CN114099473A

CN114099473A - Slow release microcapsule for inducing in vitro osteogenic differentiation of MC3T3-E1 cells and preparation thereof

Info

Publication number: CN114099473A
Application number: CN202111440790.1A
Authority: CN
Inventors: 华臻; 施冬健; 陈继伟
Original assignee: Individual
Current assignee: Individual
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-03-01
Anticipated expiration: 2041-11-30
Also published as: CN114099473B

Abstract

The invention discloses a slow-release microcapsule for inducing in-vitro osteogenic differentiation of MC3T3-E1 cells and a preparation method thereof, wherein the preparation process comprises the following steps: preparation of Alg-grafted PBA, Alg-PBA-ICA, SiO₂‑NH₂Preparing microspheres, preparing Alg-PBA-ICA vesicles and preparing Alg-PBA-ICA/BMP-2 sustained-release microcapsules; has the capability of inducing osteoblast MC3T3-E1, icariin ICA and bone morphogenetic protein BMP-2 to carry out osteogenic differentiation, and the ICA is 10 at the optimal combined concentration^‑8When mol/L, BMP-2 is 100ng/mL, the capability of the sustained-release microcapsule for inducing osteogenesis is larger than that of the sustained-release microcapsuleThe BMP is embedded in the inner layer and the ICA is embedded in the outer layer, so that the proliferation can be promoted firstly, and then the differentiation can be promoted, and the requirement of maximizing the osteogenesis can be realized; the design provides laboratory basis for the traditional Chinese medicine combined with growth factors to promote bone tissue repair and regeneration.

Description

Slow release microcapsule for inducing in vitro osteogenic differentiation of MC3T3-E1 cells and preparation thereof

Technical Field

The invention belongs to the technical field of medical materials, and particularly relates to a sustained-release microcapsule for inducing in-vitro osteogenic differentiation of MC3T3-E1 cells and a preparation method thereof.

Background

Under natural conditions, the bone repair and healing period needs 3-6 months, and the process is a complex and long process in which osteoblasts, osteoclasts and multiple growth factors participate together. Osteoblasts (OB), which are the main cells for bone formation, are responsible for synthesis, secretion and mineralization of bone matrix, are the direct source of Osteocytes (OS), and are also involved in regulating bone resorption by Osteoclasts (OC). Therefore, maintaining the amount and activity of OB is of great importance for the repair and regeneration of bone tissue damage.

Research shows that various growth factors such as Bone Morphogenetic Protein (BMP), Transforming Growth Factor (TGF), Vascular Endothelial Growth Factor (VEGF), Fibroblast Growth Factor (FGF) and the like have positive effects on the repair of bone defects, and bone morphogenetic protein-2 (BMP-2) is positioned in the center of an action field. BMP-2, the most important osteogenic regulatory signal molecule, acts on cells in vivo in an autocrine and paracrine manner, and exerts its osteogenic effect by activating Smads signaling and regulating the transcription of osteogenic genes through the binding of ligands to cell membrane surface receptors. BMP-2 can up-regulate the expression of more than 60 genes, including more than ten important osteogenesis related transcription factors such as Smad6, Smad7, Msx2 and the like; BMP-2 has strong proliferation and expression effects on most bone matrix proteins and can mineralize human osteoblasts, and the enhancement of ALP activity and PTH reactivity and the increase of cAMP production in human primary osteoblasts are the results of the action of BMP-2, which suggests that BMP-2 has great significance in callus formation. In vitro experiments also prove that BMP-2 has very remarkable effects on inducing ectopic osteogenesis, repairing bone defects and the like, and at present, BMP-2 is approved by FDA to be applied to clinic as a stable local bone formation induction growth factor and mainly exerts bone effect in carrier implantation slow release, local injection, gene transfection and other modes.

With the rapid development of the modernization of the traditional Chinese medicine, the research of preventing and treating diseases by taking the kidney-tonifying traditional Chinese medicine preparation as an inducer for promoting osteogenic differentiation is gradually developed and becomes effective initially, which provides a new idea for researching the repair and reconstruction of bone tissues. Data mining and research of related formulas show that epimedium is popular among people nowadays because of the effects of tonifying kidney and strengthening yang and strengthening tendons and bones, the use frequency is high, and a plurality of basic researches also prove that the epimedium has a positive effect on the aspects of increasing bone mass and bone quality. Related researches find that Icariin (ICA) has the most outstanding osteogenic performance, ICA can promote BMSCs to differentiate towards osteoblasts, promote osteoblast proliferation, inhibit osteoclast activity, improve osteoblast activity, increase ALP positive clone number of osteoblasts and calcify nodules, up-regulate gene expression levels of osteoblast specific transcription factors Runx-2, Osterix and the like, obviously increase the secretion of ColI and show huge osteogenesis promoting potential.

In order to make some drugs or growth factors act for a long time, researchers often perform certain packaging processing or modification on the stent material to achieve the purpose of drug slow release. The packaging process mainly adopts microsphere microcapsule wrapping and hydrogel wrapping; the modification comprises chemical modification and biological modification; at present, a plurality of spongy porous scaffolds prepared from polymer materials with good biocompatibility have been widely applied to the research of bone tissue engineering, such as Polycaprolactone (PCL), polylactic acid (PLA) and the like, but the drug carried in the material scaffold is inactivated due to the structural damage of protein during the processing, and most of the polymer materials are hydrophobic materials and are difficult to be compatible with water-soluble BMP-2 and ICA.

