Improving Antiadhesion Effect of Thermosensitive Hydrogel with

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Improving anti-adhesion effect of thermosensitive hydrogel with sustained release of tissue-type plasminogen activator in a rat repeated-injury model Tao He, Chang Zou, Linjiang Song, Ning Wang, Suleixin Yang, Yan Zeng, Qinjie Wu, Wenli Zhang, Yingtai Chen, and Changyang Gong ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b13184 • Publication Date (Web): 18 Nov 2016 Downloaded from http://pubs.acs.org on November 28, 2016

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ACS Applied Materials & Interfaces

Improving anti-adhesion effect of thermosensitive hydrogel with sustained release of tissue-type plasminogen activator in a rat repeated-injury model Tao He 1, Chang Zou 1, Linjiang Song 1, Ning Wang 1, Suleixin Yang 1, Yan Zeng 1, Qinjie Wu 1, Wenli Zhang 1, Yingtai Chen 2,*, Changyang Gong 1,* 1

State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan

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

Cancer Hospital, Chinese Academy of Medical Sciences, Peking Union Medical College, China National Cancer Center, Beijing 100021, P. R. China

* To whom correspondence should be addressed (C Gong and Y Chen). E-mail: [email protected], [email protected].

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ABSTRACT Intraperitoneal adhesion occurs frequently after pelvic and abdominal surgery, which plays an enormous burden on patients. Various drugs and barrier agents were studied and used to prevent adhesions, but few of them were satisfactory. A thermosensitive hydrogel was developed in our previous work and was effective in preventing adhesions. But in our preliminary experiment, it showed limited activity in a more rigorous rat repeated-injury adhesion model which was much closer to clinical. In this study, tissue-type plasminogen activator (tPA) loaded thermosensitive hydrogel (tPA-hydrogel) was prepared, which combined barrier functions with sustained release of anti-adhesion drug. The obtained tPA-hydrogel was injectable and degraded in vivo gradually in 4 weeks. Both hematoxylin and eosin (H&E) and Masson trichrome staining confirmed that the tPA-hydrogel exhibited excellent anti-adhesion effects on repeated-injury adhesion. Scanning electron microscopy (SEM) was used to observe the injured abdominal wall and cecum remesothelialized after treated with tPA-hydrogel for 14 days. In addition, the PAI-1 and tPA levels was measured by ELISA. Results showed the PAI-1 concentrations in peritoneal lavage fluids of tPA-hydrogel treated rats were lower than that of other groups, leading to decreased fibrin formation, while there were no significant differences observed in tPA blood levels at any point in time (P > 0.05). This study demonstrated that the effectiveness of thermosensitive hydrogel in preventing adhesions could be enhanced by delivering anti-adhesion drugs, and the tPA-hydrogel might be a promising system for clinical application.

KEYWORDS: peritoneal adhesion; hydrogel; tissue-type plasminogen activator; thermosensitive; biodegradable; repeated-injury

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1. INTRODUCTION Intraperitoneal adhesion occurs frequently after laparotomy, which could result in increased risk of surgical complications, including chronic abdominal pain, acquired infertility and small intestinal obstruction

1-3

. Formation of adhesions is primarily caused by infections,

trauma, foreign bodies, and dehydration at the injury sites

4, 5

, many of them have to be

physically separated by adhesiolysis 6. The clinical problem has troubled millions of individual all over the world, and causes enormous costs in healthcare 7. Thus, compared with a second operation to heal adhesions, prevention of intraperitoneal adhesion formation has been considered as the best way to combat adhesion-related morbidity 1, 8, 9. In the past decades, there were mainly two approaches developed to prevent adhesion 2, 10

after surgery, including barrier agents and pharmacological approaches have attracted much attention due to their remarkable advantages diversified forms: polymer solutions

12, 13

, solid membranes

. Barrier systems

11

, and have tested in

14, 15

, and cross-linking hydrogels

16-19

. Some of them have access to markets, such as oxidized regenerated cellulose membrane

(Innterceed®), hyaluronic acid carboxymethyl cellulose membrane (Seprafilm®), 4% icodextrin solution (Adept®). Although barrier systems have many considerable advantages for the adhesion prevention, the size and shape of solid membranes could not guarantee that the affected tissues were covered completely

17

. Besides, the short residue time of polymer

solutions in injured regions also limits their application in preventing adhesions

20

. Many

anti-adhesions drugs which against different therapeutic targets have been used in preventing adhesions, including dexamethasone, heparin, tissue-type plasminogen activator, mitomycin C and etc

21-23

. They may work in reducing inflammation, preventing fibrin clot formation,

stimulating fibrinolysis and inhibiting fibroblast proliferation respectively 11, but few of them have really satisfactory treatment effectiveness

16

. Thus, preventing the formation of

intraperitoneal adhesion is still a challenge. In previous studies

24, 25

, we had developed a kind of barrier system named

poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone) (PCEC) hydrogel, which was biocompatible, degradabale and non-toxic in vivo. Particularly, the PCEC solution was free-flowing in low temperature, but rapidly transformed into solid-like hydrogel at body

