CN111973549B - Bioadhesive hydrogel for the treatment of gynecological reproductive system inflammation - Google Patents

Abstract

The bioadhesive hydrogel for treating gynecological inflammation comprises a hydrogel matrix and nitric oxide nanobubbles, wherein the nitric oxide nanobubbles are uniformly dispersed in the hydrogel matrix. The hydrogel matrix consists of heparin-poloxamer, chitosan quaternary ammonium salt and epidermal cell growth factor, wherein the mass ratio of the heparin-poloxamer to the chitosan quaternary ammonium salt is 1-5:10, and the molar mass of the epidermal cell growth factor is equal to 50% of that of the heparin-poloxamer. The nanometer nitric oxide bubble consists of yolk phospholipid and poloxamer as bubble membrane material and the yolk phospholipid and poloxamer are coated with nitric oxide gas in the weight ratio of 1 to 25, particle size of 500-900 nm and concentration of 6 x 10 ⁷ ～9×10 ⁷ And each mL. The hydrogel is infused into the genital cavity and is used for treating gynecological genital system inflammation.

Description

Bioadhesive hydrogel for the treatment of gynecological reproductive system inflammation

technical field

The invention relates to a pharmaceutical preparation for treating gynecological reproductive system inflammation, in particular to a bioadhesive hydrogel for treating gynecological reproductive system inflammation.

Background technique

Gynecological reproductive system inflammation is the most common gynecological disease, such as vaginitis, endometritis, etc., can occur in all age groups, and its harm is very serious, such as affecting the quality of life and causing female infertility. After women suffer from gynecological reproductive system inflammation, they will have symptoms such as increased leucorrhea and genital itching, which seriously affect women's normal life. If a woman suffers from gynecological reproductive system inflammation during pregnancy, it will have a great impact on fetal development. Especially during childbirth, the disease may be transmitted to the fetus and affect the health of the fetus.

Studies have found that nitric oxide has a good therapeutic effect on inflammation. Through inflammation model animal experiments, it is found that in the early stage of inflammation, nitric oxide mainly plays an anti-inflammatory role, and its specific roles include: maintaining the integrity of microcirculation, relaxing vascular smooth muscle, increasing intestinal mucosal blood flow, and inhibiting the adhesion of platelets and leukocytes to the surface of endothelial cells and accumulation, prevent thrombosis, inhibit myeloperoxidase activity, protect the epithelial barrier and promote the repair of epithelial tissue. But in the later stage of inflammation, accompanied by the generation of superoxide anion oxygen, nitric oxide can synthesize with anion oxygen to produce peroxynitrite, and its biological oxidation will cause damage to normal tissues.

Nitric oxide plays a dual role in the development of gynecological reproductive system inflammation, which not only protects the mucous membrane of the reproductive system, but also kills sperm to cause infertility. In the hypoxic reproductive cavity environment, the nitric oxide molecule mainly plays an anti-inflammatory and protective role. Most of the reported methods for supplementing nitric oxide in the body are nitric oxide donor compounds. Nitric oxide donor compounds fall into two categories: non-enzymatic and enzymatic. Most of the non-enzymatic nitric oxide donor compounds come from nitro compounds, including nitroprusside, organic or inorganic nitrite and nitrate, nitrosamines, nitrogen mustard, hydrazine, etc., with small doses and large toxic and side effects. The enzymatic nitric oxide donor compound (such as arginine) needs to be decomposed by biological enzymes in the body to produce nitric oxide molecules. The nitric oxide molecules produced by the above nitric oxide donor compounds cannot be concentrated in the genital cavity to take effect, so the effect of using nitric oxide donor compounds to treat reproductive system inflammation is very poor.

The nitric oxide molecule is in the form of a gas. Nitric oxide gas (NO) is slightly soluble in water, and its solubility in water at 20°C is only 5.6×10 ^-3 g/L (equivalent to 0.186 μmol/L), which cannot be made into common dosage forms. , Play the anti-inflammatory effect of the reproductive system.

