DISULFIDE CROSSLINKED HYALURONIC ACID GEL FOR POSTOPERATIVE ABDOMINAL (PELVIC) ADHESION PREVENTION AND PREPARATION METHOD THEREOF

Abstract

A disulfide cross-linked hyaluronic acid gelatin for postoperative abdominal (pelvic) adhesion prevention and a preparation method therefor, wherein the content of disulfide cross-linked hyaluronic acid of the disulfide cross-linked hyaluronic acid gelatin is among 3-8 mg/mL. The disulfide cross-linked hyaluronic acid gelatin not only has favorable biocompatibility, but also has favorable prevention effects on tissue adhesions when being used for postoperative abdominal (pelvic) adhesion prevention.

Claims

1. A disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation, characterized in that the content of disulfide crosslinked hyaluronic acid is between 3˜8 mg/mL.

2. The disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 1, wherein the content of the disulfide crosslinked hyaluronic acid is between 4˜7 mg/mL.

3. The disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 2, wherein the content of the disulfide crosslinked hyaluronic acid is between 4.5˜6 mg/mL.

4. The disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 1, wherein the disulfide crosslinked hyaluronic acid gel is prepared by hyaluronic acid thiolated derivative.

5. The disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 4, wherein thiol content of the hyaluronic acid thiolated derivative is 10˜100 μmol/g.

6. The disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 5, wherein thiol content of the hyaluronic acid thiolated derivative is 20˜70 μmol/g.

7. A method for preparing a disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 1, wherein the disulfide-crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation is prepared through an oxidation process from a hyaluronic acid thiolated derivative aqueous solution.

8. The method for preparing a disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 7, wherein the oxidation process is performed under the action of oxygen.

9. The method for preparing a disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 8, wherein the oxygen is oxygen in the air and/or oxygen dissolved in aqueous solution.

10. A method for preparing a disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 2, wherein the disulfide-crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation is prepared through an oxidation process from a hyaluronic acid thiolated derivative aqueous solution.

11. A method for preparing a disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 3, wherein the disulfide-crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation is prepared through an oxidation process from a hyaluronic acid thiolated derivative aqueous solution.

12. A method for preparing a disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 4, wherein the disulfide-crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation is prepared through an oxidation process from a hyaluronic acid thiolated derivative aqueous solution.

13. A method for preparing a disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 5, wherein the disulfide-crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation is prepared through an oxidation process from a hyaluronic acid thiolated derivative aqueous solution.

14. A method for preparing a disulfide crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation according to claim 6, wherein the disulfide-crosslinked hyaluronic acid gel for preventing tissue adhesion after abdominal (pelvic) operation is prepared through an oxidation process from a hyaluronic acid thiolated derivative aqueous solution.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1. Effect of disulfide crosslinked hyaluronic acid content on gel dynamic viscosity.

[0025] FIG. 2. Effect of disulfide crosslinked hyaluronic acid content on the area involved in adhesion.

[0026] FIG. 3. Effect of disulfide crosslinked hyaluronic acid content on the severity of adhesions.

[0027] FIG. 4. Effect of the content of disulfide crosslinked hyaluronic acid on the adhesion length.

DETAILED DESCRIPTION

[0028] The embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention.

Example 1: Preparation of Disulfide Crosslinked Hyaluronic Acid Gels

[0029] Hyaluronic acid thiolated derivatives are made from sodium hyaluronate with a molecular weight of 180 KDa and prepared by the method reported by Shu et al. (Shu et al., Biomacromolecules 2002, 3: 1304-1311). The thiol contents of the derivatives were 24 μmol/g, 38 μmol/g and 57 μmol/g, respectively.

[0030] The above-mentioned hyaluronic acid thiolated derivatives were dissolved to obtain aqueous solutions with contents of 3 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, and 10 mg/mL respectively. The pH value of the solutions was adjusted to 7.4. After sterilization by filtration, the solutions were transferred to sterile glass containers. The solutions were kept sealed at room temperature for 4 weeks, and the solutions lose fluidity and form disulfide crosslinked hyaluronic acid gels.

Example 2: Evaluation of Dynamic Viscosity Properties of Disulfide Crosslinked Hyaluronic Acid Gels

[0031] The dynamic viscosity properties of the disulfide crosslinked hyaluronic acid gels prepared in Example 1 were evaluated with a rotational viscometer at a shear rate of no less than 0.25 Hz (25±0.1° C.) in accordance with the second method of Appendix VIG of Part II of the Chinese Pharmacopoeia (2010 edition).

