Self-Healing Hydrogels of Low Molecular Weight Poly(vinyl alcohol

Subscriber access provided by READING UNIV

Article

Self-Healing Hydrogels of Low Molecular Weight Poly(vinyl alcohol) Assembled by Host-Guest Recognition Yong-Guang Jia, Jiahong Jin, Sa Liu, Li Ren, Juntao Luo, and X. X. Zhu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01707 • Publication Date (Web): 17 Jan 2018 Downloaded from http://pubs.acs.org on January 19, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 20 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

Self-Healing Hydrogels of Low Molecular Weight Poly(vinyl alcohol) Assembled by Host-Guest Recognition Yong-Guang Jiaa,*, Jiahong Jina, Sa Liua, Li Rena,*, Juntao Luob, X. X. Zhuc,* a

School of Materials Science and Engineering, South China University of Technology,

Guangzhou, 510641, China b

Department of Pharmacology, State University of New York Upstate Medical University,

Syracuse, New York 13210, United States c

Département de Chimie, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, QC,

H3C 3J7, Canada ABSTRACT: Poly(vinyl alcohol) (PVA) is a cytocompatible synthetic polymer and has been commonly used to prepare hydrogels. Bile acids and β-cyclodextrin are both natural compounds and they form stable host-guest inclusion complexes. They are attached covalently onto a low molecular weight PVA separately. Self-healing hydrogels can be easily formed by mixing the aqueous solutions of these PVA based polymers. The mechanical properties of the hydrogels can be tuned by varying the molar fractions of bile acid units on PVA. The dynamic inclusion complexation of the host-guest pair of the hydrogel allows the self-healing rapidly under ambient atmosphere and their mechanical properties could recover their original values in 1 min after incision. These PVA based polymers exhibited the good cytocompatiblity and high hemocompatibility as shown by their biological evaluations. The use of natural compounds for host-guest interaction make such gels especially convenient to use as biomaterials, an advantage over conventional hydrogels prepared through freeze-thaw method.

ACS Paragon Plus Environment

1

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

Page 2 of 20

Introduction Hydrogels are three-dimensional networks that are similar to soft biological tissues and have highly variable mechanical properties,1 and are thus important materials for numerous biomedical and industrial applications, such as tissue engineering, drug delivery systems, and medical devices.2-5 Biomedical applications usually require a combination of proper mechanical strength and self-healing,6 along with biocompatibility.7, 8 To realize such a combination still remains a great challenge,9 since emphasis is often placed on the self-healing property of a gel with a lack of consideration of biocompatibility and toxicity, or with relatively poor mechanical strength.10 Poly(vinyl alcohol) (PVA) is the only one biodegradable synthetic polymer made via free radical polymerization11 and has been widely used to make hydrogels with good cytocompatiblity, in which the pendant hydroxyl groups can be easily modified for cross-linking with biopolymers.12 These hydrogels have been extensively studied and considered as one of the hydrogels the most suitable for cell encapsulation and drug delivery applications due to its biocompatibility, nontoxicity and tunable mechanical properties.13 Compared with the other natural polymers (such as hyaluronic acid and collagen), slow degradability of PVA-based polymers render them more advantages in bioapplications where the proper stability is essential. The main strategies for making self-healable hydrogels are based on the use of dynamic covalent bonds6, 14, 15 or supramolecular interactions.16-23 Zhao and coworkers firstly discovered that PVA hydrogel prepared using the freeze/thaw method can autonomously self-repair at room temperature, where hydrogen bonding between PVA chains is at the origin of the self-healing phenomenon.10 Though numerous supramolecular PVA hydrogels have been prepared but required the use of concentrated (>20 wt%) high molecular weight (>100 kDa) PVA through a freeze/thaw process, limiting their applications as biomaterials. Other supramolecular hydrogels are based on modified PVA,1, 15 including the use of host-guest interactions.16, 24, 25 Multiple noncovalent interactions endow the material with good binding affinity and directionality.26-28 Based on the strong host-guest ternary complexation of cucurbit[8]uril (CB[8]) with guests, PVA were modified with binding guests of methyl viologen derivatives for CB[8] and Scherman et al prepared hydrogels of very high water content (up to 99.7% water by weight).1 The dynamics of the CB[8] ternary complex cross-linking allows for rapid self-healing of the materials after damage.


