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Poly(ether-carbonate) based hydrogel with tunable mechanical strength and enhanced bioactivity prepared by Michael addition
Polymer 188 (2020) 122115
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Poly(ether-carbonate) based hydrogel with tunable mechanical strength and enhanced bioactivity prepared by Michael addition Tao Wang a, b, c, Yang Han e, Ying Bai c, d, Qingtang Zhu a, c, Daping Quan b, c, d, *, Xiaolin Liu a, c, ** a
Department of Orthopedic and Microsurgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China PCFM Lab, GD HPPC Lab, School of Chemistry, Sun Yat-sen University, Guangzhou, China c Guangdong Provincial Peripheral Nerve Tissue-engineering and Technology Research Center, Guangdong Provincial Functional Biomaterials Engineering Technology Research Center, Guangdong Provincial Soft Tissue Biofabrication Engineering Laboratory, Guangzhou, China d School of Materials Science and Engineering, Sun Yat-sen University, Guangzhou, China e Department of Obstetrics, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: poly(ether-carbonate) hydrogel Michael addition Dorsal root ganglion
Poly (ether-ester) based hydrogels have been broadly used as degradable biomaterials in drug delivery systems and tissue engineering. However, they were synthesized with uncontrollable mechanical properties and inert to most of the biological activities, such as cell adhesion and stem cell differentiation. Herein, we synthesized a water-soluble copolymer with poly (trimethylene carbonate-co-2-methyl,2-methylacrylate-bimethylene carbon ate) (PTMAc) and poly(ethylene glycol)(PEG) blocks (PCE), in which the PTMAc segments provided free active double bonds and can be further conjugated with thiol-ended peptides to improve its cytocompatibility. Meanwhile, the extra double bonds were crosslinked by Michael addition, forming hydrogels with precisely controlled mechanical strength. The chemical structure and molecular weight of the synthesized PCE were characterized by 1H NMR and gel permeation chromatography (GPC). The composition of the PCEs was controlled by changing the feed ratios of 2-methyl,2-methylacrylate-bimethylene carbonate (Ac) comonomer (fAc). It was noted that when the ratio of comonomers to the macroinitiator PEG ([M]0/[I]0 ¼ 18) and the ratio of DL-Dithiothreitol (DTT) to Ac ([thiol]/[Ac] ¼ 1/1) were fixed, the storage moduli of PCE hydrogels increased from 2 kPa to 10 kPa and the gelation time decreased with increasing fAc. The pendant double bonds were easily conjugated with CRGD to promote the neurite sprouting and axonal extension from the dorsal root ganglion (DRG) neurons. Moreover, the weaker the hydrogel matrix (G0 ¼ 0.2 kPa) was, the lower the axon extension length was, but the axon diameter became thicker. An increase in modulus is beneficial to axon extension (5 kPa), while a high modulus (10 kPa) is unfavorable. These results demonstrated that this novel poly(ethercarbonate) based hydrogel can be introduced as a suitable scaffold for fast and effective nerve regeneration.
1. Introduction A property-controlled living microenvironment often leads to spe cific behaviors of their embedded cells, such as proliferation, migration and differentiation [1]. Therefore, well-designed cytocompatible mate rials have been broadly pursued in terms of their cell-laden capability and ,clinical applications [2]. Among them, the highly water containing hydrogels exhibited great potential in cell-based regenerative thera peutics, for example in minimally invasive surgeries, which could un dergo crosslinking either physically or chemically for tunable mechanical properties [3].
So far, it has been realized that most of the polyethlene glycol (PEG) or polyester-ether based thermo-sensitive hydrogels exhibit low me chanical strength and poor bioactivity [4,5]. Chemical crosslinking has been proven to be an effective way to improve their mechanical strength. It has been reported that acrylate/vinyl sulfone-functionalized linear or star PEG hydrogels by chemical crosslinking decreased the critical gelation concentration (CGC) and resulted in highly increased mechanical properties [4]. In addition, some short peptides were introduced into the chemically crosslinked PEG hydrogels, such as acrylic modified adhesive sequence (arginine-glycine-aspartate acid sequence, RGD) and matrix metalloproteinase sensitive sequence
* Corresponding author. School of Chemistry, Sun Yat-sen University, Guangzhou, China. ** Corresponding author. Department of Orthopedic and Microsurgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. E-mail addresses: [email protected] (D. Quan), [email protected] (X. Liu). https://doi.org/10.1016/j.polymer.2019.122115 Received 13 September 2019; Received in revised form 3 December 2019; Accepted 19 December 2019 Available online 21 December 2019 0032-3861/© 2019 Published by Elsevier Ltd.
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(MMP), to improve the biological activity and also to conjugate the biodegradability of hydrogels [5]. Furthermore, the crosslinking of the diacrylated block polyether-esters often leads to hydrogels with enhanced mechanical properties, such as diacrylated polycaprolactone-PEG-polycaprolactone (PCL-PEG-PCL-DA) [6], dia crylated polylactic acid-PEG-polylactic acid (PLA-PEG-PLA-DA) [7], diacrylated poly(lactic-co-glycolic acid)-PEG-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA-DA) [8], which may find application in drug release and regenerative medicine. However, some research studies also showed that the residual catalysts from the process of free radical addition retained their cytotoxicity and inhibitory for cell growth [9, 10]. Moreover, the acrylation process usually resulted in limited amount of free double bonds after crosslinking, which brought more difficulty for further biomimetic modifications. Chemical crosslinking by Michael addition reaction has been intro duced into the hydrogel systems to effectively bypass the toxicity of residual catalysis mentioned above [11]. An injectable bioactive hybrid hydrogel, based on thiolated collagen (Col-SH) and multiple acrylate-containing oligo (acryloyl carbonate)-b-poly (ethylene glycol)-oligo (acryloyl carbonate) (OAC-PEG-OAC) copolymers, was reported to form via Michael addition [12]. As compared to the func tional group-terminated PEG gels or polyether-ester block copolymers, copolymers with multiple functional groups on the side chain are highly prospective to reach the goal of crosslinking for enhanced mechanical strength and conjugating biomolecular activities into the hydrogels simultaneously [13,14]. In this work, we report a well-designed strategy for synthesizing hydrogels with adjustable biological and mechanical properties. 2methyl,2-methylacrylate-bimethylene carbonate (Ac), was copoly merized with trimethylene carbonate (TMC), using PEG as the macro initiator. The resulted copolymers are soluble in water by controlling the length of polycarbonate blocks. Consequentially, the chemical cross linked hydrogels were performed by mild Michael addition reaction with DL-Dithiothreitol (DTT) as the crosslinker. Moreover, the me chanical strength of the crosslinked hydrogel was tuned by manipulating the addition of DTT, the bioactivities of hydrogels was induced by modification of the relative abundant double bonds with thiol-ended RGD peptides along the copolymer side chains. Finally, the ability of modified hydrogels to support outgrowth of the axones was evaluated in a rat dorsal root ganglion (DRG) culture model.
sequentially quantitative anhydrous PEG, TMC and Ac were dissolved in dried dichloromethane (DCM) and then DBU was added in a vacuum glove box (O2% < 0.1 ppm, H2O% < 0.1 ppm) in 25 mL round bottom reaction flasks. After 10 h of agitation at 28 � C, the polymerization was terminated by two drops of acetic acid. The raw product was isolated via precipitation in cold diethyl ether at room temperature and then dried in vacuo at 40 � C to constant weight. Yield: 80.1%–83.5%. 2.3. Characterization All 1H NMR and 13C NMR spectra were recorded on a Mercury-plus VARIAN (300 MHz for 1H NMR and 75 MHz for 13C NMR) spectrometer using tetramethylsilane (TMS) as an internal reference and CDCl3 or deuterated dimethyl sulphoxide (DMSO‑d6) as the solvent. A GPC system equipped with a Waters 1525 separations module, Waters 2414 RI de tector and Waters Styragel 7.8 � 300 mm HR1, HR3 and HR4 were used to determine the molecular weight (Mn and Mw, respectively) and polydispersity of the copolymers. Tetrahydrofuran (THF) was used as the eluent at a flow rate of 1.0 mL/min at 40 � C and the MWs were calibrated using polystyrene standards (MW range from 1 kDa to 240 kDa). 2.4. Gelation, mechanical and degradation properties The quantitative copolymer (50 mg) was dissolved in 0.95 mL phosphate buffered saline (PBS, pH 7.4, 0.1 M) at a concentration of 5 wt % as stock solution and then quantitative DTT ([thiol]/[Ac] ¼ 1/1) was added and incubated at 37 � C after thoroughly mixed. 1H NMR of resulting polymer was measured after dialysis and freeze drying with DMSO‑d6 as the solvent. The gelation time and viscoelasticity of the hydrogels were determined with an advanced rheology expanded sys tems (ARES/RFS, TA, USA) with a parallel plate fixture (diameter, 25 mm; gap, 1 mm) at 37 � C adjusted with a bath temperature controller (Neslab). Polymer solution was positioned in the parallel plate imme diately after DTT mixed and gradually squeezed by the top-plate. A plastic cover was used to minimize solvent evaporation. The evolution of storage modulus (G0 ) and loss modulus (G00 ) of hydrogel was recorded as a function of time. The test was set at a frequency of 10 Hz and a strain of 1% which was confirmed at the linear viscoelastic region. The gelation time was defined as the time point where G0 ¼ G00 and tested in triplicate. To determine the amount of thiol groups (the residual thiols of hydrogel system at different time during gelation) Ellman’s test had been used according to its standard procedure [16]. Briefly, Ellman’s reagent, Tris solution, and sample were mixed together in appropriate amounts and its absorbance was measured at 412 nm using UV–Vis spectrophotometer (Cary100, Varian Company). The prepared hydrogels (1 mL) were agitated to swell overnight in 5 mL PBS at 37 � C. The wet weights (Ws) of the gels at equilibrium swelling state were recorded after blotted dry with weight paper. These gels were then lyophilized for 24 h and the dry masses (Wd) were recorded. The equilibrium swelling ratio (ESR) was determined by the equation (Ws Wd)/Wd � 100%. For measurement of degradation behavior, the hydrogel sample was prepared and immersed in 1 mL distilled water after equilibrium swelling and then placed under agitation at 37 � C. The extracted solution was collected and replaced with pristine distilled water at each time increment. The collected solution was freeze-dried to measure the weight loss. Consequentially, the residual mass was calculated by sub tracting the total weight loss from the original weight of the sample. Samples were allowed to swell for at least 24 h in treatment media. For measuring of degradation via weight change, the patches were weighed at days 1, 3, 7, 10 and 14 after formation, and degradation was determined as residual weight compared to original weight at day 1.
