Injectable Polypeptide Hydrogel as Biomimetic Scaffolds with Tunable

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Injectable Polypeptide Hydrogel as Biomimetic Scaffolds with Tunable Bioactivity and Controllable Cell Adhesion Qinghua Xu, Zhen Zhang, Chunsheng Xiao, Chaoliang He, and Xuesi Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00142 • Publication Date (Web): 14 Mar 2017 Downloaded from http://pubs.acs.org on March 16, 2017

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Injectable Polypeptide Hydrogel as Biomimetic Scaffolds with Tunable Bioactivity and Controllable Cell Adhesion Qinghua Xu†, Zhen Zhang†‡, Chunsheng Xiao†, Chaoliang He*,†, Xuesi Chen*,† †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied

Chemistry, Chinese Academy of Sciences, Changchun 130022, P. R. China ‡

University of Chinese Academy of Sciences, Beijing 100039, P. R. China

1

ACS Paragon Plus Environment

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ABSTRACT: Injectable hydrogels have been widely investigated for applications in biomedical fields, for instance, as biomimetic scaffolds mimicking the extracellular matrix (ECM). In addition to as scaffolds for mechanical support and transferring of nutrients, the dynamic bioactivity of ECM is another critical factor that affects cell behavior. In this work, a novel injectable poly(L-glutamic acid)-based hydrogel decorated with RGD was fabricated. The presentation of RGD significantly enhanced the cell-matrix interaction, and promoted cell adhesion and proliferation. Moreover, the cell-adhesive RGD was conjugated to the network via a disulfide bond, so that the density of RGD and the bioactivity of hydrogel can be well controlled by tuning the RGD content through treating with glutathione. As a result, the cell behaviors on the hydrogel can be tuned on demand. The injectable hydrogel with controllable bioactivity may provide an interesting strategy to develop a scaffold mimicking ECM that can regulate cell adhesion dynamically.

KEYWORDS: Injectable hydrogel, bioactivity modification, cell-matrix interaction, controlled cell behavior, enzymatic cross-linking

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1. INTRODUCTION Hydrogels have been widely investigated for their potential in three dimensional (3D) cell culture and tissue engineering, due to their unique properties similar to native extracellular matrix (ECM).1-5 Due to the cross-linked 3D network, hydrogels can absorb large numbers of water while maintaining their shape, and nutrients, oxygen as well as metabolic products can transfer throughout the porous network. Moreover, the properties of hydrogels such as mechanical strength, porosity, degradation kinetic and bioactivity, can be well tailored and controlled through chemical or physical methods.6-10 Many kinds of hydrogel systems have been developed and used in biomedical applications.11, 12 However, when employing hydrogels as ECM mimics, it is necessary to understand how cells interact with and remodel the extracellular microenvironment. In vivo, the ECM provides mechanical support for cells.13 In addition to cell-cell interactions, the matrix-cell interactions play fundamental roles in regulating cellular functions, including adhesion, morphogenesis, migration, proliferation, differentiation and gene expression.14, 15 The binding ligands within ECM provide anchorages for cell adhesion and connect the cytoskeletons of cells to the surrounding environment so that cells can sense the mechanical, biochemical changes and other signaling process. In order to improve cell viability and function, it’s necessary to integrate bioactive ligands to interact with cells when design and construct hydrogel networks.16-21 RGD is the peptide sequence that acts as binding domain in fibronectin.22, 23 Due to the isolated short peptide still remaining high activity, RGD has been the most often employed peptide sequence for 3


