Use of taurine pretreated mesenchymal stem cells

Abstract

The application belongs to the technical field of biological medicine, and provides application of taurine pretreated mesenchymal stem cells. The application adopts taurine to pretreat mesenchymal stem cells, which can significantly improve the expression level of CXCR4 of the mesenchymal stem cells and enhance the lesion targeting ability of the mesenchymal stem cells. Meanwhile, the application can improve the SOD2 expression level of mitochondria of the mesenchymal stem cells and enhance the antioxidant capacity of the mesenchymal stem cells. By controlling the taurine pretreatment time, active taurine pretreated mesenchymal stem cells and taurine pretreated mesenchymal stem cells without proliferation activity can be obtained. The former can be used for targeted mitochondrial delivery treatment of non-tumorous diseases (such as rheumatoid arthritis), and the live cells are used as carriers to provide a stable biological microenvironment for mitochondria. The latter can be used as a targeted carrier of an anti-tumor drug for treatment of tumorous diseases. The application provides a new strategy for organelle treatment and targeted drug delivery.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the application of taurine-pretreated mesenchymal stem cells. Background Technology

[0002] In both drug therapy and cell therapy, ectopic accumulation is a significant factor limiting therapeutic efficacy. Ectopic drug accumulation not only leads to insufficient effective drug concentrations at the lesion site but can also damage other non-target organs, contributing significantly to drug toxicity. The same applies to cell therapy. Therefore, precise targeted therapy remains a major challenge that various drugs and cell therapies must overcome.

[0003] Stem cells are inherently highly targeted cells and are frequently used as drug carriers in various therapies to efficiently deliver drugs to lesion sites. Their tissue repair, anti-inflammatory, and antioxidant properties make them one of the most commonly used therapeutic cells in cell therapy. However, studies have indicated that due to their relatively large size, stem cells still carry the risk of ectopic accumulation in organs such as the lungs. Furthermore, the potential for tumorigenesis when using living stem cells in cancer treatment remains a controversial issue. The CXCR4 / SDF-1 axis is one of the important mechanisms by which stem cells exert their targeted effects.

[0004] Traditional treatment strategies for rheumatoid arthritis (RA) primarily include NSAIDs, glucocorticoids, DMARDs, immunosuppressants, and biologics, which exert their therapeutic effects mainly by inhibiting inflammatory responses and regulating immune signaling pathways. However, these drugs generally lack the ability to target and regulate mitochondrial dysfunction and pathological metabolic reprogramming, thus failing to effectively reverse the inflammatory proliferation and pathological remodeling of diseased synovial tissue. Mitochondrial dysfunction is a crucial mechanism driving the formation of inflammatory and invasive phenotypes in rheumatoid synovial tissue. In recent years, emerging RA treatment strategies centered on mitochondrial functional remodeling have gradually gained attention. These methods typically utilize nanomaterials to construct mitochondrial-targeted delivery platforms to restore cellular mitochondrial homeostasis and reverse abnormal pathological phenotypes. For example, mitochondrial transplantation (MT) can restore redox homeostasis in rheumatoid synovial fibroblasts (FLS-RA) by introducing exogenous healthy mitochondria with intact antioxidant systems, thereby inhibiting their pro-inflammatory activation state and achieving therapeutic goals. However, insufficient precision delivery remains a core issue limiting the long-term application of mitochondrial transplantation. Due to the lack of specific recognition and precise targeting capabilities for FLS-RA, its therapeutic efficiency and clinical translation potential are still somewhat limited. Furthermore, mitochondria, as highly dynamic organelles, are difficult to maintain their biological activity long-term under in vitro conditions, and are therefore prone to functional decline or even inactivation during delivery. Limited targeted delivery efficiency and heterogeneity in donor mitochondrial quality are important factors restricting the long-term application and therapeutic efficacy of MT.

