CN105457037A - Stem cell tumor targeting system with internal nano-prodrug and preparation method thereof - Google Patents

Abstract

The present invention belongs to the technical fields of biomedical medicine and nano-medicine, and relates to a stem cell tumor targeting system with internal nano prodrug and a preparation method thereof. The method uses stem cells as cell carriers to prepare an anti-tumor targeting system with general formula of Stem cells-(RGD-PPCD)n through endocytosis and inner load of nano prodrug. The nano prodrug has the characteristics of targeting, sustained release and acid sensitivity release; and the stem cell carrier can remotely target primary lesion and metastasis of tumor. In vitro experimental results show that the stem cells after drug load can retain the proliferation capacity and tumor migration characteristics; and the constructed system is stable to ensure that the drug stays in the form of nano-prodrug for several days in the cells, and can slowly release the drug. In vivo experimental results show that the constructed stem cell targeting system can significantly prolong the survival time of tumor-bearing animals and can maintain normal neurobehavioral characteristics of tumor-bearing animals in comparison with the original drug and nano prodrugs, so as to play the efficient, secure and long-lasting anti-tumor effect.

Description

A stem cell tumor targeting system loaded with nano-prodrug and its preparation method

technical field

The invention belongs to the technical field of biomedicine and nanomedicine, and relates to a stem cell tumor targeting system loaded with nanometer prodrug and a preparation method thereof.

Background technique

According to statistics, at present, malignant tumor is still a serious disease that threatens human health, and its mortality rate (37.0%) is second only to heart disease, ranking second in the cause of death. The main reason is that the tumor infiltrates and grows into the surrounding normal tissues in the early stage of the disease to form satellite lesions. After surgical resection, it is easy to relapse and the degree of malignancy increases, and it is resistant to radiotherapy and chemotherapy. Serious injury, etc., so the survival period of patients with malignant tumors has not been effectively extended. The emergence of nanotechnology provides an opportunity for the effective delivery of drugs to tumors, but there are still problems such as unstable circulatory system in the body; difficulty in crossing physiological barriers, so that they cannot effectively accumulate to achieve therapeutic concentrations; and difficulty in penetrating tumor parenchyma.

Zhu Saijie et al. (Patent No.: CN101879313B) used high-loaded PEGylated polyamide-amine dendrimers (PEG-PAMAM) as nanocarriers, and delivered doxorubicin via acid-sensitive bonds (Cis-acotonicanhydride, CA). (Doxorubicin, DOX) was covalently linked to the carrier, and a PEG-PAMAM-cis-aconityl-DOX (PPCD) macromolecular nano-prodrug with passive targeting and tumor site-localized drug release was synthesized; RGD cyclic peptide was further targeted By modifying PPCD to the head group, RGD-PPCD with active targeting function can be obtained. The two nano-prodrugs hardly released drugs in the circulatory system and normal tissues, and slowly released doxorubicin in tumors and intracellular weakly acidic environments, significantly reducing the toxicity of doxorubicin and prolonging the survival of animals; although In this way, it is difficult for nano-prodrugs to improve the degree of targeting and penetration into tumor tissue, and it is impossible to achieve the ideal tumor targeting effect.