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stimulating cell adhesion in synthetic hydrogels by far.24-28 For example, the incorporation of RGD significantly enhanced the spreading and proliferation of smooth muscle cells encapsulated in PEG based hydrogel.29 Similarly, human umbilical vein endothelial cells showed strong adhesion in RGD modified hyaluronic acid based hydrogel, and the altered cell adhesion behavior resulted in improved cell migration, proliferation and formation of capillary-like network within the hydrogel.30 Cells always dynamically restructure the surrounding microenvironment in native ECM, and cell-matrix interactions are regulated spatially and temporally.31-33 To mimic the dynamic process, temporal control of binding ligands presenting in hydrogels is increasingly important in cell biology, tissue engineering and cell-based therapy.34-36 Engineering hydrogels to control cell adhesion and adjust matrix-cell interactions at preferred time can approach the conditions for desired cellular responses. In recent studies, a variety of artificial substrates that can control cell adhesion behavior by treating of external stimuli have been developed.37-42 Among those, RGD tethered using stimuli-responsive chemical bond is a facile and effective strategy to adjust the cell-substrate interaction. However, the reports on injectable hydrogels with dynamic bioactivity as cell culture scaffolds are still limited. What’s more, the preferred approaches should be gentle and do not involve any harmful influences on the cells. Recently, we introduced disulfide bond into the crosslinks of hydrogel network, and precisely controlled the degradation process and mechanical property of the hydrogel by treating with a biomolecule, glutathione (GSH).43 The process is facile to 4


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implement and shows good cytocompatibility. In the present study, a novel injectable polypeptide hydrogel based on 4-arm poly(ethylene glycol)-block-poly(L-glutamic acid) grafted with tyramine and a cyclic RGD-containing peptide (denoted as 4a-PEG-PLG-g-TA/RGD) was synthesized through the catalyzed crosslinking using horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). In addition, RGD was conjugated by disulfide bond, leading to the possibility that the density of adhesion ligands within the hydrogel can be well tuned temporally by treating with thiol-containing molecules, such as GSH. The presentation of RGD significantly promoted the adhesion and proliferation of NIH 3T3 cells. Moreover, the cell-gel interaction was dynamically controlled, which may play an important role in affecting cell migration. This work develops a biomimetic scaffold and provides a method to design proper synthetic materials mimicking the dynamic features of native ECM.

2. EXPERIMENT SECTION 2.1

Synthesis

of

4-arm-poly(ethylene

glycol)-block-poly(L-glutamic

acid)-graft-tyramine (4a-PEG-PLG-g-TA) First of all, 4-arm-poly(ethylene glycol)-block-poly(L-glutamic acid) (4a-PEG-PLG) was

synthesized

via

the

ring-opening

γ-benzyl-L-glutamate-N-carboxyanhydride

(BLG

polymerization NCA)

(ROP) initiated

of by

amino-terminated 4arm PEG (4a-PEG-NH2, Mn = 10k).44 BLG NCA was synthesized according to the previously reported method.45 Briefly, 4a-PEG-NH2 (4 g, 0.4 mmol) was dehydrated through azeotropic distillation and dissolved in 60 mL of dry DMF, 5


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and then BLG NCA (2.63 g, 10 mmol) was added. The mixture was stirred at room temperature under a nitrogen atmosphere for 3 days. The 4a-PEG-PBLG copolymer was purified by precipitation into excessive diethyl ether for three times. Then the 4a-PEG-PLG product was obtained by deprotection of PBLG block according to the reported method. 4a-PEG-PLG was collected through lyophilization with a yield of 80%. The 4a-PEG-PLG-g-TA copolymer was synthesized by EDC-NHS activated amidation reaction. 4a-PEG-PLG (2 g, 3.7 mmol COOH), EDC (0.71 g, 3.7 mmol) and NHS (0.42 g, 3.7 mmol) were dissolved in 20 mL of DMF, and the solution was reacted for 2 h to activate the carboxyl group on the side chain of PLG. Tyramine (0.126 g, 0.46 mmol) was then added to the solution, and the mixture was stirred at room temperature for 48 h. After that, the solution was transferred to dialysis bag (MWCO 7000 Da) and dialyzed against distilled water for three days. The 4a-PEG-PLG-g-TA product was obtained by lyophilization and the yield was 88%. 2.2

