Membrane vesicular calcium ion carbon dots of bone marrow mesenchymal stem cells as well as preparation method and application of membrane vesicular calcium ion carbon dots

Abstract

The invention is applicable to the technical field of biological medicine, and provides bone marrow mesenchymal stem cell membrane vesicular calcium ion carbon dots and a preparation method and application thereof, the preparation method comprises the following steps: adding calcium gluconate into deionized water, and stirring to form a uniform mixed solution; heating the mixed solution, and then cooling; filtering and dialyzing the solution to obtain a CaCD solution; culturing and collecting mBMSC, adding an RIPA lysate, homogenizing and breaking cells, repeatedly centrifuging, and collecting to obtain an mBMSC membrane; the mBMSC membrane is repeatedly extruded through a liposome extruder, and mBMSC vesicles are obtained; and mixing the CaCD solution with the mBMSC vesicles, carrying out ultrasonic treatment to obtain a mixed solution, and repeatedly extruding the mixed solution through a liposome extruder to obtain the M-CaCD. Calcium gluconate is selected as a precursor to prepare CaCD through a one-step hydrothermal method, the process is simple, operation is easy, cost is low, and the method can be used for industrial production; an mBMSC membrane is selected to be packaged to the surface of the CaCD, so that the CaCD is endowed with a bone tissue targeting characteristic, and a new thought and a new method are expected to be provided for medicines for treating osteoporotic bone defects.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to bone marrow mesenchymal stem cell membrane vesicle calcium ion carbon dots and a preparation method and application thereof. Background Art

[0002] Osteoporosis (OP) is a systemic bone metabolic disease characterized by decreased bone mass, destruction of bone tissue microstructure, decreased bone density, and increased bone brittleness. It is common in middle-aged and elderly people, especially postmenopausal women. Osteoporosis is closely related to the occurrence and development of oral diseases, which can aggravate periodontitis and increase the risk of jaw fractures. Periodontitis is a chronic inflammatory disease with complex etiology, which is mainly manifested by the destruction of tooth supporting structures (periodontal ligament, alveolar bone, etc.), causing partial or even complete tooth loss, which has a significant impact on patients' chewing function, communication ability, and quality of life.

[0003] Drug therapy is the most common method for treating osteoporotic bone defects. Based on the mechanism of bone homeostasis, bone resorption inhibitors and bone formation promoters have been developed, including bisphosphonates, estrogens, fluorides, and parathyroid analogs. Although these drugs slow down the process of bone destruction to a certain extent, due to the low degree of bone vascularization, it is not easy for drugs to penetrate, which often leads to off-target absorption of drugs and significantly reduces the therapeutic effect. In addition, complications such as osteonecrosis of the jaw, increased risk of breast cancer, and hyperparathyroidism also limit the application of drugs in osteoporotic bone defects. Therefore, improving the bone-targeted delivery of drugs and exploring safe and effective new therapies are still major challenges currently faced.

[0004] As the target cells of bone metabolism therapeutic drugs, the number and functional changes of osteoblasts directly affect the occurrence, development and prognosis of osteoporosis. Among the many factors that affect osteoblast function, Ca 2+ Plays a vital role in the extracellular Ca 2+ Activates cell membrane Ca 2+ calcium-sensing receptor (CaSR), which promotes osteoblast proliferation and differentiation; in cells, Ca 2+ It promotes osteoblast differentiation by binding to calmodulin (CaM) and activating calmodulin dependent kinase 2 (CaMK2).

[0005] Carbon dots (CDs) are a new type of quasi-zero-dimensional carbon nanomaterials with good water solubility, biocompatibility and stability. Their small size allows them to cross various natural biological barriers in the body, which enables them to deliver Ca2+ to osteoblasts and their precursor cells.2+ Activated Ca 2+ The unique advantages of the signal are expected to achieve efficient bone regeneration. In order to ensure that the carbon dots can fully exert their biological efficacy, it is necessary to solve the problems of short circulation time and low targeting. The biomimetic drug delivery system based on biofilm wrapping can simulate the functions of endogenous substances and biological processes in the human body, and can accurately deliver drugs to the target location to achieve the purpose of precision treatment. It has the characteristics of small adverse reactions, good therapeutic effects and low immunogenicity. Studies have shown that cell membranes have the ability to target cells of the same origin, that is, homologous targeting. Based on this, the use of bone marrow mesenchymal stem cell membranes to wrap calcium ion carbon dots is expected to prolong their in vivo circulation time and enhance targeting, and efficiently promote osteoblast differentiation and bone formation. Summary of the invention

[0006] The purpose of the embodiments of the present invention is to provide a method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots, aiming to solve the problems raised in the above background technology.

