Magnesium-containing mesoporous active bioglass with drug sustained release, immunoregulation and bone metabolism regulation effects and application thereof

Abstract

The invention discloses magnesium-containing mesoporous active bioglass with drug sustained release, immunoregulation and bone metabolism regulation effects and application thereof. The drug-loaded MBGN is prepared by doping Mg ions and loading drugs, and the drug-loaded MBGN has good biocompatibility and can be decomposed in an acid environment to achieve controllable slow release of the drugs; on the other hand, released ions such as Si, Ca and Mg have a certain immunoregulation function, polarization of macrophages from M1 type to M2 type is promoted, inflammatory response is reduced, differentiation and maturation of osteoclasts are inhibited, and meanwhile, the released ions and drugs carried in mesopores play a synergistic effect, so that a good treatment effect is achieved. The drug-loaded MBGN provided by the invention has various biological properties, can overcome the defects of low drug loading rate, insufficient anti-inflammatory effect and the like when the existing mesoporous bioactive glass material is applied clinically, and has a good application prospect.

Description

Technical Field

[0001] The invention relates to magnesium-containing mesoporous active bioglass with drug sustained release, immune regulation and bone metabolism regulation functions and application thereof, belonging to the technical field of biomedicine. Background Art

[0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by invasive arthritis. The disease is characterized by symmetrical, persistent polyarthritis with small joints such as the hands and wrists being affected. Pathological manifestations include chronic inflammation of the synovial membrane, pannus formation, and cartilage and bone destruction of the joints, which eventually develop into limb disability and loss of function. Timely intervention and effective inhibition of bone erosion are the key to preventing irreversible joint damage. Traditional drug treatments for RA can control inflammation to a certain extent, but have no clear effect on regulating bone metabolism, and even have many serious adverse reactions, such as hypertension, osteoporosis, weight gain, and fluid retention (DINARELLO CA. Anti-inflammatory Agents: Present and Future [J]. Cell, 2010, 140 (6): 935-50). Therefore, seeking new treatments that can simultaneously control joint inflammatory reactions, regulate bone metabolism balance, and protect bone joints is a problem that needs to be solved in the clinic.

[0003] Since Professor Hench and others from the University of Florida successfully developed 45S5 glass in 1971 (Filip, DG, et al., Current Development in Biomaterials-Hydroxyapatite and Bioglass for Applications in Biomedical Field: A Review. J Funct Biomater, 2022.13(4)), various types of bioactive glasses have been continuously developed and have become a research hotspot for materials scientists and rheumatologists. Mesoporous bioactive glass nanoparticles (MGBN) are mesoporous bioactive glasses developed on the basis of traditional Na-Ca-Si-P glass. MBGN has been widely used in bone tissue repair engineering due to its good bioactivity, degradability, compatibility and other advantages. In physiological solution, MBGN can release Si, Ca, P and Na ions to mineralize and form bone-like hydroxyapatite, and upregulate growth factors and stimulate osteoblasts to secrete bone matrix genes to promote bone tissue repair. This characteristic can repair bone damage in RA patients. In addition, MBGN has a high specific surface area and pore volume, so it has a higher drug loading efficiency and a slow drug release rate, making it a good choice for drug carriers. However, traditional MGBN has poor dispersibility in water, weak sustained-release ability for drugs, and limited effects in anti-inflammatory and bone metabolism regulation, which greatly limits its promotion and application in clinical practice.

[0004] Therefore, a new type of bioglass material is needed that can simultaneously possess the comprehensive functions of drug sustained release, immune regulation and bone metabolism regulation. Summary of the invention

[0005] The purpose of the present invention is to provide a magnesium-containing mesoporous active bioglass with drug sustained release, immune regulation and bone metabolism regulation effects in order to address the technical problems of poor dispersibility of traditional MGBN in water and limited effects in anti-inflammatory and bone metabolism regulation. The present invention improves MGBN to increase its biological activity, improves its dispersibility, and enhances the sustained release of drugs to achieve better bone integration ability, and at the same time has excellent characteristics of inhibiting inflammatory response.

