Magnetic navigation BMSC exosome loaded with recombinant hirudin as well as preparation method and application of magnetic navigation BMSC exosome
By preparing magnetically guided BMSC exosomes loaded with recombinant hirudin, the problems of insufficient drug loading and targeting of exosomes were solved, achieving effective treatment of femoral head necrosis with significant anticoagulant and targeting effects.
Patent Information
- Application Number
- CN202511565396.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-30
- Publication Date
- 2026-01-27
AI Technical Summary
Existing exosome technology has limitations in terms of drug loading capacity and targeting, making it difficult to effectively treat avascular necrosis of the femoral head, especially due to problems such as insufficient local drug concentration and large surgical trauma.
Magnetic navigation BMSC exosomes loaded with recombinant hirudin were prepared by co-incubating magnetic iron oxide nanoparticles (IONP) with BMSC exosomes and recombinant hirudin (RH) to form IONP-BMSC-exos-RH, which endowed the exosomes with superparamagnetism and targeting, thereby improving drug loading and targeting.
It significantly reduces thrombus formation, significantly improves femoral head necrosis, has good anticoagulant effect and targeting, and provides higher drug concentration and lower risk of trauma.
Smart Images
Figure CN121406572A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biomedical technology, and in particular relates to a magnetically guided BMSC exosome loaded with recombinant hirudin, its preparation method and application. Background Technology
[0002] The main causes of avascular necrosis of the femoral head include the widespread use of glucocorticoids, alcoholism, or hip trauma, and the incidence rate is increasing year by year. Its core pathological mechanism is the formation of microthrombi in the femoral head, which leads to ischemic necrosis of bone cells and joint collapse. It is currently more common in middle-aged people, and some patients are bilaterally affected. Existing treatment options mainly include drug intervention, surgical treatment, or lifestyle intervention. Drug intervention aims to improve the microcirculation of the femoral head, but there is a problem of insufficient local concentration of the drug. Surgical treatment requires the reconstruction of blood supply, and the surgery is highly invasive. Joint replacement also carries the risk of prosthesis loosening and infection.
[0003] Exosomes are nanoscale vesicles secreted by cells, encapsulated by a lipid bilayer and carrying proteins, nucleic acids, and signaling molecules from their origin. Their biogenesis depends on the fusion and release of intracellular multivesicular bodies with the cell membrane. As natural intercellular communication carriers, exosomes participate in physiological regulation and disease processes. Their membrane marker proteins and intraluminal active substances make them highly promising in disease diagnosis (such as tumor liquid biopsy) and treatment, especially as drug carriers due to their low immunogenicity, high biocompatibility, and ability to cross barriers. However, traditional exosome technology faces three major bottlenecks: low separation and purification yield (recovery rate of less than 0.1% by ultracentrifugation), poor loading efficiency of hydrophilic drugs (generally <5%), and insufficient passive targeted accumulation at lesions.
[0004] Therefore, improving the drug loading capacity and targeting of exosomes for the treatment of avascular necrosis of the femoral head has become a research focus. Summary of the Invention
[0005] To address the shortcomings of existing technologies, this invention proposes a magnetically guided BMSC exosome loaded with recombinant hirudin, its preparation method, and its application.
[0006] The technical solution of this invention is as follows: A method for preparing magnetically guided BMSC exosomes loaded with recombinant hirudin includes the following steps: S1. After adding IONP to BMSCs and incubating, IONP-BMSCs are obtained; S2. The precipitate obtained after centrifugation and magnetic separation of IONP-BMSC is IONP-BMSC-exos; S3. After co-incubating IONP-BMSC-exos with RH, magnetic separation was performed. The resulting precipitate was the magnetically navigable BMSC exosome loaded with recombinant hirudin, denoted as IONP-BMSC-exos-RH.
[0007] Further, in step S1, the IONP: BMSC = (25-200) μg: 12 × 10 6 The concentration of IONP is 5-40 μg / mL; in step S3, the IONP-BMSC-exos : RH=1×10 9 Quantity: (0.04-0.07) μg.
[0008] Furthermore, the incubation process parameters in step S1 are: incubation at room temperature for 24 hours.
[0009] Furthermore, the centrifugation described in step S2 is differential ultracentrifugation.
