Potent anti-adhesion barrier combined biodegradable hydrogel with

redical production. In this study, the naturally occurring anti-oxidative and anti-inflammatory compounds extracted from Turkish galls (GEA) were enca...
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Biological and Medical Applications of Materials and Interfaces

Potent anti-adhesion barrier combined biodegradable hydrogel with multifunctional Turkish galls extract Xiaoling Li, Bingwen Zou, Na Zhao, Chao Wang, Ying Du, Lan Mei, Yuelong Wang, Shangzhi Ma, Xing Tian, Jun He, Aiping Tong, Liangxue Zhou, Bo Han, and Gang Guo ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b10668 • Publication Date (Web): 05 Jul 2018 Downloaded from http://pubs.acs.org on July 6, 2018

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Potent Anti-adhesion Barrier Combined Biodegradable Hydrogel with Multifunctional Turkish Galls Extract Xiaoling Li1,†, Bingwen Zou1,†, Na Zhao2, Chao Wang3, Ying Du3, Lan Mei1, Yuelong Wang1, Shangzhi Ma2, Xing Tian2, Jun He1, Aiping Tong1, Liangxue Zhou1, Bo Han2,*, Gang Guo1,* 1

State Key Laboratory of Biotherapy and Cancer Center, and Department of Neurosurgery,

West China Hospital, Sichuan University, and Collaborative Innovation Center for Biotherapy, Chengdu, 610041, P. R. China 2

School of Pharmacy, Shihezi University, and Key Laboratory of Xinjiang Phytomedicine Resource and Utilization, Ministry of Education, Shihezi, 832002, P. R. China

3

National Engineering Research Center for Synthesis of novel Rubber and Plastic Materials, Yanshan Branch, Beijing Research Institute of Chemical Industry, SINOPEC, Beijing, 102500, P. R. China

ABSTRACT Postoperative adhesions are common and serious complications in clinical, which almost always happens after abdominal or pelvic surgery. The adhesion development process is accompanied by increased inflammatory cell infiltration and oxygen free redical production. In this study, the naturally occurring anti-oxidative and anti-inflammatory compounds extracted from Turkish galls (GEA) were encapsulated into an injectable and biodegradable thermosensitive hydrogel. Anti-adhesion efficacy ∗

Corresponding author: Gang Guo, E-mail: [email protected] (G. Guo), [email protected] (B. Han). Tel: (86) 028-8516 4063, Fax: +86 28 85164060. † These authors contributed equally to this work. 1

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of the barrier system (GEA-NP/H) was tested on a rat peritoneum injury-cecum abrasion model. Upon injection, the mildly viscous liquid formed a potent physical barrier over the injured cecum and peritoneum without any additional cross-linkers or light sources. Once formed, GEA-NP/H presented as a durable wound dressing for more than 5 days, as well as a sustained drug depot of GEA. The polymer hydrogel can be degraded and absorbed gradually. After 14 days, severe adhesion occurred among rats treated with normal saline (NS) and GEA loaded nanoparticles (GEA-NP). Whereas, frequency of score 1 adhesion among the blank hydrogel group is 30%, and 90% of the rats from GEA-NP/H group exhibited no adhesion. In addition, pathological sections and SEM assay demonstrated that operative defects treated with GEA-NP/H suffered from mild oxidative stress and inflammatory damages at early days after injury, as well as accelerated wound healing and more mature mesothelial cell deposition at the 14th day in contrast to the blank hydrogel treatment. Therefore, the study provided an available biodegradable hydrogel barrier to effectively prevent postsurgical adhesion. KEYWORDS: Postoperative adhesions, In situ gelation, Biodegradable, Antioxidant, Anti-inflammatory, Turkish galls

INTRODUCTION Mesothelial damages and tissue ischemia after abdominal or pelvic surgery often developed into pathological fibrotic bands and connections that distort normal anatomy, which is so called postoperative peritoneal adhesion.1,2 The adhesions can 2

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result in sequential secondary effects like pelycalgia, acquired infertility, intestinal/ureteral obstruction, even death.3 Presently, operative adhesiolysis is the only way to eliminate the adhesions, but it has the drawback of recurrence. Therefore, preventing the formation of post-surgery adhesion is almost the only way to combat adhesion-related morbidity.4,5 Minimal invasive surgery, un-powdered gloves, gases with suitable humidity and temperature have been introduced in surgical treatment to prevent postoperative adhesion as possible.3 Furthermore, numerous pharmacological and barrier-based strategies have also been employed.6 Drugs like steroids, warfarin, urokinase, direct thrombin inhibitors, and heparin are commonly used for anti-adhesion, but none of them has been wholly satisfactory.6 Barrier systems which can physically isolate the wound tissues are widely applied, but there yet remain several problems to be solved. For example, solid membranes (Seprafilm®, hyaluronic acid carboxymethyl cellulose; Interceed®, oxidized cellulose) or pre-formed hydrogels (SprayGel®, PEG) often cannot completely cover the damaged tissues, and it is impossible to use these barriers in minimal invasive surgeries.7 Polymer solutions (Adept®, 4% icodextrin solution; Hyson®, 32% dextran solution) can poorly fixed to the wounds, and only remain at the injury site for quite a short time.8 An ideal anti-adhesion barrier are characterized as follows: (1) be facilely applied in both laparoscopic and open operations, (2) possess well biodegradability and biocompatibility, (3) cover the affected tissues completely, (4) be effective till the wounds heal. Injectable hydrogels can be easily used in laparoscopic procedures, and 3

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cover both complicated and microscopic wounds perfectly.9,10 These hydrogel formulations would be utilized as drug delivery depots or cell-growing matrix for tissue regeneration after injected into the targeted site.11-14 In particular, the thermosensitive hydrogel barriers are advantageous since they could be conveniently administrated as injectable sols without spatial restriction, then transform into semisolid gels rapidly initiated by temperature variation.15,16 Postoperative adhesions are thought to be caused by pathological wound healing process of the visceral or parietal peritoneal injury.17 Surgical trauma to peritoneum would active the surrounding mesothelial cells and even the underlying endothelial cells, lead to localized secretion of various coagulation and inflammatory mediators.18 Due to the persistent fibrinolysis imbalance, the secreted fibrin cannot be degraded, which develops into fibrin bands. Then extracellular matrix molecules especially collagen deposited gradually, leading to the formation of permanent fibrous connective tissue.6,19,20 This process is associated with elevated production of oxygen-free radicals from the activated mesothelium and the infiltrated neutrophils and macrophages.21 Thus we assumed that antioxidant agent may be beneficial to relieve the adhesion formation manifestations. Turkish galls, a common insect galls, contain several kinds of phenolic compound such as gallotannin, gallic acid, and ellagicacid,22 all of these are naturally occurring antioxidants that have been generally studied in the domain of wound care, neuroprotection, and oncology.23-25 The main components of Turkish galls ethyl acetate fraction (GEA) are phenolic acids and gallotannins.26 Previous study indicated 4

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that gallotannins exhibit potent anti-inflammatory effect through inhibiting iNOS and COX-2.27 Recently, Yu et al. demonstrated that GEA could mitigate severity of colitis through its anti-inflammatory property.28 Since the oxidative stress and aberrant inflammatory response are important precipitating factors of postoperative adhesion formation, we presumed that GEA might be efficient for anti-adhesion. Herein, GEA loaded nanoparticles (GEA-NP) and the thermosensitive copolymer hydrogel were combined as a novel biodegradable anti-adhesion barrier (GEA-NP/H), and the anti-adhesion ability of GEA-NP/H was studied on a rat peritoneum injury-cecum abrasion model.

