Precise Construction of Cell-Instructive 3D Microenvironments by

Jun 10, 2019 - Cell survival in the hydrogels was explored by a live/dead assay, and cell morphology was observed by CLSM. Cell viability and mean cel...
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Article Cite This: Chem. Mater. 2019, 31, 4710−4719

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Precise Construction of Cell-Instructive 3D Microenvironments by Photopatterning a Biodegradable Hydrogel Zhengwei Cai,† Kaiping Huang,† Chunyan Bao,*,† Xuebin Wang,† Xiangchao Sun,‡ Hong Xia,‡ Qiuning Lin,† Yi Yang,† and Linyong Zhu*,† †

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Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130# Meilong Road, Shanghai 200237, China ‡ State Key Laboratory of Integrated Optoelectric, College of Electronic Science & Engineering, Jilin University, Changchun, 130012 Jilin, China S Supporting Information *

ABSTRACT: On the basis of the complexity of the extracellular matrix, it is very important to control the threedimensional (3D) biochemical and physical properties of hydrogels at the microscale level. In this study, we demonstrated a synthetic strategy to construct biomimetic hydrogels (HA-MMP) for 3D cell culture that uses thiolMichael chemistry to encapsulate cells in situ, o-nitrobenzyl alcohol photochemistry (a photogenerated-aldehyde-amineligation sequence, which is also referred to as a PAAL sequence) to permit biochemical patterning, and an enzymecleavable reaction to facilitate cell-responsive degradation in hydrogels. The effects of biochemical and mechanical parameters on 3D cell culture were studied using a userprogrammed Boolean logic-based algorithm, which provided an “AND” logic gate for cell survival, spreading, and migration. Finally, using the spatial controllability of light, cell-instructive 3D microenvironments can be constructed to guide cell behavior in hydrogels. This approach enables the versatile nature of chemistry to create programmable niches in hydrogels, which provides valuable insights into the cell fate by changing the local hydrogel microenvironments.

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INTRODUCTION As known, the extracellular matrix (ECM) has a complex and dynamic composition that constantly provides biophysical and biochemical cues to direct cellular functions.1,2 Much effort has been made to engineer materials that recapitulate important facets of the ECM. Among them, hydrogel is recognized as one of the most competitive biomaterials and has attracted widespread interest as a scaffold for tissue engineering.3−5 A variety of chemical reactions triggered by pH,6 temperature,7 redox,8 and competitive binding partners 9 have been incorporated into hydrogels to present biochemical ligands to facilitate cell adhesion or to regulate matrix mechanics to provide sufficient space for cell spreading and migration. However, most of these strategies are limited to the construction of uniform hydrogels, which represent oversimplified mimics of the native ECM lacking the essential natural temporal and spatial complexity. The photopatterning technology provides a powerful tool to introduce biochemical signals for hydrogel in a noninvasive manner with high spatiotemporal control. Several groups, including our own, have reported to use mild photochemistry to realize protein pattern and mediate cell adhesion on the hydrogel surface.10−19 Although these studies have provided valuable insights into how biochemical signals direct cell fate, © 2019 American Chemical Society

most of these systems have been limited to two-dimensional (2D) interactions, where cells are seeded on hydrogels after photopatterning has occurred. Compared to 2D culture, cells cultured in the three-dimensional (3D) matrix with controllable biochemical and physical microenvironments can behave more as they grow in vivo.20−23 Recently, vinyl polymerization24 and thiol−ene addition25 have been successfully applied to photopattern bioactive molecules in hydrogels; however, the addition of photoinitiators and the free radicalinvolved process would increase the risks of protein denaturation and cell apoptosis.26 Photolysis reactions of phototriggers provide alternative strategies, in which coumarin or o-nitrobenzene undergoes photolysis and liberates active groups that react to pattern bioactive molecules.27−29 Although these strategies do not involve the free-radical process, the photolysis is always accompanied by the release of organic byproducts. We herein developed a photosensitive o-nitrobenzyl alcohol (NB) chemistry to pattern bioactive molecules into hydrogels, which can photogenerate an active aldehyde and subsequently Received: February 18, 2019 Revised: June 9, 2019 Published: June 10, 2019 4710

DOI: 10.1021/acs.chemmater.9b00706 Chem. Mater. 2019, 31, 4710−4719

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Chemistry of Materials

Figure 1. Schematic of (a) components of hydrogels, (b) thiol-Michael addition for hydrogel formation, and (c,d) photochemistry of NB for protein conjugation in hydrogels. Structure of BSA from PDB 4F5S.

