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Prevention of peritoneal adhesions by ferric ion-crosslinked hydrogels of hyaluronic acid modified with iminodiacetic acids Yuki Amano, Pan Qi, Yoshiyuki Nakagawa, Katsuhisa Kirita, Seiichi Ohta, and Taichi Ito ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00456 • Publication Date (Web): 27 Jul 2018 Downloaded from http://pubs.acs.org on August 1, 2018

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ACS Biomaterials Science & Engineering

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Prevention of peritoneal adhesions by ferric ion-crosslinked hydrogels of hyaluronic acid modified with iminodiacetic acids Yuki Amano†, Pan Qi‡, Yoshiyuki Nakagawa†, Katsuhisa Kirita§, Seiichi Ohta‡, and Taichi Ito†,‡, §* †

Department of Chemical System Engineering, The University of Tokyo, 7-3-1 Hongo,

Bunkyo-ku, Tokyo 113-8656, Japan ‡

Center for Disease Biology and Integrative Medicine, The University of Tokyo, 7-3-1

Hongo, Bunkyo-ku, Tokyo 113-0033, Japan §

Department of Bioengineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,

Tokyo 113-8656, Japan Corresponding Author * E-mail: [email protected]

Abstract: We fabricated ferric ion-crosslinked hydrogels of a chelating hyaluronic acid (HA) derivative. HA was conjugated with iminodiacetic acid (IDA) at 22% of a modification degree to its disaccharide unit of HA. This conjugate (HA-IDA) showed

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Page 2 degradability by hyaluronidase even after the IDA modification, and its degradation rate was almost the same as that of HA. HA-IDA and HA formed hydrogels (FeHA and FeHA-IDA) by crosslinking with ferric ions (Fe3+). The storage modulus of FeHA-IDA was ca.100 Pa, which was higher than ca. 10 Pa of FeHA. FeHA-IDA showed excellent biocompatibility to a mesothelium cell line, as well as rapid degradation. Above all, FeHA-IDA showed efficacy in reducing adhesion formation in a rat sidewall defect-bowel abrasion model.

KEYWORDS: Postoperative adhesion, Hydrogel, Hyaluronic acid, Iminodiacetic acid, Ferric ion, Rat sidewall defect-cecum abrasion model

Introduction Postoperative peritoneal adhesions can cause pelvic pain, bowel obstruction, and infertility. 1 The prevention of postoperative peritoneal adhesions is necessary in many surgeries. Hyaluronic acid (HA) is a polysaccharide composed of N-acetlyglucosamine and D-glucuronic acid, and it is abundantly contained in peritoneal fluid. 2 Thus, several anti-adhesion barrier materials based on HA have been developed3- 10 because of their biocompatibility and rapid degradation in the peritoneum. 1 For instance, a HA-containing sheet material, Seprafilm®,4-6 is widely-used in clinical settings. In addition, several injectable HA-based hydrogels have been investigated. 11-16 Because of the carboxyl groups of glucuronic acid residues of HA, HA is also widely used to form a soft hydrogel by crosslinking with ferric ions (Fe3+).7,

10, 17, 18

This

iron-crosslinked HA (FeHA) was released as Intergel® in a market in the late 1990s. 19


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Page 3 However, the product was withdrawn from the market in 200310, 20 because of an increase in the rate of infection and immunoreactivity.

9

Free Fe3+ released from FeHA was

proposed as a cause of the adverse events in a previous report21, although the detailed mechanism was not clarified yet. Another possible cause would be the toxicity of the residual fragments of FeHA, found in the peritoneal cavity.10 Therefore, smooth clearance of FeHA is a key to overcome the potential risks of FeHA. The interaction between polysaccharides and Fe3+ has been an interesting discussion topic in various fields. For example, safe iron supplements are necessary for the treatment of iron deficiency anemia. 22 Complexation between Fe3+ and polysaccharides such as dextran23, 24 and polygalacturonate25, 26 have been analyzed by Mössbauer spectroscopy, 13

