Fabrication of a Controlled in Situ Forming Polypeptide Hydrogel with

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Fabrication of Controlled in-Situ Forming Polypeptide Hydrogel with Good Biological Compatibility and Shapeable Property Kun Lei, Yunlong Sun, Chengyuan Sun, Dandan Zhu, Zhen Zheng, and Xinling Wang ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00157 • Publication Date (Web): 29 Mar 2019 Downloaded from http://pubs.acs.org on March 31, 2019

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ACS Applied Bio Materials

Fabrication of Controlled in-Situ Forming Polypeptide Hydrogel with Good Biological Compatibility and Shapeable Property Kun Lei,a Yunlong Sun,a Chengyuan Sun,a Dandan Zhu,a Zhen Zheng,a and Xinling Wang*ab a.

School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai 200240, China.

b. State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, Shanghai 200240, China.

ABSTRACT: Hydrogel is required to have good biocompatibility, permeability for nutrients, and an easy construction procedure for biomedical applications. In particular, in-situ forming hydrogels (ISFHs) have triggered considerable interest on account of their facile preparation methods.

Here,

an

enzyme-prompted

ISF,

biodegradable

poly(L-

lysine)-graft-4-

hydroxyphenylacetic acid (PLL-g-HPA) hydrogel in the condition of horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) and with good biocompatibility was developed. The gelling time varied from a couple of seconds to several minutes depending on the amounts of catalyst, H2O2, and polymer. Due to the conveniently ISF means, the fabricated hydrogel could be applied in any form according to the need. The hydrogels display good biological compatibility, as demonstrated in vitro cell culture and attachment experiments. Besides, the remaining -NH2 groups in the hydrogel could be further functionalized for various cell researches and bio-applications. KEYWORDS: PLL-g-HPA hydrogels, enzyme-catalyzed reaction, in situ gelation, biocompatibility, degradation property 

INTRODUCTION

In the past few decades, hydrogels, composed of three-dimensional (3D) hydrophilic polymer networks, have been extensively employed as biological materials for numerous bioapplications such as cell delivery carriers, 3D tissue engineering scaffolds on account of their good biological compatibility, water-retaining property, and well permeability for nutriments.1-3 *Corresponding author. E-mail address: [email protected] (X.-L. Wang).

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Particularly, ISFHs have drawn considerable interest for many bio-applications compared with conventional preformed hydrogels because of their distinct strengths, like convenient applications, and minimally invaded injection procedures without the implantation surgically and retrieval.4,5 Some bioactive therapeutic agents like drugs, cells, DNA could be easily blended with their pre-gel solutions before gelling, and subsequently, to form the hydrogels acting as carriers for localized delivery or 3D scaffolds in a target site. So far, lots of means have been made use of to prepare ISFHs. These methods were mainly composed of two types: (1) physical cross-linking systems, for instance, hydrogen bonds,6 electrostatic interaction,7 hydrophobic interaction,8 host-guest interaction,9 and (2) chemical cross-linking systems, such as photo cross-linking,10 isulfide bond formation,11 Michael-type addition reaction,12 and radical polymerization.13 Unfortunately, there still exists some defects about these systems comprising potential bio-toxicity, and relatively long gelation times. Recently, based on HRP and H2O2, an enzyme-prompted cross-linked reaction provides a desirable way for ISFHs on account of its controlled reaction rate, mild reaction condition for the hydrogels.14,15 The attained hydrogels are convenient to manipulate and competent for loading with bioactive molecules or cells homogeneously due to the relatively low viscosity of precursor polymer solutions. HRP can trigger the intermolecular conjugating of aniline or phenol derivatives through disintegration of H2O2.16 About HRP-prompted hydrogels, plenty of researches have been concentrated on natural materials, including chitosan,17 gelatin,18 hyaluronic acid,19 alginate,20 and cellulose.21 Based on their proteolytic biodegradability, and cell-interactive performances, these hydrogels showed potential bio-applications as biomaterials. However, these naturally derived materials have some limitations like the insufficient mechanical strength in vivo, too fast degradation rate, and narrowly tunable structure.22 Although lots of studies on the modification of natural polymer hydrogels have been carried out,23 it still keeps a major issue. Recently, the enzymatically cross-linked factitious polymers hydrogels have drawn considerable attention because of their strengths comprising low immunogenicity, controlled mechanical properties, and degradation rates. For example, the ISFHs composed of 4-arm-PPO-PEO-TA through HRP-prompted croos-linkage have been reported.24 The hydrogel exhibited good cytocompatibility and was fast constructed within 5 s utilizing H2O2 and HRP as promoters with tunable gelation time from about 5 s to 2 min. Chen et.al25 developed the enzymatically cross-linking hydrogel comprising the PEG capped by a dual-end tyramine (TA-PEG-TA) prompted by H2O2 and HRP.


