CN115947957A - Microsphere composite hydrogel and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the field of medical materials, and particularly relates to microsphere composite hydrogel as well as a preparation method and application thereof. The microsphere composite hydrogel comprises microspheres and hydrogel, wherein the microspheres are dispersed in the hydrogel; the microsphere comprises a degradable polyester material and a mesoporous material wrapped in the degradable polyester material. The microsphere composite hydrogel disclosed by the invention has good biocompatibility and bioactivity, and strong mechanical property and bone mineralization capacity; the microspheres comprise degradable polyester materials and mesoporous materials, the mesoporous materials contain rich mesoporous structures, active drugs (bone repair promoting factors, anti-infection/anti-tumor drugs and the like) can be loaded, and effective slow release of the drugs at the defect parts is realized, so that the microspheres have the effects of accelerating bone healing/resisting infection/anti-tumor, and are a bone defect repair hydrogel system suitable for different causes.

Description

Microsphere composite hydrogel and preparation method and application thereof

Technical Field

The invention belongs to the field of medical materials, and particularly relates to a microsphere composite hydrogel as well as a preparation method and application thereof.

Background

In recent years, patients with bone defects caused by trauma, tumor, infection and the like have increased remarkably, bone defects larger than a critical size are difficult to repair by themselves, and bone transplantation operations related to treatment of bone defects worldwide are performed more than 200 ten thousand times per year, which is the second most common tissue transplantation after blood transfusion. Autologous bone grafting is considered the most desirable option for bone defect surgery, but there are limitations in terms of donor source and donor site morbidity. Therefore, bone tissue engineering is of great interest to researchers. The ideal bone defect repairing biomaterial has biodegradability, biocompatibility and good mechanical property, and can mediate the reaction of bone repairing cells. The natural polymer material is selected to be combined with a better drug carrier model, so that the stem cell osteogenic differentiation can be continuously induced by controlled release of drugs while the bone repair cells are supported, the vascular cells are regulated and controlled to form new blood vessels, the bone repair is facilitated, and the method is a research and development hotspot of bone repair biomaterials.

Compared with artificially synthesized high molecular materials, the high molecular material constructed by natural protein and polysaccharide shows better biocompatibility. Among them, silk Fibroin (SF) is a natural protein derived from bombyx mori cocoons, has good biocompatibility, biodegradability and easy extraction, and thus SF is considered to be a better tissue regeneration biomaterial. However, the weak mechanical properties, the poor osteoinductivity and osteoconductivity limit the use of SF in bone tissue engineering. Therefore, SF is usually combined with other biomaterials such as chitosan and inorganic particles to construct a composite material, so as to enhance the mechanical property, antibacterial property and biological property of the composite material. Carboxymethyl chitosan (CMCS) is a derivative of chitosan. Compared with chitosan, CMCS has higher water solubility and can react with more Ca ²⁺ The chelation has excellent bone mineralization activity and certain antibacterial performance, reduces the infection risk after artificial bone transplantation, promotes the repair of surgical wounds in clinical practical application, but has higher degradation rate. Sodium Alginate (SA) is an anionic copolymer natural polysaccharide derived from brown seaweed, has good biocompatibility, can promote bone tissues to grow in along a scaffold material, has proper biodegradability and viscosity, and is widely applied to tissue engineering. The natural protein and the polysaccharide are combined, so that the mechanical property of the composite material can be improved, the composite material is endowed with certain antibacterial property, and the degradation rate of the scaffold material can be regulated and controlled to adapt to the osteogenesis speed. Although there is a certain electrostatic interaction between amino groups in protein and carboxyl, hydroxyl and other groups in polysaccharide, different proteins and polypeptides are mixed and molded, and a cross-linking agent is still required to be added to solidify the protein into a complex network structure.

Microspheres have long been widely used for drug delivery with their excellent controlled release capabilities. Among them, the degradable polyester microsphere-based composite material has received a great deal of attention. Among them, PLGA is a copolymer of PGA and PLA, and has a wide range of control of degradation time, and thus is widely used for drug release. But the hydrophilicity is poor, and the degradation product is acidic, so the bioactivity is poor, and serious local inflammation is easy to cause, which hinders the application of the degradable polyester biomaterial in bone tissue engineering to a certain extent. The mesoporous hydroxyapatite, MS, MBG and mesoporous silicate particles have good biocompatibility and bioactivity. The MS has a mesoporous structure and a high specific surface area, can be made into a carrier for loading drugs and molecules through surface modification, has a drug slow release function, and is a good drug carrier. How to prepare the bone repair material with excellent comprehensive properties such as biocompatibility, bioactivity, mechanical property, bone mineralization promotion and the like still has important significance.

Disclosure of Invention

The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the microsphere composite hydrogel provided by the invention has good biocompatibility and strong mechanical property, and can promote bone mineralization and accelerate bone defect repair.

The invention also provides a preparation method of the microsphere composite hydrogel.

The invention also provides a preparation method and application of the drug-loaded microsphere composite hydrogel material containing the microsphere composite hydrogel.

In a first aspect of the present invention, a microsphere composite hydrogel is provided, the microsphere composite hydrogel includes microspheres and a hydrogel, the microspheres are dispersed in the hydrogel; the microsphere comprises a degradable polyester material and a mesoporous material wrapped in the degradable polyester material.

Compared with the prior art, the mesoporous material adopted by the invention has abundant mesoporous structure and high specific surface area, can be used as a carrier of medicines and molecules, has the capability of forming mineral calcium phosphate similar to the surface of a bone, and can stimulate mesenchymal stem cells of the bone marrow to differentiate towards osteogenesis through a bone morphogenetic protein 2 pathway and an adenylate activated protein kinase pathway so as to promote the formation of new bone; the mesoporous material and the degradable polyester material are mixed to form the microsphere, so that the biocompatibility and the mechanical property of the microsphere can be effectively improved, and the drug slow-release time of the microsphere is prolonged. Meanwhile, researches show that the compressive strength of the microspheres obtained by wrapping and dispersing the mesoporous material in the degradable polyester material is obviously higher than that of the pure polyester microspheres, and the microspheres are more suitable for bone repair. Therefore, the microsphere composite hydrogel disclosed by the invention has good biocompatibility, biodegradability and strong mechanical property, and simultaneously has good bone mineralization capability, is beneficial to bone repair, and can further load drugs and molecules to realize the sustained release of the drugs and the molecules.