[0010] Further, the differential ultracentrifugation method is as follows: centrifuge the supernatant of IONP-BMSC at 300-1000×g for 10-20 min to obtain supernatant A; centrifuge supernatant A at 10000-20000×g for 20-40 min to obtain supernatant B; centrifuge supernatant B at 80000-120000×g for 60-90 min to obtain precipitate C; and centrifuge precipitate C again at 80000-120000×g for 60-90 min to obtain precipitate D.
[0011] Furthermore, the IONP-BMSC-exos particles obtained in step S2 have a particle size of 120 nm.
[0012] Furthermore, the incubation process parameters in step S3 are: co-incubation at room temperature for 24 hours.
[0013] IONP-BMSC-exos-RH was prepared according to the preparation method described above.
[0014] The application of IONP-BMSC-exos-RH in the preparation of anticoagulant drugs.
[0015] The application of IONP-BMSC-exos-RH in the preparation of drugs for repairing damaged bone tissue.
[0016] Furthermore, the damaged bone tissue includes femoral head necrosis.
[0017] Furthermore, the aforementioned femoral head necrosis is glucocorticoid-induced femoral head necrosis.
[0018] Furthermore, the effective dose of the drug is 100-200 μL.
[0019] Compared with the prior art, the present invention has at least the following advantages: 1. This invention relates to a method for preparing magnetically guided BMSC exosomes IONP-BMSC-exos-RH loaded with recombinant hirudin. First, magnetic mesenchymal stem cell exosomes IONP-BMSC-exos are prepared. Then, IONP-BMSC-exos are co-incubated with recombinant hirudin RH to obtain IONP-BMSC-exos-RH. The recombinant hirudin RH can bind to the ligand-binding domain of PAR2 in bone marrow stromal stem cells (BMSCs), increasing the RH loading. The IONP-BMSC-exos-RH prepared by the method described in this invention contains magnetic particles, endowing the exosomes with superparamagnetism and targeting properties.
[0020] 2. This invention also relates to the application of IONP-BMSC-exos-RH in the preparation of anticoagulant drugs. In vitro experiments have shown that, compared with the control group, the IONP-BMSC-exos-RH group has significantly reduced thrombus quality within 1.0-2.5 hours, indicating that IONP-BMSC-exos-RH has excellent anticoagulant effects.
[0021] 3. This invention also relates to the application of IONP-BMSC-exos-RH in the preparation of drugs for repairing damaged bone tissue. In vivo experiments have shown that IONP-BMSC-exos-RH accumulates in large quantities at the lesion site and can significantly reduce thrombus formation in the femoral head, exhibiting good anticoagulant effects. Micro-CT scans have also shown that IONP-BMSC-exos-RH can significantly reverse femoral head necrosis, demonstrating promising application prospects. Attached Figure Description
[0022] To more clearly illustrate the specific embodiments of the present invention, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below.
[0023] Figure 1 This is a schematic diagram illustrating the preparation method of IONP-BMSC-exos-RH and the principle of improving femoral head necrosis according to the present invention. Figure 2 In Test Example 1 of this invention, BMSC showed the best absorption effect on IONP at 40 μg / L; Figure 3 This is a graph showing the effect of different concentrations of IONP on the proliferation of BMSCs in Test Example 1 of this invention. Figure 4 This is a comparison diagram of the magnetic properties of IONP-BMSC-exos before and after testing in Test Example 2 of this invention; Figure 5 This is a TEM image of IONP-BMSC-exos, the third test example of this invention; Figure 6This is a particle size distribution diagram of IONP-BMSC-exos in Test Example 4 of this invention; Figure 7 This is a Western blot image of IONP-BMSC-exos and BMSC-related proteins in Test Example 5 of this invention; Figure 8 This is a graph showing thrombus formation at different time points during in vitro anticoagulation in different groups of test example six of this invention; Figure 9 This is a graph showing the quantitative results of thrombus weight in each group of test example six of the present invention; Figure 10 This is a live imaging image of MPS+IONP-BMSC-exos-RH+Magnet from Embodiment 7 of the present invention; Figure 11 This is an immunohistochemical analysis diagram from Embodiment 7 of the present invention; Figure 12 Micro-CT images of the treatment of femoral head necrosis in each group in Test Example 7 of this invention. Detailed Implementation
[0024] The present invention will now be described in further detail. It should be noted that the following specific embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.
[0025] This invention provides a general and / or specific description of the materials and experimental methods used in the experiments. Unless otherwise specified, all experimental or testing methods are conventional methods; all reagents or instruments used, unless otherwise specified, are commercially available conventional products prepared or used using conventional methods.
