Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
pubs.acs.org/journal/abseba
Naproxen Nanoparticle-Loaded Thermosensitive Chitosan Hydrogel for Prevention of Postoperative Adhesions Yu Wang,†,‡,# XianLun Pang,§,# Jia Luo,† Qian Wen,† ZhouXue Wu,† QiuXia Ding,∥ Ling Zhao,⊥ LingLin Yang,† BiQiong Wang,† and ShaoZhi Fu*,† †
Department of Oncology, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China Health Management Center, Affiliated Hospital of Southwest Medical University, Luzhou 646000, China § Health Management Center, Hospital (T.C.M.) Affiliated to Southwest Medical University, Luzhou 646000, China ∥ Department of Gynaecology and Obstetrics, Second Affiliated Hospital of Third Military Medical University, Chongqing 400000, China ⊥ Department of Pharmaceutics, School of Pharmacy of Southwest Medical University, Luzhou 646000, China
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‡
ABSTRACT: Postoperative adhesions are the most common complications of periabdominal surgery; they not only affect the patient’s quality of life but also increase the risk of a subsequent surgery. The use of implantable dressings to physically block surgical wounds is the primary solution to prevent postoperative adhesions. In this study, we prepared naproxen nanoparticles that were loaded with chitosan hydrogel (CS/Nap hydrogel) to prevent postoperative adhesions. Our data confirmed that the prepared CS/Nap hydrogel was thermosensitive and suitable for injection. The efficacy of anti-adhesion in a rat model revealed that the hydrogel effectively separated from the wounds of the abdominal wall and cecum. On day 7 postsurgery, the wounds were completely covered by a new epithelial layer, whereas wounds in the negative control group were glued together. Additionally, the in vivo toxicity study showed that the CS/ Nap hydrogel had fewer toxic and side effects on major tissues and organs, including the liver, spleen, heart, lung, and kidney. We showed that a drug delivery system based on CS/Nap hydrogel has the potential not only to prevent postoperative abdominal adhesions and relieve pain but also to contribute to the administration of the hydrophobic drug naproxen. KEYWORDS: naproxen, nanoparticle, chitosan, thermosensitive hydrogel, postoperative adhesion
1. INTRODUCTION In the clinic, abdominal adhesions are the most common postoperative complications of abdominal and pelvic surgery. The incidence of adhesions following surgery ranges from 67% to 93%, especially in pelvic surgery, and more than 90% of patients may suffer from adhesions.1−3 The occurrence of abdominal adhesions not only greatly impacts the quality of surgery but also significantly affects the recovery of patients. Although several adhesions may not display obvious symptoms in the clinic, they may still have serious consequences, including female infertility, intestinal obstruction, chronic pelvic pain, and the need for a subsequent surgery.4−6 Therefore, it is of utmost importance to investigate how to effectively prevent postsurgical adhesions. Currently, several strategies have been proposed to resolve postoperative adhesions. Besides pharmacological treatment, the other widely used method is the use of various physical barriers to cover the surfaces of injured tissues after surgery.7−10 For example, bioresorbable fibrous hyaluronic acid-carboxymethyl cellulose (HA-CMC, Seprafilm Genzyme) films and fibrous polylactide membranes have been successfully commercialized and are widely used during surgery.11−14 However, these barriers have been limited to cases in which © XXXX American Chemical Society
the incision is small and surgical access is restricted. It is challenging to cover the tissues of complex geometry with these membranes and have it aggressively adhere to the surface of surgeon’s gloves.8,15 Therefore, an injectable hydrogel may be a promising solution for anti-adhesion therapy.16−18 As a natural polysaccharide, chitosan (CS) hydrogels have attracted significant interest for their use in tissue engineering or as a drug delivery carrier due to their excellent biocompatibility, etc.19−21 In previous studies, many CS hydrogels were prepared and their effects on preventing postoperative adhesions have been investigated. 8,9,22,23 Although the data suggested that CS hydrogels may have good performance with respect to postoperative adhesions, the only use of these gel systems is as an anti-adhesion barrier. CS hydrogels are rarely considered for the prevention of postoperative adhesions while delivering therapeutic drugs to relieve postoperative pain or other intra-abdominal in situ treatments. Being aware of these concerns, in this study, we designed a naproxen-loaded CS hydrogel, which combined the Received: December 11, 2018 Accepted: February 18, 2019 Published: February 18, 2019 A
DOI: 10.1021/acsbiomaterials.8b01562 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
