Highly Bioadhesive Polymer Membrane Continuously Releases

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Highly Bioadhesive Polymer Membrane Continuously Releases Cytostatic and Anti-Inflammatory Drugs for Peritoneal Adhesion Prevention Jiannan Li, Weiguo Xu, Jinjin Chen, Di Li, Kai Zhang, Tongjun Liu, Jianxun Ding, and Xuesi Chen ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00605 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 8, 2017

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ACS Biomaterials Science & Engineering

Highly Bioadhesive Polymer Membrane Continuously Releases Cytostatic and Anti-Inflammatory Drugs for Peritoneal Adhesion Prevention Jiannan Li,†,‡ Weiguo Xu,‡ Jinjin Chen,‡ Di Li,‡ Kai Zhang,*,† Tongjun Liu,*,† Jianxun Ding,*,‡ and Xuesi Chen‡ †

Department of General Surgery, The Second Hospital of Jilin University, Changchun

130041, P. R. China ‡

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry,

Chinese Academy of Sciences, Changchun 130022, P. R. China

ACS Paragon Plus Environment


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KEYWORDS: emulsion electrospun nanofiber, cytostatic and anti-inflammatory agents,

ultraviolet-ozone treatment, bioadhesion, peritoneal adhesion prevention

ABSTRACT: Peritoneal adhesion is a complex fibrosis and inflammatory process, and it can be minimized by physical isolation by biomaterial membranes or treatment with cytostatic and/or anti-inflammatory drugs. However, the integration of physical isolation and pharmaceutical therapy in one platform faces many challenges. First, normal polymer anti-adhesion membranes are hydrophobic and with low bioadhesion to the injured tissue, which decrease their efficacies. Second, the significantly different release behaviors of various drugs owing to their different hydrophilic/hydrophobic properties limit their synergistic effects. In this study, a highly bioadhesive polymer membrane formed by core−sheath nanofiber to integrate physical isolation and pharmaceutical treatment together for the synergistic prevention of peritoneal adhesion. 10-Hydroxycamptothecin (HCPT) and diclofenac sodium (DS) were loaded in the sheath and core of nanofiber, respectively. The membrane was then treated by ultraviolet-ozone (UVO) for improvement of hydrophilicity and bioadhesion. Owing to the core−sheath structure, the two drugs both performed a sustained release behavior for the cytostatic and anti-inflammatory effects. The in vivo study demonstrated that the UVO-treated and dual-drug-coloaded membrane possessed the best anti-adhesion capacity, indicating its potential clinical application.


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1. INTRODUCTION Peritoneal adhesion is a confusing postoperative complication and always occurs after various peritoneal surgeries, which is a complex inflammatory and fibrosis process usually caused by trauma, infection, and presence of foreign materials.1-2 The exact pathophysiology of peritoneal adhesions is focused on two major factors. First, when the balance of fibrin degradation and deposition is interrupted, the peritoneal healing or peritoneal adhesion occurs. Second, the inflammation induced by peritoneal injury also promotes the fibrinous exudation and fibrin formation.3 Generally, when the collagen degradation effect is greater than the fibrinolysis effect, peritoneal adhesion forms.4 In addition to the financial burden, peritoneal adhesion also brings many other troubles to patients, as it results in numerous clinical complications, such as bowl obstruction, infertility, and chronic pelvic pain.3, 5 More seriously, peritoneal adhesion is a major cause of morbidity, and its complications manifest many years post-surgery. As a result, efficient anti-adhesion treatments at the early stage are emergent to the inhibition of peritoneal adhesion. Physical isolation and pharmaceutical treatment are two major routes to prevent or reduce the formation of postoperative adhesion.6-7 For physical isolation, lots of polymer-based membranes, e.g., Seprafilm® and Interceed®, have been developed in clinic. They inhibited the peritoneal adhesion by simply separating the abdominal wall and injured cecum. However, they exhibited few effects on the inhibition of inflammatory response and fibrin degradation. For pharmaceutical treatment, some drugs with different functions are applied to prevent the fibrin proliferation and/or inflammatory response. For example, 10-hydroxycamptothecin (HCPT) is one of the most common hydrophobic cytostatic drugs, which is often performed



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in preventing fibrin proliferation.8-9 Diclofenac sodium (DS) is a hydrophilic non-steroidal anti-inflammatory drug (NSAID) and has high potent of anti-inflammatory property.10-11 Even though the fibrin proliferation and inflammatory response are controlled by pharmaceutical treatment to some extent, some residual fibrin bands still form the pathological connection between the injured area and normal abdomen, leading to the formation of peritoneal adhesion. As a result, the application by physical isolation or pharmaceutical treatment alone could not lead to the significantly improved effect owing to their own limitations. So, the combination of physical isolation and pharmaceutical treatment in one platform might provide further improved anti-adhesion efficiency. However, the integration of the physical isolation and pharmaceutical treatment is not easy. First, though there are lots of polymer-based membranes, the bioadhesive abilities of these membranes are unsatisfying because of their hydrophobicity. As a result, these membranes always need to be fixed to the treated area by additional sutures,12 which might lead to undesired inflammatory response and fibrin degradation owing to the allogeneic reaction. Second, the hydrophilic/hydrophobic properties of anti-fibrin proliferation drugs, e.g., HCPT, and inflammatory response agents, e.g., DS, are quite different. As a result, if these drugs are loaded into the same membrane, the release profiles of these two kinds of drugs might be significantly different. For example, the hydrophilic drugs might obtain an initial burst release, while the release rate for hydrophobic drugs might be slow, which hindered their synergistic effects. Therefore, how to increase bioadhesion of membranes and how to balance the release profiles of different drugs become the two most emergent and most challenging problems for their clinic applications.