[0005] Osteosarcoma is the most common primary malignant bone tumor, mostly growing in the metaphysis of long bones. It is more common in adolescents and children, and can manifest as bone and joint pain and local masses. Traditionally, treatment primarily involved amputation, resulting in low survival rates. Current clinical practice typically involves surgical resection of the cancerous tissue, combined chemotherapy with multiple drugs, radiofrequency ablation, and radiotherapy (limb-sparing, 65%). However, some patients forgo treatment due to incomplete tumor resection, postoperative recurrence, or poor prognosis. Therefore, clinical treatment of osteosarcoma still faces limitations and challenges. Furthermore, chemotherapy is one of the main treatment options for osteosarcoma, with most chemotherapy regimens using a combination of drugs such as doxorubicin, methotrexate, and cisplatin. However, these chemotherapy drugs have poor tumor targeting and exhibit some degree of toxicity after entering the body. For example, doxorubicin (ADR), as a first-line broad-spectrum antitumor drug, plays a crucial role in the treatment of osteosarcoma, but its efficacy rate is only 15%-35%. Furthermore, because doxorubicin cannot target the tumor site after entering the body, it can cause adverse reactions and varying degrees of damage to other organs (nausea, vomiting, immunosuppression, stomatitis, fever, liver damage, impaired bone marrow hematopoietic function, and cardiotoxicity) after entering other organs. Therefore, if a drug carrier with good biocompatibility, the ability to target the tumor site, and a long circulation time in the body can be found, the drug's utilization rate can be greatly improved and the side effects of systemic administration can be reduced, potentially bringing a new breakthrough in the treatment of osteosarcoma. Summary of the Invention

[0006] The purpose of this invention is to provide the application of taurine-pretreated mesenchymal stem cells, aiming to solve the problems raised in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The application of taurine-pretreated mesenchymal stem cells in the preparation of drugs for treating non-tumor diseases, wherein the taurine pretreatment method is as follows: mesenchymal stem cells are placed in a culture medium containing taurine and incubated for a first time period, then the taurine-containing culture medium is removed and replaced with a taurine-free culture medium for continued culture, to obtain active taurine-pretreated mesenchymal stem cells.

[0009] Furthermore, the concentration of taurine in the culture medium containing taurine is 400-800 mM; the first time period is 0.5-6 h.

[0010] Furthermore, the concentration of taurine in the culture medium containing taurine is 600 mM; the first time period is 2 hours.

[0011] Furthermore, the taurine pretreatment is used to increase the expression levels of CXCR4 and / or mitochondrial SOD2 in mesenchymal stem cells.

[0012] Furthermore, the non-tumor disease is rheumatoid arthritis; the taurine-pretreated mesenchymal stem cells are used for targeted delivery of mitochondria to the diseased synovial tissue.

[0013] Another object of the present invention is to provide the application of taurine-pretreated mesenchymal stem cells in the preparation of drugs for treating neoplastic diseases, wherein the taurine pretreatment method is as follows: placing mesenchymal stem cells in a culture medium containing taurine and incubating for a second time period, collecting all the liquid after incubation, and centrifuging to obtain taurine-pretreated mesenchymal stem cells that have lost their proliferative activity.

[0014] Furthermore, the concentration of taurine in the culture medium containing taurine is 400-800 mM; the first time period is 12-48 h.

[0015] Furthermore, the concentration of taurine in the culture medium containing taurine is 600 mM; the first time period is 24 h.

[0016] Furthermore, the taurine-pretreated mesenchymal stem cells serve as carriers for anti-tumor drugs, enabling targeted delivery of these drugs to the tumor site.

[0017] Furthermore, the tumor is osteosarcoma; the antitumor drug is an anthracycline antitumor drug; the anthracycline antitumor drug includes doxorubicin.

[0018] The application of taurine-pretreated mesenchymal stem cells (MSCs) provided by this invention has been studied. Research has shown that taurine pretreatment of MSCs significantly increases the expression level of CXCR4 in MSCs, enhancing their lesion-targeting ability; simultaneously, it increases the SOD2 expression level in mitochondria of MSCs, enhancing their antioxidant capacity. Specifically, by controlling the taurine pretreatment time, active taurine-pretreated MSCs (short-term pretreatment) and taurine-pretreated MSCs with lost proliferative activity (long-term pretreatment) can be obtained. Short-term pretreatment promotes mitochondrial renewal in MSCs, improving their targeting ability and enabling targeted delivery of mitochondria. This can be used for targeted mitochondrial delivery therapy in non-tumor diseases (such as rheumatoid arthritis), providing a stable biological microenvironment for mitochondria using living cells as carriers, thus creating a novel organelle synergistic therapy strategy. Long-term pretreatment renders MSCs inactive, making them a good carrier for targeted drug delivery in tumor diseases, serving as a targeted carrier for anti-tumor drugs in the treatment of tumors. This invention provides a new strategy for organelle therapy and targeted drug delivery. Attached Figure Description

[0019] Figure 1 For MSC and MSC Tau-live Image showing the results of live and dead staining.

[0020] Figure 2 For MSC Tau-live The results of the trilineage differentiation ability test are shown in the figure.

[0021] Figure 3 For MSC Tau-live Image showing the results of CXCR4 expression detection.

[0022] Figure 4 For MSC and MSC Tau-live Image showing the results of mitochondrial SOD2 expression detection.

