CN117736465A - Injectable Hemoadhican polysaccharide hydrogel, preparation method and application thereof - Google Patents

Abstract

The invention discloses an injectable hemochitosan hydrogel, a preparation method and application thereof. According to the method, the Healdican polysaccharide is dissolved in NaOH solution, then a cross-linking agent polyethylene glycol diglycidyl ether is added, and the Healdican polysaccharide hydrogel is prepared through chemical cross-linking. The Healadhican polysaccharide hydrogel provided by the invention has good biocompatibility and biodegradability, can be injected into an operation site through a syringe, has good protein adsorption resistance and cell adhesion resistance, and has a wide application prospect in the field of preventing postoperative tissue adhesion.

Description

Injectable Hemoadhican polysaccharide hydrogel, preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and relates to an injectable Hemoadhican (HD) polysaccharide hydrogel, a preparation method and application thereof.

Background

The hydrogel has the characteristics of high moisture retention, soft mechanical property, good biocompatibility, excellent customization, physical similarity of biological tissues and the like, and is widely applied to the biomedical fields such as medicines and cell delivery carriers, biological adhesives, regenerated stents, engineering tissues, wound dressings, postoperative anti-adhesion barriers and the like.

The applicant' S prior chinese patent CN115197866B and its published paper (Kong C, chen S, wang X, et al, heatdhican, a Tissue Adhesion Hemostatic Material Independent of Blood conjugation, adv health mater.2023apr7:e2300705.) disclose a novel polysaccharide heatdhican and its preparation method, which has defined molecular weight, superior hemostatic function, good hydrophilicity, biodegradability and biocompatibility, and is an ideal material for preparing hydrogels for biomedical applications.

Postoperative tissue adhesions are a problem that medical science is urgent to address, and postoperative abdominal adhesions are a more common type of these. Abdominal adhesion refers to an abnormal adhesion phenomenon that occurs in the abdominal cavity, i.e., adhesion between different tissues or organs in the abdominal cavity. These tissues or organs should be free to move and slide, but adhere together due to tissue lesions caused by inflammation, surgery, trauma, etc. and abnormal scarring during repair. Abdominal adhesions can cause various discomfort and complications to the patient, including, for example, abdominal pain, digestive problems, intestinal obstruction, fertility problems, and the like.

Currently, the mainstream treatment of postoperative adhesions is still open surgery or laparoscopy. However, invasive and traumatic adhesion loosening is inevitably accompanied by a higher risk of recurrence of peritoneal adhesions, resulting in treatment failure. Accordingly, researchers have been trying to develop a non-surgical therapeutic strategy to completely prevent postoperative peritoneal adhesions and recurrence of peritoneal adhesions caused by adhesion loosening. Clinically, the most common non-surgical methods for preventing post-operative adhesions are mainly drug therapy and biomaterial barrier administration. However, topical or systemic medications, including anti-inflammatory agents and anticoagulants, have been demonstrated to be rapidly metabolized in the peritoneal cavity, which greatly reduces its prophylactic effect. Furthermore, existing biomaterial barrier products used in clinic show limited efficacy due to inherent drawbacks. For example, artificial membrane barrier products such as interseed and Seprafilm do not completely cover irregularly damaged wounds, and are inconvenient in practice. Injectable polymer solution barriers such as Adept (icodextrin solution) have only a short retention time in local peritoneal wounds. Therefore, there is an urgent need to develop a novel biomaterial with better performance and better anti-blocking effect.

Disclosure of Invention

The invention aims to provide an injectable Hemoadhican polysaccharide hydrogel which is degradable, excellent in mechanical property, biocompatibility and biodegradability, a preparation method and application thereof in preparation of surgical tissue adhesion prevention dressing.