EXPERIMENTAL SECTION Materials, cells, and animals Reverse Pluronic®10R5 (PPG–PEG–PPG, Mn = 2000), ε-caprolactone (ε-CL), stannous octoate [Sn(Oct)2], polyvinyl alcohol (PVA, average Mw 30000–70000), Rhodamine B (RB), 4-dimethylaminopyridine (DMAP), dicyclohexylcarbodiimide (DCC),

and

MTT

were

provided

by

Sigma-Aldrich

(USA).

2,2-diphenyl-1-picrylhydrazyl (DPPH) was provided by Xiya Reagent (China). All other chemicals were analytical reagent grade. HEK293 and NIH-3T3 cells (ATCC, USA) were cultured in Dulbecco’s Modified Eagle’s Medium supplied with 10% FBS and 1% penicillin-streptomycin (all from Gibco, USA). Female Sprague-Dawley rats (200-220 g), female BALB/c mice (18-20 g) and male BALB/c nude mice (16-18 g) were purchased from HFK Bio-Technology Company (China). The animal procedures 5

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were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Sichuan University (Chengdu, China). R-CPC copolymer synthesis Ring-opening copolymerization method was applied to synthesis the R-CPC copolymer (PCL–PPG–PEG–PPG–PCL). Accurately weighed reverse Pluronic®10R5 and ε-CL were catalyzed by Sn(Oct)2 (0.3%) under nitrogen atmosphere at 140 °C for 10 h. Then the cooling production was dissolved in dichloromethane and crystallized using pre-cooling petroleum ether. After vacuum drying at 25 °C for 48 h, the purified productions were stored in a desiccator. Chemical structure of the obtained polymers were characterized by 1H nuclear magnetic resonance spectroscopy (1H-NMR, Bruker Avance Ⅲ 400, Bruker, Germany) and Fourier transform infrared spectroscopy (FTIR, Nicolet 200 SXV, Thermo Fisher Scientific, America). Molecular weight of the polymers were measured by 1H-NMR and gel permeation chromatography (GPC, Agilent 110 HPLC, Agilent Technologies, America). Thermodynamic property of the polymers was studied using differential scanning

calorimetry

(NETZSCH

204,

NETZSCH,

Selb,

Germany)

and

thermogravimetric analysis (Netzsch TG 209 F1, Germany). Fluorescence probe method (pyrene as probe) were applied to determine the critical micelle concentration (CMC) of R-CPC.29 Turkish galls ethyl acetate fraction (GEA) preparation Turkish galls powder was sequentially extracted by distilled water and ethyl acetate. Then the extract was dissolved in distilled water. Before HPLC-MS determination, 6

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GEA (1 mg/mL) was filtered through a 0.22 µm filter membrane. Chromatographic separation and mass spectrometry were carried out following the previously reported method.28 GEA-NP formulation GEA-NP was prepared by w/o/w emulsion solvent evaporation method reported in the previous study.30 Briefly, 0.5 mg GEA dissolved in 100 µL distilled water was added dropwise into 1 mL organic phase which consisted of 10 mg R-CPC (S3) and 1 mL methylene chloride/ethyl acetate (1/1, v/v), and the mixture was emulsified by probe sonication for 1 min at 39.6 W. The obtained emulsion was dropped into 4 mL PVA (1%, w/v), sonicated for 1 min. The final emulsion was de-solvated by a rotary evaporator at 37 °C. After centrifugation (13,300 rpm, 4 °C, 1.5 h), the substratum was collected and diluted by distilled water, the supernatant was analyzed using an UV-6500 spectrophotometer (λ = 280 nm) to determinate the un-encapsulated GEA. Drug loading (DL) and encapsulation efficiency (EE) were calculated in accordance to the following formulas where WD and WP refers to the weight of drug and polymer, respectively. DL = WD / (WD + WP) × 100%

Eq.

(1) EE = Experimental DL / Theoretical DL ×100%

Eq.

(2) Particle-size distribution of GEA-NP was measured by Laser dynamic light scattering (Malvern, England). Morphology analysis of GEA-NP was conducted on an 7

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atomic force microscope (AFM, SPA-400, Japan). The samples were also characterized by FTIR and XRD. GEA-NP/H formulation The R-CPC copolymers (S1, S2, S3) were prepared into sequential aqueous solutions (15, 20, 25, 30, 35 wt%), tube-inversion method was used to determine their sol-gel transition temperatures. 4-mL vial containing 1 mL of the sample was incubated in the water bath which warmed up gradually, the phase of gel was defined as “non-flowing solid gel” in one minute. Rheometer (GEA Instruments, USA) equipped with a 40-mm parallel plate was used to measure rheology behavior of the polymer solutions (25 wt%) under the following test conditions: controlled stress = 0.5 dyn/cm2, frequency = 1.0 rad/s, heating rate = 1 °C/min, from 10 °C to 50 °C. The GEA-NP/H system was formulated by dispersing certain amount of GEA-NP into the R-CPC (S2) aqueous solution, the final concentration of S2 was adjusted to 20 wt%. Rheological measurements were carried out as the method mentioned above. Surface topography of lyophilized GEA-NP/H was imaged using a scanning electron microscopy (SEM, JSM-5900LV, JEOL, Japan). Ex vivo drug release study A modified dialysis method was applied to study the drug release profiles. In brief, dialysis bags (molecular cut off = 12 kDa) containing 500 µL of free GEA, GEA-NP or GEA-NP/H were incubated in 10 mL PBS (pH 7.4) at a shaking table (37 °C, 100 rpm). At determined time point, the drug release medium was collected and replaced by another 10 mL of PBS. Quantitative analysis of GEA was conducted using UV 8

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spectroscopy as mentioned above. Ex vivo cytotoxicity assay The sterilized R-CPC hydrogel (S2, 20 wt%) was extracted with DMEM for 2 days in a 37 °C incubator with 5% CO2, the leachate was diluted to 50%, 25%, and 12.5%. The HEK 293 and NIH-3T3 cells were respectively cultured in 96-well plate (5 × 103 per well). After 12 hours, the cells were exposed to the leachates of different concentrations (0.1% phenol as positive control) and cultured for 24 h. And the cells were treated with 20 µL of MTT. Before measuring the optical density at 570 nm, the precipitated formazan was dissolve with 150 µL of dimethyl sulfoxide. Ex vivo anti-oxidant study NIH-3T3 cells were seeded onto the acid-etched glass coverslips placed in 6-well plates (3 × 105 cells per well) and incubated for 12 h. The cells were treated with 500µM H2O2 solution, then free GEA and GEA-NP with a GEA concentration of 25 µg/mL was added into the corresponding wells. After 24 h, the cells were fixed by paraformaldehyde, stained with DAPI, imaged under a fluorescence microscopy (DM2500, LEICA, Germany). NIH-3T3 cells were also cultured in 96-well plate (5 × 103 cells per well) for 12 h. The cells were treated with 500 µM H2O2 solution, then blank NPs, free GEA and GEA-NP at different concentrations were added. After 24 h, cell viability was assessed by MTT assay. To measure the ability of GEA to scavenge DPPH radical, 20 µL of GEA or GEA-NP aqueous solutions with GEA concentration of 17, 25, 33, 42, 50, 67, 83 µg/mL, and blank NPs with an equal concentration of R-CPC copolymer to GEA-NP 9