[0007] The embodiment of the present invention is implemented as follows: a method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots comprises the following steps:

[0008] S1: adding calcium gluconate into deionized water and stirring to form a uniform mixed solution;

[0009] S2: transferring the mixed solution obtained in step S1 to a polytetrafluoroethylene autoclave for heating, and then cooling;

[0010] S3: filtering and dialyzing the solution obtained in step S2 to remove impurities to obtain a pure calcium ion carbon dot (CaCD) solution;

[0011] S4: culture and collect mouse bone marrow mesenchymal stem cells (mBMSC), add RIPA lysis buffer and homogenize to break the cells, and collect the mBMSC membrane after repeated centrifugation;

[0012] S5: repeatedly extruding the mBMSC membrane obtained in step S4 through a liposome extruder to obtain mBMSC vesicles;

[0013] S6: mixing the calcium ion carbon dot solution obtained in step S3 with the mBMSC vesicles obtained in step S5, and performing ultrasonic treatment to obtain a mixed solution;

[0014] S7: repeatedly extruding the mixed solution obtained in step S6 through a liposome extruder to obtain bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots (M-CaCD).

[0015] Preferably, in step S1, the concentration of calcium gluconate in the mixed solution is 0.45-0.55 mM.

[0016] Preferably, in step S2, the heating temperature is 170-190° C., and the heating time is 3.5-4.5 h.

[0017] Preferably, in step S3, the filtration uses a water-based microporous filter membrane with a pore size of 0.22 μm; the dialysis bag used for dialysis has a molecular weight of 1000 Da, and the dialysis time is 46-50 h.

[0018] Preferably, in step S4, the mouse is a two-week-old male C57BL / 6J mouse;

[0019] The frequency of the homogenization was 60 Hz, running for 3 min, pausing for 1 min, and running for 10 cycles in total.

[0020] Preferably, in step S5, the step of repeatedly extruding the mBMSC membrane through a liposome extruder is specifically that the mBMSC membrane is repeatedly extruded through 400 nm, 200 nm and 100 nm porous polycarbonate membranes in sequence.

[0021] Preferably, in step S6, the ultrasonication time is 4-6 minutes.

[0022] Preferably, in step S7, the number of repeated extrusions is 8-12 times.

[0023] Another object of the embodiment of the present invention is to provide a bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots, which are prepared by the above-mentioned preparation method.

[0024] Another object of the embodiment of the present invention is to provide an application of bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots in the preparation of a drug for treating osteoporotic bone defect diseases.

[0025] The method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots provided in the embodiment of the present invention selects calcium gluconate as a precursor to prepare CaCD by a one-step hydrothermal method. The process is simple, easy to operate, low in cost, and can be used for industrial production; mBMSC membrane is encapsulated on the surface of CaCD, giving CaCD bone tissue targeting properties;

[0026] The in vitro studies provided in the embodiments of the present invention demonstrate that M-CaCD can quickly and efficiently enter the target cells and can significantly promote the osteogenic differentiation of mBMSCs; in vivo studies demonstrate that M-CaCD has the effect of promoting the repair of osteoporotic bone defects. At the same time, M-CaCD shows good biocompatibility both in vivo and in vitro, and is expected to provide new ideas and methods for the treatment of osteoporotic bone defects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1A flow chart of the preparation of bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots ( provided in Example 1 of the present invention;

[0028] Figure 2 HR-TEM image (A) and particle size distribution (B) of CaCD prepared in Example 1 of the present invention;

[0029] Figure 3 PL spectrum (A) and UV-visible absorption spectrum (B) of the CaCD prepared in Example 1 of the present invention;

[0030] Figure 4 This is the XPS spectrum of CaCD prepared in Example 1 of the present invention;

[0031] Figure 5 TEM image (A) and hydrated particle size (B) of M-CaCD prepared in Example 1 of the present invention, and Zeta potential (C) of CaCD, mBMSC membrane (MM) and M-CaCD;

[0032] Figure 6 This is a result diagram of the effect of CaCD on MC3T3-E1 cell proliferation detected by MTT method provided in an embodiment of the present invention;

[0033] Figure 7 Fluorescence images of the uptake of CaCD and M-CaCD by mBMSC provided in an embodiment of the present invention;

[0034] Figure 8 The results of analyzing the effect of CaCD on the expression of osteogenesis-related genes Alp, Runx2, Sp7, Col1 and Bmp2 using RT-qPCR provided in the embodiment of the present invention;

[0035] Fig. 9 ALP staining results of mBMSC cells induced by CaGc, CaCD and M-CaCD provided in the embodiments of the present invention;