C-CP/MAS NMR, EPR, and XRD. Similar to these polysaccharides, HA forms a

complex with Fe3+ via monodentate carboxylate groups (Fe–OCOR) or µ-oxo bridges (Fe-O-Fe). 27 Moreover, the structure of Fe3+–HA complex greatly depends on reaction pH. 17, 27 In order to obtain homogenous FeHA which has smooth clearance and excellent biocompatibility, synthesizing a new derivative of HA and its ferric complex is a potential approach. Iminodiacetic acid (IDA) is a well-known chelating reagent to complex with various ions. We have previously synthesized a new HA derivative conjugated with IDA (HA-IDA).

28

HA-IDA chelated cisplatin, changed its hydrophobicity, and formed a

nanogel containing cisplatin spontaneously. 29 This cisplatin incorporating HA nanogel was applied for the treatment of peritoneal dissemination of gastric cancer. 28, 29 In this study, we fabricate a ferric ion-crosslinked HA-IDA hydrogel (FeHA-IDA) as shown in Figure 1. Their degradability by hyaluronidase (HAse), toxicity, and



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Page 4 viscoelasticity are measured. Their anti-peritoneal adhesion effect is evaluated using a rat sidewall defect-bowel abrasion model.

Figure 1. Concept of a ferric-ion-crosslinked HA-IDA hydrogel (FeHA-IDA).

Materials and Methods Materials HA (Mw = 850 kDa) was kindly gifted from Denka Co. (Tokyo, Japan). IDA was purchased

from

Tokyo

Chemical

Industry

(Tokyo,

Japan).

1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride was purchased from Peptide Institute, Inc. (Osaka, Japan). Iron (III) chloride (FeCl3), insulin, Dulbecco’s modified Eagle’s medium (DMEM), penicillin-streptomycin-amphotericin B were purchased from Wako Pure Chemical Industries (Osaka, Japan). Hyaluronidase (HAse) from bovine testes (Type I-S, lyophilized powder, 400–1000 unit/mg solid) was purchased from Sigma–Aldrich (St. Louis, MO, USA). Medium 199 was purchased from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Hydrocortisone was purchased from MP Biomedicals (Santa Ana, CA, USA). Fetal bovine serum was purchased from Biosera, Inc., (Villebon sur Yvette, France), and epidermal growth factor was purchased


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Page 5 from PeproTech Ltd. (London, UK). Dialysis membrane (Spectra/Por, MWCO = 6000–8000 Da) was purchased from Spectrum Laboratories Inc. (Rancho Dominguez, CA, USA). The human pleural mesothelial cell line, MeT-5A, was purchased from American Type Culture Collection (Rockville, MD, USA). Pentobarbital was purchased from Kyoritsuseiyaku Co. (Tokyo, Japan). Synthesis and characterization of iminodiacetic acid conjugated hyaluronic acid The HA-IDA conjugate was synthesized by carbodiimide chemistry as described previously28, 29 with slight modifications. In brief, 0.5 g of HA was dissolved in 100 mL of distilled water. 1-Ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (42 mg in 3.0 mL of distilled water) and IDA (51 mg in 0.6 mL of 1.0 N NaOH) were added dropwise to the HA solution. The pH was adjusted to 5.0 with 1 N HCl or 1 N NaOH and kept constant for 4 hours. The reaction was allowed to proceed overnight. The solution was dialyzed against pure water and then lyophilized. Synthesis of HA-IDA was confirmed by 1H NMR (αJEOL JNM-A500, JEOL, Tokyo, Japan) and FT-IR (FT/IR-4200ST, JASCO, Tokyo, Japan). The polymers were dissolved in D2O for 1H NMR analysis. FT-IR spectra of HA and HA-IDA were measured using a potassium bromide (KBr) tablet of each polymer. Molecular weight distributions of the polymers were determined via GPC. Separation was performed at room temperature using a TSK-Gel GMPWXL column (TOSOH, Tokyo, Japan) at a flow rate of 0.5 mL/min, using dextran as the standard. Sodium phosphate buffer (pH 6.7), composed of 0.05 M phosphate buffer containing 0.2 M NaCl was used as a mobile phase. Evaluation of the degradation behavior of HA-IDA by HAse



