Biotin-streptavidin mediated stem cell nano drug delivery system as well as preparation and application thereof

Abstract

The invention relates to a biotin-streptavidin mediated stem cell nano drug delivery system as well as a preparation method and application of the biotin-streptavidin mediated stem cell nano drug delivery system. According to the method, human-derived mesenchymal stem cells (MSCs) are marked as biotinylated stem cells, nano drug-loaded particles of polylactic acid-glycolic acid copolymer (PLGA) loaded anti-tumor drugs doxorubicin (DOX) and ethyl caffeate (EC) are prepared, streptavidin is functionally modified, and finally the stable stem cell nano drug-loaded compound (Bio-MSCs (at) SA (at) DOX / EC (at) PLGANPs) is formed through combination. The system utilizes the active targeting ability of MSCs and the high affinity of biotin-streptavidin to realize active targeting delivery of drugs, improve the delivery efficiency, biosafety and treatment sensitivity, provide a new and more effective drug delivery strategy for disease treatment, and have wide application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of nano-drugs, and in particular to a biotin-streptavidin-mediated stem cell nano-drug delivery system and a preparation and application thereof. Background Art

[0002] The Biotin-Streptavidin System (BSS) is a highly specific and stable non-covalent binding system that is widely used in biomedical research. Biotin is a small molecule vitamin, and streptavidin is a protein extracted from Streptomyces. The binding force between the two is extremely strong and almost irreversible. Using this property, biotin can be labeled on target molecules (such as cells, proteins or nanocarriers), and through the bridging effect of streptavidin, efficient and specific connection with other biological molecules can be achieved.

[0003] Mesenchymal stem cells (MSCs) have attracted extensive attention in the field of regenerative medicine and drug delivery due to their self-renewal ability, multidirectional differentiation potential, and low immunogenicity. MSCs can migrate to damaged tissues and promote tissue repair. They can also act as carrier cells to carry drugs or genes to specific sites. However, directly loading drugs into MSCs or combining them by physical adsorption has problems such as low efficiency and easy shedding.

[0004] Ethyl Caffeate (EC) is a natural compound with a wide range of biological activities, including anti-inflammatory, antioxidant and anti-tumor effects. In recent years, studies have shown that EC can enhance the efficacy of chemotherapy drugs and reduce the side effects of chemotherapy drugs.

[0005] In the current nano drug delivery system, although micro-nano carriers have shown great potential in drug delivery, most carriers still face the problem of lack of active targeting. Therefore, constructing a stable and efficient cell nano drug delivery system to improve the therapeutic sensitivity of chemotherapeutic drugs is of great significance for improving the overall therapeutic effect of drug delivery. Summary of the invention

[0006] One aspect of the present invention is to provide a method for preparing a biotin-streptavidin-mediated stem cell nano-drug delivery system.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] S1: Human mesenchymal stem cells (MSCs) in the logarithmic growth phase are subjected to a first labeling reaction with biotin to obtain biotin-labeled stem cells (Bio-MSCs). The labeling reaction is carried out at room temperature for 30 minutes, and then unbound biotin is removed by washing.

[0009] S2: Select polylactic acid-glycolic acid copolymer (PLGA) as the carrier material, and dissolve PLGA in dichloromethane to form a uniform PLGA solution; dissolve the anti-tumor drugs doxorubicin (DOX) and ethyl caffeate (EC) in the above PLGA solution to ensure uniform dispersion of the drugs; use the emulsification-solvent evaporation method to drop the PLGA / EC / DOX solution into the aqueous phase containing polyvinyl alcohol (PVA), and form an emulsion under ultrasound; evaporate the dichloromethane under magnetic stirring to form PLGA nanoparticles; remove the unencapsulated drugs DOX and PVA by centrifugation and washing to obtain pure DOX / EC@PLGA NPs nanoparticles.

