Targeted controlled-release delivery system based on mesenchymal stem cells and application thereof

Abstract

The invention provides a targeted controlled-release delivery system based on mesenchymal stem cells and application thereof, the delivery system takes the mesenchymal stem cells as a carrier, the mesenchymal stem cells carry a suicide system gene and a chimeric antigen receptor, the interior of the mesenchymal stem cells contains a gelatin-coated ferroferric oxide nanoparticle-drug compound, and the gelatin-coated ferroferric oxide nanoparticle-drug compound is used as a carrier. The delivery system can efficiently, safely and accurately deliver drugs in a targeted mode, controllable release is achieved, and a new choice is provided for accurate treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of delivery systems, and particularly to a targeted controlled-release delivery system based on mesenchymal stem cells and its application. Background Art

[0002] During disease treatment, due to the lack of selectivity of some drugs, they will accumulate in non-target sites, and systemic drug administration usually leads to adverse reactions and toxicity. For example, nano-drugs can utilize the overexpression of hydrogen peroxide and lactic acid in the tumor microenvironment for chemodynamic therapy of tumors. However, the efficiency of nano-drugs targeting tumors is usually less than 1%, and off-target nano-drugs are often phagocytosed by macrophages in the reticuloendothelial system and eventually accumulate in normal organs such as the liver and spleen for a long time, directly contacting and interacting with normal tissues, which will inevitably cause chronic toxicity, inflammatory reactions, fibrosis, and even cancer. Therefore, this low efficiency may hinder the safety of its clinical application.

[0003] Biological delivery systems can effectively solve these limitations and improve the therapeutic effect of drugs. Different from modification strategies such as affinity targeting and microenvironment response, biological delivery systems have developed a communication system that can interact with the pathological microenvironment, enabling them to recognize the pathological microenvironment through chemokine receptors on the cell surface and migrate to damaged or diseased tissues. Among many biological delivery systems, stem cells have attracted much attention due to their excellent safety guarantee and chemotactic properties.

[0004] Traditional stem cell drug delivery system strategies mainly include overexpressing drug genes to secrete drug proteins, transporting drug molecules using stem cell exosomes, or carrying drugs on the surface of stem cell membranes. However, these methods inevitably face some challenges. For example, overexpressed genes cannot carry non-protein drugs or finished drugs; exosomes lack a complete cell structure and cannot perform the homing function of stem cells; the membrane-based carrying strategy exposes drug molecules on the cell surface, and when these molecules interact with healthy tissue cells, side effects may occur.

[0005] Therefore, it is of great significance to provide a drug delivery system with precise targeting and controlled release. Summary of the Invention

[0006] To solve the above problems, in the first aspect of the present invention, a targeted controlled-release delivery system based on mesenchymal stem cells is provided. The delivery system uses mesenchymal stem cells as a carrier. The mesenchymal stem cells carry a suicide system gene and a chimeric antigen receptor. Inside the mesenchymal stem cells, there is a magnetite nanoparticle-drug complex, and the magnetite nanoparticle-drug complex is coated within a gelatin layer. The schematic diagram of the targeted controlled-release delivery system based on mesenchymal stem cells is shown inFigure 1 .

[0007] Mesenchymal stem cells are a fundamental component of drug delivery systems. Compared with delivery systems such as bacteria and viruses, mesenchymal stem cells rarely express major histocompatibility complex class II molecules and Fas ligand, which ensures their immune privilege and excellent biosafety. Although cell membranes and extracellular vesicles also exhibit low immunogenicity, due to their incomplete cell structure, they lack the key organelles for interpreting chemical guidance signals, generating energy, and driving force. Therefore, transport systems including cell membranes and extracellular vesicles can neither actively cross the vascular barrier through chemotaxis nor physically push themselves deeper into complex tissues according to chemical signals.

[0008] As a preferred technical solution, the mesenchymal stem cells are derived from menstrual blood endometrial stem cells. Menstrual blood endometrial stem cells can be collected due to their non-invasive collection method, and menstrual blood endometrial stem cells have good tissue compatibility and low immunogenicity. Umbilical cord mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, dental pulp stem cells, and menstrual blood-derived endometrial stem cells (MenSC) can all be potential sources of mesenchymal stem cell delivery systems. However, there are still several challenges to overcome before applying these mesenchymal stem cells to clinical translation. For example, the source of dental pulp stem cells is limited because they can only be isolated from a few teeth. Bone marrow-derived mesenchymal stem cells need to be collected by an invasive collection method, which will bring psychological burden to the donor. These factors seriously hinder the wide clinical application of dental pulp stem cells and bone marrow-derived mesenchymal stem cells. Umbilical cord mesenchymal stem cells are one of the most commonly used mesenchymal stem cells in clinical practice, but they can only be obtained during childbirth, with extremely high time limitations and limited applications. The uniqueness of menstrual blood-derived endometrial stem cells lies in that they are derived from the shed endometrium during menstruation. Therefore, the collection method of menstrual blood-derived endometrial stem cells is non-invasive and periodic, which can not only ensure the acquisition of a sufficient number of mesenchymal stem cells but also not bring psychological burden to the donor. In terms of the source of stem cells, the endometrium has a natural dual immunomodulatory function. It can not only obtain the "immune privilege" of pregnancy through immune cells infiltrating the uterus but also show immune tolerance to allogeneic antigens (fetus). Therefore, mesenchymal stem cells derived from the endometrium have better tissue compatibility and safety. Previous studies have shown that menstrual blood-derived endometrial stem cells have no tumorigenic risk. In clinical trials for ischemic myocardium and multiple sclerosis, no adverse reactions related to the use of menstrual blood-derived endometrial stem cells were observed.