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Among the synthetic polymers, polypeptides, a mono-poly-amino acid or a co-poly-amino acid, evoked considerable attraction on account of their good biodegradability, biocompatibility, and unique secondary structure.26 Meantime, the active side groups in the polypeptides contribute to their further post-modification. All the characteristics endow the polypeptides with unique advantages as biomaterials for biomedical applications. There are few literatures about in-situ enzymatically cross-linked hydrogels based polypeptides.27 A kind of ISFH composed of poly(L-glutamic acid) grafted by TA and PEG (PLG-g-PEG/TA) was fabricated via the facilitation of HRP and H2O2 in physiological condition.15 However, the hydrogels revealed high water uptake and maintained the integrality only for 7 days in vitro because of the existence of the hydrophilic PEG chain segment. On the other hand, another kind of hydrogel without the presence of PEG segments was found to display better steadiness, and good mechanical performance, which have been tested as carriers for 3D cell proliferation by encapsulation of mesenchymal stem cells (BMSCs) into the hydrogels.28 In this present study, a kind of enzyme-prompted ISFH composed of PLL-g-HPA was prepared under physiological conditions. PLL was chosen as the backbone polymer on account

of

its

excellent

solubility

in

water,

proteolytic

biodegradability,

and

cytocompatibility.29 Additionally, as an inherently anti-bacterial material, PLL possesses excellent anti-bacterial performance against Gram-negative and -positive bacterial.30 Hence, hydrogel containing PLL may be also used as inherent anti-bacterial material for wound infection prevention. The gelling time varied from a couple of seconds to several minutes depending on the amounts of catalyst, H2O2, and polymer. Due to the conveniently ISF means, the attained hydrogel could be applied in any form according to the need. The hydrogels display good biological compatibility, as demonstrated in vitro cell culture and attachment experiments. Besides, the remaining -NH2 groups in the hydrogel could be further functionalized for various cell researches and bio-applications. 

EXPERIMENTAL SECTION Materials: H-Lys(Z)-OH, purchased from GL Biochem (Shanghai) Ltd. 33 wt.%

hydrogen bromide solution in acetic acid (33 wt.% HBr/HOAc), α-Pinene, 4hydroxyphenylacetic acid (HPA), N-hydroxysuccinimide (NHS), and N-ethyl-N’-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl) were obtained from Acros. Trifluoroacetic acid (TFA), dimethylsulphoxide (DMSO), anhydrous ether, chloroform (CHCl3), and hydrogen peroxide (H2O2) were obtained from Aldrich-Sigma. Horseradish peroxidase (HRP, ≥ 300 units/mg), and elastin (≥ 30 units/mg) were purchased from



Shanghai Yuan Ye biotechnology co. Ltd. Triphosgene (BTC, Aldrich-Sigma) was recrystallized by ethyl acetate. Tetrahydrofuran (THF), n-hexane were dried by refluxing and distilled before use. N, N-Dimethylformamide (DMF), n-hexylamine were dried with CaH2, distilled under reduced pressure. Nε-benzyloxycarbonyl-L-lysine N-carboxyanhydride (ZLLNCA) was attained in accordance with the Fuchs-Farthing means utilizing BTC.31 Preparation of Poly(ε-benzyloxycarbonyl-L-lysine) (PZLL). PZLL was prepared by the ring opening polymerization (ROP) of ZLL-NCA utilizing n-hexylamine as an initiator in DMF. The following is a typical case about synthesis of PZLL75, where 75 is the polymerization degree (DP) of PZLL. Under a nitrogen atmosphere, newly attained ZLLNCA (2.448 g, 8 mmol) was dissolved in DMF (32 mL). n-hexylamine (10.1 mg, 0.1 mmol) in DMF (1 mL) was added into the above solution . The mixture was stirred for 3 d at 25 ℃ and subsequently, precipitated into excess anhydrous ether to attain white solid. The polymer was purified by dissolving in CHCl3 and subsequently precipitating with anhydrous ether for four times. PZLL75 was attained by filtration and dried in vacuum at 40 0C for 48 h (1.90 g, yield: 90%). Preparation of Poly(L-lysine) (PLL). The obtained PZLL75 was conducted to acidolysis to eliminate the ε-(benzyloxycarbonyl) groups of PZLL75 utilizing HBr. As a typical case, 33 wt.% HBr/HOAc (6.57 mL, 37.5 mmol HBr) was added to the solution of PZLL75 (1.90g, 0.1 mmol) in 25 mL TFA. The mixture was fast stirred for 1 h at 25 ℃ , and subsequently precipitated in excess anhydrous ether. Then, a pale yellow solid was obtained by filtration and dried in vacuum. The solid, re-dissolved in DI water, was dialyzed against DI water for 2 d (a dialysis membrane with a cutoff molecular weight of 3500). The final product was attained by lyophilization named PLL75 (1.50 g, yield: 95%). Preparation of Poly(L- lysine)-graft-HPA (PLL-g-HPA). PLL-g-HPA copolymer was obtained by grafting PLL75 with HPA by EDC/NHS activated amidation reaction. Typically, HPA (109 mg, 0.718 mmol) was first dissolved in 40 mL DMSO and activated for 1 h at 25 ℃ by using EDC·HCl (414 mg, 2.16 mmol) and NHS (249 mg, 2.16 mmol). PLL75 (1.51 g, 7.18 mmol of NH2 groups), dissolved in 45 mL DMSO, was then added to the above solution and the reaction proceeded for 1 d. Subsequently, the reaction mixture was poured into a seamless cellulose tubing and was dialyzed against DI water for purification. The resultant PLL-gHPA10 (the number 10 refers to 10% of the theoretical grafting ratio of HPA in the PLL-gHPA) was obtained by freeze-drying. (1.31 g, yield: 85%). PLL-g-HPA15 was synthesized through a similar method with a yield 87%.