Preferably, the mass of the microspheres is 0.1 to 100%, more preferably 0.5 to 50%, including but not limited to 2%, 6%, 10%, 20%, 30%, 40%, etc., of the mass of the hydrogel.

Preferably, the hydrogel comprises at least one of natural proteins and natural polysaccharides.

Preferably, the mass ratio of the natural protein to the natural polysaccharide is 1:1 to 8, more preferably 1:1 to 6, more preferably 1:1.5 to 4.

Preferably, the natural protein comprises at least one of fibrin, fibrinogen, silk Fibroin (SF), collagen, elastin.

Preferably, the natural polysaccharide comprises at least one of carboxymethyl chitosan (CMCS), starch, hyaluronic acid, sodium Alginate (SA), cellulose, chitosan.

Preferably, the hydrogel comprises a combination of Silk Fibroin (SF), carboxymethyl chitosan (CMCS) and Sodium Alginate (SA), wherein the mass ratio of the silk fibroin to the carboxymethyl chitosan to the sodium alginate is 1:0.1 to 5:0.1 to 5; more preferably 1:1:0.5, including but not limited to 1:1:0.5,1:1:1,1:1:1.5, etc.

Preferably, when the hydrogel comprises fibrin and chitosan, the mass ratio of the fibrin to the chitosan is 1:0.1 to 5, more preferably 1:3 to 5, including but not limited to 1:3,1:4,1:5, and the like.

Preferably, when the hydrogel comprises collagen and sodium alginate, the mass ratio of the collagen to the sodium alginate is 1:0.1 to 5, more preferably 1:3 to 5, including but not limited to 1:3,1:4,1:5, and the like.

Preferably, the hydrogel further comprises a crosslinking agent comprising at least one of a protein crosslinking agent, an ionic crosslinking agent, including but not limited to genipin, 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide, metal salts, and the like. The invention selects the protein cross-linking agent and the ion cross-linking agent with good biocompatibility, which have no cytotoxicity, do not influence the biocompatibility, have high cross-linking degree and are beneficial to improving the mechanical strength and the stability of the hydrogel.

Preferably, the cross-linking agent comprises 5 to 30% by mass of the hydrogel, more preferably 9 to 22% by mass, including but not limited to 9%, 12.5%, 13%, 22%, etc.

Preferably, the cross-linking agent comprises at least one of genipin and a metal salt; the mass ratio of the genipin to the metal salt is 1-20: 1, more preferably 1 to 15:1, including but not limited to 1.25:1,4:1,10: 1,12: 1,15: 1, etc.

Preferably, the ratio of genipin to the total mass of hydrogel (natural protein and natural polysaccharide) is 1:5 to 30, more preferably 1:5 to 20, including but not limited to 1:5,1:8,1:9,1:10,1:11,1:12,1:15,1:20, etc.

Preferably, the metal salt comprises CaCl ₂ 、SrCl ₂ 、CuCl ₂ 、ZnCl ₂ Of the metal salt to the hydrogel in a ratio of 1:10 to 150, more preferably 1:25 to 100, including but not limited to 1:35,1:37,1:40,1:42,1:45,1:50,1:100, etc. The calcium, strontium, copper and zinc ions can further promote the repair and reconstruction of tissues (such as bone tissues).

Preferably, the mesoporous material includes at least one of Mesoporous Silicon (MS), mesoporous bioglass, mesoporous calcium silicate, mesoporous zinc silicate, mesoporous strontium silicate, and mesoporous magnesium silicate. The silicon, calcium, zinc, strontium and magnesium ions in the mesoporous material can further promote the repair and reconstruction of tissues.

Preferably, the mesoporous material is a sphere or a sphere-like body, and the particle size of the mesoporous material is 100nm to 1 μm, more preferably 200nm to 600nm.

Preferably, the pore diameter of the mesoporous material is 1 to 20nm, more preferably 1 to 10nm.

Preferably, the mesoporous material is wrapped in the degradable polyester material, and the mass ratio of the mesoporous material to the degradable polyester is 1:0.2 to 20, more preferably 1:0.5 to 10, including but not limited to 1:0.5,1:3,1:6,1:10, etc.

Preferably, the microspheres have a particle size of 30 to 500. Mu.m, more preferably 90 to 150. Mu.m.

Preferably, the degradable polyester comprises at least one of polylactic-co-glycolic acid (PLGA), polyglycolic acid (polyglycolic acid), polylactic acid (PLA), polyhydroxyalkanoate (PHA), polycaprolactone (PCL), polytrimethylene carbonate, polybutylene succinate, epsilon-polylysine.

In a second aspect of the present invention, a method for preparing the microsphere composite hydrogel comprises the following steps:

and dispersing the microspheres in hydrogel to obtain the microsphere composite hydrogel.

Preferably, the preparation method of the microsphere composite hydrogel comprises the following steps: dissolving and mixing natural protein and natural polysaccharide, adding a cross-linking agent for mixing and cross-linking, and adding microspheres to obtain the microsphere composite hydrogel.

Wherein the crosslinking agent is added into the system in a solution form, the crosslinking agent solution comprises a genipin solution and a metal salt solution, and the mass concentration of the genipin is 0.1-10%, more preferably 0.25-0.5%; the volume dosage is determined according to the mass ratio of genipin to hydrogel (natural protein and natural polysaccharide) in the microsphere composite hydrogel, for example, about 1mL genipin solution is added per 100mg of mixture of natural protein and natural polysaccharide.