In situ gel is a kind of preparation that can undergo a phase transition immediately at the site of administration after administration in a solution state, and transform from a liquid state to a non-chemically cross-linked semi-solid gel. The gel has good tissue compatibility and has a long residence time at the administration site; at the same time, it can store the drug and prevent the drug from being affected by the environment. According to different formation mechanisms, in situ gels can be divided into temperature-sensitive, pH-sensitive, and ion-sensitive types. As a new type of drug dosage form, in-situ gel is widely used in new drug delivery systems such as sustained release, controlled release and pulse release. In-situ gel can be applied to skin, eyes, nasal cavity, oral cavity, vagina, rectum, etc. Multiple routes of drug delivery have become a research hotspot in the fields of pharmacy and biotechnology.

Although the in situ gel has many advantages in the administration of the genital tract and local treatment, nitric oxide is a gas, and there are no research reports on the efficient carrying of nitric oxide gas in the in situ gel. Therefore, the preparation of in situ gels that efficiently carry nitric oxide gas is the limiting bottleneck for nitric oxide to treat reproductive system inflammation.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art (i.e. lack of in-situ gels that efficiently carry nitric oxide gas) to provide a drug gel that works locally in the reproductive tract, providing a guarantee for the treatment of reproductive system inflammation. The greatest guarantee, while meeting the safety, effectiveness, convenience and economic requirements of clinical treatment.

The present inventors found that epidermal growth factor and nitric oxide molecules can produce synergistic effects in the treatment of reproductive system inflammation. Egg yolk phospholipids and poloxamers are used as bubble membrane materials to wrap nitric oxide gas into nitric oxide nanobubbles, which can convert nitric oxide gas into liquid. Heparin-poloxamer not only has good thermosensitive hydrogel properties, but also has high affinity of egg yolk phospholipid and high carrying capacity of epidermal growth factor. Both heparin-poloxamer and chitosan have the functions of antibacterial and anti-inflammatory, improving immunity, stopping bleeding and relieving pain, promoting repair and regeneration of damaged tissue, and inhibiting scar formation. However, there is no research report on how to organically combine these favorable factors such as "nitric oxide, epidermal growth factor, heparin-poloxamer and chitosan" to exert a synergistic therapeutic effect on reproductive system inflammation.

Through a large number of experiments, the inventors have obtained a bioadhesive hydrogel that can act locally in the reproductive tract to treat inflammation of the gynecological reproductive system. The hydrogel includes a hydrogel matrix and nitric oxide nanobubbles. The nitric oxide nanobubbles are uniformly dispersed in the hydrogel matrix.

The above-mentioned hydrogel matrix is composed of heparin-poloxamer, chitosan quaternary ammonium salt and epidermal growth factor, and the mass ratio of heparin-poloxamer and chitosan quaternary ammonium salt is 1-5:10, EGF is equal to 50% molar mass of heparin-poloxamer.

The preferred mass ratio of heparin-poloxamer and chitosan quaternary ammonium salt in the above hydrogel matrix is 2-3:10.

The above hydrogel matrix is further added: sugar, electrolyte salt, amino acid, pH buffer and antioxidant.

The above-mentioned nitric oxide nano bubbles are composed of egg yolk phospholipid and poloxamer as bubble membrane materials wrapped with nitric oxide gas to form vesicles.

The particle size range of the nitric oxide nanobubbles is 500-900nm.

The concentration of the above nitric oxide nanobubbles in the nitric oxide in situ hydrogel is 6×10 ⁷ -9×10 ⁷ /mL.