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Polymer Characterizations. PZLL, PLL and PLL-g-HPA copolymer were characterized by 1H NMR (400 Hz). DP was measured by comparing the integrals of signals at  0.80 and  3.67 for PZLL,  0.77 and  4.21 for PLL. The grafting ratio (GR), defined as the number of substituent per 100 monomer unit in polymer, was measured by comparing the integrals of signals at  7.06 and  4.15 for PLL-g-HPA. The content of conjugated HPA groups in the PLL-g-HPA copolymer was also tested through utilizing an ultraviolet-visible spectrometer (UV-Lambda 35, Perkin Elmer, Inc., USA). After being dissolved at 1.2 mg·mL-1 in DI water, the absorbance of the PLL-g-HPA at 275 nm was determined. HPA group content was figured up from a calibration curve acquired by testing the absorbance of HPA at different concentrations in DI water. Formation of the PLL-g-HPA Hydrogels and gelation time. The PLL-g-HPA hydrogels were constructed via enzyme-prompted cross-linking in the existence of H2O2 and HRP in PBS (0.01 M, pH 7.4). As a typical case, freshly prepared PBS solution of H2O2 (0.05 mL, molar ratio of H2O2: HPA = 0.4) and HRP (0.05 mL, molality ratio of HRP: HPA = 3) were added to a PBS solution of PLL-g-HPA10 copolymer solution (0.2 mL) with 5%, 7.5%, 10%, 12.5%, 15%, and 20% (w/v) of copolymer concentration, and the mixture was slightly vibrated. Final concentrations of PLL-g-HPA10 copolymer were 3.4%, 5.1%, 6.8%, 8.5%, 10.2%, and 13.6% (w/v), and the corresponding hydrogels were named as 3.4%, 5.1%, 6.8%, 8.5%, 10.2%, and 13.6% Gel. The corresponding H2O2 concentrations were 0.01, 0.015, 0.02, 0.025, 0.03, and 0.04 mmol/mL for 3.4%, 5.1%, 6.8%, 8.5%, 10.2%, and 13.6% Gel, respectively. Similarly, the final HRP concentrations were 0.075, 0.1125, 0.15, 0.1875, 0.225, and 0.3 mg/mL. The time to obtain a gel (called as gelling time) was examined utilizing the vial inverting means. No flow within 60 s upon tilting the vial was deemed as the gel state. Gel Content and Equilibrium Water Content. To measure the gel content (GC), the lyophilized hydrogel samples (0.3 g, 13.6% Gel) were firstly weighed (Wd). Subsequently, the dried hydrogels were intensively eluted with DMSO for 3 d to eliminate unreacted polymer. DMSO was replaced twice. Finally, the hydrogels were rinsed 4 times using ethanol, and dried under vacuo to an invariable weight (Wv). GC was calculated by the following equation: GC 

Wv 100% Wd

(1)

The equilibrium water content (EWC) of the 13.6% Gel was measured as follows. The freeze-dried hydrogels (Wd) were submerged in PBS (0.01 M, pH 7.4) at 37 ℃ for 3 days to



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achieve the swelling equilibrium. After removal of surface water of swollen samples using filter paper, the samples were weighted (Ws). The EWC of samples was expressed as follows: EWC 

Ws-Wd  100% Wd

(2)

The assay was conducted in triplicate. Swelling and degradation assays of PLL-g-HPA hydrogels. Firstly, hydrogel samples (0.3 mL) were formed in the vials at 37 ℃ in the light of the above procedure and exactly weighed (W0). PBS (0.01 M, pH 7.4) containing elastase (6 units/mL) was then added to the surface of hydrogels at 37 ℃. PBS was refreshed per day to keep the activity of the enzyme. The hydrogels incubated in PBS without the enzyme at 37 ℃ were used as controls. The PBS solution was removed from the samples at prescribed time intervals, and the remaining gels were weighed (Wt). The percent of residual gel mass was defined as follows: MR 

Wt  100% W0

(3)

The assay was operated in triplicate. Rheological analysis. Rheological assays of the PLL-g-HPA hydrogels were carried out on an AR-G2 Rheometer (TA Instruments, USA) utilizing a parallel plate (plate diameter = 25 mm, gap = 1 mm) at 37 ℃. As a typical case, 50 μL of HRP solution (0.3 mg/mL, in PBS) and 50 μL of H2O2 solution (0.04mmol/mL, in PBS) were mixed with 100 μL of PLL-gHPA10 (20 w/v%, in PBS) solution respectively, utilizing a dual syringe (1 mL, equal volume) with a mixing chamber and then, the mixtures were injected and placed on the plate of the rheometer at once. A thin layer of silicon oil was introduced around the outer edge of the sample to avoid the volatilization of water. A time sweep test was implemented to monitor the variations of loss modulus (G’’) and storage modulus (G’) over time during the crosslinking process (1 Hz, a strain of 1%.) MTT assay. According to the guidelines of the International Organization for Standardization (ISO), in vitro cytocompatibility of H2O2, PLL-g-HPA, and the extracts of the PLL-g-HPA hydrogels was evaluated by MTT test utilizing L929 mouse fibroblasts. The hydrogel was prepared in a 24-well culture plate and leached by Dulbecco’s modified Eagle’s medium (DMEM) containing 1% penicillin/streptomycin and 10% fetal bovine serum in an atmosphere of 5% CO2 at 37 ℃ for 72 h. L929 cells were cultivated in a 96-well culture plate at a density of 1×105 per well and incubated at 37 ℃ and in 5% CO2 atmosphere for 1 day. Then, the L929 cells were added and cultured with fresh media containing H2O2 or PLL-g-