2

Page 3 of 20 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

The CB-based supramolecular hydrogels have been intrinsically limited by the complicated synthesis and modification as well as potential toxicity29 of CBs in biomedical applications. The use of natural compounds to construct reversible networks is an attractive strategy in biomaterials design. Cyclodextrins (CDs) are water-soluble cyclic oligomers of D-(+)-glucose units bound to each other through α-1,4-glucose bonding and are produced from enzymatic processing of starch, ensuring their biocompatibility and availability. The CDs can be easily functionalized for the preparation of hydrogels.17,

30, 31

Meanwhile, bile acids are a group of

physiologically important steroids, play a crucial role in lipid digestion, transportation and absorption32 and can form acids can form stable inclusion complex with β-CD due to the size match with the cavity of β-CD.33-36 It would be an advantage to prepare self-healing hydrogels through the use of low molecular weight PVA and the host-guest pair of natural compounds such as β-CD and bile acids in the view of both materials design and use.37 In this work, we report host-guest hydrogels made of low molecular weight PVA modified with β-CD and cholic acid (CA), the most abundant bile acid in humans, respectively (Scheme 1). The resulting supramolecular hydrogels exhibit self-healing and good biocompatible properties, making them interesting for bio-related applications. Scheme 1. (A) Synthetic scheme of PVA based polymers PVA-CA and PVA-CD and (B) illustration of the formation of PVA-CA/PVA-CD hydrogel.


3


Page 4 of 20

Experimental Section Materials. PVA (average molar mass 13,000-23,000, 98% hydrolyzed), cholic acid, βcyclodextrin,

succinic

anhydride,

ethylenediamine,

p-toluenesulfonyl

chloride,

N-(3-

dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole hydrate (HOBt) ≥97.0% and p-toluenesulfonic acid (TSA) were purchased from Aldrich and used without further purification unless otherwise stated. Amino-CA (N-(2-aminoethyl)-3,7dihydroxycholan-24-amide) was prepared as reported previously38 and amino-CD were synthesized according to the literature.39 The L929 cells were obtained from ATCC (Maryland, USA). The cell count kit-8 (CCK-8) was purchased from Shanghai Beyotime Institute (Shanghai, China). RMPI 1640 medium, calcium- and magnesium-free phosphate buffered saline (PBS, pH 7.4, 1×), fetal bovine serum, and 0.25% trypsin/EDTA were purchased from Gibco BRL Co. Ltd. (California, USA). The 0.22 µm filter membrane was purchased from Merck Millipore Ltd. (Massachusetts, USA). All other materials and solvents were of analytical reagent grade. Polymer characterization. 1H NMR spectra in D2O were recorded on a Bruker AV400 spectrometer operating at 400 MHz. Rheological characterization of the hydrogels was done with a AR 2000 rheometer (TA Instruments) equipped with a 2° steel cone geometry of 20 mm diameter and solvent trap. Rheological gel characteristics were monitored by oscillatory time sweep, frequency sweep, strain sweep and temperature sweep experiments. During time sweep experiments the G’ (storage modulus) and G’’ (loss modulus) were measured at 25 °C for a period of 5 min. Temperature sweep experiments were done with a heating rate of 1 °C/min. After each temperature increment (1 °C) and 30 s equilibration, G’ and G’’ were measured. The point at which tan δ = G’/G’’ = 1 is considered as the gel temperature (Tgel). In both time and temperature sweep experiments, a constant strain of 10% and frequency of 1 Hz was used. For


4

Page 5 of 20 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

each test, the sample was added onto the plate above the Tgel of hydrogel. When the values of G’ and G” reached a plateau under time sweep after a slow cooling to 25 °C and an equilibrium of 30 min, each sample was tested once under different oscillatory sweeps. Synthesis of PVA-Acid. PVA (4.0 g) was first dissolved in 20 mL DMSO at 80 °C. Succinic anhydride (455 mg, 5% equiv. to hydroxyl groups) and p-toluenesulfonic acid (13 mg, 5% equiv. to succinic anhydride) as catalyst were added into the solution of PVA. The esterification reaction was carried out at 50 °C for a period of 48 h. The polymer was precipitated from diethyl ether and the other impurities were removed through dialysis for 5 days in water. PVA-Acid (3.78 g) shown in Scheme 1A was obtained by drying the dialyzed solution. The grafting ratio of carboxylic acid was estimated to be ca. 5% based on the 1H NMR integration ratio of the peaks c and b in Figure 1A. Synthesis of PVA-CA. The samples of PVA-CA were prepared by a reaction between aminoCA and the carboxylic acid groups of PVA-Acid (Scheme 1) under the same conditions. For example, to prepare PVA-CA2%, PVA-Acid (500 mg) was first dissolved in 10 mL dimethylformamide (DMF) at 80 °C. Amino-CA (96 mg, 0.4 equiv. to acid groups), EDC (53 mg, 1.3 equivs. to Amino-CA) and HOBt (5 mg, 0.15 equiv. to Amino-CA) were added sequentially into the solution. The mixture was immersed in a preheated oil bath at 70 °C for 24 h. The reaction was terminated by immersing the mixture into ice water, and the polymer was purified by precipitation in ethyl ether, filtration, and dissolution in water followed by dialysis for a week and freeze-drying. The molar fraction of CA units on PVA was estimated to be ca. 2% based on the 1H NMR integration ratio of the peaks 18 and b in Figure 1B. The sample was noted as PVA-CA2%. PVA-CA1.5% and PVA-CA1.0% was synthesized under a molar ratio of amino-CA to acid groups at 0.3 and 0.2 equiv., respectively. Synthesis of PVA-CD. PVA-Acid (500 mg) was first dissolved in 50 mL water at 80 °C. Amino-CD (254 mg, 0.4 equiv. to acid groups), EDC (53 mg, 1.3 equivs. to Amino-CD) and HOBt (5 mg, 0.15 equiv. to Amino-CD) were added sequentially into the solution. The mixture was stirred at room temperature for 1 h and immersed in a preheated oil bath at 50 °C for 24 h. The reaction was cooled to room temperature and purified by dialysis for a week and freeze-