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Page 6 HA or HA-IDA was dissolved in the sodium phosphate buffer (pH 6.7) , composed of 0.05 M phosphate buffer containing 0.2 M NaCl. HAse was dissolved in the same buffer at 20 units/mL, according to previous reports. 30, 31 Then, 1 mL of the polymer solution and 1 mL of the HAse solution were added to a 2-mL microtube. The solution was incubated at 37 °C. At each time point, the microtubes were collected and frozen with liquid nitrogen and frozen at ‒80 °C. The molecular weight distribution of the polymers at each time point was measured by GPC using the same conditions as previously mentioned. Dynamic viscoelasticity measurements Dynamic viscoelasticity measurements were conducted with a rheometer (MCR301; Anton Paar, Graz, Austria) using a parallel-plate geometry (PP25; diameter = 25 mm, gap = 1.0 mm) for hydrogel samples and a cone-plate geometry (CP50-1-27711; diameter = 50 mm, cone angle = 0.976 degree) for sol samples. For these measurements, equal amount of 1.0 wt% HA or HA-IDA solution and 5 mM FeCl3 solution were injected into silicone molds (diameter = 25 mm, depth = 1.0 mm) through a double-barreled syringe, which were incubated at room temperature overnight to obtain homogeneous hydrogel disks. The frequency dependencies of the storage modulus (G′) and loss modulus (G′′) of all of the samples were measured at a strain value of 5%. Dynamic strain sweep tests were performed for all the samples from 0.1 to 10% at 1 Hz to confirm that this strain was within the linear viscoelastic regime. Measurement of the degradation kinetics of the hydrogels The degradation of the hydrogels was measured gravimetrically in PBS, saline, DMEM and HAse (10 units/mL) in PBS at 37 °C over time. A 1.25 wt% HA or HA-IDA


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Page 7 sample in saline was prepared in glass vials, into which 25 mM FeCl3 in saline was added dropwise with the ratio of polymer solution: Fe3+ solution = 4 : 1. The pH was adjusted to 7.4 with 0.1 N NaOH. The 500 µL of obtained hydrogel was molded into cylindrical shape with the diameter of 4.6 mm and the height of 3 cm. The hydrogel was then incubated in 22 mL of PBS, saline, DMEM and HAse (10 units/mL) in PBS. At each time point, the weight of hydrogel was measured and the incubation solution was replaced. The weight of hydrogel at each time point (W) was normalized to the initial weight (W0) to evaluate the degradation behavior. Measurement of free Fe3+ release profile from FeHA and FeHA-IDA gels The 500 µL of FeHA or FeHA-IDA hydrogels were put into a dialysis membrane (MWCO=50 kDa). The tube was immersed in 80 mL of PBS (pH 7.4) at presence or absence of Fe3+ chelators, EDTA and IDA, then stirred at 37 °C. 31 µM of chelator was used. We collected 5 mL of the sample and added the same amount of fresh media at each time point during the release experiments, followed by determination of Fe3+ concentration using ICP-AES (iCAP 6000, Thermo Fisher Scientific, Yokohama, Japan). We conducted three independent experiments for each condition. The averaged values were used as data and standard deviations were used as error bars. Cytotoxicity Assay In vitro cell viability in the presence of HA-IDA polymer or crosslinked FeHA-IDA hydrogel was determined using the WST-8 assay (Cell Counting Kit-8; Dojindo Laboratories, Kumamoto, Japan) with MeT-5A human mesothelial cells. MeT-5A cells were maintained in Medium 199, which contained 25 mM HEPES, 10 % fetal bovine serum, 3.3 nM epidermal growth factor, 400 nM hydrocortisone, and 870 nM insulin, at



