CN111973550B - Nitric oxide in situ hydrogel for treating osteoarthritis - Google Patents

Abstract

The nitric oxide in-situ hydrogel for treating osteoarthritis of the present invention comprises a hydrogel matrix and nitric oxide nanobubbles, and the nitric oxide nanobubbles are uniformly dispersed in the hydrogel matrix. The hydrogel matrix is composed of catechol-terminated polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer, heparin-poloxamer and acidic fibroblast growth factor, and catechol-terminated polyoxyethylene The mass ratio of ethylene-polyoxypropylene-polyoxyethylene copolymer to heparin-poloxamer is 1-5:10, and the molar masses of acidic fibroblast growth factor and heparin-poloxamer are equal. Nitric oxide nanobubbles are composed of egg yolk phospholipids and poloxamers as vesicles wrapped with nitric oxide gas. The mass ratio of egg yolk phospholipids and poloxamers is 1:25, and the particle size ranges from 500 to 900nm. The concentration is 1×10 ⁷ ~5×10 ⁷ cells/mL. The nitric oxide in situ hydrogel of the present invention is injected into the joint cavity for treating osteoarthritis.

Description

Nitric oxide in situ hydrogel for the treatment of osteoarthritis

technical field

The invention relates to an in-situ preparation for treating osteoarthritis, in particular to a nitric oxide in-situ hydrogel for treating osteoarthritis.

Background technique

Osteoarthritis (OA) is a common chronic progressive arthritis, and its pathological features are degeneration and destruction of articular cartilage and subchondral bone sclerosis. Reactive hyperplasia of joint margins and subchondral bone, osteophyte formation. Osteoarthritis is more common in the elderly. Epidemiological surveys show that its incidence rate is 40% among people aged 55-64, and 60% among people over 65 years old. With the acceleration of the aging of the world population and the With the prosperity of sports, the incidence of osteoarthritis is also increasing year by year. Osteoarthritis is a chronic degenerative disease characterized by cartilage degeneration accompanied by synovial tissue inflammation. Osteoarthritis patients suffer from pain and joint deformation, and severe cases lead to physical disability, which reduces the quality of life of the elderly and brings both physical and mental pain, which has attracted widespread attention.

Studies have found that nitric oxide has a good therapeutic effect on osteoarthritis. Compared with traditional non-steroidal anti-inflammatory drugs, nitric oxide-donating non-steroidal anti-inflammatory drugs have fewer adverse reactions, and have become the main method of nitric oxide in the treatment of osteoarthritis. 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. Due to the "first-pass effect" in the traditional oral administration route of nitric oxide-donating non-steroidal anti-inflammatory drugs, the concentration of nitric oxide produced to reach the joint lesion is very low, and it is difficult to maintain. However, the nitric oxide-donating NSAIDs injected into the joint cavity cannot produce nitric oxide due to the lack of degrading enzymes. Therefore, the traditional intra-articular injection of nitric oxide-donating NSAIDs cannot inhibit the degeneration of articular cartilage.

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). A pharmaceutical form that exerts the therapeutic effect on osteoarthritis.

In situ hydrogel is a kind of preparation that can undergo a phase transition immediately at the drug site after administration in a solution state, and form a non-chemically cross-linked semi-solid hydrogel from a liquid state. The hydrogel 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 hydrogels can be divided into temperature-sensitive, pH-sensitive, and ion-sensitive types. As a new type of pharmaceutical dosage form, in situ hydrogel is widely used in new drug delivery systems such as sustained release, controlled release and pulse release. In situ hydrogel can be applied to skin, eyes, nasal cavity, oral cavity, vagina, Rectal and other routes of administration have become a research hotspot in the fields of pharmacy and biotechnology.

Although in situ hydrogel has many advantages in joint cavity drug delivery and local treatment, nitric oxide is a gas, and there is no research report on how to efficiently carry nitric oxide gas in in situ hydrogel . Therefore, the preparation of in situ hydrogels that can efficiently carry nitric oxide gas is a limiting bottleneck for exerting nitric oxide's therapeutic effect on osteoarthritis.