5


Page 6 of 20

drying. The molar fraction of CD units on PVA was estimated to be ca. 2% based on the 1H NMR integration ratio of the peaks 1 and b in Figure 1C. Preparation of hydrogels. To prepare the PVA-CA/PVA-CD hydrogels, PVA-CA and PVACD were individually dissolved in hot water at a concentration of 10 wt% and then mixed with a certain ratio of CA to CD units. To obtain the hydrogels with a good mechanical property, a molar ratio of [CA]/[CD] = 1.0 was used, corresponding to a PVA-CA/PVA-CD weight ratio of 0.78/1.00. Cytotoxicity Assay. L929 mouse fibroblast cells were cultured in RPMI 1640 medium (from Gibco) supplemented with 5% (v/v) fetal bovine serum (FBS, from Hyclone) in a humidified atmosphere at 37 °C and 5% CO2. Cells were seeded in 96-well plates at a density of 1500 cells/well and incubated for 24 h. The culture medium was replaced by fresh medium with different concentrations of PVA-CA/PVA-CD, and the cells were incubated further for 48 h. Fresh culture medium without polymers was used as control, and each sample was replicated in five wells. A total of 10 µL of Cell Counting Kit-8 (CCK-8, from Dojindo) solution were added to each well, and the cells were incubated further for 3.5 h in the same condition. The absorbance was measured at 450 nm with a reference wavelength of 600 nm by a BioTek synergy HT microplate reader. Cell viability (%) = (ODsample − ODblank)/(ODcontrol − ODblank) × 100%, where ODcontrol and ODsample were obtained in the absence or presence of polymer samples, respectively, and ODblank was obtained with RPMI 1640 medium and CCK-8 solution alone. Hemolysis Assay. 1 mL of fresh blood from healthy human volunteers was collected into 10 mL of PBS solution in the presence of 20 mM EDTA. Red blood cells (RBCs) were then separated by centrifugation at 1000 g for 10 min. The RBCs were washed three times with 10 mL of PBS, and re-suspended in 20 mL PBS. The diluted RBC suspension (200 µL) was mixed with PVA polymers of varying concentrations (10, 125, 250 ug/mL) by gentle vortex and then incubated at 37 °C. After 30 min, 4 h and overnight, the mixtures were centrifuged at 1000 g for 5 min. The supernatant free of hemoglobin was determined by measuring the absorbance at 540 nm using a UV-vis spectrometer. Incubations of RBCs with Triton-100 (2%) and PBS were used as the positive and negative controls, respectively. The percent hemolysis of RBCs was calculated using the following formula: RBC Hemolysis = 100% × (ODsample – ODPBS)/(ODtriton – ODPBS).


6

Page 7 of 20 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

Results and Discussion Characteristics of PVA based polymers. Low molar mass PVA was chosen as the polymer backbone due to its good biocompatibility and ease of chemical modification. Since the condensation of the hydroxyl groups with the carboxylic acid groups of CA may be restrained by steric hindrance, succinic anhydride was reacted with the OH groups of PVA to add a 4C spacer to obtain PVA-Acid with free carboxylic acid groups (Scheme 1), which was further reacted with the amino modified CA derivative to obtain PVA-CA (Scheme 1). The molar fractions of CA units in the polymers also varied from 1.0 to 1.5 and then 2.0%. Higher molar fractions of CA units than 2% make the polymer insoluble in water. An example of the 1H NMR spectrum of PVA-CA2% shows the characteristic proton peaks of CA moieties at 0.63, 0.84 and 0.91 ppm, assigned to methyl protons of CA on positions 18, 19 and 21, respectively (Figure 1).