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Page 8 37 °C in 5 % CO2. The cells were seeded in a 24-well plate with an initial density of 50000 cells/well and incubated at 37 °C in 5 % CO2 overnight. For the evaluation of HA-IDA polymer, the culture media were replaced with the media containing 0.1 or 1.0 w/v% of HA-IDA solution. For the hydrogel sample, the media were replaced with fresh media, and then Transwell cell culture inserts loaded with 1.0 wt% FeHA-IDA were inserted into each well. The WST-8 assay was performed 48 h after the treatment. 50 µL of tetrazolium salt solution (WST-8) was added to each well and incubated at 37 °C for 1 h. The absorbance at 450 nm was measured using a plate reader (2030 ARVO V3; PerkinElmer, Waltham, MA, USA). The absorbance value of each well was normalized by that of control wells in which no test materials were added to the media. Evaluation of peritoneal adhesion-preventing effect by a rat sidewall defect-bowel abrasion model These experiments were performed at the Animal Research Section, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo. The Animal Care Committee of the University of Tokyo approved all of the procedures performed in this study before it began. Sprague-Dawley rats (6 weeks old, male) weighing 200–220 g were purchased from CLEA Japan, Inc. (Tokyo, Japan). All the animals were in quarantine for 1 week prior to the experiment. HA-IDA was sterilized by UV irradiation and then FeHA-IDA hydrogel was prepared prior to the application. 4 mL of 1.25 wt% HA or HA-IDA in saline and 1 mL of 25 mM FeCl3 in saline were mixed in a glass vial. The pH was adjusted to 7.4 with 0.1 N NaOH.


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Page 9 The rat sidewall defect-bowel abrasion model was used as described in previous research32-35 with slight modification. Briefly, rats were anesthetized by pentobarbital (35 mg/kg bodyweight). The abdominal area was shaved, cleaned, and then a 4-cm incision was made in the median line. Two parietal peritoneum defects were created with an 8-mm diameter biopsy punch at 5 mm intervals in the right lateral peritoneal surface of the abdominal wall. The ventral side of the cecum was abraded with sandpaper 20 times in the area of 2×2 cm2. The damaged cecum was returned to the abdominal cavity, and the position was adjusted so that both injured surfaces were in touch in the absence of anti-adhesion material. 2 mL of the pre-formed FeHA-IDA hydrogel was applied between both injured surfaces via a syringe without assembling a needle, followed by closing of the abdominal cavity via suture. For the control group, the abdominal cavity was closed without material application. The anti-adhesion ability of FeHA-IDA was evaluated 1 week after the operation. The rats were sacrificed by intraperitoneal administration of pentobarbital (240 mg/kg body weight). The abdominal cavity was opened through a U-shaped incision, and the presence of material residue and the grade of adhesion formations were evaluated. The adhesion grade was scored on a scale of 0–3 (0 = no adhesion, 1 = loose filmy adhesions that can be separated without using any tools, 2 = adhesions requiring of blunt dissection, 3 = adhesions requiring of sharp dissection), which is a widely used standard scoring system.

3

The scoring was determined by two observers in a blinded manner. The

traumatized surfaces were sampled and fixed in a 10 % formalin solution, followed by tissue sectioning and subsequent H&E staining.



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Page 10 Results and Discussion Synthesis and characterization of HA-IDA HA-IDA was synthesized as described in our previous report, 28, 29 except for the use of HA with a higher molecular weight. Synthesis of HA-IDA was confirmed by using 1H NMR and FT-IR. In the 1H NMR spectra, the methylene protons of conjugated IDA (singlet peak at 2.92 ppm) was confirmed after the IDA modification (Figure 2A). The degree of modification was calculated from the ratio of the area of the peak for N-acetyl-D-glucosamine residue of HA (singlet peak at 2.0 ppm) to that for the methylene protons of IDA at 2.92 ppm. The degree of modification was 22 % to its disaccharide unit of HA. Figure 2B shows the FT-IR spectra of HA and HA-IDA. Although emergence of new distinctive peaks was not observed, the peak around 3100–3600 cm−1 was broadened, suggesting O–H stretching vibrations from the –COOH groups of IDA. These results suggest that IDA was successfully conjugated to HA even though a higher molecular weight HA was used compared with previous studies.