[0036] Fig.10 This is a diagram showing the results of Alizarin red staining of mBMSC cells induced by CaGc, CaCD and M-CaCD provided in an embodiment of the present invention;

[0037] Fig.11 A Micro-CT result diagram showing the effects of CaGc, CaCD and M-CaCD on femoral bone density in osteoporotic mice provided by the embodiments of the present invention;

[0038] Fig.12 Micro-CT results of treating osteoporotic periodontitis in mice with CaGc, CaCD and M-CaCD provided in the embodiments of the present invention;

[0039] Fig.13 This is a diagram showing the HE staining results of important organs of mice after treatment with CaGc, CaCD and M-CaCD provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0041] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0042] Example 1: A bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots (M-CaCD), the preparation method of which is as follows Figure 1 As shown, the specific steps include:

[0043] Step 1: Weigh 2.15 mg of calcium gluconate and add it to 10 mL of deionized water, stir to form a mixed solution, the concentration of calcium gluconate is 0.5 mM; transfer the solution to a polytetrafluoroethylene autoclave, heat the reaction at 180°C for 4 hours, and cool to room temperature; filter with a 0.22 μm water system microporous filter membrane, and then dialyze with a 1000 Da dialysis bag for 48 hours to remove impurities and obtain pure CaCD;

[0044] Step 2: 2-week-old male C57BL / 6J mice were killed by cervical dislocation and soaked in 75% ethanol for 7 minutes; the tibia and femur of the mice were separated under sterile conditions, soaked in 75% ethanol for 5 minutes, and washed away with pre-cooled phosphate buffered saline (PBS) to expose the bone marrow cavity; a 1 mL syringe was used to extract pre-cooled culture medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibody, and the bone marrow cavity was repeatedly rinsed 3 times; bone marrow mesenchymal stem cells were inoculated into T25 cell culture flasks and placed at 37°C and 5% CO 2 Culture in incubator.

[0045] Step 3: P3 mBMSCs were collected, washed with pre-cooled PBS and collected with a cell scraper, and then RIPA lysis buffer was added and the cells were broken using a cell homogenizer. The frequency was set to 60 Hz, and the mixture was run for 3 min, paused for 1 min, and run for 10 cycles in total. The cell nuclei and unbroken cells were removed by centrifugation at 700 g for 10 min at 4 °C, and the supernatant was collected and centrifuged at 14,000 g for 30 min at 4 °C to precipitate mBMSC membrane fragments.

[0046] Step 4: Resuspend the mBMSC membrane containing 0.121 mg / mL membrane protein in 1 mL PBS; use a manual liposome extruder to repeatedly extrude the MBMSC membrane solution through porous polycarbonate membranes with pore sizes of 400 nm, 200 nm and 100 nm to obtain mBMSC vesicles with a size of 100 nm; then ultrasonically treat the vesicles with 0.2 mL of 2 mg / mL CaCD for 5 min, mix and repeatedly extrude 10 times using a 100 nm porous polycarbonate membrane to obtain M-CaCD.

[0047] Performance Analysis:

[0048] The CaCD and M-CaCD prepared in Example 1 were characterized:

[0049] The CaCD was imaged using a FEI Tecnai G2F20 high-resolution transmission electron microscope (HR-TEM). Figure 2 As shown, according to Figure 2 It can be seen that CaCD presents a uniformly distributed nanoparticle structure with an average particle size of about 5.7 nm. HR-TEM shows that CaCD has a lattice structure with a lattice spacing of 0.21 nm, corresponding to the (100) crystal plane of graphite;

[0050] The photoluminescence (PL) spectrum of CaCD was measured using a JASCO FP-6500 fluorescence spectrophotometer, and the UV-visible absorption spectrum of CaCD was measured using a JASCO V-550 UV-visible spectrophotometer. Figure 3 As shown, according to Figure 3 It can be seen that CaCD has typical blue fluorescence emission under ultraviolet light and shows the excitation-dependent characteristics of carbon dots. When the excitation wavelength is 358nm, it has the maximum fluorescence intensity and the emission peak is located at 445nm. The UV-visible absorption spectrum shows two absorption peaks of the CaCD solution, namely the main absorption peak at 255nm and the weaker shoulder peak at 330nm, which correspond to the π→π* transition of the carbon core C=C bond and the n→π* transition of the surface group C=O bond.

