Mussel-Inspired Thermoresponsive Polypeptide–Pluronic Copolymers

May 5, 2017 - Inspired by marine mussel adhesive proteins, polymers with catechol side groups have been extensively explored in industrial and academi...
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Mussel-Inspired Thermoresponsive Polypeptide−Pluronic Copolymers for Versatile Surgical Adhesives and Hemostasis Dedai Lu,*,† Hongsen Wang,† Ting’e Li,† Yunfei Li,† Fajuan Dou,† Shaobo Sun,‡ Hongyun Guo,§ Shiqi Liao,§ Zhiwang Yang,† Qiangbing Wei,† and Ziqiang Lei† †

Key Laboratory of Eco-environment-related Polymer Materials, Ministry of Education, Key Laboratory of Polymer Materials of Gansu Province, College of Chemistry and Chemical Engineering, Northwest Normal University, Lanzhou 730070, China ‡ School of Basic Medical Sciences, Gansu University of Chinese Medicine, Lanzhou 730000, China § Institute of Gansu Medical Science Research, Gansu Provincial Cancer Hospital, Lanzhou 730050, China S Supporting Information *

ABSTRACT: Inspired by marine mussel adhesive proteins, polymers with catechol side groups have been extensively explored in industrial and academic research. Here, Pluronic L-31 alcoholate ions were used as the initiator to prepare a series of polypeptide−Pluronic−polypeptide triblock copolymers via ring-opening polymerization of L-DOPA-N-carboxyanhydride (DOPA-NCA), L-arginine-NCA (Arg-NCA), L-cysteine-NCA (CysNCA), and ε-N-acryloyl lysine-NCA (Ac-Lys-NCA). These copolymers demonstrated good biodegradability, biocompatibility, and thermoresponsive properties. Adhesion tests using porcine skin and bone as adherends demonstrated lap-shear adhesion strengths up to 106 kPa and tensile adhesion strengths up to 675 kPa. The antibleeding activity and tissue adhesive ability were evaluated using a rat model. These polypeptide−Pluronic copolymer glues showed superior hemostatic properties and superior effects in wound healing and osteotomy gaps. Complete healing of skin incisions and remodeling of osteotomy gaps were observed in all rats after 14 and 60 days, respectively. These copolymers have potential uses as tissue adhesives, antibleeding, and tissue engineering materials. KEYWORDS: polypeptide, thermoresponsive, mussel-inspired, biocompatible, surgical adhesive

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INTRODUCTION Surgical adhesive material is believed to be the next-generation method to replace the traditional invasive surgical closure techniques, such as sutures, staples, and wires.1−3 These adhesive materials have some fascinating characteristics, such as fast application, less traumatic closure, no suture removal, less pain, excellent cosmetic result, and localized drug release.3 As marketed adhesive material products, fibrin glue and cyanoacrylates have been extensively used in many surgical procedures, even though they have some drawbacks, such as contamination with viruses or prions, relatively low bonding strength to tissues, and poisonous degradation products.4 It is challenging to develop a new class of tissue adhesives with mechanically stable, biodegradable, and biocompatible properties. Similarly, bone adhesives have made contributions to tissue reconstruction; however, clinical bone adhesives are limited by the lack of interaction with the bone surface or poor strength.5,6 There is currently an attractive clinical target for alternative bone adhesives with high adhesion strengths and degradable properties. Marine mussels have an impressive ability to cling to various surfaces under wet conditions.7−9 The byssal plaques of mussels are composed of proteins containing L-3,4-dihydrox© 2017 American Chemical Society

yphenylalanine (L-DOPA), an unusual amino acid with a pendant o-dihydroxyphenyl (catechol) functional group that can form strong covalent and noncovalent interactions with substrates.10−12 A widely suggested mechanism for this process is the oxidative conversion of the catechol group to o-quinone at maritime (i.e., slightly alkaline) pH values and subsequent chemical crosslinking via the formation of catechol−catechol adducts.13−15 Inspired by the mussel adhesion mechanism, the development of new bioadhesive materials with low toxicity and high elasticity is being pursued. Polymers carrying catechol groups have been synthesized to investigate the nature of catechol functional groups and to develop adhesives for special applications.16 Additionally, polymers incorporating DOPA-like units have been explored in systems based on polypeptides,17,18 polyacrylates, 19−25 poly(ethylene glycol), 26−28 polystyrene,29−33 and polysaccharides.4,15,34−38 These synthetic biomimetic materials could provide the next generation of orthopedic cements and surgical adhesives.16 However, most of these adhesive materials lack tunability of their adhesion Received: December 23, 2016 Accepted: May 5, 2017 Published: May 5, 2017 16756

DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766

Research Article

ACS Applied Materials & Interfaces

(0.66 g, 4.48 mmol) to a PPD solution. Pluronic L-31-poly[(DOPA)co-(Arg-co-Ac-Lys)] (PPDAL, yield 67%) was synthesized by adding a mixture of Arg-NCA (0.90 g, 4.48 mmol) and Ac-Lys-NCA (1.01 g, 4.48 mmol) to a PPD solution. The 1H NMR and 13C NMR data of these copolymers are shown in the Supporting Information. NMR Spectra. NMR spectra were recorded on a VARIAN JNMECP 600 MHz instrument using deuterium dimethyl sulfoxide (DMSO-d6) as the solvent. Molecular Mass Test. The molecular weight distributions of the resultant polymers were characterized by light scattering (DAWN EOS, Laser wavelength: 690.0 nm). Solvent: water. Refractive index: 1.330. Flow rate: 1.0 mL min−1. Thermoresponsivity Test. The transmittances of solutions were measured by increasing the temperature every 1 °C at 500 nm on a spectrophotometer equipped with water-circulated cell holder after being thermostabilized for 10 min at each testing temperature point using a 1 cm quartz cell. Contact Angle Measurement. The surface physicochemical properties of the materials were characterized by the water contact angle using an SL200KB apparatus at ambient temperature (25 °C) and the normal temperature of the human body (37 °C). The volume of the individual water droplets in all contact angle measurements was 5 μL. At least five measurements were performed on different spots and averaged. All contact angles were measured with approximately 5 s of residence time of the water droplet on the surface. Lap-Shear Adhesion Strength Measurement on Wet Tissues. The wet tissue adhesive properties were measured using our previously reported method,49 and the results are shown in Figure S10. Excess fat was removed from fresh, shaved porcine skin, obtained directly from a local slaughterhouse, and was cut into rectangular pieces (5 cm long × 1 cm wide). The cut porcine skin pieces were controlled to 1 mm, washed with sodium chloride (0.9 wt %) aqueous solution, and soaked in PBS buffer solution (pH = 7.4) for 12 h to ensure the porcine skin samples remained moist. Different concentrations of copolymers dissolved in PBS buffer solution (pH = 7.4, 0.01 M) were added to different concentrations of HRP and H2O2 PBS buffer solution, and 50 μL of the copolymer solution was spread on two pieces (1 cm × 1 cm) of porcine skin surface. After they were pressed together for different lengths of time, they were pulled apart at a rate of 5 mm min−1. The lap-shear adhesion strength was obtained by dividing the maximum load (N) observed by the area of the adhesive overlap (m2), giving the lap-shear adhesion strength in pascals (Pa = N/m2). Tensile Adhesion Strength Measurement on Porcine Femur Bones. The bone adhesive properties were measured on porcine femur bones using a universal testing machine (UTM), as shown in Figure S10. Porcine femur bones were obtained from an abattoir and were split into rectangular blocks with dimensions of approximately 50 × 10 × 10 mm. Each block was then wet sanded with grain size 80 sandpaper until a smooth and even surface was obtained. A generic fracture was created by sawing each rod into two halves. Different concentrations of copolymers dissolved in PBS buffer solution (pH = 7.4, 0.01 M) were added to 1 mg·mL−1 HRP and 1 wt % H2O2 PBS buffer solution. Then, 50 μL of the copolymer solution was spread on the bone surfaces of the two halves. After the bone pieces were pressed together for different lengths of time, they were pulled apart at a rate of 5 mm min−1. The tensile adhesion strength was obtained by dividing the maximum load (N) observed by the area of the adhesive overlap (m2), giving the tensile adhesion strength in pascals (Pa = N/ m2). Degradation Study. The degradation of PPDAC was assessed according to our previously reported study.49 Briefly, the degradation study was performed by dialysis at 37 °C every 2 days; the specification of the dialysis membrane was 3000. The PPDAC was divided into two groups and dissolved in 50 mL of PBS buffer solution (pH = 7.4). Protamex was added to only one group. At different time points, the dialysis bags were washed with water and dried at room temperature, and the dry weights were measured. The percentage of mass loss was calculated by dividing the mass loss by the initial mass (w0), as shown in the equation. wt is the mass measured at the predetermined time points.

strength, degradation rates, and elastic modulus for specific biomedical applications. Lee et al. prepared a robust tissue adhesive material consisting of catechol-functionalized chitosan and thiolterminated Pluronic F-127.4 Because of the temperature sensitivity of Pluronic F-127, the chitosan/Pluronic composite could form a hydrogel instantaneously after injection into an aqueous solution under physiological conditions. With continued interest in bioadhesive material and inspired by Lee’s work, we designed and synthesized DOPA−Pluronic− DOPA triblock copolymers via ring-opening polymerization (ROP) of L-DOPA-NCA using thermoresponsive Pluronic L31 alcoholate ions as the initiator. Moreover, according to some literature, the guanidinium ion (Gu+) can form a salt bridge with oxyanions on the protein surface through electrostatic and hydrogen bonding interactions,39−41 and thiols are highly reactive with the oxidized quinone form of catechol and double bonds via Michael-type additions.42−45 Thus, multiple forces would form between the polymer chains and tissue surfaces, and L-arginine (Arg), L-cysteine (Cys), and ε-N-acryloyl L-lysine (Ac-Lys) were incorporated into the above copolymer chains to improve the adhesion strength and curing rates. There are no previous reports of similar research. The properties of bioadhesive materials were investigated in detail, including the hydrophilicity and hydrophobicity of the copolymers at different temperatures; the effects of side groups, curing time, and curing temperature on the adhesive properties; and the degradability and cytotoxicity by mass loss and MTT assay. Finally, an animal experiment was performed to evaluate the biocompatibility, bioadhesive properties, and hemostatic properties of these polypeptide−Pluronic copolymers.

