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Injectable Self-Healing Zwitterionic Hydrogels Based on Dynamic Benzoxaborole-Sugar Interactions with Tunable Mechanical Properties Yangjun Chen, Wenda Wang, Di Wu, Masanori Nagao, Dennis G Hall, Thomas Thundat, and Ravin Narain Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01679 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018
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Injectable Self-Healing Zwitterionic Hydrogels Based on Dynamic Benzoxaborole-Sugar Interactions with Tunable Mechanical Properties Yangjun Chen†, Wenda Wang†, Di Wu‡, Masanori Nagao#, Dennis G. Hall‡, Thomas Thundat*†, and Ravin Narain*†
†
Department of Chemical and Materials Engineering, University of Alberta, Edmonton, Alberta
T6G 2G6, Canada ‡
Department of Chemistry, Centennial Centre for Interdisciplinary Science, University of
Alberta, Edmonton, Alberta T6G 2G6, Canada #
Department of Chemical Engineering, Kyushu University, Fukuoka 819-0395, Japan
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ABSTRACT: Dynamic hydrogels based on arylboronic esters have been considered as ideal platforms for biomedical applications given their self-healing and injectable characteristics. However, there still exist some critical issues that need to be addressed or improved, including hydrogel biocompatibility, physiological usability, and tunability of mechanical properties. Here, two kinds of phospholipid bioinspired MPC copolymers, one is zwitterionic copolymer (PMB) containing a fixed 15 mol% of benzoxaborole (pKa ≈ 7.2) groups and the other is zwitterionic glycopolymers (PMG) with varied ratios of sugar groups (20%, 50%, 80%), were synthesized respectively via one-pot facile reversible addition-fragmentation chain transfer (RAFT) polymerization. PMBG hydrogels were formed spontaneously after mixing 10 wt% of PMB and PMG copolymer solutions due to dynamic benzoxaborole-sugar interactions. The mechanical properties of nine hydrogels (3×3) with different sugar contents and pHs (7.4, 8.4, 9.4) were carefully studied by rheological measurements and hydrogels with higher sugar content and higher pH were found to have higher strength. Moreover, similar to other arylboronic ester based hydrogels, PMBG hydrogels possessed not only self-healing and injectable properties, but also pH/sugar responsiveness. Additionally, in vitro cytotoxicity tests of gel extracts on both normal and cancer cells further confirmed the excellent biocompatibility of the hydrogels, which should be ascribed to the biomimetic nature of phosphorylcholine (PC) and sugar residues of the copolymers. Consequently, the zwitterionic dynamic hydrogels provide promising future for diverse biomedical applications.
KEYWORDS: zwitterionic; glycopolymer; dynamic hydrogel; boronic ester; self-healing
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1. Introduction Due to the hydrophilic nature, porous network structure, and human tissue mimicking mechanical properties, polymeric hydrogels have emerged as versatile platforms for diverse biomedical applications ranging from therapeutic drug delivery and cell culture to threedimensional (3D) bioprinting and tissue engineering.1-5 Nevertheless, conventional covalently crosslinked hydrogels have failed to meet the rapidly growing medical requirements because of the lack of self-healing and injectable properties.6 To address this issue, hydrogels based on dynamic covalent bonds7-9 have attracted more and more attention. The dynamic and reversible linkages incorporated in the polymeric architecture endow hydrogels the ability to autonomously recover from damage and regain mechanical properties, which can contribute to prolong lifetime and improve performance.9, 10 Moreover, hydrogels with shear-thinning characteristic hold the advantage of minimal invasion when used for in vivo administration of therapeutic drugs and cells.11 Additionally, these dynamic hydrogels can also respond to specific stimuli (e.g. temperature, pH, reduction, sugar, etc.),7 which enables controlled release of loaded cargoes. Arylboronic esters, which mainly refer to the reversible condensation reactions of arylboronic acids (ABAs) with 1,2- and 1,3-diols,12, 13 have been extensively studied in dynamic polymeric assemblies,14,
15
nanomedicine,16-21 and hydrogels.6,
22-26
The formation of arylboronic esters
usually occurs at pH above the pKa of ABAs and the binding affinity is highly pH-dependent.24, 25
Therefore, both the pKa of the arylboronic acids and pH of the media are vital for hydrogel
formation and strength. Anderson et al.6 found that hydrogels with lower ABA pKa and higher solution pH have higher strength by comparing nine hydrogels formed with three different ABAgroups at three different pH values. Moreover, hydrogel strength can also be adjusted by varying the diol/ABA ratio to change the crosslinking density. Ishihara et al.27-29 prepared
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hydrogels with varied storage modulus by mixing ABA-containing copolymer (PMBV) solution and poly(vinyl alcohol) (PVA) solution at different ratios and evaluated the influence of mechanical properties on the biological functions of encapsulated cells. It is noteworthy here that, for hydrogels used as mimetic extracellular matrix (ECM), the mechanical properties are of great significance to cell functions including migration, proliferation, and differentiation.5, 30 Therefore, the facile approach towards regulated cell microenvironments by hydrogels based on arylboronic esters provides great potential in the area of cell therapy and tissue regeneration. However, most of the currently reported ABAs have pKa values higher than 8,6, 25 leading to limited biomedical applications under physiological conditions (pH 7.4). Several efforts have been made to create ABAs with lower pKa25, 31 and Hall and co-workers developed unique benzoxaborole groups (pKa ≈ 7.2) which possess high binding affinity with cis-diols of monosaccharides to form stable five-membered rings at physiological pH.32,
33
Our group has pioneered the preparation of
hydrogels based on the interactions between benzoxaborole modified polymers and glycopolymers.34-36 Nevertheless, the tunability of hydrogel strength remained unexplored and the biocompatibility of those hydrogels was unsatisfied. 2-Methacryloyloxyethyl phosphorylcholine (MPC) based zwitterionic polymers, which possess cell membrane bioinspired molecular structures,37-39 have been widely studied as biocompatible materials and used in various biomedical areas such as antifouling surface modification40 and cancer nanomedicine.41-43 Hydrogels made from MPC polymers have been reported as biocompatible platforms for wound dressing, cell culture, and tissue engineering.44-46 In this study, reversible addition-fragmentation chain transfer (RAFT) polymerization was applied to synthesize two kinds of MPC polymers, i.e. poly(2-methacryloyloxyethyl phosphorylcholine-st-5-methacrylamido-1,2-benzoxaborole) (PMB) with a fixed benzoxaborole
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mol ratio of 15% and poly(2-methacryloyloxyethyl phosphorylcholine-st-2-gluconamidoethyl methacrylamide) (PMG) with different sugar mol ratios of 20%, 50%, and 80%. Benefitting from the one-step polymerization method developed by Narain and Armes,47-49 we could facilely obtain the PMG glycopolymers without complex protection/deprotection chemistries.50 In our design, hydrogels made from PMG copolymers with different sugar contents were supposed to have different mechanical properties due to varied crosslinking densities. Here, pH as another important factor for hydrogel strength was also investigated. Therefore, an array of nine hydrogels with different sugar contents and pHs were formed by simply mixing 10 wt% of PMB and PMG solutions. The mechanical properties were then carefully compared via rheological measurements. The injectable, self-healing, and pH/sugar responsive properties, which rely on the dynamic benzoxaborole-sugar interactions, were further studied in detail. Finally, the biocompatibility of hydrogels was evaluated by standard MTT assay after incubating both normal and cancer cells with gel extracts.