react with amine compounds by mild imine ligation.30,31 This photogenerated-aldehyde-amine-ligation sequence (PAAL sequence) produces no organic byproducts other than water molecules, providing a mild strategy to manipulate biochemical cues in cell-laden hydrogels. It is worth noting that the local milieu of the ECM can be degraded by cell-secreted matrix metalloproteinases (MMPs), which plays a key role in mediating cell migration.32−34 Therefore, the combination of photoregulation of biochemical cues and enzyme−stimuli degradation can construct more complex, biomimetic hydrogels to better study the influence of microenvironmental parameters on cell behavior and thus screening out suitable hydrogel platforms to recapitulate native cell growth in vitro. In this study, a hyaluronic acid (HA) derivative was used to construct the biomimetic hydrogels by means of a thiolMichael addition reaction and engineered for 3D cell culture. As shown in Figure 1, NB-based photochemistry was used to independently control biochemical cues in hydrogels, and a MMP-sensitive peptide (Ac-GCRDGPQGIWGQDRCGNH2) was used as the degradable cross-linker to achieve the degradation of the network. The chemical definability and mechanical degradability were programmed as logic combination inputs for 3D cell culture, thereby providing a valuable understanding of how their level and synergy influence the functions of embedded cells. Finally, we expect that cell behavior such as cell morphogenesis and migration can be precisely controlled in space and time by photopatterning cellinstructive cues within the biodegradable hydrogel.

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Synthesis of the HA-NB-VS Macromer. 3,3′-Dithiobis(propanoic dihydrazide) solution (2, 24.0 mg, 0.1 mmol in 1.0 mL of dimethyl sulfoxide) and dimethoxy-1,3,5-triazin-2-yl-4-methyl morpholinium chloride solution (DMTMM, 111.0 mg, 0.4 mmol in 1.0 mL of deionized water) were added dropwise to a homogeneous solution of HA-NB (400.0 mg) in 50 mL of 2-(N-morpholino)ethanesulfonic buffer (MES, 10.0 mM, pH = 5.5). The resulting solution was stirred at room temperature for 12 h. Then, a solution of tris(2-carboxyethyl)phosphine hydrochloride (TCEP-HCl, 143.0 mg, 0.5 mmol) in 5.0 mL of deionized water was added dropwise to the abovementioned gel system and stirred for 3 h to obtain a homogeneous solution. Finally, NaCl (1.0 g, 17.0 mmol) was added and the obtained HA-NB-SH solution was purified by dialysis (MWCO 7000, Spectrum) against diluted HCl (pH = 4.5) containing 0.1 M NaCl and diluted HCl (pH = 4.5) for 2 d. Then, the resulting HA-NB-SH solution was added dropwise to a solution of excessive divinyl sulfone (200 μL) in 50 mL of triethanolamine buffer (300 mM, pH = 8.0) and stirred overnight. After that, NaCl (1.0 g, 17.0 mmol) was added, and the obtained HA-NB-VS solution was purified by dialysis (MWCO 7000, Spectrum) against 0.1 M aqueous NaCl and deionized water for 2 d. Finally, the solution was lyophilized and stored at −20 °C. The substitution degree of the vinyl sulfone (VS) group was determined as 5.2% of HA disaccharide units by 1H NMR (Figure S3, Supporting Information). Synthesis and Characterization of Hydrogels. HA-NB-VS and the MMP-cleavable peptide cross-linker AcGCRDGPQGIWGQDRCG-NH2 were separately dissolved in phosphate-buffered saline (PBS) at a concentration of 20 mg mL−1 and sterilized by filtration. HA-NB-VS solution was further neutralized with aqueous NaOH (0.1 M). Then, the two solutions were mixed in different volumes in which the molar ratio between VS and thiol varied at 1.5:1, 1.25:1, 1:1, 1:1.25, and 1:1.5. After vortexing for 15 s, the pre-gel solution was poured into Teflon disk molds to form a hydrogel at 37 °C. Rheology analysis was carried out to analyze the gelation process with a HAAKE MARS III rheometer equipped with parallel-plate (P20 TiL, 20 mm diameter) geometry at 37 °C. Time-sweep oscillatory analyses were performed at a 10% strain, 1 Hz frequency, and a 0.5 mm gap (CD mode) for 2500 s. The gel point was

EXPERIMENTAL SECTION

Synthesis of HA-NB. Compound 1 and NB-modified HA (HANB) were synthesized as previously reported.30 In this study, the molar ratio of raw material input was 3:1 (HA disaccharide unit: 1, Figures S1 and S2, Supporting Information), and the substitution degree was determined as 3.6% of HA disaccharide units by 1H NMR. 4711

