Versatile Surgical Adhesive and Hemostatic Materials: Synthesis

Article pubs.acs.org/cm

Versatile Surgical Adhesive and Hemostatic Materials: Synthesis, Properties, and Application of Thermoresponsive Polypeptides Dedai Lu,*,† Hongsen Wang,† Ting’e Li,† Yunfei Li,† Xiangya Wang,† Pengfei Niu,† Hongyun Guo,‡ Shaobo Sun,§ Xiaoqi Wang,‡ Xiaolin Guan,† Hengchang Ma,† 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, Gansu 730070, P. R. China ‡ Institute of Gansu Medical Science Research, Gansu Provincial Cancer Hospital, Lanzhou, Gansu 730050, P. R. China § Gansu University of Chinese Medicine, Lanzhou, Gansu 730000, P. R. China S Supporting Information *

ABSTRACT: In this study, thermoresponsive and mussel-inspired polypeptides were synthesized using ring-opening polymerization of α-amino acid derivatives of Ncarboxyanhydride (NCA). The tissue adhesive properties of these polypeptides were evaluated using in vitro adhesive strength tests on porcine skin and bone. The results indicated that the species of the functional polypeptide side groups and the adhesive temperature have a significant influence on the adhesion strength. The maximum of the lap-shear adhesion strength on porcine skin was 101.2 kPa, and the maximum of tensile adhesion strength on bone was 603 kPa. The in vivo antibleeding activity and tissue adhesive ability were also evaluated using a rat model. These polypeptides exhibited superior hemostatic properties and healing effects in the skin incision and osteotomy gap, and the skin incision healing and osteotomy gap remodeling were completed in all rats after 2−9 weeks. These polypeptides are expected to be good candidates for surgical tissue adhesives, tissue engineering materials, and antibleeding materials, etc.

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INTRODUCTION It is well-known that the rapid closure and repair of wounds, especially of skin wounds or wounds from surgical or traumatic disruption, is the best way to prevent infection and to accelerate healing.1 Suture is the most common method for wound closure and repair due to its great tensile strength and low dehiscence rate. However, sutures are not ideal because of some drawbacks, such as suture removal scars and tissue formation.1,2 Therefore, finding materials that can replace or supplement conventional closure techniques for the repair of wounds is meaningful.1−4 As a result, tissue adhesive materials, a class of promising materials for wound healing, surgical tissue adhesives, wrinkle fillers, and hemostasis in surgical procedures, have attracted much attention and great progress has been made.3−6 Tissue adhesives that are currently available for commercial use can be divided into three traditional categories: natural or biological, synthetic and semisynthetic, and biomimetic adhesives.3 However, the existing tissue adhesive applications are limited due to their weak adhesive strength (e.g., fibrin glue) or poor biocompatibility (e.g., cyanoacrylate).1−7 Therefore, there is a continued demand for the development of biocompatible tissue adhesives with superior performance. Marine mussels are well-known for their capability to adhere to various substrates under humid conditions.8−11 The adhesive versatility of mussels can be attributed to the amino acid © 2017 American Chemical Society

composition of proteins that abound in 3,4-dihydroxy-Lphenylalanine (L-DOPA) and lysine near the plaque−substrate interface.8,9,12−15 Through bioinspiration from the mussel adhesion mechanism, the L-DOPA motif was reasonably incorporated into polymeric backbones to construct new materials with advanced adhesive properties.16−19 Considering that L-DOPA is a naturally existing α-amino acid derivative, biomedical application was the main interest of research in these materials, specifically for tissue adhesives and sealants.18,19 Consequently, a wide range of polymeric structures have been reported thus far, including natural backbones,20−26 poly(ethylene glycol)s/pluronic,27−36 poly(acrylic acid),37,38 poly(α-amino acid)s and polypeptides,39−44 step-growth polymers,45−50 and chain-growth polymers.51−56 Unfortunately, these tissue adhesives have shortcomings regarding their low wet adhesive strength or slow curing.46,57 Herein, we report a series of novel biomedical adhesive gels based on thermoresponsive polypeptides derived from LDOPA, diethylene glycol monomethyl ether-L-glutamate (EG2-Glu), L-arginine, L-cysteine, and ε-N-acryloyl-L-lysine (the synthetic route is shown in Scheme 1). Polypeptides have wide applications in various biomedical fields because of Received: January 19, 2017 Revised: June 13, 2017 Published: June 13, 2017 5493

