Injectable and Degradable pH-Responsive Hydrogels via

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Injectable and Degradable pH-Responsive Hydrogel via Spontaneous Amino-Yne Click Reaction Jiachang Huang, and Xulin Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b18141 • Publication Date (Web): 13 Dec 2017 Downloaded from http://pubs.acs.org on December 13, 2017

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ACS Applied Materials & Interfaces

Injectable and Degradable pH-Responsive Hydrogel via Spontaneous Amino-Yne Click Reaction Jiachang Huang, Xulin Jiang* Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Wuhan 430072, P. R. China

*Corresponding author: Xulin Jiang Key Laboratory of Biomedical Polymers of Ministry of Education & Department of Chemistry, Wuhan University, Luojia Hill, Wuhan 430072, P. R. China. Tel.:+86-27-68755200; Fax: +86-27-68754509. E-mail address: [email protected].

Jiachang Huang: [email protected]

ACS Paragon Plus Environment

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KEYWORDS: biodegradable; injectable hydrogel; carboxymethyl chitosan; pH-responsive; polyethylene glycol; tissue engineering

ABSTRACT

Injectable hydrogels have attracted increasing attentions in tissue regeneration and local drug delivery applications. Current click reactions for preparing injectable hydrogel often require a photoinitiator or catalyst, which may be toxic and may involve complex synthesis of precursors. Here we report a facile and inexpensive method to prepare injectable and degradable hydrogels via spontaneous amino-yne click reaction without using any initiator or catalyst under physiological conditions based on telechelic electron-deficient dipropiolate ester of polyethylene glycol and water-soluble commercially available carboxymethyl chitosan. The gelation time, mechanical property and degradation rate of the hydrogels could be adjusted by varying carboxymethyl chitosan concentrations and stoichiometric ratios. The reversible pH-induced solgel transitions of the hydrogel are presented and the pH-controlled drug release behaviors are demonstrated, of which the mechanism is discussed. In vitro cytoxicity assays and in vivo in situ injection study of the carboxymethyl chitosan based hydrogels showed favorable gel formation, non-toxicity and good tissue biocompatibility. Therefore, this biodegradable and injectable hydrogels prepared by spontaneous amino-yne click reaction hold potential for tissue engineering and other biomedical applications.


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INTRODUCTION Novel crosslinking methodology is highly desired for preparation of in situ formed injectable hydrogels, which has received significant interest in tissue engineering and biomedical fields.1-5 Various coupling processes, including physical crosslinking and covalent chemical crosslinking have been employed to construct hydrogels.6-8 For instance, the noncovalent physical crosslinked hydrogels are sensitive to the environment conditions (e.g., pH, temperature, ionic strength). These hydrogels can be prepared

conveniently through sol-gel transitions for the in situ

encapsulated drugs or cells.9,10 In comparison to physically crosslinked hydrogels, chemically crosslinked hydrogels provide better mechanical strength and long-term stability in vivo.11 However, most of traditional chemical crosslinkings require longer reaction time, harsh reaction conditions, and the potential toxicity of chemically reactive substances, which limits their potential applications in biological fields.12,13 It is desirable to develop nontoxic, fast, and specific chemical reactions for preparation of injectable hydrogel under physiological conditions. Recently, click reaction has been intensively demonstrated its potential in crosslinking hydrogel networks due to their significant features, such as high reaction speed, high selectivity, and mild reaction conditions.14-16 Several click reactions have been greatly expanded in the drug delivery and tissue engineering communities including azide−alkyne cycloadditions,17 thiolene/yne conjugation reactions18-22, Diels-Alder and inverse electron demand Diels−Alder cycloadditions,23-25 strain promoted azide−alkyne cycloadditions,26 tetrazine-norbornene reaction,27 and so on. Nevertheless, some of these click reactions require transition-metal catalysts, UV light irradiation, or complex of precursor synthetic approach, which complicates the experimental operation and restricts its applications. Additionally, the Cu(I)-free Huisgen cycloaddition reaction has received growing attention by employing electron-deficient dialkyne