2. Materials and methods 2.1. Materials Poly (ethylene glycol) (PEG, Mn ¼ 10 kDa) was purchased from Aldrich (Milwaukee, USA) and was dehydrated under vacuum at 120 � C for 4 h prior to use. Trimethylene carbonate (TMC) was donated from Huayang medical device company (Huizhou, China) and recrystallized in ethyl acetate prior to use. Acryloyl cyclic carbonate (Ac) was syn thesized as reported in the literature (1H NMR (300 MHZ, chloroform-d, CDCl3, δ): 1.14 (s, 3H; CH3), 4.17 (d, 2H; CH2), 4.19 (s, 2H; CH2), 4.33 – CH2)) (d, 2H; CH2), 5.91–6.45 (m, 3H; –CH– [15]. Cysteine-arginine-glycine-aspartate peptide (CRGD) was purchased from GL Biochem (Shanghai) Co., Ltd. 1,8-Diazabicyclo[5.4.0]unde c-7-ene (DBU, 99%), dithiothreitol (DTT, 99%) were purchased from J&K scientific (Shanghai, China) and used without further purification. Other chemical solvents were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China), dried using standard procedures and distilled before use. 2.2. Synthesis of PTMAc-PEG-PTMAc copolymer The polymers were synthesized via ring-opening polymerization (ROP) of TMC and Ac using PEG as an initiator and DBU as the catalyst. A typical experimental procedure is described as the following: 2
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2.5. Polymer modification with CRGD peptide
glutamine and 1% penicillin–streptomycin in a 37 � C incubator with 5% CO2 and 92% humidity. The medium was changed every 2 days.
Michael addition reaction was used to prepare the CRGD function alized PTMAc-PEG-PTMAc copolymer (abbreviated R-PCE). Typically, PTMAc-PEG-PTMAc copolymer (100 mg) was dissolved in 1.9 mL PBS at 37 � C overnight to prepare 5 wt% polymer solution and then quantita tive of CRGD ([CRGD]/[Ac] ¼ 3/10) were added and allowed to react for 2 h at 37 � C. The CRGD modified polymer was obtained via lyoph ilization after dialysis (MWCO 5000) in PBS at room temperature and then dried in vacuo at 40 � C to constant weight. Yield: 94.5%. The ki netics and efficiency of this Michael addition reaction were monitored by 1H NMR.
2.8. Immunofluorescence staining of DRG Cells were fixed by 4% paraformaldehyde in PBS for 20 min, rinsed by PBS, permeabilized and blocked with 0.1% Triton X-100 and 10% donkey serum in PBS for 30 min. Cells were incubated with primary antibodies against NF200 (dilution 1/150) and S100 (dilution 1/1000) with DRG culture samples and NF200 (dilution 1/150) and MBP (dilu tion 1/500) with neuron-Schwann cell co-culture samples for 2 h at room temperature, followed by secondary antibodies conjugated to Fluro 488 (dilution 1/1000) and Fluro 594 (dilution 1/1000) incubation for 1 h. The cells were then rinsed with PBS for 10 min (3 times) and stained with DAPI (dilution 1/2500) for 20 min. The fluorescence was observed and imaged under the laser scanning confocal microscope. The parameters and cells were measured by Image J® for at least 3 samples.
2.6. Characterization by scanning electron microscopy (SEM) All the samples were attached on conducting resin and coated with platinum. The micromorphology was observed and imaged by Hitachi S4800 (Japan) at 15 kv. The diameters of nanofibers were measured by image analysis software (Image J®).
2.9. Statistical analysis Data are presented as the mean � standard deviation (SD). Differ ences between groups were evaluated by one-way analysis of variance (ANOVO) followed by least significant difference posthoc tests. A pvalue < 0.05 indicates statistically significant difference.
2.7. DRG culturing The hydrogel membrane samples for DRG culture were immersed in 75% ethanol with 30 min for sterilization and then washed by PBS for 5 times (5 min for each time) and kept wet before cell culturing. The DRGs were isolated from newborn Sprague–Dawley rats (sup plied by Laboratory Animal Center of Sun Yat-sen University, China). The residual nerve roots were cut off under a stereomicroscope. Then DRGs were plated on hydrogel membrane/glass slides, which were put in a 48-well plate with neurobasal medium containing 2% B27, 0.3% L-
3. Result 3.1. Synthesized and characterization A series of PTMAc-PEG-PTMAc copolymers (PCE) with short
Scheme 1. (A) synthesis of the PCE copolymers. (B) crosslinking with DTT and bioactive post-modification with CRGD by Michael addition reaction. (C) the schematic diagram of PCE hydrogel with tunable mechanical strength and enhanced bioactivity prepared by Michael addition. 3
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polycarbonate segments were synthesized by ROP of Ac and TMC using PEG as macro-initiator and DBU as catalyst in DCM at 28 � C shown as Scheme 1. The corresponding GPC curves (Fig. 1 A and B) were unim odal, but the PEG efflux peak shifted into short elution time after polymerization reaction, and the elution time decreased gradually with the increase of carbonate monomer contents. As shown in Table 1, the block copolymers were water-soluble if their degrees of polymerization (DPs) were limited to 18–26 and the contents of Ac monomer (fAc) were changed from 20% to 50%. The conversions of TMC and Ac comonomers were more than 90% within 10 h of polymerization at 28 � C, and the measured contents of Ac (FAc, measured by 1H NMR) were very close to the feed ratios of the Ac monomers, indicating a good ROP controllability. The chemical structure of PCE was confirmed by 1H NMR and 13C NMR analyses. It was shown that the resonance absorption at 3.66 ppm (e) should be attributed to methylene in PEG, and the resonance ab sorption at 2.10 ppm (f) and 4.25 ppm (c) were assigned to PTMC segment. For the PAc segment, in addition to the methyl protons at 1.00
ppm (g) and the methylene protons at 4.10 ppm (d), a typical double bond resonance absorption was found in the range of 5.80–6.50 ppm (a, b), indicating the successful synthesis of copolymers containing double bonds in the side chain. DBU has been proven to be an efficient orga nocatalyst for ROP of cyclic carbonates and their derivatives [17,18]. The structure of the resulting copolymer and blocky regions of PTMC and PAc in the P(TMC-Ac) regions were further characterized by 13C NMR spectrum (Fig. 1 D). Three kinds of carbonyl groups were identified in the copolymer, including the pendant carbonyl of acrylate (165 ppm) and the carbonyls for TMC and Ac units (154–155 ppm). Upon enlarging the carbonyl region of h (inlet of Fig. 1 D), three resonance signals were observed (154.75, 154.85 and 155.00 ppm) and were assigned to the diad sequences of TMC unites (T) and Ac unites (A), including TT, AT, and AA, representing the regions of blocky PTMC, random P(TMC-Ac) and blocky PAc, respectively [19]. 3.2. Gelation via controllable Michael addition It was found that the aqueous solutions of PCE copolymers shown in Table 1 did not have the thermosensitive properties of sol-gel phase transition, which may be related to the weak interaction of hydrophobic chains of block copolymer [20]. Upon adding DTT, the aqueous solutions of PCE copolymers gradu ally solidified to form transparent gels via Michael addition reactions between the reactive double bonds in the Ac units and the thiol groups in the crosslinker of DTT. The dynamic rheological measurement was used to test the gelation transition of copolymer solution at 37 � C, as shown in Fig. 2 A, the G0 and G00 of PCE2 were equal after 6.5 min, suggesting the gelation transition time was about 6.5 min for PCE2. With extension of the time, the G0 of PCE2 solution increased until 30–35 min and reached a platform value of 2.2 kPa, but the G00 had little change over time. 1 H NMR measurement was used to monitor the reaction process of Michael addition (Fig. 2 B). The new resonances attributed to the methine group in the crosslinker DTT were first appeared at 5.2 ppm after 5 min of reaction, and the peak became spikier as the reaction proceeded (t ¼ 5, 15, 30, 45 and 120 min). Meanwhile, resonances of the acrylate protons (within the Ac units) in the range of 5.8–6.6 ppm gradually decreased during the Michael addition reaction and almost disappeared after 2 h. The amount of residual thiol groups during the reaction process was also monitored by Ellman’s test (Fig. 2C) [16]. It was found that only 14% of thiol was detected (compared with original thiol) at about 30 min and continued to decline to 6% after an hour reaction between PCE2 and DTT ([thiol]/[Ac] ¼ 1/1). As the Michael addition reaction went on, the content of thiol group in the system kept decreasing until reaching the plateau state, which was consistent with the increment of G0 (Fig. 2 A). Different from free radical polymerization, Michael addition reaction can control the degree of crosslinking by controlling the content of crosslinking agent, thus regulating the properties of hydrogels. It was showed in Fig. 2 D, when the mount of DTT was changed from insuffi cient to excess, the G0 of hydrogel increased first (from 0.5 kPa to 5 kPa) and then decreased obviously, indicating that the cross-linking reaction was precisely controlled by feed ratios. Accordingly, the ESR of hydro gels displayed contrary tendencies contrasted with G0 . 3.3. The influence of composition and concentration of copolymers on the properties of hydrogels As shown in Fig. 3 A, when the ratio of monomer to initiator ([M]0/ [I]0 ¼ 18) and the ratio of DTT to Ac ([thiol]/[Ac] ¼ 1/1) were fixed, by increasing fAc from 20% to 50%, the corresponding G0 increased by 5 times (from 2 kPa to 10 kPa), the ESR decreased and the gelation transition time get shorter (from 9 to 2.5 min), which was attributed to the increase of Ac contents and the crosslinking density in PCEs. When the molecular weight of PCE copolymers were increased, the G0 of