Preferably, the mass concentration of the metal salt solution is 10-20%, and the volume dosage is determined according to the mass ratio of the metal salt to the hydrogel (natural protein and natural polysaccharide) in the microsphere composite hydrogel, for example, 25 μ L of the metal salt solution is added to each 100mg of the mixture of the natural protein and the natural polysaccharide.

Preferably, the microsphere shell is prepared by one of an emulsion solvent evaporation method, a microfluidic method, a phase separation method, a salting-out method, a spray drying method, a membrane emulsification method and a nano-precipitation method, and more preferably, the emulsion solvent evaporation method.

Preferably, the microsphere is prepared by a preparation method comprising the following steps: mixing the degradable polyester material and the mesoporous material, adding a surfactant solution, stirring and separating to obtain the microsphere with the surface coated with the degradable polyester material.

Preferably, the mass ratio of the degradable polyester material to the mesoporous material is 1-10: 1, more preferably 5 to 10:1, more preferably 6 to 7:1.

preferably, the preparation method of the microsphere comprises the steps of dissolving the degradable polyester material and the mesoporous silicon material in dichloromethane to obtain a mixed solution, adding the surfactant, stirring for 12 hours, performing centrifugal separation, and cleaning to obtain the microsphere. The solvent of the mixed solution comprises at least one of dichloromethane and ethanol.

Preferably, the microspheres obtained by washing can be further subjected to freeze-drying and cryopreservation.

Preferably, the surfactant comprises at least one of polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyethylene glycol (PEG), gelatin, methyl cellulose.

Preferably, the mesoporous silicon used in the invention can be prepared from commercially available products or by self-making, and more preferably, the mesoporous silicon is prepared by a template method; specifically, dodecylamine and tetraethyl orthosilicate are dissolved in absolute ethyl alcohol/water solution and are mixed and reacted to obtain Mesoporous Silicon (MS).

Preferably, the mass volume ratio of the dodecylamine to the tetraethyl orthosilicate is 1g:3 to 5mL, more preferably 1g: 4-4.5 mL.

Preferably, the preparation method of the mesoporous silicon comprises the steps of mixing a dodecylamine solution with a tetraethyl orthosilicate solution, stirring, aging, extracting, drying and calcining to obtain the mesoporous silicon; the stirring time is 15 to 20 hours, and about 18 hours is more preferable; the aging time is 20-50 min, and is more preferably about 30 min; the solvent used for extraction is hydrochloric acid ethanol solution, and the extraction time is 3-6 h, more preferably about 4 h; the drying temperature is 70-100 ℃, more preferably about 80 ℃, and the drying time is 4-16 h, more preferably about 12 h; the calcination temperature is 400-800 ℃, more preferably 600-700 ℃, and the calcination time is 2-4 h, more preferably 2-3 h.

Preferably, the mass-to-volume ratio of the dodecylamine to the solvent in the dodecylamine solution is 1g: 1-2 mL, more preferably 1g: 1-1.5 mL; the solvent of the dodecylamine solution comprises ethanol and water, and the mass ratio of the ethanol to the water is 1:1 to 1.3, more preferably 1:1 to 1.15.

Preferably, the volume ratio of tetraethyl orthosilicate to solvent in the tetraethyl orthosilicate solution is 1-1.5: 1, more preferably 1 to 1.2:1. the solvent of the tetraethyl orthosilicate solution comprises at least one of ethanol and water.

Preferably, the source of the silk fibroin is not limited, and a commercially available product can be selected and can also be obtained by self-making; more preferably, the silk fibroin is prepared by a preparation method comprising the following steps:

step S11, placing the silkworm cocoons without the silkworm chrysalis in a sodium carbonate solution, and heating and degumming for two times;

and S12, mixing the lithium bromide solution with the degummed silkworm cocoons, and filtering to obtain the silk fibroin.

Preferably, in step S11, the mass-to-volume ratio of the silkworm cocoons from which the silkworm pupas are removed to the sodium carbonate is 1g: 40-60 mL; more preferably 1g: about 50 mL.

Preferably, in step S11, the concentration of the sodium carbonate solution used in the first heating degumming is 1.0 to 2.0wt%, and more preferably about 1.0 wt%; the concentration of the sodium carbonate solution used in the second heat degumming is 0.5 to 0.9wt%, more preferably about 0.5 wt%.

Preferably, in step S11, the step of finishing the first heating degumming further includes cleaning, the cleaning is performed for 5 to 7 times by using water, kneading is performed during the cleaning process, and the second heating degumming is performed after the cleaning is finished.

Preferably, in step S11, the temperature for twice heating degumming is independently selected from 80 to 110 ℃, more preferably 90 to 100 ℃; the heating time is 20 to 50min, more preferably about 30 min.

Preferably, in step S11, the silkworm cocoons obtained after twice degumming are further dried, wherein the drying temperature is 50 to 70 ℃, and more preferably about 60 ℃.

Preferably, in step S12, the mass ratio of the lithium bromide to the degummed silkworm cocoon is 5 to 10:1, more preferably 8 to 10:1.

preferably, 8-10 layers of filter cloth are adopted for filtering in the step S12, centrifugation, dialysis and freeze-drying treatment are further included after the filtering, and the rotation speed of the centrifugation is 6000-7000 rpm, more preferably 6700rpm; the centrifugation time is 5-20 min, and more preferably about 10 min; the cut-off molecular weight of the dialysis bag used for dialysis is 7000-9000 Da, more preferably 8000Da, and the dialysis time is 2-4 days, more preferably about 3 days.

The third aspect of the invention provides a drug-loaded microsphere composite hydrogel, which comprises the microsphere composite hydrogel and an active drug loaded in the microspheres.