DOI: 10.1021/acs.chemmater.7b00255 Chem. Mater. 2017, 29, 5493−5503

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Chemistry of Materials Scheme 1. Preparation Process of the Copolymers

Table 1. Average Molecular Weight and Its Distribution and Grafting Ratio of Copolymers polymer

Mn (kDa)a

Mw (kDa)a

PDIa

Mn (kDa)b

Mw (kDa)b

PDIb

I:E:D:A:C:Lc

I:E:D:A:C:Ld

M (kDa)e

BPED BPEDA BPEDAC BPEDAL

10.3 15.1 5.7 14.5

36.9 58.2 9.7 63.4

3.58 3.85 1.7 4.37

14.8 19.1 11.5 14.4

20.3 24.5 20.2 21.2

1.37 1.28 1.76 1.47

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

1:34:25:0:0:0 1:18:31:21:0:0 1:11:16:20:21:0 1:23:12:14:0:8

12.9 13.8 11.5 12.1

a Molecular mass and PDI were calculated by using light scattering. bMolecular mass and PDI were calculated by using GPC. cFeed ratio of monomer. dPolymers observed ratio of monomer by 1H NMR. eMolecular mass calculated by using polymer observed ratio of monomers. I, E, D, A, C, L show the numbers of initiator, EG2-Glu, DOPA, arginine, cysteine, ε-N-acryloyl lysine.

behaviors, degradability, and cytotoxicity of these polypeptides were examined as well. Finally, in vivo animal experiments were conducted to evaluate the biocompatibility, bioadhesive properties, and hemostatic properties of these poly(amino acid)s.

their excellent biocompatibility, biodegradability, and bioactivity.58 Poly(EG2-Glu) is a thermoresponsive and biodegradable polymer, and similar to Pluronic F-127, it shows a rapid and reversible sol−gel transition behavior, which is ideal for in situ gel formation aimed at improving the adhesive strength at body temperature.20,59,60 The guanidinium ion of L-arginine can form a salt bridge with oxyanions on the protein surface by electrostatic and hydrogen bonding interactions.61 At the same time, the thiol has high reactivity toward the oxidized quinone form of catechol and double bond via Michael-type additions.61 Therefore, L-arginine, L-cysteine, and ε-N-acryloyl L-lysine were incorporated into polypeptides chains to improve the adhesion strength and curing rates. The in vitro tissue adhesion properties of these polypeptides were evaluated using a universal testing machine (UTM) using porcine skin and porcine femur bones. The thermoresponsive phase transition

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EXPERIMENTAL SECTION

Materials. Acryloyl chloride and dihydroxyphenylalanine (LDOPA) were purchased from Shanghai Darui Finechem Co., Ltd. (Shanghai, People’s Republic of China). Diethylene glycol monomethyl ether(EG2), L-Cysteine, L-lysine hydrochloride, triphosgene, Nbenzyloxycarbonyl-L-arginine, 1,4-butanediol, and HRP (horse radish peroxidase) were purchased from Aladdin Reagent Company (Shanghai, People’s Republic of China). 8-Hydroxyquinoline was purchased from Shanghai Zhongqin Chemical Reagent Co., Ltd. (Shanghai, People’s Republic of China). Water was removed from tetrahydrofuran (THF) with calcium hydride. EG2-L-glutamate (EG25494