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Fortunately, the membranes composed of electrospun fibers become a potential candidate owing to their adjustable surface properties and structures.13-16 First, the hydrophilicity of polymer membranes can be enhanced by exposure to ultraviolet-ozone (UVO), which is a simple and low cost method.17-18 During the process of UVO treatment, the created ozone and molecular oxygen interact with the specimens, and it increases the amount of hydrophilic groups (e.g., mainly hydroxyl groups) on the surface of the samples, thus increasing hydrophilicity of the polymer membranes.19 With the increase of their hydrophilicity, the polymer membranes become more easily to stick to the injured sites in the abdominal cavity. Second, the structure of nanofiber could be controlled by emulsion electrospinning to form a core−sheath structure with oil phase in the sheath and aqueous phase in the core.20-21 Based on the core−sheath structure, the hydrophobic drugs and hydrophilic drugs are incorporated in the sheath and core of the emulsion electrospun fibers, respectively. The release rates of hydrophilic drugs in the core decelerate and keep in the similar degree with the hydrophobic drugs in the sheath for the sustained synergistic effect. Overall, based on the highly bioadhesive membrane formed by core−sheath nanofiber, the multi-functional drugs with different hydrophilic/hydrophobic properties release timely and continuously, which predictively achieve excellent anti-adhesion efficacy. Herein, a highly bioadhesive membrane was prepared, which was formed by core−sheath nanofiber loaded with hydrophobic HCPT in the sheath and hydrophilic DS in the core for the treatment of peritoneal adhesion (Scheme 1). The co-loaded nanofiber was obtained through electrospinning of methoxy poly(ethylene glycol)-block-poly(lactide-co-glycolide) (mPEG-b-PLGA) and dextran emulsion with HCPT in the oil phase and DS in the aqueous



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phase. UVO was then used to upregulate the surface hydrophilicity of the membrane. After the intraperitoneal surgery, the membrane was attached to the wound tightly as a benefit of improved hydrophilicity. First, the physical isolation separated the abdominal wall and injured cecum. Second, HCPT and DS were released continuously to play the anti-fibrin proliferation and anti-inflammatory actions. Owing to the rationally designed structure, the membrane integrated physical isolation and pharmaceutical treatment in one platform, which might be a feasible solution for anti-adhesion treatment.


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Scheme 1. Schematic illustration of preparation and anti-adhesion of HCPT and DS co-loaded membrane. 2. MATERIALS AND METHODS 2.1. Materials. Methoxy poly(ethylene glycol) (mPEG; number-average molecular weight



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(Mn) = 500 g mol−1) was obtained from Sigma-Aldrich (Shanghai, P. R. China). L-lactide and glycolide were donated by Zhejiang Hisun Pharmaceutical Co., Ltd. (Taizhou, P. R. China). Diblock

copolymer

mPEG-b-PLGA

was

synthesized

through

the

ring-opening

polymerization (ROP) of L-lactide and glycolide with a feeding molecular ratio of 3:1. Chloroform was bought from Beijing Chemical Works (Beijing, P. R. China). HCPT was obtained from Beijing Huafeng United Technology Co., Ltd. (Beijing, P. R. China). Dextran (weight-average molecular weight (Mw) = 100,000 Da) was purchased from Sigma-Aldrich (Shanghai, P. R. China), and used without further purification. DS was purchased from Aladdin Reagent Co., Ltd. (Shanghai, P. R. China). Triethyl benzyl ammonium chloride (TEBAC) was purchased from J&K Technology Co., Ltd. (Beijing, P. R. China). Elastase and Tween-80 used for membrane degradation and drug release experiments were bought from Aladdin Reagent Co., Ltd. (Shanghai, P. R. China) and Sigma-Aldrich (Shanghai, P. R. China), respectively. 2.2. Characterizations of mPEG-b-PLGA. mPEG-b-PLGA was characterized using proton nuclear magnetic resonance (1H NMR) and Fourier-transform infrared (FT IR) spectra. 1

H NMR spectrum of mPEG-b-PLGA was recorded on a 400 MHz Bruker spectrometer

(Bruker; Karlsruhe, Germany) at room temperature using deuterated chloroform (CDCl3) as a solvent. FT IR was also performed on a Bio-Rad Win-IR instrument (Bio-Rad Laboratories Inc., Cambridge, MA, USA). 2.3. Preparations of Emulsion Electrospun Membranes Loaded with HCPT or DS. The method to prepare emulsion electrospun nanofiber was described in detail in previous works.22-23 Generally, mPEG-b-PLGA was dissolved in chloroform at a concentration of 6.0


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wt.% and used as the oil phase. Dextran was dissolved in water at a concentration of 45.0 mg mL−1 as the aqueous phase. For the HCPT-loaded membranes, HCPT was dissolved in the oil phase at a concentration of 3.0 wt.%. For the DS-loaded membranes, DS was dissolved in aqueous phase at a concentration of 3.0 wt.%. For the dual-drug-coloaded membrane, 3.0 wt.% HCPT or 3.0 wt.% DS were dissolved in the oil phase or aqueous phase at the same time, respectively. To reduce the surface tension of oil phase, 5.0 wt.% TEBAC with respect to mPEG-b-PLGA was used as the emulsifier and added into the mPEG-b-PLGA/chloroform solution before emulsification. 1.0 mL of 45.0 mg mL−1 dextran aqueous solution was slowly dropped into 11.0 mL of above oil solution, and was emulsified at a routing rate of 6,500 rpm for about 5 min. The above emulsified solution was loaded in a 10-mL syringe and the electrospinning parameters were set as follows: electric strength: 26 kV; inner diameter of spinneret: 0.4 mm; injection rate of solution: 1 mL·h−1; the needle tilt angle from horizontal: 10°, and the air gap distance: 15 cm. The electrospun fiber was collected on a grounded aluminum sheet. The emulsion electrospun membranes of mPEG-b-PLGA without or with different drugs, i.e., HCPT, DS, and HCPT+DS, were referred as EEPM, EEPM/DS, EEPM/HCPT, and EEPM/DS+HCPT, respectively. 2.4. Surface Modifications of Emulsion Electrospun Membranes. To increase hydrophilicity of the electrospun membranes, UVO treatment was used in this study. The membranes were exposed to the UV radiation complementary by ozone. The exposed time was first set up as 10, 20, 30, 40, 50, or 60 s. Then the water contact angle analyses and the gross morphological changes were used to determine the appropriate exposure time. For the