[0009] As a preferred technical solution, the chimeric antigen receptor includes a signal peptide, an antigen recognition domain (such as an antibody fragment against Her2), and a transmembrane domain. Through gene editing techniques (such as lentiviral transduction), the chimeric antigen receptor can be integrated into mesenchymal stem cells. The chimeric antigen receptor (CAR) is an artificially designed receptor that is widely used in chimeric antigen receptor T cell (CAR-T) therapy and chimeric antigen receptor-modified cell therapy. CAR binds to target cells through its antigen recognition ability, enhancing the affinity and recognition ability of mesenchymal stem cells for specific target cells.

[0010] As a preferred technical solution, the chimeric antigen receptor is human epidermal growth factor receptor 2 (Her2).

[0011] As a preferred technical solution, the suicide system gene is herpes simplex virus-thymidine kinase. The herpes simplex virus-thymidine kinase gene is integrated into mesenchymal stem cells, enabling them to express thymidine kinase enzyme in vivo. Ganciclovir is an inducer of the suicide system. When the body is given ganciclovir, thymidine kinase will cause cell rupture, and the drugs adsorbed on the nanoparticle drug attachment platform are released at the targeted site, achieving the purpose of precise treatment and solving the problems of off-target and low drug release efficiency existing in traditional mesenchymal stem cells.

[0012] Magnetite nanoparticles have magnetic targeting properties and can guide mesenchymal stem cells to the target location under the action of an external magnetic field. Chemokine receptors (such as CCR2 and CXCR4) and adhesion ligands (such as P-selectin glycoprotein ligand 1 and sialyl Lewis X antigen) on the surface of mesenchymal stem cells can chemotax mesenchymal stem cells to diseased tissues. However, most transplanted mesenchymal stem cells will accumulate in the liver, spleen and lungs, which to some extent reduces their transport efficiency. The intact cell structure also hinders the contact between the loaded drug and the target organ, resulting in a decline in the drug treatment effect. Therefore, in the present invention, a targeted controlled-release drug delivery system is constructed through nanoengineering and genetic engineering. The magnetite nanoparticle drug attachment platform in the drug delivery system endows the drug delivery system with a powerful magnetic driving force. Under the action of the magnetic field, the drug delivery system is more accurately enriched at the target site. At the same time, the inherent homing effect of mesenchymal stem cells can also precisely guide the drug delivery system to the target site. After the drug delivery system reaches the target site, the chimeric antigen receptor embedded on the surface of mesenchymal stem cells binds firmly to the target tissue antigen, achieving precise targeted positioning and completing the delivery purpose. Drugs (such as doxorubicin DOX or other small molecule drugs) are attached by interacting with the nanoparticle drug attachment platform. Specifically, drug molecules can be encapsulated on the surface or inside of the nanoparticles, or bound to the nanoparticles by chemical bonding. Since the nanoparticles are phagocytosed by mesenchymal stem cells, the drugs also enter the cell interior, thus avoiding the direct exposure of the drugs on the cell surface and reducing potential side effects.

[0013] The magnetite nanoparticles are coated with gelatin to form a protective gelatin shell, isolating the magnetite nanoparticles from the surrounding environment, preventing the direct contact between the magnetite nanoparticles and cells, and reducing potential toxicity. Specific microenvironments, such as malignant tumors or inflammatory sites, are characterized by high expression of gelatinases (matrix metalloproteinase-2; matrix metalloproteinase-9), which can selectively degrade the gelatin shell and release the drug, and the drug only exerts a therapeutic effect in the specific microenvironment, achieving targeted, controllable and precise treatment.

[0014] In the second aspect of the present invention, a preparation method of a targeted controlled-release delivery system based on mesenchymal stem cells is provided, including the following steps:

[0015] Step S1, isolating and culturing mesenchymal stem cells;

[0016] Step S2, gene transduction: introducing a suicide system gene and a chimeric antigen receptor gene into mesenchymal stem cells through genetic technology to obtain gene-transduced mesenchymal stem cells;

[0017] Step S3, adsorption of drugs by iron oxide nanoparticles: The iron oxide nanoparticles are dispersed in a drug solution, and the drugs adhere to the iron oxide nanoparticles. The iron oxide nanoparticles are collected to form an iron oxide nanoparticle-drug complex.

[0018] Step S4, gelatin coating: The iron oxide nanoparticle-drug complex is dispersed in a gelatin solution to form a gelatin-iron oxide nanoparticle-drug complex. The gelatin-iron oxide nanoparticle-drug complex is stabilized with glutaraldehyde, and the nanoparticles are collected to obtain a gelatin-coated iron oxide nanoparticle-drug complex.

[0019] Step S5, gene transduction of mesenchymal stem cells phagocytizing the gelatin-coated iron oxide nanoparticle-drug complex: The gelatin-coated iron oxide nanoparticle-drug complex is dispersed in a culture medium, and gene-transduced mesenchymal stem cells are cultured in the culture medium. The gene-transduced mesenchymal stem cells are separated to form a targeted controlled-release delivery system based on mesenchymal stem cells.

[0020] In the present invention, the drug is only a generic term for the target molecule to be delivered, and can include all organic substances, inorganic substances, macromolecules, small molecules, and molecules for different purposes, such as drugs for therapeutic effects, tag proteins and fluorescent proteins for labeling effects, and elements for tracking effects.

[0021] As a preferred technical solution, the drug is doxorubicin. The drug (such as doxorubicin or other small molecule drugs) is attached to the nanoparticles through interaction with the nanoparticles.

[0022] As a preferred technical solution, the mesenchymal stem cells are isolated and cultured from menstrual blood.

[0023] As a preferred technical solution, the mass concentration of the gelatin solution is 0.5%.