[0032] Visual inspection showed that gels with higher contents of disulfide crosslinked hyaluronic acid had better strength, while test results also showed that gels with higher content had higher dynamic viscosity. The effect of the contents of disulfide-crosslinked hyaluronic acid on the dynamic viscosity of the gel was shown in FIG. 1. The gel with disulfide crosslinked hyaluronic acid content of 10 mg/mL had the highest dynamic viscosity (>10,0000 mPa.Math.s), which exceeded the upper limit of the measuring instrument measurement and thus was marked as greater than 10,000 mPa.Math.s in FIG. 1.

[0033] The thiol content of the hyaluronic acid thiolated derivative also has a certain effect on the dynamic viscosity of the gel. The gel prepared from the hyaluronic acid thiolated derivative with higher thiol content has a higher dynamic viscosity.

Example 3: Cytocompatibility Evaluation of Disulfide Crosslinked Hyaluronic Acid Gels

[0034] The in vitro cytotoxicity of the disulfide-linked cross-linked hyaluronic acid gels prepared in Example 1 were evaluated with reference to the standards of ISO10993.5-2009.

[0035] Rat fibroblasts (ATCC CCL1, NCTC Clone 929, Clone of Strain L) were cultured at 37° C., 5% CO.sub.2 and under saturated humidity, using RPMI 1640 medium containing antibiotics (100 u/mL penicillin, 100 μg/mL streptomycin) and 10% serum. When the cells grow to near confluence, digest with membrane protease, collect the cells and adjust the cell concentration to 5×10.sup.4/mL.

[0036] RPMI 1640 medium containing 10% serum was used as the extraction medium. 0.2 g of disulfide-linked cross-linked hyaluronic acid gel was added to each ml of the extraction medium. Leaching was performed at 37° C. for 24 hours. Then the leaching stock solution was diluted by RPMI 1640 with 10% serum to obtain four doses of diluted leaching solutions with the contents of the leaching stock solution of 100%, 50%, 25%, and 12.5%, respectively.

[0037] The above cell suspensions was added to a 96-well plate, 100 μL (5×10.sup.3 cells) per well, and incubated at 37° C., 5% CO.sub.2 for 24 hours; discard the medium, add the diluted leaching solutions by groups (four doses of 100%, 50%, 25% and 12.5%), negative control, blank control and positive control, 5 holes in each group, incubate in 37° C., 5% CO.sub.2 saturated steam incubator for 24 hours.

[0038] After the incubation, remove the cell culture plate, discard the medium, add 100 μL of RPMI1640 medium containing 10% of serum, add 50 μL (1 mg/mL) of MTT stain to each well, and incubate in 37° C., 5% CO.sub.2 saturated steam incubator for 3 hours; discard the medium in the culture plate, add 100 μL of isopropanol to each well, mix by shaking, and measure the absorption value at a wavelength of 570 nm.

[0039] The relative cell proliferation rate was calculated based on the ratio of the absorption value of each group to the absorption value of the blank control group, and the relative cell proliferation rate of the blank control group was calculated as 100%.

[0040] The relative cell proliferation rate of the negative control group was the same as that of the blank control group, and the relative cell proliferation rate of the positive control group was less than 10%, which results were in line with expectations. The relative proliferation rate of cells in each disulfide crosslinked hyaluronic acid gel test group was >90%, and cytotoxicity was not observed in the cells, indicating that the disulfide crosslinked hyaluronic acid gel tested had good cell compatibility.

Example 4: Evaluation of In Vivo Histocompatibility of Disulfide Crosslinked Hyaluronic Acide Gels

[0041] The in vivo histocompatibility of the nine disulfide crosslinked hyaluronic acid gels prepared in Example 1 was evaluated with reference to the standards of ISO10993.6-2007. The disulfide-linked cross-linked hyaluronic acid content of these 9 gels were 3 mg/mL, 6 mg/mL, and 8 mg/mL, respectively, and these nine gels were prepared by hyaluronic acid thiolated derivatives with a thiol content of 24 μmol/g, 38 μmol/g, and 57 μmol/g, respectively. A commercially available non-crosslinked hyaluronic acid gel product was used as a control sample.

