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A facile method to prepare polysaccharide-based in-situ formable hydrogels with antibacterial ability
Author’s Accepted Manuscript A facile method to prepare polysaccharide-based in-situ formable hydrogels with antibacterial ability Bihua Ye, Lu Meng, Zhiwen Li, Riwang Li, Lihua Li, Lu Lu, Shan Ding, Jinhuan Tian, Changren Zhou www.elsevier.com
PII: DOI: Reference:
S0167-577X(16)31136-3 http://dx.doi.org/10.1016/j.matlet.2016.07.039 MLBLUE21167
To appear in: Materials Letters Received date: 16 May 2016 Revised date: 27 June 2016 Accepted date: 10 July 2016 Cite this article as: Bihua Ye, Lu Meng, Zhiwen Li, Riwang Li, Lihua Li, Lu Lu, Shan Ding, Jinhuan Tian and Changren Zhou, A facile method to prepare polysaccharide-based in-situ formable hydrogels with antibacterial ability, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2016.07.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A facile method to prepare polysaccharide-based in-situ formable hydrogels with antibacterial ability Bihua Ye, Lu Meng, Zhiwen Li, Riwang Li, Lihua Li*, Lu Lu, Shan Ding, Jinhuan Tian, Changren Zhou* Department of Material Science and Engineering, Engineering Research Center of Artificial Organs and Materials, Jinan University, Guangzhou, 510632, China [email protected] [email protected] *
Corresponding authors: Tel.: +0086 20 85226663; fax: +0086 20 85223271
*
Corresponding authors:
Abstract Polysaccharide-based in-situ formable composite hydrogels were prepared by a facile one-pot approach via Michael addition reaction, with maleilated chitosan and thiol derivatised hyaluronan. The hydrogels were characterized by Fourier transform infrared spectroscopy, Scanning electron microscopy, swelling ratio, oscillatory rheology and antibacterial activity. Results showed that the hydrogels with different molar ratios of free thiol/vinyl all formed in-situ within 15min. With increasing of vinyl contents, gelation time and storage modulus increased, while porosity and swelling ratio decreased. Additionally, the hydrogels presented antibacterial activities against Staphylococcus aureus and Escherichia coli, which will be benefit for biomedical applications. Graphical abstract
1
Keywords: Biomimetic, Composite materials, Polysaccharide, In-situ formable hydrogels, Michael addition
1.
Introduction In-situ formable hydrogels have been employed as scaffolds, drug delivery systems, and cell carriers
for wide biomedical applications [1, 2] since they allow easy and homogenous distribution of bioactive molecules or cells within any defect size or shape and minimize the invasiveness of the surgical techniques [3]. In-situ formable hydrogels have been prepared by various chemical and physical crosslinking methods [4]. Photopolymerization of vinyl modified polymers is one of the most popular methods to achieve hydrogel formation at a defect site [5], irrespective of the potential cytotoxicity of photo-initiators and UV light [6, 7]. Michael addition reaction is an increasing popular method to prepare hydrogels, wherein crosslinking is achieved by addition reactions between nucleophiles (e.g. thiol groups) and
2
electrophiles (e.g. vinyl/acrylate groups) [8]. Poly(ethylene glycol) (PEG)-based hydrogel systems have been mostly studied, but the multistep of hyper-branched macromers synthesis, functionalization and purification [9, 10], undoubtedly increases the cost and complexity of preparation process. Naturally derived polymers have showed great advantages over common synthetic polymers [4, 11], such as better biocompatibility, tailored enzymatic biodegradation, etc. It is worthy to be noted that, comparing with PEG, the chemically reactive multi-groups on such water solvable macromolecules offer more crosslinking points to form large network. Therefore, the design of natural polymers with desired functional groups to provide crosslinking in situ is highly advantageous, which can create a more biomimetic microenvironment for biomedical applications. Additionally, hydrogels with inherent antibacterial activity are even more beneficial since they can absorb biological fluids, avoid water loss and microbial invasions, and offer adequate gaseous exchange when exposed to the environment [12]. Herein, we propose a novel, facile one-pot approach to prepare in-situ forming antibacterial hydrogel via self-crosslinking of functionalized polysaccharide derivatives, without using any extraneous chemical crosslinking agents. 2.