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EXPERIMENTAL SECTION

Materials. Dihydroxphenyl alanine (L-DOPA) and acryloyl chloride were purchased from Shanghai Darui Finechem Co., Ltd (Shanghai, People’s Republic of China). 8-Hydroxyquinoline was purchased from Shanghai Zhongqin Chemical Reagent Co., Ltd (Shanghai, People’s Republic of China). N-Benzyloxy carbonyl-Larginine, L-cysteine, triphosgene, L-lysine hydrochloride, horseradish peroxidase (HRP), and tetrahydrofuran (THF) were purchased from Aladdin Reagent Company (Shanghai, People’s Republic of China). Pluronic L-31 was purchased from MSC Petrochemical Plants in Jiangsu Province (Haian, People’s Republic of China). THF was removed from the water using calcium hydride. ε-N-Acryloyl lysine was synthesized according to the literature.46 Other chemicals were of analytical grade and were used without further purification. Origin 8.0 software was used to evaluate the significance of differences between results. Preparation of Monomers and Initiator. DOPA-NCA, ArgNCA, Cys-NCA, and Ac-Lys-NCA were prepared according to previously reported procedures (Supporting Information).47−50 The initiator was prepared by adding excessive metallic potassium to Pluronic L-31 until there were no air bubbles at room temperature; then, the excess metal potassium was removed. A highly transparent viscous liquid was obtained. Polymer Preparation. DOPA-NCA (1.0 g, 4.48 mmol) was dissolved in 10 mL of dry THF, and initiator (0.13 g, 0.112 mmol) and 3 mL of dry THF were added (the feed ratio of the monomer is shown in Table S1). After 3 days, we obtained a viscous yellow liquid. The resultant viscous yellow liquid was purified by washing with anhydrous ethanol, and a yellowish precipitate of Pluronic L-31-poly[DOPA] (PPD, yield 83%) was produced. Similarly, Pluronic L-31-poly[(DOPA)-co-(Arg)] (PPDA, yield 77%) was synthesized by adding Arg-NCA (0.90 g, 4.48 mmol) to a PPD solution. Pluronic L-31poly[(DOPA)-co-(Arg-co-Cys)] (PPDAC, yield 72%) was synthesized by adding a mixture of Arg-NCA (0.90 g, 4.48 mmol) and Cys-NCA 16757

DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766

Research Article

ACS Applied Materials & Interfaces Scheme 1. Preparation Process of the Copolymers

Table 1. Light Scattering Data and Grafting Ratio of Copolymers polymers

Mn (kDa)

Mw (kDa)

PDI

I/D/A/C/La

I/D/A/C/Lb

Mcc (kDa)

PPD PPDA PPDAC PPDAL

35.9 54.3 19.2 41.7

72.4 92.5 23.9 129.1

2.02 1.70 1.24 3.09

1:40:0:0:0 1:40:40:0:0 1:40:40:40:0 1:40:40:0:40

1:17:0:0:0 1:15:12:0:0 1:15:16:15:0 1:16:13:0:9

4.14 6.01 8.41 8.10

a The feed ratio of monomers. bPolymer ratios of monomers observed by 1H NMR. cMolecular mass determined based on the ratio of monomers observed by 1H NMR. I, D, A, C, and L indicate the numbers of initiator, DOPA, arginine, cysteine, and ε-N-acryloyl lysine, respectively.

Mass loss (%) =

w0 − wt × 100 w0

use of experimental animals. All experiments were approved by the local animal experiments ethical committee. Briefly, rats were anesthetized, their backs were shaved, and skin incisions (1.5 cm long and skin thickness deep) were made on the rat’s back. For this study, 20 wt % PPDAC−PPDAL PBS buffer solution containing 1 mg· mL−1 HRP and 1 wt % H2O2 was applied to the wound area (the dosage was described in Scheme 2). After 7 and 14 days, the closure skin was obtained and fixed in formaldehyde solution (3.7 wt %) for histological analysis by hematoxylin and eosin (H&E) staining. Animal Experiment for Bone Adhesion. Animal experiments for bone adhesion were performed using male white rats (150−200 g) in compliance with the guidelines of the national regulations for the use of experimental animals. All experiments were approved by the local animal experiments ethical committee. Briefly, the rats were anesthetized and their skin overlying the tibia was shaved and sterilized. After a longitudinal incision was made on the skin along the bone, the tibia was exposed by dissection of the overlying musculature. The tibia was fractured with a sharp osteotome. For the experimental group, 50 wt % PPDAC−PPDAL PBS buffer solution containing 1 mg·mL−1 HRP and 1 wt % H2O2 was applied to the osteotomy gap; in the control group, the osteotomy gap was performed without a polymer. All groups adopted external stability after surgical stitching. Standard X-rays were taken postoperatively, and animals were observed after 20, 40, and 60 days. At the 20, 40, and 60 day time

Cytotoxicity Evaluation. The cytotoxicity of the copolymer samples was evaluated using the MTT assay according to our previously reported method.49 Briefly, L929 cells (an endothelial cell line) were cultured in 96-well plates supplemented with 10% fetal bovine serum, with 1.0% penicillin−streptomycin, and 1.2% glutamine. L929 cells were seeded in wells of a 96-well plate at a density of 105 cells per well and were incubated overnight at 37 °C in a humidified atmosphere containing 5% CO2. Then, copolymers of varying concentrations (10 μL, in PBS) were added. After 24, 48, or 72 h of incubation, MTT (20 μL, 5 mg·mL−1 in PBS) solution was added to each well. After 4 h of incubation at 37 °C, the culture medium was removed and DMSO (100 μL) was added, and the dissolved solution was homogenized by shaking for approximately 10 min. The optical density (OD) of the solution was determined using a microplate reader at 490 nm. For the comparison, L-929 cells were seeded in a medium containing 0.64% phenol as positive control, and a fresh culture medium was used as the negative control. Each group was tested five times using this approach. Animal Experiment for Wound Closure. Animal experiments for wound closure were performed according to previously reported literature,51 and male white rats (150−200 g) were used, in compliance with the guidelines of the national regulations for the 16758

DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766

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Figure 1. (a) Temperature−light transmittance curves of PPD, PPDA, PPDAC, and PPDAC−PPDAL. (b) Thermoresponsive phenomenon was demonstrated in vitro by PPD aqueous solution (3 wt %). (c,d) Photographs of the water droplet shape and contact angle on the film surface of PPD. points, the rats were killed, and the bone tissue was obtained and fixed in formaldehyde solution (3.7 wt %) for histological analysis by H&E staining. In Vivo Hemostatic Ability Test. Animal experiments for hemostatic ability were performed according to previously reported literature,51 and male white rats (150−200 g) were used in compliance with the guidelines of the national regulations for the use of experimental animals. All experiments were approved by the local animal experiments ethical committee. Briefly, the rats were anesthetized and fixed on a surgical corkboard. The liver was exposed by abdominal incision, and a preweighed filter paper was placed beneath the liver. Then, a liver incision was made. Bleeding from the liver was induced to the filter paper. A 20 wt % PPDAC−PPDAL PBS buffer solution containing 1 mg·mL−1 HRP and 1 wt % H2O2 was immediately applied to the bleeding site. After 3 min, the weight of the filter paper with absorbed blood was measured and compared with a control group (no treatment).

Moreover, the solubility of PPDAC is less than PPD and PPDA because the introduction of sulfhydryl can decrease the solubility of polymers. Therefore, Rg of PPDAC is less than PPD and PPDA. According to the formula Rg2 = kMα,53 Mn and Mw of PPDAC are less than PPD and PPDA. Thermoresponsivity and Hydrophilicity. The thermoresponsivity of polymers is dependent on the effect of the hydrophobic groups or the hydrogen bonds between the polymer and water molecules.54 Temperature plays an important role in controlling the solubility of polymers. The lower critical solution temperature (LCST) was defined as the temperature when the transmittance reaches the median in the range of phase transition. LCST is one of the important characterizations of thermoresponsive polymer. The lower the temperature is below the LCST, the better the solubility of the polymer. When the temperature is higher than the LCST, the opposite result is obtained. This phenomenon can be attributed to the intermolecular hydrogen bonding and damage caused by temperature variation.55,56 As a result, the solution transmittance changed sharply as the temperature increased from 30 to 35 °C (Figure 1a). As Pluronic is a series of thermoresponsive polymers, the micellar packing through coil-to-micelle transition can lead to a sol-to-gel transition of the Pluronic aqueous solution with increasing temperature.55 It is reasonable that copolymers containing Pluronic L-31 exhibit a thermoresponsive behavior with changing temperatures. Figure 1b shows the thermoresponsive phenomenon of PPD. However, the transmittance is not identical when catechol, guanidyl, and mercapto groups were introduced.49 The relationship between the temperature and the hydrophilicity of the copolymers was investigated using the water contact angle. The water contact angle of the copolymer was approximately 34.6° at 25 °C (Figure 1c), and a larger water contact angle (approximately 62.6°) was observed at 37 °C (Figure 1d), indicating that the copolymer has switched from hydrophilic to hydrophobic. This change is caused by the weakening hydrophilicity of Pluronic as the temperature increases. The reversible transformation from hydrophilic to

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RESULTS AND DISCUSSION Polymer Synthesis and Characterization. All amino acids of NCA were synthesized under anhydrous conditions to avoid the hydrolysis of NCA. The NCA product was stored at a low temperature and kept away from moisture.48 A novel class of copolymers, including Pluronic L-31-poly[DOPA] (PPD), Pluronic L-31-poly[(DOPA)-co-(Arg)] (PPDA), Pluronic L31-poly[(DOPA)-co-(Arg-co-Cys)] (PPDAC), and Pluronic L31-poly[(DOPA)-co-(Arg-co-Ac-Lys)] (PPDAL), was synthesized via ROP of amino acid NCA derivatives using Pluronic L31 alcoholate ions as the initiator. The use of alcoholate ions as an initiator has been reported previously.52 The preparation process of the copolymers is shown in Scheme 1. A representative 1H NMR spectrum of the adhesive polymers is depicted in Figures S1−S5. Molecular Mass Analysis. The light scattering analysis shows the number-average molecular mass (Mn), weightaverage molecular mass (Mw), and polydispersity index (PDI) of the polymers containing different functional groups (Table 1). In Table 1, Mn and Mw of polymers are greater than Mc because that there may have aggregation or conformation change of polymer chains, as a result, the radius of gyration (Rg) of real chains is greater than the ideal single chain. 16759

DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766

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Figure 2. Adhesion properties of copolymers: (a−c) lap-shear adhesion strength of copolymers on porcine skin; (d) time lap-shear adhesion strength curve of PPDAC−PPDAL; (e) influence of humidity on the lap-shear adhesion strength of PPDAC−PPDAL; (f) elastic modulus of the copolymers; (g) time-elastic modulus curve of PPDAC−PPDAL; (h) tensile adhesion strength of copolymers on porcine bone after 2 h of adhesion; and (i) time-tensile adhesion strength curve of PPDAC−PPDAL on porcine bone. (j) Lap-shear adhesion strength of PPDAC−PPDAL was shown on an electronic scale. (c−g) Copolymers (20 wt %) were tested on porcine skin. All tests were conducted in PBS buffer solution containing 1 mg· mL−1 HRP and 1 wt % H2O2 at 37 °C after 24 h of adhesion, unless otherwise noted. Data are presented as the mean ± SD, n = 5. Statistically significant differences are indicated by *p < 0.05 or **p < 0.01.