[0010] S3: The prepared DOX / EC@PLGA nanoparticles were subjected to a third functionalization reaction with streptavidin to obtain streptavidin-functionalized DOX / EC@PLGA nanoparticles (SA@DOX / EC@PLGANPs). The duration was 30 minutes, after which the unbound streptavidin was removed by washing.

[0011] S4: The biotin-labeled stem cells (Bio-MSCs) were subjected to the fourth binding reaction with streptavidin-functionalized DOX / EC@PLGA nanoparticles (SA@DOX / EC@PLGANPs) to obtain a biotin-streptavidin-mediated stem cell nanoparticle drug complex (Bio-MSCs@SA@DOX / EC@PLGANPs). The binding reaction was carried out at room temperature for 30 minutes, after which the unbound nanoparticles were removed by centrifugation and washing.

[0012] Furthermore, in step S1, after the human mesenchymal stem cells (MSCs) are cultured to the logarithmic growth phase, they are digested with trypsin and collected by centrifugation. The number of cells is preferably 1×10 7 indivual.

[0013] Furthermore, the biotin in step S1 is preferably N-hydroxysuccinimide biotin (NHS-biotin), and its concentration is preferably 10 μg / mL.

[0014] Furthermore, in step S2, the mass ratio of PLGA, DOX and EC is 10:1:1, and the concentration of PLGA in dichloromethane is 5-10 mg / mL;

[0015] Furthermore, the PLGA / EC / DOX solution in step S2 is slowly added dropwise to the aqueous solution containing 1% PVA, and ultrasonicated at a frequency of 40 kHz for 10 minutes to form an emulsion.

[0016] Furthermore, the concentration of streptavidin in step S3 is preferably 20 μg / mL, and the functionalization reaction is performed at room temperature.

[0017] Another aspect of the present invention provides an application of a biotin-streptavidin-mediated stem cell nano-drug delivery system in the preparation of tumor therapeutic drugs.

[0018] Furthermore, the biotin-streptavidin-mediated stem cell nano-drug delivery system utilizes the active targeting ability of MSCs and the extremely high affinity and specificity between streptavidin and biotin, so that the binding between the nano-drug delivery particles and stem cells is stable, thereby achieving effective delivery.

[0019] Beneficial Effects

[0020] The present invention mainly solves the problems of targeting and efficiency in drug delivery systems. By using biotinylated mesenchymal stem cells (MSCs) as cell carriers and combining streptavidin bridging connection technology, nano drug-carrying particles are stably combined with stem cells, and the carrier function of mesenchymal stem cells and the synergistic effect of caffeic acid ethyl ester and doxorubicin are combined to achieve active targeted delivery of drugs and improve the therapeutic sensitivity of drugs. This system not only improves the delivery efficiency of drugs, but also makes full use of the natural tropism and low immunogenicity of stem cells, providing a new and more effective drug delivery strategy for disease treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The morphology characterization results of DOX / EC@PLGANPs and SA@DOX / EC@PLGANPs nanoparticles; Figure 1 a is the transmission electron microscopy image of DOX / EC@PLGA NPs nanoparticles. Figure 1 b is the transmission electron microscopy image of SA@DOX / EC@PLGANPs nanoparticles, scale bar: 100 nm.

[0022] Figure 2 These are the biosafety evaluation results of the biotin-streptavidin-mediated stem cell nanodrug delivery system.

[0023] Figure 3 Flow cytometry was used to evaluate the cellular uptake results of the biotin-streptavidin-mediated stem cell nanodrug delivery system.

[0024] Figure 4 Fluorescence microscopy was used to evaluate the cellular uptake results of the nano-drug delivery system mediated by phycocyanin-streptavidin in stem cells; Figure 4 a is the evaluation result of cell targeting of the nano-drug delivery system in the control group. Figure 4 b is the cell targeting evaluation result of the nanodrug delivery system in the experimental group, scale bar: 20 μm.