Experimental section Materials, specific syntheses and characterization of maleilated chitosan (CS-MA) and thiol
derivatised hyaluronan (HA-SH) are provided in the Supporting Information. Chitosan (CS) was modified with vinyl carboxylic acid groups [13], while hyaluronan (HA) was functionalized with thiol groups [9, 14]. The functionalized polysaccharide derivatives were characterized by 1H nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy (FT-IR), Zeta potential and UV-visible spectrophotometric measurements.
3
Preparation of Polysaccharide Hydrogels via Michael-type Thiol-Ene Reaction: The concentration is defined as the total dry weight of both HA-SH and CS-MA per volume of deionized water. CS-MA and HA-SH were separately dissolved in deionized water, and then were mixed in vials by vortexing for 1min at a final polymer concentration of 20 mg/mL corresponding to the molar ratio of free thiol/vinyl of 4:1, 4:2, 4:3, and 4:4, respectively. Afterwards, 58% (wt%) beta-glycerophosphate disodium salt (β-GP) aqueous solution was added to adjust the pH to 7 and the mixtures were incubated at 37°C for 24h. The time for gel formation (denoted as gelation time) was determined using the vial inverting method [8]. Rheological experiments were performed with a Kinexus Pro rheometer (Malvern, UK) using parallel plates (Ø 20 mm). The time-sweep of precursor solution was carried out at 37°C, a frequency of 1 Hz, and a strain of 1%. The modulus values versus frequency analyses of hydrogels were carried out at 37°C, a strain of 1%. Scanning electron microscopy (SEM, PHILIPS XL-30ESEM, the Netherlands) was employed to determine the morphology of freeze-dried hydrogels, which were kept at -80°C for 24h prior to be freeze-dried. Swelling ratio (Q) of the hydrogel was determined as described in literature [9], which was defined as (Ws/Wd) ×100% (the weight of swollen hydrogel/the dried weight of hydrogel). The antibacterial activities of hydrogels against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were measured as described in literature (detials in Supporting Information) [15], which was calculated by the equation: killing percentage (%)=[(Ncontrol-Nsample)/Ncontrol]×100% (Ncontrol and Nsample are the number of colony forming units at the end of the incubation period on the Luria-Bertani agar plates of the control and hydrogel samples, respectively). 3.
Results and Discussion Characterization of CS-MA: The chemical composition of CS-MA was characterized by 1H NMR
4
and FT-IR. Compared with the spectrum of pure CS, a new peak of –CH=CH– at δ=6.3 [16] was observed, as Fig. 1(a) shows. The determined substitution degree of CS-MA from 1H NMR was 65%, by comparing the integrals of signals at δ=3.0 (C2 proton of glucosamine unit in CS) and δ=6.3 (–CH=CH– protons). The result was further confirmed by a UV-visible spectrophotometric method, which was 1867.6±8.9 μmol/g. In the FT-IR spectra (Fig. 1c), several new peaks at 1700 cm-1, 1620 cm-1, and 864 cm-1 were found in the modified CS, which correspond to the free unsaturated carboxylic acid group (asterisk) and double bond of the maleoyl group (black arrows), respectively [13]. Characterization of HA-SH: HA-SH was synthesized via amide bond formation between the carboxylic acid groups of HA and amine groups of L-cysteine. 1H NMR spectra in Fig. 1(b) revealed that the new peak of methylene protons on –CH2SH at δ=2.9 indicated the successful modification [14]. The substitution degree of HA-SH determined from 1H NMR was 45%, by comparing the peak area of methylene protons on –CH2SH and N-acetyl methyl protons (δ=2.0). The modification degree of HA with free thiol groups was 330.6±18.8 μmol/g (by standard Ellman’s assay).