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initial concentration and volume, respectively, of BSA added in the pre-gel solution. Photopatterning of Proteins. BSA-Rho was used as the model protein and premixed in the pre-gel solution at a concentration of 10 μM. Gradient patterning was conducted as follows: a line-shaped mask (200 μm) was covered on an HA-MMP hydrogel (7 mm × 1 mm) and subjected to different irradiation times on each line (365 nm LED light source, 10 mW cm−2, 15−210 s from left to right with a 15 s increment time). The photopatterned hydrogel was gently washed with PBS on an orbital shaker for 12 h and analyzed by a confocal laser scanning microscope (Leica-TCS-SP8). Photomasks with designed patterns were used for 3D photopatterning on hydrogels with a diameter of 7 mm and height of 200 μm, where 2D geometric shapes defined by photomasks can be patterned throughout the gel thickness. The photomasks contained stripes with a 200 μm width and spacing, lattices with a 50 μm width and 200 μm spacing, and frames with a 100 μm extra-side length and 50 μm intraside length. After irradiating for 3 min (365 nm LED light source, 10 mW cm−2), the photopatterned hydrogel was gently washed with PBS on an orbital shaker for 12 h and analyzed by a confocal laser scanning microscope. 3D protein photopatterning in hydrogels was also fabricated using a homemade FsLDW system. The femtosecond laser beam (Spectra Physics MTEV VF-N1S, 80 MHz repetition rate, 100 fs pulse width, 800 nm central wavelength with 40 mW) was tightly focused in the HA-MMP hydrogel using a 60× oil immersion objective lens with a high-numerical aperture (Olympus, NA = 1.40). A piezo stage (Physik Instrument P-622.ZCD) and a two-galvano-mirror set were used to control the vertical and horizontal scanning movements of the focused laser spot simultaneously. A charge-coupled device was used to observe the fabricating process in real time. After photopatterning was completed, the gel was washed overnight with fresh PBS to remove the unbound protein and analyzed by confocal laser scanning microscopy. Protein-Mediated Cell Adhesion. Human dermal fibroblast (HDF) cells were obtained from the American Type Culture Collection (ATCC) and cultured in high-glucose Dulbecco’s modified Eagle medium (DMEM; HyClone) supplemented with 10% (v/v) inactivated fetal bovine serum (FBS, Sigma-Aldrich) and 1% (v/v) penicillin/streptomycin (Gibco). Cells were maintained under common cell culture conditions at 37 °C in an atmosphere of 5% CO2. Photopatterning of c(RGDfK), BSA, and BSA−c(RGDfC) was performed as described above. Then, the patterned hydrogel was placed in a confocal dish and 2 mL of culture media was added with 5 × 104 suspended cells. After 12 h of culture, the hydrogels were gently washed with PBS to remove unattached cells, and the remaining cells on the hydrogel were stained with a live/dead reagent (calcein acetoxymethyl ester/propidium iodide, Calcein-AM/PI) for 0.5 h, where live cells emitted green fluorescence and dead cells emitted red fluorescence upon excitation at a wavelength of 488 nm. The guidance for cell adhesion was observed with a confocal fluorescence microscope, and all cell images are presented from the calcein channel for clarity because there was negligible number of cells observed in the PI channel. Degradation of HA-MMP Hydrogels. Scanning electron microscopy (SEM, Hitachi S-4800) and rheological testing evaluated enzymatic degradation of the hydrogels. HA-MMP and HA-PEG hydrogels containing premixed BSA (10 μM) were irradiated for 3 min (using a 365 nm LED light source, 10 mW cm−2) and then washed with PBS for 12 h. Then, collagenase type IV in PBS (0.5 mg mL−1, 400 μL) was added to the hydrogels. SEM images were acquired for the freeze-dried samples of HA-MMP hydrogels. Meanwhile, the storage modulus (G′) of the hydrogels was monitored over time by a rheometer (HAAKE MARS III) at 37 °C using a 20 mm parallel-plate geometry. 3D Cell Encapsulation. For homogeneous encapsulation of cells, HDF cells were suspended at 2 × 106 cells mL−1 in a 20 mg mL−1 total hydrogel precursor solution in PBS. According to the combined input signals, the hydrogel precursor solutions used were divided into