Scheme 1. Schematic Illustration of Dynamic Hydrogels Based on Zwitterionic Copolymers with Pendent Benzoxaborole and Sugar Residues
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2. Experimental Section 2.1. Materials. 2-Methacryloyloxyethyl phosphorylcholine (MPC) was kindly donated by Prof. Ishihara’s lab (Tokyo University, Japan). 5-Methacrylamido-1,2-benzoxaborole (MAABO),34, 36 2-gluconamidoethyl methacrylamide (GAEMA),51 4-cyano-4-(thiobenzoylthio)pentanoic acid (CTP),52 and fluorescein isothiocyanate labeled bovine serum albumin (FITC-BSA)53 were synthesized as previously reported. 4,4’-Azobis (4-cyanovaleric acid) (ACVA), rhodamine B (RhB), glucose, fructose, and thiazolyl blue tetrazolium bromide (MTT) were purchased from Sigma-Aldrich. All cell culture products, including DMEM medium, antibiotics, fetal bovine serum (FBS), and trypsin with EDTA, were obtained from Gibco. All organic solvents, including methanol and dimethyl formamide (DMF), were purchased from Caledon Laboratories Ltd. (Canada) and used without further purification. 2.2. Characterization. 1H NMR spectra of the monomers and copolymers were recorded on a Varian 500 MHz spectrometer using D2O, DMSO-d6, or D2O/DMSO-d6 mixture as solvent. The number (Mn) and weight (Mw) average molecular weights and polydispersity (PDI = Mw/Mn) of the zwitterionic copolymers were determined by Viscotek conventional gel permeation chromatography (GPC) system equipped with two WAT011545 Waters Ultrahydrogel linear columns using 0.5 M sodium acetate/0.5 M acetic acid buffer as eluent at a flow rate of 1.0 mL/min.34 The GPC was calibrated by monodisperse pullulan standards (Mw = 5900-404,000 g/mol). The porous morphology of hydrogels was observed by field emission scanning electron microscopy (FESEM, Zeiss Sigma 300/VP). Three hydrogels with different sugar contents at the same pH of 7.4 were freeze-dried and sputter-coated with gold to provide a conductive environment prior to SEM characterization.
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2.3. Synthesis of Zwitterionic Copolymers with Pendent Benzoxaborole/Sugar Groups. The zwitterionic statistical copolymers were synthesized via RAFT polymerization method using CTP as chain transfer agent (CTA) and ACVA as initiator. We found that the polymer chain length is of great importance for the hydrogel formation and hydrogel strength.29, 34 We first tried to synthesize zwitterionic copolymers with targeted degree of polymerization of 200, which, however, could only form hydrogels at high pH solutions (usually over pH 9). Then, we synthesized zwitterionic copolymers with higher targeted DP of 400. The resulting copolymers could form hydrogels under all the pH conditions (pH 7.4, 8.4, and 9.4). The easier formation of hydrogels by copolymers with longer chain length could be explained by the higher valency (more crosslinking sites) per polymer chain which improves the formation of hydrogel networks. Therefore, only the synthesis of copolymers with DP of 400 is presented here. For the synthesis of PMB copolymer with a fixed benzoxaborole content of 15%, MPC (1.003 g, 3.4 mmol), MAABO (130.2 mg, 0.6 mmol), CTP (2.8 mg, 0.01 mmol), and ACVA (0.7 mg, 2.5 µmol) were placed in a 50 mL polymerization tube and dissolved with a mixture of methanol, DMF, and 10% of DI water. After degassing with nitrogen for 30 min, the polymerization was carried out at 70 °C for 24 h. The reaction was terminated by rapid cooling in liquid nitrogen and exposure to air and the resultant copolymer was isolated after precipitation in acetone. The product was further dialyzed against DI water for 24 h and lyophilized to get PMB as light brown solid (84.7% yield). For the sugar decorated copolymers, three copolymers with different sugar contents, i.e. 20%, 50%, and 80%, were synthesized and denoted as PMG-20%, PMG-50%, and PMG-80%, respectively. Take PMG-20% as an example, GAEMA (244.8 mg, 0.8 mmol) was first dissolved in 8 mL of DI water, then mixed with MPC (944 mg, 3.2 mmol), CTP (2.8 mg, 0.01 mmol), and
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ACVA (0.7 mg, 2.5 µmol) in 4 mL of methanol. The mixture was placed in a 50 mL polymerization tube and degassed using nitrogen for 30 min. After reacting at 70 °C for 24 h, the polymerization was stopped by rapid cooling in liquid nitrogen and exposure to air, and the resultant polymer was isolated by precipitation in acetone. The PMG-20% copolymer (85.8% yield) could be obtained after dialyzing against DI water for 24 h (MWCO of dialysis bag = 6000-8000) and freeze-drying. PMG-50% and PMG-80% were synthesized in similar way and obtained with a yield of 81.6% and 65.8%, respectively. 2.4. Fabrication and Characterization of Hydrogels. PMB and PMG (20%, 50%, and 80% sugar content) copolymers were first dissolved in PBS solutions with different pHs (pH 7.4, 8.4, and 9.4) at a fixed polymer concentration of 10 wt%. To investigate the impact of pH and sugar content on the mechanical properties of hydrogels, an array of nine hydrogels (3×3, hydrogels were denoted as PMBG-X-Y, where X represents sugar content and Y represents pH) were prepared by simply mixing the same volume of PMB and PMG stock solutions via pipetting. The gelation was observed by test tube inversion method and the hydrogels were allowed to be stored in the dark overnight to reach equilibrium before rheological tests. The mechanical and selfhealing properties were studied by an AR-G2 rheometer (TA instruments) with a 20 mm 2.008º cone plate geometry at 25 °C.36 To compare the mechanical properties, frequency sweeps of all the nine hydrogels were tested from 0.1 rad/s to 100 rad/s with a constant strain of 1%. The crossover angular frequency (ωc) of every hydrogel was recorded when storage modulus (G') is equal to loss modulus (G"). PMBG-50%-7.4 hydrogel was used as an example to study the selfhealing ability. The hydrogel was first tested by oscillatory strain amplitude sweep from 0.1% to 500% at a constant frequency of 10 rad/s to determine the critical stain required for gel failure. Then, step-stain test was performed by repeating large strain (200%, 30 s) for network disruption