Fig. 1. Synthesis schemes and 1H NMR spectra of CS-MA (a) and HA-SH (b), FT-IR spectra (c) of CS, CS-MA and composite hydrogel (nfree thiol/nvinyl=4:4), and synthesis scheme of the hydrogels (d). Hydrogel Formation and Gelation Time: CS-MA and HA-SH formed an insoluble network via Michael addition by reaction of the vinyl groups and thiolated anions [9]. Vial inverting method verified
5
the hydrogel formation (Fig. 2a,b), and the hydrogels with different molar ratios of free thiol/vinyl all formed in-situ within 15min (Supporting Information, Table 1). With increasing vinyl contents, the gelation time increased, which could be attributed to the molecular chain entanglement of CS-MA subsequently hindering the interaction of thiol and vinyl groups. Furthermore, the reaction was also confirmed by FT-IR depicted in Fig. 1(c). The characteristic peaks of free unsaturated carboxylic acid group at 1700 cm-1, the double bond of the maleoyl group at 1622 cm-1 and 864 cm-1 decreased and became a shoulder peak after reaction. Morphology and swelling behavior of hydrogels: In Fig. 2(c), the thin layer on the surface may be attributed to the collapse of surface pores during freeze-drying, and the cross-sectional part displayed a continuous and porous structure with pore size of 100-400 μm. Smaller pore sizes and tighter network structure could be observed with higher ratio of vinyl (Fig. 2d), due to higher crosslinking density. This finding indicated that the pores diameter and porosity of freeze-dried hydrogels were related with the molar ratio of thiol/vinyl. Fig. 2(e) shows that the swelling ratio (Q) decreased along with higher ratio of vinyl, due to higher crosslinking density and lower porosity. Specifically, the 4/1 hydrogel showed the largest Q of 992±15%.
Fig. 2. Sol-gel phase transition photographs (a-b) of composite hydrogel (nfree thiol/nvinyl=4:4); SEM photographs of freeze-dried hydrogels with thiol/vinyl molar ratio of 4:1 (c) and 4:4 (d); (e) Equilibrium
6
swelling ratio of hydrogels in phosphate buffered saline at 37°C (n=3); Rheological analysis: (f) G’ and G’’ analyses during gelation process (nfree thiol/nvinyl=4:4; 37°C; frequency: 1.0 Hz; strain: 1.0%), (g) G’ values of hydrogels with different thiol/ vinyl molar ratios (37°C; strain: 1.0%). Rheological Analysis of hydrogels: The time-sweep of precursor solution with thiol/vinyl ratio of 4:4 is showed in Fig. 2(f). The storage modulus (G’) gradually increased and it was observed to intersect loss modulus (G’’) at ~725s (ca. 12min), revealing the formation of hydrogel. Furthermore, measurements of the storage modulus versus frequency were carried out to analyze the effect of different molar ratios of thiol/ vinyl groups on hydrogels’ mechanical properties. As shown in Fig. 2(g), the higher G’ value was found for the hydrogels with lower thiol/ vinyl ratio in low frequency, indicating that the crosslinking density was crucial to the mechanical properties of hydrogels. Antibacterial Property of hydrogels: As shown in Fig. 3, compared to the control, the composite hydrogel prepared by thiol/vinyl molar ratio of 4:4 exhibited antibacterial activities against both S. aureus and E. coli, and its killing percentage against S. aureus (86.4±4.9%) was obviously higher than that for E. coli (63.0±4.4%). The antibacterial property of hydrogel was attributed to the electrostatic interaction of the unsubstituted, cationic amino groups of CS-MA (average zeta potential=22.15±0.99 mV, n=3) with the anionic components on bacterial biomembrane [17]. The antibacterial activities of CS were limited to acidic conditions due to its poor solubility under neutral pH, while CS-MA have improved solubility, which was tunable and rely on the amount of unsubstituted amino groups. By incorporating with HA-SH to form hydrogel in situ, may provide a facile and promising approach for fabricating a contact-active, excellent defect margin adaptative coating with microbe resistance as well as biocompatibility simultaneously.