[0024] In the third aspect of the present invention, there is provided an application of a targeted controlled-release delivery system based on mesenchymal stem cells in delivering anticancer drugs, including the following steps:

[0025] Step S1: Deliver the delivery system into the body;

[0026] Step S2: Apply a magnetic field at the targeted tissue site;

[0027] Step S3: After the delivery system reaches the target tissue site, use an inducer to induce drug release.

[0028] Efficient and safe drug delivery is achieved through the drug delivery system, providing a new option for precision treatment.

[0029] Through the above technical solutions, the present invention has produced the following technical effects:

[0030] (1) The iron oxide nanoparticles drug attachment platform in the drug delivery system endows the drug delivery system with a powerful magnetic driving force. Under the action of a magnetic field, the drug delivery system is more accurately enriched at the target site.

[0031] (2) The inherent homing effect of mesenchymal stem cells can precisely guide the drug delivery system to the target site.

[0032] (3) After the drug delivery system reaches the target site, the chimeric antigen receptor on the surface of mesenchymal stem cells firmly binds to the target tissue antigen, achieving precise targeting and completing the delivery purpose.

[0033] (4) Drug molecules can be encapsulated on the surface or inside of the nanoparticles, or combined with the nanoparticles by chemical bonding. Since the nanoparticles are phagocytosed by mesenchymal stem cells, the drugs also enter the cell interior, thus avoiding the direct exposure of the drugs on the cell surface and reducing potential side effects.

[0034] (5) Iron oxide nanoparticles are coated with gelatin to form a protective gelatin shell, isolating the iron oxide nanoparticles from the surrounding environment, preventing the direct contact of the iron oxide nanoparticles with cells, and reducing potential toxicity.

[0035] (6) In a specific microenvironment with high expression of gelatinase, the gelatin shell can be selectively degraded to release the drug, and the drug only exerts a therapeutic effect in the specific microenvironment, achieving targeted, controllable, and precise treatment.

[0036] (7) Mesenchymal stem cells are derived from menstrual blood endometrial stem cells. Menstrual blood endometrial stem cells can be collected by a non-invasive method, and menstrual blood endometrial stem cells have good tissue compatibility and low immunogenicity.

[0037] (8) Under the action of an inducer, the suicide system causes cell rupture, and the drugs adsorbed on the nanoparticle attachment platform are released at the target site, achieving the purpose of precise treatment and solving the problems of off-target and low drug release efficiency existing in traditional mesenchymal stem cells. Description of the Drawings

[0038] Figure 1 It is a schematic structural diagram of a targeted controlled release delivery system based on mesenchymal stem cells; wherein 1 represents mesenchymal stem cells, 2 represents the suicide system gene, 3 represents the chimeric antigen receptor, 4 represents the gelatin-coated iron oxide nanoparticle-drug complex, and 5 represents the inducer;

[0039] Figure 2Confocal microscopy imaging of Example 2; in the figure, MenSC represents the fluorescence imaging of BFP in the targeted controlled-release delivery system after introducing BFP, and BFP is used to label cells; CAR represents the fluorescence imaging of FITC in the targeted controlled-release delivery system after immunofluorescence staining, and Merge represents the fitting of the fluorescence imaging of MenSC and CAR.

[0040] Figure 3 Prussian blue staining diagram of Example 3; the arrow indicates iron oxide nanoparticles.

[0041] Figure 4 For Example 4 MenSC CTN and confocal microscopy imaging of the targeted controlled-release delivery system I; MenSC CTN represents MenSC carrying the label MCherry CTN imaging, and MCherry shows red fluorescence; GFP represents MenSC CTN after phagocytosing gelatin-coated magnetic nanoparticle-GFP complexes; Merge represents the fitting of the fluorescence imaging of MenSC CTN and GFP.

[0042] Figure 5 Three-dimensional imaging of the targeted controlled-release delivery system II of Example 4; GFP represents GFP fluorescence imaging, DOX represents DOX fluorescence imaging, and Merge represents the fitting of the fluorescence imaging of DOX and GFP.

[0043] Figure 6 Electrophoresis diagram of Example 5;

[0044] Figure 7 Cell morphology diagram of Example 6;

[0045] Figure 8 Multi-directional differentiation staining diagram of Example 6; among them, Adipogenic differentiation represents Oil Red O staining for adipogenic differentiation, Osteogenic differentiation represents Alizarin Red S staining for osteogenic differentiation, and Chondrogenic differentiation represents Alcian Blue staining for chondrogenic differentiation; the yellow arrow points to the lipid droplets formed inside the cells after Oil Red O staining, indicating that the cells have successfully undergone adipogenic differentiation; the white arrow points to the mineralized nodules formed outside the cells after Alizarin Red S staining, indicating that the cells have successfully undergone osteogenic differentiation; the black arrow points to the chondroitin sulfate-rich matrix deposited outside the cells after Alcian Blue staining, indicating that the cells have successfully undergone chondrogenic differentiation.

[0046] Figure 9 Results of the surface marker detection test in Example 6; the abscissa represents fluorescence intensity, and the ordinate represents cell count;

[0047] Figure 10 Results of the immunogenicity test in Example 6; in flow cytometry, the abscissa represents fluorescence intensity, and the ordinate represents cell number; R8, R9, R10, R4, and R5 in the figure represent different cell subsets, and NS indicates no significant difference between groups;

[0048] Figure 11 Staining map of the magnetic targeting test in Example 7; among them, Origin represents the starting state, Giemsa represents Giemsa staining, Prussian Blue represents Prussian blue staining, and Eosin represents eosin staining;

[0049] Figure 12 Giemsa staining of the migration ability test in Example 7; among them, the Control column represents the control, the non-concentrated medium, the Skov3-CM column represents different Skov3 concentrations, 2× represents 2-fold concentration, 5× represents 5-fold concentration, the 10× row represents observation under a 10-fold microscope, and the 20× row represents observation under a 20-fold microscope;