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Chemistry of Materials Glu) and ε-N-acryloyl lysine were synthesized according to the method outlined in the literature.60,62 Other chemicals were of analytical grade and were used without any further purification. Origin 8.0 software was used to evaluate the significance of differences between results. Preparation of Initiator and Monomers. The initiator was prepared by adding excessive potassium to 1,4-butanediol until there were no air bubbles at room temperature, and then excess metallic potassium was removed. We obtained a highly transparent viscous liquid. Levodopa-N-carboxyanhydride (DOPA-NCA), EG2-L-glutamate-N-carboxyanhydride (EG2-Glu-NCA), cysteine-N-carboxyanhydride (Cys-NCA), arginine-N-carboxyanhydride (Arg-NCA), and ε-Nacryloyl lysine-N-carboxyanhydride (Ac-Lys-NCA) were prepared according to previously reported procedures60,63−66 (Supporting Information). Synthesis of Copolymers. EG2-Glu-NCA was dissolved in 10 mL of dry THF, and the initiator that was dissolved in 3 mL of dry THF was added. After 3 days, L-DOPA-NCA was added (the feed ratio of the monomer is shown in Table 1). We obtained a yellow viscous liquid after 3 days. The resulting yellow viscous liquid was purified by washing with anhydrous ethanol, and finally yellowish precipitate 1,4butanediol-poly[(EG2-Glu)-co-(DOPA)] (BPED) was produced. Similarly, Arg-NCA, Arg-NCA and Cys-NCA, Arg-NCA and AcLys-NCA were dissolved in dry THF (10 mL). These solutions were then separately added to the BPED THF solution (the feed ratio of monomer is shown in Table 1). After 3 days, 1,4-butanediolpoly[(EG2-Glu)-co-(DOPA-co-Arg)] (BPEDA), 1,4-butanediol-poly[(EG2-Glu)-co-(DOPA-co-Arg-co-Cys)] (BPEDAC), and 1,4-butanediol-poly[(EG2-Glu)-co-(DOPA-co-Arg-co-Ac-Lys)] (BPEDAL) were produced. NMR Spectra. 1H NMR spectra of the synthesized polymers were recorded on a VARIAN JNM-ECP 600 MHz instrument at room temperature using DMSO-d6 as the solvent and TMS as the internal reference. Average Molecular Weight Testing. The average molecular weight and its distributions of the synthesized polymers were tested by light scattering (LS) (DAWN EOS, laser wavelength 690.0 nm, refractive index 1.330, water was used as the solvent at a flow rate of 1.0 mL min−1) and gel permeation chromatogram analysis (GPC) (Waters 1515-2414-2707, Agilent organic column, PMMA standard, mixed-C column, DMF added 10 mmol L−1, LiBr was used as the eluent at a flow rate of 0.5 mL min−1 at 45 °C.). Thermoresponsivity Test. Transmittance of solutions of the synthesized polymers was recorded by increasing the temperature every 5 °C at 500 nm of wavelength after thermostabilization for 10 min. Contact Angle Measurement. Water contact angle was measured with a SL200 KB apparatus at room temperature (25 °C) and normal body temperature (37 °C). The temperature was controlled by a superthermostat. Lap-Shear Adhesion Strength Measurement on Wet Tissue. Wet tissue adhesive properties were measured on porcine tissue following the procedures described in ASTM standard F2255-05 and are shown in Figure 2a. Fresh shaved porcine skin, obtained directly from a local slaughterhouse, was removed from any excess fat and prepared by cutting into rectangular pieces (5 cm × 1 cm). Different concentrations of polymers solutions in a phosphate buffer saline (PBS, pH = 7.4) were added to different concentrations of HRP and H2O2 in PBS solutions; 50 μL of the polymer solution was then added on porcine skin and spread over the lap area (1 cm × 1 cm). After the pieces were pressed together for different amounts of time, the adherends 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); the lap-shear adhesion was measured in Pascal (Pa = N/m2). Tensile Adhesion Strength Measurement on Porcine Bones. To study the adhesion characteristics of the polymers on bone, end-toend bone adhesion experiments were performed on porcine bone (Figure 2b). The porcine bones were split into approximately rectangular pieces of ∼5 cm long × 1 cm width × 1 cm thick by a