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crosslinkers to react with multivalent azide-functionalized polymers under physiological conditions to generate a hydrogel network.28,29 So far, there are few studies focusing on the use of activated alkynes as reactants for the preparation of hydrogel. More recently, a spontaneous amino-yne click polymerization at room temperature has been reported using ester-activated dialkynes and aliphatic secondary diamines in the absence of a metal catalyst.30 Chitosan has been widely studied as a renewable resource. Carboxymethyl chitosan (CMC), one important chitosan derivatives, has great potential in biomedical fields due to its excellent water solubility, nontoxicity, biocompatibility, and biodegradability.31 Thanks to the amino groups on the CMC backbone, CMC-based hydrogels prepared in most case through covalent linkages to increase their mechanical strength, have gained significant attentions, for instance, a gel network created by Schiff base formations.32 However, some of these hydrogels showing a very short gelation time (within seconds) have limited applications as injectable hydrogels.33-35 Thus, to design an ideal crosslinking system that can produce an injectable hydrogel with tunable gelation time, mechanical properties and degradation rate is highly desirable. Here, we have demonstrated the applicability of spontaneous amino-yne click reaction to synthesize an in situ crosslinking injectable hydrogel for the first time. As shown in Scheme 1, this hydrogel was prepared without using any catalyst or initiator under physiological conditions through amino-yne click reaction between amino groups of water-soluble CMC and alkyne groups of dipropiolate ester of polyethylene glycol (DA-PEG). The effects of alkynyl/amino feed molar ratios and the concentrations of the polymer precursors on the gelation time, mechanical properties and degradation rate, of the obtained hydrogels were investigated. The pH-induced sol-gel transitions of the hydrogel by the addition of acid or alkali were presented and the pH-


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controlled drug release behaviors were evaluated. Finally, the biocompatibility of these hydrogels was studied preliminarily in vitro and in vivo. Scheme 1. Illustrative Synthesis of DA-PEG/CMC Hydrogel by Crosslinking CMC with DA-PEG


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EXPERIMENTAL SECTION Materials. Carboxymethyl chitosan (CMC) powder was obtained from Zhejiang Aoxing Biotechnology (Zhejiang, China). The degree of acetylation and the degree of carboxymethyl substitution were determined by 1H NMR to be 0.04 and 0.96 (Figure S1 in the Supporting Information), respectively.36 The weight-average molecular weight (Mw) of CMC was acquired by size-exclusion chromatography (SEC) in conjunction with multiangle laser light scattering detector to be 74 kDa. Poly(ethylene glycol) (PEG, Mw = 2 kDa) and propiolic acid were obtained from Aladdin Chemistry (Shanghai, China). Doxorubicin hydrochloride (DOX·HCl), ptoluenesulfonic acid monohydrate (p-TSA), chloroform-d (CDCl3) were purchased from SigmaAldrich (Shanghai, China). Biological reagent Lysozyme (≥ 20,000 U/g) was obtained from Sinopharm Chemical. (Beijing, China). All other chemicals were analytical grade and were used as received without further purification. Synthesis of Dipropiolate PEG (DA-PEG). PEG (10.0 g, 5 mmol), propiolic acid (3.5 g, 50 mmol) and p-TSA (0.57 g, 3 mmol) were dissolved in dry toluene (150 mL). The mixture solution was stirred and refluxed for 48 h with constant removal of yielded water. Then the solution was concentrated under vacuum with removal of most of the solvent, precipitated with diethyl ether and recrystallized from isopropanol. The resulting crystals were collected and dried in vacuum. Eventually, 9.33 g of dipropiolate ester of PEG (DA-PEG) was obtained in 88% yield. In situ Hydrogel Formation. Before hydrogel preparation, the CMC (3 wt %) and DA-PEG (20 wt %) solutions were obtained by dissolving certain amounts of polymer in PBS, respectively. For a hydrogel with the molar ratio of [alkyne] : [amino] = 0.42, 0.205 g DA-PEG solution was added into a vial containing 1.0 g of CMC solution and 0.29 g PBS, in which the


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final CMC concentration in the mixture solution was set at around 2.0 wt %. Then the precursor solution was mixed homogenously, and incubated at 37 oC for 24 h. The obtained hydrogel was coded as 0.42-2%. More detailed information about the feed ratios of other hydrogels was summarized in Table 1. Structure Characterization of DA-PEG and Hydrogel. The 1H NMR spectrum of DA-PEG was measured on a Bruker Avance 500 spectrometer in deuterated chloroform. Fourier transform infrared (FT-IR) spectra of PEG, DA-PEG, CMC, and a series of freeze-dried DA-PEG/CMC xerogels were determined using Perkin-Elmer Spectrum One FT-IR spectrometer (KBr disk). Rheological Analysis. The rheological test of DA-PEG/CMC hydrogels was carried out using a stress-controlled rheometer as described in our previous works.9,12 A 40 mm parallel-plate geometry attached to a transducer and a gap of 0.5 mm were applied to all assays. The precursor solutions of hydrogels were prepared as described above, and then immediately pipetted onto the plate, which was incubated at 4 oC. Then the plate was heated up to the desired temperature. Compressive Modulus of Hydrogel. The compressive property of DA-PEG/CMC hydrogels was determined as reported previously.12 The precursor solutions of hydrogels were separate poured into cylinder molds (8 mm diameter × 7 mm height) for 24 h incubation at 37 oC to prepare cylindrical hydrogels. The compressive modulus was obtained from the slope of the stress-strain curve in the initial linear portion between 10 and 20% strain. Hydrogel Morphology. The microstructure of the representative DA-PEG/CMC hydrogel was observed as described in our previous work9 through scanning electron microscopy (SEM). The as-prepared hydrogel was snap frozen in liquid nitrogen and was subsequently lyophilized to get the dried sample coated with gold for SEM.


