WO2023123813A1

WO2023123813A1 - Drug-loaded microspheres as well as preparation method therefor and use thereof

Info

Publication number: WO2023123813A1
Application number: PCT/CN2022/092450
Authority: WO
Inventors: 许为康; 李桂香
Original assignee: 广东省科学院生物与医学工程研究所
Priority date: 2021-12-30
Filing date: 2022-05-12
Publication date: 2023-07-06
Also published as: CN114288262A; CN114288262B

Abstract

Drug-loaded microspheres as well as a preparation method therefor and the use thereof. The drug-loaded microspheres comprise a carrier loaded with a therapeutic agent, a coating layer covering the surface of the carrier and comprising an aldehyde modified biomacromolecule material layer, and an acellular matrix layer covering the surface of the aldehyde modified biomacromolecule material layer. The drug-loaded microspheres have a good drug slow-release effect, the drug release period can reach 28 days or longer, and the drug release control capability is extremely high; the drug-loaded microspheres have further improved biocompatibility and biological activity and can effectively promote tissue repair and reconstruction, thereby being suitable for treatment of diseases such as tissue defects, bacterial infection and inflammation.

Description

A drug-loaded microsphere and its preparation method and application

technical field

The invention relates to the field of biomedical materials, in particular to a drug-loaded microsphere and a preparation method and application thereof.

Background technique

With the continuous development of medicine, pharmacy, and biology, new drugs for various diseases emerge in an endless stream, but how to release drugs continuously and stably in the body is still a difficult problem. Regardless of oral administration or intravenous injection, there will be a "peak-valley" phenomenon in the change of blood drug concentration. If the drug concentration is too high, it will cause relatively large toxic and side effects, while if it is too low, the therapeutic effect will not be achieved. Although the emerging bioactive macromolecular drugs are highly effective, they have a short biological half-life and are easily inactivated, which is also a problem that plagues drug developers. At present, the main way to solve these problems is to use appropriate biological materials to embed or absorb drugs to form a drug-controlled release system, which is implanted into the tissue "focus" for local drug delivery. Biomaterials used as carriers of drug controlled release systems need to have biocompatibility and biodegradability. According to the properties of materials, they can be mainly divided into inorganic materials, natural polymer materials and synthetic polymer materials. Among them, artificially synthesized degradable polymer materials, especially degradable polyester, can design their biological response characteristics by changing the chemical composition, material structure and surface properties of their raw materials. Since the drug-loaded microsphere technology was studied, its in vivo performance research is inseparable from the compound with gel system or scaffold material.

Hydrogel is a gel with water as the dispersion medium. It introduces a part of hydrophobic groups and hydrophilic residues into water-soluble polymers with a network crosslinked structure. The hydrophilic residues combine with water molecules, and the water molecules A cross-linked polymer in which the hydrophobic residues swell when exposed to water, and are connected within the network. Hydrogels can absorb thousands of times their own mass in water and can take on virtually any shape and size. As a polymeric scaffold, hydrogels have a variety of applications in tissue repair and other disease treatments. Due to the network structure of the hydrogel, proteins or cells can be entrapped inside the network and the envelopes can be released in a controlled manner. In addition, the hydrogel is degraded and absorbed in the body, and can be perfectly integrated with the surrounding tissue, so that the complexity of surgical removal can be avoided, and the possibility of inflammatory reactions can also be reduced. Compared with artificially constructed scaffold materials, the acellular matrix can better sense the "external" environment after being implanted in the defect site, establish signal contact with surrounding tissues and cells and exchange substances, and mediate the adhesion of cells and various proteins , actively participate in the whole process of tissue repair, and have strong biological responsiveness. However, acellular matrices can only provide short-term release of drugs, and cannot release drugs for a long time to continuously stimulate the "focus" site, making it difficult for acellular matrices to treat defective tissues alone. The drug-loaded acellular matrix composite scaffold can make up for the shortcomings of these two materials used alone, and take advantage of their advantages in drug controlled release, mechanical strength, porous interconnected structure, and biocompatibility.

Contents of the invention

The present invention aims to solve at least one of the technical problems in the above-mentioned prior art. For this reason, the present invention proposes a drug-loaded microsphere, which can achieve long-term controlled drug release, further improves the biocompatibility and bioactivity of the drug-loaded microsphere, and can effectively promote tissue repair and reconstruction.

The invention also proposes the preparation method and application of the drug-loaded microspheres.

In the first aspect of the present invention, a drug-loaded microsphere is proposed, which includes a carrier loaded with a therapeutic agent and a coating layer coated on the surface of the carrier, and the coating layer includes an aldehyde modified A biomacromolecular material layer, and an acellular matrix layer coated on the surface of the aldehyde-modified biomacromolecular material layer.