The technical scheme for realizing the purpose of the invention is as follows:

the preparation method of the injectable hemochitosan polysaccharide hydrogel comprises the following steps:

(1) Dissolving HD polysaccharide in NaOH solution, and stirring at room temperature to obtain HD polysaccharide solution;

(2) Adding polyethylene glycol diglycidyl ether into the HD polysaccharide solution, stirring at room temperature, and performing chemical crosslinking reaction to obtain HD polysaccharide hydrogel;

(3) Neutralizing the HD polysaccharide hydrogel with an acidic solution, and then cleaning with water to obtain a neutral HD polysaccharide hydrogel;

(4) Sterilizing the neutral HD polysaccharide hydrogel at high temperature to obtain the sterile neutral HD polysaccharide hydrogel.

Further, in the step (1), the concentration of the HD polysaccharide solution is 6-10wt%, and the concentration of the NaOH solution is 0.1mol/L.

Further, in the step (1), the stirring speed is 400-800rpm, and the stirring time is 10-20min.

Further, in the step (2), the volume ratio of the mass of the HD polysaccharide to the polyethylene glycol diglycidyl ether crosslinking agent is 1:0.8938-1:0.4489, g: mL.

Further, in the step (2), the stirring speed is 400-800rpm, and the stirring time is 20-30min.

Further, in the step (2), the crosslinking temperature is 40 ℃, and the reaction time is 10-12 hours.

Further, in the step (3), the acidic solution is a HCl solution of 0.1mol/L.

Further, in the step (3), the washing times are 8-15 times, and the content of the HD polysaccharide in the neutral HD polysaccharide hydrogel is 10mg/mL.

Further, in the step (4), the high-temperature sterilization condition is 121 ℃ for 20min.

The invention also provides the injectable Hemoadhican polysaccharide hydrogel prepared by the preparation method.

Further, the invention provides application of the injectable hemochitosan polysaccharide hydrogel in preparing a dressing for preventing postoperative tissue adhesion.

Further, the postoperative tissue adhesion prevention dressing is a postoperative abdominal tissue adhesion prevention dressing.

Compared with the prior art, the invention has the following advantages:

(1) The microbial polysaccharide chemically crosslinked injectable hemochitosan hydrogel has good biocompatibility and biodegradability, can be injected into an operation position through a syringe, is simple and convenient to operate, can better ensure that the hydrogel is in a sterile condition, and reduces infection risk.

(2) The Healadhican polysaccharide hydrogel provided by the invention has good protein adsorption resistance, can effectively avoid fibrin deposition at a wound, and has cell adhesion resistance, so that postoperative tissue adhesion is prevented, and the application prospect in postoperative tissue adhesion prevention is wide.

Drawings

FIG. 1 is a physical form of HD hydrogel before and after gel formation.

FIG. 2 is a physical diagram showing the gel formation of HD hydrogels.

FIG. 3 is a schematic diagram of the injectability of HD hydrogels, wherein (a) is a HD hydrogel shear rate scan curve, and (b) is a schematic diagram of the injection of HD-L hydrogels and HD-H hydrogels through a 27 gauge needle.

FIG. 4 is a graph showing the effect of the HD hydrogel on protein adsorption and cell adhesion resistance, wherein (a) shows the adsorption of Bovine Serum Albumin (BSA) on the HD hydrogel surface, and (b) shows the live/dead image of the adhesion of L929 cells to the HD hydrogel over time.

Fig. 5 is a graph showing the effect of HD hydrogel on prevention of peritoneal adhesions, wherein (a) is a representative macroscopic graph of each of the groups of peritoneal adhesions on the 7 th and 14 th days after surgery, and (b) is a graph of each of the groups of adhesion scores on the 7 th and 14 th days after surgery.

FIG. 6 is a graph showing the effect of biosafety of HD hydrogels, wherein (a) is a graph showing the survival rate of L929 cells cultured for 24 hours with HD hydrogel extracts at different concentrations, and (b) is a graph showing histological evaluation of major organs (heart, liver, spleen, lung, kidney) after 1 week of subcutaneous injection of HD hydrogels.

