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In Situ “Clickable” Zwitterionic Starch Based Hydrogel for 3D Cell Encapsulation Dianyu Dong, Junjie Li, Man Cui, Jinmei Wang, Yuhang Zhou, Liu Luo, Yufei Wei, Lei Ye, Hong Sun, and Fanglian Yao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b12141 • Publication Date (Web): 28 Jan 2016 Downloaded from http://pubs.acs.org on February 5, 2016

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ACS Applied Materials & Interfaces

In Situ “Clickable” Zwitterionic Starch Based Hydrogel for 3D Cell Encapsulation Dianyu Dong a, Junjie Li c, Man Cui b, Jinmei Wang a, Yuhang Zhou a, Liu Luo a, Yufei Wei a, Lei Ye a, Hong Sun b*, Fanglian Yao a, d* a

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China

b

Department of Basic Medical Sciences, North China University of Science and Technology,

Tangshan 063000, China c

Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and

Tissue Engineering Research Center, Academy of Military Medical Science, Beijing 100850, China d

Key Laboratory of Systems Bioengineering, Ministry of Education, Tianjin University, Tianjin

300072, China

*Corresponding author at: 1. School of Chemical Engineering and Technology, Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072, China.

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Tel.: +86-22-27402893; Fax: +86-22-27403389 E-mail address: [email protected] (Fanglian Yao) 2. Department of Basic Medical Sciences, North China University of Science and Technology, Tangshan 063000, China. Tel.: +86-315-3725740; Fax: +86-315-3726552 E-mail address: [email protected] (Hong Sun)


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ABSTRACT: 3D cell encapsulation in hydrogel provides superb methods to investigate the biochemical cues in directing cellular fate and behaviors outside the organism, the primary step of which is to establish suitable “blank platform” to mimic and simplify native ECM microenvironment. In this study, zwitterionic starch based “clickable” hydrogels were fabricated via a “copper- and light- free” Michael-type “thiol-ene” addition reaction between acylatedmodified sulfobetaine derived starch (SB-ST-A) and dithiol-functionalized poly (ethylene glycol) (PEG-SH). By incorporating antifouling SB-ST and PEG, the hydrogel system would be excellently protected from non-target proteins adsorption to act as a “blank platform”. The hydrogels could rapidly gel under physiological conditions in less than 7 minutes. Dynamic rheology experiments suggested the stiffness of the hydrogel was close to the native tissues, and the mechanical properties as well as the gelation times and swelling behaviors could be easily tuned by varying the precursor proportions. The protein and cell adhesion assays demonstrated the hydrogel surface could effectively resist nonspecific protein and cell adhesion. The degradation study in vitro confirmed that the hydrogel was biodegradable. A549 cells encapsulated in the hydrogel maintained high viability (up to 93 %) and started to proliferate in number and extend in morphology after 2 days’ culture. These results indicated the hydrogel presented here could be a potential candidate as “blank platform” for 3D cell encapsulation and biochemical cues induced cellular behavior investigation in vitro.

KEYWORDS: hydrogel, blank platform, ECM, “thiol-ene” click chemistry, 3D cell encapsulation


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1. INTRODUCTION The field of tissue engineering has been taking efforts to exploit living cells to regenerate healthy tissues or organs in laboratory for lesion sites repair and transplantation.1-3 Thus, 3D cell culture in vitro presents superb opportunities to help systematical exploration of the biochemical cues in directing cellular morphology, function and behavior outside the organism. Cells in vivo interact with their complex surrounding microenvironments, comprising not only neighboring cells but also the extracellular matrix (ECM).4 Native ECM provides complex signals to direct cellular processes through specific biological media. For example, the growth factors stimulate cell proliferation and differentiation, and adhesion proteins facilitate cell attachment and influence motility, etc.5,6 It is often challenging to elucidate whether these cues affect individually or synergistically on desired cell functions and which occupies a decisive position, because countless signaling pathways confound the contributions of the key roles.7,8 Therefore, the first step of 3D cell culture in vitro is to establish specific artificial model microenvironments to mimic the native ECM in certain degree and simultaneously deconvoluting its complexity. Thus, the hydrogel represents a robust biomaterial system for satisfying these requirements.9-12 Moreover, for fundamental researches of directing cell fates and behaviors in vitro, it is also essential to construct a “blank platform” for the introduction of individual biochemical signal without other interferences to manipulate critical cellular functions.8 Hydrogels as scaffolds, similar to other materials or devices, implanted in the body always suffer immunoreactions as a natural mammalian protective mechanism.13,14 And the nonspecific protein adsorption on the hydrogel surface stemming from the complex in vivo environment is considered the first step to trigger foreign-body reactions.15 However, although cells


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encapsulated in hydrogels and culture artificially in vitro would effectively avoid the immunoreactions, the nonspecific adsorption of non-target proteins of hydrogel matrixes in complex biological culture medium would also probably confuse the experimental variables as mentioned above. Therefore, except for the minimal functionality in architecture, another key factor to establish a “blank platform” is to eliminate nonspecific protein adsorption of the hydrogel system. Recently, the excellent ultra-low-fouling property have made zwitterionic based materials, such as sulfobetaine (SB)16-18, carboxybetaine (CB)19-21 and phosphorylcholine (PC)22,23, extensively applied as antifouling biomaterials. The electrostatically induced high hydration around their opposing charges builds a high energy hydration barrier to primely overcome the nonspecific protein adsorptions.24 Although the hydrogels composed of zwitterionic polymers like poly (carboxybetaine) (PCB) analogue have been preliminarily investigated in 3D cell encapsulation25-27, the purely synthetic and non-degradable matrix disenabled the spread and migration of cells embedded, and thus, the long-term cellular fate is still concerned. We have previously reported28 a novel design of sulfobetaine derived starch (SB-ST) with excellent properties in both antifouling ability and biocompatibility. This neotype ultra-low fouling biomaterial was constituted via a Williamson etherification between a new sulfobetaine type zwitterionic etherifying agent DCAPS and the hydroxyl groups of starch. By incorporating zwitterionic moieties, the electrostatically induced high hydration endowed SB-ST preferable antifouling property than virgin starch. The corresponding SB-ST hydrogels were prepared with poly (ethylene glycol) diglycidyl ether (PEGDE) as crosslinker in NaOH aqueous solution (pH 11). Although these hydrogels were confirmed to have highly antifouling and tunable properties,