[0042] After normal disinfection of the skin of healthy SD rats, the disulfide crosslinked hyaluronic acid gels prepared in Example 1 or a control sample (0.5 mL) were implanted subcutaneously along the midline of the spine of the rats (2 cm away from the spine). Rats were sacrificed painlessly 3 days, 7 days, 10 days and 14 days after implantation. The implant and surrounding tissues were cut out for macroscopic observation. The implants and surrounding tissues were placed in 10% formalin for fixation, dehydrated from gradient alcohol, paraffin embedded, sliced, and stained with HE for histopathological observation and evaluation.

[0043] Visual observation of the gels and control samples showed slight redness and edema in the wounds of the rats 3 days after implantation, but gradually disappeared as the implantation time increased. Histopathological observation showed that the tissue response of each gel group was not more than mild, similar to the control sample. The results of this test indicate that the disulfide crosslinked hyaluronic acid gels tested have good histocompatibility.

Example 5: Preparation of Disulfide Crosslinked Hyaluronic Acid Gels

[0044] Hyaluronic acid thiolated derivatives are made from sodium hyaluronate with molecular weights of 300 KDa and 1,500 KDa, and are prepared by the method reported by Wang et al (Wang et al, J Mater Chem. B, 2015, 3:7546-7553). Their thiol contents were 103 μmol/g and 75 μmol/g, respectively.

[0045] The above-mentioned hyaluronic acid thiolated derivatives were dissolved to obtain aqueous solutions with contents of 3 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL and 10 mg/mL. Adjust the pH value of the solutions to 7.4. Transfer the solutions in to glass containers and then sterilize the solutions by moist heat. The solutions were stored in a sealed container at room temperature for 4 weeks. The solutions lose fluidity and form disulfide crosslinked hyaluronic acid gels.

Example 6: Cytocompatibility Evaluation of Disulfide Crosslinked Hyaluronic Acid Gels

[0046] The in vitro cytotoxicity of the disulfide-linked cross-linked hyaluronic acid gels prepared in Example 5 were evaluated by the same method as in Example 3.

[0047] The relative proliferation rates of cells in each disulfide crosslinked hyaluronic acid gels test group were >90%, and cytotoxicity was not observed in the cells, indicating that the disulfide crosslinked hyaluronic acid gels tested had good cell compatibility.

Example 7: Evaluation of In Vivo Histocompatibility of Disulfide Crosslinked Hyaluronic Acid Gel

[0048] The in vivo tissue compatibility of the four disulfide-linked cross-linked hyaluronic acid gels prepared in Example 5 were evaluated by the same method as in Example 4; two of the gels were prepared by hyaluronic acid thiolated derivatives with a thiol content of 75 μmol/g (1,500 KDa), the other two gels were prepared by hyaluronic acid thiolated derivatives with a thiol content of 103 μmol/g (300 KDa). The contents of the disulfide crosslinked hyaluronic acid in the gels were 3 mg/mL and 4.5 mg/mL, respectively. A commercially available non-crosslinked hyaluronic acid gel product was used as a control sample.

[0049] Visual observation of the gels and control samples showed slight redness and edema in the wounds of the animals 3 days after implantation, but gradually disappeared as the implantation time increased. Histopathological observation showed that the tissue response of each gel group was not more than mild, similar to the control sample. The results of this test indicate that the disulfide crosslinked hyaluronic acid gels tested had good histocompatibility.

Example 8: Evaluation of the Effect of Disulfide Crosslinked Hyaluronic Acid Gels on Prevention of Abdominal (pelvic) Cavity Adhesions

[0050] The classic white rabbit sidewall model (John et al., Fertil Steril. 1997, 68: 37-42) was used to evaluate effects of the disulfide-linked crosslinked hyaluronic acid gels prepared in Example 1 on preventing abdominal (pelvic) cavity adhesions. The disulfide crosslinked hyaluronic acid gels used were prepared from a hyaluronic acid thiolated derivatives with a thiol content of 38 μmol/g, and the contents of the disulfide crosslinked hyaluronic acid in the gels were 3 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, and 10 mg/mL, respectively.

[0051] Healthy female white rabbits were anesthetized by intramuscular injections of a mixture of ketamine hydrochloride (55 mg/kg) and lopon (5 mg/kg), and midline laparotomies was performed on the rabbits. Remove the cecum and small intestine, apply pressure to bleed under all serosal surfaces, and then gently rub the damaged intestine with sterile gauze until spotting bleeding is observed. Return the cecum and small intestine to their normal anatomical position. Resect the peritoneums and transverse abdominis muscles with an area of 5×3 cm.sup.2 on the right side of the abdominal wall. The resected site was coated with a disulfide crosslinked hyaluronic acid gel (˜4 mL) or a physiological saline reference. The surgical incision is closed with two layers of absorbable sutures, and be careful when operating to protect the intestine from injury.