According to the first aspect of the present invention, the present invention has at least the following beneficial effects:

The acellular matrix used in the present invention can sense the "external" environment well, establish signal contact with surrounding tissues and cells and perform material exchange, mediate the adhesion of cells and various proteins, and actively participate in the whole process of tissue repair. Strong bioresponsiveness and excellent biocompatibility. Using acellular matrix as a coating material can further delay the release of drugs, and improve the biocompatibility and bioactivity of drug-loaded microspheres, which can effectively promote tissue repair and reconstruction. At the same time, the adhesion of the acellular matrix can be improved by modifying the biomacromolecular material through the aldehyde group, so that the acellular matrix can effectively exert the slow-release effect.

Preferably, the aldehyde-modified biomacromolecular material includes at least one of oxidized chitosan quaternary ammonium salt, oxidized alginate, and aldehyde-modified gelatin, and the more preferred aldehyde-modified biomacromolecular material is Oxidized chitosan quaternary ammonium salt. Among them, the aldehyde group in the aldehyde-modified biomacromolecular material reacts with the active groups on the acellular matrix, which can enhance the adhesion of the acellular matrix on the surface of the drug-loaded microspheres. At the same time, the aldehyde-modified biomacromolecular material has A certain degree of sustained release effect further enhances the drug sustained release effect of the drug-loaded microspheres.

Preferably, the oxidation degree of the oxidized chitosan quaternary ammonium salt is 1-98%; more preferably 55%-80%, such as 55%, 60%, 75%, 80% and so on.

Preferably, the coating layer further includes a degradable material layer, the degradable material layer is coated on the surface of the carrier, and the aldehyde-modified biomacromolecular material layer is coated on the degradable material layer s surface.

Preferably, said degradable material comprises degradable polyester. The degradable polyester includes polylactic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly(3-hydroxyalkanoate), poly(3-hydroxybutyrate), poly(3-hydroxybutyrate) At least one of ester-co-3-hydroxyvalerate), polytrimethylene carbonate, polybutylene succinate.

Preferably, the molecular weight of the degradable polyester is 1-160,000 Daltons, more preferably 1-100,000 Daltons, further preferably 1-60,000 Daltons.

Preferably, the carrier is a nanoscale porous material, including at least one of a silicon-based porous carrier and a porous metal compound, and the silicon-based porous carrier includes at least one of silicon dioxide, calcium silicate, and magnesium silicate The porous metal compound includes at least one of porous titanium dioxide, porous magnesium hydroxide, and porous aluminum hydroxide.

Preferably, the support has a mesoporous structure, and the porous support is more preferably mesoporous silica.

Preferably, the average particle diameter of the carrier is 2-100 nm, more preferably 2-50 nm, further preferably 2-30 nm. The specific surface area of the carrier is 100-2200m ² /g, more preferably 300-1800m ² /g.

Preferably, the therapeutic agents include, but are not limited to, chemotherapeutic agents, biotherapeutic agents, such as antitumor agents, antibodies, anti-inflammatory agents, immunotherapeutic agents, and other natural or synthetic drugs with specific functions.

The therapeutic agents include therapeutic agents for tissue repair and regeneration. The therapeutic agent includes at least one of bone morphogenetic protein-2, bone morphogenetic protein-7, vascular endothelial growth factor, sodium alendronate, naringin, and resveratrol.

Preferably, the mass ratio of the therapeutic agent to the carrier is 1:1-30, more preferably 1:1-20.

Preferably, the drug encapsulation rate of the drug-loaded microspheres is 10-95%, more preferably 40-95%.

Preferably, the particle diameter of the drug-loaded microspheres is 0.005-5 mm, more preferably 0.01-2 mm.

In the second aspect of the present invention, a method for preparing drug-loaded microspheres is proposed. The method for preparing drug-loaded microspheres includes the following steps: preparing a coating layer on the upper surface of the carrier loaded with therapeutic agents to obtain the drug-loaded microspheres. Microsphere; the coating layer includes an aldehyde-modified biomacromolecular material layer and an acellular matrix layer coated on the surface of the aldehyde-modified biomacromolecular material layer.

Preferably, the coating layer further includes a degradable material layer.