Synthesis

of

4-arm-poly(ethylene

glycol)-block-poly(L-glutamic

acid)-graft-tyramine/c(RGDfC) (4a-PEG-PLG-g-TA/RGD) First of all, the small molecule 2-(2-pyridyldithio)ethylamine hydrochloride (PSA) was prepared according to the references.46, 47 The structure of PSA was characterized by 1H NMR and

13

C NMR spectra (Figure S2). Then the synthesized PSA was

conjugated to the side chain of PLG through amidation reaction. 4a-PEG-PLG-g-TA (0.5 g, 1.44 mmol COOH) was dissolved in 10 mL of DMF, and PyBop (0.08 g, 0.15 mmol), DIPEA (0.055 mL, 0.31 mmol) as well as PSA (0.035 g, 0.15 mmol) were 6


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added to the solution. The mixture was allowed to react for 2 days at room temperature. The product was purified through dialysis and the yield was 80%. The obtained 4a-PEG-PLG-g-TA/PSA (0.4 g, 0.086mmol PSA) was dissolved in 5 mL of DMF and 5 mL of distilled water, and then c(RGDfC) (75 mg, 0.13 mmol) was added to the solution. After stirring for 24 h, the mixture was dialyzed against distilled water for 3 days and the yield of the collected 4a-PEG-PLG-g-TA/RGD was 85%. 2.3 Determination of RGD content The number of RGD conjugated to the resulted copolymer 4a-PEG-PLG-g-TA/RGD was characterized by measuring the arginine content with fluorescence spectroscopy, and the detailed method was showed in Supporting Information.48,

49

c(RGDfC)

dissolved in water with different concentrations were used as standards to generate the calibration curve. 2.4 Preparation of the enzymatically crosslinked hydrogels Two kinds of hydrogels were prepared via the crosslinking of polymer solutions with the aid of HRP and H2O2. The hydrogel without RGD (designated as P1 Gel) was formed from the solution of 4a-PEG-PLG-g-TA, and the other hydrogel decorated with RGD (designated as P1+P1-RGD Gel) was prepared by dissolving both 4a-PEG-PLG-g-TA and 4a-PEG-PLG-g-TA/RGD (with the mass ratio of 1:1) in PBS (0.01 M, pH 7.4). Polymer solution (200 µL) with certain concentration was mixed with HRP (50 µL) and H2O2 (50 µL, 0.3 in molar ratio relative to TA). The gelation time was recorded using the vial inverting method. The rheological properties and in vitro degradation behaviors of the hydrogels were characterized following the 7


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procedures in the Supporting Information. 2.5 Release profiles of RGD from the P1+P1-RGD Gel RGD was conjugated to the hydrogel network with disulfide bond, so it could release from the hydrogel by treating with glutathione (GSH). P1+P1-RGD Gel (100 µL, total polymer concentration: 4% (w/v)) were incubated in 1 mL of PBS (0.01M, pH 7.4) containing different concentrations of GSH (1 and 5 mM). After treated for different time (0.5, 2 and 24 h), the PBS solution was collected, and the remained hydrogel was washed with 1 mL of fresh PBS. Then the 2 mL of PBS solution were mixed together, and the released content of RGD was measured by fluorescence spectroscopy. 2.6 Cell adhesion on the surface of hydrogels The in vitro cytocompatibilities of 4a-PEG-PLG-g-TA and 4a-PEG-PLG-g-TA/RGD copolymers were assessed by MTT assay against NIH 3T3 cells, and the detailed procedures were showed in the Supporting Information. For the hydrogels used in cell studies, the total polymer concentration was 4 wt%, while the concentrations of HRP and H2O2 were 1 unit/mL and 5 mM, respectively. The polymer solution was quickly mixed with HRP and H2O2 solutions by vortex and the total volume was 300 µL. Then the mixtures were immediately placed on the coverslip in a 6-well culture plate, and a layer of homogeneous hydrogel was formed on the surface of the coverslip after incubated at 37 oC for 20 minutes. NIH 3T3 cells were seeded on the surface of the two kinds of hydrogels (1.5 × 105 cells per well) and cultured at 37 oC in 5% CO2 for 12 h. NIH 3T3 cells cultured on the coverslip was used as control. Subsequently, the DMEM were removed and all samples were 8