[0050] Figure 13 Number of homing cells in the migration ability test in Example 7; among them, the Control column represents the control (the medium is not concentrated); 2× represents 2-fold concentration, 5× represents 5-fold concentration, and * represents significant difference; the ordinate is the number of MenSC CTN Cell number;

[0051] Figure 14 Results of the affinity targeting test in Example 7;

[0052] Figure 15 Schematic diagram of the gelatin-coated Fe3O4 nanoparticle complex; among them, Fe3O4 NPs represents Fe3O4 nanoparticles, Gelatin represents gelatin; Gelatin Modification represents the gelatin-coated Fe3O4 nanoparticle complex;

[0053] Figure 16 Coomassie brilliant blue staining results of Fe3O4 nanoparticles; among them, Fe3O4 NPs@Gelatin represents the staining result of the gelatin-coated Fe3O4 nanoparticle complex; Fe3O4 NPs represents the staining result of pure Fe3O4 nanoparticles;

[0054] Figure 17Results of the Fenton reaction for the drug targeted release test in Example 7; where Original represents pure iron oxide nanoparticles, Digestion represents the complex of iron oxide nanoparticles coated with gelatin after degradation by gelatinase, Gelatin represents the complex of iron oxide nanoparticles coated with gelatin; the abscissa represents the scanning wavelength (nm), and the ordinate represents the absorbance (a.u.) at the corresponding wavelength, which is used to monitor the absorption change of the nanoparticles before and after the Fenton reaction and reflect the reaction degree;

[0055] Figure 18 For the Calcein-AM / PI staining and microscopy imaging in Example 8; where MenSC CTN The Untreated column represents without ganciclovir treatment, the GCV(+) column represents with ganciclovir treatment, the Calcein-AM / PI row represents Calcein-AM / PI staining, the Bright Field row represents microscopy imaging, and the red arrows in the figure indicate the iron oxide nanoparticles inside the cells, and the blue arrows indicate the iron oxide nanoparticles released outside the cells;

[0056] Figure 19 Results of flow cytometry cell rupture detection before and after ganciclovir treatment in Example 8; where GCV represents ganciclovir treatment, blue represents before treatment, and green represents after treatment;

[0057] Figure 20 Results of the CCK8 assay in Example 8; the ordinate is the absorbance at a wavelength of 450 nm; the abscissa is the nanoparticle concentration;

[0058] Figure 21 Results of flow cytometry detection with different concentrations of ganciclovir treatment in Example 8; in flow cytometry, the abscissa represents the fluorescence intensity, and the ordinate represents MenSC CTN Cell number;

[0059] Figure 22 Results of Calcein-AM / PI staining with different concentrations of ganciclovir treatment in Example 8;

[0060] Figure 23 Results of Calcein-AM / PI staining with ganciclovir treatment for different times in Example 8;

[0061] Figure 24 Live cell imaging diagrams with ganciclovir treatment for different times in Example 8; the white arrows indicate MenSC in the medium without adding GCV CTN and the yellow arrows indicate MenSC in the medium with adding GCV CTN ;

[0062] Figure 25Fluorescence image of the cell rupture test of the lung metastasis model in Example 8. Detailed implementation mode

[0063] To make the above objects, features, and advantages of the present invention more obvious and understandable, the applicant will illustrate and analyze through examples.

[0064] Example 1, Preparation of a drug-free mesenchymal stem cell-based targeted controlled release delivery system, including the following steps:

[0065] Step S1, Isolate and culture mesenchymal stem cells; the mesenchymal stem cells are isolated and cultured from menstrual blood;

[0066] Step S2, Gene transduction: By gene technology, the suicide system gene and the chimeric antigen receptor gene are introduced into mesenchymal stem cells to obtain gene-transduced mesenchymal stem cells; the chimeric antigen receptor expressed by the chimeric antigen receptor gene includes a signal peptide, an antigen recognition domain, and a transmembrane domain, and the chimeric antigen receptor is human epidermal growth factor receptor 2; the suicide system gene is the herpes simplex virus-thymidine kinase gene; the herpes simplex virus-thymidine kinase gene is derived from the herpes simplex virus; the gene transduction technology uses lentivirus to transduce the chimeric antigen receptor gene and the herpes simplex virus-thymidine kinase gene into mesenchymal stem cells;

[0067] Step S3, Gelatin coating: Disperse iron oxide nanoparticles in a gelatin solution to form a gelatin-iron oxide nanoparticle complex; the gelatin-iron oxide nanoparticle complex is stabilized with glutaraldehyde, and the nanoparticles are collected to obtain a gelatin-coated iron oxide nanoparticle complex; the mass concentration of the gelatin solution is 0.5%;

[0068] Step S4, Gene-transduced mesenchymal stem cells phagocytize the gelatin-coated iron oxide nanoparticle complex: Disperse the gelatin-coated iron oxide nanoparticle complex in a culture medium, culture the gene-transduced mesenchymal stem cells in the culture medium, and isolate the gene-transduced mesenchymal stem cells to prepare a mesenchymal stem cell-based targeted controlled release delivery system.

[0069] The targeted controlled-release delivery system based on mesenchymal stem cells prepared in this example uses mesenchymal stem cells as carriers. The mesenchymal stem cells carry the herpes simplex virus-thymidine kinase gene suicide system gene, and this suicide system gene uses ganciclovir as an inducer. Herpes simplex virus-thymidine kinase is an enzyme that can phosphorylate thymidine into thymidine monophosphate (dTMP). When the herpes simplex virus-thymidine kinase gene is introduced into mesenchymal stem cells and stably expressed, there is thymidine kinase activity in the cells. Ganciclovir itself is a nucleoside analog and has no activity under normal circumstances. When ganciclovir enters cells expressing the herpes simplex virus-thymidine kinase gene, it will be phosphorylated under the action of herpes simplex virus-thymidine kinase. Specifically, herpes simplex virus-thymidine kinase first phosphorylates ganciclovir into ganciclovir monophosphate, and then under the action of other kinases in the cell, it is further transformed into ganciclovir diphosphate and active ganciclovir triphosphate. Ganciclovir triphosphate can competitively inhibit viral or cellular DNA polymerase, thereby hindering normal DNA synthesis, interfering with the nucleic acid metabolism and proliferation process of cells, and triggering apoptosis or necrosis of cells.