determined as the time when the storage modulus (G′) surpassed the loss modules (G″). Photolysis of NB-1, HA-NB, and HA-MMP Hydrogels. Photolysis analysis of NB-1 was performed as follows: NB-1 (2 × 10−4 mol mL−1) was dissolved in acetonitrile−water solution (4/1, v/ v) using a cuvette. Then, the homogeneous solution was irradiated with a 365 nm light-emitting diode (LED) light source (10 mW cm−2), and at specific time intervals, the solution was analyzed by UV−vis absorption and high-performance liquid chromatography (HPLC) spectra. When photolysis of NB-1 was complete, Boc-Lys was added to the photolysis solution at a concentration of 10−3 M, and the corresponding UV−vis and HPLC spectra were analyzed. Furthermore, liquid chromatography−mass spectrometry (LC−MS) spectra were used to assign the components. For HPLC, a BetaBasic18 column was used and eluted with a mixture of 50% acetonitrile and 50% water at a flow rate of 0.5 mL min−1, and the chromatogram was obtained using absorbance analysis at a wavelength of 290 nm. Photolysis of HA-NB was performed by irradiating a solution of HA-NB (2 mg mL−1) in PBS with a 365 nm LED light source (10 mW cm−2) in a quartz cuvette. Between certain time intervals, the irradiated solution was detected by the UV−vis absorption spectrum. Photolysis of the HA-MMP hydrogel was performed by irradiating a hydrogel film with a thickness of 500 μm with a 365 nm LED light source (10 mW cm−2) on a quartz slide. FTIR−ATR Analysis for Protein Photoconjugation in Hydrogels. HA-PEG hydrogels were prepared according to the procedure of HA-MMP hydrogel preparation, in which the nondegradable crosslinker HS-PEG-SH (Mw = 2000 kDa) was used, and the molar ratio between the VS and thiol groups was 1:1. Hydrogels with a size of 7 mm × 1 mm (diameter × height) were used as samples and hydrogels with or without the bovine serum albumin (BSA) premix (10 μM) were irradiated by a 365 nm LED light source (10 mW cm−2) for 3 min. After washing several times, the samples were freeze-dried in vacuum for Fourier transform infrared (FTIR)−attenuated total reflectance (ATR) spectroscopy analysis. Quantification of Photoconjugated Proteins in Hydrogels. The level of protein incorporation was quantified by a bicinchoninic acid (BCA) assay according to the instructions of the manufacturer. HA-PEG hydrogels (7 mm × 1 mm) with BSA premix (10 μM) that were prepared as mentioned above were used to quantify protein photoimmobilization in the hydrogels, and UV light (a 365 nm LED light source with an intensity of 5, 10, and 20 mW cm−2) was used for irradiation. After irradiating for certain amounts of time, the hydrogels were immersed in 1 mL of PBS for 12 h to wash out the unbound BSA, and the hydrogels without light irradiation were used as controls to subtract the possible physical adsorption of proteins in the hydrogels. The amount of photoimmobilized proteins in the hydrogels was determined by the following equations: The physical adsorption of protein for nonirradiated hydrogel Δ0 % =

(C0 × V0 − Cnonirra × Vnonirra) × 100% (C0 × V0)

The protein adsorption for irradiated hydrogel Δ1% =

(C0 × V0 − Cirra × Virra) × 100% (C0 × V0)

Then, the immobilized protein by photopatterning was calculated by the following equation Δ% = Δ1% − Δ0% =

(Cnonirra × Vnonirra − Cirra × Virra) × 100% (C0 × V0)

where Cnonirra is the unbound protein concentration in the washing solution for a hydrogel without light irradiation, Vnonirra is the volume of the washing solution for a hydrogel without light irradiation, Cirra is the unbound protein concentration in the washing solution for a hydrogel with light irradiation, Virra is the volume of the washing solution for a hydrogel with light irradiation, and C0 and V0 are the 4712

DOI: 10.1021/acs.chemmater.9b00706 Chem. Mater. 2019, 31, 4710−4719

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Figure 2. NB photochemistry and protein patterning in hydrogels. (a) Schematic of the PAAL sequence between NB-1 and Boc-Lys. The evolution of (b) UV−vis and (c) HPLC spectra of NB-1 upon 365 nm LED light irradiation (10 mW cm−2) and after the addition of Boc-Lys in acetonitrile−water solution (4/1, v/v). (d) Quantification of photoimmobilized BSA in HA-PEG hydrogels through the variations of the light intensity (5, 10, and 20 mW cm−2) and exposure time (0−180 s). Data were determined from a BCA assay and are presented as the mean values of three independent experimental replicates. (e) Precisely defined BSA-Rho patterns (200 μm lines) by predictably exposing HA-MMP hydrogel surfaces to gradients of light exposure (created by covering a line-shaped mask and subjecting each line to different irradiation times of 15−210 s from right to left with a 15 s increment time). (f) BSA-Rho 3D patterning in HA-MMP hydrogels by photomasking after 3 min of irradiation (10 mW cm−2). (g) BSA-Rho 3D patterning by two-photon photolithography. four types as follows: BSA + HA-PEG (0, 0), BSA−c(RGDfC) + HA-PEG (1, 0), BSA + HA-MMP (0, 1), and BSA−c(RGDfC) + HA-MMP (1, 1). The protein concentration was fixed at 10 μM. After homogeneous mixing, the cell containing solutions (200 μL) were pipetted in confocal dishes (diameter: 15 mm, thickness: 1 mm) and allowed to react for 40 min at 37 °C to form cell-laden hydrogel sheets. Then, all the hydrogel sheets were exposed to a 365 nm LED light source (10 mW cm−2) for 3 min and cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/ streptomycin at 37 °C in an atmosphere of 5% CO2. The medium was changed five times on the first day to remove unbound proteins and then changed every 2 days. Cell survival in the hydrogels was explored by a live/dead assay, and cell morphology was observed by CLSM. Cell viability and mean cell spreading area were quantified using ImageJ software from CLSM images. Immunofluorescence Staining. Immunofluorescence staining for F-actin and nuclei was performed as follows: the cell-laden hydrogel was fixed in 4 v/v% paraformaldehyde for 1.5 h, then permeabilized with 0.2% Triton-100 for 2 h, and finally stained with Alexa Fluor 488-phalloidin and DAPI for 2 h each. The sample was extensively rinsed and imaged on a CLSM. Cell Migration. The preparation procedure of HDF cell-laden collagen clots was described as follows. A total of 1.5 × 105 cells were suspended in 5 μL of collagen solution (2 mg mL−1, pH = 7.5, and 0.01 M PBS), and the obtained suspension was dropped onto a Teflon plate and incubated at 37 °C for 30 min. Then, the cell-laden clots were incorporated into hydrogels by placing them in the hydrogel precursor solutions with different components as described above. After hydrogel formation, protein photoimmobilization was performed upon irradiation with a 365 nm LED light source (10 mW cm−2) for 3 min. Finally, the obtained samples were cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/ streptomycin at 37 °C in an atmosphere of 5% CO2. Phase-contrast microscopy was used to analyze cell morphogenesis. The distance from the edge of the collagen clot to the leading edge of migrating cells was measured to quantify cell migration out of the collagen clot. Each data point was collected and averaged from 10 data points.

