CN113877000B

CN113877000B - Microsphere composition for injection and application thereof

Info

Publication number: CN113877000B
Application number: CN202111193560.XA
Authority: CN
Inventors: 雷磊; 王丹; 李常辉
Original assignee: Kedi Biomedical Wuxi Co ltd
Current assignee: Kedi Biomedical Wuxi Co ltd
Priority date: 2021-10-13
Filing date: 2021-10-13
Publication date: 2023-01-24
Anticipated expiration: 2041-10-13
Also published as: CN113877000A

Abstract

The invention discloses a microsphere composition for injection, which comprises the following components in part by weight: a) 10-97% by weight of microspheres a containing polylactide glycolide cosmetic and/or pharmaceutical ingredients, B) 1-40% by weight of microspheres B containing a cross-linking agent and a matrix material having a melting point of 21-45 ℃, said microspheres B melting at body temperature and releasing the cross-linking agent to cause a cross-linking reaction of hyaluronic acid or a salt thereof, c) 2-50% by weight of hyaluronic acid or a salt thereof. The application method of the invention comprises the steps of fully mixing the microspheres A, the microspheres B, hyaluronic acid (salt) and isotonic physiological saline solution or phosphate buffer solution to obtain suspension which has certain viscosity and can be smoothly injected, injecting the suspension into human tissues, melting the microspheres B at the body temperature and releasing the cross-linking agent, and crosslinking the hyaluronic acid (salt) and the cross-linking agent to generate an elastomer with higher mechanical strength, so that the lubrication and filling effects can be more stably exerted, and the beauty and/or medicinal active ingredients can be slowly released to exert double effects.

Description

Microsphere composition for injection and application thereof

Technical Field

The invention relates to a microsphere composition for injection and application thereof in the fields of medical cosmetology and medicine.

Background

Osteoarthritis is a common degenerative disease, which is caused by many factors such as age-increase, obesity, strain, trauma, congenital abnormality of joints, joint deformity, joint cartilage degeneration injury, reactive hyperplasia of joint margin and subchondral bone, and is also called osteoarthropathy, degenerative arthritis, senile arthritis, hypertrophic arthritis, etc. The clinical manifestations of joint pain often occur in morning, and the pain is relieved after activities, but can be aggravated if the activities are excessive. Another symptom is joint stiffness, which often occurs when the joint gets up in the morning or after the joint remains in a certain position for a long time during the day. When the affected joint is examined, the joint may swell, press pain, and feel fricative or "clicking" during movement, and the patient with serious disease may have muscular atrophy and joint deformity.

The main treatments for osteoarthritis are to reduce the load and burden on the joints and to perform appropriate physical activity therapies. The oral administration and external application of the non-steroidal anti-inflammatory analgesic drug can also relieve or control symptoms. Chondroprotective agents such as glucosamine sulfate have symptomatic relief and improved function, while long-term administration can delay the structural progression of the disease. In the case of advanced disease, the artificial joint replacement can be performed, which can greatly improve the life quality of the patient.

Hyaluronic acid is injected into a joint sheath, namely viscoelasticity supplementation therapy is also one of the main clinical treatment methods, and exogenous hyaluronic acid is injected into a joint cavity, so that the lubricating and buffering functions of joint synovial fluid can be restored, cartilage tissue repair is promoted, OA symptoms are relieved, and the physiological functions of joints are improved.

The current active treatment methods, mainly for the treatment of osteoarthritis, provide a mechanical lubrication support by injection of a drug-free hyaluronic acid gel, and are free of therapeutic drugs. On one hand, as hyaluronic acid is easily and quickly degraded by a human body, the hyaluronic acid is short in retention time in a joint cavity and cannot play a role in providing treatment for a long time, repeated injection administration is usually required in clinic, the treatment risk is greatly increased, the compliance of a patient is low, and the treatment effect of osteoarthritis is challenged; the cross-linked hyaluronic acid gel has stronger mechanical property than the common hyaluronic acid gel, can provide greater lubrication and support for osteoarthritis, and reduces the abrasion of periosteum and the disease degree of osteoarthritis, but the cross-linked hyaluronic acid gel has no fluidity and can not be injected. On the other hand, without active treatment with drugs, osteoarthritis is difficult to treat fundamentally close to physical lubrication. Therefore, the in-situ crosslinking microsphere gel which can release the osteoarthritis treating medicine for a long time has great clinical advantages and wide application value.