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Reagents or materials used in the examples described below may be synthesized by commercial purchase or reference to existing methods unless otherwise specified.

While the preferred embodiments of the present invention have been described in detail, it should be apparent that the described embodiments are only some, but not all embodiments of the present invention. It should be understood that numerous modifications and variations can be made in accordance with the concepts of the invention by one of ordinary skill in the art without undue burden. Therefore, all technical solutions which can be obtained by logic analysis, reasoning or limited experiments based on the prior art by the person skilled in the art according to the inventive concept shall be within the scope of protection defined by the claims.

Example 1

Preparation of injectable Healadhican polysaccharide hydrogels:

(1) Dissolving HD polysaccharide powder in NaOH water solution with concentration of 0.1mol/L, stirring at room temperature at stirring speed of 400-800rpm for 10-20min to obtain transparent solution with polysaccharide concentration of 8wt%;

(2) Adding polyethylene glycol diglycidyl ether cross-linking agent into HD polysaccharide solution according to volume ratio of 1:0.036 and 1:0.072 respectively, so that the volume ratio of the mass of HD polysaccharide to the polyethylene glycol diglycidyl ether cross-linking agent is 1:0.8938 and 1:0.4489 respectively, g: mL, stirring uniformly at room temperature under the conditions of stirring speed of 400-800rpm and stirring time of 20-30min, and performing chemical reaction cross-linking reaction at 40 ℃ for 12h to obtain HD polysaccharide hydrogel;

(3) Neutralizing the HD polysaccharide hydrogel with 0.1mol/L HCl solution, and washing with deionized water for 8-15 times to obtain neutral HD polysaccharide hydrogel and HD-H hydrogel with HD polysaccharide content of 10 mg/mL;

(4) Sterilizing the neutral HD-L hydrogel and the HD-H hydrogel at 121deg.C under high temperature and high pressure for 20min to obtain sterile neutral HD-L hydrogel and HD-H hydrogel.

Fig. 1 is a physical diagram of the morphology of the hydrogel before and after gel formation, and fig. 2 is a physical diagram of the HD-L hydrogel and the HD-H hydrogel formed by the crosslinking reaction of the HD polysaccharide.

Example 2

Determination of injectability of injectable Healadhican polysaccharide hydrogels:

the HD-L hydrogel and the HD-H hydrogel were tested sequentially and the injectability of the HD hydrogel was characterized using a rheometer. The viscosity of the hydrogels was measured as a function of shear rate (0.1 to 1000 1/s) in a complex viscosity test. The HD-L hydrogel and HD-H hydrogel were then separately loaded into 1mL syringes using 27 gauge needles for injectability testing.

As shown in FIG. 3 (a), the viscosity of the HD-L and HD-H hydrogels decreased rapidly by two orders of magnitude as the shear rate increased. Injectability was verified on a macroscopic level.

As shown in fig. 3 (b), both hydrogels were successfully extruded through a 27 gauge needle and integrated together to form a viscous whole gel, further intuitively verifying the injectability of the HD hydrogels.

Example 3

Determination of anti-adsorption protein Properties and anti-cell adhesion Properties of injectable Healoadhican polysaccharide hydrogels:

(1) Determination of anti-adsorption protein Performance:

the anti-adsorption protein performance test uses bovine serum albumin as a model protein, and tests the adsorption capacity of nonspecific proteins. The fully swollen HD hydrogel was soaked in BSA solution (2 mg/mL). After incubation of the hydrogel for 4 hours at 37 ℃, the hydrogel was washed 3 times with physiological saline and then soaked in 1% Sodium Dodecyl Sulfate (SDS) for 1 hour, releasing the adsorbed protein. Protein concentration in the eluate was determined using a micro BCA protein assay kit.