saw. The test ends of the bone samples were then sanded using sandpaper with a grain size of 80 until a smooth and even surface was achieved, and the bone samples were soaked in PBS solution 1 h before adhesive application. Different concentrations of polymers solutions in a PBS solution were added to 1 mg mL−1 HRP and 1 wt % H2O2 PBS solution, respectively; 50 μL of the polymer solution was then spread on both test end surfaces. After the pieces were pressed together for different amounts of time, the adherends 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); the tensile adhesion strength was measured in Pascal (Pa = N/m2). Degradation Study. Briefly, the polymer was divided into two groups, protamex was added to one group, two groups polymers were dissolved in 50 mL of a PBS solution. Subsequently, the polymer solutions were placed in 3000-specification dialysis membrane and immersed in PBS solution at 37 °C. At every 2 days, after PBS solution was removed, the adhesive sample was washed with distilled water, dried, and weighed again. The mass loss was calculated by comparing the initial mass (w0) with the mass measured at the predetermined time points (wt), as shown in the following equation: w − wt Mass loss (%) = 0 × 100 w0 Cytotoxicity Evaluation. The cytotoxicity of the copolymer samples was evaluated using the ISO10993-5 standard test method. L929 cells (normal healthy mammalian cell line) were cultured in 96well plates at a density of 105 cells per well containing 10% fetal bovine serum, 1.0% penicillin−streptomycin, and 1.2% glutamine at 37 °C in a 5% CO2 incubator. The polymers of varying concentration (10 μL, in PBS) were then added; after 24, 48, and 72 h of incubation, the 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, DMSO (100 μL) was added, and the dissolved solution was swirled homogeneously for approximately 10 min in a shaker. The optical density of the solution was detected by a microplate reader at 490 nm. Animal Experiments for Wound Closure. Animal experiments for wound closure were performed following the guidelines of the national regulations on experimental animals and with the approval of the local animal experiments ethical committee. Rats (female, average weight of 200 ± 50 g) were anesthetized using chloral hydrate (350 mg/kg). The fur around the surgical site was shaved and sterilized with 70% ethanol. Skin incisions 1.5 cm long and skin thickness depth were made on rats’ backs. Then 20 wt % BPEDAC−BPEDAL PBS solution containing 1 mg mL−1 HRP and 1 wt % H2O2 was immediately applied to the wound area. After 7 and 14 days, the rats were sacrificed, and skin tissue at the wound sites was obtained and fixed in a formaldehyde solution (3.7 wt %). These samples were stained with hematoxylin and eosin (H&E) for histological analysis. Animal Experiments for Bone Adhesion. Animal experiments for bone adhesion were carried out using white male rats (150−200 g) in compliance with the guidelines provided by the national regulations for the use of experimental animals and with approval from the local animal experiment ethical committee. The rats were anesthetized, and their skin overlying the femur was shaved. The surgical sites were draped according to standard sterile techniques. After a longitudinal incision on the lateral side of the leg was made on the skin and along the bone, the bone was exposed by dissecting overlying musculature. The tibia was fractured with a sharp osteotome. For the experimental group, 50 wt % BPEDAC−BPEDAL PBS solution containing 1 mg mL−1 HRP and 1 wt % H2O2 was immediately applied into the osteotomy gap; however, the polymer was not used in control group. Both groups used external fixation after surgical stitching at wound site. Standard X-rays were adopted to observe fracture healing after 20, 40, and 60 days. The rats were then sacrificed, and bone tissue was obtained and fixed with 3.7% formalin solution for histological analysis by H&E staining. In Vivo Hemostatic Ability Test. To evaluate the hemostatic potential of the polymer, a hemorrhaging liver rat model was used (150−200 g, male). All animal experiments were performed in 5495


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Chemistry of Materials compliance with the guidelines provided by the national regulations for the use of experimental animals and with approval from the local animal experiments ethical committee. In brief, a rat was anesthetized using chloral hydrate (350 mg/kg) and immobilized on a surgical corkboard. After abdominal incision, serous fluid around the liver was carefully removed to accurately evaluate the blood weight obtained by the filter paper beneath the liver. Then 20 wt % BPEDAC−BPEDAL PBS solution containing 1 mg mL−1 HRP and 1 wt % H2O2 was immediately applied to the bleeding site. After 3 min, the amount of blood absorbed on the filter paper was weighed and compared with that of a control group (no treatment after pricking the liver).