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were added into 100 µL ethanolic solution of DPPH (250 µM), respectively. After 15 min of incubation at room temperature, the absorbance value at 517 nm was measured. For the positive control group, GEA was not added. For the blank control group, DPPH solution was absence. The radical scavenging capacity of GEA was calculated by the following equation: (Inhibition %) = (A0 – At) / A0 × 100%

Eq. (3)

Where A0 is the absorbance of the positive group, At is the absorbance of tested group. Ex/in vivo degradation study For ex vivo study, 1 mL of R-CPC solutions (S2, 20 wt%) were introduced into test tubes and incubated at 37 °C. After 10 min, 10 mL of PBS (pH 7.4, 37 °C) was added. The samples were incubated in an air bath table (37 °C), PBS was replaced each three days. Sample was collected and frozen-dried at designed time point, observed under SEM, and measured by GPC. For in vivo study, BALB/c mice were subcutaneously injected with 0.5 mL of R-CPC solutions (S2, 20 wt%), and also intraperitoneally injected with 0.1 mL of GEA-NP/H (R-CPC, 20 wt%), respectively. At specified time point, three mice were subjected to cervical dislocation to observe hydrogel degradation. To study biocompatibility of the R-CPC copolymer hydrogel, muscles around the injection site were collected for hematoxylin and eosin (H&E) staining. To noninvasively determine the in vivo degradation behavior of the copolymer hydrogel, rhodamine B (RB) was conjugated to hydroxyl groups of the R-CPC copolymer

31,32

(the detailed method was appended in the supporting information), 10

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and the obtained RB/R-CPC was used as a fluorescence probe. Five BALB/c nude mice were intraperitoneally injected with 0.1 mL of R-CPC solution (S2, 20 wt%) containing 1 wt% RB/R-CPC. To detect the remaining hydrogel at determined time point, the mice were imaged under an IVIS spectrum pre-clinical in vivo imaging system (PerkinElmer, USA; excitation = 540 nm, emission = 620 nm). In vivo anti-adhesion study In the study, a rat peritoneum injury-cecum abrasion model was established. After anesthetized with chloral hydrate (10%, 3 mL/kg), abdomen of the SD rats were shaved and disinfected with Betadine. Then a midline incision of 4 cm was made to expose the peritoneum. Subsequently, sterile dry surgical gauze was used to make a 1 × 2 cm2 defect of the cecum with oozing blood. A scalpel was used to create a 1 × 2 cm2 wound of the right peritoneum with punctate hemorrhage. And the wounds were placed together by suture (5-0). The rats were treated with 1 mL of NS, hyaluronic acid (HA), blank hydrogel, GEA-NP, or GEA-NP/H. After fully gelatinize, the peritoneum and skin were closed with 3-0 and 4-0 silk suture, respectively. At specified time point, 10 rats per group were sacrificed by excessive anaesthesia. Adhesion severity was evaluated by double-blind scoring in accordance with the standard scoring system.33 The adhesion tissues, damaged cecum and peritoneum were paraffin embedded, sectioned for H&E and Masson Trichrome (MT) staining. The specimens were also fixed and gradient dehydrated for SEM analysis. In addition, 5 rats from GEA-NP/H group were dissecting at 1, 3, 5, 7, 10, and 14 days post-operation to study the wound healing status and hydrogel degradation behavior. 11

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The damaged abdominal wall and cecum were collected for histopathological examination. Statistical analysis The data were analyzed by SPSS 10.0 software (Chicago, IL, USA). For adhesion scores, statistical inferences were carried out using Manne-Whitney U-tests, or Fisher’s exact test. A p value less than 0.05 was considered statistically significant.

RESULTS AND DISCUSSION Characterization of R-CPC copolymer Poloxamers with the block structure of PEG–PPG–PEG (Pluronic®) or PPG–PEG– PPG (reverse Pluronic®) are biocompatible amphiphilic polymers, but their biomedical application was limited by the extremely high critical micelle concentration (CMC).34 Thus, poly lactic acid (PLA)30 or hydrophobic quercetin35 has been grafted onto the PPG segments of 10R5 to decrease the CMC value and improve its drug loading capacity. In contrast to increasing researches on the Pluronic® copolymers, few study pays close attention to the reverse Pluronic® copolymers. In view of this, we modified reverse Pluronic®10R5 with lipophilic poly caprolactone (PCL) to moderate its biodegradability and physical properties. Ring-opening polymerization method as exhibited in Figure S1A was applied to synthesis the R-CPC copolymer. The characterized peaks of ester bond at 1735 cm-1 was found in the FTIR spectrum of R-CPC (Figure S1B), which indicated the successful synthesis of R-CPC. Molecular mass of R-CPC was evaluated using GPC 12

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(Figure S1C) and 1H NMR (Figure S2), the relative data were shown in Table 1. TG and DTG curves in Figure S3A-B suggested that with increase of the molecular mass, R-CPC copolymers showed faster weight loss. Furthermore, the DSC curves in Figure S3C indicated that melting and crystallization temperatures of R-CPC elevated with increase of their molecular weight. Data reflected thermal behavior of the R-CPC copolymers were presented in Table 2. As shown in Figure S3D, the CMC value of R-CPC (S2) was approximate 1 µg/mL which was much smaller than that of 10R5. Rhodamine B (RB) conjugated R-CPC copolymer (RB/R-PCP) was synthesized via esterification as the pathway descripted in Figure S4A. As shown in Figure S4B, RB/R-CPC possessed absorption peak at 560 nm which was attributed to the conjugation of RB. Furthermore, there are characteristic peaks at the range of 6.5-8.5 ppm in the 1H NMR spectrum of RB/R-CPC (Figure S5). All the data indicated that RB was successfully conjugated to the R-CPC copolymer. And the content of RB determined by UV spectrum was 4.32%. Characterization of GEA-NP In the past decades, multiple bioactive compounds with anti-oxidative, anti-inflammatory, anti-bacterial, anti-tumor, or anti-mutation properties have been extracted from Turkish galls.25,36-38 However, few study focus on their use as anti-adhesion agent up to now. Accumulating studies validated that oxidative damage to the abdominal wall initiates mesothelial cell dysfunction, peritoneal fibrosis, ultimately adhesion formation.39-41 Therefore, lots of antioxidant agents have been utilized to prevent postoperative adhesion.42,43 In light of this, we assumed that the 13

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extracts from Turkish galls might facilitate postoperative adhesion prevention due to their potent antioxidant and anti-inflammatory effects. Nanoparticles can enhance stability and bioavailability of the bioactive agent, as well regulate the drug release rate to improve therapeutic effect and reduce dosage frequency.44-47 In the study, Turkish galls ethyl acetate fraction (GEA) was prepared for the further anti-adhesion study. To improve drug stability and therapeutic effects, GEA were loaded into the R-CPC nanoparticles through w/o/w emulsion solvent evaporation. GEA-NP which had a particle size of 98±2.5 nm were uniform spheres (Figure 1A). The DL and EE of GEA-NP were 4.1±0.3% and 85.3±6.5%, respectively. As shown in Figure 1B, the IR spectrum of GEA-NP presented characteristic bands of both R-CPC and GEA. Moreover, the characteristic peaks ascribed to the crystalline structure of R-CPC were disappeared in the XRD spectrum of GEA-NP (Figure 1C). These results suggested that GEA was successfully loaded into the R-CPC copolymer nanoparticles, and intermolecular interactions occurred between GEA and R-CPC. HPLC-MS analysis (Figure 2) indicated there are 18 identified compounds contained in the chromatogram of GEA, they are in sequence Gallic acid (1), Digalloyl-glucoside