Immunofluorescence staining for F-actin and nuclei was performed as mentioned above. Photomodulated 3D Cell Culture. A total of 4 × 105 HDF cells were mixed with the HA-MMP hydrogel precursor solution (20 mg mL−1 total concentration, 200 μL) with BSA−c(RGDfC) at 10 μM. Then, the cell-laden precursor solution was pipetted into a confocal dish (diameter: 15 mm, thickness: 1 mm) and incubated at 37 °C for 40 min. After hydrogel formation, a line-shaped chrome photomask with 200 μm strips was covered on the hydrogel, which was exposed to a 365 nm LED light source (10 mW cm−2) for 3 min. The photopatterned hydrogel was cultured in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin at 37 °C in an atmosphere of 5% CO2. During the process, the medium was changed five times on the first day to remove the unbound proteins and then changed every 2 days. Cell viability and morphogenesis were analyzed by a live/dead assay as mentioned above. Photomodulated 3D Cell Migration. The hydrogels with a cellladen collagen clot were prepared as described above, in which BSA− c(RGDfC) was premixed at 10 μM. A BSA−c(RGDfC) path (200 μm width and 5 mm length) was created by photopatterning. After photopatterning, the unbound proteins were removed by changing the culture medium and then cultured at 37 °C in an atmosphere of 5% CO2. Data collection and analysis were the same as mentioned above.

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RESULTS AND DISCUSSION Design and Synthesis of HA-MMP Hydrogels. HA, a natural polymer commonly found in the ECM of many tissues, was used as the backbone of the hydrogels to better mimic the native ECM while maintaining a transparent hydrogel for 3D functionalization.35 Then, the HA macromer was bifunctionally modified (HA-NB-VS, Figures S1−S3, Supporting Information), which included a VS moiety that can react with the MMP-cleavable cross-linker and a photolabile NB group that can photogenerate aldehyde to conjugate amine-containing bioactive molecules. After mixing HA-NB-VS with the MMPcleavable cross-linker in different molar ratios, hydrogels (HA4713

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Figure 3. Adhesive protein photopatterning and degradation of hydrogel. (a) Synthetic procedure and (b) MALDI-TOF and (c) SDS-PAGE analysis of BSA−c(RGDfC). (d) Precisely controlled cell adhesion on hydrogels by gradient patterning of BSA−c(RGDfC). A total of 5 × 104 cells were seeded on the hydrogels. (e) Schematic and SEM analysis of hydrogel degradation without or with collagenase type IV (0.5 mg mL−1 in PBS) stimuli. (f) Evolution of the storage modulus (G′) of HA-MMP and HA-PEG hydrogels upon stimulation with collagenase type IV. G′0 is the initial storage modulus of hydrogels without the addition of collagenase type IV. Structure of BSA from PDB 4F5S.

Then, photolysis of the NB-containing macromer (HA-NB) and hydrogel (HA-MMP) was also investigated (Figure S6, Supporting Information), and the evolution of the UV−vis absorption spectra showed results similar to NB-1 photolysis. FTIR with ATR was further performed on freeze-dried hydrogels to characterize the composition and the change in composition after light irradiation and protein conjugation (Figure S7, Supporting Information). Here, BSA was used as the protein model. To avoid interference of an MMP-cleavable cross-linker on protein IR absorption, an HA-PEG hydrogel was constructed using a nonpeptide cross-linker (HS-PEG-SH, Mw = 2000 kDa). The disappearance of IR absorption at 1320 and 1520 cm−1, ascribed to −NO2 stretching vibration from NB, verified the successful photolysis of NB in HA-PEG, and the increased absorption at 1644 cm−1 (amide δCO of protein) indicated BSA immobilization by imine ligation in the hydrogel. To further quantify the protein conjugation by photomediated imine ligation, a simple dose-dependent assay was performed and analyzed by a BCA assay. In this experiment, HA-PEG hydrogels with BSA premix (10 μM) were irradiated with different light intensities (5, 10, and 20 mW cm−2) and exposure times (0−180 s) and subsequently immersed in 1 mL of PBS to remove unbound BSA. By using the nonirradiated samples as controls, the amount of protein immobilized in the hydrogels was calculated. As illustrated in Figure 2d, the extent of BSA photoimmobilization increased with increasing exposure time and intensity, showing a dose response to the number of incident photons. Adhesive Protein Photopatterning and Degradation of Hydrogels. To highlight the spatiotemporal advantage of light manipulation, protein photopatterning was performed in HA-MMP hydrogels, in which a rhodamine B isothiocyanatelabeled protein (BSA-Rho) was used for fluorescent imaging. As illustrated in Figure 2e, complex protein patterns with line shape and dose-dependent gradients were achieved by controlling the exposure dosage. Complex 3D protein patterning was also achieved in hydrogels by traditional