Through the search of the prior art documents, the following results are found: chinese patent 201510593332.X discloses a hyaluronic acid mixed gel containing amphiphilic microspheres for injection and a preparation method thereof, wherein the microsphere material relates to amphiphilic materials such as PLLA-PEG, PLGA and PLGA-PEG. The molecular weight of PLLA or PLGA is 10,000-500,000, PEG is 1,000-10,000, PLGA, LA/GA = 90/10-10/90. The average grain diameter of the microspheres is 1-200 mu m, and the mass fraction of the microspheres in the gel is 1-50%; the gel component is hyaluronic acid or divinyl sulfone or glycidyl ether crosslinked hyaluronic acid, the molecular weight of the hyaluronic acid is 100,000-3,000,000, and the mass fraction of the hyaluronic acid is 1-50%. The gel component can also be animal-derived collagen, chitosan, amino cellulose, sodium alginate, etc. The microsphere mixed gel is prepared in sodium chloride solution or phosphate buffer solution with the osmotic pressure of 250-350 mOsm/L and the pH value of 6.5-7.5. The invention directly mixes the amphiphilic microspheres with the hyaluronic acid gel, the amphiphilic microspheres are easily and uniformly distributed in the gel, and after being injected into a human body, the amphiphilic microspheres are not easy to aggregate into blocks in human body water environment. The used cross-linked hyaluronic acid gel is cross-linked before injection, namely pre-cross-linked hyaluronic acid, the viscosity of the pre-cross-linked hyaluronic acid is ultrahigh, the injection pushing needle is difficult, and the poor fluidity in tissues easily causes uneven distribution at the injection part, so that the clinical defect exists; in addition, the microsphere does not contain medicinal components and has no effect of treating diseases. The invention has the difference that the injection contains two microspheres and non-crosslinked hyaluronate, the non-crosslinked hyaluronate gel is adopted before injection, the viscosity is low, the injection is easy to push a needle, after the injection enters tissues, the microspheres B can quickly release a crosslinking agent and a catalyst to cause the hyaluronate to be crosslinked, the hyaluronic acid is converted into the crosslinked gel with higher mechanical strength in the tissues, the crosslinked gel provides larger supporting force and longer maintaining effect for bone joints, and the microspheres A are used for slowly releasing the medicament for treating osteoarthritis, so that the long-acting release medicament is realized for treating diseases.

Chinese patent 201810348070.4 discloses a self-crosslinking injection cosmetic filling material, which consists of an agent A and an agent B, wherein the agent A and the agent B are independently stored and mixed when in injection use, and the mass ratio of the agent A to the agent B is 1; wherein, the agent A is calcium citrate microcapsule (chitosan is used as capsule wall); the agent B comprises the following components in parts by weight: 10 to 20 portions of sodium alginate, 5 to 30 portions of hyaluronic acid, 10 to 25 portions of collagen, 1 to 15 portions of vitamin C and 80 to 120 portions of normal saline. When the hyaluronic acid gel is used, the water-soluble sodium alginate is mixed with the calcium citrate sustained-release microspheres with the sustained-release function, calcium ions are slowly released in a machine body, and the calcium ions enable the sodium alginate to be self-crosslinked, so that hyaluronic acid is networked, the alginate gel and the hyaluronic acid lose rheological property, the degradation of the hyaluronic acid is effectively prevented, the filling plasticity of the machine body is stable, the shaping effect is good, the biocompatibility is good, and the defect that the use of the crosslinked hyaluronic acid easily causes biological toxicity is overcome. The filling material has a remarkable effect on filling and repairing the depression of the face part. The invention has the defects that the traditional Chinese medicine composition does not contain therapeutic drugs, cannot provide an active treatment effect for osteoarthritis, and in addition, the chitosan and the sodium alginate which are used are not materials which can be normally degraded in a human body, so that long-term retention toxicity can be generated when the traditional Chinese medicine composition is implanted into bone joints. The chitosan is used for wrapping calcium citrate, calcium ions are slowly released in vivo and are used for sodium alginate to generate self-crosslinking, and hyaluronic acid is wrapped and fixed in a calcium alginate gel network.

Chinese patent 201811491353.0 discloses a preparation method of an injectable sodium hyaluronate gel containing microspheres, which comprises the following steps: dissolving sodium hyaluronate in pure water to obtain solution A; adding microsphere materials into the solution A, and uniformly stirring to obtain uniformly dispersed suspension B; adjusting the pH value of the suspension B to obtain a suspension C; adding a cross-linking agent into the suspension C, uniformly stirring to obtain a uniformly mixed suspension D, reacting for a certain time, and then cross-linking sodium hyaluronate in the suspension D to form a colloid E containing uniformly dispersed microsphere materials; crushing the colloid E to obtain gel particles F, and passing through screens with different meshes to obtain gel particles G; removing soluble impurities and a cross-linking agent from the gel particles G in a repeated soaking or dialysis mode to obtain gel particles H; repeatedly soaking the gel particles by using a phosphate buffer solution for balancing to obtain gel particles I; the sodium hyaluronate gel is obtained through the steps of semi-dehydration, filling and sterilization. The method effectively solves the problem of the deposition nodules of the microsphere material. The invention has the defects that the invention does not contain therapeutic drugs and cannot provide active therapeutic action for osteoarthritis; in addition, the invention suspends the microspheres in the gel of the sodium alginate, then completes crosslinking in vitro to form colloid, then crushes the colloid to form gel particles, screens the gel particles, and then performs soaking dialysis and purification to obtain the gel particles, thereby solving the problem of the deposition nodules of the gel particle material.

Disclosure of Invention

Technical problem to be solved by the invention

The cross-linked sodium hyaluronate for injection which is clinically common is micro-cross-linked flowable sodium hyaluronate gel, which is convenient for injection, so that the viscosity, namely the cross-linking degree, cannot be too high. However, for patients with tissue augmentation and osteoarthritis, a higher viscosity of high strength cross-linked hyaluronic acid gel is desirable. The osteoarthritis is clinically treated by closed treatment through injecting a medicinal solution into a joint sheath, and a common injection does not have the capability of slowly releasing a medicament, so frequent injection is needed, and inconvenience and pain caused by repeated injection are brought to a patient.