Preferably, the preparation method of the drug-loaded microspheres specifically includes the following steps:

Step S1, coating the degradable material on the surface of the carrier loaded with the therapeutic agent to obtain the carrier/degradable material composite microspheres loaded with the therapeutic agent;

Step S2, coating the aldehyde-modified biomacromolecular material on the surface of the therapeutic agent-loaded carrier/degradable material composite microsphere to obtain the therapeutic agent-loaded carrier/degradable material/aldehyde-modified biomacromolecule Molecular material composite microspheres;

Step S3, coating the acellular matrix on the surface of the carrier/degradable material/aldehyde-modified biomacromolecular material composite microspheres loaded with therapeutic agents to obtain drug-loaded microspheres with acellular matrix on the surface.

Preferably, the step S1 specifically comprises: mixing the carrier loaded with the therapeutic agent and the degradable material solution, adding it into the aqueous solution containing the surfactant, and stirring to obtain the carrier/degradable material composite microspheres loaded with the therapeutic agent.

Preferably, the stirring time is 6-16 hours, more preferably 8-14 hours.

Preferably, the preparation method of the carrier/degradable material composite microspheres loaded with therapeutic agent in the step S1 is at least one of emulsified solvent volatilization method, phase separation method, emulsified solvent extraction method, spray drying method, and melting method, The emulsion solvent evaporation method is more preferable. The present invention prepares the carrier/degradable material composite microspheres loaded with therapeutic agents by the emulsion solvent volatilization method, the method is relatively simple, the requirements for equipment are not high, the source of raw materials is easy to obtain, the cost is low, and the industrialization is easy to realize.

Preferably, when the preparation method of the carrier/degradable material composite microspheres loaded with a therapeutic agent in the step S1 is the emulsion solvent volatilization method, the preparation method of the carrier/degradable material composite microspheres loaded with a therapeutic agent includes: One of /W emulsification method, S/O/W emulsification method, O ₁ /O ₂ emulsification method, double emulsion-drying method in liquid, more preferably S/O/W emulsification method.

Preferably, the step S1 adopts the S/O/W emulsification method to prepare the carrier/degradable material composite microspheres loaded with the therapeutic agent, which specifically includes the following steps: dissolving the carrier loaded with the therapeutic agent and the degradable material in a solvent to form a blend, Then the blend solution is added to the water phase containing surfactant, dispersed to form an S/O/W emulsion, and the organic solvent in the S/O/W emulsion is volatilized to obtain a carrier/degradable material composite loaded with a therapeutic agent Microspheres.

Preferably, the solvent is not limited, as long as it can dissolve the degradable material, such as dichloromethane, chloroform, ethyl acetate, ethanol, methanol or acetone; more preferably, dichloromethane is used as the solvent. Properties such as the solubility of the solvent in the water phase will affect the particle size, encapsulation efficiency and drug loading capacity of the drug-loaded microspheres, and the use of dichloromethane in the present invention can improve the encapsulation efficiency of the therapeutic agent.

Preferably, the surfactant includes at least one of gelatin, polyvinyl alcohol, and carboxymethyl cellulose. The polyvinyl alcohol includes at least one of polyvinyl alcohol 1788, polyvinyl alcohol 124, and polyvinyl alcohol 1799.

Preferably, the mass concentration of the surfactant in the aqueous solution containing the surfactant is 2-100 mg/mL, more preferably 2-50 mg/mL. The type and concentration of the surfactant are related to the droplet size in the formed emulsion, the carrier of the loaded therapeutic agent and the degree of dispersion of the water phase. The present invention adopts polyvinyl alcohol as the surfactant and selects a specific concentration to improve it to a certain extent. Encapsulation efficiency and drug loading of carrier/degradable material composite microspheres loaded with therapeutic agents.

Preferably, the mass volume ratio of the degradable material to the solvent in the degradable material solution is 1:5-50 g/mL, more preferably 1:5-30 g/mL.

Preferably, the volume ratio of the degradable material solution to the aqueous solution containing surfactant is 1:10-80, more preferably 1:20-60.

Preferably, the mass ratio of the therapeutic agent-loaded carrier to the degradable material is 1-50:100, more preferably 1-30:100.

Preferably, the dispersing method of adding the blended solution into the water phase containing the surfactant to disperse and form the S/O/W type emulsion includes mechanical stirring and ultrasonic dispersion. The dispersion method used in the process of forming the emulsion and evaporating the solvent has a certain influence on the formation of the carrier/degradable material composite microspheres loaded with therapeutic agents. The present invention adopts the above-mentioned dispersion method to increase the particle size of the finally obtained drug-loaded microspheres , The encapsulation rate is improved.

Preferably, the dispersion method is mechanical stirring; the rotational speed of the mechanical stirring is 100-1500 rpm, more preferably 200-1000 rpm.