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pH-Responsive Ability of Hydrogel. The 0.28-2.5% hydrogel (total weight 1 g) was first prepared in a vial as described above. To the hydrogel 100 µL of 6 M HCl aqueous solution was added for liquefaction in approximately 5 min by vigorous vortex mixing. Subsequently, the liquid mixture was neutralized with 6 M NaOH solution (100 µL,) to regenerate the hydrogel rapidly within 10 s. The gel-sol-gel transitions regulated through HCl or NaOH aqueous solutions could be repeated at least five cycles. Swelling and Degradation Properties of Hydrogels. The swelling capacity and degradation property of the as-prepared DA-PEG/CMC hydrogels were examined through gravimetric analysis.9 Columned hydrogels (initial weight W0 around 0.7 g, n = 3) were incubated in 10 mL PBS at 37 oC under shaking (60 rpm) in the presence of lysozyme at concentration of 0, 2 and 20 mg/L, corresponding to 0, 40 and 400 unit/mL, respectively. At select time points, the hydrogels were weighted (Wt) after their surface water removed gently. The buffer was replenished every two days. The weight percentage of the remaining hydrogel was determined as the following equation (1). Relative weight (%) = Wt / W0 × 100

(1)

Drug Loading and Release Behaviors of Hydrogel. Doxorubicin selected as model drug was loaded to the as-prepared 0.28-2.5% hydrogels using a swelling diffusion method.37,38 Dried cylindrical hydrogel (0.04 g; n = 3) was soaked in DOX aqueous solution (0.5 mM, 10.0 mL) with shaking incubator at 37 °C (60 rpm) in darkness for 24 h. Subsequently, the drug-loaded hydrogels were washed gently by water to remove the drug adsorbed on the surface of hydrogels before dipped in the release media. The residual DOX in the immersing solution was determined by absorbance at 481 nm using an UV-visible spectrophotometer (Lambda 35, PerkinElmer).


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The DOX loading content (LC) and the loading efficiency (LE) of the drug-loaded hydrogels were determined based on the equations (2) and (3). LC (wt %) = (M0 - Me) / Mx × 100

(2)

LE (wt %) = (M0 - Me) / M0 × 100

(3)

where W0 and We represent the total amounts of the drug in soaking solution before and after drug loading of the hydrogels, respectively, and Mx is the mass of the xerogels used. The above-mentioned drug-loading hydrogels (n = 3) were separately immersed in 10 mL buffer solution at pH = 2, 5, 7.4 in darkness at 37 oC with shaking (60 rpm). At certain time intervals, the 1.0 mL of released medium was taken out and 1.0 mL of fresh buffer was added. The accumulative drug release can be determined using the following equation (4).

Cumulative release (%) =

∑

× 100

(4)

where is the amount of drug in the hydrogel, is the volume of released medium ( = 1.0 mL), ( = 10 mL, and stands for the concentration of drug in the nth sample. In Vitro Cytotoxicity Assay. COS-7 cells (normal cells) and HeLa cells (cancer cells) were used to evaluate the DA-PEG toxicity using Cell Counting Kit-8 (CCK-8, Dojindo, Japan) assay. These cells with a density of 7000 cells per cell were cultured for 24 h as described previously.9 The DA-PEG solution (20 wt %) filtrated through a 0.22 µm syringe filter was added into the medium to adjust the final polymer concentration range of 0-7 mg/mL. These cells were cultured for another 48 h and then the medium was replaced by new DMEM containing 10% CCK-8 for 4 h. The relative cell viability (n=5) was calculated at 450 nm absorbance by a microplate spectrophotometer with the control wells containing only cell culture medium.