Means for solving the problems

In order to solve the technical problems, the invention provides a composition for spontaneously generating crosslinked microsphere gel at an injection site, namely, a sodium hyaluronate solution with good fluidity is crosslinked with a crosslinking agent after being injected into a target tissue, so that the injection is convenient, gel with higher crosslinking degree can be obtained, and the clinical problem is solved. In addition, the invention also provides that the osteoarthritis treating drug is injected into a joint sheath together with the hyaluronic acid solution through certain formulation design, long-acting slow release is realized, two purposes are achieved at one stroke, and one-needle injection can be used for injecting gel for filling and lubricating the joint sheath and can also be used for injecting therapeutic active ingredients. In order to achieve the effect, the invention creates the following technical scheme:

the invention provides a microsphere composition for injection, which comprises:

microspheres B comprising a cross-linking agent and a matrix material having a melting point of 21-45 ℃, and hyaluronic acid or a salt thereof.

The invention also provides a microsphere composition for injection, which comprises:

a) 10-97% by weight of microspheres A, said microspheres A comprising polylactide glycolide and a cosmetically or pharmaceutically active ingredient,

b) 1-40% by weight of microspheres B, said microspheres B comprising a cross-linking agent and a matrix material having a melting point of 21-45 ℃;

c) 2 to 50% by weight of hyaluronic acid or a salt thereof.

In the composition of the present invention, the content of the microspheres a may be 20 to 80% by weight, the content of the microspheres B may be 10 to 35% by weight, and the content of hyaluronic acid or a salt thereof may be 10 to 45% by weight.

In the composition of the present invention, the melting point of the matrix material of the microspheres B is more preferably 25 to 41 ℃ and may be, for example, 25 to 38 ℃.

In the composition of the invention, the crosslinking agent is selected from the group consisting of lysine ethyl ester, adipic acid dihydrazide, 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, 1,2,7,8-dioxirane and 1,3-diepoxybutane, ferric chloride, calcium gluconate, calcium citrate, and mixtures thereof.

The composition further comprises a crosslinking catalyst, wherein the crosslinking catalyst is selected from one or more of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS) and N-hydroxythiosuccinimide (sulfo-NHS).

In the composition of the present invention, the hyaluronic acid salt is a pharmaceutically acceptable salt.

In the composition, the active ingredient is any combination of one or more of triamcinolone acetonide, hydrocortisone, dexamethasone, prednisone, cortisone, lidocaine, tetracaine, vitamin C, vitamin E, tranexamic acid or pharmaceutically acceptable salts thereof.

In the composition of the present invention, the matrix material is C _10-35 Fatty acid esters of (a).

In the composition of the invention, the matrix material is selected from: methyl palmitate, methyl heptadecanoate, methyl octadecanoate, ethyl heptadecanoate, ethyl octadecanoate, ethyl nonadecanoate, ethyl eicosanoate, propyl octadecanoate, propyl nonadecanoate, propyl eicosanoate, butyl nonadecanoate and butyl eicosanoate.

In the composition, the average grain diameter D50 of the microspheres A is 10-200 μm, and the microspheres A contain 5-70 wt% of cosmetic or medicinal active ingredients.

In the composition of the present invention, the molecular weight of the hyaluronic acid or a salt thereof is preferably 10 to 600 ten thousand, and the hyaluronic acid salt is preferably sodium hyaluronate.

In the composition of the present invention, the content of the crosslinking agent in the microspheres B is 0.1 to 30% by weight.

The invention also provides a preparation method of the composition, the microsphere A is prepared by any one of an oil/water emulsification-solvent volatilization method, a solid/oil/water emulsification-solvent volatilization method, a spray drying method, a phase separation method and a membrane filtration method, and the microsphere B is prepared by a high-temperature melting spray low-temperature solidification method.

In the preparation method of the invention, the oil/water emulsification-solvent volatilization method for preparing the microspheres A comprises the following steps: dissolving the medicine and polylactide glycolide in a dichloromethane solvent to prepare an oil phase, adding the oil phase into a polyvinyl alcohol aqueous solution containing 0.2-2% of volume while stirring, volatilizing dichloromethane, filtering, collecting microspheres, and drying to obtain the microspheres A.

In the preparation method of the invention, the high-temperature melting spraying low-temperature curing method for preparing the microsphere B comprises the following steps: heating and mixing matrix material (such as fatty acid ester) and crosslinking agent to obtain flowable homogeneous hot mixture, spraying into cold air flow at 10 deg.C or lower, rapidly cooling atomized liquid drop, and solidifying to obtain micro-particulate to obtain microsphere B.

The compositions of the present invention may be used for tissue augmentation, as well as the treatment of osteoarthritis.

The application method of the invention comprises the steps of fully mixing the microspheres A, the microspheres B, hyaluronic acid (salt) and sterile water, isotonic physiological saline solution or phosphate buffer solution to obtain suspension which has certain viscosity and can be smoothly injected into human tissues, melting the microspheres B at body temperature and releasing the cross-linking agent, and generating the elastomer with higher mechanical strength after the hyaluronic acid (salt) and the cross-linking agent are cross-linked to play roles in tissue lubrication, filling and treatment.