Contents of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art (that is: lack of in-situ hydrogels that efficiently carry nitric oxide gas) to provide a nitric oxide in-situ hydrogel that can act locally in the joint cavity, for Guarantee the effective concentration of nitric oxide and the treatment effect of osteoarthritis to provide the greatest guarantee, and at the same time meet the requirements of safety, effectiveness, convenience and economy of clinical treatment.

Through a large number of experiments, the inventors have obtained a nitric oxide in situ hydrogel that can act locally in the joint cavity. The hydrogel includes a hydrogel matrix and nitric oxide nanobubbles, and the nitric oxide nanobubbles are uniform. dispersed in the hydrogel matrix.

The above hydrogel matrix is composed of catechol-terminated polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer, heparin-poloxamer and acidic fibroblast growth factor, and catechol-terminated The mass ratio of the polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer to the heparin-poloxamer is 1-5:10, and the molar masses of the acidic fibroblast growth factor and the heparin-poloxamer are equal.

The preferred mass ratio of catechol-terminated polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer, heparin-poloxamer and acidic fibroblast growth factor in the above hydrogel matrix is 2 to 3: 10. The molar masses of acidic fibroblast growth factor and heparin-poloxamer are equal.

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

The above nitric oxide nanobubbles are composed of egg yolk phospholipids and poloxamers as bubble membrane materials to wrap nitric oxide gas into vesicles, and the mass ratio of egg yolk phospholipids and poloxamers is 1:25.

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

The concentration of the nitric oxide nanobubbles in the nitric oxide in situ hydrogel is 1×10 ⁷ -5×10 ⁷ /mL.

A method for preparing nitric oxide in-situ hydrogel for treating osteoarthritis, comprising the following steps:

a: Disperse polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer and heparin-poloxamer in 6 times the mass of 8°C water for injection, place in a refrigerator at 4-8°C overnight, slowly dissolve, add acid Fibroblast growth factor, 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, 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 1×10 ⁷ to 5×10 ⁷ cells/mL, shake gently to mix well to prepare nitric oxide in situ hydrogel solution, and store in a sealed environment at 10°C;

d: The nitric oxide in situ hydrogel solution prepared in the above step c is exposed to an environment of 30-37° C., and rapidly gels to form a nitric oxide in situ hydrogel.

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

The nitric oxide in situ hydrogel mentioned above is injected into the joint cavity for the treatment of osteoarthritis.

The nitric oxide in situ hydrogel for treating osteoarthritis of the present invention has the following advantages: ① The in situ hydrogel has bioadhesion and sustained-release long-acting effects, and has good affinity and Biocompatibility; ②The hydrogel does not use any nitric oxide donor compound, and will not cause adverse reactions and toxic side effects on body tissues due to the nitric oxide donor compound; ③The in-situ hydrogel is easy to use, and the original Immediately after in-situ injection, it will be in the state of hydrogel, which meets the needs of controlled-release and long-acting treatment for osteoarthritis; ④The storage and transportation of the in-situ hydrogel are convenient.

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 nitric oxide in situ hydrogel

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

a: Disperse polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer and heparin-poloxamer in 6 times the mass of 8°C water for injection, place it in a refrigerator at 4-8°C overnight, dissolve slowly, and add Acidic fibroblast growth factor with equal molar mass of heparin-poloxamer, 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 , gently shake and mix to prepare a nitric oxide in situ hydrogel solution, and store in a sealed environment at 10° C. to obtain a nitric oxide in situ 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 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; KGF-2 means keratinocyte growth Factor -2; NO stands for nitric oxide gas; _O2 stands for oxygen gas; _N2 stands for nitrogen gas.

Example 2 Application Effect of Nitric Oxide In Situ Hydrogel for Treating Osteoarthritis

(1) Establishment of model animals

Taking the Diannan small-ear pig as the research object, 20% papain (papain) was injected into the right knee joint of the adult Diannan small-ear pig. After 4 weeks, the articular cartilage degeneration, osteoblast activity, and osteophyte formation were evaluated by histology. , confirm that the modeling is successful.