The sodium alginate/polylysine microcapsule technology is mature, but the application of the polylysine is severely limited by the expensive price and the complex preparation process; therefore, the chitosan with similar structure becomes an ideal substitute material of polylysine, the chitosan is non-toxic and non-irritant, has good biocompatibility, can provide a three-dimensional scaffold required by cell growth and an extracellular environment similar to a cartilage matrix, and is widely applied to the fields of orthopedic repair, colon administration, microsphere embolization, carrier gene and the like at present. Many researches show that the microcapsule membrane prepared by coating sodium alginate with chitosan has high strength, adjustable permeability, good biocompatibility and pH sensitivity, great potential in the fields of immobilized cells and drug sustained-release carriers, and good mechanical properties, and is an ideal carrier for embedding living cells and controlling a release system.

Chinese patent CN 103495209B discloses an autofluorescence bone repair magnetic sustained-release microsphere, which adopts solvothermal method to synthesize nano Fe₃O₄Adding nano Fe₃O₄Uniformly dispersing in sodium alginate solution, and simultaneously compounding icariin which is a traditional Chinese medicine having proliferation and differentiation promoting effects on osteoblast; dropping the mixture into gel bath through a microcapsule forming device to form primary microspheres; and then immersing the primary microspheres in a chitosan solution, forming a polyelectrolyte semipermeable membrane by utilizing the charge complementary characteristics of chitosan and sodium alginate, and crosslinking the composite microspheres with genipin to prepare the autofluorescence bone repair magnetic sustained-release microspheres. The obtained microsphere can control release of icariin, and can be used for treating and repairing bone cancer and bone defect diseases and promoting production of new bone tissue. The patent mainly utilizes the medicinal characteristics of ICA, but as can be seen from the foregoing, ICA and BMP can independently show reliable bone-promoting characteristics, and the effect of single use is always limited, and if the ICA and BMP can be combined and exert synergistic effect, huge social benefit can be brought.

However, considering the problems of easy inactivation and degradation of BMP-2 in vivo, how to reasonably design the microcapsule structure and effectively maintain the sustained release of ICA and BMP locally is also a problem to be solved urgently.

Disclosure of Invention

The invention aims to provide a sustained-release microcapsule for inducing in-vitro osteogenic differentiation of MC3T3-E1 cells and a preparation method thereof, which define the effect of the icariin and BMP-2 combined induction osteogenic differentiation and the relation between the icariin and BMP-2, establish a alginic acid/chitosan microcapsule capable of slowly releasing icariin and BMP phenylboronic acid, define the effect and advantages of icariin and BMP slow release osteogenesis promotion, confirm the scientificity of the theory of 'kidney bone marrow generation' in traditional Chinese medicine, and provide a laboratory basis for the traditional Chinese medicine combined cytokine treatment bone repair and regeneration.

The technical scheme of the invention is as follows: a preparation method of a sustained-release microcapsule for inducing in-vitro osteogenic differentiation of MC3T3-E1 cells specifically comprises the following steps:

(1) preparation of Alg-grafted PBA

Dissolving sodium alginate (Alg) in deionized water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 1.15g N-hydroxysuccinimide (NHS) into the solution, and activating for 30 min; and (3) dropwise adding aminobenzeneboronic acid (APBA) into the mixed solution, stirring for reaction, precipitating a product by using ethanol, purifying, repeating for three times, dialyzing, and freeze-drying to obtain Alg-PBA.

(2) Preparation of Alg-PBA-ICA: preparing a mixed solvent of PBS and DMSO, and dissolving Icariin (ICA) in the mixed solvent; dissolving the Alg-PBA obtained in the previous step in a mixed solvent, slowly dripping the ICA solution into the Alg-PBA solution, stirring for reaction at room temperature, dialyzing, and freeze-drying to obtain Alg-PBA-ICA;

(3)SiO₂-NH₂preparing microspheres: uniformly mixing absolute ethyl alcohol, distilled water and ammonia water, and marking as solution A; tetraethyl orthosilicate (TEOS) and ethanol are uniformly mixed and marked as solution B; adding the solution B into the solution A, reacting at room temperature, centrifugally washing, and drying in vacuum to obtain SiO₂Microspheres;

SiO to be prepared₂Dispersing the microsphere sample in anhydrous toluene, dropping KH550 under vigorous stirring, condensing, refluxing, standing, removing supernatant, alternately centrifuging and washing with toluene and ethanol for 3 times, and vacuum drying to obtain SiO₂-NH₂Microspheres;

(4) preparation of Alg-PBA-ICA vesicles: preparing Alg-PBA-ICA, CS solution and SiO₂Suspending the solution, ultrasonic treating the suspension with SiO₂Slowly dropping the suspension into Alg-PBA-ICA solution, washing with buffer solution for 3 times after adsorption reaction, dispersing into buffer solution, slowly dropping into CS solution, washing with buffer solution for 3 times after adsorption reaction, dispersing into buffer solution, slowly dropping into Alg-PBA-ICA solution, washing with buffer solution for 3 times after adsorption reaction, redispersing into buffer solution, dropping into calcium chloride solution for crosslinking, washing with buffer solution and centrifuging for 3 times, and freeze-drying to obtain Alg-PBA-ICA @ SiO @ in a layer-by-layer self-assembly manner₂Core-shell particles;

mixing Alg-PBA-ICA @ SiO₂Addition of NH to the core-shell particles₄F/HF buffer solutionIn the solution, stirring at room temperature after ultrasonic dispersion, washing with deionized water, centrifuging for 3 times, and freeze-drying to obtain Alg-PBA-ICA vesicles;