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the rigorous alkaline gel condition draws concerns for the further application of this material in the area of constructing “blank platform” for 3D cell encapsulation. Click chemistry defines a synthetic concept involving a wide assortment of reactions with different mechanisms but fulfill identical criteria: extremely high regiospecificity and stereospecificity under various mild conditions with high efficiency.29,30 In addition, an emerging type of “copper- and light- free” click reactions31-33, such as “thiol-ene” addition, Diels-Alder cycloaddition, strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC), satisfy all the criteria above but dispense any catalysts and external stimuli. Thereinto, the biocompatible reaction conditions have made Michael-type “thiol-ene” addition34, for example, “thiol-(meth) acrylate”35,36, “thiol-vinyl sulfone”37 and “thiol-maleimide”38 enable the fabrication of complex “clickable” hydrogels formed in situ and the optimal choice for cell encapsulation. Herein, we devote ourselves in developing in situ forming “clickable” zwitterionic starch based hydrogels via a “copper- and light- free” Michael-type “thiol-ene” addition reaction for building a 3D cell encapsulation system in vitro. Acrylated-modified sulfobetaine derived starch SB-ST-A was synthesized and hydrogels were then fabricated with dithiol-functionalized poly (ethylene glycol) (PEG-SH) as crosslinker. In order to explore the potentiality of this hydrogel system operated as a “blank platform”, excellently antifouling biomaterial SB-ST accompanied with low-fouling PEG were introduced. Therefore, the hydrogel would perform excellently in resisting the disturbances from non-target proteins and cells of complex culture environment for further discovering the truth how biochemical cues affect cellular fates. Moreover, the existence of natural polysaccharide starch would also create a more biomimetic and bioactive microenvironment for cellular growth. In this study, the hydrogel could rapidly form under biophysical conditions and its tunable gelation time, mechanical property, equilibrium swelling


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ratio and microstructure morphology were also investigated. BSA adsorption and cell adhesion assays were carried out to confirm the antifouling ability. In the end, the biocompatibility of this hydrogel system for 3D cell culture in vitro was evaluated by A549 cell encapsulation.

2. EXPERIMENTAL SECTION 2.1. Materials N,N-dimethylaminopyridine (DMAP), N,N'-dicyclohexylcarbodiimide (DCC), acrylic acid, ptoluenesulfonic acid (p-TSA), 3-mercaptopropionic acid, DL-1,4-dithiothreitol (DTT) were purchased from Alfa Aesar. Poly (ethylene glycol) (PEG, Average MW 4000 Da) was purchased from Aladdin. Bovine Serum Albumin (BSA), Bicinchoninic acid assay kit (QuantiProTM BCA assay), 3-(4,5-Dimethylthiazol-2-yl)-2,5-bromide (MTT), high glucose Dulbecco's modified Eagle medium (DMEM) with 10 % fetal bovine serum (FBS) and 1 % penicillin and streptomycin were from Sigma Aldrich. 3-Dimethyl (chloropropyl) ammonium propanesulfonate (DCAPS) and DCAPS modified starch (SB-ST) with a degree of substitution of 0.3 (DS, 30 DCAPS groups per 100 anhydroglucosidic units of starch) were synthesized as previously reported.28 All other regents were of analytical grade without further purification.

2.2. Synthesis of Acrylate-modified SB-ST (SB-ST-A) Starch was conjugated with DCAPS using Williamson etherification reaction to yield SB-ST, followed by esterification between carboxyl group of acrylic acid and residual hydroxyl groups of SB-ST with DCC/DMAP as catalytic system, yielding acrylate-modified SB-ST (SB-ST-A), as elaborated in scheme 1. The DCAPS and SB-ST were synthesized according to previously reported method of our lab.28 Different DS of acrylate group (0.2, 0.3 and 0.4) were controlled


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by changing the molar ratio between acrylic acid and anhydroglucosidic (AHG) units of SB-ST (Table 1). Typically, SB-ST (1.345 g, 6.0 mmol equivalent AHG units, DS of SBCP: 0.3) and NaCl (0.105 g, 1.8 mmol) were dispersed in 14 mL anhydrous DMSO and heated with stirring at 130 ˚C for 6 h followed by cooling to room temperature until the SB-ST was completely dissolved. DMAP (0.02 g) was then added to activate hydroxyl groups and stirred for 1 h. Acrylic acid (1.235 mL, 18 mmol) and DCC (3.714 g, 18 mmol) were dissolved in 6 mL anhydrous DMSO stirring for 30 min at room temperature and then added dropwise to the SB-ST solution above. The reaction was left stirring at room temperature and stopped after 48 h. Resulting solution was filtered to remove the solid byproduct of N,N'-dicyclohexylurea (DCU) and then dropped into excess methanol, precipitated, filtered and dried under vacuum for 24 h. The product was in a yield of 60 % and the DS of acrylate group determined via 1H NMR was 0.3. 1H NMR (400M Hz, D2O, δ/ ppm) :δ= 6.37, 5.94 (-C=CH2), 6.14 (-CO-CH=C), 5.30, 3.85, 3.73, 3.55 (starch backbone: Ha (C-CH-O), He’ (C-CH-O), Hf’ (C-CH2-O), Hb’+c’ (C-CH-C)), 3.40 (N+-CH2), 3.04 ( N+-(CH3)2), 2.89 (CH2-SO3-), 2.02 (C-CH2-C).


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Scheme 1. Synthesis of SB-ST-A

2.3. Synthesis of Dithiol-functionalized Poly (ethylene glycol) (PEG-SH) The dithiol-functionalized poly (ethylene glycol) (PEG-SH) was synthesized according to the procedure reported39, as elaborated in Scheme 2. Briefly, PEG (Average MW 4000 Da) was dried by azeotropic distillation with anhydrous toluene to remove residual water prior to use. PEG (4 g, 2 mmol OH), 3-mercaptopropionic acid (3.486 mL, 40 mmol), DTT (154 mg, 1 mmol), p-toluenesulfonic acid (76 mg, 0.4 mmol), were dissolved in 200 mL anhydrous toluene and the solution was refluxed with stirring under a nitrogen atmosphere for 24 h. Toluene was


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removed by reduced pressure distillation and the crude product was poured into excess acetone and then recrystallized under a cold temperature for three times and dried under vacuum for 24 h. The white product was recovered with a 95 % yield and stored at -35 ˚C for further use. The functionality of thiol was determined via 1H NMR was 1.98. 1H NMR (400M Hz, CDCl3, δ/ ppm): δ= 4.28 (-CH2OC(O)-), 3.86~ 3.44 (PEG backbone, -CH2CH2O-) , 2.81~ 2.62 (CH2CH2SH). 1H NMR (400M Hz, DMSO-d6, δ/ ppm): δ= 4.16 (-CH2OC(O)-), 3.71~ 3.37 (PEG backbone, -CH2CH2O-), 3.61 (-CH2-C-OC(O)-), 2.80~ 2.60 (-CH2CH2SH). 1H NMR (400M Hz, D2O, δ/ ppm): δ= 4.18 (-CH2OC(O)-), 3.75~ 3.40 (PEG backbone, -CH2CH2O-), 3.67 (-CH2-COC(O)-), 2.80~ 2.60 (-CH2CH2SH).