At present, the research on mesenchymal stem cells and neural stem cells as targeting vectors is mainly focused on the gene therapy of tumors. Mesenchymal stem cells (MSCs) have good migration ability and anti-tumor effect, and can be used as cell carriers. Experiments have shown that after MSCs enter the human body, they will preferentially gather in tumor stroma or wounds: after intravenous or intratumoral injection, MSCs transfected with viral vectors are distributed in tumor tissues and borders, and no distribution in normal organs has been found. ; Anti-cancer factors (INF, IL, TRAIL, CX3CL1 and CCL5) expressed by intratumoral MSCs significantly improved the survival time of experimental animals and inhibited tumor growth. In addition, MSCs have no ethical restrictions, are easy to isolate and culture in vitro, have low immunogenic and intrinsic mutation rates, and have no neurotoxicity or tumorigenicity. At present, the tumor treatment scheme based on MSCs has been applied to breast cancer and its lung metastasis model, ovarian cancer and its metastasis model, liver cancer and its metastasis model, brain glioma, pancreatic cancer, colorectal cancer, Kaposi's sarcoma and kidney cancer and other experimental models of malignant tumors (Tang Zhiqiang et al.: Tumor, 2010, 30(11): 980-983), and achieved good therapeutic effects. Neural stem cells (NSCs) belong to pluripotent stem cells, which have anti-tumor effects, and have the characteristics of stability and tumor tendency as cell carriers. Intravenous injection of NSCs can effectively pass through the blood-brain barrier (the transmission rate is much higher than that of nanoparticles), and can migrate throughout the brain tumor tissue and cancer cell infiltration area, and the distribution in normal brain tissue has not been found, showing that it is resistant to brain glue. Glioma has a strong tropism; after intracranial injection of gene-transfected NSCs, NSCs spread throughout the tumor and migrate to other parts with the tumor; the anti-cancer cell factors expressed by NSCs in the tumor and infiltrating areas exert a strong anti-tumor effect . However, the safety of viral vectors has always been controversial, and the transfection process may also cause tumorigenic genes and transfected genes to be inserted into the genome of stem cells, thereby causing genome breaks and malignant transformation of stem cells, which limit the further application and clinical application of stem cells. test.

Recently, studies have attempted to use MSCs as cell carriers to load chemotherapeutic drugs (doxorubicin and paclitaxel) through endocytosis or membrane surface binding. These studies have found that the combined MSCs still have their own cell activity and multi-lineage differentiation function; MSCs loaded with drugs can still be located in glioma and its infiltrating areas, improving the efficacy of drugs targeting tumors. However, it is difficult for stem cells to load small-molecule chemotherapeutic drugs to achieve acid-sensitive release of drugs, and the released small-molecule chemotherapeutic drugs still have the problem of being pumped out of the tumor parenchyma, losing their specific targeting effect; premature excretion of chemotherapeutic drugs is also difficult. It takes 2 to 4 days to ensure that the stem cells migrate to the tumor tissue, which affects the effective accumulation of drugs in the tumor and reduces the targeting and anti-tumor effects.

In view of the above status quo, the inventors of the present application intend to provide a more stable and efficient tumor-targeted drug delivery system, which combines the targeting and acid-sensitive release characteristics of nano-prodrugs with the strong tumor tropism of stem cells, and constructs an endogenous The stem cell tumor targeting system loaded with nano-prodrugs is expected to overcome the respective shortcomings of nano-prodrugs and stem cell carriers, and realize the localization and effective accumulation of drugs in tumors.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, to provide a stem cell tumor targeting system loaded with nano-prodrugs, especially the stem cell tumor-targeting system loaded with nano-prodrugs and its preparation method and the system in tumor Use in targeted therapy.

Specifically, the present invention provides a stem cell tumor targeting system loaded with nano-prodrugs. Stem cells are used as cell carriers to load nano-prodrugs through cell endocytosis to prepare a tumor-targeting system. The target The directed system has the general formula:

Stemcells—(RGD-PPCD) _n

Wherein, Stemcells are stem cells, and said stem cells are mesenchymal stem cells and neural stem cells; said mesenchymal stem cells and neural stem cells may be human-derived mesenchymal stem cells and human-derived neural stem cells.

RGD-PPCD is a multifunctional anti-tumor nano-prodrug based on dendrimers; the anti-tumor nano-prodrug is a laboratory research product (patent number: CN101879313B), which has a general formula:

RGD-PEG-Dendrimer-DOX

Among them, RGD is a cyclic polypeptide containing RGD sequence such as RGDyC, RGDyK, RGDfC, RGDfK, etc., and its structure contains free thiol or free amino groups; PEG is a bifunctional polyethylene glycol with a molecular weight of 1000-10000 Da, and one end contains Imide group, the other end contains hydroxysuccinimide activated ester; Dendrimer is a dendritic polymer with amino groups on the surface, such as polyamide-amine dendritic polymer (PAMAM) of integer generation, polypropylene imine dendritic polymer (PPI) and dendritic polylysine (PLL), etc.; DOX is doxorubicin.