Advantageous technical effects

1. Compared with the prior art, the composition contains one or two microspheres with different functions and hyaluronic acid or salt thereof, and is characterized in that the microsphere B is a temperature-sensitive microsphere, is solid at the normal storage temperature (5-25 ℃) and low temperature (less than 5 ℃), is melted into liquid at body temperature after being injected into a human body, and after the microspheres are melted, the cross-linking agent encapsulated inside is quickly released to perform cross-linking reaction with surrounding hyaluronic acid or hyaluronic acid salt gel to generate cross-linked hyaluronic acid gel or hyaluronic acid salt gel with higher mechanical strength, thereby having longer lubricating effect and stronger supporting force than common joint gel. Under the action of the cross-linking agent, the microsphere gel can be bonded with surrounding tissues to a certain extent, so that the fixing effect is realized, the displacement in the tissues is prevented, and the time for retaining and acting at a target part is prolonged. The microspheres A in the composition slowly release active ingredients, and play a role in long-acting treatment of diseases (such as osteoarthritis).

2. The composition of the invention has good fluidity before injection, can smoothly pass through a stainless steel injection needle to complete injection, and the fluid injection liquid is more easily distributed in the whole body tissue (such as a cavity of a joint sheath and the like). After the injection is completed, the joint needs a stronger support. The invention has another characteristic that the crosslinking and curing of the hyaluronic acid gel are carried out after the injection is finished, and the crosslinking is carried out in situ in the cavity, thereby effectively solving the technical defect that the pre-crosslinked hyaluronic acid gel cannot be injected. Cross-linking after injection has the further advantage that the hyaluronic acid gel can flow to fill the entire cavity before cross-linking, and continue to remain filled after cross-linking.

3. Another advantage of the present invention is that the crosslinking agent and crosslinking catalyst in the composition are encapsulated in the microspheres B prior to release and do not come into contact with the sodium hyaluronate, and therefore do not cross-link the sodium hyaluronate prior to injection. After being injected into the cavity, the microspheres B are melted at the body temperature, and the cross-linking agent and the catalyst in the microspheres B can be quickly released and diffused to the whole cavity to be finally cross-linked with the hyaluronic acid gel in the cavity to obtain the high-strength elastic gel.

4. The composition can be applied to the field of skin aesthetics, such as soft tissue or skin filling, has certain filling and shaping effects, wrinkle filling and subcutaneous water replenishing effects after being injected into intradermal or subcutaneous tissues, has good collagen hyperplasia stimulating effects, filling effects and active ingredient slow-release effects, and can simultaneously occur and synergize. The composition can be applied to the medical field, for example, the composition can prolong the retention time in vivo when being used for articular cavity injection, reduce the injection times, and make up the defects that the prior medical sodium hyaluronate gel needs to be repeatedly injected and has no active ingredient to treat diseases when being used for treating osteoarthritis.

Drawings

FIG. 1 is a scanning electron micrograph of microsphere A of example 1;

FIG. 2 shows the release profile (90 days of sustained release) of methopterin released from the injection of example 1 and comparative example 4;

FIG. 3. Cross-linker release for microspheres B of examples 1 and 2 and microspheres B of comparative example 2.

Detailed Description

The microsphere composition for injection of the present invention comprises: microspheres B comprising a cross-linking agent and a matrix material having a melting point of 21-45 ℃ and hyaluronic acid or a pharmaceutically acceptable salt thereof.

The composition of the invention can further comprise microspheres A for carrying medicinal or medical and cosmetic active ingredients according to the requirements of treatment/cosmetology, the microspheres A preferably use a material with good biocompatibility, slow and controlled release performance and biodegradability as a carrying system of medical and cosmetic active ingredients, and in the invention, the inventor preferably selects polylactide glycolide (PLGA). As the PLGA, there may be used, for example, 50: 50LA.

Before the microsphere composition for injection is used, microsphere suspension which takes hyaluronic acid or salt thereof as a thickening agent and can be smoothly injected is obtained by uniformly mixing isotonic physiological saline or phosphate buffer solution, when the injection suspension is injected into a body (particularly cavities such as joint cavities, and the like, and also skin tissues, muscle tissues, connective tissues and the like), microspheres B in the composition are melted under the body temperature environment and release a crosslinking agent (or the crosslinking agent and a crosslinking catalyst) encapsulated in the microspheres B, and the crosslinking agent and the hyaluronic acid or salt thereof generate crosslinked hyaluronic acid gel with higher mechanical strength after chemical reaction at an injection part. The poly (lactide-co-glycolide) (PLGA) contained in microspheres A in the composition can slowly release active ingredients with therapeutic/cosmetic effects, and has the effect of treating diseases (such as osteoarthritis) for a long time.

The microsphere B has the temperature-sensitive performance that the microsphere B is solid at the conventional storage temperature (5-20 ℃) and the low-temperature storage temperature (less than 5 ℃) and can be melted in the temperature range of a human body (35-41 ℃).