Scheme 2. Synthesis of PEG-SH

2.4. Preparation of “Clickable” S/P Hydrogels “Clickable” S/P hydrogels (abbreviation of SB-ST-A/PEG-SH hydrogel, similarly hereinafter without specific emphasis) were prepared via a Michael type “thiol-ene” addition between SBST-A (DS of acrylate is 0.3, used in all the experiments below unless otherwise specified) and PEG-SH, as shown in Figure 1(a). SB-ST-A solutions in PBS (1 mL, 10 mmol, pH 7.4), at four different concentrations (10.0, 5.0, 2.5 and 1.0 %, w/ v), were treated with PEG-SH solutions


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(1mL, dissolved in PBS) in amounts such that acrylate: thiol group molar ratios were 2: 1, 1: 1 and 1: 2 respectively. The mixture was thoroughly mixed before transferred into a cylindrical plastic mold (8mm in diameter and 10cm in height), allowing to gel at 37 ˚C under a humidified atmosphere for 48h until the completely gelation. Gelation time was measured by the vial-tilting method for different precursor concentrations. In brief, aqueous SB-ST-A solution (100 µL) was mixed with aqueous PEG-SH solution (100 µL) and immediately injected into a vial. The vial was then immersed in a thermostatic waterbath at 37 ˚C and frequently tilted until the mixture show no flow within 30 s, it was regarded as a gel. A timemeter was used to record the gelation time in triplicate for each sample.

Figure 1. S/P hydrogel cross-linked via “copper- and light- free” Michael-type “thiol-ene” addition between SB-ST-A and PEG-SH (a) and gelled rapidly under physiology conditions by simply mixing of SB-ST-A and PEG-SH solutions in PBS (b).


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2.5. 1H NMR and FT-IR Characterization of Polymers and Hydrogel The syntheses of SB-ST-A and PEG-SH and the chemical structures of final hydrogel were confirmed by 1H NMR (400M Hz, Avance III, Bruker, U.S.A.) and FT-IR (Magna-560, Nicolet, U.S.A.). 1H NMR spectra of samples were obtained with D2O (for SB-ST-A, PEG-SH and hydrogel), CDCl3 and DMSO-d6 (for PEG-SH) as the solvent. The samples for FT-IR were prepared in KBr pellets. The hydrogel specimen for 1H NMR characterization was prepared by mixing the precursor solutions of SB-ST-A (10.0 % w/v) and PEG-SH (acrylate: thiol group molar ratio of 2: 1) in D2O (0.25 mL for each with Na2HPO4 and NaH2PO4, 10 mM, pH 7.4) followed by injected into a test tube and was then allowed to gel at 37 ˚C for 24 h until the completely gelation before the test. The powdery hydrogel specimen for FT-IR characterization was prepared by grinding the lyophilized dry hydrogel after fully swollen and rinsed by deionized water.

2.6. SEM Characterization of Hydrogel The pore morphologies of hydrogels were observed with a field emission scanning electron microscopy (S-4800, Hitachi, Japan). Hydrogels were frozen at -40 ˚C for 24 h after swelling equilibrium in deionized water followed by lyophilization at -80 ˚C for 24 h. The lyophilized specimens were fractured after quenched in liquid nitrogen to obtain an interior cross-section and were then sputter-coated with a thin layer of gold for the investigation of internal topographies.

2.7. Swelling Characterization The dynamic swelling kinetics and equilibrium swelling ratio (ESR) of the S/P hydrogels prepared at various concentrations and acrylate: thiol group molar ratios were measured in


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deionized water at 37 ˚C. For the dynamic swelling kinetics test, hydrogel disks (8 mm in diameter and 2 mm in thickness) were allowed to swell in 3 mL deionized water and weighted immediately at regular time intervals after the removal of excess water on the surface by rolling them on filter papers, and the water was then refreshed. The test lasts for 48 h. Real-time swelling ratio (RSR) was calculated from the swollen hydrogel weight at each time point (Wt) and the dry hydrogel weight after lyophilization (Wd) as follows: =

(1)

Equilibrium swelling test was continued to perform for 48 h until the complete swelling of hydrogels. The equilibrium swelling ratio (ESR) was calculated from the equilibrium hydrogel weight (We) and the lyophilized weight (Wd): =

(2)

Each measurement was performed in triplicate.

2.8. Dynamic Rheology Experiments Gelation kinetics of hydrogel was monitored by small strain oscillatory shear experiments with a control-strain rheometer (Ar 1000, TA Instrument, U.S.A.) at 37 ˚C. The storage modulus (G’), loss modulus (G”) and gel point (sol-gel transition point, regarded as the initial gelation time) were determined by time-sweep mode at a constant oscillator frequency (0.5 Hz) and strain (0.1 % ) using a 20 mm diameter parallel plate geometry and a 500 µm gap. Frequency-sweeps at 0.1 % strain were performed on each sample to ensure the linearity of viscoelasticity. 2.9. BSA Protein Adsorption BSA protein adsorption assay was performed in 48-well tissue culture plate utilizing the standard bicinchoninic acid (BCA) method. Prior to the experiment, hydrogel disks (5 mm in


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diameter and 2 mm in thickness) were immersed in PBS overnight to reach a swelling equilibrium. Each sample was then suspended into a well containing 400 µL BSA protein solution (1 mg/mL, freshly prepared in PBS) followed by incubation at 37 ˚C for 2h. Before transferred into another clean plate, the samples were rinsed with 5× 500 µL PBS to remove dissociative BSA proteins. 400 µL of sodium dodecyl sulfate solution (SDS, 1.0 wt %) was added into each well and soaking for 1 h at room temperature to strip the proteins adsorbed on the surface of the hydrogels for detection. BCA protein assay was carried out to determine the amount of BSA proteins adsorbed on the hydrogel, and the absorbance was measured by a microplate reader at 562 nm. The amount of protein was calculated according to the calibration curve of BSA. The control sample was BSA protein adsorbed on the hydrophobic polystyrene (PS) surface. Each sample was measured in triplicate.

2.10. In Vitro Degradation Study The biodegradability of the S/P hydrogel in vitro was assessed in PBS at 37 ˚C. In brief, S/P hydrogel (500µL for each) prepared with an SB-ST-A concentration of 10.0 % (w/v) and an acrylate: thiol group molar ratio of 2: 1were incubated in 3 mL PBS (10 mM, pH 7.4) at 37 ˚C in vials with the PBS refreshed every 2 days. The hydrogels were taken out at regular time intervals and the dry hydrogel weights after lyophilization were measured. The weight remaining of the hydrogel was calculated from the dry hydrogel weight at each time point (Wd,t) and the original dry hydrogel weight (Wd,o) as follows:

ℎ (%) = , × 100

(3)

,


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2.11. Cytotoxicity Assay The cytotoxicity of S/P hydrogel was evaluated by a MTT assay using A549 cells. Hydrogel disks were first sterilized with 75 % ethanol solution for 48 h and followed by immersed in 5 mL of DMEM culture medium at 37 ˚C. After 48 h, the hydrogels were removed and the retained extract solutions were applied to the cytotoxicity assay. The A549 cells were maintained in complete DMEM at 37 ˚C and 5 % CO2, and the culture medium were refreshed every 2~3 days. Cell suspension (200 µL, at a cell density of 5× 103 cells/ well) was added into each well of a 96-well plate and incubated at 37 ˚C and 5 % CO2 overnight for cell attachment. The culture medium was then replaced by 200µL of hydrogel extracts and routinely incubated for another 1, 2, 3 days, the MTT assay was performed afterwards. Into each well, 200µL of MTT solution (0.5 mg/ mL in DMEM) was added for another 4 h of incubation at 37 ˚C in a humidified atmosphere of 5 % CO2. The purple crystal formazan secreted by mitochondria in each well was dissolved by 200 µL DMSO and then measured at 490 nm by a microplate reader. All the samples were measured in triplicate and the final absorbance values were normalized to the well without any extracts after 1 day’s culture.