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sake of briefness, the UVO-treated membranes without or with different drugs, i.e., HCPT, DS, and HCPT+DS, were referred as UEEPM, UEEPM/DS, UEEPM/HCPT, and UEEPM/DS+HCPT, respectively. 2.5. Characterizations of Membranes. In order to visualize and ensure the core−sheath structure of the emulsion electrospun nanofiber, transmission electron microscope (TEM; JEOL; Tokyo, Japan) and confocal laser scanning microscope (CLSM; Carl Zeiss, LSM 780; Jena, Germany) were used in this study. For TEM analyses, pure fibers were directly deposited on a carbon-coated copper mesh under the same electrospinning parameters mentioned above. For CLSM examination, dextran was firstly labeled with rhodamin-B (RhB), and then the emulsion electrospun nanofiber with RhB-dextran in the core was imaged. The surface morphologies of EEPM, EEPM/DS, EEPM/HCPT, EEPM/DS+HCPT, UEEPM, UEEPM/DS, UEEPM/HCPT, and UEEPM/DS+HCPT were tested by scanning electron microscope (SEM; Inspect-F, FEI; Finland). SEM was carried out at an accelerating voltage of 20 kV in high vacuum. The samples were mounted on silicon sheets separately, and vacuum-coated with gold-palladium for 30 − 60 s. The average diameters of the fibers and pore sizes of different membranes were analyzed based on SEM images using the image analysis software Adobe Photoshop CS6 (Adobe; California, USA). SEM was conducted again when different membranes were incubated in 0.2 mg mL−1 elastase phosphate for 7 and 14 days. The hydrophilicity/hydrophobicity of different membranes was analyzed by water contact angle tests. About 0.5 × 0.5 cm of membranes were grounded on glass slides and deionized


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water was dropped on the membranes. The contact angles were tested and recorded. In addition, the tissue adhesion ability of different membranes was also tested by a well-designed experiment. As shown in Figure 1E, a plastic sheet pasted with a piece of mouse-derived abdominal tissue was vertically placed under one side of the balance tray. A surgical line was used to connect the balance tray and the electrospun membrane by double-sided foam tape. The electrospun membrane was pasted onto the surface of abdominal tissue. Water was added into the breaker on the other tray of the balance at every 100.0 µL. The amount of added water was recorded once the membrane separated from the abdominal tissue. Then the relative amount of strength was calculated. Five samples of each membrane were tested and recorded. For mechanical analyses, the membranes were cut into small pieces (5.0 × 0.5 cm2 in size, approximately 0.1 mm in thickness). Tensile mechanical properties were performed using a universal test machine (Instron 4502; Instron Corporation, Springfield, NJ) at room temperature and 50% humidity. And then, the tensile strength, Young’s modulus, and the elongation at break were calculated from the stress-strain curves. 2.6. In Vitro Degradation. The biodegradability of different membranes treated without or with UVO was carried out by the weight loss of the membranes in 2.0 mL of phosphate-buffered saline (PBS) containing 0.05% Tween-80 and 0.2 mg mL−1 of elastase phosphate at 37 °C for about 30 days. To maintain the enzyme activity, the incubation media was replaced daily. At predetermined time intervals, the samples were carefully rinsed with distilled water, dried, and weighted. The weight loss of each membrane was carefully calculated by equation Eq. 1:



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Weight loss=

(W0 − Wt ) × 100% W0

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(1)

W0 represented the initial weight of membrane, whereas Wt indicated the weight of membrane after degradation at each time interval. The tests were represented for three times. 2.7.

Drug

Release

Tests.

For

drug

release

test,

EEPM/DS,

EEPM/HCPT,

EEPM/DS+HCPT, UEEPM/DS, UEEPM/HCPT, and UEEPM/DS+HCPT were also immersed in PBS containing 0.05% Tween-80 and 0.2 mg mL−1 of elastase phosphate solution as mentioned above. Tween-80 was used to improve the solubility of HCPT and DS in PBS or elastase solution. The solution was collected at the predetermined time intervals for further detection and replaced with fresh ones. The HCPT and DS concentrations in the solution were determined by ultraviolet/visible (UV/Vis) absorbances at 370 and 280 nm, respectively. The amounts of released HCPT and DS were calculated through the standard curves constructed by serial concentrations of HCPT and DS. 2.8. Animal Studies. The male Kunming mice, weighing 20 − 25 g, were obtained from the Laboratory Animal Center of Jilin University and fed at Changchun Institute of Applied Chemistry, Chinese Academy of Science. All the operations on the animals were in accordance with the NIH guidelines and approved by the Laboratory Animal Center of Jilin University. The animals were housed for two days prior to surgery and randomly divided into nine groups, i.e., control, EEPM, EEPM/DS, EEPM/HCPT, EEPM/DS+HCPT, UEEPM, UEEPM/DS, UEEPM/HCPT, and UEEPM/DS+HCPT. Animals were weighted and anesthetized by intraperitoneal injecting 0.3 − 0.5 mL of pentobarbital sodium solution at a


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concentration of 10.0 mg mL−1. The abdomen was shaved and sterilized by povidone-iodine. 1.0 − 1.5 cm of laparotomy was performed to expose the cecum. Then the ventral surface of the cecum was abraded by scalpels, until the appearance of petechial hemorrhage. Then the opposite abdominal wall was also abraded in the same way. 1.0 × 1.0 cm2 of membranes were immersed in PBS for about 10 min, and pasted on the abraded sites before abdomens were closed. The control group didn’t receive any intervention. The process of the animal study was shown in Scheme 1. The muscular layer and skin were closed using 4−0 sutures. The mice were placed in clean cages and warmed until consciousness. All the operation procedures were taken in a clean formal animal operation room.