The matrix material preferably has a melting point slightly higher than the conventional storage temperature (5 ℃ to 20 ℃) and lower than the human body temperature range (35 ℃ to 41 ℃), and more preferably has good biodegradability so as to reduce retention in the body.

In the composition of the present invention, the matrix material of the microspheres B preferably does not contain hydroxyl groups and amino groups, because the hydroxyl groups or amino groups have a possibility of undergoing a crosslinking reaction with the crosslinking agent to reduce the activity of the crosslinking agent or partially deactivate the crosslinking agent, which affects the technical effect of the invention.

The matrix material is preferably C _10-35 In the composition of the invention, the matrix material is preferably selected from: methyl palmitate, methyl heptadecanoate, methyl octadecanoate, ethyl heptadecanoate, ethyl octadecanoate, ethyl nonadecanoate, ethyl eicosanoate, propyl octadecanoate, propyl nonadecanoate, propyl eicosanoate, butyl nonadecanoate and butyl eicosanoate.

In the composition of the present invention, the crosslinking agent may be any crosslinking agent capable of crosslinking hyaluronic acid or a salt thereof, and is preferably, but not limited to, lysine ethyl ester, adipic acid dihydrazide, 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polybutylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, 1,2,7,8-diepoxyoctane and 1,3-diepoxybutane, ferric chloride, calcium gluconate, calcium citrate, and a mixture thereof.

The composition of the present invention may further contain a crosslinking catalyst for increasing the rate and efficiency of the crosslinking reaction, and the crosslinking catalyst is not particularly limited as long as it can promote the crosslinking reaction, and preferably includes one or more of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and N-hydroxythiosuccinimide (sulfo-NHS).

In the composition of the present invention, the hyaluronic acid or a salt thereof may be formulated as isotonic physiological saline or a phosphate buffer.

In the composition of the present invention, the cosmetic or pharmaceutical active ingredient is not particularly limited, and an appropriate active ingredient may be selected as needed, and for example, any combination of one or more of triamcinolone acetonide, hydrocortisone, dexamethasone, prednisone, cortisone, lidocaine, tetracaine, vitamin C, vitamin E, tranexamic acid, or a pharmaceutically acceptable salt thereof may be used.

In the composition, the average grain diameter D50 of the microspheres A is 10-200 mu m, and the microspheres A contain 5-70 wt% of cosmetic or medicinal active ingredients.

In the composition of the present invention, the molecular weight of the hyaluronic acid or a salt thereof may be sufficient to have a viscosity that can be injected into a syringe, and is preferably not higher than 600 ten thousand daltons, and more preferably 10 to 600 ten thousand daltons.

The hyaluronic acid salt is not particularly limited as long as it is a pharmaceutically acceptable salt, and sodium hyaluronate most commonly used in the art is preferred in view of economy and easy availability.

In the composition of the present invention, the content of the crosslinking agent in the microspheres B is not particularly limited, and may be appropriately selected and adjusted as needed, and is preferably 0.1 to 30% by weight.

In the present invention, the preparation method of the microspheres a and B is not particularly limited, and a suitable preparation method in the field may be selected as needed, for example, the microspheres a may be prepared by any one of an oil/water emulsion-solvent evaporation method, a solid/oil/water emulsion-solvent evaporation method, a spray drying method, a phase separation method, and a membrane filtration method. The microspheres B can be prepared by a high-temperature melting and spraying low-temperature curing method.

The composition of the invention can be used for tissue filling, and the composition containing the cosmetic and/or medicinal active ingredients also has the function of slowly releasing the active ingredients to achieve long-acting disease treatment.

Examples

The present invention will be described in detail with reference to examples. The examples, which are given to give detailed embodiments and procedures sufficient to disclose and demonstrate the practice of the invention, do not represent limitations on the invention, and the scope of the invention is not limited to the examples described below.

Experimental procedures in the examples of the specification, where specific conditions are not indicated, are generally performed according to conditions conventional in the art or according to conditions recommended by the manufacturer. In the present invention, unless otherwise specified, parts represent parts by weight, and% represents weight percent.

1. Method for preparing microsphere composition

Example 1

The preparation method of the microsphere A comprises the following steps: 1g of PLGA material (PLGA 75) with the molecular weight of 6 ten thousand Da and 0.25g of methotrexate are taken to be mixed and dissolved with 9g of dichloromethane, the mixture is fully mixed with 40g of PVA (hydrolysis degree of 88 percent and molecular weight of 4 ten thousand) aqueous solution with the high-speed homogenization to prepare emulsion, the emulsion is transferred into 500mL of water, the mixture is stirred and vacuumized for 24 hours to obtain dispersion liquid of solid microspheres, the dispersion liquid is filtered by using 80 mu m and 10 mu m pore size screens, the microspheres with the particle size of 10 mu m-80 mu m are collected, and the freeze-drying and the crushing are carried out to obtain the microsphere. The scanning electron microscope picture of microsphere A is shown in FIG. 1.

The preparation method of the microsphere B comprises the following steps: taking 45g of ethyl heptadecanoate, heating to 50 ℃ to melt the ethyl heptadecanoate, adding 5g of cross-linking agent 1,4-butanediol diglycidyl ether, fully stirring and mixing at 50 ℃, pumping into a spray dryer at a speed of 1mL/min at a temperature of 50 ℃ to atomize into ultra-small droplets, and condensing into solid particles at a cold air temperature of 5 ℃ (the air supply in the spray dryer is liquid nitrogen cold air at 5 ℃).