2.12. Cell Adhesion Assay Hydrogel disks with 5 mm in diameter and 2 mm in thickness were immersed in PBS overnight for swelling equilibrium. The swollen hydrogels were then sterilized by 75 % ethanol solution for 48 h before transferred into a 24-well plate with one disk in each well. A549 cells were seeded onto the hydrogels at a concentration of 7.5× 104 cells/ mL suspended in DMEM medium and allowed to grow for 24 h at 37 ˚C in a humidified atmosphere with 5 % CO2. Then


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the medium was removed and the hydrogels were gently washed with PBS before photographed at 10× magnification on the microscope. 2.13. Cell Encapsulation, Viability and Morphology A549 cell encapsulation in vitro by the “clickable” S/P hydrofgel was carried out utilizing the procedure similar to the gel fabrication method mentioned above, as elaborated in Figure 2. GFPtransfected A549 cells were suspended in SB-ST and PEG-SH precursors mixture (in PBS at a SB-ST concentration of 10.0 % (w/ v) and acrylate: thiol molar ratio of 2: 1) at a cell density of 1 × 106 cells/ mL followed by added into a 24-well plate (200 µL each well) and incubated at 37 ˚C and 5 % CO2 for 10 min until complete gelation. Cell-laden hydrogels were then immersed in 1mL DMEM culture medium and incubated at 37 ˚C in a humidified atmosphere of 5 % CO2 with a medium exchange every 2 days. Cell viabilities and morphologies at each time point (0, 1, 2 days) were determined by a fluorescence microscope (IX73, Olympus). For quantitative analysis of cell viability, 10 couples of micrographs (one fluorescence image with one optical image at the same horizon) were taken randomly from the hydrogels. Cell viability was then calculated from the proportion of live cells (emitted green light in fluorescence image) in the entire cells (counted by the optical image).


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Figure 2. “Thiol-ene” S/P hydrogel for 3D cell encapsulation and culture in vitro. (A) SB-ST-A, (B) PEG-SH, (C) cells and (D) cell-laden hydrogel.

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of SB-ST-A and PEG-SH The SB-ST was prepared via a Williamson etherification between the sulfobetaine etherifying agent DCAPS and the hydroxyl groups of starch.28 And the DS of sulfobetain ranging from 0 to 0.4 could be conveniently controlled through changing the feeding ratio of DCAPS/ starch AHG. The SB-ST-A was then synthesized through the homogeneous acylation of SB-ST with acrylic acid carried out in anhydrous DMSO at room temperature, as illustrated in scheme 1. SB-ST can be only dissolved in DMSO besides water, which is yet inappropriate for esterification reactions. Previously, Fabian Rodler et al.40 have elaborated the pH-switchable self-assemble behavior of serine-derived guanidiniocarbonyl pyrrole carboxylate zwitterion in DMSO. Interestingly, SBST behaved an analogous pH sensibility after dissolving in DMSO. The homogeneous DMSO


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solution of SB-ST would gradually aggregate and precipitate when organic acid, such as acrylic acid and acetic acid, was added. Additionally, sulfobetaine-based (meth) acrylic polymers, such as poly (sulfobetaine methacrylate) (PSBMA), always endow an electrostatic forces-induced upper critical solution temperature (UCST) behavior in aqueous solution.41-43 Coincidentally, SB-ST in DMSO was also thermosensitive, inversely, with a lower critical solution temperature (LCST), which depended on the DS of DCAPS moieties. For instance, the LCST reaches 77 ˚C when the DS is 0.3. However, a higher DS of 0.4 would result in a much lower LCST of 41 ˚C. Relative to the pure starch dissolved in DMSO, both pH-sensitivity and thermosensitivity of SB-ST mentioned above were all attributed to the inter- /intra- molecular electrostatic interaction between positive ammonium and negative sulfonate charges of the sulfobetaine moieties conjugated on starch backbone. Nevertheless, all these special properties would be an undesirable obstacle for a homogeneous and stable reaction system of SB-ST and acrylic acid in DMSO at room temperature, especially for SB-ST with a higher DS of sulfobetaine. While sulfobetain-based polyzwitterion always suffer different thermo- /pH- response properties in the presence of salt, such as NaCl, in aqueous solutions.44 Surprisingly, as a common charge screening agent, NaCl can also dramatically increase the LCST and weaken pH sensibility of SB-ST in DMSO. We even find out the optimum screening effect occurred at a NaCl: sulfobetaine moieties molar ratio of 1:1. For completely dissolving, the mixture of SB-ST, NaCl and anhydrous DMSO were heated at 130˚C for 6h followed by cooled to room temperature. The esterification reaction occurred between acrylic acid and residue hydroxyl group on the SB-ST backbone with DMAP/ DCC as catalytic system at room temperature. Different molar feeding ratios of acrylic acid/ SB-ST AHG were required for different substitution degrees (Table 1).


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Table 1. Synthesis of SB-ST-A and the substitution degree of acrylate group Molar feeding concentration a of SB-ST (mmol/ mL)

0.3

Molar feeding ratio of acrylic acid/ SB-ST AHG

DS b of SB-ST-A

3.0

0.2

4.0

0.3

5.0

0.4

a

The molar feeding concentration is 0.3 mmol SB-ST AHG/ ml of DMSO. acrylate group in SB-ST-A was determined by 1H NMR in D2O.

b

The DS of

The chemical structure of SB-ST-A was confirmed by 1H NMR (Figure 3) and FT-IR (Figure 4) spectra. Figure 3 displays the 1H NMR spectra of (A) virgin starch, (B) SB-ST and (C) SBST-A. Besides the signals attributed to starch, four new peaks at 3.4, 3.1, 2.9, 2.0 ppm (peaks b~ g) were all due to the DCAPS moieties. Beyond that, three extra new peaks at 5.94, 6.14, 6.37 ppm (peaks h~ j) belonging to three different allyl protons confirmed successful conjugation of acrylate groups on the SB-ST. The degree of modification is calculated according to the integral ratio of the peak from glucose units of starch (singlet peak at 5.29 ppm) and that from acrylic acid (singlet peak at 6.14 ppm) as follows: DS= Ih/ Ia. DS varies from 0.2 to 0.4 depends on the feeding ratio of acrylic acid/ SB-ST AHG (illustrated in Table 1) and the 1H NMR spectra were shown in Figure S1.