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The cytotoxicity of the hydrogels against COS-7 cells was examined using a direct contact method.9 In brief, the sterilized CMC powder and DA-PEG solution were filtrated through a 0.22 µm syringe filter, respectively. Then, 0.28-2.5% hydrogel was prepared as previously described by using Dulbecco’s phosphate buffered saline (DPBS) as solvent. The hydrogel was cut into pieces (2 mm thickness × 5 mm diameter) and immersed in DMEM prior to cell assay. COS-7 cells (n = 5, 1.0 × 104 cells/well) were seeded and incubated overnight. The culture medium was replaced with 500 µL new DMEM and the hydrogel cubes were added into the wells. The cell/hydrogel constructs were further incubated for 6 h, 24 h and 48 h, while the hydrogels pieces and culture medium were removed and replenished by fresh medium with 10% CCK-8. After 4 h incubation, 100 µL medium was measured with a microplate spectrophotometer at 450 nm. Statistical analysis of the data was similar as previous work with statistical significance set at p ≤0.05.9 The corresponding viabilities of COS-7 cells were further estimated after a Live-Dead cell staining by confocal laser scanning microscopy (CLSM).9 In Vivo Injectability and Biocompatibility of Hydrogel. The sterilized precursor mixtures (200 µL, n = 3 per time point) of 0.28-2.5% hydrogels were separately injected subcutaneously into the back of the female BALB/c mice aged 6-7 weeks (Beijing Vital River Laboratory Animal Technology, China) using a syringe with a 26-gauge needle.3,

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The mice were

euthanized and the formed hydrogels were recorded at specific time intervals by a digital camera (Cannon IXUS 125 HS). For histopathological evaluation, residual hydrogels and the surrounding tissues were isolated and fixed using 4% paraformaldehyde for hematoxylin-eosin [H&E] staining.


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RESULTS AND DISCUSSION Synthesis and Characterization of the Crosslinker. The difunctional crosslinker dipropiolate ester of PEG (DA-PEG) was prepared by a simple, one-step esterification reaction of hydroxylterminated PEG with propiolic acid (Scheme S1 in the Supporting Information) resulted in a fairly good yield (88%). As shown in Figure 1a for the 1H NMR spectrum of the DA-PEG, the protons on PEG backbone were observed at about 3.60 ppm, and new peaks appeared at 2.98 and 4.35 ppm, corresponding to the ethynyl proton and the methylene proton next to ester group, respectively, which indicated the successful incorporation of propiolic acid ester in PEG. The integration ratios among the peaks at 2.98, 4.35 and 3.6 ppm were 2: 4: 175, which indicated that most of the PEG chains were terminated with alkyne groups at both chain ends. As shown in Figure 1b, the absorption peaks of the FT-IR spectrum of the DA-PEG at 2112 and 1715 cm-1, which were assigned to the C≡C and -COO-, respectively, further confirmed the successful modification of PEG with propiolic acid.

Figure 1. (a) 1H NMR spectrum of DA-PEG in CDCl3 and (b) FT-IR spectra of DA-PEG and PEG. The solvent peak marked with asterisk.


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Preparation and Characterization of Hydrogels. As shown in Scheme 1, the reaction between the alkyne end-groups of the DA-PEG and amino groups of the carboxymethyl chitosan (CMC) in PBS buffer (pH 7.4) under physiological conditions was conducted by the formation of enamine bonds. The activation of the alkyne group through the adjacent electron withdrawing group in DA-PEG should promote the reaction between DA-PEG and CMC by amino-yne click reaction without elevated temperature or metal-based catalysts, and finally form a hydrogel.30 Indeed, such hydrogels were prepared successfully by simply mixing DA-PEG and CMC solutions with different feed ratios and incubating at 37 oC for 24 h (Table 1), which indicated the occurrence of the crosslinking reaction between alkyne and amino groups. The xerogels obtained from lyophilized hydrogels were characterized by FT-IR spectroscopy (Figure S2). Compared to the spectrum of DA-PEG, the alkyne absorbance at 2112 cm-1 was very weak in the FT-IR spectra of xerogels, which implied that the alkyne groups disappeared almost in the hydrogels.

Table 1. Composition of the precursor mixtures utilized for the preparation of DAPEG/CMC hydrogels at 37 oC Code

alkyne/aminoa

0.42-2% 0.28-2% 0.14-2% 0.11-2% 0.28-2.5% 0.28-1.5%

0.42 0.28 0.14 0.11 0.28 0.28

CMC concentration (wt %)b 2 2 2 2 2.5 1.5

Total polymer concentrationc (wt %) 4.73 3.82 2.91 2.71 4.78 2.87

Timegel (min)d 7.88 8.61 11.72 21.33 7.78 20.43

a

Molar ratio of alkyne group in DA-PEG to amino group in CMC. b The concentration of CMC used in the corresponding hydrogel. c The total polymer concentration (including both polymers DA-PEG and CMC) in the corresponding hydrogel. d Gelation time at 37 oC is identified as the time at which G′ and G″ began to crossover.