A method for preparing the above-mentioned bioadhesive hydrogel for treating gynecological reproductive system inflammation is characterized by comprising the following steps:

a: Disperse heparin-poloxamer and chitosan quaternary ammonium salt in 6 times the mass of 8°C water for injection, place in a refrigerator at 4-8°C overnight, slowly dissolve, add epidermal growth factor, mix well, forming a hydrogel matrix solution;

b: Take egg yolk phospholipids and poloxamer with a mass ratio of 1:25, mix them, dissolve them in anhydrous tert-butanol 10 times the mass at 65°C, slowly cool down until the solution solidifies, let stand overnight at -10°C, and freeze-dry to obtain The loose freeze-dried powder is transferred into a stoppered bottle, filled with nitric oxide gas to saturation, added to water for injection with 5 times the weight of the freeze-dried powder, and mixed to form a nitric oxide nanobubble solution;

c: Add the nitric oxide nanobubble solution prepared in step b above to the hydrogel matrix solution prepared in step a at 10°C until the concentration of nitric oxide nanobubbles in the hydrogel is 6×10 ⁷ to 9×10 ⁷ cells/mL, shake and mix well to prepare a bioadhesive hydrogel solution for the treatment of gynecological reproductive system inflammation, and store in a sealed environment at 10°C;

d: The bioadhesive hydrogel solution prepared in step c for treating gynecological reproductive system inflammation is exposed to an environment of 30-37° C., and rapidly gels to form a semi-solid hydrogel.

The method for preparing bioadhesive hydrogel for treating gynecological reproductive system inflammation is characterized in that: sugar, electrolyte salt, amino acid, pH buffer and antioxidant are further added to the hydrogel matrix.

The above-mentioned hydrogel is perfused into the reproductive cavity, and is used for treating inflammation of the gynecological reproductive system.

The bioadhesive hydrogel for the treatment of gynecological reproductive system inflammation of the present invention has the following advantages: ① the favorable components of "nitric oxide, epidermal growth factor, heparin-poloxamer and chitosan" are organically combined to exert Synergistic therapeutic effect on reproductive system inflammation; ②has bioadhesion and sustained-release long-acting effects, and has good affinity and biocompatibility to the epithelial mucosa of reproductive tract; ③does not use any nitric oxide donor compound, does not It will cause adverse reactions and toxic side effects to body tissues due to nitric oxide donor compounds; ④Easy to use, it will be in a gel state immediately after infusion into the reproductive cavity, and achieve the long-term treatment of gynecological reproductive system inflammation.

Detailed ways

Specific embodiments of the present invention will be described in detail below. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be regarded as isolated, and they can be combined with each other to achieve better technical effects.

Example 1 Preparation of Bioadhesive Hydrogel for Treating Gynecological Reproductive System Inflammation

According to the component ratios in Table 1, the bioadhesive hydrogel of the experimental group was prepared, which specifically included the following steps:

a: Disperse heparin-poloxamer and chitosan quaternary ammonium salt in 6 times the mass of 8°C water for injection, place it in a refrigerator at 4-8°C overnight, dissolve slowly, add heparin-poloxamer 50% epidermal growth factor with equal molar mass, mixed to form a hydrogel matrix solution;

b: Take egg yolk phospholipids and poloxamer with a mass ratio of 1:25, mix them, dissolve them in anhydrous tert-butanol 10 times the mass at 65°C, slowly cool down until the solution solidifies, let stand overnight at -10°C, and freeze-dry to obtain The loose freeze-dried powder is transferred into a stoppered bottle, filled with nitric oxide gas to saturation, added to water for injection with 5 times the weight of the freeze-dried powder, and mixed to form a nitric oxide nanobubble solution;

c: Add the nitric oxide nanobubble solution prepared in step b above to the hydrogel matrix solution prepared in step a at 10°C, and adjust the concentration of nitric oxide nanobubbles in the hydrogel according to the design in Table 1 , and gently shake and mix to prepare a nitric oxide in situ hydrogel solution, which is stored in a sealed environment at 10° C. to obtain a bioadhesive hydrogel.