(2) Cell adhesion resistance assay:

in vitro experiments observed the adhesion of L929 cells to the HD hydrogel surface. After autoclaving, the HD hydrogel was cut into circles and placed on a 6-well plate. L929 cells were grown at 1X 10 ⁴ Cell density was seeded on hydrogel sample surface at 37℃with 5% CO ₂ Culturing in the environment. Tissue culture treated polystyrene (TCPS, corning) was used as a control group. L929 cells were stained with calcein-amp at predetermined time points and observed with a fluorescent microscope.

As shown in fig. 4 (a), in bovine serum albumin solution, only a few proteins were adhered to the HD hydrogel surface, which were relatively lower than the Tissue Cultured Polystyrene Surface (TCPS).

Panel (b) in FIG. 4 is a live/dead image of L929 cells adhered to the HD hydrogel over time. To investigate fibroblast adhesion, L929 cells were seeded on TCPS as a positive control and compared to cells directly seeded on the surface of HD hydrogel. Live/dead staining images were obtained under a fluorescence microscope. In the TCPS group, green fluorescence intensity gradually increased over time. This means that living cells adhere and proliferate on TCPS, while only a small number of scattered spots were observed in the HD-L and HD-H hydrogel groups. An effective anti-adhesion barrier should not adhere to other unrelated tissues. These results indicate that HD hydrogels have good enough anti-protein adsorption and anti-cell adhesion properties to be useful for preventing post-operative tissue adhesion.

Example 4

In vivo test of injectable hemoad hican polysaccharide hydrogel to prevent postoperative abdominal adhesions:

(1) Establishment of a cecal and abdominal adhesion model after a mouse operation:

the experimental animal is female C57BL/6 mice, 20-22 g. Feeding under standard experimental conditions: light for 12 hours-dark cycle for 12 hours, water and food are freely taken. C57BL/6 mice were anesthetized with inhalable isoflurane. The laparotomy was performed by cutting 1.5cm along the midline of the abdominal wall. The cecum was exposed with a sterile cotton swab and rubbed with sterile gauze until there was a visible spot. The corresponding abdominal wall of the cecum was then scraped off with a scalpel, the area of the abdominal wall defect being approximately 1cm by 1cm. The abdominal cavity was then closed by a 40-over Kang Sixian layer-by-layer suture.

(2) The injectable hemoad hican polysaccharide hydrogel prevents the postoperative cecal abdominal adhesion of mice:

in the molding process, mice are randomly divided into 5 groups including a sham operation group, a model group, HD-L, HD-H and a positive control group, wherein in the HD-L, HD-H and the positive control group, a defect area is covered by HD hydrogel or sodium Hyaluronate (HA) gel, the model group is treated by normal saline only, the sham operation group performs an open surgery, and the abdominal wall and the cecum are not damaged. The mice were injected subcutaneously with antibiotics after abdominal suturing to prevent infection. Mice were euthanized, opened, observed for extent of adhesion formation according to standard adhesion scoring system and double-blind graded, 0 point, no adhesion, at 7 and 14 days post-surgery; 1 minute, one part is thinner and adhered; 2 minutes, more than one part is adhered; 3, the adhesion is relatively tight; 4 minutes, more than one place is tightly adhered; 5 minutes, large area vascularized adhesion.

As shown in fig. 5 (a), severe adhesions were observed at the injured site on day 7 of the model group, which means that the model of the abdominal adhesions of mice was successfully established. On day 14 post-surgery, the severity of abdominal adhesions increased further. Even nearby adipose tissue and mesentery adhere firmly to the peritoneal tissue.

As shown in figure 5 (b), the commercial HA hydrogel group reduced adhesion to some extent on day 7, but was less effective after 14 days, and still found significant adhesion, possibly with intraoperative bleeding and subsequent inflammation affecting the effectiveness of the HA hydrogel. In contrast, HD-H group was completely free of adhesions and the damage caused by day 0 of modeling was recovering; on day 14, the wound on the abdominal wall had healed, with only a small amount of hydrogel remaining in the abdominal cavity. The HD-L group showed similar preventive effect on day 7, with a blocking score of 1.0 on day 14, which was better than the HA group, but slightly lower than the HD-H group.