[0006] In order to achieve the above object, the present invention provides a method for preparing drug-loaded MBGN nanoparticles, comprising the following steps:

[0007] Step 1: dissolving the organic template in deionized water, adding ethyl acetate and mixing evenly, then adding ammonia water and stirring, and then sequentially adding silicon source, calcium source and magnesium source for stirring reaction, collecting the nanoparticles generated by the reaction by centrifugation after the reaction, and sequentially washing, drying and calcining to obtain magnesium-containing MBGN;

[0008] Step 2: The magnesium-containing MBGN prepared in step 1 is uniformly dispersed in Tris-HCL, and then the drug tofacitinib is added, stirred overnight, and centrifuged to obtain drug-loaded MBGN nanoparticles.

[0009] Preferably, the organic template in step 1 is hexadecyltrimethylammonium bromide;

[0010] And / or, the silicon source is at least one of ethyl orthosilicate, methyl orthosilicate and butyl orthosilicate.

[0011] Preferably, the calcium source in step 1 is at least one of calcium chloride, calcium nitrate, a hydrate of calcium chloride and a hydrate of calcium nitrate;

[0012] And / or, the magnesium source is at least one of magnesium chloride, magnesium nitrate, a hydrate of magnesium chloride and a hydrate of magnesium nitrate.

[0013] Preferably, the feed ratio of the silicon source, calcium source and magnesium source in step 1 is calculated according to the molar ratio of silicon, calcium and magnesium being 80:10:10.

[0014] Preferably, the calcination temperature in step 1 is 600-700° C. and the calcination time is 2-4 hours.

[0015] Preferably, in step 2, the feed mass ratio of magnesium-containing MBGN to the drug tofacitinib is 0.8-1.2:1.

[0016] The present invention also provides drug-loaded MBGN prepared by the above preparation method.

[0017] The present invention also provides the use of the drug-loaded MBGN prepared by the above preparation method in preparing a product for treating rheumatoid arthritis.

[0018] In order to endow bioactive glass with multiple biological functions, the present invention improves mesoporous bioactive glass by incorporating Mg ions, thereby endowing it with the ability to inhibit macrophage inflammatory response and osteoclast differentiation.

[0019] Specifically, the present invention improves MBGN by incorporating Mg ions and loading drugs, which can decompose in an acidic environment to achieve controlled sustained release of drugs, and release Si, Ca ions, etc., to form a hydroxycarbonate apatite (HCA) layer through mineralization deposition, promote the material to form bone bonding with host tissues, and have good biocompatibility; on the other hand, the released Si, Ca, Mg ions have certain immune regulatory functions, promote macrophage polarization from M1 type to M2 type, reduce inflammatory response, and inhibit the differentiation and maturation of osteoclasts, while playing a synergistic role with the drugs carried in the mesopores, thereby achieving a good therapeutic effect.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) The present invention improves MBGN by incorporating Mg ions and loading drugs, and successfully prepares a drug-loaded MBGN that inhibits osteoclast differentiation and maturation into osteoclasts and inhibits macrophage inflammatory response. By introducing magnesium elements in the preparation process of mesoporous active bioglass, it has the effect of releasing Mg ions to regulate immunity; the drug-loaded MBGN prepared by the present invention has good controllable sustained release ability and can release drugs in response to pH; therefore, the present invention endows MBGN with more biological functions by improving MBGN;

[0022] (2) The process of the present invention is simple, efficient and has good repeatability. The drug-loaded MBGN prepared by the present invention has good stability and multiple biological properties, which can improve the shortcomings of the current mesoporous bioactive glass materials in clinical applications, such as low drug loading rate and insufficient anti-inflammatory effect. Therefore, the drug-loaded MBGN of the present invention has good clinical application potential and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 :MBGN surface morphology under transmission electron microscope, energy spectrum mapping and EDS element analysis.

[0024] Figure 2 : Drug release curves of MBGN loaded with drug in solutions with different pH values.

[0025] Figure 3 :CCK-8 experiment verifies the safety of drug-loaded MBGN cells.