7
Fig. 3 Antibacterial result of composite hydrogels prepared by thiol/vinyl molar ratio of 4:4 against E. coli (a) and S. aureus (b) (n=3). 4.
Conclusion In-situ formable polysaccharide hydrogels with antibacterial ability were fabricated with CS-MA and
HA-SH via Michael addition reaction. The gelation time, swelling ratio, morphology and mechanical properties of hydrogels are tailorable, providing a possibility to satisfy more different requirements in biomedicine. Acknowledgements The work was supported by National Natural Science Foundation of China (31270021) and Science and Technology Project of Guangdong Province (2014A010105031).
References [1] H. Tan, C.R. Chu, K.A. Payne, K.G. Marra, Biomaterials 30 (2009) 2499-2506. [2] A.J. DeFail, C.R. Chu, N. Izzo, K.G. Marra, Biomaterials 27 (2006) 1579-1585. [3] L. Yu, J. Ding, Chem. Soc. Rev. 37 (2008) 1473-1481. [4] A. Sivashanmugam, R. Arun Kumar, M. Vishnu Priya, S.V. Nair, R. Jayakumar, Eur. Polym. J. 72 (2015) 543-565.
8
[5] J.L. Ifkovits, J.A. Burdick, Tissue Eng. 13 (2007) 2369-2385. [6] J. Elisseeff, K. Anseth, D. Sims, W. McIntosh, M. Randolph, R. Langer, Proc. Natl Acad. Sci. USA. 96 (1999) 3104-3107. [7] N.E. Fedorovich, M.H. Oudshoorn, D. van Geemen, W.E. Hennink, J. Alblas, W.J.A. Dhert, Biomaterials 30 (2009) 344-353. [8] R. Jin, L.S. Moreira Teixeira, A. Krouwels, P.J. Dijkstra, C.A. van Blitterswijk, M. Karperien, J. Feijen, Acta Biomater. 6 (2010) 1968-1977. [9] Y. Dong, A.O. Saeed, W. Hassan, C. Keigher, Y. Zheng, H. Tai, A. Pandit, W. Wang, Macromol. Rapid Commun. 33 (2012) 120-126. [10] L. Cao, B. Cao, C. Lu, G. Wang, L. Yu, J. Ding, J. Mater. Chem. B 3 (2015) 1268-1280. [11] T.C. Tseng, L. Tao, F.Y. Hsieh, Y. Wei, I.M. Chiu, S.H. Hsu, Adv. Mater. 27 (2015) 3518-3524. [12] A. Song, A.A. Rane, K.L. Christman, Acta Biomater. 8 (2012) 41-50. [13] T.M. Don, H.R. Chen, Carbohydr. Polym. 61 (2005) 334-347. [14] J. Ding, R. He, G. Zhou, C. Tang, C. Yin, Acta Biomater. 8 (2012) 3643-3651. [15] Z. Xin, S. Du, C. Zhao, H. Chen, M. Sun, S. Yan, S. Luan, J. Yin, Appl. Surf. Sci. 365 (2016) 99-107. [16] Q. Mu, Y.e. Fang, Carbohydr. Polym. 72 (2008) 308-314. [17] F. Ding, Z. Nie, H. Deng, L. Xiao, Y. Du, X. Shi, Carbohydr. Polym. 98 (2013) 1547-1552.
Highlights
A novel and facile one-pot approach is proposed to fabricate polysaccharide-based in-situ formable hydrogels.
The method proposed can provide important implications for the design of other bioinspired materials.