Example 5

Evaluation of safety of injectable Healadican polysaccharide hydrogels:

(1) Cytotoxicity experiment:

in this experiment, the in vitro cytotoxicity of HD polysaccharide and HD hydrogel was evaluated by MTT method and live/dead imaging method. L929 cells were seeded in 96-well plates and cultured for 24 hours. 1ml of the hydrogel was incubated in DMEM for 48 hours, HD hydrogel extracts were prepared at 37℃and then the culture was continued for 24 hours with conventional DMEM medium containing different concentrations of HD hydrogel extracts. After MTT treatment, cell viability was measured at 570 nm.

(2) Visceral safety evaluation:

this experiment evaluates in vivo degradation and biocompatibility of HD hydrogels by subcutaneous implantation in the back of C57bL/6 mice. 200 μl of sterile hydrogel was subcutaneously injected with a 27 gauge needle to the back side of the mice. Mice were euthanized 7 days after implantation. The main organs (heart, liver, spleen, lung and kidney) were subjected to H & E staining and Masson staining to analyze whether the HD hydrogel was toxic to the internal organs of mice.

As shown in fig. 6 (a), cell viability exceeded 90% in all ranges, and these results indicate that HD hydrogels have good in vitro biocompatibility.

As shown in fig. 6 (b), no significant inflammatory reaction or tissue necrosis was observed from the H & E staining image. This further demonstrates the low in vivo toxicity of HD hydrogels, with good biocompatibility.

Claims (10)

1. The preparation method of the injectable hemochitosan polysaccharide hydrogel is characterized by comprising the following steps of:

(1) Dissolving the Healoadhican polysaccharide in a NaOH solution, and stirring at room temperature to obtain an HD polysaccharide solution;

(2) Adding polyethylene glycol diglycidyl ether into the hemochitosan polysaccharide solution, stirring at room temperature, and performing chemical crosslinking reaction to obtain a hemochitosan polysaccharide hydrogel;

(3) Neutralizing the Hemoadhican polysaccharide hydrogel with an acidic solution, and then cleaning with water to obtain a neutral HD polysaccharide hydrogel;

(4) Sterilizing the neutral Healdican polysaccharide hydrogel at high temperature to obtain sterile neutral Healdican polysaccharide hydrogel.

2. The method according to claim 1, wherein in the step (1), the concentration of the Healadhican polysaccharide solution is 6 to 10wt%, and the concentration of the NaOH solution is 0.1mol/L; the stirring speed is 400-800rpm, and the stirring time is 10-20min.

3. The method according to claim 1, wherein in the step (2), the volume ratio of the mass of the Hemoadican polysaccharide to the polyethylene glycol diglycidyl ether crosslinking agent is 1:0.8938-1:0.4489, g: mL.

4. The method according to claim 1, wherein in the step (2), the stirring speed is 400 to 800rpm and the stirring time is 20 to 30 minutes.

5. The process according to claim 1, wherein in step (2), the crosslinking temperature is 40℃and the reaction time is 10 to 12 hours.

6. The method according to claim 1, wherein in the step (3), the acidic solution is 0.1mol/L HCl solution, the number of times of washing is 8 to 15, and the content of Headhican polysaccharide in the neutral HD polysaccharide hydrogel is 10mg/mL.

7. The method according to claim 1, wherein the high-temperature sterilization condition in the step (4) is 121 ℃ for 20min.

8. The injectable headhican polysaccharide hydrogel prepared according to any one of claims 1 to 7.

9. Use of an injectable headhican polysaccharide hydrogel according to claim 8 for the preparation of a dressing for preventing post-operative tissue adhesions.

10. The use according to claim 9, wherein the postoperative tissue adhesion prevention dressing is a postoperative abdominal tissue adhesion prevention dressing.