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HPA copolymer at different concentrations or the leaching solutions of PLL-g-HPA hydrogels for another 24 h. Subsequently, 0.02 mL MTT (6 mg/mL) was added to each well and cultured at 37 ℃ for 5 h. The attained formazan was dissolved in DMSO (200 µL for each well) and the absorbance value at 490 nm was determined with a microplate reader (Bio-rad). Polyethylenimine 10K (PEI 10K, Mw = 10,000, Aldrich) was used as the positive control. Each well without the H2O2 or PLL-g-HPA copolymer or the leaching solutions of the hydrogels was used as the negative control and the well without the cells was used as the blank control. Relative cell viability (%) was calculated in accordance with the followed equation: cell viability (%) = (Asample- Ablank)/(Anegative- Ablank) ×100%, where Asample, Ablamk, and Anegative are the absorbance values of the sample, blank control, negative control wells, respectively. The determination was operated in triplicate. Cell adhesion assays. The hydrogels were incubated using 2mL of 1× 105 cells per well suspension of L929 mouse fibroblasts in 6 well plates at 37 °C and 5% CO2. After 1, 3 d, substrate was washed with fresh medium to eliminate un-adhered cells. Cells were digested, and counted utilizing a cell counter. Subsequently, 1.5% FDA solution was used to stain the cells on the hydrogel surface, followed by immobilization with 2% glutaraldehyde. Fluorescence pictures were acquired utilizing a laser confocal microscopy (LCFM). Functionalization of PLL-g-HPA hydrogel. Rhodamine B was utilized to functionalize the samples by the amidation. PLL-g-HPA hydrogel sample was immersed in a methanol of rhodamine B (1.5 mg · mL−1) at 55 °C to perform the reaction between the residual –NH2 groups within the network and the -COOH groups of rhodamine B. Then, the sample was rinsed with CH3OH and water for 8 h with frequent solution replacement until the absorbance of rhodamine B at 554 nm disappeared by the UV-vis absorption spectrum. Subsequently, the hydrogel was directly pictured through utilizing a LCFM. Besides, a UV/vis spectrophotometer was utilized to measure the absorption spectrum of the functionalized PLL-g-HPA hydrogel. The scan range is 300-700 nm with step of 1.5 nm. PTFE was applied as an absorption standard. A hydrogel sample with diameter of 20 mm and height of 2 mm was sandwiched in two quartz plates. The content of conjugated rhodamine in the PLL-gHPA hydrogels was defined as fluorescence yield (Wti). The absorbance at 554 nm of the functionalized hydrogel with final 6.8 w/v% of PLL-g-HPA for different time was measured. The fluorescence yield (Wti) was obtained from a calibration curve attained by examining the absorbance of rhodamine at different concentrations in deionized water. Fluorescence yield (Wti) was determined as follows:



Wti=

ci 100% 68

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(4)

Here, ci refers to the concentration of rhodamine in the functionalized hydrogel for different time, calculated from a calibration curve of rhodamine, in mg/mL; the number 68 refers to the final concentration of PLL-g-HPA in hydrogel system, in mg/mL, i=0, 3, 6, 9, 18, 24 h. 

RESULTS AND DISCUSSION Preparation and characterization of the in situ cross-linking hydrogels. The hydrogel

was rapidly constructed by HRP-mediated conjugation reaction of phenol moieties in the PLL-g-HPA copolymer in PBS using a dual-mixing syringe. The synthetic route of PLL-gHPA was shown in Scheme S1. The Nε-benzyloxycarbonyl-L-lysine was firstly reacted with triphosgene to obtain ZLL-NCA in accordance to the Fuchs-Farthing strategy.31 The 1H NMR, 13C

NMR, IR spectra of ZLL-NCA are displayed in Figures S1, S2, and S3. PZLL was

prepared by ROP of ZLL-NCA in the catalysis of hexylamine, followed by deprotection of benzyloxycarbonyl groups of PZLL utilizing HBr to attain PLL. Then, HPA was grafted to the main chain of PLL via EDC/NHS-catalyzed amidation reaction. 1H NMR spectra of PZLL, PLL, and PLL-g-HPA10 were all displayed in Figure S4. The DP of PZLL, PLL could be measured as 75 by contrasting the integrals of signals a, b, respectively in Figures S4A, S4B. In comparison to the designed DP of 80, this illustrates that ROP of ZLL-NCA catalyzed by the hexylamine is a basically controlled reaction and the deprotection of PZLL utilizing HBr as catalyst didn’t lead to the breakage of the main chains of PLL, respectively. The GR of HPA residues was determined by comparing the peaks at δ 6.75 (assigned to HPA) and at δ 4.15 (assigned to PLL) in Figure S4C, determined to be 9.4%. The content of conjugated HPA groups in the PLL-g-HPA10 copolymer was also evaluated through the UVVis spectra in the PLL-g-HPA10 aqueous solution due to a specific absorbance peak at 275 nm corresponding to the absorption of phenol groups (Figure S5a). The content of HPA was 9.8 wt.%, which is approximately in accordance with the 1H NMR result. This indicates that amidation reaction of PLL using carbodiimide as a catalyst is a quantitative reaction. In addition, circular dichroism spectroscopy (CD) was utilized to examine the conformation of PLL-g-HPA10 in pH 7.4 aqueous solution. As can be seen from Figure S5b, at approximately 218 nm, a positive maximum and at approximately 203 nm, a large minimum were detected, displaying that PLL-g-HPA10 presented a random conformation.32 This was ascribed to the ionization of the remaining amino groups of PLL-g-HPA10 at pH 7.4.32 In the gelation


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process, HRP accelerates the phenol groups of PLL-g-HPA to couple with each other via the C-O bond between the phenoxy oxygens and carbons or the ortho positioned C-C bond,33 resulting in the formation of intermolecular cross-linking and the fast construction of hydrogels. The schematic illustration of the gelation using a dual syringe was displayed in Scheme 1.The dual-mixing syringe involves PLL-g-HPA and HRP solutions in one tube, and PLL-g-HPA and H2O2 solutions in the other tube. Enzymatic crosslinking of PLL-g-HPA copolymer including PLL-g-HPA10, and PLL-g-HPA15 were studied, in where different polymer concentrations (3.4-13.6 w/v%), HRP/HPA ratios (0.5-3.5 mg/mmol), and H2O2/HPA ratios (0.4-1.5 mmol/mmol) were applied.