[0025] Figure 5 The experimental results show that EC enhances the tumor killing ability of biotin-streptavidin-mediated stem cell nanodrug delivery system.

[0026] Figure 6 These are the experimental results of targeting different cell lines of biotin-streptavidin-mediated stem cell nanodrug delivery system. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. The embodiments are merely explanations of the present invention and are not limitations of the present invention. Any changes made by those skilled in the art after reading the specification of the present invention will be protected by the patent law as long as they are within the scope of the claims.

[0028] Example 1: Preparation of a stem cell nano-drug delivery system based on biotin-streptavidin S1 Biotin labeling of stem cells: Human mesenchymal stem cells (MSCs) were selected as model cells. First, MSCs were cultured to the logarithmic growth phase, then digested with trypsin and centrifuged to collect 1×10 7 cells. Next, the cells were washed twice with PBS and resuspended in 1 mL of 10 μg / mL NHS-biotin (N-hydroxysuccinimide biotin) in a PBS solution. After incubation at room temperature for 30 minutes, the cells were washed three times with PBS to remove unbound NHS-biotin. Finally, the biotin-labeled MSCs were resuspended in complete culture medium to obtain Bio-MSCs for use.

[0029] S2 Preparation of nano drug-loaded particles: Polylactic acid-co-glycolic acid (PLGA) was selected as the carrier material of nano drug-loaded particles. First, 80 mg of PLGA was dissolved in 10 mL of dichloromethane to form a uniform solution. Then, 8 mg of the anti-tumor drug doxorubicin (DOX) was dissolved in the above PLGA solution to ensure that the drug was evenly dispersed; 8 mg of ethyl caffeate was continued to be added to the drug solution; then, the nano drug-loaded particles were prepared by the emulsification-solvent evaporation method, that is, the PLGA / EC / DOX solution was slowly added dropwise to the aqueous phase containing 1% polyvinyl alcohol (PVA), and an emulsion was formed under ultrasonic action (40 kHz, 10 min). Subsequently, dichloromethane was volatilized under magnetic stirring to form PLGA / EC nano drug-loaded particles. Finally, the unencapsulated drugs DOX, EC and PVA were removed by centrifugation (5000 rpm, 10 min) and washing, and repeated three times to obtain pure DOX / EC@PLGANPs nanoparticles.

[0030] S3 Functional modification with streptavidin: The prepared DOX / EC@PLGANPs nanoparticles were dispersed in a PBS solution containing streptavidin (20 μg / mL). After incubation at room temperature for 30 minutes, the nanoparticles were washed three times with PBS to remove unbound streptavidin. In this way, streptavidin was successfully modified on the surface of the nanoparticle system to prepare SA@DOX / EC@PLGANPs nanoparticles.

[0031] Formation of S4 stem cell nano-drug delivery complex: MSCs labeled with biotin (Bio-MSCs) were mixed with streptavidin-functionalized PLGA / EC nano-drug delivery particles (SA@DOX / EC@PLGANPs) and incubated at room temperature for 30 minutes. Due to the extremely high affinity and specificity between biotin and streptavidin, they will quickly bind to form a stable stem cell nano-drug delivery complex. Finally, the complex was collected by centrifugation and washed twice with PBS to remove the unbound nano-drug delivery system. Finally, the biotin-streptavidin-mediated stem cell nano-drug delivery system Bio-MSCs@SA@DOX / EC@PLGANPs was obtained.

[0032] Comparative Example 1

[0033] The difference between this embodiment and embodiment 1 is that no caffeic acid ethyl ester is added during the preparation of S2 nano drug-loaded particles.