[0052] The experimental animals were sacrificed 21 days after the operation, and general observations were performed on the open abdomens. At the same time, the percentage of adhesion area and the severity of adhesions involved in side wall injuries were measured and evaluated. The seventies of adhesions were scored according to the quartile method: 0=no adhesions occur; 1=mild adhesions (adhesion that is easy to peel off); 2=moderate adhesions (not peelable, do not tear organs); 3=dense adhesion (not peelable, tearing organs while moving).

[0053] Macroscopic observation did not reveal any symptoms of chronic inflammation and granulomatous, indicating that the disulfide crosslinked hyaluronic acid gels tested had good histocompatibility. At the same time, no gel residue was found, indicating that the disulfide crosslinked hyaluronic acid gels tested were completely degraded and absorbed.

[0054] The evaluation results are shown in FIG. 2 (percentage of adhesion area) and FIG. 3 (severity of adhesion). The disulfide crosslinked hyaluronic acid gels tested all had adhesion prevention effects, and the gels with contents of 3 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, and 8 mg/mL have better adhesion prevention effect (less adhesion area and lower severity of adhesion) than the gel with contents of 10 mg/mL.

Example 9: Evaluation of the Effect of Disulfide Crosslinked Hyaluronic Acid Gel on Prevention of Abdominal (Pelvic) Cavity Adhesions

[0055] The classic white rabbit horn model (John et al., FertilSteril. 1997, 68: 37-42) was used to evaluate the disulfide-linked crosslinked hyaluronic acid gel prepared in Example 1 to prevent abdominal (pelvic) cavity adhesions effect. The disulfide crosslinked hyaluronic acid gels used were prepared from hyaluronic acid thiolated derivatives with a thiol content of 57 μmol/g, and the contents of the disulfide crosslinked hyaluronic acid in the gels were 3 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, 8 mg/mL, and 10 mg/mL, respectively.

[0056] Healthy female white rabbits were anesthetized by intramuscular injections of 3% sodium pentobarbital (3 mg/kg), and the abdominal cavity was opened along the ventral white line to expose the bilateral uterus and fallopian tubes, and the uterine horn was positioned. The diameter of the uterine horn is measured and recorded. Only white rabbits with a uterine horn diameter of 3 mm or more can continue the test. Using a No. 10 surgical blade, start at the uterine horn, and scrape about 20 times within a range of 1 cm from the fallopian tube and 4 cm from the uterine body until intermittent bleeding on the serous surface can be spotted with naked eyes. Put the scratched uterus and fallopian tubes back to their original natural anatomical sites, and apply 2.5 ml of disulfide-linked crosslinked hyaluronic acid gels or normal saline controls on each side of the injuries. Before closing the abdomen, use a syringe to inject 5 mL of disulfide-linked cross-linked hyaluronic acid gel or saline into the abdominal cavity from the tail end of the abdominal wall incision.

[0057] The test animals were sacrificed two weeks after the operation, and the abdominal cavities were opened to observe the positions and appearances of the main organs, as well as the general conditions such as peritoneal effusion and residual test samples. Expose the uterus and fallopian tubes, determine the adhesion of the uterine fallopian tubes or the surrounding organs, determine the length of each adhesion, and add the adhesion lengths of the uterine fallopian tubes on both sides as the postoperative adhesion length of the animal.

[0058] Rough observation did not reveal any adverse reactions, indicating that the disulfide crosslinked hyaluronic acid gels tested had good histocompatibility. At the same time, no gel residue was found, indicating that the disulfide crosslinked hyaluronic acid gels tested were completely degraded and absorbed.

[0059] See FIG. 4 for the evaluation results. The disulfide crosslinked hyaluronic acid gels tested all had adhesion prevention effects, and the gels with contents of 3 mg/mL, 4 mg/mL, 4.5 mg/mL, 5 mg/mL, 6 mg/mL, 7 mg/mL, and 8 mg/mL had a better adhesion prevention effect (lower adhesion length) than the gel with content of 10 mg/mL.

[0060] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the examples. Any other changes, modifications, combinations, substitutions and simplifications made without departing from the spirit and principle of the present invention should be equivalent replacements, and all are included in the protection scope of the present invention.