(2)–(3),

Tetragalloyl-glucoside

Digallic

acid

(10)–(12),

(4),

Trigalloyl-glucoside

Pentagalloyl-glucoside

(5)–(9), (13)–(15),

Hexagalloyl-glucoside (16)-(17), Heptagalloyl-glucoside (18).28 The characterization and identification of the compounds in the chromatogram of GEA was shown in Table S2. The main compounds in GEA were identified in which, the gallotannins was 88.03% and the phenolic acids was 11.12% by peak area calculation (Figure 2B). 14

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Characterization of GEA-NP/H The thermosensitive nature of R-CPC copolymers was validated by tube-inversion method and rheology. As shown in Figure S6, R-CPC copolymers with lager molecular weight showed higher sol-gel transition temperature and wider gel window. Arising from this, aqueous solution prepared from copolymer S2 at a concentration of 20 wt% can be utilized as an injectable in situ gel forming carrier. Compared with bare micelles or nanoparticles, hydrogel system strikingly expand the drug retention time at the administrated site, thus improve drug bioavailability.48 In view of this, GEA-NP were blended with the R-CPC copolymer hydrogel to fabricate the antioxidant and anti-inflammatory barrier system (GEA-NP/H). Figure 3A indicated that GEA-NP/H was mildly viscous solution at room temperature, but pliable and durable gel around body temperature. Furthermore, GEA-NP/H presented the 3D cross-linking network structure under SEM (Figure 3B). Ex vivo drug release profile of GEA Since GEA showed the similar UV absorbance curve (185–900 nm) as tannic acid (Figure S7), the release behavior of GEA was studied using UV-vis spectroscopy.23 GEA in the free drug form released much faster than that of GEA-NP and GEA-NP/H, due to the interaction of the hydrogel matrix, GEA-NP/H group showed delayed drug release profile compared with GEA-NP. As illustrated in Figure 3C, approximately 100% of GEA was released from the free GEA group within 12 h, while only 30% and 40% of GEA were released from GEA-NP and GEA-NP/H. Cytotoxicity of R-CPC hydrogel 15

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MTT assay was used to study the cytotoxicity of R-CPC copolymer hydrogel. HEK 293 and NIH-3T3 cells were treated with gradient diluted hydrogel leachates, after 24 h incubation the cell viability showed only slight descent with increase of the leachates concentration (Figure 3D). This suggested that R-CPC copolymer possesses high biocompatibility and will be safe for the in vivo application. Antioxidant and radical scavenging activity The ex vivo antioxidant activity of GEA were studied by qualitative morphological observations (Figure 4A). After exposed to H2O2 for 24 h, 3T3 cells in the NS group experienced severe oxidative damage, displaying shrinkage of the cell bodies and significant condensation of nuclei which were index of cell death. However, cells in free GEA and GEA-NP groups presented continuous cell layer, distinct cell boundary, flat and spindle shape. The morphological analysis show that GEA protects cells from H2O2-induced cell damage effectively.49 Protective effect of GEA was also evaluated by quantitating viable H2O2-treated 3T3 cells using MTT assay. Figure 4B demonstrated that both free GEA and GEA-NP aqueous solutions could prevent the cells from oxidative damages. When the cells were exposed to 25 µg/mL of GEA, the oxidative effects of H2O2 was completely wiped out. Furthermore, Free GEA and GEA-NP showed quite high free-radical scavenging ability (Figure 4C). Biodegradation of R-CPC hydrogel For ex vivo study, the R-CPC hydrogel was incubated with PBS (pH 7.4) at 37 °C. The samples disintegrated gradually (Figure 5A). Macroscopic observations displayed that the hydrogel gradually swelled and the surface was eroded within 2 weeks, finally 16

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resulting in disintegration of the hydrogel matrix. At designed time point, the samples were free-dried for the SEM and GPC measurement. SEM images supported that with swelling of the hydrogel, its regular 3D cross-linking network started to be destructed, and ultimately disappeared after 8 weeks’ degradation. As shown in Figure 5B, retention time of the samples were gradually extended, which reflected a steady molecular weight decrease of the R-CPC copolymer as result of hydrolysis. The R-CPC copolymer aqueous solution (20 wt%) could be easily injected at room temperature, it quickly conversed to a semisolid like gel upon dorsal subcutaneous injection. As shown in Figure S8A, the hydrogel degradation degree at week 5 almost reached 70%, and disappeared after 10 weeks. The connective and muscular tissues surround the hydrogel were collected for histopathology examination. An acute inflammatory reaction was observed at the first week, then the number of neutrophils and macrophages reduced gradually, and completely vanished at week 10 (Figure S8B). The fluorescence probe RB/R-CPC with a RB content of 4.32% was introduced into the R-CPC copolymer aqueous solution (20 wt% R-CPC, with 1 wt% RB/R-CPC). After intraperitoneal injection, the solution transformed into solid-like gel with potent fluorescence. Then the fluorescence intensity attenuated gradually due to degradation of the copolymer hydrogel (Figure 5C). Furthermore, GEA-NP/H almost disappeared at day 14 after intraperitoneal injection (Figure 5D-E). These results sufficiently certified the excellent biodegradability of the R-CPC copolymer. Anti-adhesion activity of GEA-NP/H 17

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Formation of peritoneal adhesion is a common complications following abdominal and pelvic surgeries, which can result in a lot of morbidities even death.50 Ever since a long time, physic barriers in form of solutions, solid sheets, semisolid hydrogels, have been widely studied in the prevention of post-operative adhesions.51,52 Although accumulating efforts were attempted to explore potent anti-adhesion therapeutics, there is still not effective treatment strategy in clinical. It’s essential to develop proper barrier systems to overcome the rapid clearance or non-biodegradation, and handling difficulty of the existing anti-adhesion barriers. After operative trauma, the rapid influx of neutrophils and macrophages elicits an acute inflammatory response characterized by the generation of multiple pro-inflammatory mediators like cytokine, growth factors, nitric oxide, reactive oxygen species (ROS).53,54 There is mounting evidence indicated that acute oxidative stress and disordered inflammatory response might induce intra-abdominal adhesion.39-41 Inspired by these studies, we combined the antioxidant and anti-inflammatory agent GEA with the biodegradable and injectable R-CPC copolymer hydrogel to fabricate a potential anti-adhesion barrier system. As displayed in Figure 6A, in situ gelation happened soon after application of GEA-NP/H, which formed a protective barrier conforming to the shape of the wound. Five days later, there was still some residual hydrogel on the separated abdominal wall and cecum. However, HA exhibited rapid degradation behaviors, and there was no HA hydrogel remain after 5 days. Two weeks post-surgery, severe adhesions occurred among the NS, HA and GEA-NP treated rats, whereas administration of the 18