Figure 1. 1H NMR spectra of (A) PVA-Acid, (B) PVA-CA2% and (C) PVA-CD2% in D2O at 25 °C and the assignments of their characteristic peaks. The host polymer PVA-CD shown in Scheme 1A was prepared under similar condition. Amino-CD reacted with carboxylic acid groups on PVA-Acid, resulting in PVA-CD bearing βCD pendants. The molar fraction of β-CD units on PVA-CD is estimated to be ca. 2% based on the 1H NMR integration ratio of the protons of PVA backbone versus protons at H1 on β-CD units at ca. 4.99 ppm (Figure 1).


7


Page 8 of 20

Table 1. Composition and the corresponding Tgels of PVA-CA/PVA-CD hydrogels. Hydrogels[a] Guest polymer[b] Host polymer[b] Tgel(°C)

[a]

Gel1

PVA-CA1%

PVA-CD2%

29.9

Gel2

PVA-CA1.5%

PVA-CD2%

41.0

Gel3

PVA-CA2%

PVA-CD2%

49.1

Total polymer concentration in hydrogels was kept at 10 wt% with a molar ratio of [CA]/[CD]

= 1. [b]Numbers of percentage indicate the molar fraction of CA or CD units on PVA backbones. Preparation of supramolecular hydrogels. Previous study 40, 41 showed that the high viscosity of the high molar mass PVA is inconvenient for the preparation of hydrogels and the physically crosslinked hydrogels were usually prepared through the well-known freeze/thaw cycles, limiting their applications as biomaterials. Thus, simple mixing of two low viscosity solutions seems an ideal way to prepare such hydrogels as biomaterials though rarely reported. In the present work, the low molar mass of PVA ensures the good solubility and low viscosity of PVACA and PVA-CD in water. Under time sweep, no change in G’ or G’’ is observed and G’’ always remains greater than G’ (Figure S2), suggesting the polymers are both fluids under these conditions. Such low viscosity is easy to handle in the preparation of hydrogels for bio-related applications. When PVA-CA and PVA-CD are mixed at a ratio of [CA]/[CD] = 1.0, the viscosity of such mixture sharply increases. More importantly, G’ becomes much greater than G’’ (Figure 2), indicating the formation of a hydrogel (inverted vial tests shown in Scheme 1B). In our previous work, when either [CA]/[CD] is > 1.0 or < 1, a decrease in G’ was found, thus indicating the effective formation of complementary inclusion complexes at the equimolar host-guest ratio. The affinity constant Ks between CA and CD is about 1.2 × 103 M-1 in our previous publication.36 The formation of hydrogel should be ascribed to the synergetic result of guest-host interaction and the hydrogen bonding from vinyl alcohol units. However, the PVA precursor without guesthost interaction is the only solution with low viscosity under the same conditions, indicating the guest-host interaction is the necessary to form hydrogel and the hydrogel is mainly based on such interaction. Meanwhile, the concentration of the hydrogel can be as low as 7.0 wt% (Figure S4), much lower than that of PVA hydrogels without host-guest recognition (>25 wt%).10


8

Page 9 of 20 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

Figure 2. Storage (G’, red) and loss moduli (G’’, black) of different hydrogels (Gel1, Gel2 and Gel3), PVA-CA, PVA-CD and unmodified PVA under the time sweeps (25 °C, 10 wt%). Rheological characterization of hydrogels. To study the effect of temperature on the hydrogel’s rheological properties, oscillatory temperature sweep test was performed. Figure 3A shows the variation of G’ and G’’ of the hydrogels listed in Table 1 as a function of temperature at the same concentration of 10 wt%. At low temperatures, G’ is much greater than G’’ and the higher molar fraction of CA units on PVA-CA in hydrogels leads to an increase of both G’ and G’’. Increasing temperature leads to a gradual decline of G’ and eventual crossover with G’’ at Tgel (the gelation temperature where G’ = G’’), indicating a gel−sol transition. Tgel is estimated to be at 29.9 °C for the Gel1 at a concentration of 10 wt%. Gel-sol transitions are also observed for the Gel2 and Gel3 under heating, showing an increasing trend of the Tgel value of 41.0 and 49.1 °C, respectively (Table 1). Further heating leads to a Newtonian fluid for which G’ becomes lower than G’’.