[0026] The preparation method of IONP-BMSC-exos-RH of this invention, and the principle diagram for improving femoral head necrosis are shown below. Figure 1 As shown.
[0027] Source of materials: PEG carboxyl-terminated IONP: purchased from Jiangsu Xianfeng Nanomaterials Technology Co., Ltd., item number: 1317-61-9; Recombinant hirudin: purchased from Beijing Keerhui Technology Co., Ltd., product number: BR150628.
[0028] Example 1: Preparation of IONP-BMSC-exos-RH S1. Preparation of IONP-BMSCs: Primary human BMSCs (bone marrow stromal stem cells) were purchased as the parent cells. 5 mL of PEG-modified IONP (Iron Oxide Nanoparticles, magnetic iron oxide nanoparticles, concentrations of 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 120 μg / mL, 160 μg / mL, and 200 μg / mL) were added to the culture medium. The number of BMSCs was 12 × 10⁻⁶. 6 One sample was incubated at room temperature for 24 hours to obtain IONP-BMSCs; S2. Preparation of IONP-BMSC-exos: Magnetic mesenchymal stem cell exosomes were enriched and extracted using differential ultracentrifugation and magnetic separation. The cell supernatant was centrifuged at 1000×g for 10 min, and the supernatant was collected. It was then centrifuged at 10000×g for 30 min, and the supernatant was collected again. It was then centrifuged at 100000×g for 70 min, and the precipitate was collected. The precipitate was then dissolved in PBS. The precipitate was then allowed to stand on a magnetic separator for 24 h. The supernatant was discarded, and the precipitate was collected to obtain IONP-BMSC-exos. S3. Preparation of IONP-BMSC-exos-RH: The IONP-BMSC-exos obtained in step S2 was mixed with 100 μL of 600 μg / LRH (Recombinant Hirudin) and incubated at room temperature. After 24 hours, IONP-BMSC-exos-RH was obtained by magnetic separation again.
[0029] Test Example 1 determines the saturation point of BMSC intake of IONP. The IONP-BMSCs prepared in step S1 of Example 1 were fixed with paraformaldehyde for 20 min, washed with double-distilled water, stained with Prussian blue for 30 min, and then stained with eosin for 15 s. After washing, the blue IONPs were observed under a microscope, demonstrating the uptake of IONP by BMSCs and identifying the IONP uptake saturation point. The results of the microscopic observation are as follows. Figure 2 As shown in A, Figure 2 A represents the intake of IONP by BMSCs at concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL, respectively. Figure 2 B represents the fold change in optical density of cells relative to the control group at IONP concentrations of 10 μg / mL, 20 μg / mL, 40 μg / mL, and 80 μg / mL. Figure 2 As shown in Figure B, the optimal uptake by cells is achieved when the concentration of IONP is 40 μg / mL.
[0030] The results of the CCK-8 experiment are as follows: Figure 3 As shown in the figure, IONP concentrations below 40 μg / L have no effect on the proliferation of BMSCs.
[0031] Test Example 2: IONP-BMSC-exos Magnetic Detection To verify whether the IONP-BMSC-exos prepared in this invention possesses magnetic properties, the IONP-BMSC-exos prepared in step S2 of Example 1 were subjected to magnetic detection. The prepared IONP-BMSC-exos suspension was placed on one side of a magnet for 20 minutes, and the comparison before and after placement is shown in the figure. Figure 4 As shown in the figure, after the suspension was left to stand for 20 minutes, IONP-BMSC-exos was found to be significantly enriched on the side of the magnet, indicating that IONP-BMSC-exos is magnetic.
[0032] Test Example 3: IONP-BMSC-exos Characterization The IONP-BMSC-exos prepared in step S2 of Example 1 of this invention were examined by transmission electron microscopy (TEM): IONP-BMSC-exos were loaded onto a 2 nm copper grid and dried at room temperature for 10 min. Then, the morphology and internal IONPs were observed using a Hitachi H-7650 TEM. The TEM images are shown below. Figure 5 As shown in the figure, IONP-BMSC-exos contains IONP particles, indicating successful preparation.
[0033] Test Example 4: Particle Size Testing of IONP-BMSC-exos To test the particle size of the IONP-BMSC-exos prepared in this invention, nanoparticle tracking analysis (NTA) was performed on the IONP-BMSC-exos prepared in step S2 of Example 1: the particle size distribution and concentration of IONP-BMSC-exos were measured using Nanosizer™ technology (Malvern), and the data were processed using ZetaView software. The detection results are as follows: Figure 6 As shown, NTA analysis revealed that the particle size of IONP-BMSC-exos is around 120 nm.