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Figure 3. 1H NMR spectra of (A) virgin starch, (B) SB-ST and (C) SB-ST-A in D2O. Figure 4 shows the FT-IR spectra of (A) virgin starch, (B) SB-ST and (C) SB-ST-A. As elaborated before, electron-deficient C=C double bonds are crucial for nucleophilic Michael-type “thiol-ene” addition.34 Thus, the stretching vibration of carbonyl in acrylate group at 1730 cm−1 only appears in the FT-IR spectra of SB-ST-A also indicates the success in synthesizing SB-STA and allyl C=C bond was adjoined by electron withdrawing carbonyl group. Additionally, two new absorbance at 1483 and 1209 cm-1 representing N-C stretching vibration and S=O asymmetrical stretching vibration in DCAPS moieties respectively confirmed the successful combining of DCAPS with the starch backbone.


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Figure 4. FT-IR spectra of (A) virgin starch, (B) SB-ST and (C) SB-ST-A. The straightforward synthetic route of dithiol-functionalized crosslinker PEG-SH was according to the method published before39, as illustrated in scheme 2. The functionalization was carried out on the basis of ester formation between hydroxyl-terminal liner PEG and 3mercaptopropionic acid with p-toluene sulfonic acid as the catalyst, in the anhydrous toluene under reflux condition. DTT was employed as reducing agent against disulfide bond formation during the reaction. The structure and thiol-functionality of PEG-SH was also determined by 1H NMR (Figure S2 (a) and (b)) and FT-IR spectra (Figure S3). In the 1H NMR spectra of PEG-SH in CDCl3 as shown in Figure S2 (a) , the characteristic chemical shifts of PEG-SH are readily identifiable: new peaks at 2.81~2.62, 4.28 ppm are attributed to the protons of methylenes on mercaptopropionic acid and the terminal methylenes on PEG adjoined to the ester linkages, respectively. Therefore, 1H NMR spectra confirms the successful synthesis of PEG-SH, as well as the stretching vibration of carbonyl in ester linkages at 1728 cm−1 appeared in the FT-IR spectra (Figure S3). The thiol functionality calculated via the


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spectra was nearly 2, which is also certified by the completely disappeared terminal-hydroxyl peak at 4.58 ppm of 1H NMR spectroscopy in DMSO-d6 (Figure S2 (b)).

3.2. In Situ “Clickable” S/P Hydrogels Fabrication and Gelation Time The ultimate target of this paper is the establishment of strategies to fabricate in situ SB-ST based multifunctional hydrogels gelled under mild physiological conditions for 3D cell culture. “Copper- and light- free” click chemistry becomes the optimum choice because of the excellent reaction characteristics, such as outstanding orthogonality, extremely high efficiency, without any catalyst or external stimuli like UV. Although a variety of this reaction types have been employed to construct “clickable” polysaccharide or PEG hydrogels, including tetrazinenorbornene reaction for alginate cross-linking45, cross-linking of bis(DIFO3) di-functionalized polypeptide and four-arm PEG tetra-azid46, conjugation of furan modified HA and dimaleimide PEG47 and so on, nucleophilic Michael-type “thiol-ene” additions48-50 have been most ubiquitously used for their more simplified synthetic process but tantamount superiority as mentioned above. Furthermore, the existence of electron-deficient C=C double bonds provide opportunities for incorporating various peptide sequences with cysteine residues, like cell adhesion protein RGD. Herein, the crosslinking reaction for the fabrication of S/P hydrogels were carried out via Michael-type “thiol-ene” addition between acrylate groups grafted on SB-ST backbone and terminal thiols of PEG-SH as illustrated in Figure 1(a). Upon fully mixing the same amounts of SB-ST-A and PEG-SH solutions respectively dissolved in PBS, gelation occurred in minutes at 37 ˚C. The apparent gelation process can be observed by inclining the vial shown in Figure 1(b).


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The gelations were carried out under various precursor concentrations and acrylate: thiol molar ratios to investigate their influences on the gelation time. The four concentrations of SB-ST-A were fixed at 10.0, 5.0, 2.5, 1.0 % (w/ v) and the amounts of PEG-SH varied to guarantee the acrylate: thiol molar ratios were 2: 1, 1: 1 and 1: 2 respectively for every concentration of SBST-A. As summarized in Table 2, when the SB-ST-A solution was 1.0 %, all the conditions failed leading to a hydrogel. However, when the concentration of SB-ST-A was increased to 2.5 %, viscosity of the precursor solution increased over time until the ultimate formation of a concretionary transparent hydrogel. Additionally, the gelation time could be accelerated by increasing the molar ratio of acrylate: thiol. As shown in Table 2, in this gel system, a higher proportion of acrylate would result in a faster cross-linking rate as well as a shorter gelation time for all the concentrations. When the molar ratio is 2: 1, the gelation occurred in less than 20 min. In particular, the constitutions of 10.0 %/ 2:1 (defined as 10 % of SB-ST-A concentration with a acrylate: thiol molar ratio of 2:1, similarly hereinafter) and 5.0 %/ 2:1 gelled in about 7 min. However, S/P could hardly gel within 1h with a ratio of 1: 2. On the other hand, creating a rapider gel system could also depend on the promotion of precursor concentrations with any specific acrylate: thiol ratio (except for 5.0 %/ 1: 1, and the detailed gelation times of the “1: 2” group are not shown).


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Table 2. Gelation times of S/P hydrogels with different precursor concentrations and molar ratios of acrylate: thiol

SB-ST-A

PEG-SH

Total

Molar Ratio of Acrylate: Thiol

10.0

13.4

23.4

2:1

410 ± 2

5.0

6.7

11.7

2:1

438 ± 3

2.5

3.4

5.9

2:1

1215 ± 13

1.0

1.4

2.4

2:1

——

10.0

26.8

36.8

1:1

2209 ± 4

5.0

13.4

18.4

1:1

531 ± 1

2.5

6.7

9.2

1:1

3094 ± 25

1.0

2.7

3.7

1:1

——

10.0

53.5

63.5

1:2

>3600

5.0

26.8

31.8

1:2

>3600

2.5

13.4

15.9

1:2

>3600

1.0

5.4

6.4

1:2

——

Concentration (%, w/ v)

Gelation Time (s)

3.3. Chemical Structure of Hydrogel In order to characterize the chemical structure of the final hydrogel, FT-IR and 1H NMR experiments were carried out. The S/P hydrogel 10.0 %/ 2: 1 was chosen as a model. As shown in Figure 5(a), the FT-IR spectrum of the hydrogel contains the absorption bands of both SB-STA and PEG-SH, which fully indicates that the hydrogel was composed of these two components and a completed network was formed. In order to further characterize the crosslinking reaction through Michael-type “thiol-ene” addition, the 1H NMR characterization was carried out. As