[0026] Figure 4 : Experiment on the inhibition of osteoclast differentiation induced by mouse bone marrow-derived macrophages (BMM) by drug-loaded MBGN: tartrate-resistant acid phosphatase (TRAP) staining detection.

[0027] Figure 5: Experiment on the inhibition of osteoclast differentiation induced by primary mouse bone marrow macrophages by drug-loaded MBGN: Immunofluorescence detection of osteoclast F-actin ring.

[0028] Figure 6 :Drug-loaded MBGN inhibits inflammatory response of macrophages: PCR verification of inflammatory genes.

[0029] Figure 7 :Drug-loaded MBGN inhibits macrophage inflammatory response: ELISA was used to detect the secretion of inflammatory factors in cell culture supernatant. DETAILED DESCRIPTION

[0030] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0031] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0032] Example

[0033] 1. Preparation of drug-loaded MBGN

[0034] (1) Preparation of magnesium-containing MBGN:

[0035] 0.7g CTAB (hexadecyltrimethylammonium bromide) was added to 33mL ddH2O, and stirred with a magnetic stirrer to fully dissolve the CTAB particles. 10mL ethyl acetate was added, and 7mL 1mol / mL NH3.H2O (ammonia water) was added after stirring for 30min. 3.6mL TEOS (tetraethyl silicate) was added after stirring for 15min. 1.096g calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) was added after stirring for 30min. 0.595g magnesium nitrate hexahydrate (Mg(NO3)2·6H2O) was added after stirring for at least 4h. Centrifuge at 8000rpm for 10min, discard the supernatant, wash twice with ddH2O, wash once with anhydrous ethanol, and then dry in an oven. Calcinate at 650℃ for 3h to obtain magnesium-containing MBGN.

[0036] (2) MBGN drug delivery:

[0037] 10 mg of the magnesium-containing MBGN prepared above was ultrasonically treated in Tris-HCL for 20 min, and after being evenly dispersed, an equal mass of the drug Tofacitinib was added, magnetically stirred overnight, centrifuged at 7000 rpm for 10 min, the supernatant was removed, and the precipitate was dried to obtain drug-loaded MBGN: MBGN-Tofa nanoparticles.

[0038] 2. Characterization of materials:

[0039] (I) Characterization methods

[0040] (1) TEM characterization: MBGN was sterilized by ultraviolet light and placed in 1.5 mL EP tubes with three replicate wells in each group. After being resuspended in anhydrous ethanol, the samples were ultrasonically dispersed for 20 min. The samples were then dropped onto a support film and observed using a transmission electron microscope (TEM) (JEOL JEM-F200, Japan).

[0041] (2) Energy spectrum and elemental analysis: MBGN was sterilized by ultraviolet light and placed in 1.5 mL EP tubes with three replicate wells in each group. After being resuspended in anhydrous ethanol, the samples were ultrasonically dispersed for 20 min. The samples were then dropped onto a support membrane and subjected to energy spectrum mapping scanning and EDS elemental analysis using a transmission electron microscope TEM (JEOL JEM-F200, Japan).

[0042] (II) Characterization results

[0043] Figure 1 This is the characterization of the surface morphology, energy spectrum mapping and EDS element analysis of the MBGN prepared above.

[0044] 1. Sustained release performance test of drug-loaded nanoparticles MBGN-Tofa:

[0045] 10 mg of the prepared MBGN-Tofa nanoparticles were dispersed in 2 mL of phosphate buffered saline (PBS) at pH 5.3 and 7.4 and placed in a dialysis bag (14,000 Da MWCO). The sealed bag was placed in a beaker with 50 mL of PBS as a release medium (pH 5.3 and pH 7.4), and 2 mL of the liquid in the beaker was taken at 6 h, 12 h, 24 h, 48 h, 72 h, and 5 d, and the corresponding liquid was replenished, and the absorbance was measured by a UV spectrophotometer.

[0046] Figure 2 The drug release curves of MBGN loaded with drugs in solutions with different pH values. Figure 2 The results showed that the drug release rate was higher in a low pH environment, suggesting that the drug release rate was higher in the RA joint cavity in a low pH environment.