Physical and chemical properties of the hydrogels can be tuned by the molar ratio of free thiol/vinyl groups.
The hydrogels presented antibacterial activities against Staphylococcus aureus and Escherichia coli.
9
PII: DOI: Reference:
S0167-577X(16)31136-3 http://dx.doi.org/10.1016/j.matlet.2016.07.039 MLBLUE21167
To appear in: Materials Letters Received date: 16 May 2016 Revised date: 27 June 2016 Accepted date: 10 July 2016 Cite this article as: Bihua Ye, Lu Meng, Zhiwen Li, Riwang Li, Lihua Li, Lu Lu, Shan Ding, Jinhuan Tian and Changren Zhou, A facile method to prepare polysaccharide-based in-situ formable hydrogels with antibacterial ability, Materials Letters, http://dx.doi.org/10.1016/j.matlet.2016.07.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
A facile method to prepare polysaccharide-based in-situ formable hydrogels with antibacterial ability Bihua Ye, Lu Meng, Zhiwen Li, Riwang Li, Lihua Li*, Lu Lu, Shan Ding, Jinhuan Tian, Changren Zhou* Department of Material Science and Engineering, Engineering Research Center of Artificial Organs and Materials, Jinan University, Guangzhou, 510632, China [email protected] [email protected] *
Corresponding authors: Tel.: +0086 20 85226663; fax: +0086 20 85223271
*
Corresponding authors:
Abstract Polysaccharide-based in-situ formable composite hydrogels were prepared by a facile one-pot approach via Michael addition reaction, with maleilated chitosan and thiol derivatised hyaluronan. The hydrogels were characterized by Fourier transform infrared spectroscopy, Scanning electron microscopy, swelling ratio, oscillatory rheology and antibacterial activity. Results showed that the hydrogels with different molar ratios of free thiol/vinyl all formed in-situ within 15min. With increasing of vinyl contents, gelation time and storage modulus increased, while porosity and swelling ratio decreased. Additionally, the hydrogels presented antibacterial activities against Staphylococcus aureus and Escherichia coli, which will be benefit for biomedical applications. Graphical abstract
1
Keywords: Biomimetic, Composite materials, Polysaccharide, In-situ formable hydrogels, Michael addition
1.
Introduction In-situ formable hydrogels have been employed as scaffolds, drug delivery systems, and cell carriers
for wide biomedical applications [1, 2] since they allow easy and homogenous distribution of bioactive molecules or cells within any defect size or shape and minimize the invasiveness of the surgical techniques [3]. In-situ formable hydrogels have been prepared by various chemical and physical crosslinking methods [4]. Photopolymerization of vinyl modified polymers is one of the most popular methods to achieve hydrogel formation at a defect site [5], irrespective of the potential cytotoxicity of photo-initiators and UV light [6, 7]. Michael addition reaction is an increasing popular method to prepare hydrogels, wherein crosslinking is achieved by addition reactions between nucleophiles (e.g. thiol groups) and
2
electrophiles (e.g. vinyl/acrylate groups) [8]. Poly(ethylene glycol) (PEG)-based hydrogel systems have been mostly studied, but the multistep of hyper-branched macromers synthesis, functionalization and purification [9, 10], undoubtedly increases the cost and complexity of preparation process. Naturally derived polymers have showed great advantages over common synthetic polymers [4, 11], such as better biocompatibility, tailored enzymatic biodegradation, etc. It is worthy to be noted that, comparing with PEG, the chemically reactive multi-groups on such water solvable macromolecules offer more crosslinking points to form large network. Therefore, the design of natural polymers with desired functional groups to provide crosslinking in situ is highly advantageous, which can create a more biomimetic microenvironment for biomedical applications. Additionally, hydrogels with inherent antibacterial activity are even more beneficial since they can absorb biological fluids, avoid water loss and microbial invasions, and offer adequate gaseous exchange when exposed to the environment [12]. Herein, we propose a novel, facile one-pot approach to prepare in-situ forming antibacterial hydrogel via self-crosslinking of functionalized polysaccharide derivatives, without using any extraneous chemical crosslinking agents. 2.
