreover, the ALP activity of the BMP2/NanoClik-10 group with exogenous BMP-2 in basal media was significantly higher than


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that of the BMP2/NanoClik-10 group without exogenous BMP-2, which indicates that the activity of the BMP-2 incorporated into the NanoClik film was maintained throughout the culture period and that it enhanced the osteoblastic differentiation of the MSCs.

Conclusion We developed a novel thin film for use as an aECM based on nanogel tectonics using self-assembled nanogels as building blocks. These transparent thin films were characterized for their physical properties, function as protein reservoirs, and suitability as cell culture substrates (cell adhesion, spreading, and focal adhesion). The mechanical and surface properties of the films were controlled by changing the nanogel and crosslinker concentrations. The activity of the amphiphilic nanogel in trapping and stabilizing the proteins of interest remained after crosslinking and the formation of thin films. The NanoClik films loaded with vitronectin and BMP-2 with relative stiff mechanical properties enhanced the osteogenic differentiation of hMSCs and acted as an ECM. We can select various nanogels as building blocks such as pH-, thermo- and photo-responsive nanogels to design tailor-made functional thin films. Nanogelbased thin films may be very useful for several biomedical applications, including as scaffolds for tissue engineering, carriers for protein delivery, and surface coating materials for implants.


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Figure 1. A) Self-assembly of the CHOPA nanogels by hydrophobic interactions between the cholesterol groups in aqueous media. B) Hydrodynamic diameter distribution of the CHPOA nanogels as measured by dynamic light scattering.


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Figure 2. A) Schematic of the NanoClik film preparation. Representative images of the NanoClik films prepared at crosslinker concentrations of B) 0.066%, C) 0.13%, and D) 0.67%.


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Figure 3. A) Stress–strain curves from the NanoClik films obtained through tensile loading tests at a strain rate of 10 mm min−1 at 40 °C. The inset graph shows the stress–strain curves of the NanoClik films for strains up to 1%. B) Young’s modulus, C) ultimate tensile strength, D) yield strength, and E) failure strain as a function of PEGSH concentration. The Young’s modulus was estimated from the slope of the stress–strain curve in the small strain, linear response region.


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Figure 4. A) Average contact angle (°) measured by the sessile drop method with pure water on the top of NanoClik films under both dry and wet conditions. B) Release of nanogels from the NanoClik films. These NanoClik films were incubated in PBS supplemented with 10% FBS at 37 °C.


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Figure 5. A) Protein encapsulation profiles of the NanoClik films using FITC-insulin as a model protein. B) CLSM images of the NanoClik films complexed with FITC-insulin. C) Protein release profiles of the NanoClik films loaded with FITC-insulin at an initial concentration of 80 µg mL−1 in PBS supplemented with 10% FBS at 37 °C.


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Figure 6. A–D) Differential interference contrast and E–H) immunostaining images of NIH3T3 cells adhered to a nanogel coat, NanoClik-1, NanoClik-2, and NanoClik-10 24 h after cell seeding. The nucleus (blue), filamentous actin (green), and vinculin (red) of the cells were stained. i) Average number of adherent cells per unit area (mm2) on the nanogel coat, NanoClik1, NanoClik-2, and NanoClik-10. j) Average area of adherent cells on the nanogel coat, NanoClik-1, NanoClik-2, and NanoClik-10. K) Relationship between cell adhesion area and number of adherent cells. L) Viability of the NIH3T3 cells after incubation with 0 to 5 mg mL−1 of CHPOA nanogels.


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Figure 7. A) Young’s modulus distribution of the NanoClik films calculated from the AFM force-distance measurements in a 30-µm × 30-µm area. B) Average Young’s modulus of the NanoClik films for the different PEGSH concentrations.


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Figure 8. ALP staining of hMSCs cultured on A, B) TCPS, C, D) vitronectin-loaded NanoClik10, and E, F) BMP-2- and vitronectin-loaded NanoClik-10 (BMP2/NanoClik-10) in the presence or absence of BMP-2. Scale bar: 250 µm. G) ALP activity of the hMSCs cultured on TCPS and the NanoClik films in the presence or absence of BMP-2. *: P < 0.05 and **: P < 0.005.


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Table 1. Properties of the nanogel-crosslinked (NanoClik) films. Molar ratio Group

acryloyl:/thio l

(µm)

Swelling ratio (%)

Water content (%)

16.6 ± 2.3

dissolved

dissolved

Crosslinke r conc. (%)

Thickness

0

nanogel coat

100:0

NanoClik-1

100:1

0.066

18.3 ± 2.2

587 ± 79

85.7 ± 1.5

NanoClik-2

100:2

0.13

19.0 ± 2.5

511 ± 33

83.6 ± 0.9

NanoClik-10

100:10

0.67

23.2 ± 2.9

373 ± 68

80.2 ± 2.0


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Supporting Information STED observation procedure; NMR spectra of the synthesized CHPOA; Nanogel structures in the NanoClik films by STED observation; Differential interference contrast images of NIH3T3 cells on the NanoClikfilms, and vitronectin-treated PClik films after 24 h of cell seeding

Corresponding Author Email: [email protected] Present Addresses †Department of Material-based Medical Engineering, Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, 2-3-10 Kanda-Surugadai, Chiyoda-ku Tokyo 101-0062 Japan Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally to this work. Acknowledgements We wish to thank Mr. I. Obataya of JPK Instruments AG for making the AFM measurements and Mr. H. Yamaguchi of Leica Microsystems for the STED microscopy. This work was supported by the Exploratory Research for Advanced Technology of the Japan Science and Technology Agency (JST-ERATO).

ABBREVIATIONS


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extracellular matrix; ECM, cholesterol-bearing pullulan; CHP, nanogel-crosslinked gel; NanoClik gel, bone morphogenetic protein-2; BMP-2, pentaerythritol tetra(mercaptoethyl) polyoxyethylene; PEGSH, mesenchymal stem cell; MSC, tissue culture polystyrene; TCPS.

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