[0047] 2. Bioactivity Functional Detection of MBGN-Tofa Drug-Loaded Nanoparticles

[0048] (I) Detection method

[0049] (1) Macrophage RAW264.7 cells were 10 4 / 100μl was inoculated in a 96-well plate and pre-cultured in an incubator overnight. The supernatant was discarded and washed twice with PBS; 200μl of 25, 50, 100, 200, and 500μg / mL of drug-loaded MBGN (MBGN-Tofa nanoparticles prepared above) was added, 3 replicate wells were set, and the incubator was incubated for 24h, and PBS was washed twice. 200μl of culture medium containing 10μl CCK8 was added to each well. Do not generate bubbles, and incubate in the incubator for 0.5, 1, 2, 3, and 4h (based on an OD value of about 1). Measure the OD value with an enzyme reader at 450nm.

[0050] (2) Extraction and induction of mouse primary macrophages (BMDM): C57BL / 6 mice aged 4-6 weeks were killed and soaked in 75% ethanol for 20 minutes. Subsequent operations were performed on a cell clean bench; all instruments used were sterilized or sterile. The femur and bone of the mouse hind limbs were removed with scissors and tweezers, and the muscle tissue was removed and soaked in 75% ethanol for 10 minutes. After cutting both ends of the bone, use a 1 mL insulin syringe to draw incomplete 1640 culture medium from a section of the bone shaft and pierce the bone marrow cavity, and wash the bone marrow through a 30 μm cell filter into a 50 mL centrifuge tube. Repeat this operation 5-6 times until the bone turns white; collect the cell pellet by centrifugation (1500 rpm 8 min), discard the supernatant, add red blood cell lysis solution and incubate on an ice bath for 3 min to lyse the red blood cells, discard the supernatant after centrifugation, add complete 1640 culture medium containing GM-CSF (20 ng / mL) and IL-4 (20 ng / mL) to resuspend the cells, divide them into 6-well culture plates, place them in a 5% CO2, 37°C cell culture incubator for culture, and mark them as day 0; then, 3 / 4 of the medium was replaced on the 2nd and 4th days, respectively, and the replaced fresh culture medium contained GM-CSF (20 ng / mL). On the 6th day, the culture medium was aspirated and the cells were gently blown to collect the suspended and semi-adherent cells, which were mouse BMDM cells.

[0051] (3) Experiment on the inhibition of osteoclast differentiation induced by primary bone marrow macrophages induced by MBGN: The experiment was divided into three groups: negative control group, positive control group, and MBGN treatment group. BMDMs were cultured at a rate of 1×10 4The cells were inoculated into 96-well plates at a density of 100 μL / well. 100 μL of culture medium (containing 10% FBS, 30 ng / mL M-CSF) was added to each well of the negative control group, and 100 μL of culture medium (containing 10% FBS, 30 ng / mL M-CSF and 50 ng / mL RANKL) was added to each well of the positive control group and the treatment group. After sufficient mixing, the cell culture plate was placed in a 37°C, 5% CO2 cell constant temperature incubator for overnight culture. After the cells adhered to the wall, the culture medium was discarded, and freshly prepared cell culture medium containing drug-loaded MBGN was added according to the grouping, with 3 replicates per group to induce cell osteoclast differentiation; the medium was changed every three days. On the 7th day, when multinuclear osteoclasts were formed, the culture medium was discarded, and the cells were washed 3 times with PBS buffer, and the cells were fixed with 4% paraformaldehyde for 15 minutes, and then the cells were gently washed again.

[0052] (4) Tartrate-resistant acid phosphatase (TRAP) staining to detect osteoclast differentiation: According to the instructions of the TRAP staining kit, prepare the staining working solution (preheat 45 mL of double distilled water to 37°C in advance), namely: take 0.5 mL of Fast Garnet GBCBase Solution and 0.5 mL of Sodium Nitrite Solution and slowly mix for 30 seconds, and let it stand for 2 minutes; take a 50 mL centrifuge tube, add 0.5 mL of Naphthol AS-BIPhosphate Solution, 2 mL of Acetate Solution and 45 mL of double distilled water preheated to 37°C to the mixed solution in the previous step, mix well, and heat to 37°C; add 200 μL of the staining working solution to each well, place in a 37°C incubator and continue incubating for 1 hour, then discard the staining solution and carefully wash three times with PBS buffer; observe under an optical microscope, take pictures, and count the number of TRAP-positive cells with more than three nuclei.