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temperature. The obtained hydrogel had been used in an attempt to preventing adhesion formation in our previous work. Several rat adhesion models were established to evaluate the anti-adhesion availability of PCEC hydrogel, including desiccation-induced peritoneal adhesion model, ischemia-induced peritoneal adhesion model and sidewall defect-cecum abrasion model 26-28, and the hydrogel was effective in these models. It could be explained that the PCEC hydrogel attached to the surface of injured tissues may not result in destruction of fibrin, but effectively prevents the formation of fibrinous adhesions without significant adverse effects during the healing process. From the practical point of view, these researched anti-adhesion barriers or drugs should be applicable for clinical practice. However, so many animal adhesion models developed in laboratory were not much more severe than that in clinical conditions, in which ideal conclusions were more inclined to get. Therefore, we established a repeated-injury adhesion animal model that was more difficult to heal and much closer to clinical 29. As expected, in our previous experiments, the anti-adhesion activity of the hydrogel we used before was considerable limited in the repeated-injury animal model. Beyond these, we designed a sustained drug delivery system that encapsulating the tissue-type plasminogen activator (tPA) into hydrogel to form tPA-hydrogel, which integrated the barrier functions and pharmacological therapies to treat more severe animal adhesion model.

2. MATERIALS AND METHODS 2.1 Materials and animals ɛ-Caprolactone (ɛ-CL, Alfa Aesar, USA) was purchased from Alfa Aesar (USA), tPA (Alteplase, CathfloTM Activase® ) was sourced from Genentech (USA), poly(ethylene glycol) (PEG, Mn=1000), stannous octoate (Sn(oct)2) and other materials were purchased from Sigma-Aldrich (St. Louis, MO). All other chemicals used were analytic reagent grade without further purification. Female BALB/c mice (18-22g) and female Sprague Dawley (SD) rats (210-250g) were provided by the Laboratory Animal Center of Sichuan University. All animal experiments were cared for in compliance with the Institutional Animal Care and Treatment Committee of Sichuan University (Chengdu, P.R. China), and were in quarantine for a week before treatment.

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2.2 Synthesis of PCL-PEG-PCL (PCEC) triblock copolymer Biodegradable PCEC triblock copolymer was prepared as reported elsewhere in our group 24

. In detail, PEG, ε-caprolactone, stannous octoate were introduced into a dry reaction vessel

under a nitrogen atmosphere. The reaction system was stirred gently at 130°C for 6 hours. After degassed under vacuum for one hour, the obtained PCEC copolymer was cooled to room temperature, and then precipitated from pre-cooled pereoleum ether. Finally, the copolymer was filtered and vacuum dried to a constant weight. The purified PCEC copolymer was stored in desiccators for further use. 2.3 Preparation and characterization of tPA-hydrogel PCEC hydrogel was made according to our previous work 24. Briefly, the obtained PCEC copolymer was dissolved in normal saline (20 wt.%) at 55°C to form micelle by self-assembly method and then cooled to 4°C. The sol was filtered with sterile syringe filter (0.22 µm, Millipore), and then 250 µg of sterile tPA was added into 1 mL of micelle to form homogeneous solution. The sterile stock solution would transform into hydrogel when the temperature increased to 37°C. SEM (Inspect F, FEI Company, Eindhoven, The Netherlands) was used to observe the morphology of tPA-hydrogel. In brief, the obtained tPA-hydrogel was frozen in liquid nitrogen and lyophilized, then coated with a layer of gold before observation. In addition, the test tube-inverting method was employed to investigate the sol-gel-sol phase transition behavior of the tPA-hydrogel. The sample was loaded into a 4 mL tightly screw-capped vial and then heated from 4°C to 60°C with a heating rate of 1°C /min. The sol-gel-sol phase transition behavior was visually observed by inverting the vials. 2.4 In vitro release kinetics The release kinetics of tPA from tPA-hydrogel were evaluated by using a membraneless model. Two milliliter of medium (PBS, pH 7.4) was added on the top of tPA-hydrogel (500 µl, 20 wt.% of hydrogel containing 1 mg of tPA) which was placed into the bottom of 4 mL Eppendorf tubes, and incubated at 37°C with gentle shaker. At predetermined time intervals, the medium were collected and replaced with 2 mL fresh PBS. The tPA concentration in medium was determined by BCATM Protein Assay Kit (PIERCE, USA). 2.5 In vivo degradation behavior 5



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Twelve BALB/c mice were used to evaluate the degradation of tPA-hydrogel in vivo. 500 µL of tPA-hydrogel was administered by dorsal subcutaneous injection. Three mice were sacrificed periodically (1 week, 2 week, 3 week and 4 week). The state of hydrogel in injection sites was observed by opening the skin with a surgical scissors. 2.6 Development of rat repeated-injury adhesion model and treatment A first laparotomy and sidewall defect-cecum abrasion model was established according to our previous work