Fig. 1. GPC and NMR characterization of the synthesized PCE polymers. GPC spectra of PEG and the synthesized PCE copolymers with different mole ratios (A) and DP (B) in THF at 40 � C. 1H NMR (C, CDCl3) and 13C NMR (D, DMSO‑d6) spectra of PCE2 at 26 � C. 4
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Table 1 PTMAc-PEG-PTMAc copolymers synthesized by ROP. Samplea
[M]0/[I]0b , DP
fAcc (%)
PCE1 PCE2 PCE3 PCE4 PCE5 PCE6
18:1 18:1 18:1 18:1 22:1 26:1
20 30 40 50 30 30
Conv.(%)d TMC
Ac
94 94 91 92 95 96
100 100 100 100 100 100
FAcd,e (%)
Mn, theoryf (kg/mol)
Mn, NMRg (kg/mol)
Mn, GPCh (kg/mol)
Mw/Mhn
19 30 39 47 30 33
12.2 12.4 12.5 12.7 12.9 13.4
12.1 12.4 12.5 12.6 12.9 13.5
22.0 22.2 22.3 22.8 23.1 23.9
1.20 1.21 1.23 1.23 1.20 1.19
a
All polymerizations were performed in DCM at 28 � C for 10 h, using PEG as the macroinitiator and DBU as the catalyst. PCE1 ~ PCE6 polymers were named sequentially in Table 1, varied by their DPs and Ac mole fractions, respectively. b Degree of polymerization in feed. c Molar fraction of Ac in feed (nAc/(nTMC þ nAc)). d Measured by 1H NMR spectroscopy. e Molar fraction of Ac in the isolated polymer. f Calculated with theory feed (10000 þ 198 � DP � fAcþ102 � DP � (1-fAc)). g Calculated by 1H NMR spectroscopy (10000 þ 198 � DP � FAcþ102 � DP � (1-FAc)). h Determined by GPC analysis in THF using polystyrene as the standard.
Fig. 2. The gelation and controllable Michael addi tion reaction. (A) The gel point was defined as the time point where the G0 and G00 crossed and obtained by dynamic rheological measurement (10.0 rad/s frequency, 1% strain at 37 � C). The photo of PCE2 was taken at 6.5 min. (B) The 1H NMR (DMSO‑d6) spectra of PCE2 (0.5 wt%) and DTT ([thiol]/[Ac] ¼ 1/1) re action system at different reaction time. (C) The re sidual thiols of PCE2 (0.5 wt%)/DTT ([thiol]/[Ac] ¼ 1/1) determined by Ellman’test in PBS at 37 � C at different time. (D) The G0 and ESR of PCE2 hydrogel (5 wt%) with different [thiol]/[Ac] (after reaction for 30 min) in PBS at 37 � C determined by rheology.
hydrogel also increased gradually, ESR and gelation transition time decreased. Considering the water solubility of copolymer, while the change ranges of molecular weight were limited. The concentration of water-soluble PCE2 affected the properties of hydrogels (Fig. 3 B). It was found that when the concentration of PCE2 aqueous solution was 8%, the corresponding G0 was 32 kPa. By contrast, hydrogels made from 4% of PCE solution had a G0 of only 0.21 kPa, however, 5 kPa of G0 was obtained for 5% group. ESR and gelation transition time also changed correspondingly. In vitro degradation behaviors of the PCE2 hydrogels at different concentrations (4 wt%, 5 wt% and 6 wt%) were tested (Fig. 3 D). The hydrogel at 4 wt% concentration exhibited the fastest degradation and the residual mass was only 43% � 12% after 35 days. The stability of the PCE2 hydrogel increased dramatically with small increment of polymer
concentration. The residual mass of 6 wt% hydrogel was 96% at day 35 with relatively intact shape. The morphologies of the lyophilized hydrogels were observed by SEM (Fig. 3D). With the increase of fAc and DP, the average pore diameter of hydrogel decreased from several hundred microns to several tens of microns. Considering the mechanical strength and porosity of hydrogel, 5% PCE2 was selected for in vitro cell culture. 3.4. Modification PCE with CRGD CRGD as fibronectin sequences was often used to modify the different materials and to improve their biocompatibility. Here we incorporated CRGD into the PCE copolymer by Michael addition re actions between the reactive double bonds in the Ac units and the thiol 5
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groups in the CRGD, the reaction was processed at 37 � C for 2 h. In Fig. 4 A, the new resonances attributed to CRGD peptide sequences were observed in the range of 8.0–9.0 ppm, 2.6–2.7 ppm and 1.4–1.6 ppm. The resonance absorption of acrylate protons in the range of 5.8–6.6 ppm almost disappeared contrast with spectra of PCE2 shown as Fig. 1C, which was consistent with the molar feed ratio ([CRGD]/[Ac] ¼ 1/1), indicating the CRGD modification can be precisely controlled in a quantitative manner. By adjusting the ratio of [CRGD]/[Ac] (1/10, 3/10), two kinds of hydrogels with different CRGD density were obtained and labeled as R10-PCE2 (5 wt%) and R30-PCE2 (5 wt%). The influences of CRGD density on the axon length and axon number of DRG were evaluated after 7 days’ co-culture (Fig. 4 B and C). It was showed that on the R30PCE2 hydrogel surface, the axon length come from DRG neuron was about 814 � 62 μm and the axon number was 786 � 156, while the length and number of axon of R10-PCE2 were 314 � 87 μm and 623 � 120 as control, the pure PCE2 hydrogel were 121 � 35 μm and 425 � 154, respectively. The results suggested that CRGD-modified hydrogel could significantly promote the sprouting and extension of the axons of the DRG, and the impact can be easily controlled by changing the den sity of short peptide sequence. 3.5. The responses of DRG on the tunable modulus of hydrogel In addition to control the density of CRGD, the modulus of hydrogel can also be regulated by changing the degree of crosslinking, thus affecting the behavior and function of cells. We selected three group of copolymer 5 wt% R-PCE2, 4.3 wt% R-PCE4 and 5.2 wt% R-PCE4, which have the same RGD density (2.2 mg/ml) but different G0 (0.2 kPa, 5 kPa and 10 kPa), and co-cultured embryonic DRG with this three hydrogels in vitro to investigate the effect of hydrogels’ moduli on the growth of DRG neurons. It was noted that the neurite outgrowth from DRG was relatively sensitive to the variation in G0 of hydrogel matrix (Fig. 5). On the hydrogel surface with a G0 of 0.2 kPa (Fig. 5 A and D), the axon length of DRG was lower than that of two higher G0 groups (Fig. 5 B E and C F), but the axon diameter was significantly larger than that of the other two groups (Fig. 5H). This phenomenon might be due to the stronger intercellular interactions between the neighboring neurites in response to the relatively soft matrix [21]. As compared with the 10 kPa group, the 5 kPa group had more nerve axons (Fig. 5 I), indicating too soft or too hard substrates is not conducive to neurite outgrowth. It was also observed that the Schwann cells (S100-positive) wrapped around the extending neurites (Fig. 5 D, E, and F), but the percentage of Schwann cells associated with the regenerated axons was almost the same and close to 100% between the cultured hydrogels with different moduli (Fig. 5 J), implying that the R-PCE hydrogels were beneficial for promoting axonal remyelination toward nerve fiber functionalization [22]. 4. Discussion and conclusion At present, most of the chemically crosslinked hydrogels based on poly(ether-ester) have been synthesized by free radical polymerization [23,24]. There were a few drawbacks retained from the synthesis pro cedure for biomedical applications, such as the residual toxicity of the initiators, the relatively fast polymerization usually leads to operational difficulty during in situ injection. Additionally, the free radical poly merization is often initiated by UV light, which also resulted in incon venience application in vivo. Michael addition undergo mild reaction conditions with no initiator required. For this reported PCE copolymers, the gelation time can be controlled in the range of a few minutes to more than ten minutes (Fig. 2), the injected solution could be further manipulated in situ before the sol-gel transition. More importantly, the degree of crosslinking is easily controlled by tuning the ratio of the reactive groups. In the PCE polymer, free double bonds were introduced
Fig. 3. The mechanical, degradation and morphological properties of the PCE hydrogel. The effect of composition (A, concentration of PCE hydrogels were 5%) and concentration (B, PCE2) of hydrogels on the G0 tested, ESR and gela tion time were evaluated. (C) Degradation behavior of the hydrogels with different concentrations. (D) The microstructures of lyophilized hydrogels were observed by SEM. The dynamic rheological measurements were under 10.0 rad/s frequency, 1% strain at 37 � C. [thiol]/[Ac] ¼ 1:1 was controlled at each group.