2.8.1. Gross Evaluations. All the animals were sacrificed two weeks after the surgery, and the adhesion was scored as follows: 0, no adhesion; 1, adhesion that can be easily separated by blunt dissection; 2, adhesion that cannot be separated easily; 3, dense adhesions with difficult dissection plane. The adhesion parts were photographed and the adhesion areas were analyzed by Adobe Photoshop CS6.

2.8.2. Histological Analyses. The abraded abdominal wall, abraded cecum, and adhesion parts were dissected, rinsed with PBS, fixed in 4% (W/V) PBS-buffered paraformaldehyde, and finally embedded in paraffin. The tissues were serially sectioned at 5.0 µm interval. The sections were then stained with hematoxylin and eosin (H&E) and Masson’s trichome for histopathological and adhesion formation analyses, respectively.

2.8.3. Immunofluorescent Analyses of Inflammatory Factors. In order to evaluate the inflammation status in the operated sites in different groups, inflammatory factors, i.e., tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and interleukin-6 (IL-6), were determined by



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immunofluorescent staining. Briefly, the paraffin sections were firstly incubated at 60 °C for about 2 h to melt the paraffin. The sections were dewaxed in xylene for two times and hydrated in a graded array of alcohol. Then boiling citrate solution was used for antigen retrieval until the temperature of citrate solution returned to room temperature. Goat serum was used to inhibit the non-specific immune response for about 20 min at 37 °C. After that, the primary antibody solutions of TNF-α, IL-1, and IL-6 (Abcam Company; Cambridge, USA) with suitable concentrations were used to cover the surface of the samples overnight at 4 °C, respectively. The sections were washed by PBS and covered by the fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Boster Biological Engineering Co., Ltd., Wuhan, China) for 40 min at 37 °C. Then the sections were washed again by PBS. Afterwards, 4′,6-diamino-2-phenyl indole (DAPI) (Sigma-Aldrich; Shanghai, P. R. China) was used to dye the nucleus. Finally, these sections were observed and photographed under CLSM. The nucleus was stained blue, and the inflammatory factors were stained green. After the images were obtained, semiquantitative analyses were applied to evaluate the inflammations by ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). 2.9. Statistical Analyses. Data were analyzed with Graphpad Prism 7.0 software (Graphpad Inc., San Diego, CA, USA) and the results were expressed as mean ± standard deviation (SD). Student’s test was used to analyze the statistical difference. Statistical significance was set at *P < 0.05, and highly statistical significance was set as **P < 0.01 and ***P < 0.001.


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Figure 1. Characterizations of emulsion electrospun membranes. (A) TEM image of emulsion electrospun fiber. (B) CLSM images of emulsion electrospun fiber. (C) SEM images and water contact angles of different membranes. (D) Mechanical properties of different membranes, including stress−strain curve, tensile strength, Young’s modulus, and



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elongation at break. (E) Illustration and data of tissue bioadhesive ability test. Data are presented as mean ± SD (for D and E, n = 5; *P < 0.05, **P < 0.01, ***P < 0.001). Scale bars = 500 nm. 3. RESULTS 3.1. Characterizations of Emulsion Electrospun Membranes. The triblock copolymer of mPEG-b-PLGA was successfully synthesized by the ROP of L-lactide and glycolide with mPEG (Mn = 500 g mol−1) as a macroinitiator and stannous(II) 2-ethylhexanoate (Sn(Oct)2) as a catalyst. The characterizations of mPEG-b-PLGA were provided in the Supporting Information. The membranes loaded without or with different drugs were successfully developed by emulsion electrospinning. During the electrospun process, HCPT was well dissolved in the oil phase, while DS was well dissolved in the water phase. After the emulsion electrospinning, the core−sheath structure was formed. The structure of the fiber was confirmed by both TEM and CLSM. As shown in Figure 1A, the core−sheath structure of the fiber was obviously observed by TEM owing to the difference in the contrast of the oil and aqueous phase. To better distinguish the core and sheath, the core was loaded with RhB and observed by CLSM. As shown in Figure 1B, the inner layer of the fibers emitted obvious red fluorescence, while there was no fluorescence in the outer layer. The detailed morphologies of the membranes before and after UVO treatment were characterized by SEM. As shown in Figure 1C, before the treatment with UVO, the membranes were all formed by the smooth and regular fibers. Moreover, there was no obvious drug crystal in the fibers of drug-loaded membranes. When treated by UVO for 50 s,