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To further get insight into the gelation process of the hydrogels, the rheological data of the DA-PEG/CMC precursor solutions were monitored by dynamic viscoelastic method at 37 oC. It can be seen from the inset of Figure 2a, at first the storage modulus (G') value was lower than that of the loss modulus (G"), suggesting that there was no premature formation of gel before the test. When G' was equal to G", the sol-to-gel transition occurred, which indicated the formation of hydrogel network, and the corresponding time was usually determined as the gelation time.24,29,40 As shown in Table 1, the gelation time can be tuned from about 21.3 min to 7.8 min by increasing alkyne/amino molar ratios from 0.11 to 0.42 using same CMC concentration. In addition, the samples with lower alkyne/amino molar ratio but higher CMC concentration with a certain value of the total polymer concentration (e.g. 0.28-2.5% vs 0.42-2%, 0.14-2% vs 0.281.5%) exhibited a shorter gelation time, indicating that the gelation time depended strongly on the CMC concentration. Furthermore, the gelation time can also be adjusted by changing the reaction temperature. It can be seen from Figure S3, the gelation time of 0.28-2.5% hydrogel was about 95, 22 and 7 min at 10, 25 and 37 oC, respectively, suggesting that the precursor mixture solution can be operated with plenty of time at low temperature (≤ 25 oC) as free-flowing liquid before gelation, and can form hydrogel rapidly under physiological conditions when applied in vivo.


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Figure 2. The G' and G" of the systems (a) on time with frequency of 1 Hz and (b) on frequency at 37 oC by rheological analysis with strain of 1 %. It can be seen from Figure 2a, after the gel point G' continued increasing very fast due to more junction points formed, then the increase slowed down due to the motion of CMC chains restricted and the reaction formation of more junction points limited. Finally, the G' reached a plateau, indicating the almost completion of the crosslinking reaction between CMC and DAPEG.


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In addition, the effects of the alkyne/amino molar ratio and the CMC concentration on the rheological properties of hydrogels after completely crosslinked for 6 h on parallel plate of the rheometer were also investigated via a frequency sweep test. As shown in Figure 2b, when the CMC concentration was fixed at 2%, the G' value increased markedly from 0.55 to 3.8 kPa with the increase of alkyne/amino molar ratios from 0.11 to 0.42. This is due to the increase in the concentrations of the reactive alkyne groups of DA-PEG and amino groups of CMC, which contribute to an enhanced crosslinking density. It is noteworthy that, the highest value of G' (4.8 kPa) was appeared for the 0.28-2.5% hydrogel in the studied hydrogels. These results suggest that the G' values depended strongly on the CMC concentration, which was consistent with that of the gelation time. Furthermore, the G' value exhibited two or three orders of magnitude greater than the G" value, and both moduli were independent of frequency for each hydrogel, showing a predominantly elastic composition in the hydrogel networks, which possessed nice mechanical stability and stiffness.38,41 The compressive stress-strain curves and compressive modulus by uniaxial compression tests were given to evaluate the compressive strength of as-prepared DAPEG/CMC hydrogels (Figure 3). The fracture stress of the hydrogels increased from 16 to 65 kPa with increasing the alkyne/amino molar ratio and the polymer concentration, corresponding to the compressive modulus of hydrogels from 6 to 27 kPa. This is in line with the viscoelasticity result described above. Therefore, the DA-PEG/CMC hydrogels exhibited tunable mechanical properties, which could be optimized to match the host tissues ranging from 100-10 000 Pa.23 Taking into consideration its appropriate gelation time and mechanical property, the 0.28-2.5% hydrogel was selected for the further studies.


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Figure 3. The compressive stress-strain curves (a) and the compressive modulus (b) of DAPEG/CMC hydrogels with different feed ratios (compression rate: 1 mm/min, data is presented as mean ± SD, n = 3). The microstructure of the freeze-dried hydrogel was observed by SEM, as presented in Figure 4. A porous three-dimensional interpenetrating network was observed in the cross-section of hydrogel, which may facilitate the permeability of nutrients to support cellular growth as well as drug diffusion.9