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blank hydrogel or GEA-NP/H could effectively prevent adhesion formation, significantly reduce the adhesion area (Figure 6B) and severity score (Figure 6C). In particular, 50% of the rats from HA group exhibited Score 3 adhesion, while frequency of score 1 adhesion among the blank hydrogel group and GEA-NP/H group is 30% and 10% respectively, which was mainly attributed to delayed degradation of R-CPC copolymer, the antioxidant and anti-inflammatory properties of GEA. Histological assay (Figure 7) showed that tissues from rats with adhesion severity score 0 had been completely covered with layers of neo-mesothelial cells. In contrast, rats suffering tissue adhesion manifested conglutination between musculature of the injured cecum and peritoneum with connective tissues of different degrees. H&E and MT staining sections of the blank hydrogel and GEA-NP/H groups were shown in Figure 8. Due to the antioxidant, anti-inflammatory, and antibiosis activity of GEA, accelerated wound healing process was observed among defects treated with GEA-NP/H. Moreover, GEA-NP/H group presented much mature and well-organized mesothelial cell deposition on both cecum and abdominal wall after 14 days post-operation. To observe the wound healing process, rats from GEA-NP/H treated group were sacrificed on indicated days. As shown in Figure S9A, the hydrogel were observed on the separated abdominal wall and cecum on day 5, and the hydrogel degraded gradually within 7 days, the damaged peritoneum and cecum were completely repaired without adhesion tissues after 14 days. H&E and MT staining (Figure S9B) suggested that neutrophils significantly decreased on the 5th day, while fibroblasts and 19

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endothelial cells gradually increased with little collagen deposition. On the 14th day, the inflammatory cells disappeared, well-organized fibroblasts, endothelial cells, mesothelial cells, and collagens were observed on the abdominal wall and cecum, indicating the completely remesothelialization of the wounds. In addition, remesothelialization of the damaged cecum and abdominal wall was further studied using SEM. For GEA-NP/H treated group, less inflammatory cells (red arrows) and more elongated cells (yellow arrows) could be found on the cecum at day 5 (Figure 9A), which is attributed to the anti-oxidative effect of GEA. As shown in Figure 9B, defected peritoneum treated with GEA-NP/H presented much accelerated remesothelialization manifested by the more closely arranged squamous-shaped cells with microvilli (blue arrows). H&E staining of major organs for the GEA-NP/H treated rats at day 14 post-injury did not show any pathologic changes (Figure S10). Therefore, GEA-NP/H is a potent and safe anti-adhesion barrier.

CONCLUSION Reducing and preventing peritoneal adhesion after abdominal and pelvic operations still remains a major clinical challenge. In the study, a novel injectable hydrogel barrier (GEA-NP/H) composed of natural antioxidants extracted from Turkish galls (GEA) and the biocompatible R-CPC copolymer was developed and applied on the rat peritoneum

injury-cecum

abrasion

model.

Ex/in

vivo

studies

validated

biodegradability and thermosensitivity of GEA-NP/H. The hydrogel system could completely cover the operative defect, form a durable barrier thereby isolate the 20

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injured cecum and peritoneum at the critical period of adhesion formation. Furthermore, sustained release of GEA would effectively scavenge the free radicals, suppress oxidative stress, facilitate remesothelialization, and ultimately avoid postoperative adhesion. Owing to its excellent efficacy and safety, GEA-NP/H has promising application in adhesion prevention.

ASSOCIATED CONTENT Supporting information Synthesis scheme, IR, GPC, 1H NMR spectrum and TG, DTG, DSC curves of the R-CPC copolymers; Synthesis scheme, 1H NMR spectrum and UV curves of RB/R-CPC copolymer; sol-gel transition scheme and rheology measurement of the R-CPC aqueous solutions; UV absorbance curves of tannic acid (TA) and Turkish galls ethyl acetate fraction (GEA); Degradation behavior of R-CPC hydrogel after subcutaneous injection and H&E staining of the tissue around the injected hydrogel at determined time point; Wound healing process of GEA-NP/H treated rats; H&E staining of major organs for the GEA-NP/H treated rats at day 14 post-injury.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National S&T Major 21

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Project (2011ZX09102-001-10 and 2015ZX09102010) and the National Natural Sciences Foundation of China (81560699).

REFERENCE (1) Salem, S. E.; Proudman, C. J.; Archer, D. C. Prevention of Postoperative Complications Following Surgical Treatment of Equine Colic: Current Evidence. Equine. Vet. J. 2016, 48, 143−151. (2) Uysal, B.; Demirbag, S.; Poyrazoglu, Y.; Cayci, T.; Yesildaglar, N.; Guven, A.; Sürer, I.; Korkmaz, A. Medical Ozone Therapy Decreases Postoperative Uterine Adhesion Formation in Rats. Arch. Gynecol. Obstet. 2012, 286, 1201−1207. (3) Brochhausen, C.; Schmitt, V. H.; Planck, C. N.; Rajab, T. K.; Hollemann, D.; Tapprich, C.; Krämer, B.; Wallwiener, C.; Hierlemann, H.; Zehbe, R.; Planck, H.; Kirkpatrick, C. J. Current Strategies and Future Perspectives for Intraperitoneal Adhesion Prevention. J. Gastrointest. Sur. 2012, 16, 1256−1274. (4) Ten Broek, R. P.; Kok-Krant, N.; Bakkum, E. A.; Bleichrodt, R. P.; van Goor, H. Different Surgical Techniques to Reduce Post-Operative Adhesion Formation: a Systematic Review and Meta-Analysis. Hum. Reprod. Update 2013, 19, 12−25. (5) Kutuk, M. S.; Ozgun, M. T.; Batukan, C.; Ozcelik, B.; Basbug, M.; Ozturk, A. Oral Tadalafil Reduces Intra-Abdominal Adhesion Reformation in Rats. Hum. Reprod. 2012, 27, 733−737. (6) Alonso, Jde. M.; Alves, A. L.; Watanabe, M. J.; Rodrigues, C. A.; Hussni, C. A. Peritoneal Response to Abdominal Surgery: the Role of Equine Abdominal Adhesions 22

ACS Paragon Plus Environment

Page 22 of 44

Page 23 of 44 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

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and Current Prophylactic Strategies. Vet. Med. Int. 2014, 279730. (7) Bang, S.; Lee, E.; Ko, Y. G.; Kim, W. I.; Kwon, O. H. Injectable Pullulan Hydrogel for the Prevention of Postoperative Tissue Adhesion. Int. J. Biol. Macromol. 2016, 87, 155−162. (8) Bae, S. H.; Son, S. R.; Kumar Sakar, S.; Nguyen, T. H.; Kim, S. W.; Min, Y. K.; Lee, B. T. Evaluation of the Potential Anti-Adhesion Effect of the PVA/Gelatin Membrane. J. Biomed. Mater. Res. B Appl. Biomater. 2014, 102, 840−849. (9) Posadowska, U.; Brzychczy-Włoch, M.; Drożdż, A.; Krok-Borkowicz, M.; Włodarczyk-Biegun, M.; Dobrzyński, P.; Chrzanowski, W.; Pamuła, E. Injectable Hybrid Delivery System Composed of Gellan Gum, Nanoparticles and Gentamicin for the Localized Treatment of Bone Infections. Expert. Opin. Drug. Deliv. 2016, 13, 613−620. (10) Zhang, Z.; Ni, J.; Chen, L.; Yu, L.; Xu, J.; Ding, J. Biodegradable and Thermoreversible PCLA-PEG-PCLA Hydrogel as a Barrier for Prevention of Post-Operative Adhesion. Biomaterials 2011, 32, 4725−4736. (11) Li, J.; Mooney, D. J. Designing Hydrogels for Controlled Drug Delivery. Nat. Rev. Mater. 2016, 1, 16071. (12) GuhaSarkar, S.; Pathak, K.; Sudhalkar, N.; More, P.; Goda, J. S.; Gota, V.; Banerjee, R. Synergistic Locoregional Chemoradiotherapy Using a Composite Liposome-in-gel System as an Injectable Drug Depot. Int. J. Nanomedicine 2016, 11, 6435−6448. (13) Luo, Z.; Jin, L.; Xu, L.; Zhang, Z.; Yu, J.; Shi, S.; Li, X.; Chen, H. 23