MMP) were formed in situ by a thiol-Michael addition reaction. Gelation occurred within ∼3 min of mixing, as estimated by the crossover point of the rheological storage (G′) and loss (G″) moduli, and the final moduli varied from 100 to 250 Pa by controlling the molar ratio of the reaction groups (Figure S4, Supporting Information). Because of the optimized mechanical performance, the hydrogel formed by equimolar VS and thiol groups (VS/SH = 1:1) was used for subsequent experiments. Investigation of PAAL Sequence of NB. Before protein patterning of the hydrogel, the photokinetics and PAAL sequence reactions of NB were validated in a small molecular system, in which NB-1 was used as the photosensitive molecule and t-butyloxy carbonyl-protected lysine (Boc-Lys) was used as the amine compound for imine ligation (Figure 2a). Time-resolved UV−vis absorption spectra were first analyzed, as illustrated in Figure 2b, and changed dramatically upon irradiation with a 365 nm LED light source (10 mW cm−2). With increasing exposure time, the absorbance at approximately 260 nm increased rapidly, the peak at 348 nm red-shifted to 375 nm, and an isobestic point was observed at 295 nm, exhibiting typical characteristics of NB photochemistry from NB to o-nitrosobenzaldehyde. After adding excess of Boc-Lys, the maximum absorption at 375 nm blueshifted to 330 nm, which suggested that the photogenerated aldehyde reacted with Boc-Lys by imine ligation. HPLC and LC−MS were further conducted to confirm the PAAL process of NB. As shown in Figure 2c, light irradiation induced the consumption of NB-1 as well as the generation of the aldehyde product NB-2. Then, the addition of Boc-Lys induced the consumption of NB-2 and the generation of the ligation product NB-3, which was stable even after 7 days (Figure S5, Supporting Information). Only water molecules were released during the entire reaction process, and the PAAL sequence of NB provided the premise for protein photopatterning of HA hydrogels. 4714

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Figure 4. Logic gate for in situ 3D cell culture. (a) Schematic of hydrogels with user-programmed inputs for 3D cell culture. (b) Confocal images of cells in the user-programed hydrogels at 12 d. Evaluations of (c) cell viability and (d) cell spreading area in hydrogels. (e) Confocal image for cytoskeleton observation by immunofluorescence staining for F-actin and nuclei. Structure of BSA from PDB 4F5S.

was incorporated with BSA (BSA−c(RGDfC), Figures 3a and S11, Supporting Information) and used as a biochemical signal to guide cell adhesion in the hydrogels. Both matrix-assisted laser desorption/ionization time-of-flight MS (MALDI-TOF, Figure 3b) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Figure 3c) analysis verified the efficient incorporation of the peptide into BSA, and the substitution degree of c(RGDfC) was determined as 9.4% of BSA amine units. After photopatterning of BSA−c(RGDfC) on a HA-MMP hydrogel, HDF cells were added and cocultured for 12 h. As expected, HDF cells showed distinct heterogeneous cell distribution and preferentially adhered to the patterned regions of BSA−c(RGDfC), whereas BSA patterning (Figure S10, Supporting Information) or simple photoirradiation for hydrogels (Figure S12, Supporting Information) did not facilitate cell adhesion. All these confirmed that BSA efficiently improved the density of RGD patterned in hydrogel that mediated cell adhesion. Encouragingly, gradient photopatterning of BSA−c(RGDfC) correspondingly induced gradient control on the number of adherent cells (Figure 3d), indicating the ability of this approach to guide cell behavior by constructing user-defined biochemical cues. Cells can remodel their microenvironment to allow cell spreading or migration by secreting proteinases to degrade their ECM. To demonstrate MMP-sensitive degradation, a buffer-containing collagenase type IV was added to the HAMMP hydrogels. SEM analysis (Figure 3e) indicated that the presence of collagenase type IV resulted in a sparser network structure of the HA-MMP hydrogel than that of hydrogel without collagenase type IV. To further confirm the