solution transmittance declined sharply as the temperature increased from 30 to 35 °C. This result can be explained by the formation of intramolecular hydrogen bonding that makes the polypeptide chains agglomerate as the temperature increases. A more vivid demonstration of the temperature-sensitive phenomenon of these polypeptides is shown in Figure 1b. The hydrophilic/hydrophobic properties were also examined by goniometric measurement, and the results show that the contact angle changed from 27.5° to 72.5° as the temperature increased from 25 to 37 °C (Figure 1c, 1d). In other words, a polymer’s hydrophilicity increased as the temperature increased. That means the hydrophilicity of the polypeptide containing EG2-Glu weakened as the temperature increased. Because DOPA is easily converted to DOPA-quinone and promotes intermolecular cross-linking with other groups, such as amines on tissue surface, hydrogen peroxide (H2O2) was used as the curing agent. The effect of H2O2 concentration was investigated, and the results showed that the wet lap-shear adhesion strength of BPED increased as the concentration of H2O2 increased from 0.25% to 1%, but further increases in the concentration weakened the adhesion (Figure 2c). This finding may be attributed to overoxidation that results in too much cross-linking, which causes the adhesion strength to decrease.18,56 HRP is a hemoprotein that can promote crosslinking between DOPA-quinone and amines on the tissue surface due to decomposed H2O2 molecules.70 It has been shown that the HRP concentration affects the cross-linking kinetics, while variation in the H2O2 concentration primarily affects the mechanical properties.46,71Figure 2d shows how the adhesion strength increased as the concentration of HRP increased. The reason for this result is that HRP promoted cross-linking between DOPA-quinone and the amines on the tissue surface, but the lower HRP concentration was not sufficient to fully cross-link the copolymer. The wet lap-shear adhesion strength of the different polymers was measured using a universal testing machine at 37 °C after 24 h. As shown in Figure 2e, BPED shows a high wet lap-shear adhesion strength because the dopamine becomes dopaquinone under oxidative conditions, and the dopaquinone has an affinity for diverse nucleophiles (e.g., amines, thiol) on the tissue surface via a Michael-type addition reaction (Scheme 2e).32,72−74 Moreover, a catechol functional group can trigger chemical cross-linking via the formation of catechol−catechol adducts (Scheme 2a).8,26The wet lap-shear adhesion strength of BPEDA is stronger than that of BPED due to catechol and guanidinium (Gu+) forming a salt bridge with oxoanions that exist ubiquitously in proteins because of electrostatic and hydrogen bonding interactions, which can inhibit the ATPdriven sliding motion of actomyosin (Scheme 2d).75,76 Additionally, proximal thiol groups can form disulfide bonds (Scheme 2f)77and thiol groups can cross-link with catechol groups20,55(Scheme 2c). Therefore, the increase in the adhesive strength was partly ascribed to the incorporation of sulfhydryl. Because of the thiol−ene click reaction78(Scheme 2b) or OH radical polymerization (Scheme 2g), the adhesive strength further increased when BPEDAC and BPEDAL were used at the same time. All of the copolymers have greater lap-shear adhesion strengths at 37 °C than at 25 °C due to the temperature-responsive behavior and faster evaporation of the solvent at 37 °C. The tensile adhesion strengths of polypeptides containing different functional groups were tested on porcine bone samples to demonstrate their potential application as a bone

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RESULTS AND DISCUSSION All amino acids NCA were synthesized under anhydrous conditions to avoid the hydrolysis of NCA. The NCA product was stored at a low temperature and away from moisture.64 A novel class of copolymers, including BPED, BPEDA, and BPEDAC−BPEDAL, were synthesized via ring opening polymerization (ROP) of NCA amino acid derivatives using alcoholate ions67 as the initiator. The process of preparing the copolymers is shown in Scheme 1. A representative 1H NMR spectrum analysis of the adhesive polymers is shown in the Supporting Information. The number-average molecular mass, weight-average molecular mass, and polydispersity index (PDI) of the polymers containing different functional groups were tested using light scattering and GPC (Table 1). These polypeptides are zwitterionic polymers, they have the high tendency of association of polymer chains in water.68 Therefore, the Mw from LS are significantly higher than those from GPC. Notably, the thermoresponsive polypeptides containing EG2Glu monomeric units have been extensively studied because of their promising applications in biomedical fields.58,59,69 Interestingly, thermoresponsive polypeptides can show a hydrophilic-to-hydrophobic transition with an increase in the temperature due to the formation of intramolecular hydrogen bonding. Conversely, intermolecular hydrogen bonding formed between the polypeptides and the surrounding water molecules when the temperature decreased; in turn, the polypeptide solubility increased.58,60 Figure 1a shows the temperature− transmittance curves of BPED, BPEDA, BPEDAC, and BPEDAC−BPEDAL aqueous solutions. It is clear that the