into a white powder by a freeze-drying method (the theoretical drug loading rate of Nap-loaded nanoparticles was 4%). 2.2.2. Particle Size and Morphology. The size distribution of prepared Nap nanoparticles was measured by a dynamic light scattering (DLS) analyzer (NanoBrook 90Plus Zeta, Brookhaven, NY) at 25 °C. Transmission electron microscopy (TEM) (Tecnai G2 F20, FEI, Hillsboro, OR) was used to visualize the surface morphology of Nap nanoparticles. In brief, one drop of a Nap nanoparticle suspension was placed on a carbon-coated copper grid that was covered with nitrocellulose. Prior to observation, Nap-NPs were negatively stained with a 2% aqueous phosphotungstic acid solution for 1 min and dried at room temperature. 2.2.3. Drug Loading and Encapsulation Efficiency. A highperformance liquid chromatography (HPLC) (Agilent1260, Agilent Technologies, Palo Alto, CA) instrument with a reverse phase C18 column (4.6 mm × 150 mm, 3.5 μm particle size, Agilent Technologies) was applied to determine the drug loading (DL) and encapsulation efficiency (EE). A mixture of methanol and 0.01 M potassium dihydrogen phosphate in a 75:25 (v/v) ratio was used as a mobile phase (the pH was adjusted to 3.0 with a phosphoric acid solution), and the flow rate was 1.0 mL/min. The detection wavelength was 254 nm. Exact weight Nap nanoparticles were completely dissolved in the mobile phase and filtered through a 220 nm filter. DL and EE were calculated by the following equations:
functions of anti-adhesion properties and relieving postoperative pain or anti-inflammatory. Naproxen (Nap), as a typical nonsteroidal anti-inflammatory drug (NSAID), is widely used in the clinic to relieve pain and reduce inflammation by oral and suppository forms.24 However, administration of Nap is a challenge for patients with swallowing difficulties or for patients who are unconscious.25 Therefore, in our study, we attempted to encapsulate Nap nanoparticles into thermosensitive CS hydrogels and evaluated their anti-adhesion efficacy in an abdominal cecum-adhesion model in rats.
2. MATERIALS AND METHODS 2.1. Materials. Chitosan (85% deacetylated, average molecular weight of 250 kDa, viscosity of 200000 cps) was purchased from Jinan Debei Biological Co. Ltd. (Shandong, China). β-Glycerolphosphate disodium salt pentahydrate (β-GP) was purchased from SigmaAldrich Co. (St. Louis, MO). Nap was obtained from Aladdin Bioscience-Tech. Co. Ltd. (Shanghai, China). Other chemical agents were purchased from Chengdu Kelong Co. Ltd. (Chengdu, China) and were of analytical grade and used directly without further purification. 2.2. Preparation and Characteristics of Naproxen Nanoparticles. 2.2.1. Preparation of Naproxen Nanoparticles. In this study, the copolymer used to prepare Nap nanoparticles was a biblock amphiphilic copolymer monomethoxy poly(ethylene glycol)-poly(εcaprolactone) (MPEG-PCL), which was prepared in our laboratory according to the schematic diagram shown in Figure 1A. Briefly,
DL% =
drug × 100% polymer + drug
(1)
EE% =
actual drug loading × 100% theoretical drug loading
(2)
2.3. Preparation and Characteristics of Thermosensitive Chitosan Hydrogels. 2.3.1. Preparation of Chitosan Hydrogels. The CS solution was prepared by dissolving 100 mg of CS powder in 4 mL of a 0.1 M aqueous acetic acid solution (HAC) under continuous magnetic stirring at 4 °C. A β-GP solution was obtained by dissolving 500 mg of β-GP powder in 1 mL of deionized water and stored at 4 °C. Subsequently, a homogeneous liquid CS hydrogel containing 2% (w/v) CS and 10% (w/v) β-GP was prepared by adding the cooled β-GP solution to the CS solution under mild stirring. 2.3.2. In Vitro Drug Release Study. A dialysis method was used to investigate the in vitro drug release behavior for the release of Nap from Nap-NPs. Briefly, 1.0 mL of CS/Nap hydrogel (equal to 0.5 mg of Nap) and 1.0 mL of a Nap solution in dehydrated alcohol (0.5 mg/ mL) were separately placed in two dialysis bags with cutoff molecular weights of 8.0−14 kDa. The dialysis bags were incubated in 40 mL of phosphate buffer (PBS, pH 7.4) containing Tween 80 [0.5% (w/v)] at 37 °C under gentle shaking with a rate of 100 rpm. At predetermined time points, 2 mL of incubation medium was withdrawn, which was replaced with fresh medium. The removed released medium was centrifuged for 15 min at 12000 rpm and room temperature. The collected samples were measured by HPLC to determine the concentration of released Nap. Measurements were performed at least in triplicate. 2.3.3. Cytotoxicity Assay. The cytotoxicity of CS and CS/Nap hydrogel was assessed by the MTT assay using NIH 3T3 cells. In brief, NIH 3T3 cells were seeded into 96-well plates (5 × 104 cells/ well) and incubated at 37 °C in 5% CO2 for 24 h. Then, cells were treated with 100 μL of CS hydrogel or CS/Nap hydrogel at different concentrations and incubated for 48 h. After incubation, 10 μL of the MTT solution (5 mg/mL) was added to each well, and cells were cultivated for an additional 4 h. Finally, the supernatant was carefully removed, and 150 μL of dimethyl sulfoxide was added to each well to dissolve blue formazan crystals. The absorbance was measured at a wavelength of 490 nm using a microplate reader (iMark, Bio-Rad, Hercules, CA). The cell viability of NIH 3T3 cells was calculated and normalized using cells treated with only media as a control.