In the invention, the microspheres in the microsphere composite hydrogel comprise a degradable polyester material and a mesoporous material wrapped in the degradable polyester, wherein the mesoporous material contains abundant mesoporous structures and high specific surface area, can be used as a carrier of an active drug, further loads the active drug and can prolong the release of the drug; the degradable polyester material can protect the medicine in the mesoporous structure from being damaged or inactivated, and can improve the slow release effect; the hydrogel can further prolong the release process of the drug. The drug-loaded microsphere composite hydrogel prepared by the invention has good biocompatibility, mechanical property and bone mineralization capability, and also has good drug slow release effect.

Preferably, the drug-loading rate of the drug-loaded microsphere composite hydrogel is 0.00001-10%, more preferably 0.1-5%, and even more preferably about 1.5%.

Preferably, the active drug is not limited, and different active drugs can be selected according to the required curative effect, including but not limited to bone repair promoting factors, anti-infection/anti-tumor drugs and the like.

In a fourth aspect of the invention, the preparation method of the drug-loaded microsphere composite hydrogel comprises the following steps:

s31, mixing the mesoporous material with an active drug to obtain a drug-loaded mesoporous material;

s32, mixing the drug-loaded mesoporous material with the degradable polyester material, adding a surfactant solution, stirring and separating to obtain the drug-loaded microspheres;

s33, dispersing the drug-loaded microspheres in hydrogel to obtain the drug-loaded microsphere composite hydrogel.

In step S31, the mass of the active drug is preferably 0.0001 to 50% of the mass of the mesoporous material, more preferably 0.1 to 30%, and still more preferably about 20%.

In the invention, the preparation method of the drug-loaded microsphere composite hydrogel and the microsphere composite hydrogel are mainly different in that the mesoporous material is loaded with the active drug before being coated with the degradable polyester material, and other experimental processes are similar.

In the fifth aspect of the invention, the microsphere composite hydrogel and the drug-loaded microsphere composite hydrogel are applied to preparation of bone implantation or bone repair materials, anti-infection and anti-tumor drugs.

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

the microsphere composite hydrogel disclosed by the invention has good biocompatibility, stronger mechanical property and certain bone mineralization and biological activity, and the microsphere comprises a degradable polyester material and a mesoporous material wrapped in the degradable polyester material, wherein the mesoporous material contains rich mesoporous structures, and can be loaded with active drugs (such as bone repair promoting factors, anti-infection/anti-tumor drugs and the like) to realize effective slow release of drugs at a defect part, so that the microsphere composite hydrogel can have the functions of accelerating bone healing/anti-infection/anti-tumor, and is a hydrogel system suitable for bone defect repair caused by different causes.

Drawings

The invention is further described with reference to the following figures and examples, in which:

FIG. 1 characterization of MS/PLGA microspheres of comparative example 1: (a) SEM images; (b) particle size distribution; (c) energy spectrum scanning.

FIG. 2 characterization of comparative example 2 and examples 1-3: (a) FITR spectroscopy; (b) an XRD pattern; (c) SEM image.

FIG. 3 mechanical properties of comparative example 2 and examples 1 to 3, and drug release of comparative example 3 and example 4:

(a) A stress-strain curve; (b) stress at 60% strain; (c) a compressive modulus of elasticity between 5% and 15% strain;

(d) The cumulative release amount of rhodamine.

FIG. 4 Performance of comparative example 2 and examples 1 to 3 in buffer: (a) swelling ratio, (b) saturation swelling ratio, (c) porosity, (d) degradation ratio in the absence of enzyme, and (e) degradation ratio in the presence of enzyme.

FIG. 5 evaluation of cell compatibility for comparative example 2 and examples 1 to 3: (a) live-dead staining experiments; (b) cell proliferation assay.

FIG. 6 osteogenic Properties of comparative example 2 and examples 1 to 3: (a) ALP staining and ARS staining; (b) day 7 expression of osteogenesis related genes; (c) bone formation-related gene expression at day 14.

Detailed Description

The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the objects, features and effects of the present invention. It is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments, and those skilled in the art can obtain other embodiments without inventive effort based on the embodiments of the present invention, and all embodiments are within the protection scope of the present invention.

The starting materials used in the following examples, unless otherwise specified, are available from conventional commercial sources; the adopted process adopts the conventional process in the field if no special indication is provided; the operating temperatures used, unless otherwise specified, are room temperature (20. + -. 5 ℃ C.).

Example 1

This example prepares an SF/CMCS/SA composite hydrogel containing 0.5% MS/PLGA microspheres

1. Synthesis of MS/PLGA microspheres

Preparation of MS: solution A was obtained by dissolving 10g of dodecylamine (DDA) in 70mL of absolute ethanol and 80mL of distilled water at room temperature, sonicating for a few minutes and then magnetically stirring for 1 h. Solution B was prepared by mixing 44.6mL of hot Tetraethylorthosilicate (TEOS) with 40mL of anhydrous ethanol at room temperature for about 30min with magnetic stirring. The two solutions were then mixed with stirring at room temperature for about 18h. The mixture was then aged for 30min, washed with 800mL distilled water, extracted with 600mL HCl-ethanol for 4h, then dried at 80 ℃ for 12h, and finally calcined at 600 ℃ for 3h to give MS powder.

MS/PLGA microspheres were prepared by a modified single emulsion solvent evaporation method, dissolving 1g PLGA and 150mg MS powder in 8mL dichloromethane, sonicating for several minutes, adding 1.0% PVA aqueous solution, mechanically stirring for 12h. And washing the obtained MS/PLGA microspheres for 5 times by using deionized water, and finally freeze-drying and storing at low temperature for later use.

2. Extraction of SF

Placing a certain amount of silkworm pupa-removed silkworm cocoons into 1.0wt% anhydrous sodium carbonate solution (wherein 50mL of anhydrous sodium carbonate solution is not required for 1g of silkworm cocoons), boiling at 95 ℃ for 30min, washing with deionized water for 7 times after boiling, kneading during the washing process, changing into 0.5wt% anhydrous sodium carbonate solution, boiling once again under the same conditions, and repeating the above operations. After degumming, drying the silkworm cocoons in an oven at 60 ℃, and sealing and storing.