Preferably, a step of surface treatment of the carrier/degradable material composite microspheres loaded with the therapeutic agent is also included between the step S1 and the step S2. The surface treatment is specifically to use acid, alkali or alcohol to treat the carrier/degradable material composite microspheres loaded with therapeutic agent, so that active groups are exposed on the surface. The active groups are carboxyl, hydroxyl and other groups. In the subsequent process, the surface active groups of the carrier/degradable material composite microspheres loaded with therapeutic agents react with the aldehyde-modified biomacromolecular material, which increases the impact of the aldehyde-modified biomacromolecular material on the carrier/degradable material loaded with therapeutic agent. Adhesion to the surface of degradable material composite microspheres.

Preferably, the acid includes at least one of acetic acid, formic acid, hydrochloric acid and solutions thereof. The mass concentration of the acid in the acid solution is 1-5%, such as 3%.

Preferably, the alkali includes at least one of sodium hydroxide, potassium hydroxide, ammonia water and solutions thereof. The mass concentration of the alkali in the alkali solution is 5-20%, more preferably 8-20%.

Preferably, the alcohol includes at least one of methanol, ethanol, propanol and solutions thereof. The mass concentration of alcohol in the alcohol solution is 1-10%, such as 5%.

Preferably, the step S2 is specifically to disperse the carrier/degradable material composite microspheres loaded with the therapeutic agent in the solution containing the aldehyde-modified biomacromolecular material, and coat the aldehyde-modified biomacromolecular material On the surface of the carrier/degradable material composite microsphere loaded with the therapeutic agent, the composite microsphere of the carrier loaded with the therapeutic agent/degradable material/aldehyde-based modified biomacromolecular material is obtained.

Preferably, the mass-to-volume ratio of the carrier/degradable material composite microspheres loaded with therapeutic agents to the solution of the aldehyde-modified biomacromolecular material is 1:200-2500 g/mL, more preferably 1:300-2000 g /mL.

Preferably, the mass concentration of the aldehyde group-modified biomacromolecular material in the solution containing the aldehyde group-modified biomacromolecular material is 5-40%, more preferably 10-30%.

Preferably, the pH value of the solution of the aldehyde-containing modified biomacromolecular material is 3-11, more preferably 3-10.

Preferably, the solvent used in the solution of the aldehyde-containing modified biomacromolecular material is not limited, and it can uniformly disperse the carrier/degradable material composite microspheres loaded with therapeutic agents and dissolve the aldehyde-modified biomacromolecular material. Yes, such as PBS buffer.

Preferably, the dispersion time in the step S2 is 1-15 hours, more preferably 2-10 hours. The dispersion temperature is 2-60°C, more preferably 4-40°C.

Preferably, the step S3 is specifically dissolving the acellular matrix in an aqueous solution to obtain a liquid acellular matrix, and then dispersing the carrier/degradable material/aldehyde-based modified biomacromolecular material composite microspheres loaded with therapeutic agents In the liquid decellularized matrix, the drug-loaded microspheres are obtained.

Preferably, the mass ratio of the therapeutic agent-loaded carrier/degradable material/aldehyde-modified biomacromolecular material composite microspheres to the acellular matrix is 1:0.1-3, more preferably 1:0.1-2.

Preferably, the mass volume ratio of the acellular matrix to water in the liquid acellular matrix is 1:100-15000 g/mL, more preferably 1:200-10000 g/mL.

Preferably, the dispersion time in the step S3 is 2-16 hours, more preferably 5-12 hours.

Preferably, the preparation method of the acellular matrix includes the following steps of treating bone tissue with chemical reagents and enzymes to obtain the acellular matrix.

Preferably, the chemical reagent includes at least one of hydrochloric acid, disodium edetate, sodium lauryl sulfate, and triton. More preferably, the chemical reagent is a composition comprising hydrochloric acid and at least one of disodium edetate, sodium lauryl sulfate and triton.

Preferably, the concentration of the hydrochloric acid is 0.1-1.2N, more preferably 0.4-1N.

Preferably, the mass concentrations of disodium edetate, sodium lauryl sulfate and triton are independently 0.02-0.12%, more preferably 0.02-0.1%.

Preferably, the enzyme includes at least one of trypsin, deoxyribonuclease (DNase), and ribonuclease (RNase).

Preferably, the mass concentration of the trypsin is 0.02-0.06%, such as 0.025%, 0.05%. The dosage of the deoxyribonuclease (DNase) and/or ribonuclease (RNase) is independently 100-500 U/mL.

In the third aspect of the present invention, a medical composition is proposed, which comprises the drug-loaded microspheres.