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and small stain (1%, 60 s) for mechanical recovery. The self-healing ability was also tested by putting three hydrogel cubes together for ca 20 s before lifting on its own weight and stretching. The stability of reformed hydrogel in PBS solution was further studied by immersing the reformed hydrogel in pH 7.4 PBS solution for 4 h. To study the shear-thinning property, PMBG50%-7.4 hydrogel was loaded into a syringe and injected into pH 7.4 PBS solution through a 21G needle. 2.5. pH and Sugar Responsive Behaviors of Hydrogels. PMBG-50%-7.4 hydrogel was used to study the pH and sugar responsiveness. The hydrogel was stained with RhB dye for better observation. To test the pH-responsiveness, 5 µL of 1 M HCl solution was added to the hydrogel, followed by vigorous shaking. After the hydrogel was totally dissociated into sol state, 10 µL of 1 M NaOH solution was added for regelation. The cyclic addition of HCl-NaOH was repeated for 2 times and the reversible gel-sol-gel transitions were recorded. To investigate the sugar responsiveness, hydrogels were immersed in PBS (pH 7.4) solution with or without 50 mM fructose. The changes of hydrogels were recorded at different time intervals. RhB and FITC-BSA loaded hydrogels were further used to test the potential application for pH and sugar responsive controlled drug release. To test the pH responsiveness, RhB was loaded into PMBG-50%-7.4 hydrogels at a concentration of 200 µg/mL. 0.2 g of hydrogel was placed into an Eppendorf tube (1.5 mL), and 1.2 mL of PBS (pH 7.4 and pH 6.8) was added. 0.4 mL of the supernatant was taken out at predetermined time intervals and the same amount of fresh medium was replenished. The absorbance of released RhB solutions were obtained by UV-Vis spectrometer (V-630, JASCO, Japan) and the concentrations were calculated by standard curve method. To test the sugar responsiveness, FITC-BSA was loaded into PMBG-50%-7.4 hydrogels at a concentration of 1 mg/mL. 0.2 g of hydrogel was placed into an Eppendorf tube (1.5 mL),
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and 1.2 mL of PBS (pH 7.4, with/without 4 g/L glucose) was added. 0.6 mL of the supernatant was taken out at predetermined time intervals and the same amount of fresh medium was replenished. FITC fluorescent intensity (Ex: 485 nm, Em: 535 nm) was then analysed on a TECAN Genios pro microplate reader to determine the concentration of released FITC-BSA.6 2.6. Cell Cytotoxicity. Gel extracts of PMBG-50%-7.4 hydrogel were applied to examine the biocompatibility of hydrogels using both normal skin fibroblast cells (NSFB) and HeLa cancer cells. Briefly, 400 mg of 10wt% hydrogel was incubated in 4 mL of low glucose DMEM medium for 48 h. NSFB and HeLa cells were seeded onto 96-well plates at a density of 6000 cells per well with 200 µL of DMEM medium. The cells were incubated at 37 °C in a balanced air humidified incubator with an atmosphere of 5% CO2. After incubation for 24 h, the culture media were replaced by 200 µL of fresh DMEM media or DMEM media containing gel extracts. The cells were allowed to be further incubated for 24 h or 48 h before addition of 20 µL of MTT solution (5 mg/mL). After 4 h incubation, the media were carefully removed and DMSO was added to dissolve the formazan crystals. The absorbance at 570 nm was obtained by TECAN Genios pro microplate reader and cell viability was calculated by comparing O.D. values of cells treated with/without gel extracts.
3. Results and Discussion 3.1. Synthesis and Characterization of Zwitterionic Copolymers Bearing Benzoxaborole and Sugar Groups. MAABO and GAEMA monomers were firstly synthesized according to our previous reports34, 51 and characterized by 1H NMR (Figure S1-2). The purity was confirmed by comparing the integrals of the typical phenyl protons in MAABO (δ 7.3, 7.7, and 8.1 ppm) and sugar protons in GAEMA (δ 4.0 and 4.3 ppm) with integrals of methacrylate peaks (δ 5.5 and 5.8
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ppm for MAABO, δ 5.4 and 5.6 ppm for GAEMA). Then, RAFT copolymerization of MPC with MAABO (PMB) or GAEMA (PMG) was conducted as illustrated in Scheme 2 with a targeted degree of polymerization (DP) of 400. The statistical zwitterionic copolymers were characterized by 1H NMR and GPC, and the detailed results are summarized in Table 1. Unlike the conventional protection/deprotection method to synthesize glycopolymers, Narain and Armes developed facile aqueous polymerization method to obtain various glycopolymer structures without any protection of the hydroxyl groups.47,
48
Therefore, GAEMA could be easily
copolymerized together with the hydrophilic MPC monomer to make zwitterionic glycopolymers in one step aqueous RAFT polymerization. In order to study the impact of sugar content on the mechanical properties of the hydrogels, three PMG copolymers with different targeted sugar contents (mol%) of 20%, 50%, and 80% were synthesized and denoted as PMG-20%, PMG-50%, and PMG-80%, respectively. From the 1H NMR data shown in Figure S3, higher characteristic sugar peaks (δ 3.6-3.9 ppm) were clearly observed in PMG copolymer which was designed with higher targeted sugar ratio. By comparison of the specific peak integrals of MPC (δ 3.9-4.4 ppm, 3.1-3.3 ppm) and GAEMA (δ 4.0-4.4 ppm, 3.3-3.5 ppm ), the sugar content in these three copolymers was calculated to be 23.3%, 51.7%, and 88.7%, respectively, which is quite consistent with the designed ratio. Because of the excellent hydrophilicity of PMG copolymers, aqueous GPC was applied to determine the Mn, Mw, and PDI. As shown in Table 1, these three PMG copolymers had similar Mn of ca 90 kDa and shared narrow PDI of around 1.2, which should be attributed to the well-controlled nature of RAFT. Figure S4 showed the 1H NMR spectrum of PMB copolymer, with typical peaks of both MAABO (δ 4.9-5.2 ppm, 7.3-8.0 ppm) and MPC (δ 3.0-4.5 ppm) residues clearly observed. The benzoxaborole molar ratio in PMB copolymer was calculated to be 16.6%. The Mn and PDI of PMB copolymer were also