[0023] Figure 5 For MSC and MSC Tau-live The image shows the results of in vivo targeted testing.

[0024] Figure 6 The figure shows the results of CCK8 assays to detect the proliferation activity of cells in culture media containing different concentrations of taurine.

[0025] Figure 7 For MSC Tau-dead Image showing the results of CXCR4 expression detection.

[0026] Figure 8 For MSC Tau-dead Image showing drug loading test results.

[0027] Figure 9 For MSC Tau-dead The graph shows the results of the correlation detection between MSCs and K7M2 cells; in the graph, the control group has PBS in the lower chamber; the MSC group has MSCs in the lower chamber. Tau-dead Group: Lower chamber is MSC Tau-dead .

[0028] Figure 10 For MSC and MSC Tau-live HE and Safranin-Fix Green staining results of joints in CIA mice after treatment. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0030] The study in this invention found that pretreatment of stem cells with high concentrations of taurine can increase CXCR4 expression and enhance their lesion-targeting ability. Furthermore, different treatment times have varying effects on stem cell viability. By changing the pretreatment time, the viability of pretreated stem cells can be regulated, making them suitable for the treatment of different diseases. Specifically, shorter treatment times do not affect cell viability and enhance targeting, making them suitable for treating non-tumor diseases. Longer treatment times result in cell loss of viability, making them suitable as good drug carriers for treating tumor diseases.

[0031] Specifically, in one embodiment of the present invention, the application of taurine-pretreated mesenchymal stem cells (MSCs) in the preparation of drugs for treating non-tumor diseases (such as rheumatoid arthritis) is provided. The taurine pretreatment is a short-term pretreatment, and the specific method is as follows: MSCs are incubated in a culture medium containing 400-800 mM taurine for a first time period (e.g., 0.5-6 h), then the taurine-containing culture medium is removed and replaced with ordinary culture medium without taurine for another 12-36 h, resulting in viable taurine-pretreated mesenchymal stem cells, denoted as MSCs. Tau-live .

[0032] In this embodiment of the invention, MSCs are pretreated with taurine for a short time. Tau-live Taurine can enhance lesion-targeting capabilities. It promotes selective autophagy in mitochondria, improves mitochondrial quality control, and enhances their antioxidant capacity, thereby overcoming key factors limiting the long-term nutritional and therapeutic effects of mitochondrial transplantation (targeted delivery and mitochondrial quality heterogeneity). This strategy achieves sustained maintenance of MT efficacy by enhancing mitochondrial renewal and promoting targeted mitochondrial transport. These findings establish the role of MSCs in mitochondrial transplantation. Tau-live This study explores the therapeutic potential of novel organelle synergistic therapy strategies in RA and provides a new direction for the development of next-generation cell therapy technologies.

[0033] For non-cancerous diseases such as rheumatoid arthritis (RA), short-term pretreatment of MSCs with taurine, using live cells as mitochondrial delivery carriers, can provide a stable biological microenvironment for mitochondria, thereby maintaining their biological activity during transport. In this process, MSCs... Tau-live Mitochondrial antioxidant function was further enhanced, and mitochondria with high antioxidant properties were able to exert a better therapeutic effect. Meanwhile, MSCs... Tau-live It has the ability to target lesion sites and can deliver mitochondria to lesioned joints (synovial tissue).

[0034] In another embodiment of the present invention, the application of taurine-pretreated mesenchymal stem cells (MSCs) in the preparation of drugs for treating neoplastic diseases is also provided. The taurine pretreatment is a long-term pretreatment, specifically as follows: MSCs are incubated in a culture medium containing 400-800 mM taurine for a second time period (e.g., 12-48 h). All the incubation liquid is collected, and the MSCs, having lost their proliferative activity, are obtained by centrifugation and designated as MSCs. Tau-dead .

[0035] MSC Tau-dead It can be used as a carrier for anti-tumor drugs, enabling targeted delivery of these drugs to tumor sites. Tumors include osteosarcoma, and the anti-tumor drugs are anthracycline anti-tumor drugs (such as doxorubicin).

[0036] In this embodiment of the invention, MSCs are pretreated with taurine for an extended period of time. Tau-dead This enhances the lesion-targeting ability while deactivating cells, making it a good drug carrier that efficiently targets the lesion site and releases the drug at a specific point, thereby reducing the toxic side effects of chemotherapy drugs.

[0037] It should be noted that, unless otherwise specified, the experimental methods in the following embodiments are conventional methods in the art. Unless otherwise specified, all reagents and materials used are commercially available.