Preferably, the medical composition includes a scaffold, dressing or other composite material containing drug-loaded microspheres. The medical composition utilizes the biological activity and drug controlled release effect of drug-loaded microspheres to achieve medical purposes based on specific therapeutic agents, such as tissue damage repair and regeneration.

According to the fourth aspect of the present invention, a medical device is provided, which comprises the drug-loaded microspheres and/or the medical composition.

Preferably, the medical device includes an implantable device based on a drug-controlled release system, and the repair of tissue damage is achieved through the use of the drug-loaded microspheres and/or the medical composition.

According to the fifth aspect of the present invention, the application of the drug-loaded microspheres in the preparation of materials for tissue repair and regeneration, and disease treatment.

Compared with the prior art, the present invention has at least the following beneficial effects:

The present invention uses porous materials such as mesoporous silicon as carriers for loading therapeutic agents, and uses degradable materials, aldehyde-modified biomacromolecular materials (such as oxidized chitosan quaternary ammonium salts) and acellular matrix as coating materials to prepare Drug-loaded microspheres with three coating layers were produced. The inner coating layer is a degradable material, which is directly coated on the surface of the carrier; the sub-outer coating layer is oxidized chitosan quaternary ammonium salt, which is located on the outer surface of the inner coating layer; the outer layer is an acellular matrix, which is located on the sub- The outer surface of the outer cladding. In the present invention, the carrier loaded with therapeutic agents, degradable materials, oxidized chitosan quaternary ammonium salt, and acellular matrix are used in combination, so that the drug-loaded microspheres have good drug sustained release effect at the same time, and the drug release period can reach more than 28 days. The drug release control ability is extremely strong, and the biocompatibility and bioactivity of the drug-loaded microspheres are further improved, which can effectively promote tissue repair and reconstruction, and is suitable for the treatment of tissue defects, bacterial infections, inflammation and other diseases.

Description of drawings

The present invention will be further described below in conjunction with accompanying drawing and embodiment, wherein:

Fig. 1 is a graph showing the in vitro drug release performance results of drug-loaded microspheres prepared in Examples and Comparative Examples of the present invention.

Detailed ways

The conception and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Apparently, the described embodiments are only some of the embodiments of the present invention, rather than all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts belong to The protection scope of the present invention.

Example 1

(1) Mix 20mg sodium alendronate and 20mg mesoporous silicon to obtain a mixed powder of drug and mesoporous silicon, and then disperse the above mixed powder in 10mL containing 0.4g polylactic acid-glycolic acid copolymer (molecular weight: 3 10,000) in dichloromethane solution, to obtain the drug-loaded mesoporous silicon/polylactic acid-glycolic acid copolymer blend; configure 400mL aqueous solution containing 4g of gelatin, and then slowly add the above blend into the gelatin aqueous solution at 300rpm After continuous stirring for 12 hours, the composite microspheres at the bottom of the container were separated to prepare drug-loaded mesoporous silicon/degradable polyester composite microspheres.

(2) Soak 100 mg of drug-loaded mesoporous silicon/degradable polyester composite microspheres in 50 mL of 10% sodium hydroxide solution for 6 minutes, and then wash the drug-loaded mesoporous silicon/degradable polyester composite microspheres; 37 At ℃, soak 100 mg of pretreated drug-loaded mesoporous silicon/degradable polyester composite microspheres in 20% oxidized chitosan quaternary ammonium salt (oxidation degree 80%) aqueous solution (pH=10) for 4 hours, and then Cleaning the drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium salt composite microspheres to obtain drug-loaded mesoporous silicon/degradable polyester/oxidized shell coated with oxidized chitosan quaternary ammonium salt Polysaccharide quaternary ammonium salt composite microspheres.

(3) sequentially adopt organic solvents such as 0.5N hydrochloric acid, chloroform and methanol, 0.25% trypsin, 0.1% sodium lauryl sulfate, etc. to treat the bone tissue and clean and freeze-dry to obtain the decellularized matrix; take 100mg Dissolve the acellular matrix in 300mL water to obtain a liquid acellular matrix, and then soak 100mg of the drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium composite microspheres obtained in step (2) in the liquid acellular matrix 10 h, washed and freeze-dried to obtain drug-loaded microspheres with decellularized matrix on the surface.