Figure 1. (a) Temperature−transmittance curves of BPED, BPEDA, BPEDAC, and BPEDAC−BPEDAL. (b) Thermoresponsive phenomenon was demonstrated vividly in vitro by BPED aqueous solution (3 wt %). (c, d) Photographs of the water droplet shape and contact angle on the film surface of BPED. 5496


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Chemistry of Materials

Figure 2. (a,b) Experimental setup for the adhesion strength tests on porcine skin and bone. Adhesion properties of copolymers: (c,d,e) Lap-shear adhesion strength of copolymers on porcine skin after 24 h of adhesion. (f) Tensile adhesion strength of copolymers on porcine bones after 2 h of adhesion. (g) Time−lap-shear adhesion strength curve of BPEDAC−BPEDAL at 37 °C. (h) Time−tensile adhesion strength curve of BPEDAC− BPEDAL on porcine bone. (i) Elastic modulus value of copolymers. Data are presented as the mean ± SD, n = 5. Statistically significant differences are indicated by *p < 0.05 or **p < 0.01.

concentrations of non-cross-linking polymers were greater at the start, and the oxidative and cross-linking reactions occurred easily.66 At the same time, the residual solvent is another reason that the optimal adhesion could not be obtained quicker. The existence of residual solvent may have caused the polymer chains to be mobile and allowed the chains to slip past one another easily.18 In the same manner, the effects of the cure time (0.5, 2, 4, 12, and 24 h) on the tensile adhesion strength on bone were also investigated at 37 °C (Figure 2h). The elastic modulus values of polymers were investigated at 25 and 37 °C (Figure 2i). The elastic modulus value for all polymers was greater at 37 °C than at 25 °C. Because the temperature-responsive behavior of the polypeptides containing EG2-Glu monomeric units is mainly based on the formation of closely packed individual micelles, the significantly increased mechanical strength of the polymers can be attributed to the covalent network of polypeptides containing EG2-Glu.20 Furthermore, when different functional groups, such as DOPA, arginine, cysteine, and ε-N-acryloyl lysine, were incorporated into polymers, the elastic modulus value increased significantly. It is possible that significant covalent bonds were formed among catechols with either catechols or thiols.66

adhesive. Naturally, mussels can cling to several surfaces, including rocks, using their byssus, which contains a considerable amount of catechol. Similarly, BPED has been shown to have a high adhesive strength for bone (Figure 2f). Notably, bones have an array of positive and negative charges on the surface.79 Because of the electrostatic interactions between Gu+ and the bone surface, BPEDA demonstrated a greater adhesive strength than BPED. To further enhance the adhesive strengths of the copolymer, sulfhydryl and ε-Nacryloyl lysine were also incorporated based on the similar tissue adhesion mechanism. Cure times investigations, which consisted of both short (0.5, 30, and 60 min) and long (3, 6, 12, and 24 h) cure times, were performed at two different temperatures: room temperature (25 °C) and body temperature (37 °C). In these studies, the samples were immediately placed in the oven after overlapping and tested directly once the cure time was completed and the samples were removed from the oven. The rate of adhesion strength increased rapidly during the time period from 0.5 to 60 min (Figure 2g). However, during the time period from 1 to 12 h, the increase rate in adhesion strength decelerated and became almost constant. The reason for this result was that the 5497