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Fig. 4. The modification of PCE2 with CRGD and co-culture with DRG. 1H NMR (DMSO‑d6) spectra of PCE2 modified with CRGD (A). 7 days after planted, DRGs were immunofluorescence stained (B), axon length and axon number were statistic (C). Nuclei were stained by DAPI (blue) and axon were stained by NF200 (green). Scale bar: 250 μm * indicates significant statistical difference (n ¼ 3 wells, P ˂ 0.05, Mann-Whitney test).
into the side chains of the polycarbonate segments, which can not only be employed for crosslinking and mechanical strength enhancement, but also can undergo reactions with multiple biological macromolecules for functional modifications, such as short peptide sequences [25], proteins [26], collagen [27], hyaluronic acid [28], etc. To ensure the water-solubility and crosslink ability of the PCE polymer, the oligo-carbonate chain segments were introduced into the backbone, meanwhile, the pendant double bonds on the side chains were employed for gelation. Generally, typical amphiphilic poly(etheresters) first formed “core-shell” structured micelles in aqueous solu tion due to the aggregation of hydrophobic chain segments. With increasing the temperature and/or the polymer concentration, the mi celles started to aggregate, resulting in sol-gel transformation at the macroscopic level [20]. If the double bonds were only incorporated as the end groups, though the chemical crosslinking was still triggered for gel formation, the number of reactive groups was highly restricted, and some of which might be shielded as the inner layer of the micelles. Another advantage of the Michael addition crosslinked PCE hydro gels is their controllable mechanical properties, which can be regulated by tuning the composition of the comonomers, molecular weight of the polymer and the concentration of solution. With increasing the content of comonomer Ac, the crosslinking density and the mechanical strength of the PCE copolymer increased significantly (as shown in Fig. 3, [thiol]/ [Ac] ¼ 1/1). However, when thiol-containing molecules, i.e. the cross linker DTT, was excessive, the G0 of PCE polymer was again decreased
due to the competitive thiol-ene reaction, leaving large amount of re sidual thiol groups in the hydrogel matrix. Furthermore, we have demonstrated that the chemically crosslinked PCE hydrogels can be modified into functional biomaterials to meet the individual requirements in clinical applications. As an example, we have evaluated the effect of axonal outgrowth by culturing the DRG segments on the PCE hydrogels. It was realized that the nerve cell behaviors were highly enhanced by controlling the biological and mechanical properties of hydrogel matrices towards fast and effective nerve regeneration. In summary, a series of well-defined block copolymers were syn thesized successfully from Ac and TMC upon ring-opening copolymeri zation with PEG as the macroinitiator. The composition, structure and mechanical properties of the copolymers could be controlled precisely through changing the feed ratios of Ac (fAc from 20% to 50%) with the range of DP at 18–26 in order to keep the solubility in water. Also, the pendant double bonds were easily conjugated with CRGD to promote the neurite sprouting and axonal extension from the dorsal root ganglion (DRG) neurons. An increase in modulus is beneficial to axon density (5 kPa), while too high modulus (10 kPa) is unfavorable. These results approved that this novel poly(ether-carbonate) based hydrogel can be introduced as a suitable scaffold for fast and effective nerve regeneration.
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and Technology Planning Project of Guangdong Province, China (2015B010125001, 2015B020233012, 2014B020227001, 2017A050501017); the Guangzhou Science and Technology Program (201704020145, 201604030016, 201803010091, 201807070082); the China Postdoctoral Science Foundation (Grant No. 2017M622871); National Natural Science Foundation of China (No. 31670986). References [1] R. Vink, M.R. Bullock, Traumatic brain injury: therapeutic challenges and new directions, Neurotherapeutics 7 (2010) 1, https://doi.org/10.1016/j. nurt.2009.12.001. [2] M Jane Brennan, Bridget F. Kilbride, J. Jonathan, Julie C Liu Wilker, A bioinspired elastin-based protein for a cytocompatible underwater adhesive, Biomaterials 124 (2017) 116, https://doi.org/10.1016/j.biomaterials.2017.01.034. [3] J.L. Drury, D.J. Mooney, Hydrogels for tissue engineering: scaffold design variables and applications, Biomaterials 24 (2003) 4337, https://doi.org/10.1016/s01429612(03)00340-5. [4] D.J. Munoz-Pinto, S. Samavedi, B. 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Suuronen, Z. Shen, Z. Zhong, Injectable biodegradable hybrid hydrogels based on thiolated collagen and oligo(acryloyl carbonate)-poly(ethylene glycol)-oligo(acryloyl carbonate) copolymer for functional cardiac regeneration, Acta Biomater. 15 (2015) 55, https://doi.org/ 10.1016/j.actbio.2014.12.016. [13] Yongzhi Qiu, J Lim Jeremy, Larry Scott Jr., C Adams Ryan, T. Hieu, a Bui, S Temenoff Johnna, PEG-based hydrogels with tunable degradation characteristics to control delivery of marrow stromal cells for tendon overuse injuries, Acta Biomater. 7 (2011) 959, https://doi.org/10.1016/j.actbio.2010.11.002. [14] S.Q. Liu, Q. Tian, L. Wang, J.L. Hedrick, J.H. Hui, Y.Y. Yang, P.L. Ee, Injectable biodegradable poly(ethylene glycol)/RGD peptide Hybrid Hydrogels for in vitro chondrogenesis of human mesenchymal stem cells, Macromol. Rapid Commun. 31 (2010) 1148, https://doi.org/10.1002/marc.200900818. [15] Wei Chen, Huicui Yang, Rong Wang, Ru Cheng, Fenghua Meng, Wenxiang Wei, Zhiyuan Zhong, Versatile synthesis of functional biodegradable polymers by combining ring-opening polymerization and postpolymerization modification via michael-type Addition reaction, Macromolecules 43 (2010) 201, https://doi.org/ 10.1021/ma901897y. [16] G.L. Ellman, Arch. Biochem. Biophys. 82 (1959) 70. [17] F.Y. Qiu, C.C. Song, M. Zhang, F.S. Du, Z.C. Li, Oxidation-promoted degradation of aliphatic poly(carbonate)s via sequential 1,6-elimination and intramolecular cyclization, ACS Macro Lett. 4 (2015) 1220, https://doi.org/10.1021/ acsmacrolett.5b00533. [18] S. Venkataraman, N. Veronica, Z.X. Voo, J.L. Hedrick, Y.Y. Yang, 2-Amino-1,3propane diols: a versatile platform for the synthesis of aliphatic cyclic carbonate monomers, Polym. Chem. 4 (2013) 2945, https://doi.org/10.1039/C3PY00318C. [19] Xiaojin Zhang, Hujie Mei, Chu Hu, Zhenlin Zhong, Renxi Zhuo, Amphiphilic triblock copolycarbonates with poly(glycerol carbonate) as hydrophilic blocks, Macromolecules 42 (2009) 1010, https://doi.org/10.1021/ma802350g. [20] Yu Lin, Zheng Zhang, Jiandong Ding, Influence of LA and GA sequence in the PLGA block on the properties of thermogelling PLGA-PEG-PLGA block copolymers, Biomacromolecules 12 (2011) 1290, https://doi.org/10.1021/bm101572j. [21] S. Chen, Z. Du, J. Zou, S. Qiu, Z. Rao, S. Liu, X. Sun, Y. Xu, Q. Zhu, X. Liu, H. Q. Mao, Y. Bai, D. Quan, Promoting neurite growth and Schwann cell migration by the harnessing decellularized nerve matrix onto nanofibrous guidance, ACS Appl. Mater. Interfaces 11 (2019) 17167, https://doi.org/10.1021/acsami.9b01066.