[0070] The menstrual blood-derived endometrial mesenchymal stem cells in this study were provided by the Zhongyuan Stem Cell Research Institute in Xinxiang High-tech Zone. The iron oxide nanoparticles were provided by Zhang Wenlong of Xinxiang Medical University.

[0071] Example 2. Affinity verification test of chimeric antigen receptor and antigen:

[0072] Test purpose: To determine whether the chimeric antigen receptor on the surface of mesenchymal stem cells can enhance the affinity between the targeted controlled-release delivery system and target cells.

[0073] Test method: Lentivirus was used to introduce the blue fluorescent protein (BFP) tag into the targeted controlled-release delivery system prepared in Example 1. The targeted controlled-release delivery system after the introduction of BFP is denoted as MenSC. Subsequently, MenSC was cultured in a culture dish, fixed with 4% paraformaldehyde, and blocked with 10% goat serum. The primary antibody was a rat anti-human epidermal growth factor receptor 2 antibody, and the secondary antibody was a fluorescein isothiocyanate (FITC)-labeled goat anti-rat IgG antibody for immunofluorescence staining. The targeted controlled-release delivery system after staining is denoted as CAR. MenSC and CAR will be imaged under a confocal microscope, and the fluorescence images of the two will be fitted and denoted as Merge.

[0074] Test results: The test results are shown in Figure 2 。

[0075] BFP is located inside the cell and is used to label MenSC, and blue fluorescence is shown inside the cell.

[0076] Epidermal growth factor receptor 2 antibody and fluorescent secondary antibody are used to label human epidermal growth factor receptor on the surface of MenSCs. Rat anti-human epidermal growth factor receptor 2 antibody binds to human epidermal growth factor receptor 2 on the surface of MenSC cells, and then binds to the secondary antibody labeled with fluorescein isothiocyanate (FITC), making the cell surface show green fluorescence. Figure 2 The results show that the technical solution of the present invention endows MenSCs with a chimeric antigen receptor that can recognize human epidermal growth factor receptor 2, and can enhance the binding ability of MenSCs to human epidermal growth factor on the surface of target cells.

[0077] Example 3, Mesenchymal stem cell phagocytosis verification test:

[0078] Using Prussian blue staining for the targeted controlled release delivery system prepared in Example 1, the results show that iron oxide nanoparticles are phagocytosed by mesenchymal stem cells ( Figure 3 ), and iron oxide nanoparticles have the precursor conditions to serve as a drug attachment platform.

[0079] Example 4, Drug-carrying verification test:

[0080] Test objective: To verify the ability of the targeted controlled release delivery system based on mesenchymal stem cells to carry drug molecules.

[0081] Test method:

[0082] Preparation of test sample 1: Use lentivirus to transduce the MCherry gene into menstrual blood-derived mesenchymal stem cells (MenSCs), endowing MenSCs with the label MCherry to make test sample 1; use a confocal microscope for imaging, and MCherry can show red fluorescence, represented by MenSC CTN indicated.

[0083] Preparation of test sample 2: Use green fluorescent protein (GFP) as the target molecule to simulate the drug delivered by the targeted controlled release delivery system. After incubating iron oxide nanoparticles with green fluorescent protein (GFP) overnight on a shaker at 37°C, disperse the iron oxide nanoparticles in a 0.5% gelatin solution for modification and stabilize it with glutaraldehyde to make a gelatin-coated iron oxide nano-GFP complex. Subsequently, co-culture the gelatin-coated iron oxide nanoparticle-GFP complex with test sample 1 for 12 hours to make the targeted controlled release delivery system I, and then use a confocal microscope for imaging, represented by GFP; image MenSC CTN and GFP under a confocal microscope, and fit the fluorescence images of the two, represented by Merge.

[0084] Preparation of test sample 3: After incubating iron oxide nanoparticles with green fluorescent protein (GFP) and doxorubicin (DOX) overnight on a shaker at 37 °C, the iron oxide nanoparticles were dispersed in a 0.5% gelatin solution for modification and stabilized with glutaraldehyde to form a gelatin-coated iron oxide nanoparticle-GFP-DOX complex. Subsequently, the gelatin-coated iron oxide nanoparticle-GFP-DOX complex was co-cultured with test sample 1 for 12 hours to prepare a targeted controlled-release delivery system II, and then confocal microscopy was used for imaging.

[0085] Test results: MenSCs simply carrying the label MCherry CTN showed red; the targeted controlled-release delivery system I carrying GFP showed green, indicating that mesenchymal stem cells could phagocytose iron oxide nanoparticles; iron oxide nanoparticles could efficiently adsorb GFP. The test results are shown in Figure 4 .

[0086] Confocal microscopy imaging of the targeted controlled-release delivery system II is shown in Figure 5 . The results showed that the targeted controlled-release delivery system II could simultaneously load GFP and DOX. From a three-dimensional perspective, we further confirmed that most of the drugs were located inside the targeted controlled-release delivery system II, which could effectively prevent the drugs from contacting healthy tissues.

[0087] Example 5, verification of carrying herpes simplex virus-thymidine kinase gene:

[0088] The electrophoresis results of the targeted controlled-release delivery system prepared in Example 1 showed that by using the technical solution of the present invention, the herpes simplex virus-thymidine kinase gene was successfully transduced into the targeted controlled-release delivery system. The electrophoresis results are shown in Figure 6 .