Experimental section Materials, specific syntheses and characterization of maleilated chitosan (CS-MA) and thiol
derivatised hyaluronan (HA-SH) are provided in the Supporting Information. Chitosan (CS) was modified with vinyl carboxylic acid groups [13], while hyaluronan (HA) was functionalized with thiol groups [9, 14]. The functionalized polysaccharide derivatives were characterized by 1H nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy (FT-IR), Zeta potential and UV-visible spectrophotometric measurements.
3
Preparation of Polysaccharide Hydrogels via Michael-type Thiol-Ene Reaction: The concentration is defined as the total dry weight of both HA-SH and CS-MA per volume of deionized water. CS-MA and HA-SH were separately dissolved in deionized water, and then were mixed in vials by vortexing for 1min at a final polymer concentration of 20 mg/mL corresponding to the molar ratio of free thiol/vinyl of 4:1, 4:2, 4:3, and 4:4, respectively. Afterwards, 58% (wt%) beta-glycerophosphate disodium salt (β-GP) aqueous solution was added to adjust the pH to 7 and the mixtures were incubated at 37°C for 24h. The time for gel formation (denoted as gelation time) was determined using the vial inverting method [8]. Rheological experiments were performed with a Kinexus Pro rheometer (Malvern, UK) using parallel plates (Ø 20 mm). The time-sweep of precursor solution was carried out at 37°C, a frequency of 1 Hz, and a strain of 1%. The modulus values versus frequency analyses of hydrogels were carried out at 37°C, a strain of 1%. Scanning electron microscopy (SEM, PHILIPS XL-30ESEM, the Netherlands) was employed to determine the morphology of freeze-dried hydrogels, which were kept at -80°C for 24h prior to be freeze-dried. Swelling ratio (Q) of the hydrogel was determined as described in literature [9], which was defined as (Ws/Wd) ×100% (the weight of swollen hydrogel/the dried weight of hydrogel). The antibacterial activities of hydrogels against Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were measured as described in literature (detials in Supporting Information) [15], which was calculated by the equation: killing percentage (%)=[(Ncontrol-Nsample)/Ncontrol]×100% (Ncontrol and Nsample are the number of colony forming units at the end of the incubation period on the Luria-Bertani agar plates of the control and hydrogel samples, respectively). 3.
Results and Discussion Characterization of CS-MA: The chemical composition of CS-MA was characterized by 1H NMR
4
and FT-IR. Compared with the spectrum of pure CS, a new peak of –CH=CH– at δ=6.3 [16] was observed, as Fig. 1(a) shows. The determined substitution degree of CS-MA from 1H NMR was 65%, by comparing the integrals of signals at δ=3.0 (C2 proton of glucosamine unit in CS) and δ=6.3 (–CH=CH– protons). The result was further confirmed by a UV-visible spectrophotometric method, which was 1867.6±8.9 μmol/g. In the FT-IR spectra (Fig. 1c), several new peaks at 1700 cm-1, 1620 cm-1, and 864 cm-1 were found in the modified CS, which correspond to the free unsaturated carboxylic acid group (asterisk) and double bond of the maleoyl group (black arrows), respectively [13]. Characterization of HA-SH: HA-SH was synthesized via amide bond formation between the carboxylic acid groups of HA and amine groups of L-cysteine. 1H NMR spectra in Fig. 1(b) revealed that the new peak of methylene protons on –CH2SH at δ=2.9 indicated the successful modification [14]. The substitution degree of HA-SH determined from 1H NMR was 45%, by comparing the peak area of methylene protons on –CH2SH and N-acetyl methyl protons (δ=2.0). The modification degree of HA with free thiol groups was 330.6±18.8 μmol/g (by standard Ellman’s assay).