9


Page 10 of 20

Figure 3. Variation of the storage (G’, solid) and loss moduli (G’’, open) of Gel1, Gel2 and Gel3 (A) as a function of temperature (temperature sweep) and (B) as a function of strain (strain sweep, critical strain (γc) are obtained from the crossover points between G’ and G’’) at 25 °C. The gel-sol transition indicates the importance of the reversible host-guest complexation. These Tgels of hydrogels seem to be dependent on the molar fraction of CA units on PVA-CA, higher molar fraction of CA units resulting in an increase in Tgel. Such an increasing trend of both Tgel and mechanical strength may be attributed to hydrophobic-hydrophobic interactions from CA moieties. This property may be used conveniently to tune the Tgel into a practical range for certain applications To investigate the influence of deformation on the hydrogel, oscillatory strain sweep experiments for these hydrogels were measured. For example, G’ and G’’ values of Gel3 remained parallel until the strain became larger than 180%. Further increasing the strain, G’


10

Page 11 of 20 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

decreases rapidly and crosses over with G’’ at a critical strain (γc), which is obtained from the crossover points between G’ and G’’ as shown in Figure 3B. Critical strain of Gel3 is estimated to be about 890%. Gel2 and Gel1 with the lower molar fractions of CA units on PVA show the critical strains of 490% and 230%, respectively. These results indicate that the higher grafting ratios of CA units on polymers in hydrogel result in the larger tolerance of deformations. The critical strain is associated with the energy involved in the formation of physical crosslinking between polymer chains.42

Figure 4. (A) Optical evidence of self-healing: (I) initial sample (10 wt%), (II) sliced sample, (III) sliced sample rejoined for healing, and (IV) self-healed sample with a stretchable stress. (B) G’ (triangles) and G’’ (circles) values of the PVA-CA/PVA-CD hydrogel (Gel3) (10 wt%) in continuous step strain measurements (25 oC). Small strain (10%) and large strain (1000%) were applied alternatively. Self-healing property of hydrogels. Previously, ferrocene43 and azobenzene44 guest-containing polymers are both used to prepare self-healing hydrogels through the recognition with host CDcontaining polymers. In the present study, the dynamic CA-CD inclusion complexation and the


11


Page 12 of 20

hydrogen bonding between hydroxyl groups on PVA make the hydrogels self-healable. A break test was performed as shown in Figure 4A. After the hydrogel is cut and rejoined, it heals itself within less than one minute. The hydrogel self-heals in situ and no weak point was left after the self-healing. Interestingly, the healed hydrogel can be stretched as Figure 4A, indicating high efficiency of the self-healing. The dynamic complexation between CA and CD moieties leads to a quick recovery, healing the ruptured pieces quickly at the interface. Rheological experiments were also conducted to characterize the self-healing of the PVACA/PVA-CD hydrogel. For example, the step strain measurements of Gel3 under the alternating small (10%) and large (1000%) strains were carried out as shown in Figure 4B. Under a small strain of 10%, G’ is larger than G”, indicating the formation of a self-standing hydrogel. However, the G’ and G’’ values are inverted when the strain increased to 1000%, indicating a sol state formed and the host-guest interaction between CD and CA units was disrupted under such a strain. When a strain of 10% is applied again afterwards, the value of G’ immediately becomes greater than G”. It took ca. 1 min for the value of G’ to recover to its initial value. The repeatability of this transition process is confirmed by the second cycle in Figure 4B. Cytotoxicity and hemolysis assay. MTT cytotoxicity assay is a common colorimetry method to assess the metabolism and function of the cell, reflecting the influence of materials on cell proliferation and cytotoxicity. The CCK-8 results in Figure 5A show that cell proliferation and the corresponding toxicity level of the mixture of PVA-CA and PVA-CD in aqueous solution at various concentrations. The results demonstrate that the cell toxicity of PVA based polymers (below 5 g/L) are negligible to the L929 cells, confirming that they are nontoxic and would not inhibit the growth of the cells.


12

Page 13 of 20 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

Figure 5. (A) Cytotoxicity and (B) hemolysis test of mixture between PVA-CA and PVA-CD with a weight ratio of 0.78/1.00 at the various concentrations. For the hydrogels with the chemical crosslinking, they may maintain the integrality of structure and the molecules do not directly enter the circulation. However, in this work, the low molecular weight PVA from hydrogels will dissolve into water, and hemocompatibility evaluation for these polymers is necessary. Hemolysis test is an evaluation of the vitro hemolysis of medical materials and can sensitively reflect the influence of specimen on red blood cells. A higher hemolysis ratio indicates a greater destruction of the red blood cells by the material.45 In Figure 5B, the hemolysis ratios of the mixture of PVA-CA and PVA-CD at various concentrations were less than 2% after being cultured for 24 h, indicating that these polymers meet the requirements of biomaterials.46 Hence, these PVA based polymers were suitable for use in vivo as biomaterials. This may be attributed to the good biocompatibility of PVA and of the biocompound moieties of CA and CD.