The stem cell tumor targeting system loaded with nano-prodrugs of the present invention has been tested through experiments, and the results show that the stem cells loaded with nano-prodrugs still have high tumor tropism and permeability, the stability can reach 5 days or more, and the release is slow. Compared with the original drug and nano-prodrug, the stem cell tumor targeting system loaded with nano-prodrug significantly prolongs the survival period of animals, maintains the normal neurobehavioral characteristics of tumor-bearing animals, thereby significantly enhancing the anti-tumor effect and reducing toxic side effect.

The nano-prodrug-loaded stem cell tumor targeting system prepared by the present invention not only has the advantages of nano-prodrugs, that is, the acid-sensitive drug release characteristics and the active targeting of RGD cyclic peptide modification; it also has the characteristics of stem cell carriers, That is, it can overcome physiological barriers and direct chemotaxis to the primary tumor and metastases. Nano-prodrugs loaded in stem cells, on the one hand, avoid the use of viral vectors, will not lead to malignant transformation of genes, and have better safety; The tumor tissue is released later, so that the nano-prodrug can penetrate deep into the tumor parenchyma to play a targeting and sustained release effect, so as to obtain the specific accumulation of active drugs in the tumor tissue and exert a safe and efficient anti-tumor effect.

The present invention adopts the following technical solutions to realize the present invention and achieve the above-mentioned purpose of the invention.

1. Toxicity of nano-prodrugs to stem cells

The MTT cell viability detection method was used, the incubation time was 1-96 h, and the drug concentration was 10 ^-6 -10 ⁷ μg/mL to detect the influence of doxorubicin and nano-prodrug on the cell viability of stem cells, and determine the effect of drugs on stem cell toxicity damage. concentration and time frame;

2. Investigation on optimal preparation conditions of Stemcells—(RGD-PPCD) _n

Take 10 ⁵ to 10 ⁸ stem cells in the logarithmic growth phase, and co-incubate with the drug-containing culture medium, the drug concentration is 1 μg/mL-100 μg/mL, the incubation temperature is 30-45 °C, and contains 1%-10% CO ₂ . Incubate for different times, wash with PBS after incubation, digest and centrifuge, and use flow cytometry to detect the average fluorescence intensity;

Take 10 ⁵ -10 ⁸ stem cells in the logarithmic growth phase, and co-incubate with the drug-containing culture medium for 1 h-96 h, the incubation temperature is 30-45°C, and contains 1%-10% CO ₂ . Incubate with different drug concentrations, wash with PBS after incubation, digest and centrifuge, and use flow cytometry to detect the average fluorescence intensity;

The preferred preparation conditions of the present invention are as follows: the incubation time is 1-96 hours, the incubation concentration is 1-100 μg/mL, the incubation temperature is 30-45° C., and contains 1%-10% CO ₂ .

3. Preparation of Stemcells—(RGD-PPCD) _n

Take 10 ⁵ to 10 ⁸ stem cells in the logarithmic growth phase and co-incubate with the drug-containing medium for 1-96 hours, the drug concentration is 1-100 μg/mL, the incubation temperature is 30-45°C, and 1 % to 10% of CO ₂ . After incubation, wash with PBS, digest and centrifuge to obtain Stemcells—(RGD-PPCD) _n targeting system;

The concentration of the nanometer prodrug is equivalently converted according to the concentration of doxorubicin contained;

4. Determination of Stemcells—(RGD-PPCD) _n drug loading

Take the prepared Stemcells—(RGD-PPCD) _n , ultrasonically pulverize to prepare a homogenate, add 0.5-5N HCl, and incubate in a water bath at 30-70° C. for 0.5-2 hours. After cooling to room temperature, add internal standard, PBS buffer, and 0.5-5N NaOH, then vortex extract with chloroform-methanol, and blow dry at 25-50°C with nitrogen. The residue was redissolved, and the supernatant was taken by centrifugation to determine the drug content. ;

The internal standard is daunorubicin, and the pH of the PBS buffer is 7-9;

The described sampling measuring instrument is a high performance liquid chromatograph;

5. Determination of in vitro release of Stemcells—(RGD-PPCD) _n

Using the method of bioequivalence, the drug released by Stemcells—(RGD-PPCD) _n is assayed:

①Establish a bioequivalence method for release assay

Take the prepared Stemcells—(RGD-PPCD) _n and cultivate them. After 24-96 hours, collect the conditioned medium (CM) and perform equal dilution. Use the MTT method to detect the Stemcells—(RGD-PPCD) _n —CM pair. Inhibition of tumor cell viability, the same method was used to detect the inhibition of nano-prodrug PPCD/RGD-PPCD on tumor cell viability, and draw a bioequivalence curve;

The CM equivalent dilution range is 1:128-1:1, and the concentration range of PPCD/RGD-PPCD (equiv.DOX) is 0.078-10 μg/mL;

The ordinate of the bioequivalence curve is the survival rate of tumor cells, and the abscissa is the dilution ratio of CM (upper abscissa) and the concentration range of PPCD/RGD-PPCD (lower abscissa);

In the present invention, the drug concentration contained in the CM is converted according to the bioequivalence curve;

② Stemcells—(RGD-PPCD) _n release determination

Take the prepared Stemcells—(RGD-PPCD) _n , culture them, collect CM at different times, and measure the drug concentration contained in the obtained CM by the above-mentioned bioequivalence method;

6. Stability investigation of Stemcells—(RGD-PPCD) _n

Take the prepared Stemcells—(RGD-PPCD) _n and culture them for 1-96 hours. Add Lysotracker and DAPI to locate lysosomes and nuclei respectively, and use a laser confocal microscope to observe Stemcells—(RGD-PPCD) in different phases. ) _n in the retention and distribution of doxorubicin and nano-prodrugs in stem cells, to understand the stability and stable duration of Stemcells—(RGD-PPCD) _n ;

The Lysotracker is LysotrackerGreenDND-26;

7. Investigation of Stemcells—(RGD-PPCD) _n on tumor cell penetration

Tumorspheres were cultured in vitro, co-cultured with non-drug-loaded and drug-loaded stem cells, and the permeability of Stemcells and Stemcells—(RGD-PPCD) _n to tumorspheres was investigated to understand whether the stem cells after constructing the drug delivery system maintained the ability to protect against tumors. ball penetration;

8. In vitro study on the toxicity of Stemcells—(RGD-PPCD) _n to tumor cells

Take the prepared Stemcells—(RGD-PPCD) _n , co-culture with tumor cells at a ratio of 1:1000 to 1:0.1, culture for 12 to 192 hours, use the MTT method to detect the cell viability of the tumor cells, and investigate the Stemcells - (RGD-PPCD) _n in vitro tumor cell growth inhibition;

9. In vivo pharmacodynamic testing and safety evaluation of Stemcells—(RGD-PPCD) _n

ICR tumor-bearing mice were used to establish an orthotopic glioma model, and saline, original drug, nano-prodrug and Stemcells—(RGD-PPCD) _n were injected intratumorally at different time points to investigate the survival of tumor-bearing mice , observe its neurobehavioral indicators, compare with the original drug and nano-prodrug, and evaluate the anti-tumor effect and safety of Stemcells—(RGD-PPCD) _n .

The experimental methods involved in the present invention belong to known methods in the art, and the reagents and cells used in the experiments can be obtained through commercial channels.

The beneficial effects of the present invention are that the remarkable advantages of the multifunctional anti-tumor targeting system of the present invention are:

(1) Using stem cells with tumor tropism as a carrier, loaded with nano-prodrugs PPCD and RGD-PPCD with acid-sensitive drug release and targeted delivery characteristics, it can be specifically located in the primary tumor and metastases of the tumor, and penetrate deep into the parenchyma. The slow release of drugs overcomes the shortcomings of nano-prodrugs that are difficult to penetrate into the tumor parenchyma and the defects of poor specificity of small molecule drugs.

(2) Cytotoxicity studies showed that the drug and nano-prodrug had no obvious toxicity to stem cells, and the cytotoxicity of nano-prodrug was significantly lower than that of the original drug. The drug-loaded stem cells still maintained extremely high cell viability and migration characteristics, and there was no significant difference in the chemotaxis to tumors and the infiltration of tumor spheres from those of non-drug-loaded stem cells.