Example 2

(1) Mix 10mg resveratrol and 20mg mesoporous silicon to obtain a mixed powder of drug and mesoporous silicon, and then disperse the above mixed powder in 3mL containing 0.1g polytrimethylene carbonate (molecular weight: 10,000) In the dichloromethane solution of the drug-loaded mesoporous silicon/polytrimethylene carbonate blend solution; configure 150mL aqueous solution containing 7.5g carboxymethyl cellulose, and then slowly add the above blend solution to carboxymethyl cellulose The composite microspheres at the bottom of the container were separated after continuous stirring at 450rpm for 8 hours in the plain aqueous solution to prepare the drug-loaded mesoporous silicon/degradable polyester composite microspheres.

(2) Soak 100 mg of drug-loaded mesoporous silicon/degradable polyester composite microspheres in 150 mL of 8% ammonia solution for 15 minutes, and then wash the drug-loaded mesoporous silicon/degradable polyester composite microspheres; 100 mg of pretreated drug-loaded mesoporous silicon/degradable polyester composite microspheres were soaked in 10% oxidized chitosan quaternary ammonium salt (oxidation degree 65%) aqueous solution (pH=6) for 5 h, and then loaded Drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium salt composite microspheres were cleaned to obtain drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan coated with oxidized chitosan quaternary ammonium salt Quaternary ammonium salt composite microspheres.

(3) sequentially use 0.4N hydrochloric acid, organic solvents such as ethanol and methanol, 300U/mL DNase and RNase solution, 0.05% sodium lauryl sulfate, etc. to treat the bone tissue, clean and freeze-dry to obtain the acellular matrix; Dissolve 10 mg of acellular matrix in 20 mL of water to obtain a liquid acellular matrix, and then soak 100 mg of drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium compound microspheres obtained in step (2) in the liquid decellularized matrix in the matrix for 12 hours, washed and freeze-dried to obtain drug-loaded microspheres with decellularized matrix on the surface.

Example 3

(1) Mix 1 mg of BMP-2 with 20 mg of mesoporous silicon to obtain a mixed powder of drug and mesoporous silicon, and then disperse the above mixed powder in 15 mL of bismuth containing 0.6 g of polylactic acid (molecular weight: 50,000). In the methyl chloride solution, obtain the drug-loaded mesoporous silicon/polylactic acid blend; prepare 600 mL of an aqueous solution containing 10 g of polyvinyl alcohol 1788, then slowly add the above blend into the aqueous solution of polyvinyl alcohol 1788, and continue stirring at 400 rpm for 14 hours Finally, the composite microspheres at the bottom of the container are separated to prepare the drug-loaded mesoporous silicon/degradable polyester composite microspheres.

(2) Soak 100 mg of drug-loaded mesoporous silicon/degradable polyester composite microspheres in 30 mL of 3% hydrochloric acid solution for 5 minutes, and then wash the drug-loaded mesoporous silicon/degradable polyester composite microspheres; 100 mg of pretreated drug-loaded mesoporous silicon/degradable polyester composite microspheres were soaked in 15% oxidized chitosan quaternary ammonium salt (oxidation degree 60%) aqueous solution (pH=7) for 10 h, and then loaded Drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium salt composite microspheres were cleaned to obtain drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan coated with oxidized chitosan quaternary ammonium salt Quaternary ammonium salt composite microspheres.

(3) sequentially adopt organic solvents such as 0.8N hydrochloric acid, dichloromethane and ethanol, 0.05% trypsin, 0.02% disodium edetate, etc. to treat the bone tissue and clean and freeze-dry to obtain the acellular matrix; Dissolve 50mg of acellular matrix in 600mL of water to obtain a liquid acellular matrix, and then soak 100mg of drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium composite microspheres obtained in step (2) in the liquid acellular matrix Incubate for 6 hours, wash and freeze-dry to obtain drug-loaded microspheres with acellular matrix on the surface.

Example 4

(1) Mix 0.5mg vascular endothelial growth factor and 10mg mesoporous silicon to obtain a mixed powder of drug and mesoporous silicon, and then disperse the above mixed powder in 4.5mL containing 0.9g poly(3-hydroxybutyrate-co -3-hydroxyvalerate) (molecular weight: 100,000) in the dichloromethane solution, obtain the mesoporous silicon/poly (3-hydroxybutyrate-co-3-hydroxyvalerate) blend solution of drug loading; Prepare 270mL of an aqueous solution containing 13.5g of polyvinyl alcohol 124, then slowly drop the above blend into the aqueous solution of polyvinyl alcohol 124, and continue stirring at 1000rpm for 12h, then separate the composite microspheres at the bottom of the container to prepare the drug-loaded mesoporous Silicon/degradable polyester composite microspheres.