photolithographic techniques, where the 2D geometric shapes defined by photomasks were patterned throughout the gel thickness (Figures 2f and S8, Supporting Information). These 3D protein patterns were continuous at all depths in the bulk hydrogel, and the fluorescence intensity was greatly different from the irradiated and nonirradiated areas, demonstrating the excellent spatial control of protein patterning in hydrogels. Sequential patterning of BSA-Rho and BSA-FITC resulted in a noninterfering state, and the patterns kept clear boundaries without any progressive color mixing even after soaking in PBS for 7 days (Figure S9, Supporting Information). It indicates that the imine bond for protein immobilization is stable and irreversible. Furthermore, a more complex and refined 3D photopatterning was achieved in a hydrogel by using a twophoton laser scanning lithographic approach (Figure 2g). These results confirmed the spatial and quantitative control on protein immobilization in hydrogels resulting from the PAAL sequence between NB and proteins. On the basis of the fact that cell adhesion is initiated by interactions between bioactive ligands (arginyl-glycyl-aspartic acid, RGD) in the ECM and integrins of the cell membrane,36,37 an adhesive amine-contained peptide c(RGDfK) (cyclo(Arg-Gly-Asp-D-Phe-Lys)) was chosen for photopatterning. However, its effect on cell adhesion is poor (Figure S10, Supporting Information) even when the concentration of c(RGDfK) is up to 5.6 mM. It should be attributed to the low density of immobilized RGD in hydrogel by PAAL sequence of NB. BSA is an amine-contained macromolecule and it possesses almost 60 amine groups, which makes it an excellent carrier for conjugation. Therefore, a peptide-containing RGD (cyclo(Arg-Gly-Asp-D-Phe-Cys)) 4715

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Figure 5. AND logic gate for HDF cell migration in hydrogels. (a) Microscopic images of cell migration from collagen gels to BSA−c(RGDfC) + HA-MMP hydrogels. (b) Cell migration distance was analyzed in hydrogels with different inputs. (c) Cytoskeleton image of migrated cells by immunofluorescence staining for F-actin and nuclei.

Figure 6. Photomodulated 3D HDF cell culture and migration in BSA−c(RGDfC) + HA-MMP hydrogels. (a) Schematic of protein photopatterning in cell-laden hydrogels. (b) 3D images of encapsulated cells stained by a live/dead assay after 12 d of culture. (c). Evaluation of cell spreading area in photopatterned hydrogels. **** p < 0.0001. (d) Schematic of cell migration in the patterned hydrogel. (e) Bright field image and (f) fluorescent images of cytoskeleton of migrated cells in patterned hydrogels after 12 d of culture.

biodegradability, the modulus of degradable (HA-MMP) and nondegradable (HA-PEG) hydrogels was analyzed over time. As illustrated in Figure 3f, the modulus of the HA-PEG hydrogel was stable over the 30 min study. However, HAMMP hydrogels showed a continuous decrease in modulus,

indicating that the cross-linking density of the HA-MMP hydrogel was decreased as a consequence of collagenaseinduced degradation. It indicated that the hydrogel degradation was due to the presence of the action of collagenase. 4716

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Article

Chemistry of Materials Logic Gate for 3D Cell Encapsulation and Migration. To show how the biochemical and biomechanical properties of the matrix affect the viability and morphogenesis of cells, a user-programmed Boolean logic-based algorithm was used. As illustrated in Figure 4a, the adhesive protein BSA−c(RGDfC) and biodegradability were programmed as input signals, and cell viability and spreading in hydrogels were defined as functional outputs. According to the combined inputs, four kinds of hydrogels were designed for in situ 3D cell encapsulation, including a BSA + HA-PEG hydrogel (0, 0), a BSA−c(RGDfC) + HA-PEG hydrogel (1, 0), a BSA + HAMMP hydrogel (0, 1), and a BSA−c(RGDfC) + HA-MMP hydrogel (1, 1). In this study, all the proteins were photoimmobilized in hydrogels upon irradiation using a 365 nm LED light source with a cytocompatible dosage (10 mW cm−2 for 3 min, Figure S13, Supporting Information). Considering that light attenuation will lead to inhomogeneous protein patterning in hydrogel, the variation in the longitudinal fluorescent intensity of patterned BSA-Rho in hydrogel was explored (Figure S14, Supporting Information).27,29 On the basis of the constant fluorescence intensity between 0 and 400 μm depth, the confocal images of encapsulated cells were collected with z-projection less than 400 μm to avoid the effect of protein concentration gradient. A live/dead assay was performed to evaluate the viability and spreading of cells in the hydrogels. As shown in Figure 4b (Figure S15, Supporting Information), only the HDF cells in the BSA−c(RGDfC) + HA-MMP hydrogel exhibited a spindle-like morphology at 12 d. In contrast, the cells in the other three hydrogels, where one or two of the inputs were absent, maintained a round shape, although the cells in the BSA−c(RGDfC) + HA-PEG hydrogel trended to spread. Figure 4c,d summarized the quantitative analysis of cell viability and spreading area (analyzed by ImageJ software), which indicated an “AND” logic gate of 3D cell culture. In addition, immunofluorescence staining for F-actin and nuclei (Figure 4e) further confirmed that the cells in the BSA−c(RGDfC) + HA-MMP hydrogel had disoriented and extensive actin fibers after 12 d of encapsulation, which indicated that the cells were able to attach to and locally degrade the surrounding network, leading to a spread morphology. The logical control of cell migration was further explored, in which HDF-seeded collagen clots were embedded in hydrogels and cellular migration was analyzed for 12 d. As shown in Figure 5a, microscopic observations showed that cells radially migrated to the BSA−c(RGDfC) + HA-MMP hydrogel over time with a migration rate at 69 μm/day (Figure 5b,c). In contrast, cell migration was inhibited in nonadhesive or nondegradable hydrogels (Figure S16, Supporting Information), exhibiting the same “AND” logic gate as observed in the 3D cell culture. Photomodulated 3D Cell Encapsulation and Migration. Finally, we investigated whether cell behavior in hydrogels could be spatially modulated by light manipulation. After cell encapsulation, BSA−c(RGDfC) was photopatterned in HA-MMP hydrogels to create 3D heterogeneous microenvironments for encapsulated cells by an easily handled photomask technology (Figure 6a). A live/dead assay was performed to track cell viability and morphogenesis. As shown in Figure 6b, the encapsulated HDF cells exhibited different behaviors in the patterned hydrogel after 12 d of coculture. Cells in BSA−c(RGDfC) regions exhibited higher viability (Figure S17, Supporting Information) and many cells spread