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Figure 4. SEM image of the cross-section morphology for the 0.28-2.5% hydrogel. Scale bar: 100 µm. pH-Responsive Ability of Hydrogel. The pH-induced sol-gel transitions of the as-prepared 0.28-2.5% hydrogel were investigated. As shown in Figure 5, the hydrogel was decomposed after addition of concentrated HCl solution, to be a free-flowing liquid in approximately 5 min. Subsequent addition of same mole of concentrated NaOH solution to neutralize the acid, the gel was recovered within 10 s. The second cycle regenerated hydrogel was made from the first cycle regenerated hydrogel by addition of 100 µL of 6 M HCl aqueous solution for liquefaction and subsequent addition of same mole concentrated NaOH for neutralization. This process could be repeated five times, confirming the reversibility of pH-induced gel-sol-gel transitions for the hydrogel. In order to track how much of the stiffness of the regenerated hydrogel can be restored, frequency dependence of the regenerated hydrogels for different sol-gel cycles is shown in Figure S4 and the freshly prepared 0.28-1.8% hydrogel is used as the control, because the carboxymethyl chitosan concentrations in the different regenerated hydrogels were diluted accordingly. As shown in Figure S4, the stiffness (G') of the regenerated hydrogels decreased with increasing the reversible cycles of gel-sol-gel transitions from 1 to 5 cycles. Besides, the G' value of the 5 cycles regenerated hydrogel (about 350 Pa) was much lower than that of the freshly prepared 0.28-1.8% hydrogel (about 2000 Pa) with same alkyne/amino molar ratio and


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polymer concentration. This phenomenon was similar to that of Schiff base hydrogel,33 and the possible mechanism was presumed as follows (Scheme 2). Firstly, the enamine bonds formed by crosslinking reaction are prone to form intramolecular hydrogen to construct a pseudo sixmembered ring between the N–H and C=O groups, imparting a stable network structure for the hydrogel.42,43 However, the intramolecular hydrogen bond described above can be destroyed by adding an excess of acid, and further isomerized to form imine bonds.44 Furthermore, the imine bonds are less stable and can be considered to be completely decomposed into aldehyde and amine in an acidic medium (pH value below 6.5), which leads to final liquefaction of the hydrogels.45 When the free-flowing liquid is neutralized by NaOH solution, aldehydes and amines can react to reform the imine bonds, which may be further isomerized to more stable enamine bonds and rebuild the gel network.45 Based on the above discussions, we can infer that the addition of acid or base can trigger the shift between Enamine and [Amine + Aldehyde], leading to a pH-responsive gel-sol-gel transition of the hydrogel. However, the strength of regenerated hydrogels could not be fully restored. The main reason for the stiffness reduction in regenerated hydrogels is the hydrolysis of ester bonds within hydrogel networks under strong acid and base treatment, which eventually led to decrease of the crosslinking points of the regenerated hydrogel network. To further investigate the pH-responsive property of the hydrogel, the rheological data of the regenerated hydrogel/solution at various pH values are shown in Figure S5. The G' values are greater than G" values in the range of pH 2.5 to 7, suggesting that a hydrogel network can be regenerated within this pH range. However, the stiffness (G') of the regenerated hydrogels did not fully recover. For example, the G' value (about 2200 Pa) of the regenerated hydrogel at pH 7 was lower than that of the original 0.28-2.5% hydrogel (pH 7.4, about 4800 Pa shown in Figure


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2b). It can be seen from Figure S5 that the G' values of the regenerated hydrogels decreased with decreasing pH values from 7 to 2.5, even G' was less than G" at pH = 2 (i.e. no hydrogel network was regenerated).

Figure 5. pH sensitivity of the 0.28-2.5% hydrogel. a) Original hydrogel; b) decomposition of hydrogel after adding concentrated HCl aqueous solution; c) regeneration of the hydrogel after adding concentrated NaOH aqueous solution for neutralization; d) decomposition of regenerated hydrogel after adding concentrated HCl aqueous solution; and e) regenerated hydrogel after five cycles. Scheme 2. The Mechanism of pH Sensitivity of the Hydrogel

Swelling and Degradation Behaviors of Hydrogel. The swelling and degradation properties of DA-PEG/CMC hydrogels were evaluated in PBS solutions with or without addition of lysozyme at 37 oC using weight loss method. In Figure 6a, all the hydrogels displayed a fast water sorption rate in PBS solution (pH 7.4), and the maximum swelling ratio for each hydrogel was exceed 300%. Subsequently, weight loss was observed after the certain point and eventually the hydrogel decomposed over time. The time required for the hydrogel completely decomposed was from 5 to 25 days. The initial weight increase could be attributed to the crosslinking density and the increase of the hydrogel network internal volume with more water uptake. The weight


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decrease could be due to the ester bond hydrolysis in DA-PEG within the hydrogel networks in PBS, in which the cleavage of the ester linkage broke the hydrogel network, and a more loosely network structure in turn led to a faster erosion of the hydrogel. These profiles are in line with that was reported in published papers for the hydrolysis of ester linkage within hydrogel networks.3,11,23 The presence of lysozyme can cause the hydrolysis of the β-(1→4)-glycosidic linkage in CMC main chain, which led to much faster degradation rates.39,46 Indeed as shown in Figure 6b, as lysozyme concentration increased up to 20 mg/L, the eventual degradation time of 0.28-2.5% hydrogel reduced significantly from 25 to 12 days. Hence, the DA-PEG/CMC hydrogel exhibited good biodegradability and the degradable rate could be different depending on the lysozyme concentration which may vary in different human tissue.47