[0051] The X-ray photoelectron spectroscopy (XPS) of CaCD was measured using a Thermo Fisher Scientific ESCALAB 250Xi X-ray photoelectron spectrometer. The results are as follows: Figure 4 As shown, according to Figure 4 It can be seen that C, O, and Ca elements exist in CaCD. The characteristic signal at 347.8 eV binding energy in the Ca2p spectrum and the Ca Auger electron peak (CaKLL) at 351.4 eV confirm that Ca 2+The presence of Ca, calculated by XPS peak area ratio, shows that the doping amount of Ca is 10.4%; three forms of carbon are analyzed in the C1s spectrum: 287.9eV corresponds to CC / C=C, 286.0eV corresponds to CO and 284.8eV corresponds to C=O; there are two types of oxygen at 532.8eV and 531.7eV in the O1s spectrum, corresponding to CO and C=O, respectively, indicating that CaCD is composed of a carbon core composed of sp2 / sp3 graphitic carbon and oxygen-containing functional groups on the surface, which shows that Example 1 successfully prepared a new type of calcium ion carbon dot (CaCD). During the hydrothermal reaction, glucose in calcium gluconate is carbonized as a carbon source to form a carbon core, and Ca 2+ Achieve in-situ doping and combine with the carboxylic acid groups retained on the surface of carbon dots;

[0052] The morphology of M-CaCD was observed using a JEM-2100F transmission electron microscope (TEM), the hydrated particle size of M-CaCD was measured using dynamic light scattering (DLS), and the zeta potential of CaCD, mBMSC membrane, and M-CaCD was measured using a MalvernNano ZS-90 nanoparticle size and zeta potential analyzer at 25°C. The results are as follows: Figure 5 As shown, according to Figure 5 It can be seen that there is low-density material around CaCD, which is similar to the TEM imaging of the membrane structure; DLS shows that the hydrated particle size of M-CaCD is about 105nm; Zeta potential shows that the surface potential of M-CaCD is -19.03mV, which is similar to the surface potential of mBMSC membrane, which shows that Example 1 successfully prepared a bone marrow mesenchymal stem cell membrane vesicle calcium ion carbon dot (M-CaCD).

[0053] The CaCD and M-CaCD prepared in Example 1 were tested for their in vitro biological properties:

[0054] MC3T3-E1 was used as a model cell and co-cultured with different concentrations of CaCD for 7 days. The MTT method was used to detect its effect on cell proliferation. The results are as follows: Figure 6 As shown, according to Figure 6 It can be seen that CaCD concentrations as high as 200 μg / mL still had no significant effect on cell proliferation;

[0055] mBMSCs were used as model cells and co-cultured with 200 μg / mL CaCD and M-CaCD for 6 h, respectively. The uptake of the two by mBMSCs was observed using an inverted fluorescence microscope. The results are as follows: Figure 7 As shown, according to Figure 7 It can be seen that the blue fluorescence intensity and area in the M-CaCD group were significantly higher than those in the CaCD group, indicating that the efficiency of CaCD entering the cells after being encapsulated by mBMSCs was significantly increased;

[0056] mBMSCs were used as model cells and co-cultured with different concentrations of CaCD. The expression levels of osteogenic-related genes Alp, Runx2, Sp7, Col1, and Bmp2 were analyzed by RT-qPCR. The results were as follows: Figure 8 As shown, according to Figure 8 It can be seen that after 7 days of co-culture, different concentrations of CaCD can significantly increase the expression of osteogenesis-related genes; at lower concentrations (25 μg / mL and 50 μg / mL), only Bmp2 and Col1 were upregulated, indicating that the osteogenic induction ability of CaCD is limited at low concentrations; while at high concentrations (100 μg / mL and 200 μg / mL), CaCD can upregulate the expression of Alp, Runx2, Sp7, Col1 and Bmp2;

[0057] mBMSCs were used as model cells and co-cultured with 200 μg / mL calcium gluconate (CaGc), CaCD, and M-CaCD, respectively. Alkaline phosphatase (ALP) staining and Alizarin red staining were used to detect their ability to promote osteogenic differentiation. The results are as follows: Fig. 9 , Fig.10 As shown, according to Fig. 9 It can be seen that compared with the CaGc group, the CaCD and M-CaCD groups showed higher ALP activity induction ability, and the M-CaCD group was significantly higher than the other groups. The ALP staining results confirmed that CaCD had good osteogenic induction activity, and its ability to induce ALP activity was significantly improved after being encapsulated by mBMSC vesicles; according to Fig.10 It can be seen that compared with the CaGc group, the CaCD and M-CaCD groups induced more calcium nodules to form, and the number of positively stained calcium nodules in the M-CaCD group was significantly higher than that in the other groups, indicating that CaCD can promote osteoblast differentiation well, and CaCD encapsulated by mBMSC has a stronger ability to promote osteoblast differentiation.