(5) preparing the Alg-PBA-ICA/BMP-2 sustained-release microcapsule: dissolving BMP-2 in PBS solution, adding Alg-PBA-ICA vesicles, stirring at room temperature, washing, centrifuging, and freeze-drying to obtain the Alg-PBA-ICA/BMP-2 sustained-release microcapsule.

Further, in step (1), in terms of molar ratio, Alg: EDC: NHS ═ 1:1: 2.

Further, in the step (1), the ratio of the active groups of Alg to APBA is 1: 1.5.

Further, in the step (1), the stirring reaction temperature is 25-30 ℃, and the reaction time is 20-30 h.

Further, in step (2), the molar concentration of the PBS solution was 0.001M, the pH was 7.8, and the volume ratio of the PBS solution to DMSO was 2:1

Further, in the step (3), the volume ratio of the absolute ethyl alcohol, the distilled water and the ammonia water is 14:2:1, and the volume ratio of the TEOS to the ethyl alcohol is 1: 6.

Further, in step (5), the PBS solution had a pH of 7.4 and a concentration of 0.1 mol/L.

The sustained-release microcapsule prepared by the preparation method of the sustained-release microcapsule for inducing the in-vitro osteogenic differentiation of the MC3T3-E1 cells has the ICA concentration of 10^-8The concentration of mol/L, BMP-2 is 100 ng/mL.

Compared with the prior art, the invention has the following advantages:

1. the application uses the combination of the traditional Chinese medicine monomer icariin and BMP-2 to induce osteogenic differentiation, constructs a drug-loaded stent with high biocompatibility, degradability and low toxicity, and realizes the optimal combined concentration (ICA: 10)^-8mol/L, BMP-2:100ng/mL), the capability of inducing osteogenesis by the sustained-release microcapsules is higher than that of direct administration, which preliminarily proves that icariin and BMP-2 have obvious curative effect when jointly inducing osteogenic differentiation of MC3T3-E1 cells, and the icariin and the BMP-2 have a certain synergistic effect, provide a laboratory basis for promoting bone tissue repair and regeneration by combining growth factors with traditional Chinese medicines, provide an effective thought for treating bone fracture, nonunion of tendon and bone, osteoporosis, bone defect and other orthopedic difficult diseases, and have great social benefits;

2. according to the application, after a preliminary experiment, the BMP has a strong cell osteogenesis promoting capacity, and the icariin has a strong cell proliferation promoting capacity, so that when the microcapsule is designed, the BMP is embedded in an inner layer, and the icariin is arranged in an outer layer, so that the purpose of promoting proliferation and differentiation is achieved firstly, the using amount of the BMP is reduced, and the requirement of maximizing osteogenesis is met; in addition, the use amount of expensive BMP is reduced, the overall preparation cost of the microcapsule can be obviously reduced, the universality and the clinical application feasibility of the material are favorably improved, the characteristic advantages of the traditional Chinese medicine are highlighted, and the practical value of the development of the microcapsule material is improved.

3. According to the application, the amino phenylboronic acid is used for modifying the biological macromolecules when the sustained-release capsule is prepared, the icariin is embedded into the microcapsule by utilizing the interaction between the phenylboronic acid and the icariin, and the drug can realize sugar response controllable release in a patient body by utilizing the competitive reaction among PBA, the drug and glucose through the stimulation of glucose, so that the release process is effectively regulated and controlled;

4. 3-aminophenylboronic acid (APBA) used in the preparation of the microcapsule can effectively reduce the pKa value due to the existence of amino, so that PBA can realize sugar response under physiological conditions;

5. in order to further deepen the treatment concept of the traditional Chinese medicine combined cytokine, the traditional Chinese medicine monomer is used for research, the defects of complex traditional Chinese medicine compound components, excessive interference factors and the like are overcome, and a laboratory basis is provided for the traditional Chinese medicine combined cytokine in the field of bone repair and reconstruction.

Drawings

FIG. 1 is a graph showing the results of half inhibitory concentration IC50 of ICA on MC3T 3-E1;

FIG. 2 is a graph showing the effect of incubation of ICA with BMP-2 for various periods of time on the proliferation of MC3T3-E1 cells;

FIG. 3 is a statistical plot of the effect of combination dosing on ALP level variation;

FIG. 4 is a nuclear magnetic resonance spectrum of Alg-PBA;

FIG. 5 is a standard curve of ICA in a mixed solution;

FIG. 6 shows SiO obtained in example 1₂Microspheres, SiO₂-NH₂Microspheres and Alg-PBA-ICA @ SiO₂Particle size analysis of core-shell particles;

FIG. 7 shows, in sequence from left to right, SiO obtained in example 1₂Microspheres, SiO₂-NH₂Microspheres and Alg-PBA-ICA @ SiO₂SEM image of core-shell particles;

FIG. 8 is a diagram showing the mechanism of preparation of Alg-PBA-ICA/BMP-2 vesicles;

FIG. 9 is a diagram of the mechanism of controlled release of Alg-PBA-ICA/BMP-2 vesicles;

FIG. 10 is a graph comparing the results of ICA/BMP-2-loaded sustained release microcapsules and direct administration on the proliferation of MC3T3-E1 cells;

FIG. 11 is a diagram showing the expression effect of osteogenesis related genes p38, OCN, RUNX2 and Erk detected by RT-PCR;

FIG. 12 is a graph showing the results of Western Blot detection on the expression of the proteins of osteogenesis-related RUNX2, ERK1/2 and p 38;

FIG. 13 is a comparison of lycopene staining to detect mineralized nodules.