[0038] Example 1: This example provides a method for short-term taurine pretreatment of MSCs, specifically as follows: MSCs are incubated in a culture medium containing 600 mM taurine for 2 hours, then the taurine-containing medium is removed and replaced with ordinary culture medium without taurine for another 24 hours to obtain viable taurine-pretreated mesenchymal stem cells, denoted as MSCs. Tau-live .

[0039] like Figures 1-3 As shown, after the above series of operations, MSC Tau-live Cell viability was unaffected, but CXCR4 expression was increased: MSCs Tau-live Still normal living cells ( Figure 1 ), possessing the ability to differentiate into three lines ( Figure 2 This indicates that taurine pretreatment did not alter its cellular characteristics (it did not affect its viability or stem cell-related properties); however, its CXCR4 expression increased. Figure 3 ).

[0040] SOD2 protein is a manganese-containing superoxide dismutase, mainly located in the mitochondria of cells, responsible for scavenging harmful reactive oxygen species and protecting cells from oxidative damage. Figure 4As shown, after the above taurine pretreatment, MSC Tau-live Compared with untreated MSCs, the expression of SOD2 in their mitochondria was significantly increased. Mitochondria with high SOD2 expression were transferred into FLS-RA and were able to exert a more significant antioxidant effect, promoting the reconstruction of the antioxidant network of FLS-RA.

[0041] Example 2: This example provides a method for short-term taurine pretreatment of MSCs, specifically as follows: MSCs are incubated in a culture medium containing 600 mM taurine for 24 hours. All the incubation liquid is collected, and the taurine-pretreated mesenchymal stem cells that have lost their proliferative activity are obtained by centrifugation, denoted as MSCs. Tau-dead .

[0042] like Figures 6-7 As shown, after the above series of operations, MSC Tau-dead The cells have lost their ability to proliferate and are inactive. Figure 6 ), while its CXCR4 expression is elevated ( Figure 7 ).

[0043] The above MSC Tau-dead After co-incubation with doxorubicin for 2 hours, if Figure 8 As shown, doxorubicin can be successfully loaded into MSCs. Tau -dead The results showed that MSC Tau-dead It is an excellent drug carrier.

[0044] In addition, Transwell assays were used to further detect MSCs. Tau-dead Targeting of osteosarcoma cells, such as Figure 9 As shown, the results indicate that, compared to the control group and untreated MSCs, MSCs Tau-dead The strongest correlation was observed with K7M2 cells. It should be noted that MSCs... Tau-dead Having lost their cell viability, K7M2 cells can colonize tumor sites in vivo through hemodynamic interactions with osteosarcoma cells. Since blood flow cannot be simulated in vitro, the ability of K7M2 cells to migrate to different MSCs was examined to reflect the overall MSC population. Tau-dead Targeting capabilities.

[0045] Example 3: To further evaluate MSC Tau-live To assess the therapeutic effect, this embodiment constructed a CIA mouse model (rheumatoid arthritis model) and, after randomization, administered mouse umbilical cord mesenchymal stem cells, either untreated or treated with taurine (using the method in Example 1), for in vivo treatment. Figure 10As shown, HE staining revealed significantly aggravated synovial hyperplasia in CIA mice, accompanied by rough joint surfaces and irregular structures. Safranin / Fix Green staining showed severe damage to the hyaline cartilage of the CIA mice joints, impairment of the calcified cartilage layer, discontinuity of the cartilage, and further aggravation of subchondral structural defects. After treatment, all of the above indicators in the CIA mice showed some recovery, and MSCs... Tau-live The treatment effect was better than that of untreated MSCs.

[0046] Furthermore, in this embodiment, MSCs and MSCs labeled with mitochondrial fluorescence (labeled with Mito-mScarlet) were injected via the tail vein in a CIA mouse model. Tau-live And its distribution in the synovium of the joint was assessed after 48 hours. Figure 5 As shown, after labeling synovial fibroblasts with Vimentin (a marker for synovial fibroblasts), red mitochondrial signals were detected in Vimentin-positive synovial tissue of the knee joint, and MSCs were also found to be positive. Tau-live The treatment group showed stronger Mito-mScarlet signal in the synovium. This result further illustrates the role of MSCs. Tau-live The ability of mitochondria to be delivered to the synovial membrane in rheumatoid arthritis lesions is enhanced.