Fig. 5. The influences of G0 of hydrogel on the DRG behaviors. The density of DRG in the three group hydrogel of R-PCE was 2.2 mg/mL, and the G0 of 5 wt% R-PCE2 (A, D), 4.3 wt% R-PCE4 (B, E) and 5.2 wt% R-PCE4 (C, F) was 0.2 kPa, 5 kPa and 10 kPa, respectively. After 7 days co-cultured, representative neurite outgrowth on the substrates was immunofluorescence stained and taken by confocal microscopy, the statistical results were shown as axon length (G), axon diameter (H), axon number (I) and the percentage of Schwann cells attached to the neurites was represented in (J). Neurons, Schwann cells and nuclei were stained against NF200 (green), S100 (red) and DAPI (blue) respectively. * in dicates significant statistical difference (n ¼ 3 wells, P ˂ 0.05, Mann–Whitney test). Scale bars ¼ 1000 μm in (A)-(C) and 200 μm in (D)-(F).
Author contributions section Tao Wang: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft. Yang Han: Methodology, Formal analysis, Investigation, Writing - original draft. Ying Bai: Validation, Investigation, Data curation, Writing - review & editing. Qingtang Zhu: Methodology, Resources. Daping Quan: Conceptualization, Resources, Writing - review & editing, Project administration, Funding acquisition. Xiaolin Liu: Conceptualization, Resources, Writing - review & editing, Project administration Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by National Key Research and Develop ment Plan of China (No. 2016YFC1101603, 2016YFC1100103); Science 8
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[22] C. Radtke, M. Spies, M. Sasaki, P.M. Vogt, J.D. Kocsis, Demyelinating diseases and potential repair strategies, Int. J. Dev. Neurosci. 25 (2007) 149, https://doi.org/ 10.1016/j.ijdevneu.2007.02.002. [23] G. Kowalski, K. Kijowska, M. Witczak, Kuterasi� nski Ł, M. Łukasiewicz, Synthesis and effect of structure on swelling properties of hydrogels based on high methylated pectin and acrylic polymers, Polymers (1) (2019 Jan 10) 11, https:// doi.org/10.3390/polym11010114. [24] G. Yilmaz, M.U. Kahveci, Y. Yagci, A one pot, one step method for the preparation of clickable hydrogels by photoinitiated polymerization, Macromol. Rapid Commun. 32 (2011) 1906, https://doi.org/10.1002/marc.201100470. [25] Tao Wang, Mingfa Yan, Xiumin Sun, Daping Quan, The mechanical and biological properties of polycarbonate-modified F127 hydrogels after incorporating active pendent double-bonds, Polymer 57 (2015) 21, https://doi.org/10.1016/j. polymer.2014.12.004.
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Polymer journal homepage: http://www.elsevier.com/locate/polymer
Poly(ether-carbonate) based hydrogel with tunable mechanical strength and enhanced bioactivity prepared by Michael addition Tao Wang a, b, c, Yang Han e, Ying Bai c, d, Qingtang Zhu a, c, Daping Quan b, c, d, *, Xiaolin Liu a, c, ** a
Department of Orthopedic and Microsurgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China PCFM Lab, GD HPPC Lab, School of Chemistry, Sun Yat-sen University, Guangzhou, China c Guangdong Provincial Peripheral Nerve Tissue-engineering and Technology Research Center, Guangdong Provincial Functional Biomaterials Engineering Technology Research Center, Guangdong Provincial Soft Tissue Biofabrication Engineering Laboratory, Guangzhou, China d School of Materials Science and Engineering, Sun Yat-sen University, Guangzhou, China e Department of Obstetrics, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China b
A R T I C L E I N F O
A B S T R A C T
Keywords: poly(ether-carbonate) hydrogel Michael addition Dorsal root ganglion
Poly (ether-ester) based hydrogels have been broadly used as degradable biomaterials in drug delivery systems and tissue engineering. However, they were synthesized with uncontrollable mechanical properties and inert to most of the biological activities, such as cell adhesion and stem cell differentiation. Herein, we synthesized a water-soluble copolymer with poly (trimethylene carbonate-co-2-methyl,2-methylacrylate-bimethylene carbon ate) (PTMAc) and poly(ethylene glycol)(PEG) blocks (PCE), in which the PTMAc segments provided free active double bonds and can be further conjugated with thiol-ended peptides to improve its cytocompatibility. Meanwhile, the extra double bonds were crosslinked by Michael addition, forming hydrogels with precisely controlled mechanical strength. The chemical structure and molecular weight of the synthesized PCE were characterized by 1H NMR and gel permeation chromatography (GPC). The composition of the PCEs was controlled by changing the feed ratios of 2-methyl,2-methylacrylate-bimethylene carbonate (Ac) comonomer (fAc). It was noted that when the ratio of comonomers to the macroinitiator PEG ([M]0/[I]0 ¼ 18) and the ratio of DL-Dithiothreitol (DTT) to Ac ([thiol]/[Ac] ¼ 1/1) were fixed, the storage moduli of PCE hydrogels increased from 2 kPa to 10 kPa and the gelation time decreased with increasing fAc. The pendant double bonds were easily conjugated with CRGD to promote the neurite sprouting and axonal extension from the dorsal root ganglion (DRG) neurons. Moreover, the weaker the hydrogel matrix (G0 ¼ 0.2 kPa) was, the lower the axon extension length was, but the axon diameter became thicker. An increase in modulus is beneficial to axon extension (5 kPa), while a high modulus (10 kPa) is unfavorable. These results demonstrated that this novel poly(ethercarbonate) based hydrogel can be introduced as a suitable scaffold for fast and effective nerve regeneration.
1. Introduction A property-controlled living microenvironment often leads to spe cific behaviors of their embedded cells, such as proliferation, migration and differentiation [1]. Therefore, well-designed cytocompatible mate rials have been broadly pursued in terms of their cell-laden capability and ,clinical applications [2]. Among them, the highly water containing hydrogels exhibited great potential in cell-based regenerative thera peutics, for example in minimally invasive surgeries, which could un dergo crosslinking either physically or chemically for tunable mechanical properties [3].
So far, it has been realized that most of the polyethlene glycol (PEG) or polyester-ether based thermo-sensitive hydrogels exhibit low me chanical strength and poor bioactivity [4,5]. Chemical crosslinking has been proven to be an effective way to improve their mechanical strength. It has been reported that acrylate/vinyl sulfone-functionalized linear or star PEG hydrogels by chemical crosslinking decreased the critical gelation concentration (CGC) and resulted in highly increased mechanical properties [4]. In addition, some short peptides were introduced into the chemically crosslinked PEG hydrogels, such as acrylic modified adhesive sequence (arginine-glycine-aspartate acid sequence, RGD) and matrix metalloproteinase sensitive sequence
* Corresponding author. School of Chemistry, Sun Yat-sen University, Guangzhou, China. ** Corresponding author. Department of Orthopedic and Microsurgery, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. E-mail addresses: [email protected] (D. Quan), [email protected] (X. Liu). https://doi.org/10.1016/j.polymer.2019.122115 Received 13 September 2019; Received in revised form 3 December 2019; Accepted 19 December 2019 Available online 21 December 2019 0032-3861/© 2019 Published by Elsevier Ltd.
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(MMP), to improve the biological activity and also to conjugate the biodegradability of hydrogels [5]. Furthermore, the crosslinking of the diacrylated block polyether-esters often leads to hydrogels with enhanced mechanical properties, such as diacrylated polycaprolactone-PEG-polycaprolactone (PCL-PEG-PCL-DA) [6], dia crylated polylactic acid-PEG-polylactic acid (PLA-PEG-PLA-DA) [7], diacrylated poly(lactic-co-glycolic acid)-PEG-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA-DA) [8], which may find application in drug release and regenerative medicine. However, some research studies also showed that the residual catalysts from the process of free radical addition retained their cytotoxicity and inhibitory for cell growth [9, 10]. Moreover, the acrylation process usually resulted in limited amount of free double bonds after crosslinking, which brought more difficulty for further biomimetic modifications. Chemical crosslinking by Michael addition reaction has been intro duced into the hydrogel systems to effectively bypass the toxicity of residual catalysis mentioned above [11]. An injectable bioactive hybrid hydrogel, based on thiolated collagen (Col-SH) and multiple acrylate-containing oligo (acryloyl carbonate)-b-poly (ethylene glycol)-oligo (acryloyl carbonate) (OAC-PEG-OAC) copolymers, was reported to form via Michael addition [12]. As compared to the func tional group-terminated PEG gels or polyether-ester block copolymers, copolymers with multiple functional groups on the side chain are highly prospective to reach the goal of crosslinking for enhanced mechanical strength and conjugating biomolecular activities into the hydrogels simultaneously [13,14]. In this work, we report a well-designed strategy for synthesizing hydrogels with adjustable biological and mechanical properties. 2methyl,2-methylacrylate-bimethylene carbonate (Ac), was copoly merized with trimethylene carbonate (TMC), using PEG as the macro initiator. The resulted copolymers are soluble in water by controlling the length of polycarbonate blocks. Consequentially, the chemical cross linked hydrogels were performed by mild Michael addition reaction with DL-Dithiothreitol (DTT) as the crosslinker. Moreover, the me chanical strength of the crosslinked hydrogel was tuned by manipulating the addition of DTT, the bioactivities of hydrogels was induced by modification of the relative abundant double bonds with thiol-ended RGD peptides along the copolymer side chains. Finally, the ability of modified hydrogels to support outgrowth of the axones was evaluated in a rat dorsal root ganglion (DRG) culture model.