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. In brief, the rats were anesthetized with sodium pentobarbital (3%,

30mg/kg via intraperitoneal injection), upon which the abdomen was opened by a scalpel, and then sterile dry surgical gauze was used to make a defect on the cecum with puncatate hemorrhage. Subsequently, the surface of abdominal wall was also damaged by a scalpel to create a peritoneal defect with an area of 1 cm2. The surface of chafed cecum and damaged peritoneal wall were placed together by fixing with 5/0 surgical silk suture. Repeated-injury modal was performed after one week. The adhesion sites were cut by a blunt or sharp dissection according to the need. Then, separate cecum and abdominal wall were re-abraded monodirectionally until a bleeding surface was generated. Experimental materials which contained 1mL of normal saline, blank hydrogel, tPA solution, tPA-hydrogel, arg-hydrogel (arginine was loaded in PCEC hydrogel) or in-tPA-hydrogel (inactivated tPA was made by boiling tPA for 20 minutes, and then loaded in PCEC hydrogel) were respectively used to coat the injured areas. Finally, the incision of the abdomen was closed with 5/0 surgical silk suture. 2.7 Evaluation of anti-adhesion activity of tPA-hydrogel in the repeated-injury adhesion model All rats were euthanized by injecting excess sodium pentobarbital on day 14 26. The extent of adhesion was scored using the following standard adhesion scoring system: score 0, no adhesion; score 1, one thin filmy adhesion; score 2, more than one thin adhesions; score 3, thick adhesion with focal point; score 4, thick adhesion with plantar attachment or more than one thick adhesion with focal point; score 5, very thick vascularized adhesion or more than one plantar adhesion. The separated adhesion sites were measured and the adhesion proportion of each rat was calculated to make a quantitative evaluation. For histopathological examination, the adhesion tissues, damaged abdominal wall and cecum from each group were fixed by 4% paraformaldehyde in phosphate-buffered solution for 6


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48 hours, then embedded in paraffin, sectioned and stained with H&E and Masson trichrome. The prepared slides were observed and analyzed by two pathologists in a blinded manner. For SEM analysis, the collected cecum and abdominal wall were fixed with 2.5% glutaraldehyde in PBS immediately, and then coated with a gold sputter before examined by SEM (JSM-5900LV, JEOL, Japan). 2.8 Measurement of tPA and PAI-1 levels Blood samples were collected from each rat at determined time, and centrifuged at 2000 g for 20 min to separate plasma, then stored at -80°C until analysis. Peritoneal lavage fluids were collected at the same time with drawing blood. In brief, the abdomen was gently pressed for 5 minutes after intraperitoneal injection of 5 mL PBS, and then the liquid was sucked out and centrifuged. The supernatant was collected and frozen at -80°C. tPA levels in plasma were measured by tPA elisa kit (HaiTaiTongDa Tech Co., Ltd Beijing China), and PAI-1 levels in peritoneal lavage fluids were determined by PAI-1 elisa kit (HaiTaiTongDa Tech Co., Ltd Beijing China) according to the manufacturer’s methods. 2.9 Statistical analysis The statistical analysis was performed using Graphpad Prism and Microsoft Excel. Adhesion scores did not follow a normal distribution, therefore statistical differences were made using Mann–Whitney U-tests or Fisher’s exact test. A P value < 0.05 on a 2-tailed test was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1 Preparation and characterization of tPA-hydrogel Fig. 1A showed the schematic gelling process of tPA-hydrogel. The PCEC solution (20 wt.%) was heated to 55°C to form micelle by self-assembly method, and then tPA was added into micelle to form homogeneous solution at low temperature which was flowable and injectable. A larger size of PCEC micelle appeared as the temperature increased, and the non-flowing hydrogel would form at the sol-gel transition temperature due to the further growth and aggregation of micelles. As reported in our previous work

24

, the PCEC triblock

copolymer was amphipathic that led to a special temperature-dependent sol-gel-sol transition behavior (Fig. 1B). After tPA was added into PCEC solution, the phase transition behavior was

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determined. As shown in Fig. 1C, when the concentration of PCEC solution was up to CGC (critical gelation concentration), the solution was free flowing at low temperature, but formed non-flowing gel at higher temperature such as body temperature. With the increase of temperature continuously, the gel converted into a free-flowing sol again because of the destruction of the micellar structure. In addition, the lower critical gelation temperature decreased and higher one increased with the increasing of PCEC concentration. The appropriate phase inversion temperature makes tPA-micelle transform into tPA-hydrogel in a short time period in vivo, which may form a larger coverage on injury sites upon application. For observing the morphology of tPA-hydrogel, SEM was used, and tPA-hydrogel displayed a three-dimensional network structure (Fig. 2A). In comparison with other anti-adhesion agents, the gelation is mediated by an increase in temperature, and occurs with no need for additional cross-linking agents such as initiators or light sources. 3.2 In vitro tPA release study In the past three decades, several drugs in combination with barrier agents were applied in preventing adhesions, however, none of them have been adopted for standard therapy 30. One of the challenges in developing barrier agents into an anti-adhesion drug delivery system was the rapid release of drugs. In this study, we selected PCEC hydrogel to delivery tPA in situ, as we had shown the lower drug release rate of macromolecular in it