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washed with PBS for three times. The remained cells were fixed using 4% of paraformaldehyde and permeated by 0.1% Triton X-100. After that, cell nuclei were stained with DAPI (0.1% v/v, 3 min) and F-actin filaments were stained with Alexa Fluor 488 phalloidin (0.1% w/w, 45 min). Cells were washed with PBS for five times after each step. All the stained cells on the surface of hydrogels or coverslips were observed by using the confocal laser scanning microscope (CLSM, Carl Zeiss, LSM 700). 2.7 3D Proliferation of NIH 3T3 Cells within the Hydrogels in vitro NIH 3T3 cells were encapsulated into P1 Gel and P1+P1-RGD Gel respectively and cultured for 7 days in vitro. During the process, cell viability was evaluated by using a Live/Dead cell staining kit, and cell proliferation rate was measured by the cell counting kit-8 (CCK-8) method. The detailed procedures were supplied in the Supporting Information. 2.8 Controlled cell detachment from the P1+P1-RGD Gel The precursor solutions (150 µL for each well) of P1 Gel and P1+P1-RGD Gel which contained copolymers, HRP and H2O2 were placed in a 48-well culture plate, and incubated for 20 minutes to form stable hydrogels. NIH 3T3 cells were seeded on the top of the hydrogels with the density of 2 × 104 cells per well. NIH 3T3 cells directly seeded on the cell culture plate were used as control. The cell culture media (DMEM) were removed and 0.5 mL of fresh DMEM which contained 1 mM GSH were added after cultured for 12 h. DMEM without GSH were added for the control groups. After incubated for 30 minutes, DMEM were removed and all samples were washed with 9


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PBS for three times. Cell morphologies were observed by microscope (Nikon Eclipse Ti, Optical Apparatus Co., Ardmore, PA, USA) under bright field. The relative cell adhesions compared with cells cultured in cell culture plate were measured using CCK-8 method.

3. RESULTS AND DISCUSSION Synthesis

and

Characterization

of

4a-PEG-PLG-g-TA

and

4a-PEG-PLG-g-TA/RGD copolymers. As illustrated in Scheme 1A, 4a-PEG-PLG was synthesized through the ring opening polymerization of BLG NCA with 4-arm PEG-NH2 as an initiator, followed by the deprotection of the PBLG blocks. 4a-PEG-PLG-g-TA was then prepared by conjugating tyramine (TA) to the side chain of PLG block. The 1H NMR spectra of the copolymers are shown in Figure 1, and all peaks were well-assigned. The average degree of polymerization of PLG block was estimated to be 6 for each arm of PEG, and the molecular weight of 4a-PEG-PLG was also characterized by GPC (Figure S1). The grafting number of TA to every PEG-PLG copolymer was calculated to be ~6 by comparing the integration of phenyl peaks (δ 6.93 and 6.72 ppm) with that of the methylene peaks in PEG (δ 3.75 ppm) (Figure 1A).

Scheme

1.

Synthetic

routes

of

(A)

4a-PEG-PLG-g-TA

4a-PEG-PLG-g-TA/RGD copolymers.

10


and

(B)