[0034] S2 Preparation of Nanoparticles with Drug Loading: Polylactic acid-co-glycolic acid (PLGA) was selected as the carrier material for nanoparticles with drug loading. First, 80 mg of PLGA was dissolved in 10 mL of dichloromethane to form a uniform solution. Then, 8 mg of the anti-tumor drug doxorubicin (DOX) was dissolved in the above PLGA solution to ensure uniform dispersion of the drug; then, the nanoparticles with drug loading were prepared by the emulsification-solvent evaporation method, that is, the PLGA / DOX solution was slowly added dropwise to the aqueous phase containing 1% polyvinyl alcohol (PVA), and an emulsion was formed under ultrasonic action (40 kHz, 10 min). Subsequently, dichloromethane was volatilized under magnetic stirring to form PLGA nanoparticles with drug loading. Finally, the unencapsulated drugs DOX and PVA were removed by centrifugation (5000 rpm, 10 min) and washing, and the process was repeated three times to obtain pure DOX@PLGANPs nanoparticles.

[0035] Example 2: Characterization of DOX / EC@PLGANPs and SA@DOX / EC@PLGANPs Nanoparticles

[0036] The microscopic morphological characteristics of DOX / EC@PLGANPs and SA@DOX / EC@PLGANPs drug-loaded nanoparticles were characterized by transmission electron microscopy (TEM) with a test voltage of 120 kV. DOX / EC@PLGA NPs and SA@DOX / EC@PLGANPs drug-loaded nanoparticles were dispersed in ultrapure water, 30 μL of sample was drawn and dropped on the surface of the carbon film supported by a copper mesh dedicated to the electron microscope, and then photographed after natural air drying.

[0037] like Figure 1 As shown in Figure 2, TEM observations showed that the particle sizes of DOX / EC@PLGANPs and SA@DOX / EC@PLGANPs were uniform and similarly spherical ( Figure 1 a. Figure 1 b). The particle size of DOX / EC@PLGA NPs modified with streptavidin slightly increased to about 100 nm for DOX / EC@PLGANPs and about 115 nm for SA@DOX / EC@PLGANPs, indicating that streptavidin was successfully modified on the surface of PLGA nanoparticles to form a functionalized modification layer.

[0038] Example 3: Biosafety of the Stem Cell Nano-drug Delivery System Mediated by Biotin-Streptavidin

[0039] MSCs and Bio-MSCs in the logarithmic growth phase were plated at 1×10 per well. 4 Cells were seeded in 96-well plates, 100 μL DMEM high-glucose complete medium per well, and cultured for 24 hours to make the cell confluence 70-80%. The medium was aspirated and the cells were divided into several groups. Each group was added with 100 μL of medium containing different concentrations of SA@DOX / EC@PLGANPs (0, 20, 40, 60, 80, 100 μM). After 24 hours of culture, 10 μL CCK8 solution was added to each well, and incubated at 37°C for 1.5 hours. The absorbance was measured at 450 nm using a multifunctional microplate reader. The MSCs group without any treatment was used as the control group to calculate the cell viability.

[0040] like Figure 2As shown in the figure, after treating these two cells with different concentrations of SA@DOX / EC@PLGANPs, the cell viability of both MSCs and Bio-MSCs was not less than 80%. It is worth noting that the inhibition rate of SA@DOX / EC@PLGANPs on MSCs was slightly higher than that on Bio-MSCs. This result not only reflects the biosafety of the biotin-streptavidin-mediated stem cell nano-drug delivery system, but also confirms to a certain extent that the nano-drug delivery system has a certain targeting effect on Bio-MSCs. In summary, the biotin-streptavidin-mediated stem cell nano-drug delivery system SA@DOX / EC@PLGANPs significantly reduces the toxic effect on non-target cells (MSCs) while maintaining effective drug delivery to target cells (Bio-MSCs), showing good biosafety.

[0041] Example 4: Cellular uptake experiment of biotin-streptavidin mediated stem cell nano-drug delivery system

[0042] FITC green fluorescence-labeled DOX was used to prepare the PLGA nano-drug delivery system, and the uptake of the nano-drug delivery system by MSCs was observed by flow cytometry and fluorescence microscopy to verify whether the biotin-streptavidin-mediated binding effectively improved the uptake efficiency of the nano-drug delivery system by MSCs.