[0089] Example 6, verification of the mesenchymal stem cell characteristics of the targeted controlled-release delivery system:

[0090] Test objective: To prove that the targeted controlled-release delivery system has the characteristics of mesenchymal stem cells.

[0091] Test method:

[0092] In this example, the targeted controlled-release delivery system prepared in Example 1 is denoted as MenSC CTN ; the mesenchymal stem cells obtained in step S1 of Example 1 are denoted as MenSC.

[0093] 1. Morphological observation: Those denoted as MenSC CTN and those denoted as MenSC were inoculated in a culture dish, and imaging was performed when the cells reached 80%-90% confluence.

[0094] 2. Multidirectional differentiation potential assay: MenSCs CTN and MenSCs were respectively seeded in 24-well plates and induced to differentiate using adipogenic, osteogenic, and chondrogenic differentiation kits. After induction, Oil Red O, Alizarin Red S, and Alcian Blue (05412, 05465, 05455, StemCell Technologies) staining were performed respectively.

[0095] 3. Surface marker detection: MenSCs TM and MenSCs were stained according to the mesenchymal stem cell identification kit (562245; BD Stemflow CTN ) and then analyzed using flow cytometry.

[0096] 4. Immunogenicity assay: Peripheral blood mononuclear cells (PBMCs) were extracted using lymphocyte separation medium and then co-cultured with MenSCs and MenSCs CTN respectively at a ratio of 20:1 for 48 hours. Subsequently, staining was performed using flow cytometry detection kits (Z6410002 and Z6410003, TransGen Biotech Co., Ltd., Beijing).

[0097] Test results:

[0098] 1. Consistent with untreated MenSCs, MenSCs CTN retained the spindle-shaped or polygonal structure of mesenchymal stem cells ( Figure 7 ).

[0099] 2. Showed similar multidirectional differentiation potential under the induction of adipogenic, osteogenic, and chondrogenic induction media ( Figure 8 ). Oil Red O staining showed that lipid droplets were formed inside the cells, indicating that the cells had successfully undergone adipogenic differentiation. Alizarin Red S staining showed that mineralized nodules were formed outside the cells, indicating that the cells had successfully undergone osteogenic differentiation. Alcian Blue staining showed that a matrix rich in chondroitin sulfate was deposited outside the cells, indicating that the cells had successfully undergone chondrogenic differentiation.

[0100] 3. CD44, CD73, CD90, and CD105 were continuously positively expressed in MenSCs CTN , while CD11b, CD19, CD34, CD45, and human leukocyte antigen-DR (HLA-DR) were negatively expressed, which was basically consistent with the expression of MenSCs ( Figure 9 ). These research results confirmed that MenSCs CTN met the international standards of mesenchymal stem cells, and MenSCs CTN retained the characteristics of mesenchymal stem cells.

[0101] 4. Low immunogenicity is a key property of mesenchymal stem cells, protecting them from recognition and attack by the immune system. To verify whether MenSCs CTN still retain low immunogenicity, MenSCs CTN were co-cultured with peripheral blood mononuclear cells (PBMCs). The results showed that both MenSCs CTN and untreated MenSCs exhibited fairly low immunogenicity. Among the co-cultured peripheral blood mononuclear cells, no significant differences were observed in the proportions of CD3+, CD3+ / CD4+, CD3+ / CD8+, CD3- / CD19+, and CD3- / CD16+ / CD56+ cells between the MenSCs CTN group and the untreated MenSCs group ( Figure 10 ). These research results indicate that both MenSCs CTN and MenSCs exhibit similar low immunogenicity.

[0102] Example 7. Verification of the targeting of the drug delivery system:

[0103] Test objective: To prove that the drug delivery system has triple targeting.

[0104] Test method:

[0105] In this example, the targeted controlled-release delivery system prepared in Example 1 is denoted as MenSCs CTN ; the mesenchymal stem cells obtained in step S1 of Example 1 are denoted as MenSCs.

[0106] 1. Magnetic targeting test method: MenSCs CTN were used. Excess nanoparticles were washed away with phosphate-buffered saline (PBS), the cells were digested with trypsin, and transferred to a new culture dish. A neodymium magnet was placed under the culture dish and cultured for another 12 hours. Subsequently, the cells were fixed with 4% paraformaldehyde and stained with Giemsa stain. Then, the cells were rinsed with 75% alcohol to remove the excess Giemsa stain, and further stained with Prussian blue and eosin.

[0107] 2. Migration ability test method: When ovarian cancer cells Skov3 reached 75% confluence, serum-free Dulbecco's modified Eagle's medium (DMEM) was added and cultured for another 48 hours. The prepared conditioned medium was concentrated 2-fold and 5-fold using ultrafiltration centrifugal tubes. MenSCs CTN were added to the Transwell chamber, and the concentrated medium was added to the lower layer of the chamber. After culturing for 24 hours, Giemsa staining was performed.

[0108] 3. Affinity targeting test method: When Skov3 cells reach 100% confluence in a 96-well plate, fix the cells with 4% paraformaldehyde. Stain MenSCs and MenSCs CTN with DiI dye. Then add the cells to a 96-well plate coated with Skov3 cells and incubate for 30 minutes. Wash away the unadhered cells with PBS and then perform fluorescence microscopy imaging.

[0109] 4. Drug targeted release test method: Use Coomassie Brilliant Blue R250 staining kit to stain the gelatin-coated iron oxide nanoparticles complex and evaluate the coverage of the gelatin-coated iron oxide nanoparticles complex.