Preparing 40g of lithium bromide into 50mL of solution, adding 5g of degummed silkworm cocoon into the solution, and putting the solution into a shaking table until the solution is completely dissolved. The solution was filtered using 8 layers of gauze and centrifuged at 6700rpm for 10min, transferred to a dialysis bag (8000 Da) after centrifugation for 3 days and finally lyophilized for use.

3. Preparation of 0.5% by weight MS/PLGA microsphere-loaded SF-CMCS-SA hydrogel

120mg of SF is placed in 2.925mL of deionized water to be stirred and dissolved, and 120mg of CMCS is added to be stirred and dissolved after the SF is completely dissolved. After dissolution, 60mg of SA was added and dissolved with stirring. After dissolution, 3mL of 1% genipin solution and 75. Mu.L of 10% CaCl were added ₂ And (3) uniformly stirring the solution, taking out a magnetic stirrer, adding the MS/PLGA microspheres into the prepared solution according to the proportion of 0.5%, finally placing the solution into a mould, curing the solution at 37 ℃ for 30min, freezing the cured solution in a refrigerator, freeze-drying and storing the frozen solution. Final SF, CMCS, SA, microspheres, genipin and CaCl ₂ The final concentrations of (A) were 2%,2%,1%,0.5%,0.5% and 0.125%, respectively.

Example 2

This example is a method for preparing a SF/CMCS/SA composite gel containing 1% MS/PLGA microspheres comprising the steps of:

after synthesizing MS/PLGA microspheres and extracting SF according to the method of example 1, SF, SA, CMCS and microspheres are mixed according to the final concentration ratio of 2%,1%, 2% and 1%, after stirring uniformly, genipin solution and CaCl are added ₂ Placing the solution in a mold, solidifying at 37 deg.C for 30min, freezing in refrigerator, lyophilizing, and storing. Genipin solution and CaCl ₂ The final concentration of the solution was 0.25% and 0.2%.

Example 3

This example is a method for preparing SF/CMCS/SA composite gel containing 2% MS/PLGA microspheres comprising the steps of:

after synthesizing MS/PLGA microspheres and extracting SF according to the method of example 1, SF, SA, CMCS and microspheres are mixed according to the final concentration ratio of 2%,1%, 2% and 2%, after stirring uniformly, genipin solution and CaCl are added ₂ Placing the solution in a mold, solidifying at 37 deg.C for 30min, freezing in refrigerator, lyophilizing, and storing. Genipin solution and CaCl ₂ The final concentration of the solution was 0.25% and 0.2%.

Example 4

In the embodiment, rhodamine B is used as a small molecule drug model, and rhodamine B is loaded on the 1% microsphere composite hydrogel in embodiment 2 as an example for explanation, and the specific process is as follows:

1. drug-loaded MS/PLGA microspheres: putting 1g of MS (prepared in example 1) in 200mg of rhodamine solution, centrifuging to remove supernatant, and drying to obtain drug-loaded MS powder; PLGA and drug-loaded MS powder are dissolved in dichloromethane to obtain solution A, PVA solution is prepared to obtain solution B, and then solution A and solution B are mixed and mechanically stirred for 12 hours. Then washing by deionized water, and finally freeze-drying and storing at low temperature for later use to obtain the microspheres with the drug loading of 1.5%.

2.1% drug-loaded MS/PLGA composite hydrogel: extracting SF according to the method of example 1, mixing SF, SA and CMCS solutions according to the final concentration ratio of 2%,1% and 2%, adding medicine-carrying MS/PLGA microspheres into the prepared solution after uniformly mixing, adding genipin solution and CaCl after uniformly stirring ₂ Placing the solution in a mold, solidifying at 37 deg.C for 30min, freezing in refrigerator, lyophilizing, and storing. Genipin solution and CaCl ₂ The final concentration of the solution was 0.25% and 0.2%.

Example 5

The embodiment prepares the SF/CMCS/SA composite hydrogel containing 1% mesoporous bioglass/PLGA microspheres, and the specific process is as follows:

mixing SF, SA and CMCS solutions according to the final concentration ratio of 2 percent, 1 percent and 2 percent, adding 1 percent of mesoporous bioglass/polylactic acid microspheres into the prepared solution after uniformly mixing, adding genipin solution and SrCl after uniformly stirring ₂ Placing the solution in a mold, solidifying at 45 deg.C for 10min, freezing in refrigerator, lyophilizing, and storing. Genipin solution and SrCl ₂ The final concentration of the solution was 0.25% and 0.2%.

Example 6

The embodiment prepares a fibrin/chitosan composite hydrogel containing 1% mesoporous calcium silicate/PLGA microspheres, and the specific process comprises the following steps:

mixing fibrin and chitosan according to the final concentration ratio of 1% and 4%, adding 1% mesoporous calcium silicate/PLGA microspheres into the prepared solution after uniformly mixing, adding genipin and ZnCl after uniformly stirring ₂ Placing the solution in a mold, solidifying at 45 deg.C for 10min, freezing in refrigerator, lyophilizing, and storing. Genipin solution and ZnCl ₂ The final concentration of the solution was 1% and 0.1%.

Example 7

In this example, a collagen/SA composite hydrogel containing 1% mesoporous calcium silicate/PLGA microspheres is prepared, and the specific process is as follows:

mixing collagen and SA at a final concentration ratio of 1% to 4%, adding 1% mesoporous calcium silicate/PLGA microspheres into the prepared solution, stirring, and adding genipin solution and CuCl ₂ Placing the solution in a mold, solidifying at 45 deg.C for 10min, freezing in refrigerator, lyophilizing, and storing. Genipin solution and CuCl ₂ The final concentrations of the solutions were 0.6% and 0.05%.

Comparative example 1

MS/PLGA microspheres were synthesized according to the method of example 1.