(2) The application effect of nitric oxide in situ hydrogel in the treatment of osteoarthritis

The adult Diannan mini-ear pigs with right knee osteoarthritis successfully modeled were selected and divided into several groups according to the design in Table 1. They were injected with 2 mL of nitric oxide in situ hydrogel into the right knee joint cavity, reared routinely, and sacrificed after 2 months. , through histological staining, the pathophysiological conditions of the cartilage were observed, and the effect of each group of hydrogels on osteoarthritis 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 hydrogel in the treatment of osteoarthritis in the experimental group and the control group

From the experimental results in Table 2, it can be seen that the nitric oxide in situ hydrogel in the experimental group has a good therapeutic effect on osteoarthritis, especially in the experimental group 6, the cartilage at the site of osteoarthritis recovered very well. Compared with the experimental group, the therapeutic effect of the control group on osteoarthritis is significantly poorer, especially the control group 1, 2, 3, 14 and 15, which proves that lacking any component and condition in the technical protection scheme of the present invention will cause It has a significant impact on the effect of cartilage repair. The results in Table 2 prove that the nitric oxide in situ hydrogel of the present invention can obviously repair cartilage and treat osteoarthritis, and has a good application prospect.

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 (7)

1. Nitric oxide in situ hydrogel for joint cavity injection for the treatment of osteoarthritis, characterized in that: the nitric oxide in situ hydrogel includes a hydrogel matrix and nitric oxide nanobubbles, the nitric oxide The nitrogen nanobubbles are uniformly dispersed in the hydrogel matrix; the hydrogel matrix is composed of catechol-terminated polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer, heparin-poloxamer and acid Composition of fibroblast growth factor, the mass ratio of catechol-terminated polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer and heparin-poloxamer is 1-5:10, acidic fibroblast growth factor The molar mass is equal to that of heparin-poloxamer; the nitric oxide nanobubbles are composed of egg yolk phospholipids and poloxamers as bubble membrane materials to wrap nitric oxide gas into vesicles, and the egg yolk phospholipids and poloxamers The mass ratio is 1:25; the particle size range of the nitric oxide nanobubbles is 500-900nm; the concentration of the nitric oxide nanobubbles in the nitric oxide in-situ hydrogel is 1×10 ⁷ ~ 5×10 ⁷ cells/mL. 2. The hydrogel according to claim 1, characterized in that: polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer, heparin-polyoxyethylene copolymer, heparin-polyoxyethylene, catechol-terminated in the hydrogel matrix The mass ratio of the shamer is 2-3:10, and the molar masses of the acidic fibroblast growth factor and the heparin-poloxamer are equal. 3. The hydrogel according to claim 1, characterized in that: sugar, electrolyte salt, amino acid, pH buffer and antioxidant are further added to the hydrogel matrix. 4. The hydrogel according to claim 1, characterized in that: the particle diameter of the nitric oxide nanobubbles is 700nm. 5. The hydrogel according to claim 1, characterized in that: the concentration of the nitric oxide nanobubbles in the nitric oxide in situ hydrogel is 3×10 ⁷ /mL. 6. A preparation method of the hydrogel according to claim 1, characterized in that: comprising the following steps: a: Disperse catechol-terminated polyoxyethylene-polyoxypropylene-polyoxyethylene copolymer and heparin-poloxamer in 6 times the mass of water for injection at 8°C, and place it in a refrigerator at 4-8°C Overnight, dissolve slowly, add acidic fibroblast growth factor, and mix well 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 until the concentration of nitric oxide nanobubbles in the hydrogel is 1×10 ⁷ to 5×10 ⁷ cells/mL, shake gently to mix well to prepare nitric oxide in situ hydrogel solution, and store in a sealed environment at 10°C; d: The nitric oxide in situ hydrogel solution prepared in step c is exposed to an environment of 30-37° C., and rapidly gels to form a nitric oxide in situ hydrogel. 7. The hydrogel preparation method according to claim 6, characterized in that: sugar, electrolyte salt, amino acid, pH buffer and antioxidant are further added to the hydrogel matrix solution.