Fig. 1. Synthesis schemes and 1H NMR spectra of CS-MA (a) and HA-SH (b), FT-IR spectra (c) of CS, CS-MA and composite hydrogel (nfree thiol/nvinyl=4:4), and synthesis scheme of the hydrogels (d). Hydrogel Formation and Gelation Time: CS-MA and HA-SH formed an insoluble network via Michael addition by reaction of the vinyl groups and thiolated anions [9]. Vial inverting method verified
5
the hydrogel formation (Fig. 2a,b), and the hydrogels with different molar ratios of free thiol/vinyl all formed in-situ within 15min (Supporting Information, Table 1). With increasing vinyl contents, the gelation time increased, which could be attributed to the molecular chain entanglement of CS-MA subsequently hindering the interaction of thiol and vinyl groups. Furthermore, the reaction was also confirmed by FT-IR depicted in Fig. 1(c). The characteristic peaks of free unsaturated carboxylic acid group at 1700 cm-1, the double bond of the maleoyl group at 1622 cm-1 and 864 cm-1 decreased and became a shoulder peak after reaction. Morphology and swelling behavior of hydrogels: In Fig. 2(c), the thin layer on the surface may be attributed to the collapse of surface pores during freeze-drying, and the cross-sectional part displayed a continuous and porous structure with pore size of 100-400 μm. Smaller pore sizes and tighter network structure could be observed with higher ratio of vinyl (Fig. 2d), due to higher crosslinking density. This finding indicated that the pores diameter and porosity of freeze-dried hydrogels were related with the molar ratio of thiol/vinyl. Fig. 2(e) shows that the swelling ratio (Q) decreased along with higher ratio of vinyl, due to higher crosslinking density and lower porosity. Specifically, the 4/1 hydrogel showed the largest Q of 992±15%.
Fig. 2. Sol-gel phase transition photographs (a-b) of composite hydrogel (nfree thiol/nvinyl=4:4); SEM photographs of freeze-dried hydrogels with thiol/vinyl molar ratio of 4:1 (c) and 4:4 (d); (e) Equilibrium
6
swelling ratio of hydrogels in phosphate buffered saline at 37°C (n=3); Rheological analysis: (f) G’ and G’’ analyses during gelation process (nfree thiol/nvinyl=4:4; 37°C; frequency: 1.0 Hz; strain: 1.0%), (g) G’ values of hydrogels with different thiol/ vinyl molar ratios (37°C; strain: 1.0%). Rheological Analysis of hydrogels: The time-sweep of precursor solution with thiol/vinyl ratio of 4:4 is showed in Fig. 2(f). The storage modulus (G’) gradually increased and it was observed to intersect loss modulus (G’’) at ~725s (ca. 12min), revealing the formation of hydrogel. Furthermore, measurements of the storage modulus versus frequency were carried out to analyze the effect of different molar ratios of thiol/ vinyl groups on hydrogels’ mechanical properties. As shown in Fig. 2(g), the higher G’ value was found for the hydrogels with lower thiol/ vinyl ratio in low frequency, indicating that the crosslinking density was crucial to the mechanical properties of hydrogels. Antibacterial Property of hydrogels: As shown in Fig. 3, compared to the control, the composite hydrogel prepared by thiol/vinyl molar ratio of 4:4 exhibited antibacterial activities against both S. aureus and E. coli, and its killing percentage against S. aureus (86.4±4.9%) was obviously higher than that for E. coli (63.0±4.4%). The antibacterial property of hydrogel was attributed to the electrostatic interaction of the unsubstituted, cationic amino groups of CS-MA (average zeta potential=22.15±0.99 mV, n=3) with the anionic components on bacterial biomembrane [17]. The antibacterial activities of CS were limited to acidic conditions due to its poor solubility under neutral pH, while CS-MA have improved solubility, which was tunable and rely on the amount of unsubstituted amino groups. By incorporating with HA-SH to form hydrogel in situ, may provide a facile and promising approach for fabricating a contact-active, excellent defect margin adaptative coating with microbe resistance as well as biocompatibility simultaneously.