Figure 6. Weight profiles of DA-PEG/CMC hydrogels in PBS solution (pH 7.4) at 37 oC (a) and with different lysozyme concentration (b). Data is presented as mean ± SD, n = 3. Drug Loading and Release. To assess the potential application of DA-PEG/CMC hydrogel as drug carriers, DOX as a model anticancer drug was used to explore the loading and controlled release behavior of the pH-responsive hydrogels. The DOX encapsulation was carried out by swelling diffusion to the dried 0.28-2.5% hydrogels under physiological conditions for 24 h. The


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drug loading content and the loading efficiency of the DOX in the hydrogels were 5.6% and 78.6%, respectively. The different release behaviors of the DOX under various pH conditions were observed in Figure 7a. Only 8% of DOX was released after 45 h in PBS at pH 7.4, indicating that the designed vehicles exhibited enough stability under physiological conditions. At pH 5, approximately 20% of DOX was released under the same period with a slight burst release, which is attributed to the enhance of hydrophilicity of DOX in acidic media.37 When the pH value of the release medium was further decreased to 2, the vehicles exhibited a sharp burst release, and the cumulative release percentage came to 100% after 2 h. To better understand the drug release mechanism at pH 2, the hydrogel was investigated at the same conditions by using weight loss method. As shown in Figure 7b, the hydrogel disappeared completely after around 2 h. This is probably attributed to the hydrolysis of ester bonds within hydrogel networks, as well as the enamine bonds decomposed into aldehyde and amine in an acidic medium as described in Scheme 2 (the pH-responsive nature of the hydrogels). Interestingly, compared to most of the reported hydrogel carriers for drug delivery (e.g. Schiff-based hydrogel), which were stable under physiological conditions and unstable with the drug released rapidly at pH 5,3,11,37 the prepared DA-PEG/CMC hydrogels loading DOX exhibited reduced premature drug loss at both pH 7.4 and 5. Thus, the DA-PEG/CMC hydrogels are suitable for use as drug vehicles to improve the therapeutic outcome at desired locales that have lower pH (e.g. stomach, pH 2, due to the limitations of the gastric emptying time).


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Figure 7. (a) In vitro DOX release behaviors from the 0.28-2.5% hydrogel under different pH buffers at 37 oC. (b) Degradation profiles of 0.28-2.5% hydrogels in buffer solution at pH 2 (Data is presented as mean ± SD, n = 3). In Vitro Biocompatibility Evaluation. The toxicity of DA-PEG to COS-7 cells (normal cells) and Hela cells (cancer cells) was evaluated. As presented in Figure 8a, both cells showed good viability after 48 h incubation, indicating that DA-PEG had no obvious toxicity even up to the concentration of 5 mg/mL, and could be utilized as a biocompatible crosslinker. To further investigate the potential toxicity of the crosslinked DA-PEG/CMC hydrogels, we used a direct contact method by incubating COS-7 cells with the crosslinked DA-PEG/CMC hydrogel pieces. The proliferation of the cells was determined by CCK-8 assay. In Figure 8b, the COS-7 cells cultured on tissue culture polystyrene in the presence and absence (as control) of the 0.28-2.5%


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hydrogel displayed continuous proliferation, and the proliferation rate of hydrogel group (~ 540%) was a little higher than that of the control blank group without addition of hydrogel pieces (~ 410%) after 48 h. The representative CLSM images were showed in Figure 8c, in which the live cells was stained green and dead cells red for COS-7 cells. Almost no obvious dead cells were observed in both groups and there was no side effect on cell viability and morphology in the hydrogel group throughout the incubation time. Hence, the DA-PEG/CMC hydrogel has shown good biocompatibility for the potential biomedical applications.

Figure 8. In vitro cytotoxicity test: (a) cell viability with different concentrations of DA-PEG solutions on COS-7 and Hela cells after 48 h incubation; (b) proliferation rate of COS-7 cells after different incubation culture time with the 0.28-2.5% hydrogel pieces (data was determined by CCK-8 assay as mean ± SD, *p≤0.05); (c) CLSM images of corresponding COS-7 cells cultured with the 0.28-2.5% hydrogel piece stained with live (green) /dead (red) assay. Scale bar: 100 µm.