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the fibers were not as straight as the non-UVO-treated ones. Moreover, some fused points were also found among different fibers. The average fiber diameters, pore sizes, and their frequency distributions were further analyzed and shown in Supplementary Figure S2A and S2B. The average fiber diameters in EEPM, EEPM/DS, EEPM/HCPT, and EEPM/DS+HCPT were 87.9 ± 21.5, 90.2 ± 20.3, 90.1 ± 22.3, and 93.5 ± 25.8 nm, respectively. The average pore sizes were 154.5 ± 44.4, 118.6 ± 35.9, 147.7 ± 46.3, and 151.5 ± 45.9 nm, respectively. After the treatment with UVO, the average fiber diameters of UEEPM, UEEPM/DS, UEEPM/HCPT, and UEEPM/DS+HCPT were increased to 121.0 ± 37.3, 190.8 ± 49.9, 102.1 ± 23.2, and 98.4 ± 22.9 nm, respectively. The average pore sizes were also increased to 132.0 ± 40.3, 271.0 ± 80.9, 135.0 ± 45.5, and 160.5 ± 56.5 nm, respectively. The stress−strain curves of EEPM and UEEPM were also evaluated and shown in Figure 1D. The strain of UEEPM was higher than that of EEPM under the same stress, which indicated that UEEPM possessed a stronger deformation resistant ability than that of EEPM. In addition, there was almost no difference between membranes treated without or with UVO in tensile strength, Young’s modulus, and elongation at break (Figure 1D). In order to evaluate the hydrophilicity of different membranes, water contact angle analyses and tissue bioadhesive ability tests were performed. As shown in Figure 1C, the water contact angles were all more than 100° for the membranes without UVO treatment. However, the water contact angles of the membranes decreased to about 80° after UVO treatment, indicating the increased hydrophilicity of membranes. The increased bioadhesion after UVO treatment was confirmed by the tissue bioadhesive



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ability test. The friction forces needed to separate the membranes and abdominal walls were 1.6 ± 0.2, 1.6 ± 0.3, 1.9 ± 0.2, 1.8 ± 0.3, 2.7 ± 0.3, 2.8 ± 0.4, 3.6 ± 0.4, and 3.0 ± 0.4 (10−2 N) in the EEPM, EEPM/DS, EEPM/HCPT, EEPM/DS+HCPT, UEEPM, UEEPM/DS, UEEPM/HCPT, and UEEPM/DS+HCPT groups, respectively. There were significant differences in bioadhesive abilities between the UVO-treated membranes and the non-UVO-treated ones (P < 0.001) (Figure 1E). 3.2. In Vitro Degradation. As shown in Figure 2A, there was no statistical difference in the degradation between EEPM and UEEPM either in elastase or PBS for 32 days. However, the degradation rates of the membranes in elastase solution were much higher compared with those of the membranes in PBS. After incubation for 32 days, the weight loss of EEPM and UEEPM in PBS were 19.7 ± 1.5 wt.% and 14.5 ± 0.5 wt.%. Whereas, the weight losses of EEPM and UEEPM in the elastase solution were 75.0 ± 5.0 wt.% and 67.3 ± 5.1 wt.%, respectively. Representative SEM images of different membranes incubated in elastase solution for 7 and 14 days were shown in Figure 2B and 2C, respectively. After incubation for seven days, the fibers still remained their basic structures, even though eroded surfaces and breaking points were observed. In addition, the degradation degrees were a bit more serious in the non-UVO-treated groups. However, when incubated for 14 days, the surface of the fibers became obscure in all groups. The basic fibrous structures were hard to be observed and the fibers fused into each other seriously in all groups.


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Figure 2. Degradation and drug release behaviors of membranes. (A) Degradation behaviors of EEPM and UEEPM in PBS or elastase solution. (B, C) Representative SEM images of different membranes in elastase solution for 7 (B) and 14 days (C). Scale bar is 500 nm for B and 2 µm for C. (D, F) HCPT release behaviors of EEPM/HCPT, EEPM/DS+HCPT, UEEPM/HCPT, and UEEPM/DS+HCPT in PBS (D) and elastase solution (F). (E, G) DS release behaviors of EEPM/DS, EEPM/DS+HCPT, UEEPM/DS, and UEEPM/DS+HCPT in PBS (E) and elastase solution (G). Data are presented as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001). 3.3. In Vitro Drug Release. The HCPT release profiles from EEPM/HCPT, EEPM/DS+HCPT, UEEPM/HCPT, and UEEPM/DS+HCPT in PBS were shown in Figure 2D. There were no statistical differences in the release profiles of HCPT among these four groups. A burst release of 55.7% ± 0.9%, 48.6% ± 4.8%, 54.3% ± 2.8%, and 54.7% ± 5.1%



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were

detected

in

the

EEPM/HCPT,

EEPM/DS+HCPT,

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UEEPM/HCPT,

and

UEEPM/DS+HCPT groups for the first three days, respectively. A constant fast release was observed in the first five days and a slow release of HCPT was found to be sustained for up to 13 days. After 13 days, the released HCPT were 90.2% ± 3.3%, 89.3% ± 8.2%, 94.4% ± 0.3%, and 91.9% ± 1.3% in the EEPM/HCPT, EEPM/DS+HCPT, UEEPM/HCPT, and UEEPM/DS+HCPT groups, respectively. In Figure 2E, accumulative DS release profiles were shown from EEPM/DS, EEPM/DS+HCPT, UEEPM/DS, and UEEPM/DS+HCPT in PBS. There was also no statistical differences in the release profiles of DS among these four groups. A small amount of burst release of 31.4% ± 2.1%, 39.7% ± 3.2%, 42.0% ± 2.5%, and 42.5% ± 2.7% was detected in the EEPM/DS, EEPM/DS+HCPT, UEEPM/DS, and UEEPM/DS+HCPT groups for the first four days. A constant fast release was followed by the initial burst release for six days, and then a slow release for another three days. After release for 13 days, the released DS were 86.3% ± 2.2%, 85.4% ± 1.1%, 91.0% ± 3.0%, and 90.7% ± 2.8% in the EEPM/DS, EEPM/DS+HCPT, UEEPM/DS, and UEEPM/DS+HCPT groups. As shown in Figure 2F and 2G, both HCPT and DS released faster in elastase solution than that in PBS. The total amount of released HCPT was 90.8% ± 1.5%, 90.3% ± 1.7%, 90.5% ± 0.6%, and 89.9% ± 1.4% in the EEPM/HCPT, EEPM/DS+HCPT, UEEPM/HCPT, and UEEPM/DS+HCPT groups for the first six days. After 10 days, the released DS were 91.5% ± 0.5%, 91.3% ± 2.4%, 90.2% ± 2.0%, and 90.4% ± 1.5% in the EEPM/DS, EEPM/DS+HCPT, UEEPM/DS, and UEEPM/DS+HCPT groups, respectively. 3.4. In Vivo Animal Studies. As shown in Supplementary Figure S3, the abdomen-cecum