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A

4a-PEG-PLG

4a-PEG-PLG-g-TA

B

4a-PEG-PLG-g-TA/RGD

To prepare 4a-PEG-PLG-g-TA/RGD, the synthesized PSA was first grafted to side chain of 4a-PEG-PLG-g-TA. The structure of the obtained 4a-PEG-PLG-g-TA/PSA was confirmed by the appearance of the typical pyridine resonance peaks (-C5H4-) at 7.6-8.65 ppm, as shown in Figure 1B. Then c(RGDfC) which contained thiol group was reacted with 4a-PEG-PLG-g-TA/PSA and conjugated to the copolymer via disulfide bond. In the 1H NMR spectroscopy of the obtained 4a-PEG-PLG-g-TA/RGD (Figure 1C), the signals for pyridine group disappeared and the newly appeared peaks at 7.15 ppm corresponded to the phenylalanine residue in RGD cyclic pentapeptide. The number of bonded RGD for each copolymer was calculated to be ~2 by comparing the integration of peaks at 7.15 ppm with that of the methylene resonance of PEG at 3.75 ppm. In addition, the RGD content in 4a-PEG-PLG-g-TA/RGD copolymer was also determined by measuring arginine residue using fluorimetric assay (Scheme S1 and Figure S3). After reacting with 9, 10-phenan-threnequinone, the arginine residues converted to a strongly fluorescent small molecule with maximum emission wavelength at 390 nm (λex = 312 nm). The calculated result was 11


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consistent with that characterized using 1H NMR spectroscopy.

Figure 1. 1H NMR spectra of the copolymers, (A) 4a-PEG-PLG, (B) 4a-PEG-PLG-g-TA, (C) 4a-PEG-PLG-g-TA/PSA, (D) 4a-PEG-PLG-g-TA/RGD.

Preparation and Characterization of the Injectable Hydrogels. The RGD contained hydrogel was fabricated from the mixed solution of 4a-PEG-PLG-g-TA and 4a-PEG-PLG-g-TA/RGD with the mass ratio of 1:1, and the hydrogel was denoted as P1+P1-RGD Gel. Phenol groups on the side chain of the copolymers reacted with each other in the presence of HRP and H2O2, resulting in the intermolecular cross-links and the transition from solution to hydrogel (Scheme 2). The hydrogel without RGD was prepared from the solution of 4a-PEG-PLG-g-TA and denoted as P1 Gel. During the crosslinking process, HRP catalyzed the reaction and H2O2, which was fixed at 0.3 molar ratio relative to the phenol groups, contributed to the formation 12


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of oxidized phenol groups. As a result, the gelation time depended on the concentration of HRP. As demonstrated in Figure 2A, the gelation time decreased from 74 to 15 s for the 4% (w/v) P1 Gel as the HRP concentration increased from 0.5 to 2 units/mL. The gelation time was also closely related to the copolymer concentration, and it was obviously decreased as the polymer concentration increased from 2% (w/v) to 6% (w/v) for both P1 Gel and P1+P1-RGD Gel (final concentration of HRP: 1 unit/mL) (Figure 2B). Additionally, the gelation time for P1 Gel and P1+P1-RGD Gel was similar with each other under the same condition.

Scheme 2. (A) Photographs of the solution to hydrogel transition, (B) The network structure of the in situ formed P1+P1-RGD Gel.

The rheological properties of the two kinds of hydrogels were examined during the gelation process. As seen in Figure 2C, the storage modulus (G’) of the hydrogels increased rapidly at the initial stage and then reached a plateau within 15 min, indicating the almost completion of the gelation reaction. It can be observed that the mechanical strength of the hydrogels significantly strengthened as the polymer concentration increased. It is worth mentioning that the G’ values of P1 Gel and 13


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P1+P1-RGD Gel were comparable under the same conditions. It’s well known that mechanical cues play crucial roles in manipulating cell behaviors, and it’s essential to maintain the consistency in mechanical property for the two kinds of hydrogels except for RGD presentation.50,

51

Moreover, considering the stability of the hydrogel

network and the feasibility of injection process, the final total concentrations of copolymers, HRP and H2O2 were chosen to be 4% (w/v), 1 unit/mL and 5 mM (H2O2/TA = 0.3 mol/mol), respectively, for both hydrogels in the following tests. Degradation is an important factor that should be taken into account during the design and preparation of a scaffold for biomedical applications. In vitro degradation profiles of the polypeptide based hydrogels were assessed by incubating the hydrogels in Tris-HCl buffer solution containing elastase (5 units/mL). Due to the similarities in backbone structures and mechanical properties of both types of hydrogels, only P1 Gel was used for the degradation test. As demonstrated in Figure 2D, it was observed that the mass of the hydrogel increased continuously at the initial stage and reached the maximum at the 8 day after incubation in the medium containing elastase. This should be ascribed to the gradual breakdown of the PLG blocks, resulting in the decrease in crosslinking density and increase in swelling ratio at the initial stage. Notably, after 8 days of incubation, the mass of the hydrogel decreased dramatically and disintegrated completely after 14 days of incubation. Compared to the rapid degradation process in the elastase-containing solution, the hydrogel was quite stable and maintained its integrity up to 27 days in Tris-HCl buffer solution without proteinase. 14