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illustrated in Figure 5(b), the spectrum of hydrogel shows both the peaks of SB-ST-A and PEGSH, which is in accordance with the FT-IR results. However, the three peaks at 5.94, 6.14, 6.37 ppm (peaks h~ j) belonging to three different allyl protons of the acrylate groups on SB-ST-A almost disappeared, which confirms the occurrence of the Michael addition reactions between the acrylate groups on SB-ST-A and the thiols on PEG-SH. The residual DS of acrylate groups calculated from the integration of the peaks h~ j is about 0.14 because the original acrylate: thiol group molar ratio is 2: 1 (the original DS of acrylate is 0.3). In addition, three new peaks at 2.77, 2.67, 2.62 ppm (peaks k~ n) also demonstrates the formation of the thioether bonds. These results confirm that the S/P hydrogel comprises of SB-ST-A and PEG-SH and crosslinked by “thiol-ene” addition between acrylate and thiol groups. (a)


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(b)

Figure 5. FT-IR spectra (a) and 1H NMR spectra in D2O (b) of (A) PEG-SH, (B) SB-ST-A and (C) S/P Hydrogel (10.0 %/ 2: 1).

3.4. Gelation Kinetics and Mechanical Properties by Oscillatory Rheology The hydrogels were rapidly prepared by mixing aqueous SB-ST-A and PEG-SH precursor solutions at 37 ˚C and underwent a gradual sol-gel transformation. The final mechanical property of hydrogels primarily depends on the cross-linking degree of the network, which could also be tailored through altering precursor concentrations, functional group molar ratios and even


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substitution degree of acrylate. Recent studies51-53 have shown that matrix stiffness would influence cell growth and morphogenesis in 3D and tunable mechanical property is essential for the universality of the platform used in different cell types. Herein, the gelation processes and mechanical properties were measured by dynamic time sweep rheological experiments using a control-strain oscillatory rheometer at pH 7.4 and 37 ˚C, as shown in Figure 6. (a)

(b)


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(c)

(d)

Figure 6. Dynamic time sweep rheological experiments of different S/P hydrogel systems. Storage modulus G’ and loss modulus G’’ curves for (a) 5.0 %/ 1: 1 S/P hydrogel, (b) different group molar ratios with 5.0 % SB-ST-A, (c) different SB-ST-A concentrations with group molar ratio of 2: 1 and (d) different acrylate DS of SB-ST-A with 5.0 % SB-ST-A and group molar ratio of 2: 1.

For illustration, Figure 6(a) present typical modulus curves of hydrogel 5.0 %/ 1: 1 as an example. Immediately after mixing the two components, G’ and G’’ sharply increased over time


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and the crossover is defined as the gelation point where the material is more gel-like than being a liquid. Ultimately, both of the G’ and G’’ curves approached a plateau within 45 min, suggesting that hydrogels were already stably formed and the constant value of equilibrium storage modulus G’ reflects the final mechanical strength of hydrogel. Besides, frequency-sweeps at 0.1 % strain were performed on each sample to ensure the linearity of viscoelasticity. In order to verify the influence of precursor concentration and functional group ratio to the mechanical property and gelation time, Figure 6(b) and (c) showed the dynamic curves of storage modulus G’ for different SB-ST-A solution concentrations and acrylate: thiol molar ratios respectively. From the data in Figure 6(b), we can easily find out when the group ratio was fixed at 2: 1, the hydrogel formed at low concentration (2.5 %) only has lowest equilibrium G’ value of 0.82 kPa and the G’ was too low to be measured within 650 s. However, the G’ increased with the promotion of solid content and approached to 7.19 kPa and 18.32 kPa respectively with concentrations of 5.0 % and 10.0 %. It is accessible that the significant leaps of mechanical strength stem from the higher cross-linking degree and more perfect 3D network structure. Thus hydrogels with various stiffnesses could be fabricated simply through changing the precursor concentrations. From the partial enlarged views of three G’ and G’’ crossover in the right, a higher concentration would lead to a shorter gelation time, which is in accordance with the description in Table 2. It is noteworthy that the 10.0 % / 2: 1 system, as an example, gelled at 77 s through the modulus curves. However, the vial-tilting test showed a much longer gelation time of about 7 min (Table 2). This is because a certain yield stress is necessary for the zero flow at vial tilting54 and the gelation time of vial-tilting are in close proximity to the time G’ curve begins to reach a plateau, which represents the initial time to form a complete macroscopic


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hydrogel. So the dynamic time sweep rheological experiments provided a more accurate measurement of sol-gel transformation points in microcosmic. Likewise, as shown in Figure 6(c), when the concentration of SB-ST-A was fixed at 5.0 %, the 5.0 %/ 1: 2 hydrogel was endowed a longest gelation time of about 27 min. Owing to the long gelation time by vial-tilting (in Table 2) outdistance 1h, the G’ curve of 5.0 %/ 1: 2 was still far from reaching a plateau after the test time of 45 min and the G’ (0.19 kPa) cannot represent the final mechanical strength. The gelation time was shortened with the increase of SB-ST-A proportion that hydrogel 5.0 %/ 1: 1 gelled in 297 s and 5.0 %/ 2: 1 in 127 s, which was in accordance with the variation tendency shown in Table 2. While hydrogel 5.0 %/ 1: 1 achieved the highest G’ value of 8.16 kPa and was 0.97 kPa higher than that of 5.0 %/ 2: 1 for the reason that slightly higher solid content (total concentration in Table 2) and equal amount of acrylate and thiol groups would construct more adjunction points for the network in spite of a slightly lower reaction rate. In order to further explore other influence factors for the mechanical property and gelation time, the change of the DS of acrylate group was also considered and the result was shown in Figure 6(d). We fixed the gelation component at 5.0 %/ 2: 1. As expected, the storage modulus G’ value of DS 0.2 was 4.53 kPa and when the DS was increased to 0.3 and 0.4, the G’ also promoted to 7.19 kPa and 9.78 kPa respectively because the total concentration of both acrylate and thiol groups as well as the final cross-linking points got increased. We chose 0.3 as the only substitute degree of acrylate covering the whole research because of its rapidest gel rate of 127 s. The elastic modulus of native human tissues range from near 100 Pa for the softest organs, such as the mammary gland and brain, to several thousands of Pascal for stiffer tissues like kidney, lung and muscle55. Moreover, many research results have shown that the mechanical


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property of hydrogel matrix would dramatically affect cellular fate cultured in vitro. For example, round-shaped spheroids formation and growth of ovarian cancer cells in GelMA-based hydrogel preferred a medium stiffness about 3.7 kPa56 and a rigid matrix would promote osteogenic differentiation of C2C12 encapsulated in TG-Gels57, and so on. As shown from the results of the rheology tests above, the storage modulus of different S/P hydrogels ranged from about 800 Pa to about 20 kPa, and many softer or stiffer hydrogels could also be obtained via choosing other hydrogel compositions. These results indicated that our S/P hydrogel could easily regulate the stiffness to mimic various native tissues and could also satisfy various mechanical requirements for different cell types as the 3D culture platform.