Scheme 1. Schematic of the PLL-g-HPA hydrogel formation system constructed by mixing PLL-gHPA/HRP and PLL-g-HPA/H2O2 in PBS solution using a dual-mixing syringe

The vial inversion means was applied to determine the gel time of hydrogels and was displayed to be controllable by tuning the concentrations of polymer, H2O2, and HRP as presented in Figure 1. Figure 1a shows that the gelation time reduces from 257 to 14 s for PLL-g-HPA10 when increasing the HRP/HPA ratio from 0.5 to 3.5 mg/mmol at an invariable polymer concentration (13.6 w/v%) and H2O2/HPA molar ratio (0.4 mmol/mmol). The phenomenon was ascribed to the accelerated coupling rate of generating the phenoxy radicals with HRP/HPA ratio increasing. Notably, when HRP/HPA ratio was not lower than 1.5 mg/mmol, PLL-g-HPA10 hydrogels were constructed within 60 s. On the contrary, increasing the H2O2/HPA ratio with an invariable polymer concentration (13.6 w/v%) and HRP/HPA ratio (3 mg/mmol) brought about an increase of the gel time (Figure 1b). The phenomenon was due to the fact that HRP can be oxidized to an inactivated configuration in the existence of excess H2O2.15 In this study, the optimal H2O2/HPA molar ratio was 0.4



wherein the gelation occurred within 6 min for all PLL-g-HPA with different concentrations. This exhibits that the H2O2 concentrations from 0.01 to 0.06 mmol/mL, determined by polymer concentration and GR, are necessary. In consideration of cytotoxicity problems induced by the remaining H2O2, the usage of low concentrations of H2O2 is beneficial. As has been previously demonstrated, HA-TA hydrogels constructed in the existence of 0.07 mmol/mL H2O2 are no cytotoxicity with good biocompatibility.34 The cytotoxicity of PLL-gHPA hydrogels, and different concentrations of H2O2 will be discussed in our subsequent studies. As the polymer concentration went up from 3.4 to 13.6 w/v% at a HRP/HPA molality ratio of 3 and a H2O2/HPA molar ratio of 0.4, the gel time reduced from 242 to 18 s (Figure 1c). In particular, when polymer concentrations were at or above 8.5 w/v%, the gelation took place within 1 min for the two PLL-g-HPA copolymers. This result was attributed to the reason that more phenol groups leaded to an increased possibility of crosslinkage between every phenol group. In addition, it is worth noting that at low polymer concentration, hydrogels could be also constructed quickly. For instance, PLL-g-HPA10 gelled after around 4 min with a polymer concentration 3.4 w/v%.

Figure 1. Gelling time of PLL-g-HPA hydrogels at different conditions: (a) Effect of HRP/HPA ratio on gelling time of 13.6 w/v% PLL-g-HPA10 and PLL-g-HPA15 with H2O2/HPA=0.4 mmol/mmol; (b) Effect of H2O2/HPA ratio on gelling time of 13.6 w/v% PLL-g-HPA10 and PLL-g-HPA15 with HRP/HPA=3


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mg/mmol; (c) Effect of polymer concentration on gelling time of PLL-g-HPA10 and PLL-g-HPA15 with HRP/HPA=3 mg/mmol and H2O2/HPA=0.4 mmol/mmol.

Fast gelation and polymer pre-gel solution with a low concentration are advantageous especially for ISFHs as injectable scaffold and cell delivery carrier material. This is because that slow gelling in vivo may bring about diffusion of bioactive agents or polymer pre-gel solution to surroundings and polymer pre-gel solution with a low concentration can conveniently encapsulate bioactive agents or cells into the hydrogel. Compared with the gelation time of many previously mentioned ISFHs, the enzymatic crosslinking of PLL-gHPA copolymers may generate relatively fast gelation. Chu et al. mentioned that acrylated dextran hydrogel was constructed by photo-crosslinking within 300 s with polymer concentration of 40 w/v %.35 Zhang et al. reported that dextran hydroxyethyl methacrylate hydrogel was fabricated with gelling time of 12 min utilizing a redox catalyst, and rapid gelling could be attained by greatly raising the amount of catalyst.36 The hydrogel constructed by Michael type addition of functionalized PEG and peptides completed the gelling process within 12 min under physiological surroundings.37 The constructing process of hydrogel was also measured by performing the loss modulus (G’’) and storage modulus (G’) in a time-tuned oscillatory rheology with different polymer and HRP concentrations. Figure 2a represents the change of G’ and G’’ of the PLL-g-HPA10 hydrogel over time with different HRP/HPA ratio. During the initial 40 s, the G’ of the hydrogels boosted fast with a HRP/HPA ratio of 1 mg/mmol. Gel point, treated as the point of intersection of G’ and G’’, was presented about 20 s. The G’ rapidly leveled out after 2.5 min, manifesting the completed crosslinking process. With a higher HRP/HPA ratio of 3 mg/mmol, an instant gelling after injection was formed with G’ being more than G’’ at the beginning. The change of G’ and G’’ of the PLL-g-HPA10 hydrogel over time with different polymer concentration was also measured in Figure 2b. With polymer concentration of 6.8 w/v%, the G’ of the hydrogel went up quickly during the initial 2 min. Gel point was observed about 25 s and a platform was reached within 5 min, indicating the finished crosslinking reaction. At a higher polymer concentration of 13.6 w/v%, an immediate gelling after injection was formed with G’ being more than G’’ at the beginning.