sequentially quantitative anhydrous PEG, TMC and Ac were dissolved in dried dichloromethane (DCM) and then DBU was added in a vacuum glove box (O2% < 0.1 ppm, H2O% < 0.1 ppm) in 25 mL round bottom reaction flasks. After 10 h of agitation at 28 � C, the polymerization was terminated by two drops of acetic acid. The raw product was isolated via precipitation in cold diethyl ether at room temperature and then dried in vacuo at 40 � C to constant weight. Yield: 80.1%–83.5%. 2.3. Characterization All 1H NMR and 13C NMR spectra were recorded on a Mercury-plus VARIAN (300 MHz for 1H NMR and 75 MHz for 13C NMR) spectrometer using tetramethylsilane (TMS) as an internal reference and CDCl3 or deuterated dimethyl sulphoxide (DMSO‑d6) as the solvent. A GPC system equipped with a Waters 1525 separations module, Waters 2414 RI de tector and Waters Styragel 7.8 � 300 mm HR1, HR3 and HR4 were used to determine the molecular weight (Mn and Mw, respectively) and polydispersity of the copolymers. Tetrahydrofuran (THF) was used as the eluent at a flow rate of 1.0 mL/min at 40 � C and the MWs were calibrated using polystyrene standards (MW range from 1 kDa to 240 kDa). 2.4. Gelation, mechanical and degradation properties The quantitative copolymer (50 mg) was dissolved in 0.95 mL phosphate buffered saline (PBS, pH 7.4, 0.1 M) at a concentration of 5 wt % as stock solution and then quantitative DTT ([thiol]/[Ac] ¼ 1/1) was added and incubated at 37 � C after thoroughly mixed. 1H NMR of resulting polymer was measured after dialysis and freeze drying with DMSO‑d6 as the solvent. The gelation time and viscoelasticity of the hydrogels were determined with an advanced rheology expanded sys tems (ARES/RFS, TA, USA) with a parallel plate fixture (diameter, 25 mm; gap, 1 mm) at 37 � C adjusted with a bath temperature controller (Neslab). Polymer solution was positioned in the parallel plate imme diately after DTT mixed and gradually squeezed by the top-plate. A plastic cover was used to minimize solvent evaporation. The evolution of storage modulus (G0 ) and loss modulus (G00 ) of hydrogel was recorded as a function of time. The test was set at a frequency of 10 Hz and a strain of 1% which was confirmed at the linear viscoelastic region. The gelation time was defined as the time point where G0 ¼ G00 and tested in triplicate. To determine the amount of thiol groups (the residual thiols of hydrogel system at different time during gelation) Ellman’s test had been used according to its standard procedure [16]. Briefly, Ellman’s reagent, Tris solution, and sample were mixed together in appropriate amounts and its absorbance was measured at 412 nm using UV–Vis spectrophotometer (Cary100, Varian Company). The prepared hydrogels (1 mL) were agitated to swell overnight in 5 mL PBS at 37 � C. The wet weights (Ws) of the gels at equilibrium swelling state were recorded after blotted dry with weight paper. These gels were then lyophilized for 24 h and the dry masses (Wd) were recorded. The equilibrium swelling ratio (ESR) was determined by the equation (Ws Wd)/Wd � 100%. For measurement of degradation behavior, the hydrogel sample was prepared and immersed in 1 mL distilled water after equilibrium swelling and then placed under agitation at 37 � C. The extracted solution was collected and replaced with pristine distilled water at each time increment. The collected solution was freeze-dried to measure the weight loss. Consequentially, the residual mass was calculated by sub tracting the total weight loss from the original weight of the sample. Samples were allowed to swell for at least 24 h in treatment media. For measuring of degradation via weight change, the patches were weighed at days 1, 3, 7, 10 and 14 after formation, and degradation was determined as residual weight compared to original weight at day 1.
2. Materials and methods 2.1. Materials Poly (ethylene glycol) (PEG, Mn ¼ 10 kDa) was purchased from Aldrich (Milwaukee, USA) and was dehydrated under vacuum at 120 � C for 4 h prior to use. Trimethylene carbonate (TMC) was donated from Huayang medical device company (Huizhou, China) and recrystallized in ethyl acetate prior to use. Acryloyl cyclic carbonate (Ac) was syn thesized as reported in the literature (1H NMR (300 MHZ, chloroform-d, CDCl3, δ): 1.14 (s, 3H; CH3), 4.17 (d, 2H; CH2), 4.19 (s, 2H; CH2), 4.33 – CH2)) (d, 2H; CH2), 5.91–6.45 (m, 3H; –CH– [15]. Cysteine-arginine-glycine-aspartate peptide (CRGD) was purchased from GL Biochem (Shanghai) Co., Ltd. 1,8-Diazabicyclo[5.4.0]unde c-7-ene (DBU, 99%), dithiothreitol (DTT, 99%) were purchased from J&K scientific (Shanghai, China) and used without further purification. Other chemical solvents were purchased from Guangzhou Chemical Reagent Factory (Guangzhou, China), dried using standard procedures and distilled before use. 2.2. Synthesis of PTMAc-PEG-PTMAc copolymer The polymers were synthesized via ring-opening polymerization (ROP) of TMC and Ac using PEG as an initiator and DBU as the catalyst. A typical experimental procedure is described as the following: 2
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2.5. Polymer modification with CRGD peptide
glutamine and 1% penicillin–streptomycin in a 37 � C incubator with 5% CO2 and 92% humidity. The medium was changed every 2 days.
Michael addition reaction was used to prepare the CRGD function alized PTMAc-PEG-PTMAc copolymer (abbreviated R-PCE). Typically, PTMAc-PEG-PTMAc copolymer (100 mg) was dissolved in 1.9 mL PBS at 37 � C overnight to prepare 5 wt% polymer solution and then quantita tive of CRGD ([CRGD]/[Ac] ¼ 3/10) were added and allowed to react for 2 h at 37 � C. The CRGD modified polymer was obtained via lyoph ilization after dialysis (MWCO 5000) in PBS at room temperature and then dried in vacuo at 40 � C to constant weight. Yield: 94.5%. The ki netics and efficiency of this Michael addition reaction were monitored by 1H NMR.
2.8. Immunofluorescence staining of DRG Cells were fixed by 4% paraformaldehyde in PBS for 20 min, rinsed by PBS, permeabilized and blocked with 0.1% Triton X-100 and 10% donkey serum in PBS for 30 min. Cells were incubated with primary antibodies against NF200 (dilution 1/150) and S100 (dilution 1/1000) with DRG culture samples and NF200 (dilution 1/150) and MBP (dilu tion 1/500) with neuron-Schwann cell co-culture samples for 2 h at room temperature, followed by secondary antibodies conjugated to Fluro 488 (dilution 1/1000) and Fluro 594 (dilution 1/1000) incubation for 1 h. The cells were then rinsed with PBS for 10 min (3 times) and stained with DAPI (dilution 1/2500) for 20 min. The fluorescence was observed and imaged under the laser scanning confocal microscope. The parameters and cells were measured by Image J® for at least 3 samples.
2.6. Characterization by scanning electron microscopy (SEM) All the samples were attached on conducting resin and coated with platinum. The micromorphology was observed and imaged by Hitachi S4800 (Japan) at 15 kv. The diameters of nanofibers were measured by image analysis software (Image J®).
2.9. Statistical analysis Data are presented as the mean � standard deviation (SD). Differ ences between groups were evaluated by one-way analysis of variance (ANOVO) followed by least significant difference posthoc tests. A pvalue < 0.05 indicates statistically significant difference.