13


Page 14 of 20

Conclusion In the present study, low molar mass cytocompatible PVA was selected as a polymer backbone and a natural host-guest pair of β-CD and CA were grafted onto PVA separately. Mixing the solutions of these two polymers resulted in hydrogels with a concentration as low as 7 wt%. The high molar fractions of CA on PVA lead to the strong mechanical properties of these hydrogels. The reversible host−guest networks render them self-healable with quick recovery within 1 min without diminishing their mechanical properties. These hydrogels are easier and more convenient to use than the other PVA based hydrogels so far. The host-guest pair made of natural compounds also ensures the good cytocompatiblity and high hemocompatibility of the new PVA hydrogels, as shown by the biological evaluations. The combination of cytocompatible PVA and natural host-guest pair make these simple self-healing hydrogels attractive candidates in biomedical and pharmaceutical applications. We anticipate that these hydrogels will find uses as the smart infill biomaterials for a range of biomedical applications where self-healing behavior and long stability are essential.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. 1H NMR spectra of PVA-CA, variation of storage and loss moduli of PVA-CA, PVA-CD, PVA and gel of PVA-CA1.5%/PVA-CD as a function of time, variation of storage and loss moduli of Gel3 as a function of stress and Gel1, Gel2 and Gel3 as a function of frequency. AUTHOR INFORMATION Corresponding Author Y.-G. Jia, E-mail: [email protected], L. Ren, E-mail: [email protected], X. X. Zhu, E-mail: [email protected]

Notes


14

Page 15 of 20 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

The authors declare no competing financial interests. ACKNOWLEDGMENT This work was supported by National Natural Science Foundation of China (21704026) and Foundation for Xinghua scholar of South China University of Technology. Financial support from NSERC of Canada and FQRNT of Quebec is also gratefully acknowledged. REFERENCES (1) Appel, E. A., Loh, X. J., Jones, S. T., Biedermann, F., Dreiss, C. A. and Scherman, O. A., Ultrahigh-Water-Content Supramolecular Hydrogels Exhibiting Multistimuli Responsiveness. J Am. Chem. Soc., 2012, 134, 11767-11773. (2) Drury, J. L. and Mooney, D. J., Hydrogels for Tissue Engineering: Scaffold Design Variables and Applications. Biomaterials 2003, 24, 4337-4351 (3) Bodugoz-Senturk, H., Macias, C. E., Kung, J. H. and Muratoglu, O. K., Poly(Vinyl Alcohol)Acrylamide Hydrogels as Load-Bearing Cartilage Substitute. Biomaterials, 2009, 30, 589-596. (4) Zeng, Q., Desai, M. S., Jin, H.-E., Lee, J. H., Chang, J. and Lee, S.-W., Self-Healing Elastin– Bioglass Hydrogels. Biomacromolecules, 2016, 17, 2619-2625. (5) Sun, S., Mao, L. B., Lei, Z., Yu, S. H. and Colfen, H., Hydrogels from Amorphous Calcium Carbonate and Polyacrylic Acid: Bio-Inspired Materials for "Mineral Plastics". Angew. Chem. Int. Ed., 2016, 55, 11765-11769. (6) Zhang, X. and Waymouth, R. M., 1,2-Dithiolane-Derived Dynamic, Covalent Materials: Cooperative Self-Assembly and Reversible Cross-Linking. J. Am. Chem. Soc., 2017, 139, 38223833. (7) Moutos, F. T., Freed, L. E. and Guilak, F. A., Biomimetic Three-Dimensional Woven Composite Scaffold for Functional Tissue Engineering of Cartilage, Nature Mater., 2007, 6, 162167. (8) Fung, Y. C., Biomechanics: Mechanical Properties of Living Tissues 2nd edn (Springer, 1993). (9) Sun, T. L., Kurokawa, T., Kuroda, S., Ihsan, A. B., Akasaki, T., Sato, K., Haque, M. A., Nakajima, T. and Gong, J. P., Physical Hydrogels Composed of Polyampholytes Demonstrate High Toughness and Viscoelasticity. Nature Mater., 2013, 12, 932-937.