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A

B P1 Gel P1+P1-RGD Gel

90

Gelation Time (s)

Gelation Time (s)

75 60 45 30 15

75 60 45 30

0.5

1.0

1.5

2.0

2%

4%

HRP Concentration (U/mL)

6%

Concentration (w/v)

C

D

Tris-HCl Elastase

150

Mass Remaining (%)

1000

Modulus (Pa)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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100 G', P1 Gel, 2% G', P1 Gel, 4% G'', P1 Gel, 2% G'', P1 Gel, 4%

10

G', P1+P1-RGD Gel, 2% G', P1+P1-RGD Gel, 4% G'', P1+P1-RGD Gel, 2% G'', P1+P1-RGD Gel, 4%

1

120 90 60 30 0

0.1 0

400

800

1200

1600

0

5

10

15

20

25

Time (day)

Time (s)

Figure 2. (A) Dependence of gelation time on HRP concentrations of P1 Gel, (B) Gelation time of P1 Gel and P1+P1-RGD Gel at different total polymer concentrations (HRP: 1 unit/mL, H2O2/TA = 0.3), (C) Rheological properties of P1 Gel and P1+P1-RGD Gel, (D) In vitro degradation of P1 Gel (total polymer concentration: 4% (w/v)) in Tris-HCl buffer (0.05 M, pH 7.4) and Tris-HCl buffer with 5 units/mL elastase (n = 3).

Controlled RGD release from the RGD-conjugated hydrogel. The cell-adhesive RGD was conjugated to P1+P1-RGD Gel through a disulfide bond that can be cleaved in the presence of thiol-containing reducing agents. Glutathione (GSH) is one of the most crucial thiol compounds in organism and was used to temporally regulate the RGD content within the hydrogel backbone. The hydrogels were immersed in PBS with 1 or 5 mM of GSH, and then washed with fresh PBS after incubation for different time (0.5, 2 or 24 h). The contents of released RGD under different 15


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conditions were assessed using fluorescence spectroscopy. The disulfide bond is sensitive to GSH, and the release profiles of RGD were correlated to the concentration of GSH and the treating time. As seen in Figure 3, 50% of bonded RGD was released after treating with 1 mM of GSH for 0.5 h, and 78% of RGD was removed from the hydrogel when the treating time was extended to 2 h. Nearly 90% of the RGD could be removed from the hydrogel after incubation with GSH for 24 h. Meanwhile, the release rate of RGD could be accelerated obviously by increasing the concentration of GSH. For example, the released content reached to 80% after incubation in 5 mM of GSH for only 0.5 h. Based on this result, the presentation of bioactive RGD could be controlled effectively by the treatment with GSH, so that the present hydrogel may provide an approach to mimic the dynamic native cell microenvironment and guide cell fate in turn.

Cumulative Release of RGD (%)

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100

0.5 hour

2 hour

24 hour

80 60 40 20 0 1

5

GSH Concentration (mM)

Figure 3. RGD release from P1+P1-RGD Gel by treating with different concentrations of GSH.