3.5. Hydrogel Swelling and Morphology Analysis The dynamic equilibrium swelling measurements of the S/P hydrogels in deionized water were also investigated to better understand the effect of compositional variations on the swelling kinetics and equilibrium swelling ratio (ESR). As expected, Figure 7(a) and (b) separately give the swelling kinetics and equilibrium swelling ratios of hydrogels in different precursor solution concentrations and acrylate: thiol molar ratios. As shown in Figure 7(a), the real-time swelling ratio (RSR) of the completely gelled hydrogels increased sharply after immersed in deionized water and reached equilibrium within 8 hours for all of the compositions tested. While when the group ratio was fixed at 1: 1, hydrogel swelled much faster and achieved higher water content with a lower initial SB-ST-A concentration. The ESRs are also exhibited in Figure 7(b): for hydrogel 10.0 %/ 1: 1, the ESR was 35.65. However, the ESR reached at a considerably high value of 149.63 for hydrogel 2.5 %/ 1: 1 because of a looser network. In Figure 7(b), the ESRs of varied group ratios with certain SB-ST-A concentration of 5.0 % were also studied. Undoubtedly, hydrogel 5.0 %/ 1: 1 absorbed the least water with an ESR of 54.05 for the densest


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cross-linking network and hydrogel 5.0 %/ 2: 1 and 5.0 %/ 1: 2 absorbed more for a looser structure. However, hydrogel 5.0 %/ 2: 1 achieved a much higher ESR of 189.60 because the extremely hydrophilic SB-ST-A was predominant in the composition and the ionic hydration could combine more water molecules. On the contrary, hydrogel 5.0 %/ 1: 2 could only get a lower ESR of 58.47. The high water content of S/P hydrogel would be advantageous for the transportation of nutrient, signal factors and even cell metabolic waste and then promote the growth of cells encapsulated.


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Figure 7. Swelling kinetic curves (a) and equilibrium swelling ratios (b) of hydrogels with different precursor solution concentrations and acrylate: thiol molar ratios. SEM images in two different magnifications (100× and 30×) characterized the fracture surface microstructure of freeze-dried hydrogel 10.0 %/ 2: 1 after swelling equilibrium (the dynamic swelling curve is shown in Figure 7(a)), as shown in Figure 8(a) and (b). During the lyophilization step, water absorbed in hydrogel was first frozen into ice crystals and then sublimated by negative-pressure drying process, and afterwards, the pores originally occupied by water were observed. Figure 8(a) displays a sponge like and microporous morphology inside the network and the pores were homogeneous in both size and distribution, which is in favor of cell dwelling and migration. The average size of the pores, can be easily measured from Figure 8(b), were about 100- 200µm.

Figure 8. SEM photographs of 10.0 %/ 2: 1 S/P hydrogel in two different magnifications of (a) 30× and (b) 100×, the scale bars represent 1 mm and 400 µm respectively.

3.6. Protein Adsorption


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The growth and physiological behaviors of autologous cells in native tissues are regulated by multiple signal factors transmitted from surrounding ECM. It is necessary to ascertain how and whether these factors affect cell fate individually or synergistically for 3D cell culture and the artificial induction of tissue formation in vitro. The first step is constructing “blank platform” to exclude the interference of non-target factors. Thus we should focus on solving the nonspecific adsorption of the proteins, which the traditional biomaterials often suffered, on the surface of the hydrogel. A protein adsorption assay was carried out to verify the S/P hydrogel held the ability to resist nonspecific protein adsorption. Bovine serum albumin (BSA), a globular protein, was selected as the model protein for adsorption assay. As shown in Figure 9, hydrophobic interaction between protein and hydrophobic surface of polystyrene (PS) made BSA energetically favorable to adhere. Zwitterionic polymers bearing both positively and negatively charged groups on the same monomer residue have been confirmed to generate higher hydration free energy and bind water molecules more strongly through electrostatically induced hydration than that of PEG-type materials through hydrogen bonding.24,58 Thus, poly (sulfobetaine methacrylate) (PSBMA) hydrogel possessed apparently superior antifouling property than PEG hydrogel, which although have been widely used as nonfouling material. Although the natural polysaccharide starch is also low-fouling because of the hydrogen bonding induced hydration via large amounts of hydroxyl groups, the pure starch hydrogel also got a relatively higher protein adsorption content. However, after binding sulfobetaine groups, the SB-ST hydrogel adsorbed less protein in accordance to the result of IgG, pepsin and lysozyme reported before.28 Importantly, our S/P hydrogel (with a composition of 10.0 %/ 2: 1) behaved excellently in resisting protein adsorption although somewhat inferior to that of PSBMA, but a denser grafted of sulfobetaine moieties would dramatically remedy this (the S0.4/ P hoydrogel in Figure 9,


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represents the DS of DCAPS in SB-ST-A is 0.4 and the same composition with S/P). It is suggested that the S/P hydrogel we developed here would be a suitable candidate to act as a “blank platform”.

Figure 9. BSA adsorption on the surface of different hydrogels in PBS.

3.7. Biodegradability Through the efforts above, we have established rapidly formed “clickable” S/P hydrogels based on sulfobetaine-derived starch with PEG-SH as crosslinker under mild physiological conditions. And tunable mechanical properties and high equilibrium swelling ratio make them suitable to mimic ECM biomicroenvironment for cell encapsulation and culture. At last, we selected hydrogel 10.0 % /2: 1 as the model gel for all the subsequent cell experiments in consideration of the fast gelation time of 7 min (by vial-tilting, or 77s by rheology) and moderate stiffness as well as water content.