15


Page 16 of 20

(10) Zhang, H., Xia, H. and Zhao, Y., Poly(Vinyl Alcohol) Hydrogel Can Autonomously SelfHeal. ACS Macro. Lett., 2012, 1, 1233-1236. (11) Sakai, K., Hamada, N. and Watanabe, Y., Purification and Properties of Secondary Alcohol Oxidase with an Acidic Isoelectric Point, Agri. Biol. Chem., 1985, 49, 817-825. (12) Lim, K. S., Kundu, J., Reeves, A., Poole-Warren, L. A., Kundu, S. C. and Martens, P. J., The Influence of Silkworm Species on Cellular Interactions with Novel PVA/Silk Sericin Hydrogels. Macromol. Biosci., 2012, 12, 322-332. (13) Hassan, C. M. and Peppas, N. A., Structure and Applications of Poly(vinyl alcohol) Hydrogels Produced by Conventional Crosslinking or by Freezing/Thawing Methods. Adv. Polym. Sci., 2000, 153, 37-65. (14) Deng, G., Li, F., Yu, H., Liu, F., Liu, C., Sun, W., Jiang, H. and Chen, Y., Dynamic Hydrogels with an Environmental Adaptive Self-Healing Ability and Dual Responsive Sol–Gel Transitions. ACS Macro Lett., 2012, 1, 275-279. (15) Guo, R., Su, Q., Zhang, J., Dong, A., Lin, C. and Zhang, J., Facile Access to Multisensitive and Self-Healing Hydrogels with Reversible and Dynamic Boronic Ester and Disulfide Linkages, Biomacromolecules, 2017, 18, 1356-1364. (16) Clarke, D. E., Pashuck, E. T., Bertazzo, S., Weaver, J. V. M. and Stevens, M. M., SelfHealing, Self-Assembled Beta-Sheet Peptide-Poly(Gamma-Glutamic Acid) Hybrid Hydrogels. J. Am. Chem. Soc., 2017, 139, 7250-7255. (17) Taylor, D. L. and In Het Panhuis, M., Self-Healing Hydrogels. Adv. Mater., 2016, 28, 90609093. (18) Jing, L., Li, H., Tay, R. Y., Sun, B., Tsang, S. H., Cometto, O., Lin, J., Teo, E. H. T. and Tok, A. I. Y., Biocompatible Hydroxylated Boron Nitride Nanosheets/Poly(Vinyl Alcohol) Interpenetrating Hydrogels with Enhanced Mechanical and Thermal Responses. ACS Nano, 2017, 11, 3742-3751. (19) Appel, E. A., Biedermann, F., Rauwald, U., Jones, S. T., Zayed, J. M. and Scherman, O. A., Supramolecular Cross-Linked Networks Via Host-Guest Complexation with Cucurbit[8]Uril, J. Am. Chem. Soc., 2010, 132, 14251–14260. (20) Loebel, C., Rodell, C. B., Chen, M. H. and Burdick, J. A., Shear-Thinning and Self-Healing Hydrogels as Injectable Therapeutics and for 3d-Printing, Nature Protocols, 2017, 12, 15211541.


16

Page 17 of 20 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

(21) Park, K. M., Yang, J. A., Jung, H., Yeom, J., Park, J. S., Park, K. H., Hoffman, A. S., Hahn, S. K. and Kim, K., In Situ Supramolecular Assembly and Modularmodification of Hyaluronic Acid Hydrogels for 3d Cellular Engineering, ACS Nano, 2012, 6, 2960–2968. (22) van de Manakker, F., van der Pot, M., Vermonden, T., van Nostrum, C. F. and Hennink, W. E., Self-Assembling Hydrogels Based on β-Cyclodextrin/Cholesterol Inclusion Complexes, Macromolecules 2008, 41, 1766-1773. (23) Rodell, C. B., Kaminski, A. L. and Burdick, J. A., Rational Design of Network Properties in Guest−Host Assembled and Shear-Thinning Hyaluronic Acid Hydrogels, Biomacromolecules, 2013, 14, 4125−4134. (24) Miyamae, K., Nakahata, M., Takashima, Y. and Harada, A., Self-Healing, ExpansionContraction, and Shape-Memory Properties of a Preorganized Supramolecular Hydrogel through Host-Guest Interactions. Angew. Chem. Int. Ed., 2015, 54, 8984-8987. (25) Li, G., Wu, J., Wang, B., Yan, S., Zhang, K., Ding, J. and Yin, J., Self-Healing Supramolecular

Self-Assembled

Hydrogels

Based

on

Poly(L-Glutamic

Acid.

Biomacromolecules, 2015, 16, 3508-3518. (26) Rekharsky, M. V. and Inoue, Y., Complexation Thermodynamics of Cyclodextrins. Chem. Rev., 1998, 98, 1875-1918 (27) Zheng, B., Wang, F., Dong S. and Huang, F., Supramolecular Polymers Constructed by Crown Ether-based Molecular Recognition. Chem. Soc. Rev., 2012, 41, 1621 – 1636. (28) Zhang, M., Xu, D., Yan, X., Chen, J., Dong, S., Zheng, B. and Huang, F., Self-Healing Supramolecular Gels Formed by Crown Ether Based Host-Guest Interactions. Angew. Chem. Int. Ed., 2012, 51, 7011-7015. (29) Uzunova, V. D., Cullinane, C., Brix, K., Nau, W. M. and Day, A. I., Toxicity of Cucurbit[7]Uril and Cucurbit[8]Uril: An Exploratory in Vitro and in Vivo Study. Org. Biomol. Chem., 2010, 8, 2037-2042. (30) Wei, K., Zhu, M., Sun, Y., Xu, J., Feng, Q., Lin, S., Wu, T., Xu, J., Tian, F., Xia, J., Li, G. and Bian, L., Robust Biopolymeric Supramolecular “Host−Guest Macromer” Hydrogels Reinforced

Byin

Situformed

Multivalent

Nanoclusters

for

Cartilage

Regeneration.