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determined by aqueous GPC to be 79 kDa and 1.54 (Table 1), respectively. The slightly higher PDI than PMG copolymers may be due to the existence of partial crosslinking via boroxine formation.54
Scheme 2. Synthesis of Zwitterionic Copolymers a) poly(MPC-st-GAEMA) (PMG) and b) poly(MPC-st-MAABO)
(PMB)
by
RAFT
Polymerization
Using
4-Cyano-4-
(thiobenzoylthio)pentanoic acid (CTP) as Chain Transfer Agent (CTA) and ACVA as Initiator. Table 1. Synthetic Results of PMG and PMB Copolymers composition (mol%) a
molecular weight b
polymers
MPC
GAEMA
MAABO
Mn (103)
Mw (103)
PDI (Mw/Mn)
PMG-20%
76.7
23.3
−
94.3
115.0
1.22
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a
PMG-50%
48.3
51.7
−
91.9
112.0
1.22
PMG-80%
11.3
88.7
−
90.6
112.8
1.25
PMB
83.4
−
16.6
79.0
121.4
1.54
Calculated from 1H NMR results using D2O or D2O/DMSO-d6 mixture as solvent. bObtained
from aqueous GPC using 0.5 M sodium acetate/acetic acid buffer as eluent. 3.2. Fabrication and Adjustable Mechanical Properties of Hydrogels. Prior to hydrogel preparation, PMB and PMG (20%, 50%, and 80%) copolymers were dissolved in PBS solutions (pH 7.4, 8.4, and 9.4) at the same weight ratio of 10 wt%. After sonication for 10 min, the same amount of PMB and PMG solutions were mixed together to form an array of nine hydrogels. The hydrogel formation was confirmed by test tube inversion method as illustrated in Scheme 1. All the hydrogels could be formed within ~15 s due to the efficient formation of dynamic fivemembered rings between benzoxaborole and sugar moieties. The porous network structure of PMBG hydrogels at pH 7.4 was then observed by SEM. As shown in Figure 1a and Figure S5, all the hydrogels possessed interconnected porous network structure, which could provide adequate space for cell proliferation and facilitate diffusion of oxygen and nutrients. The average pore size was about 33 µm for PMBG-20% hydrogel while smaller average pore size of around 28 µm was observed in PMBG-50% and PMBG-80% hydrogels. Moreover, the network structure of PMBG-20% hydrogel seems different from that of the other two hydrogels as looser structure with less polymers between the pores was observed for the former. Though the change of pore size by tuning sugar content of PMG copolymers (crosslinking density) was not as significant as that by the widely reported method of tuning the polymer weight ratio in the hydrogels,55,
56
the current study still shows potential as another approach to design tailored
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porous microenvironment of hydrogels for specific biomedical applications by further tuning the sugar : benzoxaborole ratio. As is well-known, the formation of arylboronic esters is highly pH-dependent, which could be used to easily adjust the hydrogel strength. In addition, previous reports have also shown the impact of the molar ratio between diol groups and ABA groups on the mechanical properties of hydrogels.27-29 However, the highest ABA ratio in the designed polymers is limited, usually lower than 20%,29 mainly due to the hydrophobicity of most ABAs. In this regard, it will be advantageous to tune the sugar ratio in the polymer chain as sugar ratio can be designed as high as 100% (glyco-homopolymer) without the concern of solubility. Therefore, nine hydrogels with different sugar contents (20%, 50%, and 80%) at different pH buffers (7.4, 8.4, and 9.4) were prepared and compared via dynamic rheological measurements. The storage modulus (G') and loss modulus (G") of nine hydrogels as functions of frequency at a constant strain of 1% were displayed in Figure 1b-d. Unlike hydrogels formed by permanent covalent bonds which exhibit frequency-independent G', all the nine hydrogels showed frequency-dependent viscoelastic behavior, which is a typical feature for dynamic hydrogels and consistent with previous reports.6, 26
At low frequencies, the hydrogels behaved like liquid with G" > G' while at high frequencies,
solid-like state was observed with G' > G". The crossover angular frequencies (ωc, when G' = G") of all the nine hydrogels were summarized in Figure 1e. As expected, both sugar content and pH influenced the mechanical properties, with lower ωc observed in hydrogels with higher sugar content and higher pH. For example, hydrogel with 20% sugar content at pH 7.4 had the highest ωc of 9.6 rad/s while hydrogel with 80% sugar content at pH 9.4 displayed the lowest ωc of 1.6 rad/s. It was also found that the influence of sugar content to ωc at higher pHs of 8.4 and 9.4 was less significant than that at pH 7.4, which could possibly be explained by the enormously
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enhanced binding affinity of benzoxaborole with the sugar at higher pH. Hydrogels with lower ωc are considered to be more elastic and stronger as the mechanical properties are dominated by the elastic component G' over a wider range of angular frequency.6 To better understand the impact of the sugar content and pH on the mechanical properties, the G' values of the nine hydrogels at a fixed frequency of 10 rad/s were collected in Figure 1f. Higher G' values were observed in hydrogels with higher sugar content and higher pH, which coincided well with ωc results in Figure 1e. The G' value could be tuned in a broad modulus range from 261.2 Pa (20% sugar, pH 7.4) to 2638.1 Pa (80% sugar, pH 9.4), indicating great potential for designing hydrogels with tailored mechanical properties to serve as mimetic ECM for cell culture and tissue engineering.
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Figure 1. a) SEM image of PMBG-50%-7.4 hydrogel. Dynamic oscillatory frequency sweeps of 10 w% PMBG hydrogels formed at a) pH 7.4, b) pH 8.4, and c) pH 9.4. e) Crossover frequencies (ωc) and f) storage modulus data (G' at γ = 1%, ω = 10 rad/s) obtained from b-d.