[0034] Test Example 5 involved Western blotting of relevant proteins in IONP-BMSC-exos precipitates. To detect the integrity of exosomes in the IONP-BMSC-exos prepared in this invention and to exclude organelle contamination, this test example performed CD63 / HSP70 / TSG101 positive marker and calnexin positive marker detection on the IONP-BMSC-exos prepared in step S2 of Example 1.
[0035] The WB (Western Blotting) results of each protein are as follows: Figure 7 As shown, Figure 7 The first column shows the Western blot (WB) images of whole-cell lysate of BMSCs, and the second column shows the WB images of IONP-BMSC-exos. As can be seen from the images, the expression of HSP70, CD63, and TSG101 in IONP-BMSC-exos is increased relative to that in MSC lysate, while the expression of the endoplasmic reticulum marker calnexin is decreased relative to that in MSC lysate. This indicates that IONP-BMSC-exos is intact, structurally pure, and safe for subsequent drug loading (RH) and targeted therapy.
[0036] Test Example 6: IONP-BMSC-exos-RH In Vitro Anticoagulation Experiment To verify the anticoagulant and thrombotic effect of the IONP-BMSC-exos-RH prepared in this invention, this test case used the IONP-BMSC-exos-RH prepared in Example 1 for an in vitro anticoagulant experiment. It was divided into four groups: a blank control group (platelet-rich plasma PRP + CaCl2 + PBS), a free RH group (PRP + CaCl2 + RH), a carrier control group (PRP + CaCl2 + IONP-BMSC-exos), and an experimental group (PRP + CaCl2 + IONP-BMSC-exos-RH).
[0037] ①Preparation of platelet-rich plasma (PRP) is carried out through the following steps.
[0038] S1. Fasting venous blood from healthy volunteers was mixed with 3.2% sodium citrate anticoagulant (venous blood: anticoagulant = 9:1) and immediately gently inverted to avoid platelet activation. S2. First centrifugation: Centrifuge at 1000×g, 22℃ for 15min, collect the upper layer of anemic platelet plasma (PPP) for later use, and discard the lower layer of red blood cells; S3. Second centrifugation: Centrifuge the PPP layer from S2 at 1500×g, 22℃ for 15min, discard the supernatant, and the remaining precipitate is platelet clumps; resuspend the platelets in PPP again to adjust the platelet concentration to 200-400×10⁻⁶. 9 / L (standard PRP concentration); S4. Quality control: Platelet count (fully automated blood cell analyzer), the purpose of which is to confirm the uniformity of platelet concentration.
[0039] ②Induce thrombus formation, then treat in groups.
[0040] S1. Take 300 μL of PRP prepared in group ① and add it to an EP tube. Add 100 μL of PBS, 100 μL of RH (0.6 μg / mL), and 100 μL of IONP-BMSC-exos (1*10) to the EP tube according to the group. 9 100 μL IONP-BMSC-exos-RH (1×10⁻⁶) 9 (a few exosomes); S2. Add 100 μL of 0.025 M CaCl2 solution (final concentration 5 mM Ca) to each of the four EP tubes from step S1. 2+ Incubate at 37℃ for 0.5h, 1h, 1.5h, 2h, and 2.5h to form a thrombus; S3. Add EDTA solution to each tube to stop the reaction, then gently aspirate the supernatant to avoid damaging the thrombus. Transfer the thrombus to pre-weighing filter paper, aspirate the surface liquid, and weigh.
[0041] After the reaction was terminated, the thrombi in each group were as follows: Figure 8 As shown, the quantitative analysis results of thrombus weight are as follows: Figure 9 As shown in the figure, no thrombus was formed in the IONP-BMSC-exos-RH group at 0.5h and 1h, and the quality of the thrombus formed at 1.5h, 2h and 2.5h was significantly lower than that in the control group. This indicates that the IONP-BMSC-exos-RH prepared by the method described in this invention has good anticoagulant effect and broad application prospects.