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Before the S/P hydrogel was employed as a “blank platform” for 3D cell encapsulation and culture, the biodegradability of hydrogel 10.0 %/ 2: 1 in vitro was evaluated in PBS at 37 ˚C. As shown in Figure 10, the degradation of the S/P hydrogel could be obviously divided into two stages. In the first stage, no significant weight loss of the hydrogel was monitored and the dry weight remained up to about 94 % after 9 days’ incubation. The tiny weight loss was caused by the incomplete conversion of the “thiol-ene” Michael addition and then the extraction of the unreacted fraction in the PBS. The hydrogel still kept its almost intact network structure. While in the second stage, the weight of the hydrogel began to decrease dramatically and continuously and it completely degraded after 21 days. During this period, plentiful active ester bonds pervading inside the hydrogel gradually hydrolyzed because of the alkalescency of PBS environment. The network structure of the hydrogel started to disrupt and it leaded to an accelerated degradation rate. As a contrast, SB-ST hydrogel crosslinked by poly (ethylene glycol) diglycidyl ether (PEGDE)28 possessed similar chemical component except that the active ester bonds in S/P hydrogel were replaced by stable ether bonds. Thus, the SB-ST hydrogel and starch hydrogel crosslinked by PEGDE as well could hardly degrade and their slight weight loss after 21 days’ incubation was apparently due to the unreacted fraction extracted by the PBS and the instinctive slow biodegradability of natural polysaccharide. Similarly, the PSBMA hydrogel crosslinked by methylene-bis-acrylamide (MBAA) experienced almost no degradation because of the stable carbon backbone. These results indicates that the S/P hydrogel had tunable biodegradability in vitro and showed faster degradation rate than the SB-ST, starch and PSBMA hydrogels, which is essential for cells encapsulated inside to remodel their surrounding microenvironment and replace the existing structure with their own ECM for normal cellular proliferation and behaviors.


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Figure 10. Degradation of different hydrogels in PBS at 37˚C 3.8. Cytotoxicity Cytocompatibility is one of essential prerequisites for biomaterials before use and thus a MTT assay was carried out to detect the cytotoxicity of hydrogel extract solutions in DMEM medium, the results were shown in Figure 11. During the 3 days of culture, the numbers of A549 cells cultured in the extracts increased over time for our S/P hydrogel as well as the SB-ST and PSBMA hydrogels which had been confirmed to be biocompatible. Moreover, the proliferation behavior of cells in extracts was similar to that in the control of DMEM medium. The MTT results demonstrate that the S/P hydrogels have low cell cytotoxicity and, thus, are biocompatible.


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Figure 11. Viability of A549 cells cultured in the extracts of different hydrogels for 1, 2, 3 days with complete DMEM culture medium as control. The cell viabilities of each group were normalized to the control cultured after 1 day.

3.9. Cell Adhesion It is generally believed that cells adhesion was mediated by the interactions with the proteins preadsorbed on the material surface.59 In view of the excellent ability of nonspecific protein resistance, S/P hydrogel should also effectively prevent cell attachment. In order to further testify the potentiality of our S/P hydrogel as a “blank platform”, a cell adhesion assay was carried out. A549 cells in same amounts were seeded onto sterilized S/P, SB-ST and PSBMA hydrogels and tissue culture plates (TCPs) respectively. After incubation for 24 h, Figure 12 shows that A549 cells were able to normally grow on the surface of TCPs, but S/P hydrogels did not support almost any cell adhesion like the SB-ST and PSBMA hydrogels acting as the negative controls. This low cell adhesion indicates that our S/P hydrogel is antifouling and holds the ability to


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protect hydrogels from the interference of other non-target cells in 3D cell culture and acts as a “blank platform”.

Figure 12. Microscopic images of A549 cells cultured on the surfaces of different hydrogels for 1 day with TCPs as control. Scale bars represent 200µm.

3.10. Cell Viability and Morphology inside S/P Hydrogel. To demonstrate the gelation process used here is cytocompatible for cell encapsulation, A549 cells labeled with green fluorescent protein (GFP) for monitoring the living cell tracer under the observation of fluorescence microscope were encapsulated in S/P hydrogel with a composition of 10.0 % / 2:1 and the result was shown in Figure 13. Accompanied with the photograph of optical microscope for the same horizon, we can easily qualitatively identify and quantificationally calculate the cell viability. It was determined that almost all the cells were alive after encapsulation (Figure 13(a)) and the initial viability was over 95 % (Figure 13(b)). After the cellladen hydrogels were continually incubated in vitro under standard condition for 2 days, cell proliferation was apparently observed by the increasing cell population density from microscope photographs (Figure 13(a)). The quantitative result shows no significant cell death occurred in


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this period and the viability maintained up to 93 % after 2 days’ culture. Interestingly, from the cell morphologies shown in Figure 14, A549 cells maintained round shape till 1 day after encapsulation because the cells were embedded in the gel without redundant space for cell movement and adhesion site for cell attachment. However, after 2 days’ culture, most cells start to stretch into fusiform shape and the budding of cells from the existing cells could also be observed. That is highly probably because the plentiful active ester bonds pervading inside the hydrogel gradually hydrolyzed in the DMEM medium containing amounts of NaCO3 and NaHCO3 and cells could remold the circumstance around. And together with the high vitality and division speed of A549 cells, we can fortunately observe the proliferation and morphology change in only 2 days and without adding any adhesion proteins (e.g. RGD). However, for further investigation of different types of cells culture in the future, especially the stem cells, RGD for cell adhesion and enzymatic cleavage sites should be considered to be introduced into the S/P system.


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Figure 13. (a) The optical, fluorescence and merged images of A549 cells encapsulated in 10.0 %/ 2:1 S/P hydrogel immediately and after 1 day and 2 days. Cell density 1× 106 cells/ mL. The scale bars represent 200 µm. (b) Cell viability quantificationally calculated from optical and fluorescence images.


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Figure 14. The fluorescence images of A549 cells encapsulated in 10.0 % / 2:1 S/P hydrogel immediately and cultured after 1 day and 2 days with TCPs as control. The scale bars represent 200µm.

4. CONCLUSIONS We have successfully synthesized acrylated-modified sulfobetaine derived starch SB-ST-A and in situ “clickable” hydrogels were prepared with PEG-SH as crosslinker via a “copper- and light- free” Michael-type “thiol-ene” addition reaction. The hydrogels could be conveniently fabricated by simply mixing the PBS solutions of SB-ST-A and PEG-SH and the gelation would rapidly occur within 7 min under physiological conditions. Moreover, the gelation time, mechanical properties and swelling behavior of the hydrogels were tunable by simply varying the precursor solution concentration and molar ratio of acrylate and thiol. The resistance of the hydrogel to nonspecific protein adsorption and cell adhesion was confirmed and attributed to the


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excellent antifouling properties of zwitterionic-derived polysaccharide and PEG moieties. The hydrogel was biodegradable and could completely degrade in vitro with 21 days. A549 cells encapsulated in hydrogel maintained high viability and surprisingly started to proliferate in number and extend in morphology after 2 days’ culture, which is probably because of the high vitality of A549 cells and the gradual hydrolysis of ester bonds all over the hydrogel. This easy handling and biocompatible hydrogel potentially provides a versatile “blank platform” for 3D cell encapsulation and culture in vitro and studying how biochemical signals affect cellular fate and behaviors individually.

ASSOCIATED CONTENT Supporting Information Figure S1, 1H NMR spectra of SB-ST-A in different DS of acrylate group. Figure S2, 1H NMR spectra of PEG-SH in CDCl3 and D2O. Figure S3, FT-IR spectra of PEG-SH. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected] (F. Yao). * E-mail: [email protected] (H. Sun). Notes The authors declare no competing financial interest.


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