Sodium hyaluronate lyophilisate: taking 200mg of sodium hyaluronate with the molecular weight of 160 ten thousand Da, fully mixing and dissolving the sodium hyaluronate with 5mL of 0.1M phosphate buffer solution with the pH value of 7.8, placing the mixture in a sterile vial after sterilization, and freeze-drying the mixture to obtain the sodium hyaluronate.

The preparation method of the microsphere composition for injection comprises the following steps: and adding 1000mg of microspheres A and 55mg of microspheres B into a penicillin bottle containing 200mg of sodium hyaluronate freeze-dried substance, and sealing to obtain the composition 1 of the embodiment. Before use, 5mL of sterile water is taken and injected into a penicillin bottle, and the mixture is shaken uniformly and can be used for injection.

Example 2

An injection was prepared as in example 1, using propyl eicosanoate instead of ethyl heptadecanoate to prepare microspheres B.

Comparative example 1

Comparative composition 1 was prepared as in example 1, microspheres B were prepared without the addition of the crosslinking agent 1,4-butanediol diglycidyl ether, the amount of crosslinking agent being replaced with ethyl heptadecanoate.

Comparative example 2

Comparative composition 2 was prepared as in example 1, microspheres B were prepared by replacing ethyl heptadecanoate with cetyl alcohol (melting point 50 ℃ C.) as the matrix material and changing the heat melting temperature to 60 ℃.

Comparative example 3

Comparative composition 3 was prepared as in example 1, replacing the sodium hyaluronate solution with crosslinked sodium hyaluronate (2% concentration) previously crosslinked with an equivalent amount of crosslinking agent from example 1 (i.e., 1,4-butanediol diglycidyl ether). The pre-crosslinked sodium hyaluronate was prepared as follows: dissolving 30g of sodium hyaluronate in 100mL of alkaline 0.5N sodium hydroxide solution containing 0.2 w/v1, 4-butanediol diglycidyl ether, stirring and reacting at 40 ℃ for 8 hours, adding 10mL of 5N hydrochloric acid solution to neutralize to neutrality, adding 1000mL of ethanol for precipitation, continuously washing with 1000mL of ethanol for 3 times, performing suction filtration to obtain white precipitate, and sufficiently stirring and dissolving with 1470mL of 0.1M phosphate buffer solution with pH 7.8 to obtain the cross-linked sodium hyaluronate solution. The rest remained the same as in example 1.

Comparative example 4

The preparation method of the microsphere B comprises the following steps: taking 45g of ethyl heptadecanoate, heating to 50 ℃ to melt the ethyl heptadecanoate, adding 1,4-butanediol diglycidyl ether 5g, fully stirring and mixing at 50 ℃, pumping into a spray dryer at a speed of 1mL/min under the condition of keeping the temperature of 50 ℃ to atomize into ultra-small liquid drops, and condensing into solid particles at a cold air temperature of 5 ℃ (air supply in the spray dryer is liquid nitrogen cold air at 5 ℃).

The preparation method of the sodium hyaluronate solution comprises the following steps: 200mg of sodium hyaluronate with a molecular weight of 160 ten thousand was taken, thoroughly mixed and dissolved with 5mL of 0.1M phosphate buffer solution with pH 7.8, and packaged in a sterilized prefilled glass syringe.

The preparation method of the injection comprises the following steps: placing 0.25g of methotrexate and 55mg of microspheres B in a penicillin bottle, injecting 5mL of sodium hyaluronate solution into the penicillin bottle, shaking uniformly, and sucking back to the syringe to obtain the comparative composition 4.

2. Simulated in vivo evaluation

1. Sticking machineEvaluation of elasticity

Viscoelasticity measurements were carried out by means of a rotary rheometer (model DHR-1 from TA) at 37 ℃ using a jig of 25mm diameter plate geometry, with measurement frequencies ranging from 0.01Hz to 10Hz. To determine the linear viscoelastic range of the material, a strain sweep was performed at an oscillation frequency of 1 Hz.

The compositions of example 1 and comparative example 1 were placed in glass bottles and, after standing for 72 hours at a temperature of 37 c, the viscoelasticity parameters of the compositions were determined and are shown in table 2. The results show that example 1 (invention) has an elastic modulus increased by 50 times and a viscosity increased by 40 times compared to comparative example 1 in which no crosslinking agent is used, indicating that the invention can produce a gel with high mechanical strength in vivo.

TABLE 2 comparison of viscoelastic parameters of the different examples

2. Evaluation of injectability

Using 2 disposable plastic syringes of 1mL, the stainless steel needles were removed, the injectates of example 1 and comparative example 3 were aspirated, 25G stainless steel needles (inner diameter 0.26 mm) were mounted, and the syringes were injected using an Instron ^TM The universal tester fixture fixes the injector sleeve and the push rod, keeps the advancing speed of the piston at 1mm/s (simulating the advancing speed of manual injection), and measures the average Dynamic Glide Force (DGF) of an object to be tested with the volume of 0.5mL extruded from the injector and contrasts the injection resistance. As shown in table 3, the injection resistance of example 1 (of the present invention) was about 8 times lower than that of comparative example 3 (pre-crosslinked sodium hyaluronate), greatly reducing the difficulty of clinical operation, and facilitating the control of the injection dose and injection site by the doctor.