[0043] 1. Flow cytometry to assess cellular uptake

[0044] In order to study the cell targeting of Bio-MSCs@SA@DOX / EC@PLGANPs nanodrug delivery system, the cells were grouped as follows.

[0045] Control group: MSCs in the logarithmic growth phase and in good growth condition were mixed with unfunctionalized FITC-DOX / EC@PLGANPs in a certain proportion to ensure sufficient contact between breast cancer cells MCF-7 and nanoparticles.

[0046] Experimental group: Bio-MSCs in the logarithmic growth phase and in good growth condition were combined with SA@FITC-DOX / EC@PLGANPs (i.e., streptavidin-functionalized PLGA nanodrug delivery system loaded with FITC-labeled DOX) according to the method in Example 1 to form a Bio-MSCs@SA@FITC-DOX / EC@PLGANPs complex.

[0047] MCF-7 cells were cultured under appropriate conditions (37°C, 5% CO 2) for 4 hours to allow the cells to take up the nano-drug delivery system. After the incubation, the cells were gently washed 3 times with PBS to remove unbound nanoparticles. The cells were digested with trypsin and the cell pellet was collected by centrifugation. The cells were resuspended in an appropriate amount of PBS and the FITC fluorescence intensity was detected by flow cytometry to evaluate the cell uptake of the nano-drug delivery system. Flowjo v10 was used to analyze the fluorescence intensity of the cells under the FITC channel.

[0048] Flow cytometry analysis showed that the FITC fluorescence intensity of the experimental group (Bio-MSCs@SA@FITC-DOX / EC@PLGANPs) was significantly higher than that of the control group (MSCs mixed with FITC-DOX / EC@PLGANPs), indicating that biotin-streptavidin-mediated binding significantly improved the uptake efficiency of the nano-drug delivery system by MSCs ( Figure 3 ). The distribution of the Bio-MSCs@SA@FITC-DOX / EC@PLGANPs group shifted to the right as a whole, indicating that the Bio-MSCs stem cells phagocytized more nanoparticles in this group, which was achieved through the high-affinity specific binding between biotin and streptavidin, confirming the effectiveness of the biotin-streptavidin-mediated stem cell nanodrug delivery system.

[0049] 2. Observation of Cellular Uptake by Fluorescence Microscopy

[0050] Cell grouping was the same as that of flow cytometry. MCF-7 cells were cultured under appropriate conditions (37°C, 5% CO 2 ) and incubate for 4 hours to allow MCF-7 cells to absorb the nano-drug delivery system. After the incubation, wash the cells with PBS and then fix the cells with 4% paraformaldehyde for 15-30 minutes. After fixation, wash again with PBS and stain the cell nuclei with DAPI to observe the cell morphology more clearly. Seal the cell slides with anti-fluorescence quenching sealing agents and observe under a fluorescence microscope. Select the FITC fluorescence channel and the DAPI fluorescence channel to observe and take images of the cell uptake of the nano-drug delivery system.

[0051] Fluorescence microscopy further confirmed this result. Figure 4 As shown in Figure 2, obvious green fluorescence signals were observed on the surface and inside of Bio-MSCs in the experimental group, indicating that FITC-labeled DOX had been successfully taken up by biotin-modified human mesenchymal stem cells (Bio-MSCs) ( Figure 4 b). In contrast, the green fluorescence signal in the control group was weak and dispersed, indicating that the cellular uptake efficiency of the nanodrug delivery system without biotin-streptavidin mediation was low ( Figure 4a). In addition, DAPI staining of the cell nucleus showed good cell morphology in both the MSCs group and the Bio-MSCs group, which was not affected by the treatment of the nano-drug delivery system, further verifying the biocompatibility of this method.