Detailed Description

The technical solution of the present invention is further described below with reference to the accompanying drawings, but not limited thereto, and any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be covered by the protection scope of the present invention.

Example one

1. Screening of optimal drug concentrations of ICA and BMP-2 for inducing osteogenic differentiation of MC3T3-E1 cells

1.1 drug concentration of ICA and BMP-2 on MC3T3-E1 proliferation

The MC3T3-E1 cell line was cultured in alpha-MEM medium containing 10% by volume fetal bovine serum, 100U/mL penicillin and 100mg/L streptomycin at 37 ℃ with 5% by volume CO₂And culturing under saturated humidity condition.

Cells were seeded at 4000 cells/well in 96-well plates and incubated 24h later with 7 ICA doses set, 10 each at 7 doses^-3/10^-4/10^-5/10^-6/10^-7/10^-8/10^-9mol/L, 6 per dose groupAnd (4) carrying out re-well, and detecting the growth inhibition rate of ICA on cells by using a CCK-8 kit after one day of incubation.

Inoculating the cells into a 96-well plate according to the density of 2000 cells/well, replacing the original culture medium with ICA and BMP-2 with different concentrations after 24h for incubation, detecting the cell proliferation rate by using a CCK-8 kit on the 1 st day, the 3 rd day and the 7 th day of incubation respectively, and changing the solution every 3 days in the administration incubation process. ICA and BMP-2 administration concentrations are shown below

As shown in FIG. 1, the half inhibitory concentration IC50 of ICA against MC3T3-E1 resulted in 30379nM, about 10^-4.48mol/L。

Effect of ICA on proliferation of MC3T3-E1 cells

The effect of incubation of MC3T3-E1 cells with ICA at various concentrations and for various times on cell proliferation is shown in FIG. 2. Wherein, the detection result after 1 day of incubation shows that ICA at each concentration has no significant difference on cell proliferation, and the OD value range is 0.35-0.45; after 3 days of incubation, a dose of 10 was administered^-8mol/L and 10^-7The mol/L has significant difference, and the OD value range is 0.95-1.40; after 7 days of incubation, a dose of 10 was administered^-8The concentration of mol/L has significant difference, and the OD value ranges from 1.5 to 2.0.

Effect of BMP-2 on proliferation of MC3T3-E1 cells

The effect of incubation with different concentrations of BMP-2 in MC3T3-E1 cells for different periods of time on cell proliferation is shown in FIG. 2. Wherein, the detection result after 7 days of incubation shows that BMP-2 with each concentration has no significant difference on cell proliferation. The OD value range of 1 day of incubation is 0.3-0.5; the OD value after 3 days of incubation ranged from 0.75 to 1.30.

1.2 drug concentrations of ICA and BMP-2 to induce MC3T3-E1 alkaline phosphatase expression

The cells were seeded in 12-well plates at 10000 cells/well, 24h later, the media was replaced with ICA and BMP-2 containing 5% fetal calf serum at different concentrations for incubation, and the ICA and BMP-2 dosing concentrations were combined with the cell proliferation design as shown below

The liquid change is carried out every 3 days during the administration incubation process. Respectively using the Biyun day cell lysate (P0013J) to lyse cells on the 1 st day, the 3 th day and the 7 th day of incubation, then using a Biyun day alkaline phosphatase detection kit (P0321) to detect the activity of ALP of each group, using a Thermo protein detection kit (23227) to detect the protein concentration, and calculating the alkaline phosphatase activity according to the enzyme activity definition.

The combined administration was performed according to the designed combined concentration, and after 7 days, cell lysates were collected and total protein content and ALP activity were measured, which indicates that the combined concentration did not significantly stimulate the expression of ALP in the cells compared to the single administration. Then, according to the slow release concept, the administration of ICA to stimulate cell proliferation, the administration of ICA and BMP-2 to induce differentiation, and the collection and detection operation after 7 days were the same as above, and the results are shown in FIG. 3, and it is known that the combination administration can increase the expression of ALP compared to the single administration, but cannot specifically enhance the expression of ALP in osteoblasts, and the combination administration can inhibit the expression of alkaline phosphatase in osteoblasts to some extent. According to experimental statistics, the administration of BMP-2 in combination with the administration of ICA alone can relatively specifically increase the expression of ALP in osteoblasts as compared with the administration of ICA alone.

And found that ICA is 10 by experiment^-7mol/L、10^-8mol/L and 10^-9At mol/L, BMP-2 can remarkably induce osteoblast proliferation at 50ng/mL and 100ng/mL, and promote ALP expression, so ICA and BMP-2 at the above concentrations are designed into sustained-release microcapsules for administration.