TABLE 3 comparison of viscous injection resistances of the various examples

Examples	Dynamic glide force (N)
		Example 1	41N
Comparative example 3	310N

3. Microsphere A drug release evaluation

As shown in fig. 2, the drug in the microsphere a of example 1 can be slowly released for about 90 days, and can achieve the purposes of persistently reducing osteoarthritis and persistently reducing pain of patients; in contrast, in comparative example 4, the drug was not prepared as microspheres, and was completely released within 1 day without sustained release.

4. Evaluation of Release of crosslinking Agents from microspheres B

The microspheres B from example 1, the microspheres B from example 2 and the microspheres B from comparative example 2, each 0.5g, were placed in stoppered tubes, 10mL of pH 7.4 phosphate buffer was added, the release of the cross-linking agent was investigated in a shaker water bath (shaking speed 120 rpm) at 37 ℃ after sealing with a sealing membrane, and the content of epoxide functional groups in the hydrolysate was determined according to the method of Nelis and Sinheimer, taking 1mL of phosphate buffer at different time points. The method of Nelis and Sinsheimer uses highly sensitive fluorescence photometry to determine the content of aliphatic epoxy compounds under physiological conditions. The measurement wavelength was an absorption wavelength at 370 nm. (A Sensitive Fluorimetric for the Determination of antigenic Epoxides under Physiological Conditions, analytical Biochemistry, volume 115, issue 1,15July 1981, pages 151-157).

As shown in fig. 3, both example 1 and example 2 can release most of the crosslinker rapidly within 72 hours; whereas microspheres B of comparative example 2 hardly released the internal cross-linking agent. Therefore, the invention can realize the effects of releasing the cross-linking agent at the body temperature and generating the cross-linked hyaluronic acid at the injection site after being injected into the human body.

3. In vivo evaluation in animals

1. Evaluation of injectable microsphere compositions in collagenase-induced osteoarthritis animal models

Rabbits (New Zealand white rabbits) were acclimatized for one week, and 1.25mL of collagenase (4 mg/mL, sigm, USA) was injected into the synovial cavity of the right knee of the rabbit, and after the collagenase acted in the knee cavity for 48 hours, sodium iodoacetate (12mg, 1mL) was injected again, and injection of the test article was started after molding was completed on day 14. The group without any treatment after successful molding was used as a blank group.

The freshly prepared injectables of example 1 and comparative example 1 were taken, 3mL were injected into the knee joint cavity, treated for 4 weeks, and blood samples were collected. The right knee was excised and fixed with 10% formalin solution. Clinical symptoms such as walking behavior, range of motion, swelling, etc. were observed during the experiment.

The applied pressure at which the avoidance response occurred was measured using a pain tester Analgesy-Meter (Ugobasile, italy) to squeeze the knee joint of a rabbit. Pain inhibition rate = (test group avoidance pressure-blank group avoidance pressure)/(positive group avoidance pressure-blank group avoidance pressure) × 100%. The pain inhibition rate was determined as 100% in the intra-articular injection positive group (intra-articular injection methotrexate injection 3ml 50mg) 2 hours after injection.

As shown in table 4, example 1 (invention) can provide sustained relief of osteoarthritis pain, significantly better than both the positive group and comparative example 1 over the 2 month study period; while the positive group provided only short-term pain relief, with no pain suppression effect for 1 week and later; the pain inhibition rate of comparative example 1 was comparable to that of example at 2 hours, but was significantly decreased with time because it did not contain a cross-linking agent, the effect of example 1 of generating cross-linked hyaluronic acid with high strength in situ was not achieved, and non-cross-linked hyaluronic acid was consumed by tissue absorption with time, failing to play a role in continuously lubricating joint cavities and relieving pain. Therefore, the present invention can realize release of the cross-linking agent at body temperature after injection into a human body, generation of cross-linked hyaluronic acid at the injection site, and longer-lasting and stronger pain reduction effect.

TABLE 4 pain inhibition rates of example 1, positive group, comparative example 1 on rabbit osteoarthritis model

2. Evaluation of recovery Rate of sodium hyaluronate in articular Cavity

To verify whether the present invention has completed in situ cross-linking in vivo, at different time points in the rabbit osteoarthritis animal study, the knee joint cavities of the treatment groups were dissected open, the cavity fluid contents were removed, viscoelasticity measurements were performed with a rotational rheometer (model TA DHR-1, manufactured by TA) at 37 ℃ using a jig of 25mm diameter plate geometry, and shear rate-viscosity scans were performed. As can be seen from the results of table 5, the hyaluronic acid recovered from the joint cavity treated in example 1 (the present invention) has a significantly higher viscosity than that of comparative example 1 containing no crosslinking agent, indicating that the present invention achieves the effect of releasing the crosslinking agent in vivo to complete the crosslinking of hyaluronic acid.