Figure 1. Schematic diagram of (A) the synthesis of the MPEG-PCL copolymer and (B) the formation of CS/Nap hydrogel. MPEG-PCL was prepared by ring-opening polymerization of ε-CL in the presence of MPEG as an initiator with Sn(Oct)2 as a catalyst. First, a calculated amount of MPEG (10.0 g, 2 mmol), ε-CL (10.0 g, 0.088 mol), and Sn(Oct)2 (0.1 g, 0.5 wt % of total reactants) was added to a three-neck round-bottom flask under the protection of a nitrogen flow. The mixture was maintained at 130 °C while being mechanically stirred for 6 h, and then, the reaction system was cooled to room temperature, dissolved in dichloromethane, and reprecipitated with an excess of precooled petroleum ether. The final product was vacuum-dried to a constant weight and stored in an airtight bag for further use. For the preparation of Nap nanoparticles, 96 mg of MPEG-PCL copolymer [theoretical molecular weight of 4000 (MPEG2000PCL2000)] and 4 mg of Nap were dissolved in 10 mL of absolute alcohol under magnetic stirring at 40 °C. After the content was dissolved completely, the solution was evaporated using a rotary evaporator at 60 °C. After evaporation of the alcohol, a uniform film formed on the walls of the flask. The film was then dissolved in prewarmed deionized water at 65 °C to create a Nap/MPEG-PCL solution, which was filtered using a 220 nm filter to obtain a clear solution with a light-blue opalescence. The solution was lyophilized B
DOI: 10.1021/acsbiomaterials.8b01562 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 2. In vitro characterization of Nap-NPs and CS/Nap hydrogel: (A) freeze-dried Nap-NP powder, (B) resolved Nap-NP solution (left) and deionized water (right), (C) particle size distribution of Nap-NPs, (D) TEM photo of Nap-NPs, (E) in vitro release behavior for release of Nap from free Nap and the CS/Nap hydrogel, and (F) in vitro cytotoxicity of CS and CS/Nap hydrogels. 2.3.4. Rheological Analysis of Chitosan Hydrogels. The thermosensitivity of the CS solution and CS/Nap hydrogels was investigated with an AR2000 ex rheometer (TA Instruments, New Castle, DE). In brief, 1.0 mL of CS or CS/Nap hydrogels was placed between parallel plates (40 mm diameter and gap size of 1 mm). A strain amplitude of 1% was applied to maintain a linear viscoelastic region. The storage modulus (G′) and loss modulus (G″) were measured by heating the systems from 10 to 60 °C at a rate of 2 °C/ min. The time dependence of G′ and G″ of the CS/Nap hydrogel was investigated at 37 °C over the range from 0 to 300 s. 2.4. In Vivo Anti-Adhesion Experiments. Forty female Wistar rats, weighting 150 ± 20 g, were supplied by the Animal Experimental Center of Southwest Medical University (Luzhou, China). All rats were housed and given free access to food and water at a temperature of 25 ± 2 °C and a relative humidity of 50−60% under natural 12 h light−dark cycles for 7 days. After the adaptation period, the general conditions of rats, such as activity, energy, hair, feces, behavior pattern, and other clinical signs, were continuously monitored for 1 week. Prevention of postoperative adhesion in vivo was carried out by establishing an abdominal wall defect-cecum abrasion model, which was adopted from ref 2. In this study, the female Wistar rats were randomly divided into four groups (n = 10 per group): the control group, the commercialized HA hydrogel, the CS hydrogel, and the CS/Nap hydrogel. All animal studies were approved by the Institutional Animal Care and Use Committee of Southwest Medical University. For the surgery involving the defect-cecum abrasion, an aseptic technique was applied throughout the entire experimental process. The animals were anesthetized by intraperitoneal injection of 3 mL of ketamine hydrochloride (10 mg/mL)/kg and 4 mg of xylazine/kg, and a 5 cm incision was made along the linea alba to access the peritoneum. Abdominal adhesions were induced according to a previous report.7 In brief, a peritoneal defect with an area of 1 cm × 2 cm was made on the right lateral abdominal wall by using a scalpel blade until it was damaged. Another defect (1 cm × 2 cm) in the cecal haustra was abraded using surgical gauze until the serosal surface was disrupted and hemorrhaging but not perforated. Then, the two injured wounds were pulled together and partially sutured with 3-0 silk sutures to induce adhesions. In the negative control group, defects were created but not treated. In the positive control group, defects were treated with 2 mL of a commercialized anti-adhesion agent with