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3.3. Self-Healing and Injectable Properties. Hydrogels with self-healing ability can offer advantages of extended lifetime and improved performance in potential biomedical applications.9 To investigate self-healing properties, PMBG-50%-7.4 hydrogel was selected as an example and firstly tested by oscillatory stain sweep (γ = 0.1% to 500%) to determine the linear viscoelastic region and crossover strain for gel-sol transition. As shown in Figure 2a, beyond the linear viscoelastic plateau, the G' value decreased dramatically to be lower than the G" value above a critical strain of 170%, indicating disruption of gel network and liquid-like state. Afterwards, step strain tests (γ = 1% or 200%) were performed on the hydrogel to test the self-healing ability. When a large strain of 200% was forced on the hydrogel, remarkable decrease of the G' value from ≈ 1100 Pa to ≈ 400 Pa was immediately observed in Figure 2b, together with the exceeding of G" over G' which demonstrated gel failure. However, the G' value could recover to around 95% of the initial value in 60 s under a small strain of 1%. Moreover, the recovery behaviour was reproducible over cyclic tests, which is highly desired in practical applications. In addition, selfhealing of PMBG-50%-7.4 hydrogel was further testified visually for more straightforward observation. As shown in Figure 2c and Movie S1, three pieces of hydrogel cubes were put together without external force and allowed to be healed for about 20 s. The reformed hydrogel was found to be strong enough to support its own weight when lifted and could even be stretched by forceps without rupture at the joints. The excellent self-repair ability should be attributed to the efficient rearrangement of benzoxaborole-sugar interactions across the attached interfaces. The stability of reformed hydrogel in PBS solution was further investigated as shown in Figure S6. The reformed hydrogel from two pieces of hydrogel was immersed in pH 7.4 PBS solution and incubated for 4 h. The reformed hydrogel kept good stability with only some leakage of encapsulated RhB dye and some extent of swelling observed. The hydrogel could be further
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stretched without rupture and the stretched hydrogel still exhibited excellent self-healing ability, which was confirmed by lifting on its own weight. Therefore, the good stability of reformed hydrogel further indicated its great potential for biomedical applications. The injectable property of PMBG-50%-7.4 hydrogel was further confirmed by extruding the hydrogel into pH 7.4 PBS solution through a 21-G needle. Continuous extrusion of RhB dye incorporated hydrogel was easily achieved as shown in Figure 3a and Movie S2. Moreover, the hydrogel could serve as ink to write typical letters of “U of A” (Figure 3b), indicating the promising application in 3D bioprinting.
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Figure 2. a) Oscillatory stain sweep, b) step-strain test, and c) demonstration of self-healing property of PMBG-50%-7.4 hydrogel.
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Figure 3. Injectable properties of PMBG-50%-7.4 hydrogel. a) Extrusion of hydrogel into pH 7.4 PBS by a 21-G needle. b) Writing “U of A” by using hydrogel as ink.
3.4. pH and Sugar Responsiveness. The binding affinity of arylboronic ester is well-known to be pH-dependent with reversible formation and dissociation occurring at pH higher and lower than the pKa, respectively. Therefore, hydrogels based on arylboronic esters could undergo reversible gel-sol-gel transitions upon change of pH.24 As shown in Figure 4a, by acidifying the solution (addition of 5 µL of 1 M HCl), PMBG-50%-7.4 hydrogel turned rapidly into sol state, indicating the fast decrosslinking of the hydrogel network induced by dissociation of benzoxaborole-sugar interactions. However, the hydrogel could be reformed after the addition of NaOH, which demonstrated a network regenerating process. Moreover, the gel-sol-gel phase transition can be carried out on several cycles without any detrimental effect on the gel intrinsic properties.
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The dynamic arylboronic ester can also be dissociated by the addition of competitive molecules containing diol moieties (such as saccharides and dopamine).24 Here, fructose was chosen as the competitive molecule due to its higher binding affinity with ABAs than glucose.24 As shown in Figure 4b, two pieces of RhB dyed hydrogel cubes were immersed in pH 7.4 PBS solutions with and without 50 mM fructose, respectively. It could be clearly observed that hydrogel in the fructose solution (50 mM) degraded over time and complete disappearance of the solid gel substance was realized in 80 min. However, hydrogel incubated in PBS solution without fructose was quite stable with no obvious volume change observed. The pH/sugar responsiveness of PMBG hydrogels could offer promising application in controlled therapeutic delivery. The unique physiological conditions of certain diseases, such as the weakly acidic (pH ≈ 6.8, lower than pKa of benzoxaborole) microenvironment in tumor tissues57 and high blood glucose level (400 mg/dL) in diabetic patients,58 could be utilized to trigger dissociation of PMBG hydrogels and subsequent therapeutic drug release. Here, we applied RhB as a hydrophilic small molecular model drug and FITC-BSA as a macromolecular protein model drug. As shown in Figure S7a, faster release of RhB at pH 6.8 than at pH 7.4 was observed, with 58.8% and 48.6% RhB released within the first 24 h at pH 6.8 and pH 7.4, respectively. To verify the glucose responsiveness, the hydrogel was incubated in 4g/L glucose solution which mimics hyperglycemia in diabetic patients. As shown in Figure S7b, 63.0% of FITC-BSA was released in PBS solution with glucose in 48 h, while for glucose-free solution, the value was only 53.6%. The difference in release rate of drugs in the current study may not be very significant but still correspond to the results of some published reports,6, 59 which could be influenced by both swelling and hydrolysis of hydrogels.60 Nevertheless, we found that under the same condition of pH 7.4, the release rate of RhB was faster than that of FITC-BSA, which
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revealed the effect of molecular weight on the release kinetics, that is, drugs with smaller molecular weight could have higher release rate probably due to easier diffusion across the porous gel network. These results could contribute to more optimized design of arylboronic ester based hydrogels for controlled drug delivery in future.
Figure 4. a) Reversible gel-sol-gel transition by changing pH. b) Hydrogel stability in PBS with (left vial) and without (right vial) 50 mM of fructose. Hydrogels were stained with RhB dye for better observation.
3.5. Cell Cytotoxicity. Biocompatibility is one of the essential requirements for biomedical applications of hydrogels. Therefore, both normal skin fibroblast cells (NSFB) and cancerous Hela cells were incubated with PMBG-50%-7.4 gel extracts and cell viabilities were achieved by standard MTT assay as shown in Figure 5. After 24 h incubation, cell viabilities of 99.6% and 92.8% were retained for NSFB and Hela cells, respectively. For longer incubation time of 48 h, a
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little bit lower yet still high cell viabilities of 92.5% and 88.8% were obtained correspondingly. In spite of the slight difference of cell viabilities between NSFB and Hela cells, which may be due to the difference of cell lines, the gel extracts showed low cytotoxicity to both cell lines. The good biocompatibility should be ascribed to the fact that the hydrogel was formed by copolymers with cell membrane biomimetic phosphorylcholine and sugar residues which have been widely recognized as safe biomaterials.37, 50 Therefore, the zwitterionic PMBG hydrogels could serve as an ideal platform for cell encapsulation and in vivo bio-applications.