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Figure 2. Synthesis of HA-IDA. (A) 1H NMR spectrum and (B) FT-IR spectrum of HA-IDA. Spectra of native HA were also shown for comparison.

HA-IDA can be degraded by HAse even after modification with IDA We measured molecular weight of HA and HA-IDA using GPC relative to dextran standards (Figure 3A). The resulting weight-average molecular weight of HA and HA-IDA were 4.0×107 Da and 3.1×107 Da. The molecular weight distributions of HA before and after IDA conjugation was almost the same, but that of HA-IDA slightly shifted to a lower molecular weight range. The molecular weight may have decreased during the conjugation of IDA conducted at pH 5. Subsequently, we measured the degradation behavior of HA and HA-IDA by HAse.



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Page 12 HAse is present in the peritoneum and can degrade any applied HA-based materials. Therefore, investigating the degradation behavior of HA-IDA for evaluating its property as an anti-adhesion material is important. There was no significant difference in the trend of degradation behavior between HA and HA-IDA (Figure 3B). In both polymers, the molecular weight was decreased until 6 hours when it reached a plateau value, which was almost 0.2 % of initial molecular weight. To further analyze the degradation kinetics, apparent pseudo first order rate constants (k′) were determined from plots of 1/Mw versus incubation time (t) using the expression as follows36; 1 1 = + k ' t (1) Mw Mw , 0 As shown in the plot data (Figure S1), the rate constant for the degradation of HA was 2.25×10−6 Da−1 h−1, whereas that for HA-IDA was 2.00×10−6 Da−1 h−1. This result suggests that the IDA conjugation to HA had little effect on degradation by HAse, indicating that HA-IDA can be cleared from the peritoneum.


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Figure 3. Molecular weight of HA-IDA. (A) Molecular weight distribution before and after IDA conjugation to HA. (B) Reduction of molecular weight of HA and HA-IDA via HAse degradation.

Comparison of hydrogel formation and viscoelasticity between FeHA and FeHA-IDA Figure 4 shows the optical images of FeHA (Figure 4A) and FeHA-IDA (Figure 4B). FeHA-IDA hydrogel was more self-supporting than FeHA hydrogel. Dynamic viscoelastic properties of FeHA and FeHA-IDA are shown in Figure 4C and Figure 4D. Although the storage modulus (G′) was always lower than the loss modulus (G′′) in HA, G′ was always higher than G′′ in FeHA, suggesting that the mixing of HA and Fe formed



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Page 14 a hydrogel. The same trend was observed in FeHA-IDA. The G′ of FeHA-IDA was around 100 Pa, which was higher than that of FeHA (ca. 10 Pa). In addition, G′ and G′′ increased with frequency in FeHA, and G′ and G′′ remained largely unchanged across wide range of frequencies in FeHA-IDA. These results suggest that IDA conjugation to HA resulted in a stiffer hydrogel formation with Fe than that without IDA conjugation, because of the increased crosslinking density caused by enhanced chelating affinity. It is expected that FeHA-IDA can be formed with lower Fe3+ than FeHA, which can decrease the usage of Fe3+ and resultant potential toxicity. Additionally, we tried to prepare FeHA and FeHA-IDA by using in situ two-liquid mixing. As shown in Figure S2, FeHA-IDA could be formed in situ with neutral pH via mixing FeCl3 solution with HA-IDA solution, pH of which was pre-adjusted to 8.0. On the other hand, unmodified HA did not form hydrogels via in situ mixing, which indicated that FeHA-IDA can be administrated by a double-barreled syringe.


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Figure 4. Viscoelasticity of FeHA and FeHA-IDA. Pictures of FeHA, (A), and FeHA-IDA, (B). Frequency dependency of the storage modulus G′ (closed circle) and loss modulus G′′ (open circle) of HA and FeHA, (C), and HA-IDA and FeHA-IDA, (D).