TABLE 5 hyaluronic acid recovery (%). From rabbit articular cavities at different time points after injection

In conclusion, the microsphere composition for injection can effectively control the microspheres B to release the cross-linking agent after injection is completed and perform cross-linking on sodium hyaluronate of an injection part to obtain in-situ cross-linked gel with high mechanical strength, and can provide better and longer-time mechanical supporting force for the joint part when applied to joint injection. The sodium hyaluronate is not crosslinked before and during injection, so that the injection resistance of the sodium hyaluronate is only one eighth of that of the crosslinked sodium hyaluronate, and the injectability is greatly improved. The microspheres A can release the drug for 3 months in a long-acting manner. The intra-articular injection of the present invention continuously and effectively reduces joint pain.

The protection content of the present invention is not limited to the above embodiments. Variations and substitutions which may occur to those skilled in the art are intended to be included within the invention without departing from the spirit and scope of the inventive concept and the scope of the appended claims is intended to be limited only by the following claims.

Claims

1. A microsphere composition for injection comprising:

microspheres B containing a crosslinking agent and a matrix material, and

a hyaluronic acid or a salt thereof, wherein the hyaluronic acid or the salt thereof,

the matrix material is a fatty acid ester with a melting point of 21-41 ℃ and a carbon number of 10-35, and is selected from: methyl palmitate, methyl heptadecanoate, methyl octadecanoate, ethyl heptadecanoate, ethyl octadecanoate, ethyl nonadecanoate, propyl octadecanoate, propyl nonadecanoate and butyl nonadecanoate.

2. A microsphere composition for injection comprising:

a) 10-97% by weight of microspheres A, said microspheres A comprising polylactide glycolide and cosmetically and/or pharmaceutically active ingredients,

b) 1-40% by weight of microspheres B, said microspheres B comprising a cross-linking agent and a matrix material, said matrix material being a fatty acid ester having a melting point of 21-41 ℃ and a carbon number of 10-35, said matrix material being selected from the group consisting of: one or more of methyl hexadecanoate, methyl heptadecanoate, methyl octadecanoate, ethyl heptadecanoate, ethyl octadecanoate, ethyl nonadecanoate, propyl octadecanoate, propyl nonadecanoate and butyl nonadecanoate;

c) 2 to 50% by weight of hyaluronic acid or a salt thereof.

3. The composition of claim 1 or 2, wherein the melting point of the matrix material in microspheres B is between 25 and 41 ℃.

4. The composition of claim 1 or 2, the crosslinker selected from the group consisting of lysine ethyl ester, adipic acid dihydrazide, 1,4-butanediol diglycidyl ether, ethylene glycol diglycidyl ether, 1,6-hexanediol diglycidyl ether, polypropylene glycol diglycidyl ether, polytetramethylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, sorbitol polyglycidyl ether, 1,2,7,8-diepoxyoctane and 1,3-diepoxybutane, ferric trichloride, calcium chloride, calcium gluconate, calcium citrate, and mixtures thereof.

5. The composition of claim 1 or 2, further comprising a crosslinking catalyst selected from one or more of 1-ethyl- (3-dimethylaminopropyl) carbodiimide, N-hydroxysuccinimide, N-hydroxythiosuccinimide.

6. The composition according to claim 1 or 2, wherein the hyaluronic acid salt is any combination of one or more of pharmaceutically acceptable salts of hyaluronic acid.

7. The composition according to claim 2, wherein the active ingredient is any combination of one or more of triamcinolone acetonide or a pharmaceutically acceptable salt thereof, hydrocortisone or a pharmaceutically acceptable salt thereof, dexamethasone or a pharmaceutically acceptable salt thereof, prednisone or a pharmaceutically acceptable salt thereof, cortisone or a pharmaceutically acceptable salt thereof, lidocaine or a pharmaceutically acceptable salt thereof, tetracaine or a pharmaceutically acceptable salt thereof, tranexamic acid or a pharmaceutically acceptable salt thereof, vitamin C, and vitamin E.

8. Composition according to claim 1, the microspheres a having a mean particle size D50 of between 10 μ ι η and 200 μ ι η and containing between 5% and 70% by weight of a cosmetically or therapeutically active ingredient.

9. The composition according to claim 1 or 2, wherein the molecular weight of the hyaluronic acid or salt thereof is 10 to 600 ten thousand, and the hyaluronic acid salt is sodium hyaluronate.

10. The composition according to claim 1 or 2, wherein the content of the crosslinking agent in the microspheres B is 0.1 to 30% by weight.

11. The method for preparing the composition of any one of claims 2 to 10, wherein the microspheres a are prepared by any one of an oil/water emulsion-solvent evaporation method, a solid/oil/water emulsion-solvent evaporation method, a spray drying method, a phase separation method and a membrane filtration method, and the microspheres B are prepared by a high-temperature melting spray low-temperature solidification method.

12. A method for preparing the composition of any one of claims 2 to 10, wherein the method for preparing the microspheres a comprises: dissolving the active ingredients and polylactide glycolide in a dichloromethane solvent to prepare an oil phase, adding the oil phase into a polyvinyl alcohol aqueous solution containing 0.2-2% of volume while stirring, volatilizing dichloromethane, filtering, collecting microspheres, and further drying to obtain the microspheres A.

13. Use of a composition according to any one of claims 1 to 10 for the preparation of a tissue bulking agent.