Bioglass composite material for treating rheumatoid arthritis through macrophage targeting and photoacoustic / fluorescent dual-mode tracing

Abstract

The invention discloses a bioglass composite material for treating rheumatoid arthritis through macrophage targeting and photoacoustic / fluorescent dual-mode tracing. According to the invention, the nano-scale mesoporous bioactive glass is coated with polydopamine, and folic acid and fluorescent dye are modified on the surface by utilizing the biological adhesion efficiency of the polydopamine, so that good arthritis targeting and in-vitro tracing ability are achieved, and the drug targeting delivery and treatment effect can be evaluated more accurately. By improving the bioactive glass, the bioactive glass is endowed with various biological functions, so that the MBGN has various functions of inflammation targeting, pH responsive release, immunoregulation and photoacoustic / fluorescence imaging integration, the material accurately targets RA inflammatory joints, has good potential for treating rheumatoid arthritis, and brings a new thought for accurate treatment of RA.

Description

Technical Field

[0001] The invention relates to a bioglass composite material for treating rheumatoid arthritis through macrophage targeting and photoacoustic / fluorescence dual-mode tracing, and belongs to the technical field of biomaterials. Background Art

[0002] In rheumatoid arthritis (RA), a large number of innate immune cells such as macrophages and synovial fibroblasts are activated, which can lead to irreversible damage and degeneration of the synovium and cartilage in the joints. M1 macrophages are activated in inflamed joints and release a large number of proinflammatory cytokines, aggravating synovitis and bone erosion; while M2 macrophages secrete anti-inflammatory cytokines, inhibit the occurrence of inflammation, promote inflammation relief and tissue repair. Therefore, targeting RA joint synovial macrophages and regulating the ratio of M1 / M2 macrophages is an important way to relieve arthritis.

[0003] Mesoporous bioactive glass nanoparticles (MGBN) are a new type of bio-nanomaterial with mesoporous structure and bioactivity, usually composed of components such as silicon oxide. It is characterized by a highly ordered pore network, the size of which ranges from 2 to 50 nanometers and is evenly distributed. This structure gives mesoporous bioactive glass nanoparticles a large specific surface area and abundant surface active sites, making them useful as drug delivery carriers. Drugs can be loaded into them and released in a controlled manner using their pore structure, thereby improving the bioavailability and therapeutic effect of drugs. In addition, these nanoparticles are bioactive, and the ions released can promote osteocyte proliferation and bone tissue regeneration, and are expected to play an important role in drug delivery, bone tissue engineering and other fields. However, bioactive glass has poor dispersibility in water, and after entering the body, it cannot accurately target the inflammatory site of the joint, nor can it display the concentration accumulated in the body in real time, which greatly limits its wide clinical application.

[0004] PDA is formed by the oxidation and self-polymerization of dopamine (DA) in an alkaline solution. As a mussel biomimetic material, PDA has excellent adhesion properties and can be wrapped on the surface of nanomaterials to perform biomimetic modification on them. PDA forms a cross-linked network structure through hydrogen bonds and π-π interactions. The aromatic rings and imine bonds in these structures have strong light absorption properties, and the absorption spectrum covers almost the entire ultraviolet-visible light and near-infrared light region. Its excellent photothermal conversion ability makes PDA have an excellent role in generating photoacoustic signals in photoacoustic imaging. It is often used as a photoacoustic imaging (PAI) contrast agent. PDA encapsulated MGBN can achieve the tracking of nanomaterials in vivo. Cy5 is a cyanine (CY) dye that is often used to label proteins, antibodies and small molecule compounds. Under in vivo fluorescence imaging of small animals, the modification of Cy5 can achieve the tracking of nanomaterials in vivo, thereby evaluating the distribution of materials in vivo. In addition, macrophages in inflammatory sites highly express folate receptors. Modifying folate on the surface of nanomaterials can give the materials the ability to actively target inflammatory sites. Summary of the invention

[0005] The purpose of the present invention is to improve bioactive glass and give it multiple biological functions. The present invention improves mesoporous bioactive glass by incorporating externally coated PDA and modifying folic acid and fluorescent dye Cy5 on the surface, thereby giving it macrophage targeting and tracing capabilities.