(3) The present invention adopts the endocytosis method to prepare the targeting system, that is, co-incubating the stem cells and the nano-prodrug in the stem cell culture medium, and the method is simple and easy to operate. By examining different incubation times and concentrations, the optimal preparation scheme was optimized, so that the maximum drug loading capacity can be obtained and the drug loading requirements can be guaranteed.

(4) In vitro release and stability experiments showed that Stemcells—(RGD-PPCD) _n can release the drug slowly, and can ensure the effective delivery of the drug in the form of nano-prodrug, and the stability can be maintained for 5 days or more, ensuring It takes 2 to 4 days for the stem cells to migrate to the tumor tissue.

(5) The results of in vivo experiments show that, compared with doxorubicin original drug and nano-prodrug, Stemcells—(RGD-PPCD) _n can significantly prolong the survival period of mice, significantly reduce the neurobehavioral toxicity, and significantly improve the survival of mice Quality, significant anti-tumor effect, reduced toxic and side effects.

Description of drawings

Figure 1.1 is the cytotoxicity curve of the original drug and nano-prodrug to mesenchymal stem cells (MSCs); Figure 1.2 is the cytotoxicity curve of the original drug and nano-prodrug to neural stem cells (NSCs),

Neither doxorubicin nor nanoprodrugs showed obvious toxic effects on MSCs and NSCs.

Figure 2 shows the change of the average fluorescence intensity of MSCs and NSCs to RGD-PPCD uptake with incubation time.

Figure 3.1 shows the variation of the average fluorescence intensity of MSCs uptake of drugs and nano-prodrugs with concentration; Figure 3.2 shows the variation of the average fluorescence intensity of NSCs uptake of drugs and nano-prodrugs with concentration.

Fig. 4 is a schematic structural diagram of the prepared Stemcells-(RGD-PPCD) _n tumor targeting system.

Fig. 5 is the bioequivalence curve of the toxicity of RGD-PPCD and MSCs—(RGD-PPCD) _n —CM to C6 cells, and the two show an excellent correlation.

Fig. 6 is the in vitro release curve of MSCs—(RGD-PPCD) _n .

Figure 7 shows the stability results of MSCs—(RGD-PPCD) _n observed at different time points by confocal laser microscopy,

After 120h, that is, after 5d, the drug can still exist stably in the form of nano-prodrug in stem cells.

Figure 8 is the penetration result of Stemcells—(RGD-PPCD) _n tumor targeting system to tumor spheres,

The infiltration ability of stem cells loaded with doxorubicin was significantly different from that of non-drug-loaded stem cells, and there was no significant difference in the infiltration ability of stem cells loaded with nano-prodrug and non-drug-loaded stem cells to tumor spheres.

Fig. 9 is the result of the investigation of B16 cytotoxicity in vitro.

Figure 10 is the survival curve of tumor-bearing mice.

Figure 11 shows the neurobehavioral evaluation results of tumor-bearing mice.

detailed description

The present invention will be further described below in conjunction with specific examples, but the content of the present invention will not be limited.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: Toxicity investigation of the original drug and nano-prodrug on MSCs:

MSCs cells in the logarithmic growth phase ^were inoculated into 96-well plate at 104 cells/well, 100 μL per well, cultured at 37°C for 24 h, and then different concentrations of DOX, PPCD and RGD-PPCD (DOXequiv.) were added respectively, and continued After culturing for 24 hours, add 0.2 mL of serum-free culture solution containing 0.5 mg/mL MTT to each well, continue to incubate for 2 hours, absorb the culture solution, add 0.1 mL of DMSO, and measure the absorbance value (λ=570nm) with a microplate reader after the solution is uniform. Inhibition of cell viability was calculated.

Embodiment 2: the investigation of the time frame of MSCs toxicity by former drug and nano-prodrug:

With the test method of Example 1, the drug concentrations of DOX and PPCD/RGD-PPCD were fixed, and the incubation time was changed to 36h, 48h, and 60h to obtain the inhibition of MSCs cell viability by drugs at different incubation times.