Figure 2. The change of storage modulus (G’) and loss modulus (G’’) of the PLL-g-HPA10 hydrogels with (a) different HRP/HPA ratio (H2O2/HPA=0.4 mmol/mmol, 13.6% Gel) and (b) different polymer concentration (H2O2/HPA=0.4 mmol/mmol, HRP/HPA=3 mg/mmol) over time.

The influences of HRP/HPA ratio and polymer concentration on the G’, G’’, and damping factor (tanδ) of the hydrogel were studied at a H2O2/HPA ratio of 0.4 mmol/mmol. Figure 3a exhibits that the G’ of the hydrogels strengthened from 9.8 to 17 kPa when HRP/HPA ratio was increased from 1.0 to 3.0 mg/mmol. Based on this results, it seems that the HRP/HPA ratio has a marked impact on the mechanical performances of the hydrogel. As shown in Figure 3b, the G’ of hydrogels formed from PLL-g-HPA10 increased with increasing polymer concentration at a fixed H2O2/HPA and HRP/HPA ratio. For instance, the G’ of PLL-g-HPA10 hydrogels went up from 0.82 to 17 kPa when polymer concentrations was increased from 5.1 to 13.6 w/v%, ascribed to more compact cross-linking network structure of hydrogel fabricated at a higher polymer concentration.38 The tanδ of the hydrogel reduced with the polymer concentration increasing, also demonstrating better network compactedness at higher polymer concentrations. Compared with the reported PLG-g-PEG/TA hydrogel,15 the PLL-g-HPA hydrogels exhibited a higher G’. What is noteworthy is that the storage modulus of hydrogels cannot be precisely determined because of the instantaneous gelling when the PLL-g-HPA concentration was set to 15 w/v%. The results of rheological analysis indicated that the mechanical performances of the hydrogel can be easily adjusted by altering the polymer and HRP concentrations.


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Figure 3. The change of loss modulus (G’’, black bar), storage modulus (G’, gray bar) and damping factor (tanδ, solid circle) of PLL-g-HPA10 hydrogel over (a) the HRP/HPA ratio (H2O2/HPA=0.4 mmol/mmol, 13.6% Gel), and (b) the polymer concentration (H2O2/HPA=0.4 mmol/mmol, HRP/HPA=3 mg/mmol) (f=1 Hz, ε=1%, 37 ℃ )

According to the result of the gelation time test (Figure 1), the gelling time of PLL-g-HPA hydrogel can be flexibly modulated from 14 to 332 s in the process of hydrogel formation and thus, PLL-g-HPA hydrogels could be utilized as injectable material. To demonstrate this, PLL-g-HPA hydrogel was formed by a double-mixing syringe. As presented in Figure 4a, the hydrogel was injected and formed in the culture dish; the inset refers to the formation hydrogel clinging on the standing culture dish. In addition, a smooth letter “W” was successfully written (Figure 4b). These results confirmed the injectability of PLL-g-HPA hydrogel. The extruded polymer solutions converged together to form an intact piece hydrogel on account of its fast gelation ability based on enzymatically catalytic reaction. This indicates the hydrogel can be utilized to encapsulate drugs/cells homogeneously and implant to a target site based on a minimally invaded method. Besides, based on the facile fabrication of the hydrogel by utilizing a dual syringe, it can be endowed with free-shapeable properties, fabricated in any mold with required shapes, such as (c) triangle, (d) heart, and (e) cross.



Figure 4. The injectable process of the in-situ formation PLL-g-HPA10 ((H2O2/HPA=0.4 mmol/mmol, HRP/HPA=3.5 mg/mmol, 13.6 w/v%) hydrogel. (a) the graph of hydrogel formation using a dual-mixing syringe without clogging; (b) a smooth letter “W” successfully written. PLL-g-HPA hydrogels could be molded freely by using a dual-mixing syringe: (c) triangle; (d) heart; (e) cross.

The hydrogel materials used as biomedical 3D tissue scaffolds are required to have certain degradation property. The in vitro swelling and degradation assays of PLL-g-HPA hydrogels were performed in PBS (0.01 M and pH 7.4) at 37 °C for preconcerted time intervals, as indicated in Figure 5. In our studies, the effects of the HRP concentrations, H2O2 concentrations, polymer concentrations, and elastase on the degradation tests of hydrogels were assessed.

Figure 5. The degradation tests of PLL-g-HPA10 hydrogels in PBS: (a) HRP/HPA=3.0 mg/mmol, H2O2/HPA=0.4, 0.8, 1.2, 1.5 mmol/mmol, 13.6 w/v%; (b) HRP/HPA=3 mg/mmol, H2O2/HPA=0.4 mmol/mmol, at different polymer concentration 6.8 w/v%, and 13.6 w/v% with 5.0 units/mL elastase (control without elastase). (c) The degradation appearance of PLL-g-HPA10 hydrogels (formed at an H2O2/HPA ratio of 0.4 mmol/mmol and an HRP/HPA ratio of 3 mg/mmol) in different time with 5.0 units/mL elastase at 37 °C in pH 7.4 PBS).