[0053] (5) Experiment on the inhibition of osteoclast differentiation induced by primary mouse bone marrow macrophages by drug-loaded MBGN. Immunofluorescence detection of the integrity of the osteoclast F-actin ring: mature osteoclasts can form a complete F-actin ring and can be specifically stained by rhodamine-labeled phalloidin. The experimental operation is as follows. The experiment is divided into four groups: negative control group, positive control group, and drug-loaded MBGN treatment group. Osteoclast precursor cells BMDMs were cultured at 1×10 4The cells were inoculated into 96-well plates at a density of 100 μL / well. 100 μL of culture medium (containing 10% FBS, 30 ng / mL M-CSF) was added to each well of the negative control group, and 100 μL of culture medium (containing 10% FBS, 30 ng / mL M-CSF and 50 ng / mL RANKL) was added to each well of the positive control group and the treatment group. After sufficient mixing, the cell culture plate was placed in a 37°C, 5% CO2 cell constant temperature incubator for overnight culture. After the cells adhered to the wall, the culture medium was discarded, and freshly prepared cell culture medium containing MBGN was added according to the grouping, with 3 duplicate wells per group to induce cell osteoclast differentiation; the medium was changed every three days. On the 7th day, when multinuclear osteoclasts were formed, the culture medium was discarded, and the cells were washed 3 times with PBS buffer, and 4% paraformaldehyde was added to fix the cells for 15 minutes, and then the cells were gently washed again, 0.1% TritonX-100 was added to break the cell membrane for 5 minutes, and then washed 3 times with PBS. Add rhodamine-labeled phalloidin (1:500 dilution) and incubate in the dark for 1 hour, then wash 3 times with PBS. Add DAPI staining solution, incubate in the dark for 15 minutes, and counterstain the cell nucleus. Use a fluorescence microscope to observe the integrity of the osteoclast F-actin ring after staining.

[0054] (6) PCR detection of macrophage inflammatory response: The experiment was divided into four groups: negative control group, positive control group, and MBGN-loaded treatment group. RAW264.7 cells from mice were used as the source of macrophages. RAW264.7 cells were cultured at 10 5 The cells were inoculated with 1 mL of complete culture medium on a 12-well plate at a density of 10 cells / well. After overnight culture at 37°C and 5% CO2, the culture medium was aspirated. The negative control group was added with blank complete culture medium, and the positive control group was added with complete culture medium containing 100 ng / mL LPS and 20 ng / mL IFN-γ. After 30 minutes of ultraviolet disinfection of MBGN, the MBGN treatment group was added with complete culture medium containing 100 ng / mL LPS and 20 ng / mL IFN-γ at a concentration of 100 μg / mL, 1 mL per well, and 3 replicates were set for each group. The stimulation lasted for 8 hours. After 8 hours, the culture medium was aspirated, the cells were rinsed three times with PBS, RNA was extracted, and PCR was performed to detect the expression of inflammatory genes.

[0055] (7) ELISA to detect macrophage inflammatory response: The experiment was also divided into four groups: negative control group, positive control group, and MBGN-treated group. RAW264.7 cells from mice were used as the source of macrophages. RAW264.7 cells were cultured at 10 5The cells were inoculated with 1 mL of complete culture medium at a density of 100 cells / well on a 12-well plate. After overnight culture at 37°C and 5% CO2, the culture medium was aspirated. The negative control group was added with blank complete culture medium, and the positive control group was added with complete culture medium containing 100 ng / mL LPS and 20 ng / mL IFN-γ. After 30 minutes of ultraviolet disinfection of MBGN, the drug-loaded MBGN treatment group was added with complete culture medium containing 100 ng / mL LPS and 20 ng / mL IFN-γ at a concentration of 100 μg / mL, 1 mL per well, and 3 replicates were set for each group for 24 hours of stimulation. After 24 hours, the culture supernatant was aspirated, centrifuged at 2500 rpm for 10 minutes, and the supernatant was taken for ELISA to detect the secretion of inflammatory factors.