Embodiment 3: the investigation of MSCs—(RGD-PPCD) _n optimal incubation time:

MSCs cells in the logarithmic growth phase were taken and co-incubated with DOX, PPCD and RGD-PPCD in culture medium, the drug concentration was 50 μg/mL (DOXequiv.), and incubated in a 37°C incubator for 0h, 1h, 2h, 4h , 8h, 24h, 48h, washed and digested with PBS (pH7.4) at 4°C, and measured the average fluorescence intensity with a flow cytometer (Ex488nm; Em550nm).

Embodiment 4: the investigation of MSCs—(RGD-PPCD) _n optimal incubation concentration:

MSCs cells in the logarithmic growth phase were taken, incubated with different concentrations of DOX, PPCD and RGD-PPCD in the culture medium, incubated in a 37°C incubator for 12h, washed and digested with 4°C PBS (pH7.4), The average fluorescence intensity was measured with a flow cytometer (Ex488nm; Em550nm).

Example 5: Preparation of MSCs—(RGD-PPCD) _n :

Take 10 ⁶ to 10 ⁷ MSCs in the logarithmic growth phase, and incubate with DOX, PPCD and RGD-PPCD in the culture medium for 12 hours, the drug concentration is 50 μg/mL, the incubation temperature is 37°C, and the incubation is completed. After washing with PBS for three times, MSCs—(DOX) _n , MSCs—(PPCD) _n , and MSCs—(RGD-PPCD) _n prodrug systems were obtained after digestion and centrifugation.

Embodiment 6: the determination of MSCs—(RGD-PPCD) _n drug loading:

Take the prepared MSCs—(DOX) _n , MSCs—(PPCD) _n , MSCs—(RGD-PPCD) _n , add 5 mL of deionized water, and ultrasonically pulverize to prepare a homogenate (working power 200w, ultrasonic time 2s, interval 1 s, ultrasonication 30 times), take 0.2 mL, add 5N HCl 0.05 mL, and incubate in a 50°C water bath for 1.5 h. After cooling to room temperature, 0.02 mL internal standard, 0.05 mL 0.5MPBS7.4, and 0.05 mL 5NNaOH were added. Add 2 mL of chloroform-methanol (4/1, v/v), vortex extract for 1 min, 10000 rpm, and centrifuge for 5 min to get 1.4 mL of the lower layer liquid, and dry it with nitrogen at 37 °C. The residue was reconstituted by vortexing with 0.2 mL mobile phase for 1 min, centrifuged at 10,000 rpm for 5 min, and 20 μL of the supernatant was injected.

Example 7: Research on the in vitro release of MSCs—(RGD-PPCD) _n :

C6 cells in the logarithmic growth phase were inoculated in 96-well plates at 5000 cells/well, 100 μL per well, and incubated with DOX, PPCD, and RGD-PPCD at different concentrations (0.078-10 μg/mL) for 72 h, and then each Add 0.2mL serum-free culture medium containing 0.5mg/mL MTT to the well, continue to incubate for 2h, then absorb the culture medium, add 0.1mL DMSO, measure the absorbance value (λ=570nm) with a microplate reader after the dissolution is uniform, and draw the cell survival rate curve , and the concentration at which cell viability was inhibited by 50% (IC ₅₀ ) was calculated.

The prepared MSCs—(DOX) _n , MSCs—(PPCD) _n , MSCs—(RGD-PPCD) _n were cultured (37°C, 5% CO ₂ ), and the conditioned medium (CM) was collected after 48 hours. Perform equal dilution (1:128-1:1), add to 96-well plate, 100 μL per well; take C6 cells in logarithmic growth phase, inoculate 5000 cells/well in 96-well plate, 100 μL per well, after 72 hours The absorbance of each well was measured by the same method as above, the cell viability curve was drawn, and the concentration at which the cell viability was inhibited by 50% (IC ₅₀ ) was calculated.