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The degradation property is one of the unique features of our adhesives. Theoretically speaking, these copolymers should degrade under protease conditions via hydrolysis of polypeptides, thus yielding small molecule amino acids within a limited period of time. For instance, the mass loss of BPEDAC was greater than 70% (Figure 3a), indicating that those copolymers have excellent biodegradability. Toxicity has an important effect on cell viability, which can be evaluated using the MTT assay. The optical density (OD) can determine cell viability as the OD is directly proportional to the number of living cells. The OD of BPEDAC was not significantly different from that of the negative control because the nontoxic degradation products included EG2-Glu, DOPA, Arg, and Cys (Figure 3b). When samples were incubated for 72 h, cell viability was still greater than 80% (Figure 3c), which demonstrated that BPEDAC had no significant influence on cell growth and proliferation. Meanwhile, no qualitative difference was observed in the attached cells by microscopy (Figure 3d). This indicates that BPEDAC has low cytotoxicity and can be used as a bioadhesive. Hemostatic materials have extensive uses in hemorrhaging tissues or organs. A number of materials reported have preventive bleeding properties but their hemostasis properties lack effectiveness. For example, some materials cannot become solid rapidly in the presence of body fluids or at body temperature. In this work, BPEDAC−BPEDAL was further studied as a hemostatic material based on its excellent adhesive property. Photographs in Figure 4a and 4b show the untreated bleeding liver control and the BPEDAC−BPEDAL-treated bleeding liver. The total blood loss after the application of BPEDAC−BPEDAL was 0.54 ± 0.11 g; the total blood loss in the control group was 1.76 ± 0.56 g (Figure 4c). The reduction in blood loss may be attributed to the fact that our polymers can rapidly solidify at body temperature. Therefore, these

Scheme 2. Schematic Illustration Showing a Potential Mechanism of the Tissue Adhesiona

a

Different functional group are capable of forming different types of cross-links in this system: (a) catechol chemistry cross-linking via the formation of catechol-catechol adducts; (b) thiol−ene click reaction; (c) covalent cross-linking between catechol and sulfydryl; (d) electrostatic interaction between guanidinium (Gu+) and oxoanions that exist ubiquitously in proteins; (e) dopamine turns into dopaquinone under oxidative conditions, the dopaquinone can have affinity to diverse nucleophiles (e.g., amines, thiol) on tissue surface via a Michael-type addition reaction; (f) covalent cross-linking by forming disulfide; (g) OH radical polymerization.

Figure 3. (a)Time−Mass loss curves of BPEDAC. (b) MTT assay OD value of L929 cells which were cultured with the extraction media from BPEDAC (a: negative Control, b: 2.5 mg mL−1, c: 5 mg mL−1, d: 10 mg mL−1, e: positive Control), p > 0.05 predicted no statistically significant differences when compared to the negative control. (c) Cell relative proliferation (b: 2.5 mg mL−1, c: 5 mg mL−1, d: 10 mg mL−1). (d) Microscopic pictures (10×) of L929 cells after incubation with BPEDAC. 5498


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tissue surface, fibrosis, and inflammation around the incision remained, which demonstrated the healing process was still incomplete after 14 days. Compared with the cyanoacrylate and sutures, BPEDAC−BPEDAL showed only a minor inflammation during wound healing after 7 days, and the incision completely recovered with a newly formed dermis and no visible fibrosis after 14 days. To the best of our knowledge, BPEDAC−BPEDAL is a rare polymer that possesses many excellent properties; it is temperature-responsive, biocompatible, and biodegradable, and it has strong wet-tissue adhesion. More importantly, BPEDAC−BPEDAL can accelerate wound healing without the help of other invasive surgical closure techniques, such as sutures, staples, or wires. Interestingly, this sutureless wound healing has a remarkable efficacy in terms of skin care because there is no needle hole. Therefore, this technique may have promising applications in wound cosmetology. Gluing technique is attractive when applied in fixing small bone fragments in orthopedic and trauma surgery.80 Here, we also attempt to use these copolymeric glues for fracture healing. An X-ray photograph of rat bones and the H&E histological examination results after different time periods are shown in Figure 6. After 20 days, despite a significant increase in osteoblasts and woven bone formation, the fracture hematoma and trabecular fragments started to decrease, but inflammatory tissue reactions were not seen in either group. The adhesion did not become an obstacle to cellular migration and did not obstruct bone-in growth or osteogenesis. Different degrees of hyaline cartilage and osteotylus formation were found in both groups after 20 days (Figure 6a). Histologically, osteogenesis was achieved by ossifying cartilage and woven bone. The osteoblasts and osteoclasts with high activity were found around trabeculae (Figure 6b). Multi-nucleated giant cells and mononuclear macrophages were found in both groups. After 40 days, the woven bone was partly transformed to the new lamellar bone in both groups. After 60 days, the former osteotomy was not detectable in the BPEDAC−BPEDAL group. The bone adhesion did not represent a barrier for bone ingrowth and neither macrophages nor giant cells remained. When new vital lamellar bone was found close to normal bone