into a spindle-like morphology. By contrast, cells in nonadhesive regions maintained a rounded morphology. Figure 6c illustrated that the cells in the protein immobilization showed much higher spreading areas than those in the areas without light exposure. These results demonstrate that cell morphogenesis can be limited to the adhesive ligands provided in a cell-responsive hydrogel matrix. Cell migration, especially for directional cell migration, is a hallmark of the tissue remodeling events, which leads to embryonic morphogenesis, wound repair, and cancer invasion.38 We herein explore if light-mediated spatial patterning of adhesive protein allows 3D directional cell migration in hydrogels. For this purpose, a HDF-seeded collagen clot was embedded in the hydrogel with premixed BSA−c(RGDfC) (Figure 6d), and a cell-adhesive pathway was created by photopatterning. The directional migration process of cells from collagen to the patterned hydrogel was monitored by means of microscopic observations. As expected, HDF cells radially migrated into HA-MMP hydrogel over time and the migration was spatially restricted within the linearly patterned region (Figures 6e,f, Figure S18, Supporting Information). The tip cells forged a path while maintaining a connection with the trailing cells. The migration distance for the tip cells arrived 1.6 mm after 12 d of culture. The migration rate was around 133 μm/day, which was twice that of the nonspatially patterned hydrogel as mentioned above. It might be attributed to the confined path that accelerated cell migration in hydrogels. By contrast, when BSA was used instead of adhesive BSA− c(RGDfC), we did not observe any directional cell migration (Figure S19, Supporting Information). Considering that the mesh size of the hydrogels is much smaller than cells, cell migration is directed by cell adhesion and localized proteolysis, thus forging a path.39 All these results verified that cell viability, spreading, and migration in 3D hydrogels are jointly controlled by photoregulated adhesion and cell-mediated proteolysis. The photopatterned adhesive ligands provide biochemical cues for regulating cell adhesion and directional migration, and the change in matrix mechanics, especially cell-remodeled biodegradability, provides sufficient space for cell spreading and migration.

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CONCLUSIONS

In summary, we developed a new platform for the fabrication of internally complex hydrogels by controllable photopatterning and enzyme stimulus degradation. The photochemistry of NB promotes the spatiotemporal and dosage control of bioactive ligands in 3D hydrogels by the specific photogeneration of active aldehyde and subsequent imine ligation with proteins without any release of organic byproducts. Through a combination of MMP-cleavable cross-linkers, cellladen hydrogels exhibited an “AND” logic gate control of cell behaviors. Finally, heterogeneous microenvironments can be programmed by protein photopatterning, allowing for the direct regulation of cell survival, spreading, and migration in 3D hydrogels. Although amine-selective photopatterning may have some limitations, we anticipate that this photochemistry of the NB PAAL sequence will provide a new opportunity to construct cell-instructive biomimetic hydrogels, thus directing cell fate similar to native ECMs. 4717

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.9b00706. Materials and characterizations, synthesis of compounds and hydrogels, photolysis of NB-1, HA-NB, and HAMMP hydrogel, protein and cell patterning, and 3D cell culture and migration (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (C.B.). *E-mail: [email protected] (L.Z.)... ORCID

Chunyan Bao: 0000-0002-7160-3007 Qiuning Lin: 0000-0002-9418-9590 Yi Yang: 0000-0001-7896-1184 Linyong Zhu: 0000-0002-0398-7213 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by NSFC (grant nos. 21472044 and 21425311). The authors thank the Research Center of Analysis and Test of East China University of Science and Technology for the help on the characterizations.

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REFERENCES

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