[0056] (II) Experimental results

[0057] (1) Figure 3 The results of CCK-8 experiment verify the safety of drug-loaded MBGN cells; Figure 4 This is the MBGN inhibition experiment on osteoclast differentiation induced by mouse bone marrow macrophages (BMM): Tartrate-resistant acid phosphatase (TRAP) staining test results; Figure 5 This is an experiment to inhibit the differentiation of osteoclasts induced by MBGN in primary macrophages of mouse bone marrow: the results of immunofluorescence detection of osteoclast F-actin ring; Figure 3 The results showed that MBGN has good biocompatibility. Figures 4-5 These results indicate that MBGN can significantly inhibit osteoclast differentiation and regulate bone homeostasis in RA.

[0058] (2) Figure 6 After MBGN and macrophages were co-cultured, PCR was used to verify the expression of inflammatory genes. It can be seen that after LPS and IFN-γ stimulation, the expression levels of M1 macrophage-related inflammatory factors CXCL-10, IL-6, and iNOS genes increased significantly, and the level of M2 macrophage-related gene CD206 decreased. After MBGN treatment, the expression of inflammatory genes was inhibited and the level of CD206 increased. *P<0.05.

[0059] (3) Figure 7 The results show that the secretion levels of inflammatory factors CXCL10, IL-6, TNF-α and iNOS after co-culture of each group of materials with macrophages. From the results, it can be seen that the positive control group stimulated by LPS secreted more CXCL10, IL-6, TNF-α and iNOS. The modified MBGN can inhibit the secretion of CXCL10, IL-6, TNF-α and iNOS. *P<0.05.

[0060] The above description is only a preferred embodiment of the present invention and is not any formal or substantial limitation of the present invention. It should be pointed out that a person skilled in the art can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. A method for preparing drug-loaded MBGN nanoparticles, characterized in that: The following steps are involved: Step 1: dissolving the organic template in deionized water, adding ethyl acetate and mixing evenly, then adding ammonia water and stirring, and then sequentially adding silicon source, calcium source and magnesium source for stirring reaction, collecting the nanoparticles generated by the reaction by centrifugation after the reaction, and sequentially washing, drying and calcining to obtain magnesium-containing MBGN; Step 2: The magnesium-containing MBGN prepared in step 1 is uniformly dispersed in Tris-HCL, and then the drug tofacitinib is added, stirred overnight, and centrifuged to obtain drug-loaded MBGN nanoparticles.

2. The preparation method according to claim 1, characterized in that The organic template in step 1 is hexadecyltrimethylammonium bromide; And / or, the silicon source is at least one of ethyl orthosilicate, methyl orthosilicate and butyl orthosilicate.

3. The preparation method according to claim 1, characterized in that: The calcium source in step 1 is at least one of calcium chloride, calcium nitrate, a hydrate of calcium chloride and a hydrate of calcium nitrate; And / or, the magnesium source is at least one of magnesium chloride, magnesium nitrate, a hydrate of magnesium chloride and a hydrate of magnesium nitrate.

4. The preparation method according to claim 1, characterized in that: The feed ratio of the silicon source, calcium source and magnesium source in step 1 is calculated according to the molar ratio of silicon, calcium and magnesium being 80:10:

10.

5. The preparation method according to claim 1, characterized in that: The calcination temperature in step 1 is 600-700° C. and the calcination time is 2-4 hours.

6. The preparation method according to claim 1, characterized in that: In the step 2, the feed mass ratio of magnesium-containing MBGN and the drug tofacitinib is 0.8-1.2:

1.

7. The drug-loaded MBGN prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the drug-loaded MBGN prepared by the preparation method according to any one of claims 1 to 6 in preparing products for treating rheumatoid arthritis.

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