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ACS Applied Materials & Interfaces 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

Thermosensitive PEG–PCL–PEG (PECE) Hydrogel as an in situ Gelling System for Ocular Drug Delivery of Diclofenac Sodium. Drug. Deliv. 2016, 23, 63−68. (14) Yun, Q.; Wang, S.; Xu, S.; Yang, J.; Fan, J.; Yang, L.; Chen, Y.; Fu, S.; Wu, J. Use of 5-Fluorouracil Loaded Micelles and Cisplatin in Thermosensitive Chitosan Hydrogel as an Efficient Therapy against Colorectal Peritoneal Carcinomatosis. Macromol. Biosci. 2017, 17, 1600262. (15) Sivashanmugam, A.; Kumar, R. A.; Priya, M. V.; Nair, S. V.; Jayakumar, R. An Overview of Injectable Polymeric Hydrogels for Tissue Engineering. Eur. Polym. J. 2015, 72, 543−565. (16) Ghasemi Tahrir, F.; Ganji, F.; Mani, A. R.; Khodaverdi, E. Ex vivo and Evaluation of Thermosensitive Chitosan Hydrogel for Sustained Release of Insulin. Drug Deliv. 2016, 23, 1038−1046. (17) Ouaïssi, M.; Gaujoux, S.; Veyrie, N.; Denève, E.; Brigand, C.; Castel, B.; Duron, J. J.; Rault, A.; Slim, K.; Nocca, D. Post-Operative Adhesions after Digestive Surgery: Their Incidence and Prevention: Review of the Literature. J. Visc. Surg. 2012, 149, e104−e114. (18) Jomezadeh, V.; Mohammadpour, A. H.; Rajabi, O.; Tavassoli, A.; Maddah, G. Evaluation of Curcumin Effects on Post-Operative Peritoneal Adhesion in Rats. Iran. J. Basic Med. Sci. 2012, 15, 1162−1167. (19) Mutsaers, S. E.; Birnie, K.; Lansley, S.; Herrick, S. E.; Lim, C. B.; Prêle, C. M. Mesothelial Cells in Tissue Repair and Fibrosis. Front Pharmacol. 2015, 6, 113. (20) Fortin, C. N.; Saed, G. M.; Diamond, M. P. Predisposing Factors to 24

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Page 24 of 44

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ACS Applied Materials & Interfaces

Post-Operative Adhesion Development. Hum. Reprod. Update 2015, 21, 536−551. (21) ten Raa, S.; van den Tol, M. P.; Sluiter, W.; Hofland, L. J.; van Eijck, C. H.; Jeekel, H. The Role of Neutrophils and Oxygen Free Radicals in Post-Operative Adhesions. J. Surg. Res. 2006, 136, 45−52. (22) Yang, J.; Zhou, Y.; Liu, H.; Wang, J.; Hu, J. MCI Extraction from Turkish Galls Played Protective Roles against X-Ray-Induced Damage in AHH-1 Cells. Int. J. Clin. Exp. Pathol. 2015, 8, 8122−8128. (23) Ninan, N.; Forget, A.; Shastri, V. P.; Voelcker, N. H.; Blencowe, A. Antibacterial and Anti-Inflammatory pH-Responsive Tannic Acid-Carboxylated Agarose Composite Hydrogels for Wound Healing. ACS Appl. Mater. Interfaces 2016, 8, 28511−28521. (24) Yang, Y. H.; Wang, Z.; Zheng, J.; Wang, R. Protective Effects of Gallic Acid against Spinal Cord InjuryⅢInduced Oxidative Stress. Mol. Med. Rep. 2015, 12, 3017−3024. (25) Han, H. J.; Kwon, H. Y.; Sohn, E. J.; Ko, H.; Kim, B.; Jung, K.; Lew, J. H.; Kim, S. H. Suppression of E-cadherin Mediates Gallotannin Induced Apoptosis in HepG2 Hepatocelluar Carcinoma Cells. Int. J. Biol. Sci. 2014, 10, 490−499. (26) Li, B.; Bhandari, D. R.; Römpp, A.; Spengler, B. High-Resolution MALDI Mass Spectrometry Imaging of Gallotannins and Monoterpene Glucosides in the Root of Paeonia Lactiflor. Sci. Rep. 2016, 6, 36074. (27) Yang, M. H.; Ali, Z.; Khan, I. A.; Khan, S. I. Anti-Inflammatory Activity of Constituents Isolated from Terminalia Chebula. Nat. Prod. Commun. 2014, 9, 25

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Page 26 of 44

965-968. (28) Yu, W.; Li, Z.; Long, F.; Chen, W.; Geng, Y.; Xie, Z.; Yao, M.; Han, B.; Liu, T. A Systems Pharmacology Approach to Determine Active Compounds and Action Mechanisms of Xipayi KuiJie’an Enema for Treatment of Ulcerative Colitis. Sci. Rep. 2017, 7, 1189. (29) Topel, Ö.; Çakır, B. A.; Budama, L.; Hoda, N. Determination of Critical Micelle

Concentration

of

Polybutadiene-Block-Poly(Ethyleneoxide)

Diblock

Copolymer by Fuorescence Spectroscopy and Dynamic Light Scattering. J. Mol. Liq. 2013, 177, 40−43. (30) Li, X.; Ye, X.; Qi, J.; Fan, R.; Gao, X.; Wu, Y.; Zhou, L.; Tong, A.; Guo, G. EGF and Curcumin co-Encapsulated Nanoparticle/Hydrogel System as Potent Skin Regeneration Agent. Int. J. Nanomedicine 2016, 11, 3993−4009. (31) Wang, W.; Liu, J.; Li, C.; Zhang, J.; Liu, J.; Dong A.; Kong D. Real-time and Non-Invasive Fluorescence Tracking of in vivo Degradation of the Thermosensitive PEGlyated Polyester Hydrogel. J. Mater. Chem. B 2014, 2, 4185–4192. (32)Shen, W.; Chen, X.; Luan, J.; Wang, D.; Yu, L.; Ding, J. Sustained Codelivery of Cisplatin and Paclitaxel via an Injectable Prodrug Hydrogel for Ovarian Cancer Treatment. ACS Appl Mater Interfaces 2017, 9, 40031−40046. (33) Yu, L.; Hu, H.; Chen, L.; Bao, X.; Li, Y.; Chen, L.; Xu, G.; Ye, X.; Ding, J. Comparative Studies of Thermogels in Preventing Post-Operative Adhesions and Corresponding Mechanisms, Biomater. Sci. 2014, 2, 1100−1109. (34) Zhao, Y. Z.; Sun, C. Z.; Lu, C. T.; Dai, D. D.; Lv, H. F.; Wu, Y.; Wan, C. W.; 26

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Chen, L. J.; Lin, M.; Li, X. K. Characterization and Anti-Tumor Activity of Chemical Conjugation of Doxorubicin in Polymeric Micelles (DOX-P) ex vivo. Cancer Lett. 2011, 311, 187−194. (35)

Hassanzadeh,

S.;

Feng,

Z.;

Pettersson,

T.;

Hakkarainen,

M.