In vitro Cell Culture in 2D and 3D. First of all, the in vitro cytocompatibility of the 4a-PEG-PLG-g-TA and 4a-PEG-PLG-g-TA/RGD copolymers were evaluated by 16


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MTT assay against NIH 3T3 cells. The NIH 3T3 cells incubated with 4a-PEG-PLG-g-TA showed high viability (> 90%) at the polymer concentrations up to 0.25 mg/mL (Figure S4). As for 4a-PEG-PLG-g-TA/RGD, the cells also maintained high viability (> 90%) at similar polymer concentration range (0.031 - 0.25 mg/mL). The results showed that both copolymers had no detectable cytotoxicity toward NIH 3T3 cells. In order to evaluate the influence of the method which was used to control the RGD release from the hydrogel, the cytotoxicity of GSH which acted as the mediator of the cell-adhesive RGD ligand was also assessed by MTT assay (Figure S5). NIH 3T3 cells were cultured with GSH for different period (10, 30, 60 and 120 min), and the GSH concentration changed from 0.1 to 5 mM. The cell viability retained over 90% in all groups, implying that our strategy to regulate the bioactivity of hydrogel will not affect the cell activity. P1 Gel and P1+P1-RGD Gel should have similar chemical and physical properties except for that P1+P1-RGD Gel was decorated with cell-adhesive RGD peptide. RGD has widespread distribution in organism, and it can interact with cells through binding with transmembrane integrin. Furthermore, integrin activation leads to specific intracellular signaling pathway and then regulates cell-matrix interaction. The precursor solutions of P1 Gel and P1+P1-RGD Gel were added onto the surface of coverslips and formed homogeneous layer of hydrogels in situ. NIH 3T3 cells were seeded on the hydrogel and cultured for 12 h. NIH 3T3 cells cultured on the coverslip directly were used as control. After that, the cell nuclei were stained with DAPI and F-actin filaments of NIH 3T3 cells were stained with Alexa Fluor 488 phalloidin. As 17


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illustrated in Figure 4, the reticulate F-actin filaments of cells were observed on the surface of P1+P1-RGD Gel and the cell morphology were similar with those cultured on coverslip. NIH 3T3 cells could efficiently adhere and well spread on the surface of P1+P1-RGD Gel. In comparison, only a few cells attached on the surface of P1 Gel and the attached cells remained a relatively round shape. The differences indicated that the P1 Gel provided non-adhesive background and RGD ligand within P1+P1-RGD Gel could enhance the cell-hydrogel interaction.

Figure 4. CLSM images of NIH 3T3 cells on the surface of P1 Gel and P1+P1-RGD Gel after culturing for 12 h. NIH 3T3 cells cultured on coverslip were used as control.

In order to explore the feasibility of the injectable hydrogel as a cell culture scaffold, NIH 3T3 cells were encapsulated in P1 Gel and P1+P1-RGD Gel during the sol-gel transition process and incubated in 3D for a week. The cell survival status was assessed by the live-dead cell staining kit. As showed in Figure 5A, most of the cells within both hydrogels were stained green with calcein-AM after culturing for 1 and 7 days, implying the satisfactory cytocompatibility of the prepared hydrogels. Additionally, we measured the proliferation of NIH 3T3 cells within the hydrogels 18


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using CCK-8 method, as shown in Figure 5B. For P1 Gel, the number of cells kept increasing after culturing for 1 and 4 days, but it slightly decreased after 7 days of incubation. This might be attributed to the fact that the major component of P1 Gel was bioinert PEG which was not conducive to long-term cell growth and long-term cell growth was suppressed.19, 52 In contrast, NIH 3T3 cells within P1+P1-RGD Gel kept proliferating during the period of culturing, and the proliferation rate was higher than those in P1 Gel. It was worth to mention that the number of cells in P1+P1-RGD Gel was 1.7 times higher that in P1 Gel after incubation for 7 days. This indicated that the modification of cell adhesive RGD ligands can promote cell proliferation within the hydrogel scaffold.

Figure 5. In vitro 3D culture of NIH 3T3 cells. (A) Viability of NIH 3T3 cells in P1 19


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Gel and P1+P1-RGD Gel after culturing for 1 and 7 days. Live cells were stained with calcein-AM and showed green fluorescence, while dead cells were stained with PI and showed red fluorescence. (B) The proliferation of NIH 3T3 cells within P1 Gel and P1+P1-RGD Gel analyzed by CCK-8 method (n=3, **p < 0.01, ***p < 0.001).