[0052] In summary, streptavidin-functionalized nanoparticles can be specifically bound to the surface of stem cells by incubating with mesenchymal stem cells labeled with biotin. This binding mode allows each stem cell to carry multiple nanoparticles, thereby increasing the efficiency of drug delivery. This example confirms that the biotin-streptavidin-mediated stem cell nanoparticle drug delivery system (Bio-MSCs@SA@DOX / EC@PLGANPs) can significantly improve the uptake efficiency of MSCs to the nanoparticle drug delivery system by both flow cytometry and fluorescence microscopy, providing strong support for subsequent drug delivery and disease treatment research.

[0053] Example 5: Anti-tumor performance of biotin-streptavidin-mediated stem cell nano-drug delivery system 1. EC enhances the tumor killing ability of biotin-streptavidin-mediated stem cell nano-drug delivery system

[0054] MSCs and Bio-MSCs in the logarithmic growth phase were selected and 1×10 4 The cells were seeded in a 96-well plate at a density of 100 μL DMEM high-glucose complete medium was added to each well to ensure that MCF-7 cells grow in a suitable environment. The 96-well plate was placed at 37°C and 5% CO. 2 The cells were cultured in a cell culture incubator for 24 hours to reach a confluence of about 70-80%. The original culture medium was aspirated and the cells were divided into two groups. One group was added with a culture medium containing SA@DOX / EC@PLGANPs; the other group was added with a culture medium containing SA@DOX@PLGANPs nanoparticles prepared in Comparative Example 1. After continuing to culture for 24 hours, 10 μL of CCK8 solution was added to each well. The 96-well plate was returned to the cell culture incubator and incubated at 37°C for another 1.5 hours to allow the CCK8 solution to react with the dehydrogenase in the living cells to produce a colored product. The absorbance value of each well was measured at a wavelength of 450 nm using a multifunctional microplate reader to calculate the cell viability.

[0055] like Figure 5As shown, in the MSCs and Bio-MSCs groups treated with culture medium containing SA@DOX / EC@PLGA NPs, cell viability showed a significant downward trend. Especially in the Bio-MSCs group, due to the specific interaction of biotin-streptavidin, SA@DOX / EC@PLGANPs can more effectively target and bind to cells, thereby showing a stronger tumor killing effect. In contrast, in the MSCs group, due to the lack of biotin labeling, the targeting of the nanoparticles was reduced, and the decrease in cell viability was relatively small. However, in cells treated with the culture medium containing SA@DOX@PLGANPs nanoparticles prepared in Comparative Example 1, the decrease in cell viability was significantly lower in both MSCs and Bio-MSCs than in the SA@DOX / EC@PLGANPs treatment group. This shows that the introduction of EC significantly enhances the tumor killing ability of the nanodrug delivery system, especially when targeting Bio-MSCs.

[0056] 2. Targeting of biotin-streptavidin-mediated stem cell nano-drug delivery system

[0057] Breast cancer cells MCF-7, neuroblastoma cells SH-SY5Y, and liver cancer cells HepG2 were selected and 1×10 4 The cells were inoculated in a 96-well plate at a density of 100 μL DMEM high-glucose complete medium per well and cultured for 24 hours to make the cell confluence 70-80%. The three different tumor cells were treated with the same concentration of medium containing the Bio-MSCs@SA@DOX / EC@PLGANPs stem cell nano-drug delivery system. After culturing for another 24 hours, 10 μL CCK8 solution was added to each well. The 96-well plate was returned to the cell culture incubator and incubated at 37°C for another 1.5 hours. The absorbance value of each well was measured at a wavelength of 450 nm using a multifunctional microplate reader to calculate the cell viability.