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adhesion animal model was successfully performed and the membranes were attached to the wounds directly. All the animals were fed in a clean and ventilated environment, and they were also under necessary and careful human care. After 14 days, the peritoneal adhesions of different groups were evaluated by gross evaluations and histological analyses. 3.4.1. Gross Evaluations. The surgical wounds in the abdomen of all animals were recovered well, and none of them was infected. The evaluation of anti-adhesion efficiency was based on a score method according to the adhesion scores 0, 1, 2, and 3 in Supplementary Figure S4. The scores indicated different degrees of anti-adhesion efficiencies as follows: Score 0, no adhesion; Score 1, a thin filmy adhesion that was easily separated; Score 2, definite adhesions which were separable by blunt dissection; and Score 3, dense adhesions separable only by sharp dissection. The distributions of adhesion scores in different groups were shown in Figure 3A. As shown in Figure 3B, the average adhesion scores of EEPM, EEPM/DS, EEPM/HCPT, EEPM/DS+HCPT, UEEPM, UEEPM/DS, UEEPM/HCPT, UEEPM/DS+HCPT, and control groups were 2.2 ± 0.5, 1.7 ± 0.5, 1.7 ± 0.5, 0.5 ± 0.6, 1.5 ± 0.6, 1.3 ± 0.5, 0.7 ± 0.5, 0.3 ± 0.5, and 2.8 ± 0.4, respectively. Even though the adhesion scores of EEPM and UEEPM groups were less than that in the control group, it was still difficult to dissect the fibrous bonds bridging the abdominal wall and the cecum wall. The other six drug-loaded membranes all suppressed the adhesion scores significantly. It was easy to separate the adhesion tissues in these groups, especially in the EEPM/DS+HCPT and UEEPM/DS+HCPT groups. The dual-drug-coloaded membranes treated without or with UVO induced less adhesion formation among all groups. Moreover, the adhesion scores of UVO-treated membranes



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slightly decreased than that in the non-UVO-treated ones. However, as for the groups of DS-loaded and dual-drug-coloaded membranes, UVO treatment had no significant difference in adhesion prevention.

Figure 3. Anti-adhesion properties of different membranes. (A) Distribution of adhesion scores in different groups. (B) Adhesion scores in different groups. (C) Adhesion areas in different groups. Data are presented as mean ± SD (n = 6; *P < 0.05, **P < 0.01, ***P < 0.001). The adhesion parts were dissected and the adhesion areas were photographed and analyzed in all groups. As shown in Figure 3C, the adhesion areas of EEPM, EEPM/DS, EEPM/HCPT, EEPM/DS+HCPT, UEEPM, UEEPM/DS, UEEPM/HCPT, UEEPM/DS+HCPT, and control groups were 0.3 ± 0.1, 0.3 ± 0.1, 0.4 ± 0.1, 0.1 ± 0.1, 0.3 ± 0.1, 0.1 ± 0.0, 0.1 ± 0.1, 0.0 ± 0.0, and 0.5 ± 0.1 cm2, respectively. Compared with that in the control group, the adhesion areas in all the other groups decreased. The dual-drug-coloaded membranes possessed best ability


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in adhesion prevention, both for the UVO- and non-UVO-treated membranes. There were no statistical differences in adhesion areas among

the EEPM and

UEEPM, and

EEPM/DS+HCPT and UEEPM/DS+HCPT groups. However, in the other groups, the adhesion areas in the UVO-treated membranes were less than that in the non-UVO-treated groups. 3.4.2. Histological Analyses. To further explore the adhesion formation and collagen distribution, H&E and Masson’s trichome staining were carried out in each group (Figure 4A). In the control group, a large area of fibrous adhesion tissue appeared between the abdominal wall and surrounded cecum. In the EEPM and UEEPM groups, the collagen tissues were also obvious. However, in the EEPM/DS, EEPM/HCPT, UEEPM/DS, and UEEPM/HCPT groups, collagen tissues were well separated by the membranes and less adhesion tissues were found, compared with that in the control group. The EEPM/DS+HCPT and UEEPM/DS+HCPT groups exhibited the best anti-adhesion efficiency with almost no adhesion formation and the least collagen degradation. 3.4.3. Inflammatory Estimations. Inflammatory estimations were carried out in all groups by immunofluorescence staining for inflammatory factors, e.g., TNF-α, IL-1, and IL-6. The CLSM images of each section were shown in Figure 4A. In the control group, large amounts of inflammatory factors were observed in the abdomen, cecum, and regenerated collagen tissues. Serious inflammatory responses were also observed in the EEPM and UEEPM groups. As for the EEPM/DS, EEPM/HCPT, UEEPM/DS, and UEEPM/HCPT groups, inflammatory

factors

decreased

dramatically.

In

the

EEPM/DS+HCPT

and

UEEPM/DS+HCPT groups, inflammatory responses were significantly suppressed,



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especially in the UEEPM/DS+HCPT group.