Degradation kinetics of FeHA-IDA Figure 5A shows the degradation behaviors of FeHA and FeHA-IDA in saline, PBS, and DMEM, which are representative media close to peritoneal fluid. Peritoneal fluid



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Page 16 exists in the abdominal cavity, whose pH is 7.5-8.0 and ionic strength is almost the same value with that of serum.

1

In addition, it contains some Fe3+ chelators, such as

transferrin, phosphate ion, and amino acids.

1

Because saline, PBS, and DMEM have

almost equal ionic strength to peritoneal fluid but different Fe3+ chelator contents, they are suitable for investigating the effect of Fe3+ chelators on degradation behavior of FeHA and FeHA-IDA. Saline contains no Fe3+ chelator. PBS contains phosphate ions (9.5 mM) that can chelate Fe3+. DMEM contains phosphate ions (0.9 mM), as well as amino acids (11 mM), some of which can potentially work as Fe3+ chelators. In all of these media, hydrogels showed initial swelling, followed by gradual degradation to polymer solution. The magnified figure for initial days is also shown in Supporting Information (Figure S3). In saline, although FeHA completely degraded in 22 days, it took 34 days for the complete degradation of FeHA-IDA, which would be due to higher chelating stability of IDA with Fe3+. The degradation of both hydrogels proceeded faster when DMEM was used as the immersion medium. The degradation in PBS was even faster than DMEM. This accelerated degradation may be due to phosphate ions (PO43−) contained in DMEM and PBS. Phosphate ions would exchange counter ion of Fe3+, resulting in the accelerated hydrogel degradation. Therefore, degradation rate depended on phosphate ion concentration, PBS > DMEM > saline. Figure 5B further shows the degradation kinetics of FeHA and FeHA-IDA in PBS containing HAse. HAse significantly accelerated the degradation, and only 3 hours were required for apparently complete degradation of both hydrogels without burst swelling. Comparing FeHA and FeHA-IDA, the degradation of FeHA-IDA was slightly slower than that of FeHA. The increase of chelating ability by IDA modification led to increased


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Page 17 crosslinking density, which would sterically prevent the degradation by HAse. In addition, after apparent degradation of hydrogel, further incubation with high concentration HAse (700 units/mL) was conducted to proceed complete degradation of HA backbone. As a result, it was found that FeHA formed a lot of large degradation products after 3 days incubation, while FeHA-IDA did not (Figure S4). These results suggested that FeHA formed irreversible crosslinking. Figure 5C shows the release profile of Fe3+ from FeHA or FeHA-IDA gels in PBS at presence or absence of EDTA and IDA, strong Fe3+ chelators. Both hydrogels hardly released free Fe3+ in PBS (Figure 5C), although the hydrogels were degraded (Figure 5A); FeHA and FeHA-IDA released only 2.3 % and 3.2 % of loaded Fe3+ in 4 days. Even by the addition of free IDA, release of Fe3+ did not change so much, suggesting that the coordination of Fe3+ with HA-IDA was more stable than that with free IDA. On the other hand, addition of EDTA accelerated the Fe3+ release, and FeHA-IDA showed slower Fe3+ release than FeHA; after 4 days, 37.9 % and 30.8 % of loaded Fe3+ was released from FeHA and FeHA-IDA, respectively. These results suggested that Fe3+ are coordinated to HA and HA-IDA even after the hydrogel degradation, resulting in negligible free Fe3+ release unless strong iron chelators such as EDTA existed. Based on these results, it was suggested that most of Fe3+ ions were not released from FeHA during degradation, and consequently a lot of large degradation products were formed. These degradation products of FeHA would be a cause of serious problems found in previous clinical use9, for example increased infection and immunoreactivity. On the other hand, although Fe3+ ions were also hardly released from FeHA-IDA, such large degradation products were not found after degradation, suggesting the potential for