[0006] In order to achieve the above object, the present invention provides a method for preparing a bioglass composite material MBGN@PDA-FA for treating rheumatoid arthritis by targeting macrophages, 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 evenly dispersed in Tris-HCL, dopamine is added, and the reaction is stirred. Folic acid is added and stirred overnight, and then the supernatant is removed by centrifugation. The precipitate is collected and dried to obtain MBGN@PDA-FA.

[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, the mass ratio of magnesium-containing MBGN and dopamine in step 2 is 0.8-1.2:1;

[0016] And / or, the feed mass ratio of the magnesium-containing MBGN to folic acid is 1:2-4.

[0017] The present invention also provides a method for preparing a bioglass composite material Cy5-MBGN@PDA-FA, comprising: first preparing MBGN@PDA-FA by the aforementioned preparation method, then incubating the prepared MBGN@PDA-FA with the fluorescent dye Cy5, collecting the precipitate and drying it to obtain Cy5-MBGN@PDA-FA.

[0018] The present invention also provides the use of the bioglass composite material MBGN@PDA-FA prepared by the above-mentioned preparation method in preparing a product for treating rheumatoid arthritis.

[0019] The present invention also provides the use of the bioglass composite material Cy5-MBGN@PDA-FA prepared by the aforementioned preparation method in preparing a product for visual treatment of rheumatoid arthritis.

[0020] The principle of the present invention is as follows:

[0021] By modifying folic acid, the material can target macrophages in the inflammatory site of the RA joint cavity, allowing the nanomaterial to accumulate more efficiently in the inflammatory site of the RA joint synovium; the responsive decomposition of PDA in the low pH environment of the RA joint cavity is used to expose the internally wrapped MBGN, and MBGN can quickly form a hydroxycarbonate apatite (HCA) layer through mineralization deposition in the physiological environment, promoting the material to form a bone bond with the host tissue, and has good biocompatibility; on the other hand, the Si, Ca, Mg ions released by it have certain immunoregulatory functions, promoting the polarization of macrophages from M1 type to M2 type, thereby reducing inflammatory reactions and achieving good therapeutic effects. In addition, after being modified with PDA and Cy5, MBGN can achieve visual monitoring under photoacoustic imaging and fluorescence imaging, realize the tracing of nanomaterials in the body, and thus evaluate the distribution of materials in the body. In summary, the MBGN@PDA-FA-Cy5 prepared by the present invention has multiple functions including inflammation targeting, pH-responsive release, immunomodulation, and integrated photoacoustic / fluorescence imaging. Through real-time monitoring by ultrasound / photoacoustic / fluorescence imaging, the therapeutic effect of the nanosystem can be evaluated and an individualized treatment plan can be formulated, bringing new ideas for the precise treatment of RA.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. After synthesizing MBGN, the present invention wraps a layer of PDA material on its surface to make it have pH responsiveness and photoacoustic imaging performance; and utilizes the adhesion ability of PDA to modify folic acid and Cy5 on the surface to make it target inflammatory macrophages and achieve in vitro tracing; the Cy5-MBGN@PDA-FA material provided by the present invention has good stability and multiple biological properties, and can improve the shortcomings of current mesoporous bioactive glass materials, such as poor targeting, insufficient anti-inflammatory effect, and difficulty in real-time visual tracing;

[0024] 2. The present invention improves bioactive glass and endows it with multiple biological functions, so that MBGN has multiple functions including inflammation targeting, pH-responsive release, immunomodulation, and integrated photoacoustic / fluorescence imaging. The material precisely targets RA inflamed joints and has good potential for treating rheumatoid arthritis, bringing new ideas for the precise treatment of RA.

[0025] 3. The preparation process of the present invention is simple, efficient, and has good repeatability, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 :Surface morphology of MBGN@PDA-FA under transmission electron microscope (A), energy spectrum mapping and EDS element analysis (B).

[0027] Figure 2 : CLSM observation of RAW264.7 uptake of nanoparticles in each group.

[0028] Figure 3 : Flow cytometric analysis (A) and quantitative statistical results (B) of RAW264.7 uptake of nanoparticles in each group; ***P<0.001, ****P<0.0001.

[0029] Figure 4 :Nanoparticles in each group inhibited the inflammatory response of macrophages: PCR verified the expression level of inflammatory genes; ****P<0.0001.

[0030] Figure 5 : In vivo imaging of small animals showed in vitro tracking of CIA mice after tail vein injection of nanoparticles in each group; **P<0.01.