Comparative example 2

The embodiment is a preparation method of SF/CMCS/SA composite gel without microspheres, which comprises the following steps:

after extracting SF according to the method of example 1, the SF, SA and CMCS solutions are mixed according to the final concentration ratio of 2%,1% and 2%, and after mixing uniformly, the genipin solution and CaCl are added ₂ Stirring the solution, placing in a mold, solidifying at 37 deg.C for 30min, freezing in refrigerator, lyophilizing, and storing. Genipin solution and CaCl ₂ The final concentration of the solution was 00.5% and 0.125%.

Comparative example 3

The comparative example is a preparation method of a pure drug-loaded microsphere, and MS/PLGA is synthesized by using rhodamine as a model drug according to the method of the example 4.

Test examples

The experimental example tests the performance of the microsphere composite hydrogel prepared in the example and the comparative example, and the performance of the drug-loaded composite hydrogel. Wherein:

(1) Morphological characterization of microspheres and microsphere composite hydrogels

Microsphere morphology of comparative example 1 and composite gels of examples 1-3 characterization: adhering microsphere (non-drug-loaded microsphere and drug-loaded microsphere) powder and 4 microsphere gels with different solid contents to a sample table by using conductive adhesive, spraying gold on the sample table, and observing the shapes of the microsphere and the gel by using a table type scanning electron microscope and carrying out energy spectrum scanning analysis.

As shown in FIG. 1, the microspheres prepared in comparative example 1 have good effect of forming spheres, have rough surfaces and MS particle coverage, and are good in uniformity of the diameter of the microspheres, which is 113.79. + -. 23.87. Mu.m, due to the dispersion of particles on the polyester surface during the emulsification process. EDS scanning results show that the microsphere contains C, O and Si elements, and is consistent with raw material elements used for preparing the microsphere.

The microspheres with different contents are loaded into an SF/CMCS/SA gel system, and by observing the morphology of the composite gel, the microspheres are more uniformly distributed in a gel network and are tightly combined with the gel as shown in figure 2 b. Although the pores of the composite gel show a reduction trend along with the increase of the content of the microspheres, the macroporous structure of the gel is still maintained. EDS energy spectrum scanning analysis shows that C, N, O, na, cl and Ca exist in the composite gel; since the 0.5%, 1%, and 2% groups contain microspheres with different solid contents, the spectral scan results show that the composite gel also contains Si element.

(2) Infrared scanning of microsphere composite hydrogels

Infrared scanning of comparative example 2 and examples 1 to 3: loading microspheres with different contents in comparative example 1 into SF, CMCS and SA composite gel, freeze-drying, and testing with ATR-FTIR mode of Fourier infrared spectrum converter, wherein specific parameters are set to 600-4000cm ^-1 The resolution ratio of the range test is 4cm ^-1 And scanning is carried out for 32 times in an accumulated way, and finally the infrared absorption spectrum is obtained.

The results are shown in FIG. 2a, and the infrared spectrum shows 3283cm ^-1 The stretching vibration peak at-OH is at 1644, 1529 and 1231cm ^-1 Characteristic absorption bands of the amide I, amide II and amide III bands of SF, respectively, are observed, which are related to the beta sheet conformation of SF. The amide I belt mainly comprises C = O stretching vibration and C-N stretching vibration, the amide II belt mainly comprises N-H in-plane bending vibration and C-N stretching vibration, and the amide III belt mainly comprises C-N stretching vibration and N-H in-plane bending vibration. 1024. 1308, 1419 and 1616cm ^-1 Stretching vibration, C-N stretching vibration, symmetrical stretching vibration of-COO and asymmetrical stretching vibration of-COO of primary alcohol C-O bond of CMCS, 1419 cm and 1616cm ^-1 And the symmetric stretching vibration peak and the asymmetric stretching vibration peak of-COO of SA are respectively positioned at the position. The infrared energy of microsphere gels with different solid contents corresponds to characteristic absorption peaks on SF, CMCS and SA.

(3) X-ray diffraction (XRD) characterization of microsphere composite hydrogels

Characterization by X-ray diffraction (XRD) for comparative example 2 and examples 1 to 3: after freeze-drying the prepared gel, testing the gel by using an X-ray diffractometer, and measuring the content of CuK alpha rays and the X-ray wavelength:

the super-energy detection counter records a diffraction intensity curve between 2 theta = 10-60 degrees, and the scanning speed is 2 degrees/min. And finally obtaining the XRD pattern of the sample. As a result, as shown in FIG. 2c, the XRD characteristic diffraction peaks of gels containing different amounts of MS/PLGA microspheres were approximately the same, and were all around 20 °, which is consistent with the XRD characteristic peaks of CMCS and SF. Due to the addition of CaCl ₂ The solution underwent cross-linking curing, so its characteristic main peak appeared at 0% of the set (comparative example 2). This indicates that SF/CMCS/SA gels of different solids MS/PLGA microspheres did not change the respective secondary structures.

(4) Mechanical property analysis of microsphere composite hydrogel

Mechanical property analysis of comparative example 2 and examples 1 to 3: and soaking the gel in PBS buffer solution, performing a compression test on a mechanical testing machine after the gel reaches the swelling balance, and testing the mechanical properties of the four groups of bracket materials. The diameter of the bracket is about 6-7 mm, and the height is 4-6 mm. The test was set at 2mm/min. The stress (σ) at 5% to 15% strain is calculated according to equation 1:

σ = F/sx100 formula 1

F and S represent the load area and the compression area, respectively.

The results are shown in fig. 3 a-c, which respectively show the stress-strain curve, the average compressive strength at 60% strain and the average compressive modulus of microsphere composite gels with different solid contents. With the increase of the solid content of the microspheres, the mechanical properties of the composite gel are gradually enhanced and then weakened, which may be related to the sedimentation and uneven distribution of the microspheres, and compared with other groups, 1% of the microsphere composite hydrogel (example 2) has the strongest mechanical properties. The average compressive strength and modulus of elasticity at 60% strain of example 2 were 415.24. + -. 3.72kPa and 13.13. + -. 1.38kPa, respectively, which are much higher than those of comparative example 1 (179.36. + -. 13.47kPa, 1.48. + -. 0.61 kPa).