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In Vivo Injectability and Tissue Biocompatibility of Hydrogel. To evaluate the injectable ability in vivo and the effect of the DA-PEG/CMC hydrogel on surrounding tissues, 200 µL precursor solution was injected directly into the subcutaneous region of the mouse back. The transparent hydrogel formed rapidly one hour after injection was found in the subcutaneous region of the mice (Figure 9a) and the hydrogel remained integrity morphology one week after injection (Figure 9b), indicating that the hydrogel has favorable in situ injectable property. The formed 0.28-2.5% hydrogel gradually degraded over time and disappeared completely after 3 weeks. Histological overview of the tissue response at the site of the injection was examined by H&E staining. As shown in Figure 9c, the infiltrated inflammatory cells showed rather low levels at early time points (at day 1 and 4), and gradually increase until one week, which mainly localized at the boundary between surrounding tissue and the hydrogel. Thereafter, the inflammation reaction was found to decrease gradually along with the degradation of the hydrogels, until it disappeared after one month. Meanwhile the infiltration of inflammatory cells was toward inner areas of the hydrogel, suggesting that local acute inflammatory reaction induced by the hydrogel at early stages does not cause systemic inflammatory reaction.48 In addition, no obvious tissue necrosis and damage was observed in all tissues surrounding the hydrogels. Thus the in situ forming DA-PEG/CMC hydrogel possesses acceptable tissue biocompatibility and biodegradability, and could be used potentially in tissue engineering.


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Figure 9. Evaluation of the 0.28-2.5% hydrogel formation in vivo and tissue biocompatibility. (a-b) Hydrogel formed subcutaneously in the BALB/c female mouse. The image was taken one hour (a) and one week (b) after subcutaneous injection of the precursor mixture, respectively. (c) H&E staining after injection of the precursor mixture at desired times. Scale bar: 100 µm.


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CONCLUSIONS A novel facile and inexpensive method to prepare in situ forming injectable and degradable hydrogels under physiological conditions from dipropiolate ester PEG (DA-PEG) and carboxymethyl chitosan (CMC) was developed by spontaneous amino-yne click reaction without using any initiator or catalyst. The amino-yne click reaction involved the formation of enamine bonds, which are sensitive to acidic stimulus to undergo the balance between enamine and amine plus aldehyde groups. This resulted in reversible pH-induced sol-gel transitions for the obtained DA-PEG/CMC hydrogels. The gelation time, mechanical property, swelling capacity and degradation rate of the DA-PEG/CMC hydrogels could be adjusted by varying the feed ratio of DA-PEG and CMC precursors and the polymer concentrations. The hydrogel degradation rate could be promoted in the presence of lysozyme. The precursor mixture solution can be operated with plenty of time at low temperature (≤ 25 oC) as free-flowing liquid before gelation, and can form hydrogel rapidly under physiological conditions when applied in vivo. The pH-controlled DOX release behaviors of the DOX-loaded hydrogels were also presented. In vitro cytoxicity assays and in vivo in situ injection study of the DA-PEG/CMC hydrogels demonstrated favorable fast gel formation and good cell and tissue biocompatibility. This study highlights the potential biomedical application of the spontaneous amino-yne click reaction to prepare biodegradable and injectable hydrogels, as well as to prepare other environmentally friendly biomaterials. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website.


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The 1H NMR spectrum of CMC in 20% DCl in D2O; The illustrative synthesis of DA-PEG; The FT-IR spectra of (a) CMC, (b) 0.11-2% xerogel, (c) 0.14-2% xerogel, (d) 0.28-2.5% xerogel, (e) 0.28-2% xerogel, (f) 0.42-2% xerogel, (g) DA-PEG; Gelation time (Timegel) of the DAPEG/CMC hydrogel at various temperatures; Frequency dependence of the regenerated hydrogels after different sol-gel reversible cycles; The effect of pH on rheological properties of the regenerated hydrogel. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was financially supported by the National Natural Science Foundation of China (21674083, 21374083 and 51533006) and the Natural Science Foundation of Jiangsu Province of China (BK20151249). ABBREVIATIONS CMC, carboxymethyl chitosan; PEG, Poly(ethylene glycol); DA-PEG, Dipropiolate PEG; p-TSA, p-toluenesulfonic acid monohydrate; DOX·HCl, doxorubicin hydrochloride; PBS, phosphate buffer saline; DCl, deuterium chloride; FT-IR, fourier transform infrared spectra; G', storage modulus; G", loss modulus; SEM, scanning electron microscopy; CCK-8, Cell Counting Kit-8; CLSM, confocal laser scanning microscopy.


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Injectable and Degradable pH-Responsive Hydrogels via

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