(2) Soak 100 mg of drug-loaded mesoporous silicon/degradable polyester composite microspheres in 200 mL of 20% potassium hydroxide aqueous solution for 8 minutes, and then wash the drug-loaded mesoporous silicon/degradable polyester composite microspheres; 30 At ℃, 100 mg of pretreated drug-loaded mesoporous silicon/degradable polyester composite microspheres were soaked in 15% oxidized chitosan quaternary ammonium salt (oxidation degree 75%) aqueous solution (pH=9) for 2 h, and then Cleaning the drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium salt composite microspheres to obtain drug-loaded mesoporous silicon/degradable polyester/oxidized shell coated with oxidized chitosan quaternary ammonium salt Polysaccharide quaternary ammonium salt composite microspheres.

(3) sequentially adopt organic solvents such as 1N hydrochloric acid, ethyl acetate and methanol, 100 U/mL DNase and RNase solution, 0.05% Triton 100, etc. to treat the bone tissue and clean and freeze-dry to obtain the acellular matrix; Dissolve 200mg of acellular matrix in 1000mL of water to obtain a liquid acellular matrix, and then soak 100mg of drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium composite microspheres obtained in step (2) in the liquid acellular matrix Incubate for 8 hours, wash and freeze-dry to obtain drug-loaded microspheres with acellular matrix on the surface.

Example 5

(1) Mix 1mg bone morphogenetic protein-7 and 10mg mesoporous silicon to obtain a mixed powder of drug and mesoporous silicon, and then disperse the above mixed powder in 30mL containing 1.1g polycaprolactone (molecular weight: 60,000) In the methylene chloride solution of the drug-loaded mesoporous silicon/polycaprolactone, prepare 600 mL of an aqueous solution containing 1.2 g of polyvinyl alcohol 1799, and then slowly add the above blend into the aqueous solution of polyvinyl alcohol 1799, After continuous stirring at 200 rpm for 10 h, the composite microspheres at the bottom of the container were separated to prepare drug-loaded mesoporous silicon/degradable polyester composite microspheres.

(2) Soak 100mg of drug-loaded mesoporous silicon/degradable polyester composite microspheres in 100mL of 5% methanol aqueous solution for 4min, then wash the drug-loaded mesoporous silicon/degradable polyester composite microspheres; 25°C Next, soak 100 mg of pretreated drug-loaded mesoporous silicon/degradable polyester composite microspheres in 30% oxidized chitosan quaternary ammonium salt (oxidation degree 55%) aqueous solution (pH=3) for 8 h, and then Drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium salt composite microspheres were cleaned to obtain drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan coated with oxidized chitosan quaternary ammonium salt Sugar quaternary ammonium compound microspheres.

(3) sequentially use 0.6N hydrochloric acid, organic solvents such as dichloromethane and methanol, 500U/mL DNase and RNase solution, 0.05% disodium ethylenediaminetetraacetic acid, etc. to treat bone tissue, wash and freeze-dry, etc. to obtain Acellular matrix: Dissolve 150mg of acellular matrix in 500mL of water to obtain a liquid acellular matrix, then soak 100mg of drug-loaded mesoporous silicon/degradable polyester/oxidized chitosan quaternary ammonium salt composite microspheres obtained in step (2) Put in the liquid acellular matrix for 5 hours, wash and freeze-dry to obtain the drug-loaded microspheres with the acellular matrix on the surface.

Comparative example 1

The difference of this comparative example and embodiment 1 is not to comprise oxidized chitosan quaternary ammonium salt, and concrete process is:

(2) Soak 100 mg of drug-loaded mesoporous silicon/degradable polyester composite microspheres in 50 mL of 10% sodium hydroxide solution for 6 minutes, and then wash the mesoporous silicon/degradable polyester composite microspheres to obtain a surface pretreated Treated mesoporous silica/degradable polyester composite microspheres.

(3) sequentially adopt organic solvents such as 0.5N hydrochloric acid, chloroform and methanol, 0.25% trypsin, 0.1% sodium lauryl sulfate, etc. to treat the bone tissue and clean and freeze-dry to obtain the decellularized matrix; take 100mg Dissolve the acellular matrix in 300mL water to obtain a liquid acellular matrix, then soak 100mg of the mesoporous silicon/degradable polyester composite microspheres whose surface was pretreated in step (2) in the liquid acellular matrix for 10h, wash and freeze-dry to obtain a surface Drug-loaded microspheres with acellular matrix.