[0058] The experimental results are as follows Figure 6 The stem cell nanodrug delivery system containing Bio-MSCs@SA@DOX / EC@PLGANPs showed different degrees of cell killing effects when treating breast cancer cells MCF-7, neuroblastoma cells SH-SY5Y and liver cancer cells HepG2. Among them, MCF-7 cells are most sensitive to the nanodrug delivery system, SH-SY5Y cells are relatively less sensitive, and the sensitivity of HepG2 cells is between the two. These results fully prove that Bio-MSCs@SA@DOX / EC@PLGANPs has targeting properties for specific tumor cell types.

[0059] The embodiments described above are only some of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative work are within the scope of protection of the present invention.

Claims

1. A method for preparing a biotin-streptavidin-mediated stem cell nano-drug delivery system, characterized in that: The following steps are involved: S1: Performing a first labeling reaction on human mesenchymal stem cells MSCs in the logarithmic growth phase with biotin to obtain biotin-labeled stem cells Bio-MSCs; the labeling reaction is carried out at room temperature for 30 minutes, and then unbound biotin is removed by washing; S2: Select polylactic acid-glycolic acid copolymer PLGA as the carrier material, and dissolve PLGA in dichloromethane to form a uniform PLGA solution; dissolve the anti-tumor drug doxorubicin DOX and ethyl caffeate EC in the above PLGA solution to ensure that the drugs are evenly dispersed; The PLGA / EC / DOX solution was added dropwise to the aqueous phase containing polyvinyl alcohol (PVA) by the emulsification-solvent evaporation method, and an emulsion was formed under the action of ultrasound. The dichloromethane was evaporated under magnetic stirring to form PLGA nanoparticles. The unencapsulated drugs DOX, EC, and PVA were removed by centrifugation and washing to obtain pure DOX / EC@PLGANPs nanoparticles; S3: The prepared DOX / EC@PLGA nanoparticles were subjected to a third functionalization reaction with streptavidin to obtain streptavidin-functionalized DOX / EC@PLGA nanoparticles SA@DOX / EC@PLGANPs; the duration was 30 minutes, after which the unbound streptavidin was removed by washing; S4: The biotin-labeled stem cells Bio-MSCs were subjected to a fourth binding reaction with streptavidin-functionalized DOX / EC@PLGA nanoparticles SA@DOX / EC@PLGANPs to obtain the biotin-streptavidin-mediated stem cell nanoparticle drug complex Bio-MSCs@SA@DOX / EC@PLGANPs; the binding reaction was carried out at room temperature for 30 minutes, after which the unbound nanoparticles were removed by centrifugation and washing.

2. The preparation method according to claim 1, characterized in that: Step S1: After the human mesenchymal stem cells (MSCs) are cultured to the logarithmic growth phase, they are digested with trypsin and collected by centrifugation. The number of cells is 1×10 7 indivual.

3. The preparation method according to claim 1, characterized in that: The biotin in step S1 is N-hydroxysuccinimide biotin NHS-biotin, and its concentration is 10 μg / mL.

4. The preparation method according to claim 1, characterized in that: Step S2: the mass ratio of PLGA, DOX and EC is 10:1:1, and the concentration of PLGA in dichloromethane is 5-10 mg / mL; The PLGA / EC / DOX solution was slowly added dropwise to the aqueous phase solution containing 1% PVA, and ultrasonicated at a frequency of 40 kHz for 10 minutes to form an emulsion.

5. The preparation method according to claim 1, characterized in that: The concentration of streptavidin in step S3 is preferably 20 μg / mL, and the functionalization reaction is carried out at room temperature.

6. Application of a biotin-streptavidin-mediated stem cell nano-drug delivery system in the preparation of tumor therapeutic drugs.

7. The use according to claim 6, characterized in that: The biotin-streptavidin-mediated stem cell nano-drug delivery system utilizes the active targeting ability of MSCs and the affinity and specificity between streptavidin and biotin to construct a stable stem cell nano-drug delivery system.

8. The use according to claim 6, characterized in that: EC enhances the tumor killing ability of biotin-streptavidin-mediated stem cell nanodrug delivery system.

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