24

. The sustained release

behavior of tPA from tPA-hydrogel was determined by a membraneless model. As shown in Fig. 2B, 64.07 ± 5.22% of tPA was released in 14 days. It is expected that the slow release performance assure the accumulation of tPA in injury sites at a suitable concentration. 3.3 In vivo degradation behavior of tPA-hydrogel In vivo degradation behavior of tPA-hydrogel was assessed by injecting subcutaneously into the back of BALB/c mice. On gross observation, the amount of tPA-hydrogel became less and less over time, and disappeared by the end of 4 weeks (Fig. 3). Besides, the time fame of the degradation of tPA-hydrogel was longer than that of drug release, which resulted in a lower drug release rate in vivo to some extent. Meanwhile, it might keep injured sites separated efficiently during the period of wound healing as the function of barrier agent. Sustained release and degradation behaviors made tPA-hydrogel a potential formulation in preventing adhesions. 8


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3.4 Assessment of anti-adhesion activity of tPA-hydrogel in the rat repeated-injury adhesion model In our previous work

26-28

, the PCEC hydrogel was used to prevent several single-injury

adhesion models, and showed exciting effects. However, there were no satisfactory results arising when PCEC hydrogel was applied to the repeated-injury adhesion model which was closer to clinic 29. Some drugs had been used in post-surgical adhesion, but they were rapidly eliminated from the peritoneum. In order to clarify the concrete advantages of tPA-hydrogel over the previous agent, the rat repeated-injury adhesion model was establishment. The peritoneal defect and cecum with puncatate hemorrhage were produced firstly (Fig. 4A), and then adhesion was visible with scores of 5 (very thick vascularized adhesion or more than one plantar adhesion) after one week (Fig. 4B). Adhesion sites were cut by a blunt dissection and a second injury was performed to induce more severe adhesion. As shown in Fig. 4C, the surface of separated cecum and abdominal wall were re-abraded to result in more intense bleeding than the initial surgery. Before complete suture, the injured area was covered with 1 mL of each formulation. Two weeks later, all the animals were euthanized, and a typical adhesion from each group after treatment was shown in Fig. 5. In NS group, all rats developed adhesions with scores of 5 (very thick vascularized adhesion or more than one plantar adhesion), which demonstrated the repeated-injury model was successfully established (Fig. 6C). On gross observation, there was no adhesion formation in tPA-hydrogel treated rats that the injured abdominal wall and cecum of which were separated completely (Fig. 5D). While the rats of other groups suffered varying degree adhesions which were not only visible in injured sits but also involved the uninjured surface of cecum and proximal colon (Fig. 5). The change of body weight, adhesion area and frequency of adhesion score are summarized in Fig. 6. There is no significant difference in weight changes observed between the groups (P > 0.05) (Fig. 6A). However, in the group of NS (n=6), all rats developed score 5 adhesion (Fig. 6C), and the median area was 3.86 cm2 which reflected the severity of the adhesion. Single tPA solution group showed limited effect in adhesion prevention, 4 of 6 rats developed score 5 adhesions and the other two were 4 and 3, which may due to a short residence time in injured sites. The median adhesion score in blank hydrogel group was 4, and the median area of adhesions was significantly lower than that in NS group (P < 0.01), 9



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suggesting the anti-adhesion activity of hydrogel as a barrier agent. However, it was much greater than that in rats treated with tPA-hydrogel (P = 0.045). To eliminate some specific effects in reducing adhesion formation, the inactivated tPA and arginine encapsulated in PCEC hydrogel respectively were used, in which tPA-hydrogel may work in preventing adhesion formation attributed to the existence of a protein, and arginine was contained in tPA solution as a cosolvent. The median adhesion area of animals treated with arg-hydrogel and In-tPA-hydrogel were 1.14 and 0.82 cm2 (Fig. 6B), respectively. Compared with the tPA-hydrogel group, the areas of adhesion were greater, and the median adhesion severity scores of arg-hydrogel and In-tPA-hydrogel group were 4 and 4.5, which were dramatically greater than that of tPA-hydrogel group (0, P < 0.05, Mann-Whitney U-tests). These results showed that non-specific protein and cosolvent did not play a major role in this process of preventing adhesions, and the dramatic anti-adhesion activity was not depend on the pharmacologic action of tPA alone, nor on the barrier effect of thermosensitive hydrogel, but a combined effect of them and a sustained release behavior of tPA. 3.5 Histopathological evaluation The specimens taken from healed abdominal walls and cecum of tPA-hydrogel treated group and adhesion tissues from NS group were stained with H&E and Masson trichrome (Fig. 7). In NS group, it was clearly that the smooth muscles of injured cecum and the skeletal muscle of abdominal wall were fused with connective tissues, which was composed of fibrocytes, inflammatory cells and collagen deposition. However, there was no large amount of fibrous tissues observed on the surface of abdominal wall and cecum in tPA-hydrogel group. In contrast, a layer of neo-mesothelial cell emerged with subjacent fibrosis. These results prove that the tPA-hydrogel have prevented the adhesion formation. 3.6 Remesothelialization of injured tissues Normal mesothelial cells provided lubrication for sliding viscera, protected against adhesions and thromboses, but injured mesothelial surfaces caused fibrin deposition, and the fibrin served as a tissue bridge for adhesion development 2. In order to further study the remesothelialization of injured tissues, SEM was used to observe the surface of healed abdominal wall and cecum in tPA-hydrogel group. As displayed in Fig. 8A and C, the abdominal wall and cecum were covered with numerous flattened and squamous-like cells 10