2. Preparation of slow-release microcapsule for inducing in vitro osteogenic differentiation of MC3T3-E1 cells

2.1 preparation of Alg-grafted PBA

Dissolving 2.0g of sodium alginate (Alg) in deionized water, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl) and 1.15g of N-hydroxysuccinimide (NHS) into the solution according to the molar ratio of 1:1:2(Alg: EDC: NHS), and activating for 30 min; dropwise adding a certain amount of aminobenzeneboronic acid (APBA) into the mixed solution (the active group ratio of Alg to APBA is 1:1.5), and stirring and reacting for 24 hours at the temperature of 25 ℃; after the reaction is finished, precipitating the product by using ethanol, dissolving and purifying by using deionized water, repeating for three times, dialyzing in the deionized water for one week, and freeze-drying to obtain Alg-PBA;

2.2 preparation of Alg-PBA-ICA

Preparing a PBS solution with the molar concentration of 0.001M and the pH value of 7.8, and mixing the prepared PBS solution and DMSO according to the volume ratio of 2:1 to obtain a mixed solvent; dissolving 0.2g Icariin (ICA) in mixed solvent to obtain 20g solution; dissolving 0.4g of Alg-PBA in the mixed solvent to obtain 20g of solution; similarly, slowly dropping the ICA solution into the Alg-PBA solution, stirring the solution at room temperature for reaction for 24h, dialyzing the solution in the mixed solution for 12h, removing the Alg-PBA and the ICA which do not participate in the reaction, and dialyzing the solution in the PBS solution for 24h to remove DMSO; lyophilizing to obtain Alg-PBA-ICA;

the scaffold structure can contain drugs to achieve the sustained release result, but the research on the stimulus response type controlled release has less reports. On the one hand, the addition of the stimulus-responsive agent is highly limited due to biological application, and on the other hand, the biological environment is not favorable for the selection of stimulus sources. The release rate of the medicine is adjusted by reasonably adopting small changes of pH, ion concentration and the like in human body, so that the release of the medicine can be changed according to human metabolism, and an ideal effect can be more effectively achieved. Phenylboronic acids (PBAs) exist in an ionic state under basic conditions and can react with polyols to link with reversible ester bonds, modify biomacromolecules with PBAs, and bind them to drugs with ortho-dihydroxy structures. By utilizing the competitive reaction among PBA, the drug and glucose, the drug can realize the sugar response controllable release in the body of a patient

Alginic acid (Alg) is a linear polymer formed by connecting a G-block (alpha-L-gulonic acid) and an M-block (beta-D-mannuronic acid) through 1,4 glycosidic bonds, has good biocompatibility and water solubility, and is widely used for constructing a stent material. Grafting of PBA onto Alg can confer a carbohydrate-responsive function. However, since phenylboronic acid (PBA) is a weak lewis acid, there is an ionization equilibrium in water, and it can be combined with a compound having an ortho-dihydroxy structure only when it is ionized into an ionic state, thereby achieving a sugar response. However, pH in physiological environment is low, ionization degree of PBA is not high, and sugar response function cannot be fully exerted. And 3-aminophenylboronic acid (APBA) can effectively reduce the pKa value thereof due to the existence of amino groups, so that the compound can realize sugar response under physiological conditions. Therefore, the APBA grafted Alg not only can realize drug loading, but also can effectively realize sugar response.

The grafting procedure for Alg-PBA was as follows

Under the activation of EDC/NHS, carboxyl in alginic acid reacts with amino of APBA to form amido bond, thereby grafting PBA on Alg. The structure of the compound is characterized by 1H-NMR, as shown in figure 4, the chemical shift is the proton peak of C1-4 hydrogen on a benzene ring at about 8, the chemical shift is assigned to the proton peaks of C5 and C6 on Alg at 5-5.5, and the chemical shift is assigned to the proton peak on Alg at about 4. The appearance of the proton peak on the benzene ring indicates that the phenylboronic acid is successfully grafted to the sodium alginate. The grafting rate of PBA can be obtained by calculating the area ratio of the hydrogen proton peak of C5 and C6 of Alg at the chemical shift of 5-5.5 to the hydrogen proton peak of benzene ring at the chemical shift of 7.5-8.5, so that the grafting rate of Alg-PBA is 30.3%.

Since ICA is not readily soluble in water, whereas Alg-PBA is a water-soluble substance, PBS was selected in a volume ratio of 2: 1: the DMSO solution is used as a mixed solvent to further graft ICA onto Alg-PBA, and the specific process is as follows

Since ICA has a high absorbance at 321nm light irradiation, the grafting rate of ICA can be obtained by measuring the absorbance of Alg-PBA-ICA at 321nm by an ultraviolet-visible spectrophotometer (UV-vis). Therefore, a standard curve of ICA in the mixed solution was established by accurately preparing a solution of ICA at a certain concentration (as shown in FIG. 5), and then the grafting ratio of ICA in Alg-PBA-ICA was measured to be 0.54%.