Figure 5. Cell viability of HeLa cancer cells and NSFB normal cells after 24 and 48 h incubation with gel extracts.
4. Conclusions In summary, zwitterionic MPC based copolymers bearing sugar and benzoxaborole groups were synthesized via RAFT polymerization. Hydrogels were formed spontaneously after mixing these two copolymers based on dynamic benzoxaborole-sugar interactions. The influence of pH and sugar content in the polymer chain were investigated by rheological measurements and it was found that hydrogels with higher pH and higher sugar content possessed higher gel strength. The
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dynamic covalent chemistry based hydrogel exhibited efficient self-healing ability and could be injected as ink to write the letters “U of A”. Moreover, the hydrogel could be dissociated by reducing the solution pH (pH < 7.2) or adding competitive sugar molecules, which further realized sustained and responsive drug release. Additionally, the hydrogel exhibited good biocompatibility due to the cell membrane bioinspired polymer structure. The zwitterionic selfhealing hydrogel could have great potential in biomedical applications such as controlled therapeutic drug delivery as well as 3D cell culture and bioprinting for regenerative medicine. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. 1
H NMR spectra of MAABO, GAEMA, PMG, and PMB. SEM images of PMBG-20%-7.4 and
PMBG-80%-7.4 hydrogels. Stability of reformed hydrogel after being immersed in PBS. Release profiles of RhB and FITC-BSA. (word) Self-healing and injectable properties of PMBG-50%-7.4 hydrogel. (video) AUTHOR INFORMATION Corresponding Author R. Narain*: E-mail:
[email protected]; T. Thundat*: E-mail:
[email protected] Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest.
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Acknowledgments This work was supported by Canada Excellent Research Chair (CERC), Natural Sciences and Engineering Research Council of Canada (NSERC) and the Canada Foundation for Innovation (CFI). Professor Hongbo Zeng is thanked for the use of the rheometer. REFERENCES (1)
Zhang, Y. S.; Khademhosseini, A. Advances in Engineering Hydrogels. Science 2017,
356. (2)
Li, J.; Mooney, D. J. Designing Hydrogels for Controlled Drug Delivery. Nat. Rev. Mater.
2016, 1, 16071. (3)
Wang, H.; Heilshorn, S. C. Adaptable Hydrogel Networks with Reversible Linkages for
Tissue Engineering. Adv. Mater. 2015, 27, 3717-3736. (4)
Jungst, T.; Smolan, W.; Schacht, K.; Scheibel, T.; Groll, J. Strategies and Molecular
Design Criteria for 3D Printable Hydrogels. Chem. Rev. 2016, 116, 1496-1539. (5)
Brown, T. E.; Anseth, K. S. Spatiotemporal Hydrogel Biomaterials for Regenerative
Medicine. Chem. Soc. Rev. 2017, 46, 6532-6552. (6)
Yesilyurt, V.; Webber, M. J.; Appel, E. A.; Godwin, C.; Langer, R.; Anderson, D. G.
Injectable Self-Healing Glucose-Responsive Hydrogels with pH-Regulated Mechanical Properties. Adv. Mater. 2016, 28, 86-91. (7)
Wei, Z.; Yang, J. H.; Zhou, J.; Xu, F.; Zrinyi, M.; Dussault, P. H.; Osada, Y.; Chen, Y. M.
Self-Healing Gels Based on Constitutional Dynamic Chemistry and Their Potential Applications. Chem. Soc. Rev. 2014, 43, 8114-8131.
ACS Paragon Plus Environment
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Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(8)
Page 26 of 33
Wang, W.; Zhang, Y.; Liu, W. Bioinspired Fabrication of High Strength Hydrogels from
Non-Covalent Interactions. Prog. Polym. Sci. 2017, 71, 1-25. (9)
Li, Q. W.; Liu, C. L.; Wen, J. R.; Wu, Y. Z.; Shan, Y.; Liao, J. F. The Design,
Mechanism and Biomedical Application of Self-Healing Hydrogels. Chin. Chem. Lett. 2017, 28, 1857-1874. (10)
Wang, Y.; Adokoh, C. K.; Narain, R. Recent Development and Biomedical Applications
of Self-Healing Hydrogels. Expert Opin. Drug Delivery 2018, 15, 1-15. (11)
Li, Y.; Rodrigues, J.; Tomas, H. Injectable and Biodegradable Hydrogels: Gelation,
Bbiodegradation and Biomedical Applications. Chem. Soc. Rev. 2012, 41, 2193-2221. (12)
Guan, Y.; Zhang, Y. Boronic Acid-Containing Hydrogels: Synthesis and Their
Applications. Chem. Soc. Rev. 2013, 42, 8106-8121. (13)
Brooks, W. L.; Sumerlin, B. S. Synthesis and Applications of Boronic Acid-Containing
Polymers: From Materials to Medicine. Chem. Rev. 2016, 116, 1375-1397. (14)
Coumes, F.; Woisel, P.; Fournier, D. Facile Access to Multistimuli-Responsive Self-
Assembled Block Copolymers via a Catechol/Boronic Acid Ligation. Macromolecules 2016, 49, 8925-8932. (15)
Deng, R.; Derry, M. J.; Mable, C. J.; Ning, Y.; Armes, S. P. Using Dynamic Covalent
Chemistry To Drive Morphological Transitions: Controlled Release of Encapsulated Nanoparticles from Block Copolymer Vesicles. J. Am. Chem. Soc. 2017, 139, 7616-7623. (16)
Naito, M.; Ishii, T.; Matsumoto, A.; Miyata, K.; Miyahara, Y.; Kataoka, K. A
Phenylboronate-Functionalized Polyion Complex Micelle for ATP-Triggered Release of siRNA. Angew. Chem. Int. Ed. 2012, 51, 10751-10755.