[0047] In summary, the technical solution provided by the embodiments of the present invention can significantly improve SOD2 expression in mitochondria, creating an organelle with high antioxidant capacity, while enhancing MSCs. Tau-live The expression of CXCR4 in MSCs was demonstrated through mitochondrial delivery using live cells as a carrier, a departure from previous organelle therapy strategies that relied solely on cell membranes or nanomaterials. Using live cells as a carrier provides a stable environment for organelles, preventing functional impairment or inactivation during delivery; thus establishing the role of MSCs in mitochondrial delivery. Tau-live This invention explores the therapeutic potential of novel organelle synergistic therapy strategies in RA and provides a new direction for the development of next-generation cell therapy technologies. Furthermore, this embodiment of the invention also utilizes long-term pretreatment of MSCs (MSCs) with taurine. Tau-dead This enhances the lesion-targeting ability while deactivating cells, making them a good drug carrier that can efficiently target lesions and release drugs at specific points, overcoming the defects of ectopic accumulation of chemotherapy drugs in previous treatments and improving the efficiency of drug therapy.

[0048] It should be noted that, in this embodiment of the invention, the function of SOD2 is as follows:

[0049] (1) Scavenging reactive oxygen species: SOD2 is the only antioxidant enzyme in the body that can scavenge superoxide anions and is indispensable in all organisms.

[0050] (2) Protecting cells: It can protect cells from the toxic damage of reactive oxygen species (ROS) produced during normal metabolism and is the most important one in the SOD family.

[0051] (3) Mitochondrial localization: SOD2 in human cells is located in mitochondria and is synthesized as a precursor protein composed of the N-terminal leader peptide mitochondrial targeting sequence and mature SOD2.

[0052] Organelle transplantation, as an emerging precision medicine strategy, has received widespread attention in the fields of regenerative medicine and disease intervention in recent years. Among them, mitochondrial transplantation, due to its ability to directly reconstruct the cellular energy metabolism network, has become the most extensively studied and widely applied form of organelle transplantation. The higher the quality of the transferred mitochondria, the better the therapeutic effect. In this embodiment of the invention, taurine pretreatment promotes the enhancement of mitochondrial antioxidant capacity and increases SOD2 expression.

[0053] Mesenchymal stem cells (MSCs) are pluripotent cells with unique regenerative capacity and immunomodulatory properties. They possess unique tumor homing characteristics and can serve as drug carriers for targeted therapy of tumors and metastatic diseases. These unique characteristics of MSCs are primarily due to the various receptors on their cell membranes, including growth factor receptors, cytokine receptors, chemokine receptors, cell-matrix receptors, and receptors for cell-cell interactions.

[0054] Based on the above-described preferred embodiments of the present invention, and through the foregoing description, those skilled in the art can make various changes and modifications without departing from the inventive concept. The technical scope of this invention is not limited to the contents of the specification.

Claims

1. The application of taurine-pretreated mesenchymal stem cells in the preparation of drugs for treating non-tumor diseases, characterized in that, The method of taurine pretreatment is as follows: mesenchymal stem cells are placed in a culture medium containing taurine and incubated for a first time period, then the taurine-containing culture medium is removed and replaced with a taurine-free culture medium for continued culture, so as to obtain active taurine-pretreated mesenchymal stem cells.

2. The application according to claim 1, characterized in that, The concentration of taurine in the culture medium containing taurine is 400-800 mM; the first time period is 0.5-6 h.

3. The application according to claim 2, characterized in that, The taurine concentration in the culture medium containing taurine is 600 mM; the first time period is 2 hours.

4. The application according to claim 1, characterized in that, The taurine pretreatment was used to increase the expression levels of CXCR4 and / or mitochondrial SOD2 in mesenchymal stem cells.

5. The application according to claim 1, characterized in that, The non-tumor disease is rheumatoid arthritis; the taurine-pretreated mesenchymal stem cells are used for targeted delivery of mitochondria to the diseased synovial tissue.

6. The application of taurine-pretreated mesenchymal stem cells in the preparation of drugs for treating neoplastic diseases, characterized in that, The method of taurine pretreatment is as follows: mesenchymal stem cells are placed in a culture medium containing taurine and incubated for a second time period. All the liquid after incubation is collected and centrifuged to obtain taurine pretreated mesenchymal stem cells that have lost their proliferative activity.

7. The application according to claim 6, characterized in that, The concentration of taurine in the culture medium containing taurine is 400-800 mM; the first time period is 12-48 h.

8. The application according to claim 7, characterized in that, The taurine concentration in the culture medium containing taurine is 600 mM; the first time period is 24 h.

9. The application according to claim 6, characterized in that, The taurine-pretreated mesenchymal stem cells serve as carriers for anti-tumor drugs, enabling targeted delivery of these drugs to the tumor site.

10. The application according to claim 9, characterized in that, The tumor is osteosarcoma; the antitumor drug is an anthracycline antitumor drug; the anthracycline antitumor drug includes doxorubicin.

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