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Page 18 smooth clearance from the peritoneal cavity. We consider that the difference of degradation behavior between FeHA and FeHA-IDA was due to their different structure of crosslinking points. FeHA is considered to form irreversible tight crosslinking points through monodentate coordination of three HA molecules

17, 27

, in which counter ion

exchange with other iron chelators, such as phosphate ions, would be difficult to occur. On the other hand, HA-IDA would preferentially coordinate to Fe3+ through µ-oxo bridges

37

which would enable reversible and faster counter ion exchange with iron

chelators. (Figure S5). This difference in Fe3+ chelation would cause faster degradation of FeHA-IDA than FeHA in phosphate-containing media, including PBS and DMEM as well as peritoneal fluid, which would result in faster clearance of degradation products that might induce the adverse effects. In peritoneal fluid, transferrin exists as a strong Fe3+ chelator. Because stability constant of Fe3+-transferrin (logK = 20) is high, transferrin is considered to promote the degradation of FeHA-IDA and clear Fe3+ from the peritoneal cavity based on the Fe3+ release under the existence of EDTA. Since Fe3+ chelated by transferrin is reported to be nontoxic, it is expected that Fe3+ contained in FeHA-IDA hardly induce toxicity.


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Figure 5. In vitro degradation of FeHA and FeHA-IDA. (A) Time course of normalized weight of FeHA and FeHA-IDA by the initial weight in PBS, saline, and DMEM. (B) Time course of the normalized weight of FeHA and FeHA-IDA in HAse containing media. (C) Release profile of Fe3+ from FeHA and FeHA-IDA hydrogels in PBS at the presence or absence of IDA or EDTA.

Biocompatibility of FeHA-IDA in mesothelial cells In vitro cell viability was evaluated using a WST-8 assay and human mesothelial cells (MeT-5A). Mesothelial cells were cultured in the presence of HA-IDA solutions or FeHA-IDA hydrogels. In order to avoid physical damage to cells caused by contact with hydrogels, we applied FeHA-IDA hydrogels via Transwell culture inserts. Cell viability was measured 2 days after the treatment with materials. As shown in Figure 6, cell viability with uncrosslinked HA-IDA at 0.1 w/v% and 1.0 w/v% was 80 % and 50 %, respectively, suggesting mild dose-dependent cytotoxicity. This toxicity of HA-IDA would be caused by high osmotic pressure and it can be observed even in the case of uncrosslinked and unmodified HA.38 However, FeHA-IDA did not exhibit any significant cytotoxicity; cell viability with 1.0 wt% FeHA-IDA hydrogel was 95 %. This result suggests that Fe3+ chelated in the hydrogel had no significant effect on cell viability, and the effect of disassociated Fe3+ could be minimal within 2 days. Cytotoxicity of degradation products of FeHA-IDA was also evaluated in Figure S6. The degradation products were applied to cells after inactivation HAse by heating. The results showed slight toxicity. However, because these degradation products would be smoothly cleared from peritoneal cavity in vivo as discussed before, their toxicity is expected to be not so


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Page 21 significant. Based on these in vitro results, we evaluated the anti-adhesion ability of FeHA-IDA hydrogel using an animal experiment and the same polymer concentration.

Figure 6. The effect of HA-IDA and FeHA-IDA on cell viability of mesothelium cells.

Prevention of peritoneal adhesions by FeHA-IDA Rats (n = 8) received cecum abrasions and adjacent abdominal wall excisions as described in the experimental section. The damaged cecum was treated with FeHA-IDA, and the abdominal cavity of control animals was closed without any treatment. As shown in Table 1, the body weight was increased in all rats regardless of treatment during the first week after the surgery. This result suggests that the surgical model did not induce severe postoperative burden. Animals were sacrificed at 1 week after the surgery and assessed for adhesion formation. Figure 7A shows a representative photograph of the trauma site treated with FeHA-IDA 1 week after the surgery. In all animals, FeHA-IDA was completely cleared. No adhesion formation was observed in 6 out of 8 rats. However, 6 out of 8 rats developed adhesions in the control group. Adhesions were observed