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which arranged densely. Furthermore, in a higher magnification, lots of obvious microvilli appeared on these cells (Fig. 8B and D), suggesting that the mesothelial cell layer was rebuilded on the surface of abdominal wall and cecum. Remesothelialization of injured tissues reveals that the rats in tPA-hydrogel group have fully recovered. 3.7 Measurement of tPA and PAI-1 levels Plasminogen activator inhibitor-1 (PAI-1) plays an antagonism role compared with tPA. The balance between tPA and PAI-1 is critical in the process of adhesion formation or prevention. Therefore, peritoneal lavage fluids were collected at determined time, and the concentration of PAI-1 was measured by ELSIA. As displayed in Fig. 9A, the PAI-1 concentrations of NS, hydrogel and In-tPA-hydrogel increased in one day after treatment, and then fell. In contrast, the group of tPA, Arg-hydrogel and tPA-hydrogel had a peak concentration on day 3. In addition, the PAI-1 concentration of tPA-hydrogel group was lower than that of other groups at each time point, especially on days 1, 3 and 5. Statistical analysis revealed that the PAI-1 levels of NS and tPA groups was significantly higher than that of tPA-hydrogel group (P < 0.05) on day 1. Similarly, the same significant differences appeared on days 3 and 5 when compared with the group of tPA and tPA-hydrogel. These results indicate adhesion formation might be reduced by enhancing fibrinolysis activity relatively in rats due to the lower expression of PAI-1. Previous studies

31

have showed that the tissue plasminogen activator (tPA) plays vital

roles in preventing adhesions formation, which is expressed by mesothelisl cells, macrophages and endothelial cells. However, a large dose of tPA may cause undesirable side effects such as excessive bleeding. To confirm if the chance of systemic bleeding would be increased after treatment of tPA-hydrogel, the plasma tPA levels were determined by ELISA. As shown in Fig. 9B, between these groups, there are no significant differences observed in tPA levels at any point in time (P > 0.05), indicating that the dosages used in our study was safe. On the other hand, internal bleeding was not observed in tPA-hydrogel treated mice. These findings show that the side effects of tPA do not develop with intraperitoneally applied tPA-hydrogel.

5. Conclusions In this study, thermosensitive tPA-hydrogel was prepared successfully, which was an

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anti-adhesion system consisted of a barrier agent (thermosensitive PCEC hydrogel) and pharmacological approach (tPA). It is a kind of safe material with biodegradability and biocompatibility. The sustained tPA release behavior improves the anti-adhesion effects of tPA-hydrogel in a more rigorous adhesion model. It can down-regulate the express of PAI-1 with no side effects in vivo. The high-efficiency in preventing repeated-injury adhesion formation may make tPA-hydrogel a promising system in clinical practice.

ACKNOWLEDGEMENT This work was financially supported by National Natural Science Foundation of China (NSFC31400814), Beijing Nova Program No. xxjh2015A090, Grant No. LC2015L11 from Cancer Hospital of Chinese Academy of Medical Sciences, and National Young Top-notch Talent Program.

DECLARATION OF INTEREST STATEMENT The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

ABBREVIATIONS PCEC, poly(ε-caprolactone)-poly(ethylene glycol)-poly(ε-caprolactone); tPA, tissue-type plasminogen activator; H&E, hematoxylin and eosin; SEM, scanning electron microscopy; PAI-1, plasminogen activator inhibitor-1; ELSIA, enzyme-linked immuno sorbent assay; NS, normal saline.

REFERENCES (1) 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. (2) Brochhausen, C.; Schmitt, V. H.; Planck, C. N.; Rajab, T. K.; Hollemann, D.; Tapprich, C.; Kramer, B.; Wallwiener, C.; Hierlemann, H.; Zehbe, R.; Planck, H.; Kirkpatrick, C. J. Current Strategies and Future Perspectives for Intraperitoneal Adhesion Prevention. J. Gastrointest Surg. 2012, 16, 1256-1274.