[0058] The CaCD and M-CaCD prepared in Example 1 were used to treat osteoporotic periodontitis mouse models to study their therapeutic effects in treating osteoporotic periodontitis. The modeling process was as follows:

[0059] Eight-week-old female C57BL / 6J mice were selected and the osteoporosis model was established by ovariectomy. The model group underwent bilateral ovarian removal, while the sham operation group retained both ovaries and only removed the fat tissue around the ovarian tissue that was equivalent to the weight of the ovaries. Five weeks after modeling, the model group was randomly divided into four groups: PBS group, CaGc group, CaCD group, and M-CaCD group. A 5-0 silk thread was placed between the first and second molars of the maxillary teeth of the five groups of mice to establish a periodontitis model. The silk thread was removed after 7 days of ligation, and different drugs were injected into the tail vein at a dose of 10 μg / g body weight.

[0060] After 2 weeks of administration, the mice were euthanized, and the femur and alveolar bones were collected. Micro-CT was used to detect the therapeutic effects of CaCD and M-CaCD on osteoporotic periodontitis. The results were as follows: Fig.11 , Fig.12 As shown, according to Fig.11 It can be seen that compared with the sham operation group, the bone density of the femur of the model group mice was significantly decreased, and compared with the PBS group, CaGc group and CaCD group, the bone density of the M-CaCD group was significantly increased, indicating that M-CaCD has a good therapeutic effect on osteoporosis; Fig.12 It can be seen that compared with the PBS group, CaGc group, and CaCD group, the distance from the cementoenamel junction to the alveolar crest (CEJ-ABC) in the M-CaCD group was significantly reduced, indicating that the therapeutic effect of M-CaCD on osteoporotic periodontitis was significantly better than that of CaGc and CaCD;

[0061] The important organs of mice were collected, fixed, dehydrated, embedded and sliced, and HE staining was used to observe the in vivo biosafety of CaCD and M-CaCD. The results were as follows: Fig.13 As shown, according to Fig.13 It can be seen that the histological structures of the mouse heart, liver, spleen, lung, and kidney were normal, and no inflammatory response was observed, indicating that CaCD and M-CaCD have good in vivo biosafety.

[0062] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots, characterized in that: The following steps are involved: S1: adding calcium gluconate into deionized water and stirring to form a uniform mixed solution; S2: transferring the mixed solution obtained in step S1 to a polytetrafluoroethylene autoclave for heating, and then cooling; S3: filtering and dialyzing the solution obtained in step S2 to remove impurities to obtain a pure calcium ion carbon dot solution; S4: Culture and collect mouse bone marrow mesenchymal stem cells, add RIPA lysis buffer and homogenize to break the cells, and collect mBMSC membranes after repeated centrifugation; S5: repeatedly extruding the mBMSC membrane obtained in step S4 through a liposome extruder to obtain mBMSC vesicles; S6: mixing the calcium ion carbon dot solution obtained in step S3 with the mBMSC vesicles obtained in step S5, and performing ultrasonic treatment to obtain a mixed solution; S7: repeatedly extruding the mixed solution obtained in step S6 through a liposome extruder to obtain bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots.

2. The method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots according to claim 1, characterized in that: In step S1, the concentration of calcium gluconate in the mixed solution is 0.45-0.55 mM.

3. The method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots according to claim 1, characterized in that: In step S2, the heating temperature is 170-190°C, and the heating time is 3.5-4.5h.

4. The method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots according to claim 1, characterized in that: In step S3, the filtration uses a water-based microporous filter membrane with a pore size of 0.22 μm; the dialysis bag used for dialysis has a molecular weight of 1000 Da, and the dialysis time is 46-50 h.

5. The method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots according to claim 1, characterized in that: In step S4, the mice are two-week-old male C57BL / 6J mice; The frequency of the homogenization was 60 Hz, running for 3 min, pausing for 1 min, and running for 10 cycles in total.

6. The method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots according to claim 1, characterized in that: In step S5, the mBMSC membrane is repeatedly extruded through a liposome extruder, specifically, the mBMSC membrane is repeatedly extruded through 400 nm, 200 nm and 100 nm porous polycarbonate membranes in sequence.

7. The method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots according to claim 1, characterized in that: In step S6, the ultrasonication time is 4-6 minutes.

8. The method for preparing bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots according to claim 1, characterized in that: In step S7, the number of repeated extrusion is 8-12 times.

9. A bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots, characterized in that: The invention is prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the bone marrow mesenchymal stem cell membrane encapsulated calcium ion carbon dots as claimed in claim 9 in the preparation of a drug for treating osteoporotic bone defect diseases.

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