Figure 4. Histological analyses of repaired sites of the injured abdomen-cecum. (A) H&E and Masson’s trichome staining, and immunofluorescent presentation of TNF-α, IL-1, and IL-6 in repaired sites. (B, C, and D) The semi-quantitative analyses of TNF-α (B), IL-1 (C), and IL-6 (D). Data are presented as mean ± SD (n = 3; *P < 0.05, **P < 0.01, ***P < 0.001). Scale bars in (A) represent 100 µm. The relative fluorescence intensities of these inflammatory factors in above CLSM images were calculated with Image-pro plus Software (Media Cybernetics, Inc. Warrendale, USA). The fluorescence intensity of control group was defined as “1”, and the relative intensity was defined as the ratios of the fluorescence intensity of treated samples and control group. The fluorescence densities of TNF-α (P < 0.01) (Figure 4B), IL-6 (P < 0.001) (Figure 4C), and


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IL-6 (P < 0.001) (Figure 4D) in the control group were significantly higher than those in all the other groups. Moreover, the fluorescence intensities of TNF-α, IL-1, and IL-6 in the EEPM/DS+HCPT group were significantly lower than those in the EEPM, EEPM/DS, and EEPM/HCPT groups (P < 0.001). Similar results were also found in the UVO-treated groups. For all the three inflammatory factors, the fluorescence densities in the UVO-treated groups were significantly lower than that in the non-UVO-treated ones. 4. DISCUSSION Peritoneal surgeries are pretty common in clinic. As a result, peritoneal adhesions often occur and are very hard to prevent for many years. Once peritoneal adhesions form, lots of confusing complications and organ dysfunctions will appear and need reoperation. As peritoneal adhesions have attracted more and more attentions nowadays, many techniques, such as improved and more careful surgery skills, physical isolation, and pharmaceutical treatment, have been well studied and applied. Among the barrier-based devices for physical isolation, polymer membranes formed by electrospun fibers have been extensively explored in adhesion formation prevention. In addition, many commercial physical electrospun membranes, including SeprafilmTM (Genzyme; Cambridge, MA, USA),24-25 InterceedTM (TC-7; Johnson & Johnson, USA),26-27 and AdeptTM (Baxter; Deerfield, IL),28-29 have already shown their satisfactory effects in preventing adhesion formation not only in the animals but also in the human body. However, one problem of many solid electrospun membranes is their low bioadhesion owing to their hydrophobicity, which leads to additional sutures to fix the barriers to the abdomen. UVO treatment of the membranes in a chamber with high ozone concentrations can improve their



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hydrophilicity and then further increases their bioadhesion. Moreover, to achieve a more satisfactory effect in adhesion prevention, numerous drugs were loaded within the electrospun membranes or the hydrogels to integrate the physical isolation and pharmaceutical treatment. These drugs mainly include HCPT,30 mitomycin C,31 celecoxib,32 and epigallocatechin-3-O-gallate,33 etc.. Due to the core−sheath structure, emulsion elelctrospun fibers provide the possibility to load dual drugs and achieve the sustained release behaviors. Herein, HCPT mainly inhibits fibroblast proliferation while DS performs its anti-inflammatory effect to synergistically prevent peritoneal adhesion. In this study, we integrated the physical isolation and pharmaceutical treatment in one platform using a highly bioadhesive membrane formed by the core−sheath nanofiber co-loaded with HCPT in the sheath and DS in the core to prevent abdominal adhesion formation in mice. The core−sheath structure of emulsion electrospun fibers was confirmed by TEM and CLSM. As shown in Figure 1A and 1B, owing to the different contrasts of core and sheath, the structure of nanofiber was easily observed. In the CLSM images, the core was distinguished form the sheath by staining with RhB. Both TEM and CLSM confirmed the successful preparation of the core−sheath nanofiber. Moreover, the surface was smooth and there was no drug crystals detected in the SEM images, which indicated that HCPT and DS were incorporated homogeneously into the sheath and core, respectively. The excellent drug encapsulation might be attributed to the good solubility of HCPT in oil phase and DS in aqueous phase. In order to increase the bioadhesion of the membranes, UVO treatment was applied.


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Previous studies have already demonstrated that the duration of exposure from UVO source has an important effect on the hydrophilicity and surface structure of the electrospun membranes, which contributes to the bioadhesion of the membranes.34 The exposure time is a key factor to the hydrophilicity improvement of the membranes. The influence of UVO treatment time (i.e., 10, 20, 30, 40, 50, and 60 s) on the hydrophilicity of the membrane was firstly evaluated. With the increase of the exposure time, the water contact angles decreased. The decreased water contact angles caused by UVO treatment indicated that the treatment indeed increased the hydrophilicity of different membranes. However, when the exposure time was extended to 60 s, the surface morphologies of the membranes changed dramatically. 50 s of UVO treatment didn’t significantly change the morphologies of the membranes and was chosen for the further experiments in this study. Besides, the average diameters of the fibers and pore sizes of different membranes also didn’t show statistical difference before and after UVO treatment. The stress-strain curves, tensile strength, Young’s modulus, and elongation at break of different membranes were all measured to evaluate the effects of UVO treatment on the mechanical properties. As shown in Figure 1D, UEEPM had stronger deformation resistant ability compared with that of EEPM, which might resulted from the slight morphology changes after UVO treatment, including increased softness and fused points. However, all the other mechanical properties of the membranes were not influenced by UVO treatment. The increased bioadhesion after UVO treatment was further confirmed by the tissue bioadhesive ability test. As shown in Figure 1E, for different drug-loaded membranes, the bioadhesive abilities significantly increased after UVO treatment compared with those of the