[0031] Figure 6 : In vitro tracking images of CIA mice after tail vein injection of each group of nanoparticles under ultrasound / photoacoustic mode (A) and quantitative statistical results of nanoparticles accumulated in inflamed joints (B); ****P<0.0001. DETAILED DESCRIPTION

[0032] In order to make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0033] The experimental methods in the following examples without specifying specific conditions were performed according to conventional methods and conditions, or selected according to the product instructions; the materials and reagents used were conventional commercially available products unless otherwise specified; the quantitative tests involved were repeated three times and the results were averaged.

[0034] Example

[0035] 1. Preparation of MBGN@PDA-FA-Cy5 nanomaterials:

[0036] (1) Preparation of MBGN:

[0037] 0.7g CTAB was added to 33mL ddH2O, stirred with a magnetic stirrer to fully dissolve the CTAB particles, 10mL ethyl acetate was added, stirred for 30min, 7mL 1mol / mL NH3.H2O (ammonia water) was added, 3.6mL TEOS 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 dry in an oven, calcined at 650℃ for 3h.

[0038] (2) Preparation of MBGN@PDA-FA:

[0039] Add 10 mL of 1% Tris-HCL to 10 mg of MBGN, disperse ultrasonically for 20 min, add an equal amount of dopamine, stir with a magnetic stirrer for 4 h, add 30 mg of folic acid, stir overnight, centrifuge at 7000 rpm for 10 min, remove the supernatant, and dry the precipitate.

[0040] (3) Preparation of Cy5-MBGN@PDA-FA:

[0041] Take 10 mg MBGN@PDA-FA and disperse it in 10 mL 10% DMSO, add 2 mg Cy5, stir overnight in the dark, centrifuge at 8000 rpm for 10 min, discard the supernatant, wash twice with ddH2O, and dry the precipitate.

[0042] 2. Characterization of Materials

[0043] Figure 1 The surface morphology, energy spectrum mapping and EDS element analysis of MBGN@PDA-FA under transmission electron microscopy. The material was sterilized by ultraviolet light, placed in a 1.5mL EP tube with 3 replicates per group, resuspended in anhydrous ethanol and ultrasonically dispersed for 20 minutes, then the sample was dropped on the support film and observed, energy spectrum mapping and EDS element analysis were performed using a transmission electron microscope TEM (JEOL JEM-F200, Japan).

[0044] 3. Material biological activity function detection

[0045] (1) CLSM (confocal laser scanning microscopy) observation of RAW264.7 uptake of nanoparticles in each group

[0046] The RAW264.7 cells used in this example are mouse-derived macrophages, and the prepared Cy5-MBGN@PDA and Cy5-MBGN@PDA-FA were cultured together with RAW264.7 cells to conduct a cell uptake experiment.

[0047] RAW264.7 cells were cultured at 10 5 The cells were plated on a confocal dish at a density of 10 cells / well with 400 μL complete culture medium and cultured at 37°C in 5% CO2. After overnight culture, the culture medium was aspirated and 400 μL 100 μg / mL LPS (or 1 ng / mL TNF-α) complete culture medium was added for stimulation for 8 h. The control group used complete culture medium. After 8 h, the culture medium was aspirated and rinsed three times with PBS. 400 μl 100 μg / mL complete culture medium of Cy5-MBGN@PDA and Cy5-MBGN@PDA-FA nanoparticles was added and incubated for 3 hours. Then the culture medium was removed and the cells were washed three times with PBS to remove non-endocytic particles. Cell uptake was observed under CLSM. The results are shown in Figure 2. Figure 2 As shown, compared with RAW264.7 cells without LPS stimulation, the cells had a stronger ability to take up nanoparticles after LPS stimulation; compared with MBGN@PDA, the macrophage uptake rate of MBGN@PDA-FA after LPS stimulation was higher.