Figure 4. Evaluation of hemostatic ability of BPEDAC−BPEDAL: (a) control and (b) BPEDAC−BPEDAL. (c) Total blood loss from the damaged livers after 3 min. Statistically significant differences are indicated by **p < 0.01.

polymers could serve as a more effective antihemorrhage material in practical applications. Because of the excellent adhesion and hemostasis properties of BPEDAC−BPEDAL, bleeding from the surgical site stopped within a few minutes when BPEDAC−BPEDAL was applied to the wound incision during the animal wound closure experiment. The proposed tissue adhesion mechanisms of BPEDAC−BPEDAL are schematically shown in Scheme 2. Moreover, BPEDAC−BPEDAL has been shown to be highly efficient in the wound healing process compared with sutured or cyanoacrylate-treated wounds during a different period (Figure 5a). In short, compared with sutures, BPEDAC− BPEDAL led to improved and accelerated wound healing. The histological evaluation, conducted 7 days after application of the suture, showed a large gap and blood clots at the incision site (Figure 5b). The emergence of multinuclear giant cells suggested that the suture induced a significant inflammatory response at the incision site. After 14 days, the existence of inflammatory cells suggested that there was also inflammation even though the large gap was negligible. Cyanoacrylate had slightly better efficacy on wound healing than suture, but the emergence of hyperkeratosis and inflammatory cells also suggested that there was significant inflammation after 7 days. Additionally, hyperkeratosis on the

Figure 5. Photographs of wound closures and H&E histological examination during different time periods. The sites of wounds are indicated by red stars. 5499


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Figure 6. Photographs of rats’ bones’ X-ray and H&E histological examination during different time periods. The arrows indicate fracture site.

ORCID

tissue, the osteotylus reconstructed into normal bone (Figure 6a).

Dedai Lu: 0000-0002-4161-5373 Xiaolin Guan: 0000-0002-1537-406X Hengchang Ma: 0000-0003-2348-9585 Ziqiang Lei: 0000-0001-9195-4472

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CONCLUSION In this work, we have designed and successfully synthesized a series of novel temperature-sensitive polypeptides based on different functional side groups (EG2-Glu, catechol, guanidyl, sulfhydryl, and double bond). When used as thermosensitive tissue adhesives, these polymers have shown not only good biodegradability, biocompatibility, and thermoresponsive properties, but also excellent mechanical properties in in vitro adhesion tests with no cytotoxicity. The wet adhesive strength has a better performance in grafting with various monomers, and BPEDAC−BPEDAL achieved the maximum strength due to multifarious cross-linking reactions. In vivo antibleeding activity and tissue adhesive ability demonstrated the superior hemostatic properties and healing effects in skin incision and osteotomy gap models. These results listed above indicate that this novel class of biomimetic adhesives hold high potential for medical applications, such as tissue adhesive and arresting bleeding.

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Author Contributions

Dedai Lu designed and supervised the whole work and wrote the article with Hongsen Wang. Hongsen Wang synthesized most of the copolymers, participated in most of the relevant experiment work, and wrote the article with Dedai Lu. Ting’e Li participated in the copolymers synthetic work. Yunfei Li participated in toxicity test and animal experiment. Xiangya Wang and Pengfei Niu participated in animal experiment. Hongyun Guo participated in toxicity test and in skin and bone tissue histological analysis. Shaobo Sun participated in bone adhesion experiment. Xiaoqi Wang completed skin and bone tissue H&E stain. Xiaolin Guan gave advice in the article writing. Hengchang Ma polished the article. Ziqiang Lei gave advice in experimental design and article writing work. Funding

National Natural Science Foundation of China (51103118, 21504070).

ASSOCIATED CONTENT

S Supporting Information *

Notes

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemmater.7b00255. Synthesis of monomers, statistical analysis, 1H NMR spectra of polymers, relative peak area of polymers, and GPC traces of polymers (PDF)

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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 (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(NWNU-LKQN-16-2). We also thank Key Laboratory of Eco-Environment-Related Polymer Materials Ministry

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. 5500


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of Education, Key Lab of Polymer Materials of Gansu Province, for financial support.

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