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Page 22 between cecum and abdominal wall excision and between cecum and adipose tissue. The adhesion scores are shown in Table 1. The adhesion score was decreased by the treatment with FeHA-IDA, although there was no statistical significance; the mean adhesion score for the control rats was 1.50 ± 0.93, whereas that for FeHA-IDA-treated rats was 0.50 ± 0.93. Figure 7B shows the representative H&E staining image of a sample taken from the trauma site in FeHA-IDA-treated rats without adhesion formation. The mesothelial cell layer showed recovery at the traumatized sidewall. However, samples taken from the trauma site in the control animals showed active inflammatory cells, and the recovery of mesothelial cell layer was not be observed (Figure S7). The above results demonstrate the potential of FeHA-IDA as an anti-peritoneal adhesion material. We consider that the advantages of FeHA-IDA revealed in this research are potential for low Fe3+ release, decreased Fe3+ usage for gelation, smooth elimination from the peritoneal cavity, and in situ injectability that enables better operability. Therefore, FeHA-IDA would be a potential anti-adhesion material in clinical use.


Page 23 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 23 Table 1. Adhesion score in a rat cecum abrasion and sidewall defect model

FeHA-IDA

Control

% Weight change

+ 25 ± 6.5

+ 21 ± 3.7

Score 3

0

0

Score 2

2

6

Score 1

0

0

Score 0

6

2

0

2

None

-

(No adhesion) Median adhesion score Material residue



Page 24 of 32

Page 24

Figure 7. Intraperitoneal administration of FeHA-IDA to a rat sidewall defect-bowel abrasion model. (A) Representative photograph of a FeHA-IDA-treated rat 1 week after the injury. Recovered peritoneum defects were indicated by white arrows. (B) Recovered mesothelium of traumatized sidewall shown in H&E staining picture. Scale bar = 500 µm

Conclusion We fabricated Fe3+-crosslinked hydrogels composed of a chelating HA derivative. Iminodiacetic acid-modified HA forms a hydrogel with Fe3+ that has a storage modulus 10 times higher than Fe3+-crosslinked native HA, because of the Fe3+ chelating ability of IDA. Therefore, it is expected to be formed with lower Fe3+, which can decrease the usage of Fe3+ and resultant potential toxicity. Both HA-IDA and FeHA-IDA hydrogels


Page 25 of 32 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Page 25 can be effectively degraded by HAse in vitro, suggesting there can be a smooth clearance if used in the peritoneum. The anti-adhesion efficacy of FeHA-IDA was demonstrated in a rat sidewall defect-bowel abrasion model. These results suggest the usefulness of FeHA-IDA as an anti-adhesion material.

Associated Content Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Figure S1 shows 1/Mw values plotted as a function of incubation time. Figure S2 shows the image of hydrogels fabricated by using a double-barreled syringe. Figure S3 shows the magnified figure for initial days of Figure 5A. Figure S4 shows the degradation products of hydrogels incubation with high concentration HAse. Figure S5 shows the hypothesis for crosslinking structure of hydrogels. Figure S6 shows the effect of degradation products on cell viability of mesothelial cells. Figure S7 shows the picture of adhesion site of a rat sidewall defect-bowel abrasion model.

AUTHOR INFORMATION

Notes The authors declare no competing financial interests.

Acknowledgments Hyaluronic acid was kindly gifted by Denka Co. We also thank Professor Tei / Chung and Associate Professor Sakai at The University of Tokyo for providing access to a



Page 26 of 32

Page 26 rheometer. This work was supported by JSPS KAKENHI Grant Number JP26420792 (Grant-in-Aid for Scientific Research(C)).

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for Table of Contents use only Prevention of peritoneal adhesions using ferric ion-crosslinked hydrogels of hyaluronic acid modified with iminodiacetic acids Yuki Amano, Pan Qi, Yoshiyuki Nakagawa, Katsuhisa Kirita, Seiichi Ohta, and Taichi Ito


Prevention of Peritoneal Adhesions by Ferric Ion ... - ACS Publications

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