2.7. DRG culturing The hydrogel membrane samples for DRG culture were immersed in 75% ethanol with 30 min for sterilization and then washed by PBS for 5 times (5 min for each time) and kept wet before cell culturing. The DRGs were isolated from newborn Sprague–Dawley rats (sup plied by Laboratory Animal Center of Sun Yat-sen University, China). The residual nerve roots were cut off under a stereomicroscope. Then DRGs were plated on hydrogel membrane/glass slides, which were put in a 48-well plate with neurobasal medium containing 2% B27, 0.3% L-
3. Result 3.1. Synthesized and characterization A series of PTMAc-PEG-PTMAc copolymers (PCE) with short
Scheme 1. (A) synthesis of the PCE copolymers. (B) crosslinking with DTT and bioactive post-modification with CRGD by Michael addition reaction. (C) the schematic diagram of PCE hydrogel with tunable mechanical strength and enhanced bioactivity prepared by Michael addition. 3
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polycarbonate segments were synthesized by ROP of Ac and TMC using PEG as macro-initiator and DBU as catalyst in DCM at 28 � C shown as Scheme 1. The corresponding GPC curves (Fig. 1 A and B) were unim odal, but the PEG efflux peak shifted into short elution time after polymerization reaction, and the elution time decreased gradually with the increase of carbonate monomer contents. As shown in Table 1, the block copolymers were water-soluble if their degrees of polymerization (DPs) were limited to 18–26 and the contents of Ac monomer (fAc) were changed from 20% to 50%. The conversions of TMC and Ac comonomers were more than 90% within 10 h of polymerization at 28 � C, and the measured contents of Ac (FAc, measured by 1H NMR) were very close to the feed ratios of the Ac monomers, indicating a good ROP controllability. The chemical structure of PCE was confirmed by 1H NMR and 13C NMR analyses. It was shown that the resonance absorption at 3.66 ppm (e) should be attributed to methylene in PEG, and the resonance ab sorption at 2.10 ppm (f) and 4.25 ppm (c) were assigned to PTMC segment. For the PAc segment, in addition to the methyl protons at 1.00
ppm (g) and the methylene protons at 4.10 ppm (d), a typical double bond resonance absorption was found in the range of 5.80–6.50 ppm (a, b), indicating the successful synthesis of copolymers containing double bonds in the side chain. DBU has been proven to be an efficient orga nocatalyst for ROP of cyclic carbonates and their derivatives [17,18]. The structure of the resulting copolymer and blocky regions of PTMC and PAc in the P(TMC-Ac) regions were further characterized by 13C NMR spectrum (Fig. 1 D). Three kinds of carbonyl groups were identified in the copolymer, including the pendant carbonyl of acrylate (165 ppm) and the carbonyls for TMC and Ac units (154–155 ppm). Upon enlarging the carbonyl region of h (inlet of Fig. 1 D), three resonance signals were observed (154.75, 154.85 and 155.00 ppm) and were assigned to the diad sequences of TMC unites (T) and Ac unites (A), including TT, AT, and AA, representing the regions of blocky PTMC, random P(TMC-Ac) and blocky PAc, respectively [19]. 3.2. Gelation via controllable Michael addition It was found that the aqueous solutions of PCE copolymers shown in Table 1 did not have the thermosensitive properties of sol-gel phase transition, which may be related to the weak interaction of hydrophobic chains of block copolymer [20]. Upon adding DTT, the aqueous solutions of PCE copolymers gradu ally solidified to form transparent gels via Michael addition reactions between the reactive double bonds in the Ac units and the thiol groups in the crosslinker of DTT. The dynamic rheological measurement was used to test the gelation transition of copolymer solution at 37 � C, as shown in Fig. 2 A, the G0 and G00 of PCE2 were equal after 6.5 min, suggesting the gelation transition time was about 6.5 min for PCE2. With extension of the time, the G0 of PCE2 solution increased until 30–35 min and reached a platform value of 2.2 kPa, but the G00 had little change over time. 1 H NMR measurement was used to monitor the reaction process of Michael addition (Fig. 2 B). The new resonances attributed to the methine group in the crosslinker DTT were first appeared at 5.2 ppm after 5 min of reaction, and the peak became spikier as the reaction proceeded (t ¼ 5, 15, 30, 45 and 120 min). Meanwhile, resonances of the acrylate protons (within the Ac units) in the range of 5.8–6.6 ppm gradually decreased during the Michael addition reaction and almost disappeared after 2 h. The amount of residual thiol groups during the reaction process was also monitored by Ellman’s test (Fig. 2C) [16]. It was found that only 14% of thiol was detected (compared with original thiol) at about 30 min and continued to decline to 6% after an hour reaction between PCE2 and DTT ([thiol]/[Ac] ¼ 1/1). As the Michael addition reaction went on, the content of thiol group in the system kept decreasing until reaching the plateau state, which was consistent with the increment of G0 (Fig. 2 A). Different from free radical polymerization, Michael addition reaction can control the degree of crosslinking by controlling the content of crosslinking agent, thus regulating the properties of hydrogels. It was showed in Fig. 2 D, when the mount of DTT was changed from insuffi cient to excess, the G0 of hydrogel increased first (from 0.5 kPa to 5 kPa) and then decreased obviously, indicating that the cross-linking reaction was precisely controlled by feed ratios. Accordingly, the ESR of hydro gels displayed contrary tendencies contrasted with G0 . 3.3. The influence of composition and concentration of copolymers on the properties of hydrogels As shown in Fig. 3 A, when the ratio of monomer to initiator ([M]0/ [I]0 ¼ 18) and the ratio of DTT to Ac ([thiol]/[Ac] ¼ 1/1) were fixed, by increasing fAc from 20% to 50%, the corresponding G0 increased by 5 times (from 2 kPa to 10 kPa), the ESR decreased and the gelation transition time get shorter (from 9 to 2.5 min), which was attributed to the increase of Ac contents and the crosslinking density in PCEs. When the molecular weight of PCE copolymers were increased, the G0 of
Fig. 1. GPC and NMR characterization of the synthesized PCE polymers. GPC spectra of PEG and the synthesized PCE copolymers with different mole ratios (A) and DP (B) in THF at 40 � C. 1H NMR (C, CDCl3) and 13C NMR (D, DMSO‑d6) spectra of PCE2 at 26 � C. 4
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Table 1 PTMAc-PEG-PTMAc copolymers synthesized by ROP. Samplea
[M]0/[I]0b , DP
fAcc (%)
PCE1 PCE2 PCE3 PCE4 PCE5 PCE6
18:1 18:1 18:1 18:1 22:1 26:1
20 30 40 50 30 30
Conv.(%)d TMC
Ac
94 94 91 92 95 96
100 100 100 100 100 100
FAcd,e (%)
Mn, theoryf (kg/mol)
Mn, NMRg (kg/mol)
Mn, GPCh (kg/mol)
Mw/Mhn
19 30 39 47 30 33
12.2 12.4 12.5 12.7 12.9 13.4
12.1 12.4 12.5 12.6 12.9 13.5
22.0 22.2 22.3 22.8 23.1 23.9
1.20 1.21 1.23 1.23 1.20 1.19
a
All polymerizations were performed in DCM at 28 � C for 10 h, using PEG as the macroinitiator and DBU as the catalyst. PCE1 ~ PCE6 polymers were named sequentially in Table 1, varied by their DPs and Ac mole fractions, respectively. b Degree of polymerization in feed. c Molar fraction of Ac in feed (nAc/(nTMC þ nAc)). d Measured by 1H NMR spectroscopy. e Molar fraction of Ac in the isolated polymer. f Calculated with theory feed (10000 þ 198 � DP � fAcþ102 � DP � (1-fAc)). g Calculated by 1H NMR spectroscopy (10000 þ 198 � DP � FAcþ102 � DP � (1-FAc)). h Determined by GPC analysis in THF using polystyrene as the standard.
Fig. 2. The gelation and controllable Michael addi tion reaction. (A) The gel point was defined as the time point where the G0 and G00 crossed and obtained by dynamic rheological measurement (10.0 rad/s frequency, 1% strain at 37 � C). The photo of PCE2 was taken at 6.5 min. (B) The 1H NMR (DMSO‑d6) spectra of PCE2 (0.5 wt%) and DTT ([thiol]/[Ac] ¼ 1/1) re action system at different reaction time. (C) The re sidual thiols of PCE2 (0.5 wt%)/DTT ([thiol]/[Ac] ¼ 1/1) determined by Ellman’test in PBS at 37 � C at different time. (D) The G0 and ESR of PCE2 hydrogel (5 wt%) with different [thiol]/[Ac] (after reaction for 30 min) in PBS at 37 � C determined by rheology.