The CM of 12h, 24h, 48h, 72h and 144h were collected respectively, and the conversion and determination of the released drug content were carried out according to the following formula,

$\mathrm{<span\; class="notranslate">\; DOX</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; Og</span>}<span\; class="notranslate">\; /</span>\mathrm{<span\; class="notranslate">\; mL</span>}<span\; class="notranslate">\; )</span><span\; class="notranslate">\; =</span>\frac{\mathrm{<span\; class="notranslate">\; IC</span>}\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; DOX</span>}<span\; class="notranslate">\; )</span>}{\mathrm{<span\; class="notranslate">\; IC</span>}\mathrm{<span\; class="notranslate">\; 50</span>}<span\; class="notranslate">\; (</span>\mathrm{<span\; class="notranslate">\; dilutionfactors</span>}<span\; class="notranslate">\; )</span>}$

Among them, IC ₅₀ (DOX) is the IC ₅₀ value determined by the original drug, and IC ₅₀ (dilutionfactors) is the IC ₅₀ value of the dilution factor of CM.

Embodiment 8: The stability investigation of MSCs—(RGD-PPCD) _n :

Take prepared MSCs—(DOX) _n , MSCs—(PPCD) _n , MSCs—(RGD-PPCD) _n , add serum-free medium containing 1 μM LysotrackerGreenDND-26, incubate for 30 min, and then add DAPI (final concentration 1 μg/ mL), and continue to incubate for 60 min. Cells were washed three times with PBS (pH7.4), and then observed by confocal. After observation, replace the fresh stem cell culture medium, continue to culture at 37°C, repeat the above process at 12h, 48h, 72h, and 120h respectively, and observe the retention and distribution of DOX and nano-prodrugs in MSCs in different phases.

Example 9: In vitro investigation of the effect of MSCs—(RGD-PPCD) _n on tumor penetration:

48-well plate for 3D tumorsphere culture. Prepare a serum-free culture solution containing 2% agarose in a water bath at 80°C. After autoclaving, add 150 μL to each well of a 48-well plate, and after 30 minutes of ultraviolet light, add 400 μL of cell suspension to each well, including C61000, MEF500, HUVEC500 , Shake by hand for 5 minutes to make it completely dispersed. Co-incubated at 37°C, 5% CO ₂ environment for 5 days, and grew to form tumor spheres.

Take the prepared MSCs—(DOX) _n , MSCs—(PPCD) _n , MSCs—(RGD-PPCD) _n , wash them three times with PBS (pH 7.4), add DAPI (final concentration: 5 μg/mL) and continue to incubate After 2 hours, PBS was washed and digested, and added to the formed tumor spheres, 2000 per well. Co-incubate for 3 h at 37° C. in a 5% CO ₂ environment. After washing with PBS, 200 μL paraformaldehyde was added to each well to fix for 30 min. After washing with PBS, the tumor spheres were transferred to laser confocal culture dishes for confocal observation.

Embodiment 10: Cytotoxicity experiment of MSCs—(RGD-PPCD) _n :

Take the B16 cells in the logarithmic growth phase, inoculate 1000 cells/well in a 96-well plate, 100 μL per well; take the prepared MSCs—(DOX) _n , MSCs—(PPCD) _n , MSCs—(RGD-PPCD) _n , add different quantities to 96-well plates (MSCs/B16: 1:1000～1:1), 100 μL per well; after culturing at 37°C for 6 days, absorb the drug-containing culture medium, and then add 0.2 mL containing 0.5 mg per well /mL MTT serum-free culture solution, continue to incubate for 2 hours, then absorb the culture solution, add 0.1mL DMSO, measure the absorbance value (λ=570nm) with a microplate reader after the dissolution is uniform, and calculate the concentration when the cell viability is inhibited by 50% (IC ₅₀ ).

Embodiment 11: Pharmacodynamic experiment of MSCs—(RGD-PPCD) _n :

Tumor-bearing mice were taken to establish a C6 orthotopic glioma model, and local drug administration was performed on a brain stereotaxic instrument on 5 days, 11 days and 18 days after the tumor was planted. Nanoprodrugs equivalent to DOX-equiv.0.3mg/kg were injected, MSCs-(DOX) _n , MSCs-(PPCD) _n and MSCs-(RGD-PPCD) _n . The survival status and neurobehavior of the mice were observed every day, and survival curves were drawn to evaluate neurobehavioral indicators. Table 1 is the neurobehavioral scoring criteria.