Comparative example 2

The difference between this comparative example and Example 1 is that it does not contain acellular matrix, and the specific process is:

Comparative example 3

The difference between this comparative example and Example 1 is that it does not contain oxidized chitosan quaternary ammonium salt and decellularized matrix, and the specific process is:

(2) Soak 100 mg of drug-loaded mesoporous silicon/degradable polyester composite microspheres in 50 mL of 10% sodium hydroxide solution for 6 minutes, and then wash the drug-loaded mesoporous silicon/degradable polyester composite microspheres to obtain Surface pretreated drug-loaded mesoporous silicon/degradable polyester composite microspheres.

Test case

This test example tested the properties of the drug-loaded microspheres prepared in the examples and comparative examples. Wherein, the test methods and parameters of in vitro cytotoxicity are shown in Table 1, and the test results are shown in Table 2.

Table 1. Cytotoxicity assay methods and parameters

Table 2. In vitro cytotoxicity evaluation results

The results of in vitro cytotoxicity evaluation are shown in Table 2. It can be seen from the table that none of the drug-loaded microspheres prepared by the present invention has cytotoxicity.

The test results of in vitro drug release performance are shown in Figure 1. Example 1 and Comparative Examples 1, 2, and 3 all contain drug-loaded microspheres loaded with alendronate sodium; but Comparative Example 1 does not contain oxidized chitosan quaternary ammonium salt , the adhesion of the acellular matrix on the surface of the drug-loaded mesoporous silicon/degradable polyester composite microspheres is low, resulting in less acellular matrix on the surface of the final drug-loaded microspheres, so it is difficult for the drug release of the drug-loaded microspheres The slowing effect of speed is weaker, and compared with Example 1, its drug release rate is larger, burst release is higher; Comparative Example 2 does not contain acellular matrix, compared with Comparative Example 1, its drug release rate is larger, burst release is higher; The release rate of the drug is higher, indicating that the acellular matrix can effectively delay the release of the drug; Comparative Example 3 does not contain oxidized chitosan quaternary ammonium salt and the acellular matrix, and its drug release rate is the highest, and the burst release is the highest, indicating that the drug-loaded mesoporous The oxidized chitosan quaternary ammonium salt on the surface of silicon/degradable polyester composite microspheres also has the effect of delaying the drug release rate.

Based on the above results, it can be seen that the drug-loaded microspheres prepared by the present invention can maintain good biocompatibility after compounding mesoporous silicon, degradable polyester, oxidized chitosan quaternary ammonium salt and acellular matrix, and endow the drug-loaded The good slow and controlled release function of the drug loaded on the microsphere makes it more suitable for repairing and regenerating tissues, and is also more suitable for the treatment of diseases such as tissue defect, bacterial infection, and inflammation.

The embodiments of the present invention have been described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments, and within the scope of knowledge of those of ordinary skill in the art, various modifications can be made without departing from the gist of the present invention. kind of change. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other if there is no conflict.

Claims

A drug-loaded microsphere, characterized in that the drug-loaded microsphere includes a carrier loaded with a therapeutic agent and a coating layer coated on the surface of the carrier, and the coating layer includes an aldehyde-modified biomacromolecular material layer, and an acellular matrix layer coated on the surface of the aldehyde-modified biomacromolecular material layer.
The drug-loaded microsphere according to claim 1, wherein the coating layer further comprises a degradable material layer, the degradable material layer is coated on the surface of the carrier, and the aldehyde-based modified organism The macromolecular material layer is coated on the surface of the degradable material layer.
The drug-loaded microsphere according to claim 1, wherein the aldehyde-modified biomacromolecular material comprises at least one of oxidized chitosan quaternary ammonium salt, oxidized alginate, and aldehyde-modified gelatin .
The drug-loaded microsphere according to claim 1, characterized in that the particle diameter of the drug-loaded microsphere is 0.005-5 mm.
The drug-loaded microsphere according to claim 1, characterized in that the drug encapsulation rate of the drug-loaded microsphere is 10-95%.
The drug-loaded microsphere according to claim 1, wherein the mass ratio of the therapeutic agent to the carrier is 1:1-30.
The method for preparing drug-loaded microspheres according to any one of claims 1 to 6, characterized in that it comprises the steps of:

Prepare a coating layer on the upper surface of the carrier loaded with therapeutic agent to obtain the drug-loaded microspheres; Acellular matrix layer on the surface of molecular material layer.
A medical composition, characterized in that the medical composition comprises the drug-loaded microspheres according to any one of claims 1 to 6.
A medical device, characterized in that the medical device comprises the drug-loaded microsphere according to any one of claims 1 to 6 and/or the medical composition according to claim 8.
Application of the drug-loaded microsphere according to any one of claims 1 to 6 in the preparation of materials for tissue repair and regeneration, and disease treatment.