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(3) Rajab, T. K.; Wallwiener, M.; Talukdar, S.; Kraemer, B. Adhesion-Related Complications are Common, but Rarely Discussed in Preoperative Consent: A Multicenter Study. World J. Surg. 2009, 33, 748-750. (4) Na, S. Y.; Oh, S. H.; Song, K. S.; Lee, J. H. Hyaluronic Acid/Mildly Crosslinked Alginate Hydrogel as an Injectable Tissue Adhesion Barrier. J. Mater. Sci: Mater. Med. 2012, 23, 2303-2313. (5) Zhou, Y.; Zhang, L.; Zhao, W.; Wu, Y.; Zhu, C.; Yang, Y. Nanoparticle-Mediated Aelivery of TGF-Beta1 miRNA Plasmid for Preventing Flexor Tendon Adhesion Formation. Biomaterials 2013, 34, 8269-8278. (6) Ward, B. C.; Panitch, A. Abdominal Adhesions: Current and Novel Therapies. J. Surg. Res. 2011, 165, 91-111. (7) Yan, S.; Yue, Y. Z.; Zeng, L.; Yue, J.; Li, W. L.; Mao, C. Q.; Yang, L. Effect of Intra-Abdominal Administration of Ligustrazine Nanoparticles Nano Spray on Postoperative Peritoneal Adhesion in Rat Model. J. Obstet. Gynaecol. Res. 2015, 41, 1942-1950. (8) Ouaissi, M.; Gaujoux, S.; Veyrie, N.; Deneve, 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. (9) Vogels, R. R.; van Barneveld, K. W.; Bosmans, J. W.; Beets, G.; Gijbels, M. J.; Schreinemacher, M. H.; Bouvy, N. D. Long-Term Evaluation of Adhesion Formation and Foreign Body Response to Three New Meshes. Surg. Endosc. 2015, 29, 2251-2259. (10) Sakai, S.; Ueda, K.; Taya, M. Peritoneal Adhesion Prevention by a Biodegradable Hyaluronic Acid-Based Hydrogel Formed in Situ Through a Cascade Enzyme Reaction Initiated by Contact with Body Fluid on Tissue Surfaces. Acta Biomater. 2015, 24, 152-158. (11) Yeo, Y.; Kohane, D. S. Polymers in the Prevention of Peritoneal Adhesions. Eur. J. Pharm. Biopharm. 2008, 68, 57-66. (12) Zheng, Z.; Zhang, W.; Sun, W.; Li, X.; Duan, J.; Cui, J.; Feng, Z.; Mansour, H. M. Influence of the Carboxymethyl Chitosan Anti-Adhesion Solution on the TGF-Beta1 in a Postoperative Peritoneal Adhesion Rat. J. Mater. Sci: Mater. Med. 2013, 24, 2549-2559. (13) Christian, D. K.; Partrick, S.; Marcel, B.; Stefan, J.; Rafael, R.; Rene, T.; Ulf, P. N.; Uwe, K. Influence of 4% Icodextrin Solution on Peritoneal. BMC Surg. 2013, 13, 1471-1482. 13



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(14) Shahram, E.; Sadraie, S. H.; Kaka, G.; Khoshmohabat, H.; Hosseinalipour, M.; Panahi, F.; Naimi-Jamal, M. R. Evaluation of Chitosan-Gelatin Films for Use as Postoperative Adhesion Barrier in Rat Cecum Model. Int. J. Surg. 2013, 11, 1097-1102. (15) Pryor, H. I., 2nd; O'Doherty, E.; Hart, A.; Owens, G.; Hoganson, D.; Vacanti, J. P.; Masiakos, P. T.; Sundback, C. A. Poly(glycerol sebacate) Films Prevent Postoperative Adhesions and Allow Laparoscopic Placement. Surgery 2009, 146, 490-497. (16) Li, L.; Wang, N.; Jin, X.; Deng, R.; Nie, S.; Sun, L.; Wu, Q.; Wei, Y.; Gong, C. Biodegradable and Injectable in Situ Cross-Linking Chitosan-Hyaluronic Acid Based Hydrogels for Postoperative Adhesion Prevention. Biomaterials 2014, 35, 3903-3917. (17) Takahashi, A.; Suzuki, Y.; Suhara, T.; Omichi, K.; Shimizu, A.; Hasegawa, K.; Kokudo, N.; Ohta, S.; Ito, T. In Situ Cross-Linkable Hydrogel of Hyaluronan Produced Via Copper-Free Click Chemistry. Biomacromolecules 2013, 14, 3581-3588. (18) Wei, C. Z.; Hou, C. L.; Gu, Q. S.; Jiang, L. X.; Zhu, B.; Sheng, A. L. A Thermosensitive Chitosan-Based Hydrogel Barrier for Post-Operative Adhesions' Prevention. Biomaterials 2009, 30, 5534-5540. (19) Ito, T.; Yeo, Y.; Highley, C. B.; Bellas, E.; Kohane, D. S. Dextran-Based in Situ Cross-Linked Injectable Hydrogels to Prevent Peritoneal Adhesions. Biomaterials 2007, 28, 3418-3426. (20) 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. (21) Tuncay, K.; Bayezit, E.; Dilek, U.; Cemal, G. Prevention of Adhesion by Sodium Chromoglycate, Dexamthasone, Saline and Appotinin after Pelvic Surgery. Aust. N. Z. J. Surg. 2004, 74, 1111-1115. (22) Reid, R. L.; Spence, J. E.; Tulandi, T.; Yuzpe, A. Clinical Evaluation of the Efficacy of Heparin-Saturated Interceed for Prevention of Adhesion Reformation in the Pelvic Sidewall of the Human. Prog. Clin. Biol. Res. 1993, 381, 261-264. (23) Hill-West, J. L.; Dunn, R. C.; Hubbell, J. A. Local Release of Fibrinolytic Agents for Adhesion Prevention. J. Surg. Res. 1995, 59, 759-763.