Controlled cell-hydrogel interaction and cell adhesion behavior. From the above results, it was observed that the bioactive modification of hydrogel scaffold is of great importance in affecting cell behaviors, including adhesion and proliferation. Additionally, for many biological basic research and biomedical applications, it may require to precisely controlling when and where cells should adhere, migrate or release. In our system, the content and density of RGD ligands in P1+P1-RGD Gel could be well regulated by treating with GSH. The process was biocompatible and easy to implement. To verify this process, P1 Gel and P1+P1-RGD Gel were prepared in situ in the 48-well culture plate, and NIH 3T3 cells were cultured on the surface of hydrogels. After culturing for 12 h, cells were incubated in cell culture media without or with 1 mM GSH for 30 min, and then the cell morphologies were observed (Figure 6A). NIH 3T3 cells cultured on P1 Gel showed a round shape and aggregated, and the interaction between cells and hydrogels were relatively weak. In contrast, cells cultured on the surface of P1+P1-RGD Gel adhered efficiently and spread very well. Notably, after treating with GSH, the cells on the P1+P1-RGD Gel turned into a round shape, and a majority of cells were found to be released from the hydrogel. The relative cell adhesions on the surface of hydrogels were measured by CCK-8 method. 20


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As illustrated in Figure 6B, the relative cell adhesion on P1+P1-RGD Gel was ~ 31%, and it decreased to 16% after treating with GSH. The biomolecule, GSH, was used as the mediator to control the RGD presentation and cell adhesion in situ. The detachment of cells from the P1+P1-RGD Gel can be reasonably attributed to the decrease of RGD ligands within the hydrogel network. Cells always dynamically restructure the surrounding microenvironment in native ECM, and cell-matrix interactions are regulated spatially and temporally. Engineering hydrogels to control cell adhesion and adjust matrix-cell interactions at preferred time can approach the conditions for desired cellular responses.34, 35

Figure 6. GSH-induced cell release from the surface of RGD-conjugated hydrogel. (A) The images of NIH 3T3 cells on the surface of hydrogels after treating with 1 mM GSH for 30 min. (B) The change of cell numbers after treating with GSH (n=3, **p < 21


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0.01, ***p < 0.001).

4. CONCLUSION In this study, a new type of enzymatically cross-linked bioactive hydrogel was developed. Cell adhesion RGD ligand was conjugated to the hydrogel network via disulfide bond, leading to a system with adjustable bioactivity through releasing RGD from the hydrogel by treating with GSH. The RGD-conjugated hydrogel showed good cytocompatibility, and led to enhanced cell proliferation when used as a 3D cell culture scaffold compared to the hydrogel without RGD. Meanwhile, the interaction between NIH 3T3 cells and hydrogel network could be dynamically changed, thus cell behavior such as adhesion and migration could be regulated on demand. The polypeptide-based hydrogel with tunable bioactivity and controlled cell-matrix interaction may provide meaningful platform mimicking cell-interactive ECM for 3D cell culture, tissue engineering and cell-based therapy.

ASSOCIATED CONTENT Supporting Information Reagents, characterizations and additional testing methods; GPC trace of PEG-PLG, 1

H NMR and

13

C NMR of the synthesized PSA compound, fluorescence spectra of

4a-PEG-PLG-g-TA and 4a-PEG-PLG-g-TA/RGD copolymers, as well as in vitro cytocompatibilities of 4a-PEG-PLG-g-TA, 4a-PEG-PLG-g-TA/RGD and GSH. 22


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AUTHOR INFORMATION Corresponding Authors *Email: [email protected]; [email protected]

ACKNOWLEDGEMENTS The financial supports from the National Natural Science Foundation of China (projects 51622307, 21574127 and 51390484), and Chinese Academy of Sciences (STS project: KFJ-SW-STS-166) are gratefully thanked.

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Injectable Polypeptide Hydrogel as Biomimetic Scaffolds with Tunable

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