2.3、SiO₂-NH₂Preparation of microspheres

Uniformly mixing 140mL of absolute ethyl alcohol, 20mL of distilled water and 10mL of ammonia water, and marking as solution A; mixing 10mL tetraethyl orthosilicate (TEOS) and 60mL ethanol uniformly, and marking as solution B; adding the solution B into the solution A, reacting for 2h at room temperature, centrifugally washing for 3 times by using ethanol, and drying in vacuum to obtain SiO₂Microspheres, as shown in FIG. 6, SiO₂The particle size of the microspheres is 111.0 +/-8.1 nm, and the particle size is uniform;

in order to better coat Alg-PBA-ICA on SiO₂Surface, 1g of prepared SiO₂Dispersing the microsphere sample into 100mL of anhydrous toluene, and dropwise adding 5mL of KH550 p-SiO under vigorous stirring₂Surface modification, condensing reflux reaction at 110 deg.C for 12 hr, standing, removing supernatant, alternately centrifuging and washing with toluene and ethanol for 3 times, and vacuum drying for 24 hr to obtain SiO₂-NH₂Microspheres, modified SiO₂-NH₂The particle size of the microspheres is 148.1 +/-2.3 nm (figure 6);

2.4 preparation of Alg-PBA-ICA vesicles

PBS solution with pH of 5 and concentration of 0.001M is prepared as solvent, then Alg-PBA-ICA solution and Chitosan (CS) solution with concentration of 2mg/mL are respectively prepared, SiO solution with concentration of 2mg/mL is prepared in the same way₂Suspending liquid, ultrasonic treating for 30min, and mixing 20mL SiO₂Slowly dropping the suspension into 20mL of Alg-PBA-ICA solution, carrying out adsorption reaction for 30min, and washing with a buffer solution with pH of 5 for 3 times to remove unreacted Alg-PBA-ICA; then re-dispersing into 20mL of buffer solution with pH being 5, slowly dropping into 20mL of CS solution, adsorbing for reaction for 30min, and washing with buffer solution with pH being 5 for 3 times to remove unreacted CS; similarly, the solution was redispersed in 20mL of a buffer solution having pH of 5, slowly dropped into 20mL of an Alg-PBA-ICA solution, and after adsorption reaction for 30min, washed 3 times with a buffer solution having pH of 5 to remove unreacted Alg-PBA-ICA; then re-dispersing into 20mL of buffer solution with pH value of 5, dripping 5mL of calcium chloride solution with concentration of 1mol/L for full crosslinking, washing with the buffer solution with pH value of 5, centrifuging for 3 times, and freeze-drying to obtain Alg-PBA-ICA @ SiO @ which is self-assembled layer by layer₂Core-shell particles.

As shown in FIG. 7, after the self-assembly of the layers, the particle size of the particles reached 210.4. + -. 2.0nm, which indicates that SiO₂-NH₂The microspheres were coated with Alg-PBA-ICA. In addition, SiO is found by testing the surface potential of the microspheres and the vesicles through Zeta potential₂-NH₂The potential of the microsphere surface is 22.67 +/-3.46 mV due to the ionization effect of-NH 2, and the surface potential of the Alg-PBA-ICA @ SiO2 core-shell particle coated with the Alg-PBA-ICA is-15.70 +/-2.12 mV, further showing that the Alg-PBA-ICA is coated on the surface of the SiO2-NH2 microsphere.

Taking 0.5g of the prepared Alg-PBA-ICA @ SiO₂Core-shell particles, 100mL of NH were added₄Dispersing in F/HF (8M/2M) buffer solution by ultrasonic, stirring at room temperature for 12h, and dissolving SiO with HF₂Etching, washing with deionized water, centrifuging for 3 times to remove residual impurities, and freeze-drying to obtain Alg-PBA-ICA vesicles;

2.5 preparation of Alg-PBA-ICA/BMP-2 vesicles

Preparing PBS solution with pH of 7.4 and concentration of 0.1mol/L, dissolving BMP-2 in the PBS solution to prepare 100mL BMP-2 solution with concentration of 1mg/mL, adding 0.5g of prepared Alg-PBA-ICA vesicles, stirring at room temperature for 12h, washing with the PBS solution for 3 times, centrifuging, and freeze-drying to prepare Alg-PBA-ICA/BMP-2 vesicles, wherein the preparation mechanism is shown in figure 8, and the Alg-PBA-ICA @ SiO is₂SiO in core-shell particles₂The nuclei were etched away, forming Alg-PBA-ICA vesicles. Due to the hydrophobic cross-linking of CS and the ionic cross-linking of Alg-Ca, the vesicles can maintain a stable cavity structure. By further soaking the Alg-PBA-ICA vesicles in a BMP-2-containing solution, BMP-2 can diffuse into the vesicles, thereby achieving dual drug entrapment.

The microcapsule prepared by the application can realize the common release of the two medicines when being externally stimulated because different medicines are respectively loaded on and in the vesicle, thereby achieving the purpose of synergistic treatment; in addition, because the release routes on the vesicle and in the vesicle are different, the release speed of the drug in the vesicle is slower, and therefore, the effect of sequential release can be achieved, and the intelligent control treatment is further realized. Because phenylboronic acid can be dissociated under the stimulation of glucose, phenylboronic acid is grafted on ICA to serve as a vesicle, in order to further improve the strength of the vesicle, biomass materials Alg and CS are selected as main framework chain segments, and ionic crosslinking bonding of Alg and calcium ions is adopted as a crosslinking structure, so that the strength of the vesicle is improved, and the self-supporting capacity is improved.