hydrogel also increased gradually, ESR and gelation transition time decreased. Considering the water solubility of copolymer, while the change ranges of molecular weight were limited. The concentration of water-soluble PCE2 affected the properties of hydrogels (Fig. 3 B). It was found that when the concentration of PCE2 aqueous solution was 8%, the corresponding G0 was 32 kPa. By contrast, hydrogels made from 4% of PCE solution had a G0 of only 0.21 kPa, however, 5 kPa of G0 was obtained for 5% group. ESR and gelation transition time also changed correspondingly. In vitro degradation behaviors of the PCE2 hydrogels at different concentrations (4 wt%, 5 wt% and 6 wt%) were tested (Fig. 3 D). The hydrogel at 4 wt% concentration exhibited the fastest degradation and the residual mass was only 43% � 12% after 35 days. The stability of the PCE2 hydrogel increased dramatically with small increment of polymer
concentration. The residual mass of 6 wt% hydrogel was 96% at day 35 with relatively intact shape. The morphologies of the lyophilized hydrogels were observed by SEM (Fig. 3D). With the increase of fAc and DP, the average pore diameter of hydrogel decreased from several hundred microns to several tens of microns. Considering the mechanical strength and porosity of hydrogel, 5% PCE2 was selected for in vitro cell culture. 3.4. Modification PCE with CRGD CRGD as fibronectin sequences was often used to modify the different materials and to improve their biocompatibility. Here we incorporated CRGD into the PCE copolymer by Michael addition re actions between the reactive double bonds in the Ac units and the thiol 5
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groups in the CRGD, the reaction was processed at 37 � C for 2 h. In Fig. 4 A, the new resonances attributed to CRGD peptide sequences were observed in the range of 8.0–9.0 ppm, 2.6–2.7 ppm and 1.4–1.6 ppm. The resonance absorption of acrylate protons in the range of 5.8–6.6 ppm almost disappeared contrast with spectra of PCE2 shown as Fig. 1C, which was consistent with the molar feed ratio ([CRGD]/[Ac] ¼ 1/1), indicating the CRGD modification can be precisely controlled in a quantitative manner. By adjusting the ratio of [CRGD]/[Ac] (1/10, 3/10), two kinds of hydrogels with different CRGD density were obtained and labeled as R10-PCE2 (5 wt%) and R30-PCE2 (5 wt%). The influences of CRGD density on the axon length and axon number of DRG were evaluated after 7 days’ co-culture (Fig. 4 B and C). It was showed that on the R30PCE2 hydrogel surface, the axon length come from DRG neuron was about 814 � 62 μm and the axon number was 786 � 156, while the length and number of axon of R10-PCE2 were 314 � 87 μm and 623 � 120 as control, the pure PCE2 hydrogel were 121 � 35 μm and 425 � 154, respectively. The results suggested that CRGD-modified hydrogel could significantly promote the sprouting and extension of the axons of the DRG, and the impact can be easily controlled by changing the den sity of short peptide sequence. 3.5. The responses of DRG on the tunable modulus of hydrogel In addition to control the density of CRGD, the modulus of hydrogel can also be regulated by changing the degree of crosslinking, thus affecting the behavior and function of cells. We selected three group of copolymer 5 wt% R-PCE2, 4.3 wt% R-PCE4 and 5.2 wt% R-PCE4, which have the same RGD density (2.2 mg/ml) but different G0 (0.2 kPa, 5 kPa and 10 kPa), and co-cultured embryonic DRG with this three hydrogels in vitro to investigate the effect of hydrogels’ moduli on the growth of DRG neurons. It was noted that the neurite outgrowth from DRG was relatively sensitive to the variation in G0 of hydrogel matrix (Fig. 5). On the hydrogel surface with a G0 of 0.2 kPa (Fig. 5 A and D), the axon length of DRG was lower than that of two higher G0 groups (Fig. 5 B E and C F), but the axon diameter was significantly larger than that of the other two groups (Fig. 5H). This phenomenon might be due to the stronger intercellular interactions between the neighboring neurites in response to the relatively soft matrix [21]. As compared with the 10 kPa group, the 5 kPa group had more nerve axons (Fig. 5 I), indicating too soft or too hard substrates is not conducive to neurite outgrowth. It was also observed that the Schwann cells (S100-positive) wrapped around the extending neurites (Fig. 5 D, E, and F), but the percentage of Schwann cells associated with the regenerated axons was almost the same and close to 100% between the cultured hydrogels with different moduli (Fig. 5 J), implying that the R-PCE hydrogels were beneficial for promoting axonal remyelination toward nerve fiber functionalization [22]. 4. Discussion and conclusion At present, most of the chemically crosslinked hydrogels based on poly(ether-ester) have been synthesized by free radical polymerization [23,24]. There were a few drawbacks retained from the synthesis pro cedure for biomedical applications, such as the residual toxicity of the initiators, the relatively fast polymerization usually leads to operational difficulty during in situ injection. Additionally, the free radical poly merization is often initiated by UV light, which also resulted in incon venience application in vivo. Michael addition undergo mild reaction conditions with no initiator required. For this reported PCE copolymers, the gelation time can be controlled in the range of a few minutes to more than ten minutes (Fig. 2), the injected solution could be further manipulated in situ before the sol-gel transition. More importantly, the degree of crosslinking is easily controlled by tuning the ratio of the reactive groups. In the PCE polymer, free double bonds were introduced
Fig. 3. The mechanical, degradation and morphological properties of the PCE hydrogel. The effect of composition (A, concentration of PCE hydrogels were 5%) and concentration (B, PCE2) of hydrogels on the G0 tested, ESR and gela tion time were evaluated. (C) Degradation behavior of the hydrogels with different concentrations. (D) The microstructures of lyophilized hydrogels were observed by SEM. The dynamic rheological measurements were under 10.0 rad/s frequency, 1% strain at 37 � C. [thiol]/[Ac] ¼ 1:1 was controlled at each group.
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Fig. 4. The modification of PCE2 with CRGD and co-culture with DRG. 1H NMR (DMSO‑d6) spectra of PCE2 modified with CRGD (A). 7 days after planted, DRGs were immunofluorescence stained (B), axon length and axon number were statistic (C). Nuclei were stained by DAPI (blue) and axon were stained by NF200 (green). Scale bar: 250 μm * indicates significant statistical difference (n ¼ 3 wells, P ˂ 0.05, Mann-Whitney test).
into the side chains of the polycarbonate segments, which can not only be employed for crosslinking and mechanical strength enhancement, but also can undergo reactions with multiple biological macromolecules for functional modifications, such as short peptide sequences [25], proteins [26], collagen [27], hyaluronic acid [28], etc. To ensure the water-solubility and crosslink ability of the PCE polymer, the oligo-carbonate chain segments were introduced into the backbone, meanwhile, the pendant double bonds on the side chains were employed for gelation. Generally, typical amphiphilic poly(etheresters) first formed “core-shell” structured micelles in aqueous solu tion due to the aggregation of hydrophobic chain segments. With increasing the temperature and/or the polymer concentration, the mi celles started to aggregate, resulting in sol-gel transformation at the macroscopic level [20]. If the double bonds were only incorporated as the end groups, though the chemical crosslinking was still triggered for gel formation, the number of reactive groups was highly restricted, and some of which might be shielded as the inner layer of the micelles. Another advantage of the Michael addition crosslinked PCE hydro gels is their controllable mechanical properties, which can be regulated by tuning the composition of the comonomers, molecular weight of the polymer and the concentration of solution. With increasing the content of comonomer Ac, the crosslinking density and the mechanical strength of the PCE copolymer increased significantly (as shown in Fig. 3, [thiol]/ [Ac] ¼ 1/1). However, when thiol-containing molecules, i.e. the cross linker DTT, was excessive, the G0 of PCE polymer was again decreased
due to the competitive thiol-ene reaction, leaving large amount of re sidual thiol groups in the hydrogel matrix. Furthermore, we have demonstrated that the chemically crosslinked PCE hydrogels can be modified into functional biomaterials to meet the individual requirements in clinical applications. As an example, we have evaluated the effect of axonal outgrowth by culturing the DRG segments on the PCE hydrogels. It was realized that the nerve cell behaviors were highly enhanced by controlling the biological and mechanical properties of hydrogel matrices towards fast and effective nerve regeneration. In summary, a series of well-defined block copolymers were syn thesized successfully from Ac and TMC upon ring-opening copolymeri zation with PEG as the macroinitiator. The composition, structure and mechanical properties of the copolymers could be controlled precisely through changing the feed ratios of Ac (fAc from 20% to 50%) with the range of DP at 18–26 in order to keep the solubility in water. Also, the pendant double bonds were easily conjugated with CRGD to promote the neurite sprouting and axonal extension from the dorsal root ganglion (DRG) neurons. An increase in modulus is beneficial to axon density (5 kPa), while too high modulus (10 kPa) is unfavorable. These results approved that this novel poly(ether-carbonate) based hydrogel can be introduced as a suitable scaffold for fast and effective nerve regeneration.
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and Technology Planning Project of Guangdong Province, China (2015B010125001, 2015B020233012, 2014B020227001, 2017A050501017); the Guangzhou Science and Technology Program (201704020145, 201604030016, 201803010091, 201807070082); the China Postdoctoral Science Foundation (Grant No. 2017M622871); National Natural Science Foundation of China (No. 31670986). References [1] R. Vink, M.R. Bullock, Traumatic brain injury: therapeutic challenges and new directions, Neurotherapeutics 7 (2010) 1, https://doi.org/10.1016/j. nurt.2009.12.001. [2] M Jane Brennan, Bridget F. Kilbride, J. Jonathan, Julie C Liu Wilker, A bioinspired elastin-based protein for a cytocompatible underwater adhesive, Biomaterials 124 (2017) 116, https://doi.org/10.1016/j.biomaterials.2017.01.034. [3] J.L. Drury, D.J. Mooney, Hydrogels for tissue engineering: scaffold design variables and applications, Biomaterials 24 (2003) 4337, https://doi.org/10.1016/s01429612(03)00340-5. [4] D.J. Munoz-Pinto, S. Samavedi, B. 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Fig. 5. The influences of G0 of hydrogel on the DRG behaviors. The density of DRG in the three group hydrogel of R-PCE was 2.2 mg/mL, and the G0 of 5 wt% R-PCE2 (A, D), 4.3 wt% R-PCE4 (B, E) and 5.2 wt% R-PCE4 (C, F) was 0.2 kPa, 5 kPa and 10 kPa, respectively. After 7 days co-cultured, representative neurite outgrowth on the substrates was immunofluorescence stained and taken by confocal microscopy, the statistical results were shown as axon length (G), axon diameter (H), axon number (I) and the percentage of Schwann cells attached to the neurites was represented in (J). Neurons, Schwann cells and nuclei were stained against NF200 (green), S100 (red) and DAPI (blue) respectively. * in dicates significant statistical difference (n ¼ 3 wells, P ˂ 0.05, Mann–Whitney test). Scale bars ¼ 1000 μm in (A)-(C) and 200 μm in (D)-(F).
Author contributions section Tao Wang: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft. Yang Han: Methodology, Formal analysis, Investigation, Writing - original draft. Ying Bai: Validation, Investigation, Data curation, Writing - review & editing. Qingtang Zhu: Methodology, Resources. Daping Quan: Conceptualization, Resources, Writing - review & editing, Project administration, Funding acquisition. Xiaolin Liu: Conceptualization, Resources, Writing - review & editing, Project administration Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was supported by National Key Research and Develop ment Plan of China (No. 2016YFC1101603, 2016YFC1100103); Science 8
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