influences the wet lap-shear adhesion strength. The adhesion strength increased with increasing polymer concentration (Figure 2a). At higher concentrations, the overlap area contains more polymer than it does at a lower concentration.31 HRP is a hemoprotein that can catalyze the crosslinking of phenol and aniline derivatives via decomposition of H2O2.51 These polymers containing catechol can be crosslinked by an enzymatic reaction in the presence of HRP and H2O2. In this enzymatic reaction process, HRP can combine with H2O2 to produce a complex (HRP−H2O2) that can oxidize polymers containing catechol. Polymers containing catechol can then be crosslinked under the catalyst.57 Figure 2b shows how the adhesion strength increases with increasing HRP concentration. This can be explained by the HRP-catalyzed conjugation of catechol groups and decomposition of H2O2 molecules. Obviously, the lower HRP concentration is not sufficient to catalyze catechol group crosslinking. Influence of Functional Groups on the Adhesion Strength on Wet Tissues. The adhesion strength of the

hydrophobic by changing the temperature is an important characteristic of surgical adhesives. Influence of Different Concentrations of H2O2 and HRP on the Lap-Shear Adhesion Strength on Wet Tissues. When using the strong oxidant hydrogen peroxide (H2O2) as a curing agent, DOPA was immediately converted to DOPA-quinone, which triggers intermolecular crosslinking, promoting the cohesive strength of the polymer. The DOPA oxidation reaction was accompanied by the reduction of H2O2 to water, a nontoxic product. Figure 2a shows how the wet lapshear adhesion strength of PPD changes with varying concentrations of PPD and H 2 O 2 . When the H 2 O 2 concentration was increased from 0.25 to 1%, the wet lapshear adhesion strength of the PPD was increased. This result suggested that a lower H2O2 concentration is not sufficient to oxidize all catechol. However, a further increase in the concentrations weakened the adhesion. Too much oxidation reaction results in too much crosslinking, which reduces the adhesion strength.31 Moreover, the polymer concentration also 16760

DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766

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ACS Applied Materials & Interfaces Scheme 2. Schematic Illustration Showing a Potential Mechanism of Tissue Adhesiona

a

Different functional groups are capable of forming different types of crosslinks in this system: (a,b) interfacial covalent crosslinking between dopamine and functional groups (e.g., −NH2) found on tissue surface, (c) covalent crosslinking and polymerization between dopamine moieties, (d) electrostatic interaction between guanidinium (Gu+) and oxoanions that exist ubiquitously in proteins, (e) covalent crosslinking by forming disulfide, (f) covalent crosslinking between the catechol and sulfydryl, and (g) thiol−ene click reaction.

Figure 3. (a) Time-mass loss curves of PPDAC. (b) MTT assay OD value of L929 cells was cultured with the extraction media from PPDAC. (c) Relative proliferation of L929 cells at 0.6 mg·mL−1 PPDAC.

Figure 4. (a) Photographs of wound closures. (b) H&E histological examination at different time periods. The site of the wound is indicated by red stars.

different polymers was measured using a UTM at 37 °C after 24 h. To demonstrate the wet lap-shear adhesion of these polymers, the wet lap-shear adhesion strength of PPDAC− PPDAL was shown on an electronic scale (Figure 2j). Figure 2c shows the adhesion strength of different copolymers on wet tissues. The PPD shows a high wet lap-shear adhesion strength, approximately 69.9 ± 7.24 kPa. The catechol group has a high binding affinity to diverse nucleophiles (e.g., amines and thiol) in these copolymers and can be anchored to peptides and

proteins on the tissue surface, which provides a potential mechanism for tissue adhesion of these copolymers (Scheme 2a).58−61 Moreover, it contains a catechol functional group that can be turned into a quinine under oxidizing conditions, resulting in chemical crosslinking via the formation of catechol−catechol adducts (Scheme 2b,c).10,15 To examine the influence of functional group ratios on the wet adhesion strength, the DOPA unit ratio in PPD was varied. As the DOPA ratio increased, the increase in adhesion strength decelerated 16761

DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766

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eventually stopped, suggesting that the thiol−ene click reaction reached saturation between the catechol and thiol groups (Table S1). Furthermore, because of the thiol−ene click reaction (Scheme 2g),43 the adhesion strength further increased when PPDAC and PPDAL were used at the same time. Influence of Functional Groups on the Adhesion Strength on Bone. We also tested the adhesion strengths of different polymers on bone samples to demonstrate their potential application as bone adhesives. Marine mussels affix themselves to inorganic surfaces using byssal plaques composed of proteins containing DOPA. It is not surprising that PPD, which contains abundant DOPA, has a high tensile adhesion strength on bone (Figure 2 h), which is composed of 70% inorganic matter and 30% organic matter. Additionally, 10% PPD showed a tensile adhesion strength of 51.4 ± 17.9 kPa, 25% PPD showed a tensile adhesion strength of 155.2 ± 62.8 kPa, and 50% PPD showed a tensile adhesion strength of 295.4 ± 74.1 kPa. This result suggests that the tensile adhesion strength increases with the increasing polymer concentration. The tensile adhesion strength of 10% PPDA was 93 ± 33.9 kPa, an increase compared with 10% PPD (51.4 ± 17.9 kPa). It is likely that bones have an array of positive and negative charges on the surface,6 which can form strong electrostatic interactions with Gu+. Therefore, it is reasonable that the tensile adhesion strength of PPDA is stronger than that of PPD. When thiol groups and ethylene were incorporated, the tensile adhesion strength on bone increased for a reason similar to that of wet tissue adhesion. Influence of Curing Time and Curing Temperature on the Adhesion Strength on Wet Tissues and Bones. Short (0.5, 30, and 60 min) and long (3, 6, 12, and 24 h) curing times were investigated at two temperatures (room temperature, 25 °C and body temperature, 37 °C). For these studies, samples were immediately placed in the oven after overlapping and were tested immediately after removal from the oven when the curing was completed. Figure 2d shows that the adhesion strength increased rapidly during 0.5−60 min, whereas the rate of increase in the adhesion strength decelerated and remained almost constant from 1−12 h because the concentrations of DOPA and noncrosslinking polymers were larger at the start and the oxidative and noncrosslinking reactions occurred easily initially49 but were gradually depressed after 3 h. In addition, the adhesion strength was approximately 74.8 ± 6.96 kPa before 3 h at 25 °C and approximately 82.4 ± 11.8 kPa before 3 h at 37 °C. There was a trend of increasing adhesion strength

Figure 5. (a) Photographs of rat bone X-rays. (b) H&E histological examination during different time periods. The arrows indicate the fracture site.

and became almost constant. In general, the wet adhesion strength does not increase linearly with an increasing DOPA ratio. The NMR study showed that the initiator:DOPA ratio had a maximum value of 1:45. When the ratio was larger than this value, the adhesion strength no longer increased due to excessive crosslinking (Table S1).33 When Gu+ was introduced into PPD, PPDA was obtained. The wet lap-shear adhesion strength of PPDA was 83.2 ± 8.72 kPa, an increase compared with PPD (69.9 ± 7.24 kPa) due to the presence of Gu+, which can form strong electrostatic and hydrogen bonding interactions with the oxoanions that are ubiquitous in proteins (Scheme 2d).62,63 However, the adhesion strength of the polymer increased more slowly when excessive Gu+ was added because the maximum ratio of Gu+ to oxoanion was reached (Table S1). Unfortunately, DOPA can be auto-oxidized to DOPA-quinone, decreasing the adhesion strength of DOPA. How can mussels maintain the equilibrium between DOPA and DOPA-quinone? The secret is two different mussel foot proteins (mfps): mfp-3, which is rich in DOPA, and mfp-6, which is rich in thiol contents. When DOPA is oxidized in mfp3, the thiols in mfp-6 can serve as antioxidants and protect DOPA from being oxidized too rapidly, which helps to preserve the adhesion properties in the initial adhesive formation period.64 Disulfide bonds are usually formed between the thiol groups of two nearby cysteine residues (Scheme 2e),65 and the catechol groups also cross-link with the thiol groups (Scheme 2f).4,66 Therefore, the introduction of sulfhydryl increased the adhesion strength. As the cysteine ratio increases, the increasing rate of the adhesion strength decelerated and

Figure 6. Evaluation of the hemostatic ability of PPDAC−PPDAL: (a) control and (b) PPDAC−PPDAL. (c) Total blood loss from damaged livers after 3 min. 16762

DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766

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ACS Applied Materials & Interfaces

different time points demonstrated the high efficiency of PPDAC−PPDAL in the wound-healing process. Histological examination was adopted to further investigate the wound-healing effects. At 7 and 14 days of adhesion, the rats were killed, and tissue specimens were obtained and fixed with formalin (3.7 wt %) for histological analysis by H&E staining. Figure 4b shows the H&E stained histology of normal skin, suture, cyanoacrylate, and PPDAC−PPDAL after 7 and 14 days. After 7 days, a large gap and blood clots were observed in the suture group, suggesting that the sutures induced a significant inflammatory response at the incision site, such as the emergence of multinuclear giant cells. After 14 days, wound healing was also incomplete. Cyanoacrylate performed much better than sutures after 7 and 14 days. Compared with the cyanoacrylate and suture groups, the PPDAC−PPDAL group showed a minor wound after 7 and 14 days. These results suggest that these polymers accelerated wound healing. Animal Experiment for Bone Adhesion. Bone adhesives have demonstrated remarkable efficacy in trauma surgery and in fixing small bone fragments in orthopedics.69 We tested the copolymeric glues for fracture healing. Figure 5 shows bone Xray photographs of rats and the H&E histological examination at different time periods. After 20 days, the woven bone began to form and trabecular fragments started to decrease (Figure 5b). Different degrees of hyaline cartilage and osteotylus formation were found in both groups after 20 days (Figure 5a,b). Histologically, osteogenesis was achieved by the woven bone and ossifying cartilage.69 After 40 days, the new lamellar bones were partially formed in both groups; osteotomy gap of PPDAC−PPDAL group was smaller than the control (Figure 5b). After 60 days, new lamellar bone was found close to the normal bone tissue (Figure 5b). The osteotylus was reconstructed to normal bone (Figure 5a). Osteotomy gap of PPDAC−PPDAL group has disappeared almost in comparison with the control group (Figure 5b). In summary, the PPDAC− PPDAL group showed accelerated bone healing in comparison with the control group. Hemostatic Ability in Vivo. Hemostatic materials are widely applied to bleeding sites of tissues and organs.56 Many materials have been explored for their properties to prevent bleeding, but they have not always shown remarkable efficacy on hemostasis, for instance, the materials cannot rapidly solidify at body temperature. In this work, PPDAC−PPDAL was studied as a hemostatic material. Photographs of the control group and PPDAC−PPDAL group are shown in Figure 6a,b. The total blood loss after applying PPDAC−PPDAL was 0.36 ± 0.13 g, and the blood loss of the control was 1.72 ± 0.63 g (Figure 6c). The most likely reason is that the polymer rapidly solidified at body temperature, reducing blood loss. Therefore, this polymer could serve as an effective antihemorrhagic agent.