Figure 5a displays the swelling and degradation tests of the hydrogel constructed at different H2O2/HPA ratio (0.4, 0.8, 1.2, 1.5 mmol/mmol) with a constant ratio of HRP/HPA=3 mg/mmol. The hydrogels formed at higher H2O2/HPA ratio were degraded faster. The PLL-g-HPA hydrogels constructed at a H2O2/HPA ratio of 1.2 and 1.5


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mmol/mmol finished the whole degradation within 25, and 20 days, respectively. However, when the H2O2/HPA ratios were 0.4, and 0.8 mmol/mmol, up to 40 days, the hydrogels still maintained the 54%, and 35% of mass remaining, respectively. In the initial 2 days, an increase of MR value was observed with the MR value varying from 266% to 352% and this was ascribed to the initial swelling of the hydrogels. The swelling degree of the hydrogels constructed at higher H2O2 concentration was higher. The swelling degrees of the hydrogels formed at an H2O2/HPA ratios of 0.4 and 1.5 mmol/mmol were 266% and 352%, respectively, in 2 days. The faster degradation and higher swelling ratio of PLL-g-HPA hydrogels at higher H2O2/HPA ratio are due to lower cross-linkage densities in the networks, which was in contrast to the result of the effect of HRP on the degradation and swelling ratio of the hydrogels (Figure S11). In addition, the effects of the polymer concentrations, and elastase on the swelling and degradation of PLL-g-HPA hydrogels were also investigated, as shown in Figure 5b. The 6.8 w/v% hydrogel finished the whole degradation within 18 days via the breakage of the peptide chains in the existence of elastase, while the 13.6 w/v% hydrogel kept its completeness up to 27 days in the same condition. A research was carried out by Chen et al., demonstrating that the polyglutamic acid hydrogel completely disintegrated within 22 days in the existence of the papain.28 The degradation appearance of PLL-g-HPA10 (13.6 w/v%) hydrogels in different time with elastase at 37 °C in pH 7.4 PBS was shown in Figure 5c. It could be obviously seen that there was a slow degradation for the first 15 days followed by a relatively rapid degradation, up to the complete disappearance of the hydrogels in 27 days. This degradation period was in accordance with the result presented in Figure 5b. The proteolysis performance of the artificial matrix was considerably necessary because for the cell or tissue cultivation, the rebuilding of the artificial extracellular matrix (ECM) is required by the proteolysis. Some biodegradable hydrogels adjusted by the proteolysis have been extensively researched.38,39 In comparison with the relatively fast degradation under the condition of the existence of elastase, the hydrogels maintained for much longer time without the enzyme. In the starting 2 days, an evident increase of MR value was observed after immersion in PBS and this was due to the initial swelling of the hydrogels. Subsequently, the hydrogels displayed a comparatively slow degradation in PBS and in the meantime, the degradation rate of 6.8 w/v% hydrogels was faster than that of the 13.6 w/v% hydrogels in the buffer solutions in the absence of elastase. As is well-known, the variation of hydrogel weight in the degradation process in vitro was due to the dual effects of both degradation and swelling.40 The degradation rate was considerably influenced by the crosslinking degree of



hydrogels.14,41 Therefore, the PLL-g-HPA hydrogels formed at a higher polymer concentration displayed lower degradation rate because of higher crosslinking degree. Furthermore, the existence of enzyme could accelerate the degradation process, attributed to the proteolysis of the polypeptide chains. All these conclusions above illustrated that the degradation process of the polypeptide hydrogels could be easily regulated through adjusting the H2O2, HRP, copolymer concentrations, and adding the enzyme. Cytocompatibility of PLL-g-HPA hydrogels. The in vitro cytotoxicity of PLL-g-HPA hydrogels was examined by MTT test using L929 mouse fibroblasts. In consideration of the very short time for the cells mixed with the pre-gel solution due to the rapid gelling process, the cells were finally mixed with the leachable solutions of hydrogels, instead of the polymer solution of high concentration. Hence, the L929 were cultivated with the polymer solution of different concentrations or leaching solutions of the hydrogels to assess the cytotoxicity of PLL-g-HPA hydrogels. As can be seen from Figure 6(a), the PLL-g-HPA copolymer did not display obvious biotoxicity on the cells, and the cell viabilities were kept above 80% when the concentration of copolymer changes from 0.015 to 0.12 mg/mL, whereas PLL exhibited moderate to high cytotoxicity with approximately 60% of cell viability.42 Besides, the in vitro cytocompatibility of PEI as positive control was also evaluated and compared with that of the PLL-g-HPA, PEI showed higher cytotoxicity. Additionally, the in vitro cytocompatibility of leaching solutions of the PLL-g-HPA hydrogels against L929 cells was shown in Figure 6(b) (3.4% Gel, 5.1% Gel, and 6.8% Gel). The L929 cells exhibited high viability (>74%), revealing low cytotoxicity of PLL-g-HPA copolymer after gel formation. This may be because the gelatination morphology decreases the cytotoxicity, and improves the cytocompatibility.

Figure 6. (a) In vitro cytocompatibility test of the PLL-g-HPA against L929 cells evaluated by MTT test. PEI 10K against L929 cells was used as positive control. (b) In vitro cytocompatibility test of leaching


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solutions of the PLL-g-HPA hydrogels against L929 cells. PEI 25K (0.12 mg/mL) was utilized as positive control.