Table 1

Example 12: Preparation and investigation of NSCs—(RGD-PPCD) _n :

The test method was the same as in Examples 1-11, with fixed test conditions and reagents, changing MSCs to NSCs, and preparing and investigating the NSCs—(RGD-PPCD) _n system.

The above experimental results show that: in vitro experimental results show that the drug-loaded stem cells can maintain their ability to divide and proliferate and tumor migration characteristics, and the constructed system is stable, which can ensure that the drug exists in the cells in the form of nano-prodrugs for several days. And the drug can be released slowly; in vivo experiment results show that compared with the original drug and nano-prodrug, the stem cell targeting system constructed can significantly prolong the survival period of tumor-bearing animals, and can maintain the normal neurobehavioral behavior of tumor-bearing animals. Features, to play its efficient, safe and durable anti-tumor efficacy.

Claims (10)

1. A stem cell tumor targeting system loaded with nano-prodrug, characterized in that, the targeting system uses stem cells as a cell carrier to load nano-prodrug, and the loading method is endocytosis, which has the following general formula: Stemcells—(RGD-PPCD) _n Among them, Stemcells are stem cells; RGD-PPCD is a dendrimer-based nanoprodrug of doxorubicin. 2. The nano-prodrug-loaded stem cell tumor targeting system according to claim 1, wherein the stem cells are mesenchymal stem cells or neural stem cells. 3. The stem cell tumor targeting system loaded with nano-prodrugs according to claim 2, wherein said mesenchymal stem cells are human-derived mesenchymal stem cells; said neural stem cells are human-derived neural stem cells. 4. The stem cell tumor targeting system loaded with nano-prodrugs according to claim 3, wherein said human-derived mesenchymal stem cells are derived from human bone marrow, fat, bone parenchyma, synovium, Muscle, lung, liver, pancreas, placenta, amniotic fluid, umbilical cord or cord blood; the human-derived neural stem cells are derived from human brain, spinal cord, bone marrow, fat, umbilical cord blood or umbilical cord tissue. 5. The stem cell tumor targeting system loaded with nano-prodrugs according to claim 3 or 4, wherein said human-derived mesenchymal stem cells are derived from human bone marrow; said human-derived mesenchymal stem cells neural stem cells derived from human brain tissue. 6. The stem cell tumor targeting system loaded with nano-prodrugs according to claim 1, wherein the nano-prodrug of doxorubicin based on dendrimers has the following general formula: RGD-PEG-Dendrimer-DOX Wherein, RGD is a cyclic polypeptide RGDyC, RGDyK, RGDfC or RGDfK containing the RGD sequence, and the structure of the cyclic polypeptide contains free sulfhydryl groups or free amino groups; PEG is a bifunctional polyethylene glycol with a molecular weight of 1000–10000Da, which contains a maleimide group at one end and a hydroxysuccinimide activated ester at the other end; Dendrimer is an integer generation polyamide-amine dendrimer, polypropyleneimine dendrimer or dendritic polylysine; DOX is the compounds CAD-1 and CAD-2 produced by the aminolysis reaction of the sugar amino group of doxorubicin and cis-aconitic anhydride. 7. The preparation method of the stem cell tumor targeting system loaded with nano-prodrugs according to claim 1, characterized in that, the endocytosis method is adopted, which comprises the steps of: co-incubating the stem cells and nano-prodrugs in the stem cell culture medium , the number of stem cells is 10 ⁵ to 10 ⁸ , the incubation time is 1 h to 96 h, the drug concentration is 1 μg/mL to 100 μg/mL, the incubation temperature is 30°C to 45°C, and other incubation conditions include 1% to 10% CO ₂ . 8 . The method according to claim 7 , wherein the number of stem cells is 10 ⁶ to 10 ⁷ , the incubation time is 12 hours, the drug concentration is 50 μg/mL, the incubation temperature is 37° C., and other incubation conditions are: Contains 5% CO ₂ . 9. Use of the stem cell tumor targeting system loaded with nanometer prodrugs according to claim 1 in the preparation of tumor targeting therapy drugs. 10. The use according to claim 9, characterized in that the tumor is glioma, melanoma, breast cancer or ovarian cancer.