with increasing temperature. After 12 h, the adhesion strength reached the maximum value. Optimal adhesion cannot be obtained at shorter times because of residual solvent. In general, the residual solvent might cause the polymer chains to be mobile, allowing them to slip past one another easily.31 Similarly, the effect of the curing time (0.5, 2, 4, 12, and 24 h) on the tensile adhesion strength on bone was also investigated at 37 °C (Figure 2i). Influence of Humidity on the Wet Lap-Shear Adhesion Strength. Figure 2e shows the time-adhesion strength curves for a high humidity environment. When soaked in water immediately after adhesion, the adhesion strength increased from 3 to 8 h. However, the adhesion strength decreased and remained almost constant from 8 to 24 h. It is likely that the concentrations of copolymers were initially high, and the oxidative crosslinking reactions occurred easily. However, the incorporation of Gu+ increased the solubility of the copolymer. Therefore, a portion of the copolymer dissolved in water, decreasing the adhesion strength from 8 to 24 h. When soaked in water after 24 h of adhesion, the adhesion strength decreased and remained almost constant because the copolymer, which contained Gu+, dissolved in water, decreasing the lap-shear adhesion strength. In summary, this type of polymer has good adhesion strength in high humidity environments. The elastic moduli of all types of polymers were higher at 37 °C than at 25 °C (Figure 2f). Because the temperatureresponsive behavior of Pluronic is mainly based on the formation of closely packed individual micelles, the significantly increased mechanical strength of polymers can be attributed to the covalent network of Pluronic micelles.4 The elastic moduli of the polymers increased gradually over time (Figure 2g); however, after 720 min, the elastic modulus increasing process decelerated and eventually stopped, possibly because of the formation of significant covalent bonds between the catechols and thiols.49 Degradation. We studied the degradation properties of polymers by measuring the mass. Degradation was assessed as the percentage of weight loss every 2 days. Figure 3a shows that the mass loss of PPDAC with Protamex was much greater than that without Protamex because Protamex promoted the hydrolysis of PPDAC. Protamex is a Bacillus protease complex consisting of subtilisin and a neutral protease,67 which can promote the hydrolysis of peptides or proteins. The results demonstrate that the copolymers are highly biodegradable. Toxicity. Toxicity has an important effect on the viability of cells; an ideal biomaterial should not have adverse reactions or release toxic products.68 DOPA, arginine, and cysteine do not have a prominent effect on cell proliferation and growth. Figure 3b shows the OD of PPDAC obtained via an MTT assay of cultured mammalian cells (L929) compared with positive and negative controls. The viability of L929 cells was investigated for PPDAC after 24, 48, and 72 h, and the cells maintained viability greater than 85% (Figure 3c). No significant difference was observed in the viabilities of cells treated with PPDAC, suggesting that PPDAC presented no cytotoxicity and had excellent biocompatibility. Animal Experiment for Wound Closure. To evaluate the performance of polymers as wound closure materials, PPDAC− PPDAL was applied to skin incisions on the backs of rats as the experimental group, and sutures and cyanoacrylate were applied in the control group (Figure 4a). The visual comparison at

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CONCLUSIONS In this study, a series of thermoresponsive polypeptide− Pluronic−polypeptide triblock copolymers with different functional side groups (catechol, guanidyl, sulfhydryl, and double bonds) were designed and synthesized by ROP of functional Ncarboxy-α-amino acid anhydride (NCA). These thermoresponsive tissue adhesives showed good biodegradability, biocompatibility, and thermoresponsive properties. In vitro adhesion tests with porcine skin and porcine bone demonstrated that these glues have excellent wet adhesive properties. In vivo antibleeding and tissue adhesives showed superior hemostatic properties and the superior healing effects in skin 16763

DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766

Research Article

ACS Applied Materials & Interfaces

(10) Lee, H.; Dellatore, S. M.; Miller, W. M.; Messersmith, P. B. Mussel-inspired Surface Chemistry for Multifunctional Coatings. Science 2007, 318, 426−430. (11) Li, Y.; Meng, H.; Liu, Y.; Narkar, A.; Lee, B. P. Gelatin Microgel Incorporated Poly(ethylene glycol)-Based Bioadhesive with Enhanced Adhesive Property and Bioactivity. ACS Appl. Mater. Interfaces 2016, 8, 11980−11989. (12) Cho, J. H.; Shanmuganathan, K.; Ellison, C. J. Bioinspired Catecholic Copolymers for Antifouling Surface Coatings. ACS Appl. Mater. Interfaces 2013, 5, 3794−3802. (13) Lee, H.; Lee, Y.; Statz, A. R.; Rho, J.; Park, T. G.; Messersmith, P. B. Substrate-Independent Layer-by-Layer Assembly by Using Mussel-Adhesive-Inspired Polymers. Adv. Mater. 2008, 20, 1619− 1623. (14) Vatankhah-Varnoosfaderani, M.; GhavamiNejad, A.; Hashmi, S.; Stadler, F. J. Hydrogen Bonding in Aprotic Solvents, a New Strategy for Gelation of Bioinspired Catecholic Copolymers with Nisopropylamide. Macromol. Rapid Commun. 2015, 36, 447−452. (15) Shin, J.; Lee, J. S.; Lee, C.; Park, H.-J.; Yang, K.; Jin, Y.; Ryu, J. H.; Hong, K. S.; Moon, S.-H.; Chung, H.-M.; Yang, H. S.; Um, S. H.; Oh, J.-W.; Kim, D.-I.; Lee, H.; Cho, S.-W. Tissue Adhesive CatecholModified Hyaluronic Acid Hydrogel for Effective, Minimally Invasive Cell Therapy. Adv. Funct. Mater. 2015, 25, 3814−3824. (16) Zhou, J.; Defante, A. P.; Lin, F.; Xu, Y.; Yu, J.; Gao, Y.; Childers, E.; Dhinojwala, A.; Becker, M. L. Adhesion Properties of Catecholbased Biodegradable Amino Acid-Based Poly(ester urea) Copolymers Inspired from Mussel Proteins. Biomacromolecules 2015, 16, 266−274. (17) Yu, M.; Deming, T. J. Synthetic Polypeptide Mimics of Marine Adhesives. Macromolecules 1998, 31, 4739−4745. (18) Wang, J.; Liu, C.; Lu, X.; Yin, M. Co-polypeptides of 3,4dihydroxyphenylalanine and L-lysine to Mimic Marine Adhesive Protein. Biomaterials 2007, 28, 3456−3468. (19) Nishida, J.; Kobayashi, M.; Takahara, A. Light-Triggered Adhesion of Water-Soluble Polymers with a Caged Catechol Group. ACS Macro Lett. 2013, 2, 112−115. (20) Min, Y.; Hammond, P. T. Catechol-Modified Polyions in Layerby-Layer Assembly to Enhance Stability and Sustain Release of Biomolecules: A Bioinspired Approach. Chem. Mater. 2011, 23, 5349− 5357. (21) Shao, H.; Stewart, R. J. Biomimetic Underwater Adhesives with Environmentally Triggered Setting Mechanisms. Adv. Mater. 2010, 22, 729−733. (22) Shao, H.; Bachus, K. N.; Stewart, R. J. A Water-Borne Adhesive Modeled after the Sandcastle Glue of P. Californica. Macromol. Biosci. 2009, 9, 464−471. (23) Nishida, J.; Kobayashi, M.; Takahara, A. Gelation and Adhesion Behavior of Mussel Adhesive Protein Mimetic Polymer. J. Polym. Sci., Part A: Polym. Chem. 2013, 51, 1058−1065. (24) Guvendiren, M.; Messersmith, P. B.; Shull, K. R. Self-Assembly and Adhesion of DOPA-Modified Methacrylic Triblock Hydrogels. Biomacromolecules 2008, 9, 122−128. (25) Chung, H.; Grubbs, R. H. Rapidly Cross-Linkable DOPA Containing Terpolymer Adhesives and PEG-Based Cross-Linkers for Biomedical Applications. Macromolecules 2012, 45, 9666−9673. (26) Podsiadlo, P.; Liu, Z.; Paterson, D.; Messersmith, P. B.; Kotov, N. A. Fusion of Seashell Nacre and Marine Bioadhesive Analogs: HighStrength Nanocomposite by Layer-by-Layer Assembly of Clay and L3,4-Dihydroxyphenylalanine Polymer. Adv. Mater. 2007, 19, 949−955. (27) Brubaker, C. E.; Messersmith, P. B. Enzymatically Degradable Mussel-Inspired Adhesive Hydrogel. Biomacromolecules 2011, 12, 4326−4334. (28) Jonker, A. M.; Borrmann, A.; van Eck, E. R. H.; van Delft, F. L.; Löwik, D. W. P.M.; van Hest, J. C. M. A Fast and Activatable CrossLinking Strategy for Hydrogel Formation. Adv. Mater. 2015, 27, 1235−1240. (29) Jenkins, C. L.; Meredith, H. J.; Wilker, J. J. Molecular Weight Effects upon the Adhesive Bonding of a Mussel Mimetic Polymer. ACS Appl. Mater. Interfaces 2013, 5, 5091−5096.

incisions and osteotomy gaps. These results indicate that the synthesized copolymers can be used not only as soft tissue and bone adhesives but also as antibleeding materials with high performance.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b16575. Synthesis of monomers 1H NMR spectra of polymers, the relative peak area of polymers, LS data of polymers, and statistical analysis (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ORCID

Dedai Lu: 0000-0002-4161-5373 Ziqiang Lei: 0000-0001-9195-4472 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by the National Natural Science Foundation of China (nos. 51103118, 21504070), the Research Project of Universities of Gansu Province (2015A005), Innovation Team Basic Scientific Research Project of Gansu Province (1606RJIA324), and Innovation Team Project of NWNU. We also thank Key Laboratory of Eco-Environment-Related Polymer Materials Ministry of Education and Key Lab of Polymer Materials of Gansu Province for financial support.

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REFERENCES

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DOI: 10.1021/acsami.6b16575 ACS Appl. Mater. Interfaces 2017, 9, 16756−16766