[0110] In the presence of hydrogen peroxide (H2O2), using 3,3',5,5'-tetramethylbenzidine (TMB, purchased from Aladdin Chemical Co., Ltd.) as the substrate, evaluate the peroxidase-like catalytic activity of CCFCB. Add CCFCB (2000 ppm) and TMB (15 mM) to 4.43 mL of PBS (pH = 5.5). Obtain the absorption peak (652 nm) of TMB by ultraviolet-visible spectroscopy.

[0111] Test results:

[0112] 1. Magnetic targeting test results: Iron oxide nanoparticles not only act as a drug attachment platform but also endow MenSCs CTN with magnetotactic properties. Magnetic targeting generates a powerful driving force to guide a large number of MenSCs CTN to the target site ( Figure 11 ).

[0113] 2. Migration ability test results: To verify whether MenSCs CTN retain their homing ability, we conducted a Transwell migration experiment. The results showed that MenSCs CTN were able to recognize the conditioned medium of ovarian cancer cell line Skov3 tumor cells with different concentration gradients, and MenSCs CTN retained the ability to respond specifically to chemical gradients and generate a driving force for cell migration ( Figure 12 , Figure 13 ). Different from magnetic targeting that guides MenSCs CTN to the tissue-level target site, the cell homing effect can provide a more precise guidance for the treatment site.

[0114] 3. Affinity targeting test results: The affinity experiment between MenSCs CTN and human epidermal growth factor receptor-positive tumor cells showed that MenSCs CTNThe chimeric antigen receptor structure on the surface significantly enhances its binding ability to the target site( Figure 14 ).

[0115] 4. Results of drug targeted release test: To prevent the drug attached to the surface of iron oxide nanoparticles from directly contacting MenSCs CTN and thus reduce the possible toxicity of the drug to MenSCs CTN , we modified the surface of iron oxide nanoparticles with gelatin to form a protective gelatin shell( Figure 15 ). The results of Coomassie brilliant blue staining test showed that the iron oxide nanoparticles wrapped with gelatin presented light blue, while the color of pure iron oxide nanoparticles remained unchanged( Figure 16 ).

[0116] Specific microenvironments, such as malignant tumors or inflammatory sites, are characterized by the high expression of gelatinases (matrix metalloproteinase-2; matrix metalloproteinase-9), which can selectively degrade the gelatin shell. Since iron oxide nanoparticles have peroxidase (POD) activity, we used the Fenton reaction to evaluate the effectiveness of the gelatin "shell".

[0117] This Fenton reaction experiment was used to detect the integrity of the gelatin shell wrapping the nanoparticles and its specific degradation ability in the gelatinase microenvironment. Gelatin effectively isolates the nanoparticles from the surrounding environment. When in a microenvironment rich in gelatinase, the gelatinase degrades the gelatin shell, the iron oxide nanoparticles are exposed, triggering a strong Fenton reaction and restoring peroxidase activity( Figure 17 ). Therefore, a microenvironment rich in gelatinase can effectively degrade the gelatin shell on the surface of the nanoparticles and release the drug, enabling it to play a therapeutic role only in specific microenvironments.

[0118] Example 8, verification of drug release by herpes simplex virus-thymidine kinase suicide system:

[0119] Purpose of the test: To prove that ganciclovir induces the MenSC CTN suicide system and ruptures cells to release drugs.

[0120] Test method:

[0121] 1. Detection of the rupture of MenSCs was performed using the calcein-acetoxymethyl ester (Calcein-AM) / propidium iodide (PI) detection method CTN . Detection of the rupture of MenSCs CTNThe cells were cultured in 6-well plates and treated with 50 ng / mL of GCV for 0, 3, 6, 9 and 12 h, respectively. Cells not treated with GCV were used as controls. Calcein-AM / PI (C2015L; Biotech) staining and fluorescence microscopy imaging were performed, and flow cytometry was used to detect cell rupture.

[0122] 2. Evaluation of cell proliferation and survival: MenSC CTN Cells were cultured in 6-well plates and treated with different concentrations of ganciclovir (GCV concentrations were 0, 5, 25, 50, and 100 ng / mL) for 12 hours. CCK-8 (Cell Counting Kit-8) was used to measure absorbance to assess cell proliferation and survival.

[0123] 3. Lung metastasis model: Female BALB / c mice aged 6 to 8 weeks (weight 18-25 g) were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. The mice were randomly divided into cages and housed in a specific pathogen-free environment with an ambient temperature of 25°C, a humidity of 50±5%, and a light-dark cycle of 12 h.

[0124] Labeling of MenSC and MenSC with green fluorescent protein (GFP) CTN After that, 1×10 6 The cells were transplanted into BALB / c mice via tail vein injection. Twelve hours later, GCV (50 mg / kg) was administered via tail vein injection. After another 24 hours, the lungs of the mice were removed and imaged using a stereo microscope.

[0125] All experimental procedures in this study were approved by the Ethics Committee of Xinxiang Medical College and were performed in accordance with the approved guidelines.

[0126] Test results:

[0127] 1. Integration into MenSC CTN The herpes simplex virus-thymidine kinase suicide system in the drug-nanoparticle complex can achieve controlled release. Ganciclovir is a drug used clinically to treat herpes simplex and is approved by MenSC CTN Thymidine kinase expressed in the cells catalyzes the formation of GCV-TP, which in turn blocks DNA synthesis, leading to cell rupture and death, while releasing the gelatin-encapsulated ferric oxide nanoparticle-drug complex. CTN The controlled release of the delivered drug is dependent on the action of ganciclovir, which provides a promising target for MenSC CTN The delivery system provides another "safety lock" ( Figure 18 , Figure 19 ).

[0128] 2. CCK8 assays, flow cytometry, and live / dead cell staining experiments showed that only about 50 ng / ml of ganciclovir was sufficient to completely rupture MenSCCTN ( Figure 20 , Figure 21 , Figure 22 ).