A

Proof-of-Concept for Folate-Conjugated and Quercetin-Anchored Pluronic Mixed Micelles as Molecularly Modulated Polymeric Carriers for Doxorubicin. Polymer 2015, 74, 193−204. (36) Rattanata, N.; Klaynongsruang, S.; Leelayuwat, C.; Limpaiboon, T.; Lulitanond, A.; Boonsiri, P.; Chio-Srichan, S.; Soontaranon, S.; Rugmai, S.; Daduang, J. Gallic Acid Conjugated with Gold Nanoparticles: Antibacterial Activity and Mechanism of Action on Foodborne Pathogens. Int. J. Nanomedicine 2016, 11, 3347−3356. (37) Sahiner, N.; Sagbas, S.; Sahiner, M.; Silan, C.; Aktas, N.; Turk, M. Biocompatible and Biodegradable Poly(tannic acid) Hydrogel with Antimicrobial and Antioxidant Properties. Int. J. Biol. Macromol. 2016, 82, 150−159. (38) Braidy, N.; Grant, R.; Adams, S.; Guillemin, G. J. Neuroprotective Effects of Naturally Occurring Polyphenols on Quinolinic Acid-Induced Excitotoxicity in Human Neurons. FEBS J. 2010, 277, 368−382. (39) Gotloib, L.; Wajsbrot, V.; Cuperman, Y.; Shostak, A. Acute Oxidative Stress induces Peritoneal Hyperpermeability, Mesothelial Loss, and Fibrosis. J. Lab. Clin. Med. 2004, 143, 31−40. (40) Shavell, V. I.; Saed, G. M.; Diamond, M. P. Cellular Metabolism: Contribution 27

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to Postoperative Adhesion Development. Reprod. Sci. 2009, 16, 627−634. (41) Pol, H. W.; Sibma, E.; Zeebregts, C. J.; Pierik, E. G.; Meerwaldt, R. Increased Skin Autofluorescence after Colorectal Operation Reflects Surgical Stress and Postoperative Outcome. Am. J. Surg. 2011, 202, 583−589. (42) Chu, D. I.; Lim, R.; Heydrick, S.; Gainsbury, M. L.; Abdou, R.; D'Addese, L.; Reed, K. L.; Stucchi, A. F.; Becker, J. M. N-acetyl-L-cysteine Decreases Intra-Abdominal Adhesion Formation through the Upregulation of Peritoneal Fibrinolytic Activity and Antioxidant Defenses. Surgery 2011, 149, 801−812. (43) Reed, K. L.; Heydrick, S. J.; Aarons, C. B.; Prushik, S.; Gower, A. C.; Stucchi, A. F.; Becker, J. M. A Neurokinin-1 Receptor Antagonist that Reduces Intra-Abdominal Adhesion Formation Decreases Oxidative Stress in the Peritoneum. Am. J. Physiol. Gastrointest. Liver. Physiol. 2007, 293, G544−G551. (44) Shao, J.; Xie, H.; Huang, H.; Li, Z.; Sun, Z.; Xu, Y.; Xiao, Q.; Yu, X.; Zhao, Y.; Zhang, H.; Wang, H.; Chu, P. K. Biodegradable Black Phosphorus-Based Nanospheres for in vivo Photothermal Cancer Therapy. Nature Communications 2016, 7, 12967. (45) Fan, J.; He, Q.; Liu, Y.; Zhang, F.; Yang, X.; Wang, Z.; Lu, N.; Fan, W.; Lin, L.; Niu, G.; He, N.; Song, J.; Chen, X. Light-Responsive Biodegradable Nanomedicine Overcomes Multidrug Resistance via NO-Enhanced Chemosensitization. ACS Appl. Mater. Interfaces 2016, 8, 13804−13811. (46) Fan, R.; Mei, L.; Gao, X.; Wang, Y.; Xiang, M.; Zheng, Y.; Tong, A.; Zhang, X.; Han, B.; Zhou, L.; Mi, P.; You, C.; Qian, Z.; Wei, Y.; Guo, G. Self-Assembled 28

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Bifunctional Peptide as Effective Drug Delivery Vector with Powerful Antitumor Activity. Adv. Sci. 2017, 4, 1600285. (47) Wang, M.; Yang, X.; Zhang, P.; Cai, L.; Yang, X.; Chen, Y.; Jing, Y.; Kong, J.; Yang,

X.;

Sun

F.

Polyethyleneimine-Modified

Sustained

Delivery

Nanoparticles

Promote

Growth

Factors

Embryonic

Stem

with Cells

Differentiation and Liver Regeneration. Adv. Sci. 2016, 3, 1500393. (48) Zhang, Z.; He, Z.; Liang, R.; Ma, Y.; Huang, W.; Jiang, R.; Shi, S.; Chen, H.; Li, X. Fabrication of a Micellar Supramolecular Hydrogel for Ocular Drug Delivery. Biomacromolecules 2016, 17, 798−807. (49) Cao, Y.; He, W. Water-Soluble Antioxidant Derivative Poly(Triethylene Glycol Methyl Acrylate-co-α-Tocopheryl Acrylate) as a Potential Prodrug to Enable Localized Neuroprotection. Acta Biomater. 2013, 9, 4558−4568. (50) Rajab, T. K.; Ahmad, U. N.; Kelly, E. Implications of Late Complications from Adhesions for Preoperative Informed Consent. J. R. Soc. Med., 2010, 103, 317−321. (51) Rizzo, A.; Spedicato, M.; Mutinati, M.; Minoia, G.; Angioni, S.; Jirillo, F.; Pantaleo, M.; Sciorsci, R. L. Peritoneal Adhesions in Human and Veterinary Medicine: from Pathogenesis to Therapy. A Review. Immunopharmacol. Immunotoxicol. 2010, 32, 481−494. (52) Zhang, Y.; Gao, C.; Li, X.; Xu, C.; Zhang, Y.; Sun, Z.; Liu, Y.; Gao, J. Thermosensitive Methyl Cellulose-Based Injectable Hydrogels for Post-Operation Anti-Adhesion. Carbohydr. Polym. 2014, 101, 171−178. (53) Suzuki, T.; Kono, T.; Bochimoto, H.; Hira, Y.; Watanabe, T.; Furukawa, H. An 29

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Injured Tissue Affects the Opposite Intact Peritoneum During Postoperative Adhesion Formation. Sci. Rep. 2015, 5, 7668. (54) Tzianabos, A. O.; Holsti, M. A.; Zheng, X. X.; Stucchi, A. F.; Kuchroo, V. K.; Strom, T. B.; Glimcher, L. H.; Cruikshank, W. W. Functional Th1 Cells are Required for Surgical Adhesion Formation in a Murine Model. J. Immunol. 2008, 180, 6970−6976.

Figure Caption Figure 1. (A) AFM images of GEA-NP; (B) IR spectrum of R-CPC, GEA, the physic mixture of R-CPC and GEA, GEA-NP; (C)XRD curves of the blank NP, free GEA, GEA-NP, and the physic mixture of R-CPC and GEA. Figure 2. (A) MS analysis of GEA in negative ion mode. (B) HPLC analysis of GEA at 269 nm. Figure 3. (A) Rheology analysis of GEA-NP/H and grass images of GEA-NP/H which exhibited sol-gel transition around body temperature; (B) SEM images of freeze-dried GEA-NP/H; (C) Ex vivo GEA release profiles in PBS (pH 7.4, 37 °C) from free GEA solutions, GEA-NP, and GEA-NP/H (n=3); (D) Cytotoxicity of the R-CPC hydrogel

on HEK 293 and NIH-3T3 cells (n=6).