Macromolecules, 2016, 49, 866-875.


17


Page 18 of 20

(31) Rodell, C. B., Dusaj, N. N., Highley, C. B. and Burdick, J. A., Injectable and Cytocompatible Tough Double-Network Hydrogels through Tandem Supramolecular and Covalent Crosslinking. Adv. Mater., 2016, 28, 8419-8424. (32) Nakai, K., Tazuma, S., Nishioka, T. and Chayama, K. B., Inhibition of Cholesterol Crystallization under Bilirubin Deconjugation: Partial Characterization of Mechanisms whereby Infected Bile Accelerates Pigment Stone Formation. Biophys. Acta 2003, 1632, 48–54. (33) Chen, Y., Li, F., Liu, B.-W., Jiang, B.-P., Zhang, H.-Y., Wang, L.-H. and Liu, Y., Thermodynamic Origin of Selective Binding of

β-Cyclodextrin Derivatives with Chiral

Chromophoric Substituents toward Steroids. J. Phys. Chem. B, 2010, 114, 16147–16155. (34) Tan, Z. J., Zhu, X. X. and Brown, G. R., Formation of Inclusion Complexes of Cyclodextrins with Bile Salt Anions as Determined by NMR Titration Studies. Langmuir, 1994, 10, 1034-1039. (35) Jia, Y.-G. and Zhu, X. X., Self-Healing Supramolecular Hydrogel Made of Polymers Bearing Cholic Acid and β-Cyclodextrin Pendants. Chem. Mater., 2015, 27, 387-393. (36) Jia, Y. G., Malveau, C., Mezour, M. A., Perepichka, D. F. and Zhu, X. X., A Molecular Necklace: Threading Beta-Cyclodextrins onto Polymers Derived from Bile Acids. Angew. Chem. Int. Ed., 2016, 55, 11979-11983. (37) Tschan, M. J. L., Brulé, E., Haquette, P. and Thomas, C. M., Synthesis of Biodegradable Polymers from Renewable Resources. Polym. Chem., 2012, 3, 836-851. (38) Liu, H., Avoce, D., Song, Z. and Zhu, X. X., N-Isopropylacrylamide Copolymers with Derivatives of Cholic Acid: Synthesis and Characterization. Macromol. Rapid Commun., 2001, 22, 675-680. (39) Ohashi, H., Hiraoka, Y. and Yamaguchi, T., An Autonomous Phase TransitionComplexation/Decomplexation Polymer System with a Molecular Recognition Property. Macromolecules 2006, 39, 2614-2620. (40) H. Zhi, X. Fei, J. Tian, M. Jing, L. Xu, X. Wang, D. Liu, Y. Wang and Liu, J., A Novel Transparent Luminous Hydrogel with Self-Healing Property. J. Mater. Chem. B, 2017, 5, 57385744. (41) Y. Hou, C. Chen, K. Liu, Y. Tu, L. Zhang and Li, Y., Preparation of PVA Hydrogel with High Transparence and Investigations of Its Transparent Mechanism. RSC Adv., 2015, 5, 24023– 24030.


18

Page 19 of 20 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

(42) Wilhelm, M., Reinheimer, P. and Ortseifer, M., High sensitivity Fourier-transform rheology. Rheol. Acta 1999, 38, 349-356. (43) Nakahata, M., Takashima, Y., Yamaguchi, H. and Harada, A., Redox-Responsive SelfHealing Materials Formed from Host-Guest Polymers. Nat. Commun., 2011, 2, 511. (44) Chen, H., Ma, X., Wu, S. and Tian, H., A Rapidly Self-Healing Supramolecular Polymer Hydrogel with Photostimulated Room-Temperature Phosphorescence Responsiveness. Angew. Chem. Int. Ed., 2014, 53, 14149-14152. (45) Qiu, W., Wang, J. and Li, L., Preparation and Biological Performance of Poly(Vinyl Alcohol)/Hydroxyapatite Porous Composites Used for Cartilage Repair. RSC Adv., 2016, 6, 99940-99947. (46) Xiong, D., Deng, Y., Wang, N. and Yang, Y., Influence of Surface PMPC Brushes on Tribological and Biocompatibility Properties of UHMWPE. Appl. Surf. Sci., 2014, 298, 56-61.


19


Page 20 of 20

TOC used only


20

Self-Healing Hydrogels of Low Molecular Weight Poly(vinyl alcohol

Recommend Documents