a the brand name of Xinkeling in a sodium hyaluronate solution (HA hydrogel, Hangzhou Singclean Medical Products Co. Ltd., Hangzhou, China). For the two treatment groups, 2 mL of a viscous CS solution or CS/Nap hydrogel was sprayed to completely cover the defect sites. Finally, the incisions were closed in two layers using 3-0 silk sutures. Rats were returned to their cages and maintained with normal food and water. Rats received water containing 10000 units of penicillin per day for 3 days after surgery, except for the CS/Nap hydrogel group, in which rats did not receive treatment. On day 7 postsurgery, rats were sacrificed using an overdose of intravenous sodium pentobarbital. The sutured abdomen was reopened to assess the adhesions. The anti-adhesion efficacy was evaluated by two independent observers in a double-blind manner according to the following standard scoring system: score 0, no adhesions; score 1, mild, easily separable intestinal adhesions; score 2, moderate intestinal adhesions, separable by blunt dissection; score 3, severe intestinal adhesions, which required sharp dissection.2 To identify possible side effects in rats that were given different treatments, animals were observed, including activity, energy, hair, feces, behavior pattern, and other clinical signs. After mice were sacrificed, several major organs (heart, liver, spleen, lung, and kidney) and specimens of the abdominal wall were harvested and fixed in 4% paraformaldehyde in PBS. Tissues were sectioned, stained with hematoxylin and eosin (H&E), and evaluated in a blinded manner by two pathologists. 2.5. Statistical Analysis. The adhesion scores did not always follow a normal distribution. Therefore, statistical inferences were made using the Mann−Whitney μ test or Fisher’s exact test using SPSS 10.0 software (SPSS, Chicago, IL). P < 0.05 was considered statistically significant.
3. RESULTS 3.1. Preparation and Properties of Naproxen Nanoparticles. In this study, Nap-loaded polymeric nanoparticles were prepared using biblock copolymer MPEG-PCL by a thinfilm hydration method. As shown in panels A and B of Figure 2, aqueous Nap-NPs could be lyophilized into a loose white powder, and the powder could be redissolved in deionized water with blue opalescence. The prepared Nap-NPs had a narrow size distribution with an average diameter of ∼33 nm and a polydispersity index (PDI) of 0.16 (Figure 2C). C
DOI: 10.1021/acsbiomaterials.8b01562 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX
Article
ACS Biomaterials Science & Engineering
Figure 3. Thermosensitive properties of CS hydrogels and CS/Nap hydrogels: (A and C) room temperature and (B and D) 37 °C. (E and F) Temperature dependence of the storage (G′) and loss (G″) moduli for the CS hydrogel and CS/Nap hydrogel, respectively, upon heating from 0 to 60 °C. (G) Time dependence of G′ and G″ for the CS/Nap hydrogel at 37 °C.
According to the HPLC measurements, DL and EE of NapNPs were 3.86% and 98.4%, respectively. TEM analysis showed the Nap-NPs had a smooth surface and a spherical shape (Figure 2D). The in vitro release pattern for release of Nap from the CS/ Nap hydrogel followed a time-dependent pattern and is shown in Figure 2E. Approximately 76% of free Nap was released within the first 24 h, whereas when Nap was released from the CS/Nap hydrogel, only 32% was released within 24 h. Six days later, only 68% of Nap was released from the CS/Nap hydrogel. Figure 2F shows the in vitro cell viability of NIH 3T3 cells, which were cultured with CS and CS/Nap hydrogel for 48 h. At lower concentrations ( 0.05), which may be due to the addition of Nap-NPs. 3.2. Preparation and Thermosensitive Properties of Chitosan Hydrogels. Thermosensitive CS hydrogels were prepared by mixing a CS solution with an aqueous β-GP solution. The thermoresponse of blank CS hydrogels and CS/ Nap hydrogels was verified and is shown in Figure 3. At room temperature, the aqueous CS solution (Figure 3A) could be converted into a nonflowable hydrogel state at 37 °C (Figure 3B). After the loading of Nap-NPs, the CS/Nap hydrogel composite presented a thermosensitivity that was similar to that of the blank CS hydrogel (Figure 3C,D). Moreover, the results of rheological analysis confirmed the changes in the storage modulus (G′) and loss modulus (G″) during the heating process as shown in panels E and F of Figure 3. When the temperature was