The cell attachment assay of hydrogels has been widely utilized to evaluate for preclinical tests and basic study, like stem cell differentiation research, cancer cell behaviors.43,44 As presented in Figure 7a, most of the L929 fibroblasts on the hydrogels exhibited green fluorescence after staining with 2% FDA solution, revealing high cell viability and good biocompatibility of the hydrogels. Besides, compared with the amount of stained cells cultured for 1 days, the cells adhering on the hydrogels were obviously more after being cultured for 3 days, indicating that with the culture time increasing, L929 cells little by little proliferated on PLL-g-HPA hydrogels. Figure 7b showed that the numbers of L929 cells on the hydrogel surface incubated for 1, 3 days are 1.4x104, and 2.6x104/mL counted by a cell counter, respectively. Based on the cell attachment tests, it is concluded that the PLL-g-HPA hydrogel may be hopeful biomaterials for many applications, such as cell therapy, tissue engineering, and biological diagnosis.

Figure 7. (a) The LCFM pictures of L929 cells incubated on PLL-g-HPA hydrogels with the 6.8 w/v% of polymer concentration for 1, 3 days (bar scale: 50 μm). (b) The amount of L929 fibroblasts after culturing on PLL-g-HPA hydrogels for 1, 3 days.

Functionalization of the hydrogels. As reported in the literatures, amino groups could be conveniently decorated for numerous bio-applications like fluorescence probing, cell therapy, and cell diagnosis.45,46 Because of the existence of large amounts of un-grafted amino groups in the PLL-g-HPA, the PLL-g-HPA hydrogels could be simply post-functionalized by the reaction between the amino and other active groups. Here, the rhodamine B was used to functionalize the hydrogel by the amidation between carboxyl and amino groups in methanol. Subsequently, the PLL-g-HPA hydrogel was directly subject to the LCFM (Figure 8a). The



apparent red color among the hydrogels texture due to the excitation of the rhodamine B exhibited the consummate functionalization. Besides, the ultraviolet-visible spectra of the PLL-g-HPA hydrogel after post-functionalization for different time were shown in Figure 8b. The intensity of the absorption peaks of rhodamine at 554 nm gradually enhanced with the time lengthening, and after post-functionalization for 18 h, the intensity remained almost unchanged, indicating the complete reaction of COOH-NH2 amidation. The corresponding fluorescence yields (Wti) of rhodamine in PLL-g-HPA hydrogels functionalized for different time were summarized in Figure 8c. After the complete functionalization of PLL-g-HPA hydrogel for 18 h, the fluorescence yield was approximately 13.2x10-5. Images of the hydrogels before and after functionalization were displayed in Figure 8d. The amino may also interact with lots of other active groups, like aldehyde group, maleic anhydride, and so on. Based on these reactions, the PLL-g-HPA hydrogels could be conveniently functionalized with bioactive drugs or proteins.

Figure 8. (a) The LCFM pictures of 6.8% PLL-g-HPA hydrogels after being post-functionalized by rhodamine B. (b) Ultraviolet-visible spectra of the hydrogel after post-functionalization for 0, 3, 6, 9, 18, 24h. (c) Fluorescence yield (Wti) of Rhodamine B in PLL-g-HPA hydrogels functionalized for different time. (d) Images of the hydrogels before and after functionalization.



CONCLUSIONS

In this paper, a novel in situ cross-linkable enzyme-mediated hydrogel composed of PLL-gHPA with designable shapes and good biocompatibility was successfully prepared. The


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hydrogels were fast formed by an enzyme-mediated cross-linking under physiological environments utilizing H2O2 and HRP. The gelation time could be well tuned resting with the HRP, H2O2, and polymer concentration. In addition, the degradation time of the hydrogels can be easily regulated by adjusting the HRP, H2O2, polymer concentrations or adding the enzyme to attain either a fast degradable PLL-g-HPA hydrogel or a relatively stable PLL-gHPA hydrogel. The PLL-g-HPA hydrogels possess good biological compatibility, as demonstrated in vitro cell cultivation and attachment experiments. Furthermore, the remaining amino groups in the hydrogel could be further functionalized for various cell researches and bio-applications. 

ASSOCIATED CONTENT

Supporting Information The Supporting Information is available free of charge on the ACS Publications Website at DOI: xxx. Additional detailed information including synthetic route of the PLL-g-HPA copolymer; the 1H

NMR,

13C

NMR, and FTIR of the ZLL-NCA; 1H NMR spectra of PZLL in DMSO-d6,

PLL in D2O, and PLL-g-HPA copolymer in D2O; UV-Vis absorbance, and circular dichroism spectra of PLL-g-HPA aqueous solution (pH 7.4); the GPC of the PLL75; the 1H NMR of the PLL-g-HPA15; the UV-vis of the PLL-g-HPA15; gel content and equilibrium water content of hydrogels prepared from 13.6 w/v% PLL-g-HPA10 in PBS; the storage modulus (G’) and loss modulus (G’’) of the PLL-g-HPA10 hydrogels as a function of frequency; the swelling and degradation assays of PLL-g-HPA10 hydrogels in PBS (0.01 M and pH 7.4) at 37 °C at different HRP concentration; the FTIR of the ZLL-NCA, PZLL, PLL; the In vitro cytocompatibility of H2O2 against L929 cells detected by MTT assay (n= 3); calibration curve of the absorbance at 554 nm of rhodamine B at different concentrations. The content of conjugated rhodamine groups in the PLL-g-HPA hydrogel was calculated by using the fitting formula, y=122.74x+0.08, R2=0.9999 (PDF). 

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected] Tel: (+86) 021-54745817. ORCID Xinling Wang: 0000-0001-7158-5737 Notes



The authors declare no competing financial interest. 

ACKNOWLEDGMENTS

The work was financially supported by the National Natural Science Foundation of China (21274085) and Shanghai Leading Academic Discipline Project (No. B202). 

REFERENCES

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99x94mm (300 x 300 DPI)


Fabrication of a Controlled in Situ Forming Polypeptide Hydrogel with

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