(5) Drug release properties of microsphere composite hydrogels

Drug release of example 4 and comparative example 3: rhodamine B is used as a small molecule drug model, and in-vitro drug release performance tests are performed on drug-loaded microspheres (comparative example 3) and 1% drug-loaded microsphere composite gel (example 4). The drug-loaded microspheres and 1% drug-loaded microsphere composite gel are respectively put into a 10mL centrifuge tube, the volume is determined to be 5mL by PBS buffer solution, and the centrifuge tube is placed in a shaking table at 37 ℃ for slight oscillation to carry out in-vitro release test. And taking 1.5mL of supernatant respectively at preset time, filtering and detecting the concentration of rhodamine B by using an ultraviolet-visible spectrophotometry. 1.5mL of fresh PBS solution was immediately supplemented after each sampling, and the cumulative release rate was calculated.

The results are shown in fig. 3d, and comparative example 3 and example 4 did not produce burst release of the drug at the previous stage, indicating that the microspheres have better drug sustained release effect. The drug of comparative example 3 began to be slowly released at 7 days as the microspheres degraded, whereas the microsphere/gel composite of example 4 released more than comparative example 3 before 7 days, which is that the microspheres released a portion of the drug during the preparation of the microsphere composite gel. After 14 days, the drug amount released by the microspheres is greatly increased, the microsphere composite gel is slowly released, the swelling performance and the degradation performance of the gel are not separated, the drug of the microspheres in the gel shallow layer network is firstly released, and the gel can form a coating layer on the surfaces of the microspheres, so that the controlled release performance of the gel is favorably improved. The drug release time of each group of materials reaches more than 2 months, which shows that the constructed microspheres and gel have good controlled release and slow release performance.

(6) Swelling property, porosity and degradation property of microsphere composite hydrogel

Swelling, porosity and degradation properties of comparative example 2 and examples 1 to 3: lyophilizate material (weight W ₁ ) Soak in 10ml EP tube, add 5ml PBS solution (pH = 7.4), placed in 37 degrees C in 90rpm shaking table, until the gel swelling to reach the balance. During which the excess water on the surface is adsorbed by a filter paper every 2 hours, and the gel is weighed as W ₂ Measuring the weight of the swollen gel until it no longer changes, recording the weight of the fully swollen gelAnd (4) weight. The swelling ratio SI was then calculated according to the formula.

SI＝ (W ₂ - W ₁ ) / W ₁ *100% of formula 2

The freeze-dried microsphere gel with different solid contents is immersed in absolute ethyl alcohol until the microsphere gel is saturated, and no air bubbles come out. Before immersion in absolute ethanol (W) ₃ ) And thereafter (W) ₄ ) The gel was weighed. The porosity P is calculated using the following formula:

P＝(W ₄ -W ₃ )/ρ _ethanol V _Gel *100% of formula 3

Wherein, W ₃ And W ₄ Respectively, the weight of the sample before and after immersion in alcohol, V _Gel Is the volume of the gel, p _Ethanol Is a constant (density of absolute ethanol at room temperature).

The degradation behavior of the scaffold material was tested in PBS (volume ratio 1: 100) with or without protease type XIV (2U/mL) in a shaker at 37 ℃ at 90 rpm. The initial weight (W) of the dried sample was determined ₅ ). Samples were washed with water at regular intervals, lyophilized and weighed (W) ₆ ). And calculating the degradation rate according to a formula.

Degradation rate (%) = (W) ₅ –W ₆ )/W ₅ *100% equation 4

The results are shown in FIGS. 4 a-b, showing the swelling properties of the composite gel. With the increase of time, the four groups of composite gels show the fastest water absorption within 0.h, the water absorption amount is still increased after 0.5h, but the water absorption rate is small and slowly stable, and the equilibrium swelling level is reached after 24 h. The equilibrium swelling of the microsphere composite gel is reduced with the increase of the solid content of the microspheres, and the water absorption capacity is reduced, and the equilibrium swelling ratios of 0% (comparative example 1), 0.5% (example 1), 1% (example 2) and 2% (example 3) are 1573.56 +/-2.85%, 1272.99 +/-4.05%, 1116.02 +/-27.25 and 952.58 +/-42.32%, respectively. Fig. 4c shows the porosity of each set of composite gels, with all microsphere gels having a porosity of over 80%, indicating that the composite gels have good porosity. The porosity is reduced along with the increase of the solid content of the microspheres, which shows that the larger the solid content of the microspheres is, the more compact the formed three-dimensional network structure is. Fig. 4d and 4e present the degradation performance of the sets of complex gels in the presence and absence of enzyme. With the increase of the solid content of the microspheres, the degradation rate of each group of composite gel is in a descending trend, but the degradation rate is approximately the same and is approximately stable after being accelerated. The degradation rate of the composite gel under the enzyme condition is obviously faster than that under the enzyme-free condition, however, after 28 days, each group of composite gel still remains about half, which shows that the composite gel has good degradability.

(7) Cytotoxicity test and live-dead staining of microsphere composite hydrogel

Cytotoxicity experiments: firstly, preparing a leaching liquor of the microsphere composite gel, mixing the leaching liquor with a complete culture medium 1:1, mixing the suspension with cells to obtain cell suspension, placing the cell suspension in the wells of a 48-well culture plate, wherein 2 ten thousand cells are placed in each well, and replacing the culture medium every 2 days. Cell proliferation was detected by the CCK-8 method at 1 st, 3 nd and 7d of the culture, respectively.

Cell live and dead staining experiment: respectively culturing the cells in a pore plate for 1d and 7d, and then detecting the activity of the mesenchymal stem cells in the microsphere gel with different solid contents by using a live/dead cell staining kit

The results are shown in FIG. 5, and the CCK-8 results show that each group of microsphere/gel materials has good cell compatibility, and the cell activity of example 3 is slightly lower than that of comparative example 2. While the staining results of the live and dead cells showed that the cells of comparative example 2 and examples 1-2 were very active and proliferated well, the cells of example 3 were less numerous than those of comparative example 2, and the results of CCK-8 were consistent.