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(24) Gong, C. Y.; Shi, S.; Dong, P. W.; Yang, B.; Qi, X. R.; Guo, G.; Gu, Y. C.; Zhao, X.; Wei, Y. Q.; Qian, Z. Y. Biodegradable in Situ Gel-Forming Controlled Drug Delivery System Based on Thermosensitive PCL-PEG-PCL Hydrogel: Part 1--Synthesis, Characterization, and Acute Toxicity Evaluation. J. Pharm. Sci. 2009, 98, 4684-4694. (25) Gong, C.; Shi, S.; Wu, L.; Gou, M.; Yin, Q.; Guo, Q.; Dong, P.; Zhang, F.; Luo, F.; Zhao, X.; Wei, Y.; Qian, Z. Biodegradable in Situ Gel-Forming Controlled Drug Delivery System Based on Thermosensitive PCL-PEG-PCL Hydrogel. Part 2: Sol-Gel-Sol Transition and Drug Delivery Behavior. Acta Biomater. 2009, 5, 3358-3370. (26) Gao, X.; Deng, X.; Wei, X.; Shi, H.; Wang, F.; Ye, T.; Shao, B.; Nie, W.; Li, Y.; Luo, M.; Gong, C.; Huang, N. Novel Thermosensitive Hydrogel for Preventing Formation of Abdominal Adhesions. Int. J. Nanomed. 2013, 8, 2453-2463. (27) Wu, Q.; Li, L.; Wang, N.; Gao, X.; Wang, B.; Liu, X.; Qian, Z.; Wei, Y.; Gong, C. Biodegradable and Thermosensitive Micelles Inhibit Ischemia-Induced Postoperative Peritoneal Adhesion. Int. J. Nanomed. 2014, 9, 727-734. (28) Zhang, W.; Wu, Q.; Li, L.; Cui, T.; Sun, L.; Wang, N.; Liu, L.; Li, X.; Gong, C. Prevention of Desiccation Induced Postsurgical Adhesion by Thermosensitive Micelles. Colloids Surf., B 2014, 122, 309-315. (29) Wu, Q.; Wang, N.; He, T.; Shang, J.; Li, L.; Song, L.; Yang, X.; Li, X.; Luo, N.; Zhang, W.; Gong, C. Thermosensitive Hydrogel Containing Dexamethasone Micelles for Preventing Postsurgical Adhesion in a Repeated-Injury Model. Sci. Rep. 2015, 5, 13553. (30) Ward, B. C.; Panitch, A. Abdominal Adhesions: Current and Novel Therapies. J. Surg. Res. 2011, 165, 91-111. (31)

Alkhamesi, N. A.; Schlachta, C. M. The Role of Aerosolized Intraperitoneal Heparin

and Hyaluronic Acid in the Prevention of Postoperative Abdominal Adhesions. Surg. Endosc. 2013, 27, 4663-4669.

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FIGURE LEGEND Figure 1 Preparation and phase transition behavior of tPA-hydrogel. (A) Schematic illustration of the formation of tPA-hydrogel. Micelle was formed by self-assembly method at 55°C, then cooled to 4°C, the micelle became larger with increased temperature and transformed into hydrogel at sol-gel transition temperature; (B) Photograph of tPA-hydrogel at 37°C (left) and tPA- micelle at 4°C (right); (C) Sol-gel-sol phase transition behavior of tPA-hydrogel. PCEC, poly(ε-caprolactone)-poly(ethyleneglycol)-poly(ε-caprolactone); tPA, tissue-type plasminogen activator Figure 2 tPA release behavior and characterization of tPA-hydrogel. (A) Cumulative release of tPA from tPA-hydrogel; (B) Scanning electron micrograph of tPA-hydrogel. Figure 3 In vivo degradation of tPA-hydrogel. Mice were euthanized after subcutaneous injection of tPA-hydrogel for 0 (A), 1 (B), 2 (C), 3 (D), 4 (E) weeks. The state of tPA-hydrogel was observed. Figure 4 Establishment of repeated-injury adhesion model in rat. (A) Injury abdominal sidewall and cecum was establishment at the first surgery; (B) The single-injury adhesion model was established; (C) Repeated-injury adhesion model was performed after one week, the detached cecum and abdominal wall were re-abraded monodirectionally. Figure 5 Typical adhesions in group of NS (A), hydrogel (B), tPA (C), tPA-hydrogel (D), In-tPA-hydrogel (E) and Arg-hydrogel (F) on postoperative Day 14. Black arrows indicate the adhesions. Figure 6 (A) Weight loss after the secondary operation for 14 days (as percentage of preoperative body weight); (B) Adhesion area of each group after treatment (*P < 0.05; **P < 0.01; ***P < 0.001); (C) The frequency of rats in each group with adhesion score. Figure 7 Histological observations of tissues treated with tPA-hydrogel and NS 14 days after secondary operation. H&E staining of healed abdominal wall (A) and cecum (B) in the tPA-hydrogel group; H&E staining of adhesive tissue from NS group (C); Masson’s trichrome staining of healed abdominal wall (D) and cecum (E) in the tPA-hydrogel group; (F) Masson’s trichrome staining of adhesive tissue from NS group. AW: abdominal wall; Me: mesothelial cells; CE: cecal mucosa; SK: skeletal muscle. Figure 8 SEM images of the surface of peritoneum (A) and cecum (C) in tPA-hydrogel group 16