According to the component ratios in Table 1, the hydrogel of the control group was prepared with reference to the experimental group. Each experimental group is configured according to the components and proportions within the protection scope of the claims of the present application, while each control group is missing a certain component or the mass percentage of components exceeds the protection scope of the claims of the present application.

Table 1 Composition of bioadhesive hydrogels in experimental group and control group

Note: "√" means that the item is prepared according to the concentration and method of the experimental group in Example 1; "/" means that the item does not exist; * means that the component of this item is replaced by the component in brackets; TGF-α means transforming growth factor -α; NO stands for nitric oxide gas; _O2 stands for oxygen; _N2 stands for nitrogen gas.

Example 2 Application effect of bioadhesive hydrogel for treating gynecological reproductive system inflammation

(1) Establishment of reproductive system inflammation model animals

Reference [Construction of Rat Reproductive Tract Inflammation Animal Model. Sichuan Journal of Physiological Sciences, 2007, 29(001): 1-3], using phenol mucilage to construct a rat vaginitis animal model. The method is briefly described as follows: remove the ovaries of bilateral rats, inject estradiol benzoate and hydrocortisone to cause pseudoestrus and systemic immune dysfunction, and then inject 0.1 mL of phenol glue into the vagina of each mouse, once every other day for a total of 3 days. Every day, observe and record the changes of the vulva. After weighing on the 15th day after the operation, blood was collected from the eyeball, and the total number of white blood cells was determined by microscopic counting. When the total number of white blood cells in rats is significantly increased, and the vaginal opening is red and swollen with purulent discharge, it is determined that the modeling is successful.

(2) The effect of each group of hydrogels in the treatment of vaginitis

Rats with successful vaginitis modeling were selected and divided into several groups on average according to the design in Table 1. On day 1, 3, 5, 7, and 9, 100 μL of hydrogel was instilled into the vagina, and they were reared routinely. Changes in the vulva were recorded on day 14, and blood was collected from the eyeballs. The total number of leukocytes was measured by microscopic counting method, the neck was cut off, and the vaginal tissue was taken for pathological examination. Through histological staining, the pathophysiological conditions of the vagina were observed, especially whether there was degeneration and necrosis of the epithelial cells of the vaginal mucosa, whether there were ulcers in the vagina, etc., and the effect of each group of hydrogels on vaginitis was evaluated.

The above evaluation results of each group are shown in Table 2. Based on each evaluation index, the total score of the application effect of each group of hydrogel is given.

Table 2 The effect of bioadhesive hydrogel in the treatment of vaginitis in the experimental group and the control group

It can be seen from the experimental results in Table 2 that the bioadhesive hydrogel of the experimental group has a good therapeutic effect on vaginitis, especially in the experimental group 6, the vulva of the rats was normal in shape, the total number of white blood cells was close to the normal value, the epithelial cells of the vaginal mucosa were close to normal, and the vaginal epithelial cells were close to normal. The treatment of inflammation is very good. Compared with the experimental group, the curative effect of the hydrogel in the control group on vaginitis was significantly worse, especially in the control groups 1, 2, 3, 14 and 15, the vulva of the rats was obviously red and swollen, the total number of white blood cells was significantly increased, and the epithelial cells of the vaginal mucosa Degeneration and necrosis, obvious ulceration of the vagina, and poor treatment of vaginitis.

The experimental results in table 2 prove that any component and condition in the technical protection scheme of the present invention are all synergistic and indispensable, and the lack of any component and condition in the technical protection scheme of the present invention will affect reproductive system inflammation. significantly affected the therapeutic effect. The bioadhesive hydrogel of the present invention organically combines favorable factors such as "nitric oxide, epidermal growth factor, heparin-poloxamer and chitosan" to play a synergistic therapeutic effect on gynecological reproductive system inflammation, and has good application prospects.