[0048] (2) Flow cytometry analysis was used to detect the uptake of nanoparticles in each group by RAW264.7

[0049] Figure 3 Flow cytometry analysis of the uptake of nanoparticles in each group by RAW264.7 cells. 6 The cells were plated on a 12-well plate at a density of 1 cell / well with 1 mL of complete culture medium and cultured at 37°C in 5% CO2. After overnight culture, the culture medium was aspirated and 1 mL of 100 μg / mL LPS (or 1 ng / mL TNF-α) complete culture medium was added for stimulation for 8 h. The control group used complete culture medium. After 8 h, the culture medium was aspirated and the cells were rinsed three times with PBS. 1 mL of 100 μg / mL complete culture medium of Cy5-MBGN@PDA and Cy5-MBGN@PDA-FA nanoparticles was added and incubated for 3 hours. The culture medium was then removed and the cells were washed three times with PBS to remove non-endocytic particles. Cell uptake was detected under the PE-Cy5 channel of a flow cytometer (BD, FortessaX20). The results are shown in Figure 3 As shown, the results have the same trend as the CLSM observation results. Compared with RAW264.7 cells without LPS stimulation, the cells have a stronger ability to take up nanoparticles after LPS stimulation; compared with MBGN@PDA, the macrophage uptake rate of MBGN@PDA-FA after LPS stimulation is higher.

[0050] (3) Macrophage inflammatory response by PCR

[0051] The experiment was also divided into four groups: negative control group, positive control group, MBGN treatment group, and MBGN@PDA treatment group. RAW264.7 cells from mice were used as the source of macrophages. RAW264.7 cells were cultured at 10 6 The cells were inoculated in 12-well plates at a density of 100 cells / well with 1 mL of complete culture medium. After overnight culture at 37°C and 5% CO2, the culture medium was aspirated. The negative control group was added with complete culture medium, and the positive control group was added with complete culture medium containing 100 ng / mL LPS. After UV disinfection for 30 minutes, the MBGN treatment group and the MBGN@PDA treatment group were added with complete culture medium containing 100 ng / mL LPS 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. The culture medium was aspirated, washed three times with PBS, RNA was extracted, and PCR was performed to detect the expression of inflammatory genes.

[0052] Figure 4 The results show that the secretion levels of inflammatory factors iNOS and CXCL10 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 iNOS and CXCL10. The modified MBGN@PDA can inhibit the secretion of iNOS and CXCL10, and MBGN@PDA-FA has a stronger anti-inflammatory effect.

[0053] (4) Animal in vivo experiments

[0054] The in vivo experiment was verified using collagen-induced arthritis (CIA) mice. CIA mouse arthritis shares common immunological and pathological characteristics with human rheumatoid arthritis, so the CIA model is widely used in the study of pathogenic mechanisms and treatment options. CIA mouse modeling uses 7-8 week old DBA / 1 strain mice.

[0055] Figure 5 In vitro tracing of each group of nanoparticles after tail vein injection of CIA mice for small animal in vivo fluorescence imaging. In vivo and in vitro fluorescence imaging of CIA mice 24 hours after tail vein injection of Cy5, Cy5-MBGN@PDA, and Cy5-MBGN@PDA-FA. The results showed that Cy5-MBGN@PDA-FA has a stronger targeting effect on inflamed joints.

[0056] Figure 6The ultrasound / photoacoustic mode is used to track the nanoparticles in vitro after the tail vein of CIA mice was injected. The ultrasound / photoacoustic imaging of healthy mice and CIA mice was performed 24 hours after the tail vein injection of MBGN@PDA and MBGN@PDA-FA. The results under PA (photoacoustic) mode showed that compared with MBGN@PDA, MBGN@PDA-FA has a stronger targeting ability in inflamed joints.

[0057] 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 a bioglass composite material MBGN@PDA-FA for treating rheumatoid arthritis by targeting macrophages, 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 evenly dispersed in Tris-HCL, dopamine is added, and the reaction is stirred. Folic acid is added and stirred overnight, and then the supernatant is removed by centrifugation. The precipitate is collected and dried to obtain MBGN@PDA-FA.

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 mass ratio of magnesium-containing MBGN to dopamine is 0.8-1.2:1; And / or, the feed mass ratio of the magnesium-containing MBGN to folic acid is 1:2-4.

7. A method for preparing a bioglass composite material Cy5-MBGN@PDA-FA, characterized in that: include: First, MBGN@PDA-FA is prepared by the preparation method described in any one of claims 1 to 6, and then the prepared MBGN@PDA-FA is incubated with the fluorescent dye Cy5, and the precipitate is collected and dried to obtain Cy5-MBGN@PDA-FA.

8. Use of the bioglass composite material MBGN@PDA-FA prepared by the preparation method according to any one of claims 1 to 6 in preparing products for treating rheumatoid arthritis.

9. Use of the bioglass composite material Cy5-MBGN@PDA-FA prepared by the preparation method described in claim 7 in preparing a product for visual treatment of rheumatoid arthritis.

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