[0129] The release process of the drug from MenSC CTN was both progressive and bursty. The rupture and death process of MenSC CTN lasted for more than 12 hours, and most MenSC CTN ruptured concentratedly around 12 hours ( Figure 23 ), and live cell imaging further confirmed this phenomenon ( Figure 24 ).

[0130] 3. To verify the rupture process of MenSC CTN in vivo, a lung metastasis experiment was conducted after adding green fluorescent protein (GFP) labeling to MenSC CTN . The results showed that 12 hours after exposure to 50 mg / kg of ganciclovir, the fluorescence intensity of the green fluorescent protein in the MenSC CTN group decreased significantly, while no obvious change in fluorescence intensity was observed in the untreated MenSC because of the absence of the herpes simplex virus-thymidine kinase suicide system ( Figure 25 ). Based on these rupture characteristics, doctors can regulate the time point when the drug concentration reaches its peak in the body.

[0131] Example 9. Preparation of a mesenchymal stem cell-based targeted controlled release delivery system containing a drug, comprising the following steps:

[0132] Step S1. Isolate and culture mesenchymal stem cells; the mesenchymal stem cells are isolated and cultured from menstrual blood;

[0133] Step S2. Gene transduction: By genetic techniques, a suicide system gene and a chimeric antigen receptor gene are introduced into mesenchymal stem cells to obtain gene-transduced mesenchymal stem cells; the chimeric antigen receptor expressed by the chimeric antigen receptor gene includes a signal peptide, an antigen recognition domain, a transmembrane domain, and a co-stimulatory signal domain, and the chimeric antigen receptor is human epidermal growth factor receptor 2; the suicide system gene is the herpes simplex virus-thymidine kinase gene; the herpes simplex virus-thymidine kinase gene is derived from herpes simplex virus; the gene transduction technique uses lentivirus to transduce the chimeric antigen receptor gene and the herpes simplex virus-thymidine kinase gene into mesenchymal stem cells;

[0134] Step S3, adsorption of drugs by iron oxide nanoparticles: The iron oxide nanoparticles are dispersed in a drug solution, and the drugs adhere to the iron oxide nanoparticles. The iron oxide nanoparticles are collected to form an iron oxide nanoparticle-drug complex; the drugs are GFP and DOX;

[0135] Step S4, gelatin coating: The iron oxide nanoparticle-drug complex is dispersed in a gelatin solution to form a gelatin-iron oxide nanoparticle-drug complex; the gelatin-iron oxide nanoparticle-drug complex is stabilized with glutaraldehyde, and the nanoparticles are collected to obtain a gelatin-coated iron oxide nanoparticle-drug complex; the mass concentration of the gelatin solution is 0.5%;

[0136] Step S5, gene transduction of mesenchymal stem cells to phagocytize the gelatin-coated iron oxide nanoparticle-drug complex: The gelatin-coated iron oxide nanoparticle-drug complex is dispersed in a culture medium, and gene-transduced mesenchymal stem cells are cultured in the culture medium. The gene-transduced mesenchymal stem cells are separated to form a targeted controlled-release delivery system based on mesenchymal stem cells.

Claims

1. A targeted controlled release delivery system based on mesenchymal stem cells, characterized in that, The delivery system uses mesenchymal stem cells as a carrier. The mesenchymal stem cells carry a suicide system gene and a chimeric antigen receptor. Inside the mesenchymal stem cells, there are iron oxide nanoparticles-drug complexes, and the iron oxide nanoparticles-drug complexes are coated within a gelatin layer.

2. The delivery system according to claim 1, wherein The mesenchymal stem cells are derived from menstrual blood endometrial stem cells.

3. The delivery system according to claim 2, wherein, The chimeric antigen receptor is human epidermal growth factor receptor 2.

4. The delivery system according to claim 3, wherein The suicide system gene is the herpes simplex virus-thymidine kinase gene.

5. The delivery system according to claim 3, wherein The chimeric antigen receptor includes a signal peptide, an antigen recognition domain, and a transmembrane domain.

6. A preparation method of a targeted controlled-release delivery system based on mesenchymal stem cells, characterized in that, It includes the following steps: Step S1: Isolate and culture mesenchymal stem cells; Step S2: Gene transduction: Through genetic techniques, introduce the suicide system gene and the chimeric antigen receptor gene into the mesenchymal stem cells to obtain gene-transduced mesenchymal stem cells; Step S3: Adsorb drugs onto iron oxide nanoparticles: Disperse the iron oxide nanoparticles in a drug solution, and the drugs attach to the iron oxide nanoparticles. Collect the iron oxide nanoparticles to form iron oxide nanoparticles-drug complexes; Step S4: Gelatin coating: Disperse the iron oxide nanoparticles-drug complexes in a gelatin solution to form a gelatin-iron oxide nanoparticles-drug complex; The gelatin-iron oxide nanoparticles-drug complex is stabilized with glutaraldehyde, and the nanoparticles are collected to obtain gelatin-coated iron oxide nanoparticles-drug complexes; Step S5: Gene-transduced mesenchymal stem cells phagocytize the gelatin-coated iron oxide nanoparticles-drug complexes: Disperse the gelatin-coated iron oxide nanoparticles-drug complexes in a culture medium. After culturing the gene-transduced mesenchymal stem cells in the culture medium, separate the gene-transduced mesenchymal stem cells to prepare a targeted controlled-release delivery system based on mesenchymal stem cells.

7. The preparation method according to claim 6, characterized in that, The mass concentration of the gelatin solution is 0.5%.

8. Use of the delivery system according to claim 1 in delivering anticancer drugs.

9. The application according to claim 8, characterized in that, It includes the following steps: Step S1: Deliver the delivery system into the body; Step S2: Apply a magnetic field at the targeted tissue site; Step S3: After the delivery system reaches the targeted tissue site, use an inducer to induce drug release.

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