ACS Paragon Plus Environment
26
Page 27 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
(17)
Jia, H. Z.; Zhu, J. Y.; Wang, X. L.; Cheng, H.; Chen, G.; Zhao, Y. F.; Zeng, X.; Feng, J.;
Zhang, X. Z.; Zhuo, R. X. A Boronate-Linked Linear-Hyperbranched Polymeric Nanovehicle for pH-Dependent Tumor-Targeted Drug Delivery. Biomaterials 2014, 35, 5240-5249. (18)
Kim, J.; Lee, Y. M.; Kim, H.; Park, D.; Kim, J.; Kim, W. J. Phenylboronic Acid-Sugar
Grafted Polymer Aarchitecture as a Dual Stimuli-Responsive Gene Carrier for Targeted AntiAngiogenic Tumor Therapy. Biomaterials 2016, 75, 102-111. (19)
Scarano, W.; Duong, H. T.; Lu, H.; De Souza, P. L.; Stenzel, M. H. Folate Conjugation to
Polymeric Micelles via Boronic Acid Ester to Deliver Platinum Drugs to Ovarian Cancer Cell Lines. Biomacromolecules 2013, 14, 962-975. (20)
Diaz-Dussan, D.; Nakagawa, Y.; Peng, Y.-Y.; C, L. V. S.; Ebara, M.; Kumar, P.; Narain,
R. Effective and Specific Gene Silencing of Epidermal Growth Factor Receptors Mediated by Conjugated Oxaborole and Galactose-Based Polymers. ACS Macro Lett. 2017, 6, 768-774. (21)
Deng, Z.; Qian, Y.; Yu, Y.; Liu, G.; Hu, J.; Zhang, G.; Liu, S. Engineering Intracellular
Delivery Nanocarriers and Nanoreactors from Oxidation-Responsive Polymersomes via Synchronized Bilayer Cross-Linking and Permeabilizing Inside Live Cells. J. Am. Chem. Soc. 2016, 138, 10452-10466. (22)
Tao, F.; Qin, L.; Wang, Z.; Pan, Q. Self-Healable and Cold-Resistant Supercapacitor
Based on a Multifunctional Hydrogel Electrolyte. ACS Appl. Mater. Interfaces 2017, 9, 1554115548. (23)
He, L.; Fullenkamp, D. E.; Rivera, J. G.; Messersmith, P. B. pH Responsive Self-Healing
Hydrogels Formed by Boronate-Catechol Complexation. Chem. Commun. 2011, 47, 7497-7499.
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Biomacromolecules 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
(24)
Page 28 of 33
Pettignano, A.; Grijalvo, S.; Haring, M.; Eritja, R.; Tanchoux, N.; Quignard, F.; Diaz
Diaz, D. Boronic Acid-Modified Alginate Enables Direct Formation of Injectable, Self-Healing and Multistimuli-Responsive Hydrogels. Chem. Commun. 2017, 53, 3350-3353. (25)
Deng, C. C.; Brooks, W. L. A.; Abboud, K. A.; Sumerlin, B. S. Boronic Acid-Based
Hydrogels Undergo Self-Healing at Neutral and Acidic pH. ACS Macro Lett. 2015, 4, 220-224. (26)
Guo, R.; Su, Q.; Zhang, J.; Dong, A.; Lin, C.; Zhang, J. Facile Access to Multisensitive
and Self-Healing Hydrogels with Reversible and Dynamic Boronic Ester and Disulfide Linkages. Biomacromolecules 2017, 18, 1356-1364. (27)
Oda, H.; Konno, T.; Ishihara, K. The Use of the Mechanical Microenvironment of
Phospholipid Polymer Hydrogels to Control Cell Behavior. Biomaterials 2013, 34, 5891-5896. (28)
Oda, H.; Konno, T.; Ishihara, K. Efficient Differentiation of Stem Cells Eencapsulated in
a Cytocompatible Pphospholipid Polymer Hydrogel with Tunable Physical Pproperties. Biomaterials 2015, 56, 86-91. (29)
Chantasirichot, S.; Inoue, Y.; Ishihara, K. Amphiphilic Triblock Phospholipid
Copolymers Bearing Phenylboronic Acid Groups for Spontaneous Formation of Hydrogels with Tunable Mechanical Properties. Macromolecules 2014, 47, 3128-3135. (30)
Huang, Q.; Zou, Y.; Arno, M. C.; Chen, S.; Wang, T.; Gao, J.; Dove, A. P.; Du, J.
Hydrogel Scaffolds for Differentiation of Adipose-Derived Stem Cells. Chem. Soc. Rev. 2017, 46, 6255-6275. (31)
Ma, R.; Shi, L. Phenylboronic Acid-Based Glucose-Responsive Polymeric Nanoparticles:
Synthesis and Applications in Drug Delivery. Polym. Chem. 2014, 5, 1503-1518.
ACS Paragon Plus Environment
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Page 29 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Biomacromolecules
(32)
Dowlut, M.; Hall, D. G. An Improved Class of Sugar-Binding Boronic Acids, Soluble
and Capable of Complexing Glycosides in Neutral Water. J. Am. Chem. Soc. 2006, 128, 42264227. (33)
Pal, A.; Berube, M.; Hall, D. G. Design, Synthesis, and Screening of a Library of
Peptidyl Bis(Boroxoles) as Oligosaccharide Receptors in Water: Identification of a Receptor for the Tumor Marker TF-Antigen Disaccharide. Angew. Chem. Int. Ed. 2010, 49, 1492-1495. (34)
Kotsuchibashi, Y.; Agustin, R. V. C.; Lu, J.-Y.; Hall, D. G.; Narain, R. Temperature, pH,
and Glucose Responsive Gels via Simple Mixing of Boroxole- and Glyco-Based Polymers. ACS Macro Lett. 2013, 2, 260-264. (35)
Kotsuchibashi, Y.; Ebara, M.; Sato, T.; Wang, Y.; Rajender, R.; Hall, D. G.; Narain, R.;
Aoyagi, T. Spatiotemporal Control of Synergistic Gel Disintegration Consisting of Boroxoleand Glyco-Based Polymers via Photoinduced Proton Transfer. J. Phys. Chem. B 2015, 119, 2323-2329. (36)
Wang, Y. N.; Li, L.; Kotsuchibashi, Y.; Vshyvenko, S.; Liu, Y.; Hall, D.; Zeng, H. B.;
Narain, R. Self-Healing and Injectable Shear Thinning Hydrogels Based on Dynamic OxaboroleDiol Covalent Cross-Linking. ACS Biomater. Sci. Eng. 2016, 2, 2315-2323. (37)
Matsuno, R.; Ishihara, K. Integrated Functional Nanocolloids Covered with Artificial
Cell Membranes for Biomedical Applications. Nano Today 2011, 6, 61-74. (38)
Chen, Y.; Li, Z.; Wang, H.; Wang, Y.; Han, H.; Jin, Q.; Ji, J. IR-780 Loaded
Phospholipid Mimicking Homopolymeric Micelles for Near-IR Imaging and Photothermal Therapy of Pancreatic Cancer. ACS Appl. Mater. Interfaces 2016, 8, 6852-6858.