[0042] Test Example 7: IONP-BMSC-exos-RH In Vivo Anticoagulation Experiment Preparation method of DIR (lipid-soluble near-infrared fluorescent dye) labeled IONP-BMSC-exos-RH: Take IONP-BMSC-exos-RH prepared in Example 1 and DIR dye (final concentration of 0.5 μM) and add them to PBS solution. Incubate at 37°C in the dark for 30 min to obtain DIR labeled IONP-BMSC-exos-RH.
[0043] Establishment of a femoral head necrosis model: Rats were first divided into four groups: ① PBS group (blank control), ② MPS group (methylprednisolone-induced femoral head necrosis), ③ MPS+IONP-BMSC-exos-RH group, and ④ MPS+IONP-BMSC-exos-RH+Magnet group (methylprednisolone + IONP-BMSC-exos-RH + magnet). PBS control group: 100 μL PBS was injected via tail vein. Methylprednisolone injection groups (②, ③, and ④): SD rats were intraperitoneally injected with 40 μg / kg lipopolysaccharide (LPS) for 3 consecutive days to upregulate in vivo inflammation levels and improve the success rate of femoral head necrosis modeling; then, SD rats were injected with methylprednisolone via tail vein once daily for 4 consecutive days to induce femoral head necrosis; simultaneously, sterile neodymium magnets were implanted subcutaneously around the hip joint of SD rats in group ④. Rats in groups ③ and ④ were simultaneously injected with methylprednisolone and 100 μL of IONP-BMSC-exos-RH via the tail vein.
[0044] One day after intervention, in vivo imaging was performed on the MPS+IONP-BMSC-exos-RH+Magnet group, and the results were as follows: Figure 10 As shown, DIR-tagged IONP-BMSC-exos-RH was found to be attracted by a magnet, indicating that IONP-BMSC-exos-RH is magnetic and accumulates near the hip joint.
[0045] Figure 11 The image shows an immunohistochemical analysis. As can be seen from the image, compared with the MPS group, the MPS+IONP-BMSC-exos-RH+Magnet group can significantly reduce the formation of thrombi in the femoral head.
[0046] Figure 12 Micro-CT images of the femoral head of rats in each group were obtained one day after intervention. The images showed that the femoral head bone density of the IONP-BMSC-exos-RH+magnet group was significantly increased and the necrotic area was reduced. It was found that IONP-BMSC-exos-RH can significantly improve femoral head necrosis under the action of a magnet.
[0047] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be covered within the scope of the claims and specification of the present invention.
Claims
1. A method for preparing magnetically guided BMSC exosomes loaded with recombinant hirudin, characterized in that, Includes the following steps: S1. After adding IONP to BMSCs and incubating, IONP-BMSCs are obtained; S2. The precipitate obtained after centrifugation and magnetic separation of IONP-BMSC is IONP-BMSC-exos; S3. After co-incubating IONP-BMSC-exos with RH, magnetic separation was performed. The resulting precipitate was the magnetically navigable BMSC exosome loaded with recombinant hirudin, denoted as IONP-BMSC-exos-RH.
2. The preparation method according to claim 1, characterized in that, In step S1, the IONP:BMSC = (25-200) μg : 12 × 10 6 The concentration of IONP is 5-40 μg / mL; in step S3, the IONP-BMSC-exos : RH=1×10 9 Quantity: (0.04-0.07) μg.
3. The preparation method according to claim 1, characterized in that, The centrifugation described in step S2 is differential ultracentrifugation.
4. The preparation method according to claim 3, characterized in that, The differential ultracentrifugation method is as follows: centrifuge the supernatant of IONP-BMSC at 300-1000×g for 10-20 min to obtain supernatant A; centrifuge supernatant A at 10000-20000×g for 20-40 min to obtain supernatant B; centrifuge supernatant B at 80000-120000×g for 60-90 min to obtain precipitate C; and centrifuge precipitate C again at 80000-120000×g for 60-90 min to obtain precipitate D.
5. IONP-BMSC-exos-RH prepared by the preparation method according to any one of claims 1-4.
6. The use of IONP-BMSC-exos-RH as described in claim 5 in the preparation of anticoagulant drugs.
7. The use of the IONP-BMSC-exos-RH of claim 5 in the preparation of a medicament for repairing damaged bone tissue.
8. The application according to claim 7, characterized in that, The damaged bone tissue includes femoral head necrosis.
9. The application according to claim 8, characterized in that, The avascular necrosis of the femoral head mentioned is glucocorticoid-induced avascular necrosis of the femoral head.
10. The application according to claim 9, characterized in that, The effective dose of the drug is 100-200 μL.