(8) Evaluation of osteogenic Properties of microsphere composite hydrogels

The osteogenic differentiation performance of comparative example 2 and examples 1 to 3 was characterized: firstly, preparing a leaching liquor of the microsphere composite gel, mixing the leaching liquor with a complete culture medium 1:1, mixing the suspension with cells to form cell suspension, and placing the cell suspension in 48 holes for culturing to evaluate the osteogenesis performance.

Alkaline phosphatase (ALP) staining experiment: placing microsphere gels with different solid contents into the wells of 24-well culture plate, wherein each well is 2.5 × 10 ⁴ Rat BMSCs. After 24h of culture, changing the cell culture medium for osteogenic induction differentiation cultureAnd culturing the culture medium for 7d and 14d respectively, and replacing the culture medium every 2 d. After termination of the incubation, the gel samples were washed three times with pre-cooled PBS and fixed with 4% paraformaldehyde fixing solution, then stained using ALP staining kit according to the instructions and visualized by photography.

Alizarin Red (ARS) staining experiment: placing microsphere gels with different solid contents into the wells of a 24-well culture plate, wherein each well is 2.5 × 10 ⁴ Rat BMSCs. After 24h of culture, the cell culture medium was changed to osteogenic induction differentiation medium, and the osteogenic induction medium was changed every 2 d. And (3) after culturing for 7d and 21d, removing the original culture medium, washing for 2 times by using a PBS (phosphate buffer solution), fixing each group of cells by using an alizarin red staining fixing solution, adding the alizarin red staining solution into each hole after washing by using the PBS to cover the cells, incubating and staining for 30min at room temperature, washing the staining solution by using deionized water, photographing under an inverted microscope, and observing the formation amount of calcium nodules.

Quantitative real-time polymerase chain reaction: placing microsphere gels with different solid contents into the wells of 24-well culture plate, wherein each well is 2.5 × 10 ⁴ Rat BMSCs. After 24h of culture, the cell culture medium was changed to osteogenic induction differentiation medium, and the medium was changed every 2 d. Collecting cells in the pore plates at 7d and 14d, extracting total RNA of each group of cells, and detecting osteogenic related genes OPN and RunX2 by a real-time quantitative RT-PCR method by taking the cDNA of a reverse transcription reaction product as a template.

FIG. 6a shows that examples 1 to 3 all have good osteogenic properties. From ALP staining it can be seen that each of the composite gels showed some osteogenic properties, with 1% (example 2) and 2% (example 3) groups being significantly stronger than 0% (comparative example 2) and 0.5% (example 1). As can be seen from the ARS staining results, more red-stained calcium nodules can be observed under a microscope with the increase of the solid content of the microspheres, which indicates that the osteogenic differentiation performance is also obviously enhanced.

FIGS. 6b and 6c show the expression of OPN and RunX2 genes at 7day and 14day, respectively, and OPN and RunX-2 are osteogenesis-related genes. From the figure, it can be found that the OPN and RunX2 gene expression of 14day cultured is significantly higher than that of 7day cultured, and the osteogenic gene expression of 1% and 2% of the groups is significantly higher than that of 0% and 0.5% of the groups. As described above, the composite gel of example 2, which contains 1% of MS/PLGA microspheres, has the best osteogenic properties.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and should not be taken as limiting the scope of the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. The microsphere composite hydrogel is characterized by comprising microspheres and hydrogel, wherein the microspheres are dispersed in the hydrogel; the microsphere comprises a degradable polyester material and a mesoporous material wrapped in the degradable polyester material.

2. The microsphere composite hydrogel of claim 1, wherein the mass of the microspheres is 0.1 to 100% of the mass of the hydrogel.

3. The microsphere composite hydrogel of claim 1, wherein said hydrogel comprises at least one of native proteins, native polysaccharide molecules.

4. The microsphere composite hydrogel of claim 1, wherein said hydrogel further comprises a crosslinking agent;

the cross-linking agent comprises at least one of genipin and metal salt.

5. The microsphere composite hydrogel of claim 1, wherein the mesoporous material comprises at least one of mesoporous silicon, mesoporous bioglass, mesoporous calcium silicate, mesoporous zinc silicate, mesoporous strontium silicate, and mesoporous magnesium silicate.

6. The microsphere composite hydrogel of claim 1, wherein said degradable polyester material comprises at least one of polylactic acid-glycolic acid copolymer, polyglycolic acid, polylactic acid, polyhydroxyalkanoate, polycaprolactone, polytrimethylene carbonate, polybutylene succinate, and epsilon-polylysine.

7. The method for preparing microsphere composite hydrogel according to any one of claims 1 to 6, which is characterized by comprising the following steps:

and dispersing the microspheres in hydrogel to obtain the microsphere composite hydrogel.

8. A drug-loaded microsphere composite hydrogel, which is characterized by comprising the microsphere composite hydrogel of any one of claims 1 to 6 and an active drug loaded in the microspheres.

9. The preparation method of the drug-loaded microsphere composite hydrogel of claim 8, which is characterized by comprising the following steps:

s31, mixing the mesoporous material with an active drug to obtain a drug-loaded mesoporous material;

s32, mixing the drug-loaded mesoporous material with the degradable polyester material, adding a surfactant solution, stirring and separating to obtain the drug-loaded microspheres;

s33, dispersing the drug-loaded microspheres in hydrogel to obtain the drug-loaded microsphere composite hydrogel.

10. Use of the microsphere composite hydrogel as claimed in any one of claims 1 to 6 and the drug-loaded microsphere composite hydrogel as claimed in claim 8 in the preparation of bone implant or bone repair materials, anti-infection and anti-tumor drugs.

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