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after treatment for 14 days. The higher magnification of peritoneum (B) and cecum (D) were observed. Figure 9 Plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) levels over time. The changes of (A) PAI-1 levels in peritoneal fluids and (B) tPA blood levels after secondary surgery for one week. (*P < 0.05, indicates significant differences between the tPA group and tPA-hydrogel group)

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Table of Contents

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Preparation and phase transition behavior of tPA-hydrogel. (A) Schematic illustration of the formation of tPA-hydrogel. Micelle was formed by self-assembly method at 55°C, then cooled to 4°C, the micelle became larger with increased temperature and transformed into hydrogel at sol-gel transition temperature; (B) Photograph of tPA-hydrogel at 37°C (left) and tPA- micelle at 4°C (right); (C) Sol-gel-sol phase transition behavior of tPA-hydrogel. PCEC, poly(ε-caprolactone)-poly(ethyleneglycol)-poly(ε-caprolactone); tPA, tissuetype plasminogen activator. Figure 1 53x58mm (600 x 600 DPI)



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tPA release behavior and characterization of tPA-hydrogel. (A) Cumulative release of tPA from tPA-hydrogel; (B) Scanning electron micrograph of tPA-hydrogel. Figure 2 127x46mm (300 x 300 DPI)


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In vivo degradation of tPA-hydrogel. Mice were euthanized after subcutaneous injection of tPA-hydrogel for 0 (A), 1 (B), 2 (C), 3 (D), 4 (E) weeks. The state of tPA-hydrogel was observed. Figure 3 127x122mm (300 x 300 DPI)



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Establishment of repeated-injury adhesion model in rat. (A) Injury abdominal sidewall and cecum was establishment at the first surgery; (B) The single-injury adhesion model was established; (C) Repeatedinjury adhesion model was performed after one week, the detached cecum and abdominal wall were reabraded monodirectionally. Figure 4 127x95mm (300 x 300 DPI)


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Typical adhesions in group of NS (A), hydrogel (B), tPA (C), tPA-hydrogel (D), In-tPA-hydrogel (E) and Arghydrogel (F) on postoperative Day 14. Black arrows indicate the adhesions. Figure 5 127x144mm (300 x 300 DPI)



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(A) Weight loss after the secondary operation for 14 days (as percentage of preoperative body weight); (B) Adhesion area of each group after treatment (*P < 0.05; **P < 0.01; ***P < 0.001); (C) The frequency of rats in each group with adhesion score. Figure 6 127x201mm (300 x 300 DPI)


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Histological observations of tissues treated with tPA-hydrogel and NS 14 days after secondary operation. H&E staining of healed abdominal wall (A) and cecum (B) in the tPA-hydrogel group; H&E staining of adhesive tissue from NS group (C); Masson’s trichrome staining of healed abdominal wall (D) and cecum (E) in the tPA-hydrogel group; Masson’s trichrome staining of adhesive tissue from NS group. AW: abdominal wall; Me: mesothelial cells; CE: cecal mucosa; SK: skeletal muscle. Figure 7 127x64mm (300 x 300 DPI)



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SEM images of the surface of peritoneum (A) and cecum (C) in tPA-hydrogel group after treamtment for 14 days. The higher magnification of peritoneum (B) and cecum (D) were observed. Figure 8 127x117mm (300 x 300 DPI)


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Figure 9. Plasminogen activator inhibitor-1 (PAI-1) and tissue plasminogen activator (tPA) levels over time. (A) PAI-1 levels in peritoneal fluids and (B) tPA blood levels were changed after secondary surgery for one week. (*P < 0.05, indicates significant differences between the tPA group and tPA-hydrogel group) Figure 9 127x50mm (300 x 300 DPI)


Improving Antiadhesion Effect of Thermosensitive Hydrogel with

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