Glucose is widely present in human bodies, is an important product of life metabolism, is the dynamic basis of life activities, and is closely related to human life activities. The phenylboronic acid contains glucose sensitive units, and dissociation can occur under the stimulation of glucose. Therefore, the Alg-PBA-ICA/BMP-2 vesicles can be dissociated from outside to inside under the action of glucose, the grafted ICA in the vesicles is gradually released in the dissociation process, the vesicles are gradually decomposed into fragments along with the further deepening of the stimulation effect, and the BMP-2 entrapped in the vesicles is gradually released to form the slow release effect of the second medicament (as shown in figure 9).

Correlation performance testing and characterization

1. Effect of ICA/BMP-2-carrying sustained-release microcapsules on MC3T3-E1 cell proliferation

Adopting a CCK-8 kit to prepare 3 multiple wells per group, taking out the well plate after 72h, adding 10 mu L of CCK-8 reagent into each well according to the specification of the cell proliferation and activity detection kit (CCK-8), incubating for 2h, measuring the absorbance (A) value of each well at 450nm by using a full-automatic enzyme labeling instrument, and calculating the cell proliferation rate by using the A value. The cell proliferation rate was defined as experimental group a/control group a.

Compared with the ICA/BMP-2-loaded sustained-release microcapsule and the ICA/BMP-2 with the same concentration, the results of detecting and analyzing the light absorption value after 72 hours are shown in figure 10, and compared with the direct administration, the drug-loaded microcapsule and the drug-loaded microcapsule have no significant difference on the cell proliferation.

RT-PCR detection of expression of osteogenesis related genes p38, OCN, RUNX2 and Erk under microcapsule administration

Cells were plated at 2X 10 per well⁵Inoculating the cells in 6-well plate, stimulating and culturing for 7d, removing culture medium, washing with PBS for 3 times, extracting total RNA from TRIZOL, detecting gene concentration and integrity, and synthesizing corresponding cDNA by reverse transcription reaction, wherein gapdh is used as housekeeping gene. Preparing real-time PCR 20 muL reaction system for qRT-PCR detection(reaction parameters are all according to the kit instructions), and after the reaction is finished, analysis of an amplification curve and a melting curve is carried out. The expression level of the gene is expressed as the amount of the initial template for detecting the gene (2)^-ΔΔCt)。

As shown in FIG. 11, all osteogenic genes were upregulated after microencapsulation compared to the blank group, but the degree of upregulation was different, and subsequent experiments were discarded to contain ICA at a concentration of 10 based on differences in gene expression at the mRNA level^-9And (3) the microcapsules in mol/L.

Western Blot detection of expression of osteogenesis-related RUNX2, ERK1/2 and p38 proteins

(1) Cells were harvested on ice after induction culture for 7 d. Total protein is extracted, and protein concentration is determined by a BCA method and then quantified. Mixing a certain amount of loading buffer with the sample, uniformly mixing, quickly centrifuging, and carrying out metal bath at 90 ℃ for 5 min;

(2) the rubber plate is cleaned, and the rubber plate is installed and fixed after being dried by a blower. The slab rubber was prepared according to the following recipe, with the concentrated gum being placed at the end.

Preparation of 10% separation gel (. times.4) preparation of 5% concentrated gel (. times.4)

(3) After the rubber plate is prepared, carefully pull out the comb. Mounting a rubber plate, preparing for sample loading, loading a marker at first, and then loading the prepared sample;

(4) after the loading is finished, the electrophoresis tank is arranged, and enough running buffer is added. Pressing the glue by using 80V voltage, and then running the glue by using 100V voltage;

(5) and (3) after the electrophoresis is finished, carrying out mold transfer, cutting off the concentrated gel, spreading the separation gel in a membrane transfer tank, and covering an equal volume of PVDF membrane on the separation gel. Carefully driving the glue and bubbles in the film away, installing a die rotating machine, and working for 100min at 100V;

(5) after the mold is turned, proteins on the gel are turned to the PVDF membrane, and the membrane is cut according to the molecular weight of the target protein, so that the closure is convenient. As a confining liquid, TBST is selected to be matched with 5% skimmed milk, the membrane is placed in the confining liquid, shaking is carried out for incubation, and the process lasts for 1 h;

(6) sequentially hatching the antibodies according to the instruction;

(7) and (4) adjusting the machine to obtain a clear strip chart and taking a picture of the strip in a dark environment.

From the results recorded after ECL development exposure (fig. 12), the optimal combined concentration for inducing osteogenesis, i.e. BMP-2:100ng/mL, ICA 10^-8mol/L, the non-sustained release administration can obviously induce the expression of the bone-related protein, and after the microcapsule is prepared, the inducing effect of the optimal combination concentration can be increased.

4. Lycopene staining observation of mineralized nodule content

Cells were induced for 28 days, fixed in cold methanol for 15 min, rinsed 2 times with deionized water, and stained with 1% alizarin red solution (Macklin, China) for 30min at room temperature. Representative images were obtained by light microscopy.

The results showed (FIG. 13) that all the administered groups formed calcified nodules, and the area of calcified nodules formed in the 100ng/mL BMP-2 combined with the 10nM ICA microencapsulated group was larger than in the other groups.