7
Fig. 3 Antibacterial result of composite hydrogels prepared by thiol/vinyl molar ratio of 4:4 against E. coli (a) and S. aureus (b) (n=3). 4.
Conclusion In-situ formable polysaccharide hydrogels with antibacterial ability were fabricated with CS-MA and
HA-SH via Michael addition reaction. The gelation time, swelling ratio, morphology and mechanical properties of hydrogels are tailorable, providing a possibility to satisfy more different requirements in biomedicine. Acknowledgements The work was supported by National Natural Science Foundation of China (31270021) and Science and Technology Project of Guangdong Province (2014A010105031).
References [1] H. Tan, C.R. Chu, K.A. Payne, K.G. Marra, Biomaterials 30 (2009) 2499-2506. [2] A.J. DeFail, C.R. Chu, N. Izzo, K.G. Marra, Biomaterials 27 (2006) 1579-1585. [3] L. Yu, J. Ding, Chem. Soc. Rev. 37 (2008) 1473-1481. [4] A. Sivashanmugam, R. Arun Kumar, M. Vishnu Priya, S.V. Nair, R. Jayakumar, Eur. Polym. J. 72 (2015) 543-565.
8
[5] J.L. Ifkovits, J.A. Burdick, Tissue Eng. 13 (2007) 2369-2385. [6] J. Elisseeff, K. Anseth, D. Sims, W. McIntosh, M. Randolph, R. Langer, Proc. Natl Acad. Sci. USA. 96 (1999) 3104-3107. [7] N.E. Fedorovich, M.H. Oudshoorn, D. van Geemen, W.E. Hennink, J. Alblas, W.J.A. Dhert, Biomaterials 30 (2009) 344-353. [8] R. Jin, L.S. Moreira Teixeira, A. Krouwels, P.J. Dijkstra, C.A. van Blitterswijk, M. Karperien, J. Feijen, Acta Biomater. 6 (2010) 1968-1977. [9] Y. Dong, A.O. Saeed, W. Hassan, C. Keigher, Y. Zheng, H. Tai, A. Pandit, W. Wang, Macromol. Rapid Commun. 33 (2012) 120-126. [10] L. Cao, B. Cao, C. Lu, G. Wang, L. Yu, J. Ding, J. Mater. Chem. B 3 (2015) 1268-1280. [11] T.C. Tseng, L. Tao, F.Y. Hsieh, Y. Wei, I.M. Chiu, S.H. Hsu, Adv. Mater. 27 (2015) 3518-3524. [12] A. Song, A.A. Rane, K.L. Christman, Acta Biomater. 8 (2012) 41-50. [13] T.M. Don, H.R. Chen, Carbohydr. Polym. 61 (2005) 334-347. [14] J. Ding, R. He, G. Zhou, C. Tang, C. Yin, Acta Biomater. 8 (2012) 3643-3651. [15] Z. Xin, S. Du, C. Zhao, H. Chen, M. Sun, S. Yan, S. Luan, J. Yin, Appl. Surf. Sci. 365 (2016) 99-107. [16] Q. Mu, Y.e. Fang, Carbohydr. Polym. 72 (2008) 308-314. [17] F. Ding, Z. Nie, H. Deng, L. Xiao, Y. Du, X. Shi, Carbohydr. Polym. 98 (2013) 1547-1552.
Highlights
A novel and facile one-pot approach is proposed to fabricate polysaccharide-based in-situ formable hydrogels.
The method proposed can provide important implications for the design of other bioinspired materials.
Physical and chemical properties of the hydrogels can be tuned by the molar ratio of free thiol/vinyl groups.
The hydrogels presented antibacterial activities against Staphylococcus aureus and Escherichia coli.
9