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non-UVO-treated membranes (P < 0.001). In addition, according to the tissue bioadhesive ability test, the suitable bioadhesion ability of electrospun membranes in preventing peritoneal adhesion was 3.0 ± 0.5 (10−2 N). The biodegradability was also important for the clinical applications of these membranes. The biodegradation of these membranes were measured by their weight loss after incubated with PBS or elastase solution. As shown in Figure 2A, UVO treatment didn’t have any effect on the in vitro degradation of different membranes, while the elastase did. The weight loss of the membrane in the elastase solution (more than 70%) was much higher than that in PBS (about 20%) after incubation for 32 days. Consistent with their weight loss rates, the membranes became more obscure and irregular when degraded for 14 days, compared with that degraded for seven days (Figure 2B and 2C). The sustained drug release of both HCPT and DS from the membrane was significant for the long-term pharmaceutical treatment. Drug release behaviors of HCPT and DS were also evaluated in PBS and elastase solution. Consistent with the in vitro degradation observation, UVO also had no influence on release of drugs. In the first three days, about 50% of HCPT released from the single-drug-loaded or dual-drug-coloaded membranes in PBS. Then HCPT released continuously to about 90% till 13 days. The release of DS was slightly slower than that of HCPT for the first five days and also exhibited continuous release behavior till 13 days. In the elastase solution, though the two drugs released faster than that in PBS, the sustained release were still maintained for more than six days. Both of the sustained release behaviors of HCPT and DS were contributed from the core−sheath structure of the fibers. First, HCPT released slowly from the outer sheath owing


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to the slow degradation of mPEG-b-PLGA. Second, the release of hydrophilic DS was decelerated resulted from the retention effect of the inner core. The rationally designed core−sheath structure balanced the release profiles of both hydrophobic and hydrophilic drugs, which would predictively benefit the long-term anti-adhesion effect. The anti-adhesion capabilities of different membranes were carried out in a cecum-abdominal animal model. The gross evaluation revealed that the blank membrane only slightly decreased the adhesion scores (Figure 3B) and adhesion area (Figure 3C) compared with the control group (P < 0.05), because the single treatment by physical isolation was not enough for the ideal anti-adhesion effect. With respect to the non-UVO-treated membranes, even though EEPM/DS and EEPM/HCPT slightly reduced the adhesion formation compared to the control group, there was no significant difference between the drug-loaded EEPM group and the EEPM group (P > 0.05) in adhesion scores and areas. The unsatisfied anti-adhesion effects of non-UVO-treated membranes were mainly caused by their poor bioadhesion to the wound. For the UVO-treated and drug-loaded membranes, the anti-adhesion effects were more obvious. As shown in Figure 3B and 3C, the adhesion scores and adhesion areas decreased dramatically in the UEEPM/DS, UEEPM/HCPT, and UEEPM/DS+HCPT groups, especially in the UEEPM/DS+HCPT group. The excellent anti-adhesion effect of UEEPM/DS+HCPT was owing to the successful integration of physical isolation and pharmaceutical treatment. First, after UVO treatment, the membrane attached to the wound tightly. Under this circumstance, movement of these membranes from the injured sites was less possible. Second, based on the core−sheath structure, both the hydrophobic HCPT and hydrophilic DS



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continuously released from the fibers, which maintained long-term cytostatic and anti-inflammatory effect. Finally, the synergistic effect of HCPT and DS well reduced the adhesion formation to a relative low extent. As a result, the physical isolation and pharmaceutical treatment were both necessary to the upregulated anti-adhesion effect. Further H&E and Masson’s trichome staining were also carried out in Figure 4A. There was no obvious fibrosis formation in the UEEPM/DS+HCPT group. All these results further confirmed the superiority of UEEPM/DS+HCPT. The anti-inflammatory behaviors of these membranes were also important to their anti-adhesion effect. TNF-α, IL-1, and IL-6 are the three main inflammatory factors in the process of inflammation. TNF-α is an important factor in the association of specific immune and inflammatory responses, which effects on many kinds of cells and induce the formation of IL-1 or IL-6. These three factors can increase the permeability of capillaries, activate other inflammatory cells, and stimulate the formation of oxygen radicals, which lead to more severe inflammatory responses and damaging normal tissues.35 The secretion of TNF-α, IL-1, and IL-6 was evaluated by CLSM in Figure 4A. Moreover, the semi-quantitative analyses were also listed in Figure 4B, 4C, and 4D. In detail, all UVO-treated membranes showed better anti-inflammatory effects than those of non-UVO-treated ones, resulting from the enhanced

bioadhesive

abilities

of

membranes

after

UVO

treatment.

Besides,

single-drug-loaded membranes revealed their effects of anti-inflammatory ability compared to the control and EEPM groups owing to pharmaceutical treatment (P < 0.001). However, the best anti-inflammatory effect was observed in the UVO-treated and dual-drug-coloaded group, UEEPM/DS+HCPT. The expression of these factors could be significantly suppressed


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compared to the other groups (Figure 4A). 5. CONCLUSIONS In this study, a highly bioadhesive membrane composed of HCPT and DS coloaded nanofiber was successfully prepared through emulsion electrospinning. HCPT was loaded in the sheath and DS was loaded in the core for both sustained release profiles. The membrane rationally integrated the physical isolation and pharmaceutical treatment in one platform for highly effective anti-adhesion. First, UVO treatment was used to increase bioadhesion of the membrane, which benefited the physical isolation effect of the membrane. Second, the release behaviors of both HCPT and DS were continuous and maintained for several days, owing to the well-designed core−sheath structure. More interestingly, the UVO-treated and dual-drug-coloaded membrane exhibited the best anti-adhesion efficiency. Further histological staining also proved that UEEPM/DS+HCPT not only decreased the fibrosis formation but also inhibited the inflammatory response. All these results indicated the superiority of the rationally-designed membrane in the application of anti-adhesion postsurgery.

ASSOCIATED CONTENT

Supporting Information. The following files are available free of charge. Characterizations of mPEG-b-PLGA; 1H NMR and FT IR spectra of mPEG-b-PLGA; distribution of diameters and pore sizes of different membranes; establishment of cecum-abdomen animal model.



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AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This study was financially supported by the National Natural Science Foundation of China (Grant Nos. 51673190, 51603204, 51673187, 51390484, 51473165, and 51520105004) and the Science and Technology Development Program of Jilin Province (Grant Nos. 20160204015SF and 20160204018SF).

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ToC Graphic


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Highly Bioadhesive Polymer Membrane Continuously Releases

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