The above detailed description is a specific description of a feasible embodiment of the invention. This embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or change that does not deviate from the present invention shall be included in the patent scope of the present invention. In addition, those skilled in the art can also make various modifications, additions and substitutions in other forms and details within the scope and spirit disclosed in the claims of the present invention. Certainly, the various modifications, additions, substitutions and other changes made according to the spirit of the present invention shall all be included within the scope of protection claimed by the present invention.

Claims (8)

1. A bioadhesive hydrogel for treating inflammation of the gynaecological reproductive system, characterized in that: the hydrogel bagThe nitric oxide nano-bubbles are uniformly dispersed in the hydrogel matrix; the hydrogel matrix consists of heparin-poloxamer, chitosan quaternary ammonium salt and epidermal cell growth factor, wherein the mass ratio of the heparin-poloxamer to the chitosan quaternary ammonium salt is 1-5:10, and the molar mass of the epidermal cell growth factor is equal to 50% of that of the heparin-poloxamer; the nitric oxide nanobubble is composed of a vesicle formed by wrapping nitric oxide gas by taking egg yolk phospholipid and poloxamer as bubble membrane materials, wherein the mass ratio of the egg yolk phospholipid to the poloxamer is 1:25; the particle size range of the nitric oxide nanobubble is 500-900 nm; the concentration of the nitric oxide nanobubble in the nitric oxide in-situ hydrogel is 6 multiplied by 10 ⁷ ～9×10 ⁷ individual/mL; the hydrogel is infused into genital cavity and is used for treating gynecological genital system inflammation.

2. The hydrogel of claim 1, characterized in that: the mass ratio of the heparin-poloxamer to the chitosan quaternary ammonium salt in the hydrogel matrix is 2-3:10, and the molar mass of the epidermal cell growth factor is equal to 50% of that of the heparin-poloxamer.

3. The hydrogel of claim 1, characterized in that: the hydrogel matrix is further added with: sugar, electrolyte salt, amino acid, pH buffer and antioxidant.

4. The hydrogel of claim 1, characterized in that: the particle size of the nitric oxide nanobubble is 700nm.

5. The hydrogel of claim 1, characterized in that: the concentration of the nitric oxide nanobubble in the nitric oxide in-situ hydrogel is 7.5X10 ⁷ And each mL.

6. A method of preparing the hydrogel of claim 1, comprising: the method comprises the following steps:

a: dispersing heparin-poloxamer and chitosan quaternary ammonium salt in 6 times of 8 ℃ water for injection, placing the mixture in a refrigerator at 4-8 ℃ for overnight, slowly dissolving, adding epidermal cell growth factor, and uniformly mixing to form hydrogel matrix solution;

b: mixing yolk phospholipids and poloxamer in a mass ratio of 1:25, dissolving in anhydrous tertiary butanol with a mass of 10 times of that of 65 ℃, slowly cooling to solidify the solution, standing overnight at-10 ℃, freeze-drying to obtain loose freeze-dried powder, transferring into a bottle with a plug, filling nitric oxide gas to saturation, adding into water for injection with a mass of 5 times of that of the freeze-dried powder, and uniformly mixing to form a nitric oxide nanobubble solution;

c: adding the nitric oxide nanobubble solution prepared in the step b into the hydrogel matrix solution prepared in the step a at 10 ℃ until the concentration of the nitric oxide nanobubbles in the hydrogel is 6 multiplied by 10 ⁷ ～9×10 ⁷ Mixing with small amount of water at a ratio of one mL, and sealing and preserving at 10deg.C;

d: the bioadhesive hydrogel solution for treating gynecological inflammation prepared in the step c is exposed to the environment of 30-37 ℃ to rapidly gel to form semisolid hydrogel.

7. The method for producing a hydrogel according to claim 6, characterized in that: the hydrogel matrix is further added with: sugar, electrolyte salt, amino acid, pH buffer and antioxidant.

8. The method for producing a hydrogel according to claim 6, characterized in that: the hydrogel is infused into genital cavity and is used for treating gynecological genital system inflammation.