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(39)
Page 30 of 33
Ishihara, K.; Mu, M.; Konno, T.; Inoue, Y.; Fukazawa, K. The Unique Hydration State of
Poly(2-Methacryloyloxyethyl Phosphorylcholine). J. Biomater. Sci. Polym. Ed. 2017, 28, 884899. (40)
Iwasaki, Y.; Ishihara, K. Phosphorylcholine-Containing Polymers for Biomedical
Applications. Anal. Bioanal. Chem. 2005, 381, 534-546. (41)
Jin, Q.; Chen, Y.; Wang, Y.; Ji, J. Zwitterionic Drug Nanocarriers: A Biomimetic
Strategy for Drug Delivery. Colloids Surf. B. Biointerfaces 2014, 124, 80-86. (42)
Chen, Y.; Han, H.; Tong, H.; Chen, T.; Wang, H.; Ji, J.; Jin, Q. Zwitterionic
Phosphorylcholine-TPE Conjugate for pH-Responsive Drug Delivery and AIE Active Imaging. ACS Appl. Mater. Interfaces 2016, 8, 21185-21192. (43)
Tong, H.; Chen, Y.; Li, Z.; Li, H.; Chen, T.; Jin, Q.; Ji, J. Glutathione Activatable
Photosensitizer-Conjugated Pseudopolyrotaxane Nanocarriers for Photodynamic Theranostics. Small 2016, 12, 6223-6232. (44)
Harvey, A. C.; Madsen, J.; Douglas, C. W.; MacNeil, S.; Armes, S. P. Antimicrobial
Graft Copolymer Gels. Biomacromolecules 2016, 17, 2710-2718. (45)
Su, T.; Liu, Y.; He, H.; Li, J.; Lv, Y.; Zhang, L.; Sun, Y.; Hu, C. Strong Bioinspired
Polymer Hydrogel with Tunable Stiffness and Toughness for Mimicking the Extracellular Matrix. ACS Macro Lett. 2016, 5, 1217-1221. (46)
McRae Page, S.; Parelkar, S.; Gerasimenko, A.; Shin, D. Y.; Peyton, S. R.; Emrick, T.
Promoting Cell Adhesion on Slippery Phosphorylcholine Hydrogel Surfaces. J. Mater. Chem. B 2014, 2, 620-624. (47)
Narain, R.; Armes, S. P. Direct Synthesis and Aqueous Solution Properties of Well-
Defined Cyclic Sugar Methacrylate Polymers. Macromolecules 2003, 36, 4675-4678.
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Biomacromolecules
(48)
Narain, R.; Armes, S. P. Synthesis of Low Polydispersity, Controlled-Structure Sugar
Methacrylate Polymers under Mild Conditions without Protecting Group Chemistry. Chem. Commun. 2002, 2776-2777. (49)
Quan, S.; Kumar, P.; Narain, R. Cationic Galactose-Conjugated Copolymers for
Epidermal Growth Factor (EGFR) Knockdown in Cervical Adenocarcinoma. ACS Biomater. Sci. Eng. 2016, 2, 853-859. (50)
Miura, Y.; Hoshino, Y.; Seto, H. Glycopolymer Nanobiotechnology. Chem. Rev. 2016,
116, 1673-1692. (51)
Bhuchar, N.; Deng, Z.; Ishihara, K.; Narain, R. Detailed Study of the Reversible
Addition–Fragmentation Chain Transfer Polymerization and Co-Polymerization of 2Methacryloyloxyethyl Phosphorylcholine. Polym. Chem. 2011, 2, 632-639. (52)
Chen, D.; Xia, X.; Gu, H.; Xu, Q.; Ge, J.; Li, Y.; Li, N.; Lu, J. pH-Responsive Polymeric
Carrier Encapsulated Magnetic Nanoparticles for Cancer Targeted Imaging and Delivery. J. Mater. Chem. 2011, 21, 12682-12690. (53)
Ahmed, M.; Narain, R. Intracellular Delivery of DNA and Enzyme in Active Form Using
Degradable Carbohydrate-Based Nanogels. Mol. Pharmaceutics 2012, 9, 3160-3170. (54)
Zou, J.; Zhang, S.; Shrestha, R.; Seetho, K.; Donley, C. L.; Wooley, K. L. pH-Triggered
Reversible
Morphological
Inversion
of
Orthogonally-Addressable
Poly(3-
Acrylamidophenylboronic Acid)-Block-Poly(Acrylamidoethylamine) Micelles and Their Shell Crosslinked Nanoparticles. Polym. Chem. 2012, 3, 3146-3156. (55)
Russell, L. N.; Lampe, K. J. Oligodendrocyte Precursor Cell Viability, Proliferation, and
Morphology is Dependent on Mesh Size and Storage Modulus in 3D Poly(ethylene glycol)Based Hydrogels. ACS Biomater. Sci. Eng. 2017, 3, 3459–3468.
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Ren, K.; Cui, H.; Xu, Q.; He, C.; Li, G.; Chen, X. Injectable Polypeptide Hydrogels with
Tunable Microenvironment for 3D Spreading and Chondrogenic Differentiation of BoneMarrow-Derived Mesenchymal Stem Cells. Biomacromolecules 2016, 17, 3862-3871. (57)
Ge, Z.; Liu, S. Functional Block Copolymer Assemblies Responsive to Tumor and
Intracellular Mmicroenvironments for Site-Specific Drug Delivery and Enhanced Imaging Performance. Chem. Soc. Rev. 2013, 42, 7289-7325. (58)
Hu, X.; Yu, J.; Qian, C.; Lu, Y.; Kahkoska, A. R.; Xie, Z.; Jing, X.; Buse, J. B.; Gu, Z.
H2O2-Responsive Vesicles Integrated with Transcutaneous Patches for Glucose-Mediated Insulin Delivery. ACS Nano 2017, 11, 613-620. (59)
Shan, M.; Gong, C.; Li, B.; Wu, G. A pH, Glucose, and Dopamine Triple-Responsive,
Self-Healable Adhesive Hydrogel Formed by Phenylborate–Catechol Complexation. Polym. Chem. 2017, 8, 2997-3005. (60)
Zhao, L.; Niu, L.; Liang, H.; Tan, H.; Liu, C.; Zhu, F. pH and Glucose Dual-Responsive
Injectable Hydrogels with Insulin and Fibroblasts as Bioactive Dressings for Diabetic Wound Healing. ACS Appl. Mater. Interfaces 2017, 9, 37563-37574.
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Injectable Self-Healing Zwitterionic Hydrogels Based on Dynamic Benzoxaborole-Sugar Interactions with Tunable Mechanical Properties Yangjun Chen†, Wenda Wang†, Di Wu‡, Masanori Nagao#, Dennis G. Hall‡, Thomas Thundat*†, and Ravin Narain*†
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