As can be seen from the above, it was found that osteoblasts MC3T3-E1, icariin ICA and BMP-2 all had the ability to induce their osteogenic differentiation, and ICA:10 was found to be the optimum combined concentration^-8When the concentration is mol/L, BMP-2:100ng/mL, the capability of the sustained-release microcapsule for inducing osteogenesis is larger than that of direct administration. The sustained-release microcapsule has a microcapsule skeleton formed by sodium alginate, and has effects of resisting gastric juice digestion, and increasing small intestine absorption, thereby improving osteogenic differentiation inducing ability. Cell level experiments prove that the sustained-release microcapsule coated with ICA and BMP2 can be used as a preparation for research on inducing osteogenic differentiation.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes performed by the present specification and drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.

Claims

1. The preparation method of the slow release microcapsule for inducing the in vitro osteogenic differentiation of the MC3T3-E1 cells is characterized by comprising the following steps:

(1) preparation of Alg grafted PBA: dissolving Alg in deionized water, adding EDC & HCl and NHS into the solution, dropwise adding APBA into the solution after activation, stirring for reaction, precipitating the product with ethanol, purifying, repeating for three times, dialyzing, and freeze-drying to obtain Alg-PBA;

(2) preparation of Alg-PBA-ICA: preparing a mixed solvent of PBS and DMSO, and dissolving ICA in the mixed solvent; dissolving the Alg-PBA obtained in the previous step in a mixed solvent, slowly dripping the ICA solution into the Alg-PBA solution, stirring at room temperature, dialyzing, and freeze-drying to obtain Alg-PBA-ICA;

(3)SiO₂-NH₂preparing microspheres: uniformly mixing absolute ethyl alcohol, distilled water and ammonia water, and marking as solution A; mixing TEOS and ethanol uniformly, and marking as liquid B; adding the solution B into the solution A, reacting at room temperature, centrifugally washing, and drying in vacuum to obtain SiO₂Microspheres;

SiO to be prepared₂Dispersing the microsphere sample into anhydrous toluene, dropwise adding KH550 while violently stirring, condensing, refluxing, reacting, standing, removing supernatant, alternately centrifuging and washing with toluene and ethanol, and vacuum drying to obtain SiO₂-NH₂Microspheres;

(4) preparation of Alg-PBA-ICA vesicles: preparing Alg-PBA-ICA, CS solution and SiO₂Suspending the solution, ultrasonic treating the suspension with SiO₂Slowly dropping the suspension into an Alg-PBA-ICA solution, washing with a buffer solution after adsorption reaction, dispersing into the buffer solution, slowly dropping into a CS solution, washing with the buffer solution after adsorption reaction, dispersing into the buffer solution, slowly dropping into the Alg-PBA-ICA solution, washing with the buffer solution after adsorption reaction, dispersing into the buffer solution, dropping into a calcium chloride solution for crosslinking, washing with the buffer solution, centrifuging, and freeze-drying to obtain the layer-by-layer self-assembled Alg-PBA-ICA @ SiO @ ICA₂Core-shell particles;

mixing Alg-PBA-ICA @ SiO₂Addition of NH to the core-shell particles₄Stirring at room temperature after ultrasonic dispersion in an F/HF buffer solution, washing with deionized water, centrifuging, and freeze-drying to obtain Alg-PBA-ICA vesicles;

2. The method for preparing sustained-release microcapsules for inducing osteogenic differentiation in vitro of MC3T3-E1 cells according to claim 1, wherein in step (1), the molar ratio of Alg to EDC to NHS is 1:1: 2.

3. The method for preparing the sustained-release microcapsule for inducing in vitro osteogenic differentiation of MC3T3-E1 cells according to claim 1, wherein in the step (1), the ratio of active groups of Alg to APBA is 1: 1.5.

4. The method for preparing sustained-release microcapsules for inducing in vitro osteogenic differentiation of MC3T3-E1 cells according to claim 1, wherein the temperature of the stirring reaction in step (1) is 25-30 ℃ and the reaction time is 20-30 h.

5. The method for preparing sustained-release microcapsules for inducing osteogenic differentiation in vitro of MC3T3-E1 cells according to claim 1, wherein in step (2), the molar concentration of the PBS solution is 0.001M, the pH is 7.8, and the volume ratio of the PBS solution to the DMSO is 2: 1.

6. The method for preparing sustained-release microcapsules for inducing osteogenic differentiation in vitro of MC3T3-E1 cells according to claim 1, wherein in the step (3), the volume ratio of absolute ethanol, distilled water and ammonia water is 14:2:1, and the volume ratio of TEOS to ethanol is 1: 6.

7. The method for preparing sustained-release microcapsules for inducing osteogenic differentiation in vitro of MC3T3-E1 cells according to claim 1, wherein in step (5), the pH of the PBS solution is 7.4 and the concentration is 0.1 mol/L.

8. The sustained-release microcapsule prepared by the method for preparing the sustained-release microcapsule for inducing in vitro osteogenic differentiation of MC3T3-E1 cells according to any one of claims 1 to 7, wherein the sustained-release microcapsule is prepared by a method for preparing the sustained-release microcapsuleThe concentration of the intermediate ICA is 10^-8The concentration of mol/L, BMP-2 is 100 ng/mL.