Figure 4. (A) Representative images of H2O2-treated NIH-3T3 cells exposed to NS, free GEA, and GEA-NP, nuclei were stained blue with DAPI, scale bar = 50 µm, 30

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magnification 20×; (B) Cell viability of the H2O2-treated NIH-3T3 cells exposed to NS, free GEA, and GEA-NP (n=6); (C) Free redical scavenging activity of GEA, GEA-NP, and the blank NP (n=6). Figure 5. Ex vivo and in vivo degradation study of R-CPC polymer hydrogel. (A) SEM images and macroscopic observations of R-CPC polymer hydrogel degraded in 10 mL PBS (pH 7.4, 37 °C) at different time point; (B) Molecular mass changes of R-CPC polymer at determined degradation time; (C) Fluorescence IVIS imaging of the BABL/c nude mice after intraperitoneal injection of R-CPC hydrogel containing RB/R-CPC (n=5); (D) Macroscopic observations of GEA-NP/H degradation after intraperitoneal injection to BABL/c mice at designed time point (n=3); (E) Mass loss of GEA-NP/H during the degradation in abdomen (n = 3). Figure 6. Prevention of postoperative abdominal adhesion in a rat cecum-defect abrasion model. (A) GEA-NP/H formed a durable barrier over the defects upon injection, and the hydrogel barrier still remained on the 5th day. 14 days post-surgery, the cecum and peritoneum of rats treated with GEA-NP/H and the blank hydrogel were well separated, while severe adhesions were observed in the NS, HA and GEA-NP group; (B) Adhesion area of the rats treated with NS, HA, GEA-NP, blank hydrogel, and GEA-NP/H (n=10); (C) Adhesion severity scoring of the rats treated with NS, HA, GEA-NP, blank hydrogel, and GEA-NP/H (n=10). Figure 7. Adhesion severity scoring. Score 0, no adhesion; score 1, mild adhesion, easily separable intestinal adhesion; score 2, moderate intestinal adhesion, separable by blunt dissection; score 3, severe intestinal adhesion, adhesion requiring sharp 31

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dissection. Figure 8. (A) H&E and (B) Masson trichrome’s staining of peritoneum (line 1 and line 3), cecum (line 2 and line 4) from hydrogel and GEA-NP/H groups at day 5 and 14. Figure 9. SEM images of cecum (A) and (B) abdominal wall of the hydrogel and GEA-NP/H treated rats at day 5 and 14. Line 3 was high magnifications of line 2. Red arrows indicated the inflammatory cells, yellow arrows indicated the elongated cells, and blue arrows indicated the squamous-shaped cells with microvilli.

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Table 1 The synthesized R-CPC copolymers Code

PCL/10R5

Mna(×103) Mnb(×103) Mnc(×103) Mwc(×103) Mw/Mnc

(w/w)a S1

2000:2000

4

4.1

5.8

7.7

1.32

S2

3000:2000

5

5.3

7.1

9.6

1.37

S3

4000:2000

6

6.1

8.7

12.4

1.42

Notes: the Superscript “a”, “b”, “c” indicated results calculated from feed ratio, 1H NMR, and GPC, respectively.

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Table 2 Thermal behavior of the R-CPC copolymers Code

Tm (oC)

Tc (oC)

Td,50%(oC)

Td,95% (oC)

S1

31.8

41.5

7.8

356

401

S2

40.1

48.8

16.2

324

384

S3

45.2

51.4

22.8

321

374

Notes: Tm, melting temperature; Tc, crystallization temperature; Td,50%/95%, temperature at which 50% or 95% weight reduced.

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Graphical Abstract

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Figure 1. (A) AFM images of GEA-NP; (B) IR spectrum of R-CPC, GEA, the physic mixture of R-CPC and GEA, GEA-NP; (C)XRD curves of the blank NP, free GEA, GEA-NP, and the physic mixture of R-CPC and GEA. 99x62mm (300 x 300 DPI)

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Figure 2. (A) MS analysis of GEA in negative ion mode. (B) HPLC analysis of GEA at 269 nm. 99x72mm (300 x 300 DPI)

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Figure 3. (A) Rheology analysis of GEA-NP/H and grass images of GEA-NP/H which exhibited sol-gel transition around body temperature; (B) SEM images of freeze-dried GEA-NP/H; (C) Ex vivo GEA release profiles in PBS (pH 7.4, 37 °C) from free GEA solutions, GEA-NP, and GEA-NP/H (n=3); (D) Cytotoxicity of the R-CPC hydrogel on HEK 293 and NIH-3T3 cells (n=6). 99x78mm (300 x 300 DPI)

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Figure 4. (A) Representative images of H2O2-treated NIH-3T3 cells exposed to NS, free GEA, and GEA-NP, nuclei were stained blue with DAPI, scale bar = 50 µm, magnification 20×; (B) Cell viability of the H2O2treated NIH-3T3 cells exposed to NS, free GEA, and GEA-NP (n=6); (C) Free redical scavenging activity of GEA, GEA-NP, and the blank NP (n=6). 99x86mm (300 x 300 DPI)

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Figure 5. Ex vivo and in vivo degradation study of R-CPC polymer hydrogel. (A) SEM images and macroscopic observations of R-CPC polymer hydrogel degraded in 10 mL PBS (pH 7.4, 37 °C) at different time point; (B) Molecular mass changes of R-CPC polymer at determined degradation time; (C) Fluorescence IVIS imaging of the BABL/c nude mice after intraperitoneal injection of R-CPC hydrogel containing RB/R-CPC (n=5); (D) Macroscopic observations of GEA-NP/H degradation after intraperitoneal injection to BABL/c mice at designed time point (n=3); (E) Mass loss of GEA-NP/H during the degradation in abdomen (n = 3). 44x19mm (300 x 300 DPI)

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Figure 6. Prevention of postoperative abdominal adhesion in a rat cecum-defect abrasion model. (A) GEANP/H formed a durable barrier over the defects upon injection, and the hydrogel barrier still remained on the 5th day. 14 days post-surgery, the cecum and peritoneum of rats treated with GEA-NP/H and the blank hydrogel were well separated, while severe adhesions were observed in the NS, HA and GEA-NP group; (B) Adhesion area of the rats treated with NS, HA, GEA-NP, blank hydrogel, and GEA-NP/H (n=10); (C) Adhesion severity scoring of the rats treated with NS, HA, GEA-NP, blank hydrogel, and GEA-NP/H (n=10). 99x64mm (300 x 300 DPI)

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Figure 7. Adhesion severity scoring. Score 0, no adhesion; score 1, mild adhesion, easily separable intestinal adhesion; score 2, moderate intestinal adhesion, separable by blunt dissection; score 3, severe intestinal adhesion, adhesion requiring sharp dissection. 56x32mm (300 x 300 DPI)

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Figure 8. (A) H&E and (B) Masson trichrome’s staining of peritoneum (line 1 and line 3), cecum (line 2 and line 4) from hydrogel and GEA-NP/H groups at day 5 and 14. 75x56mm (300 x 300 DPI)

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Figure 9. SEM images of cecum (A) and (B) abdominal wall of the hydrogel and GEA-NP/H treated rats at day 5 and 14. Line 3 was high magnifications of line 2. Red arrows indicated the inflammatory cells, yellow arrows indicated the elongated cells, and blue arrows indicated the squamous-shaped cells with microvilli. 99x96mm (300 x 300 DPI)

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