Tumor targeted therapy intelligent delivery system and method based on stem cell exosome

Abstract

The invention discloses an intelligent delivery system and method for tumor targeted therapy based on stem cell exosomes. The system comprises engineered exosomes secreted by engineered stem cells, and the engineered stem cells over-express cell-penetrating peptide-nuclear localization signal peptide fusion protein; a chemotherapeutic drug and a prodrug with tumor microenvironment responsiveness are entrapped in the engineered exosome. Genetic engineering modification is performed on stem cells, so that cell-penetrating peptide-nuclear localization signal peptide fusion protein is displayed on an exosome membrane secreted by the stem cells. The fusion protein not only endows the exosome with efficient transmembrane ability, but also can guide the exosome to be further enriched to the nucleus after entering the tumor cell by virtue of the nuclear localization signal (NLS) characteristic of the fusion protein, so that dual active targeting of tissue targeting (tumor) and organelle targeting (nucleus) is realized, and the concentration of a drug at an action site is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a tumor-targeted therapy intelligent delivery system and method based on stem cell exosomes. BACKGROUND

[0002] Malignant tumor is one of the major diseases that seriously threaten human health. Chemotherapy is the main means of current tumor treatment, but traditional chemotherapy drugs have poor targeting, large systemic toxicity, and easy drug resistance. In order to overcome these difficulties, targeted drug delivery system (DDS) has become a research hotspot.

[0003] Exosomes are nanoscale vesicles secreted by cells, which have good biocompatibility, low immunogenicity and the ability to cross biological barriers (such as blood brain barrier), and are ideal drug delivery carriers. In particular, mesenchymal stem cell (MSC) derived exosomes are of great concern due to their natural tumor homing properties. MSCs can be recruited by inflammatory factors and chemotactic factors in the tumor microenvironment, and their derived exosomes to some extent inherit this property of enrichment in tumor sites.

[0004] However, the existing stem cell exosome-based delivery system still has many limitations. First, the tumor targeting efficiency of natural exosomes still needs to be improved, and its homing ability is limited by the state of the donor cells and the type of tumor, sometimes not enough to achieve effective drug concentration at the lesion site. Second, traditional drug loading methods such as electroporation and ultrasonic extrusion can easily damage the membrane structure of exosomes, resulting in low drug loading efficiency and possibly affecting their natural functions. Finally, and most importantly, the existing system mostly lacks the ability to release drugs "intelligently". Drugs are usually released passively after exosomes reach the tumor site, which limits the maximization of therapeutic effect and may cause "off-target" toxicity to normal tissues.

[0005] Therefore, developing an intelligent exosome delivery system with high efficiency targeting, high drug loading capacity and controllable release function is an urgent need to solve the current dilemma of tumor treatment. SUMMARY

[0006] The purpose of the present application is to solve the problems mentioned in the background art, and the present application provides a tumor-targeted therapy intelligent delivery system and method based on stem cell exosomes.

[0007] In order to achieve the above purpose, the present application specifically adopts the following technical solutions: The application discloses a stem cell exosome-based intelligent delivery system for tumor targeted therapy, which comprises: engineered exosomes secreted by engineered stem cells, wherein the engineered stem cells overexpress a transmembrane peptide-nuclear localization signal peptide fusion protein; and the engineered exosomes contain a chemotherapeutic drug and a prodrug with tumor microenvironment responsiveness.

[0008] Further, the transmembrane peptide is a TAT peptide, and the nuclear localization signal peptide is an SV40 large T antigen NLS peptide.

[0009] Further, the chemotherapeutic drug is selected from one or more of doxorubicin, paclitaxel or cisplatin.

[0010] Further, the prodrug with tumor microenvironment responsiveness is a compound in which a chemotherapeutic drug is connected with a quenching group or a shielding group through a matrix metalloproteinase-cleavable peptide chain.

[0011] The application further discloses a stem cell exosome-based intelligent delivery method for tumor targeted therapy, which comprises the following steps: S1: constructing a gene vector encoding a transmembrane peptide-nuclear localization signal peptide fusion protein, transfecting the gene vector into stem cells, and screening to obtain engineered stem cells stably expressing the fusion protein; S2: culturing the engineered stem cells in a culture medium containing a chemotherapeutic drug and a prodrug with tumor microenvironment responsiveness; S3: collecting cell supernatant after culture, separating and purifying the cell supernatant to obtain engineered exosomes containing the chemotherapeutic drug and the prodrug.

[0012] Further, in step S2, the chemotherapeutic drug and the prodrug are simultaneously or sequentially added into the culture medium.

[0013] Further, in step S3, the separation and purification are performed by using a differential ultracentrifugation method.

[0014] Further, the stem cell exosome-based intelligent delivery system and method for tumor targeted therapy are applied to the preparation of a drug for treating tumors.

[0015] A pharmaceutical composition comprises the intelligent delivery system and a pharmaceutically acceptable carrier.

[0016] Further, the tumor is breast cancer, liver cancer or brain glioma.

[0017] The beneficial effects of the present application are as follows: double active targeting, improving tumor enrichment efficiency: the present application genetically engineers stem cells to secrete exosomes with a fusion protein of a cell-penetrating peptide-nuclear localization signal peptide on the exosome membrane. The fusion protein not only gives the exosome high penetration ability, but also its nuclear localization signal (NLS) characteristics can guide the exosome to further enrich in the nucleus after entering the tumor cell, realizing double active targeting of "tissue targeting (tumor)" and "organelle targeting (nucleus)", greatly improving the concentration of drugs at the action site. "Nuclear drug loading" mechanism, efficient and safe drug loading: traditional methods directly load drugs on exosomes, which can easily damage exosomes. The present application innovatively uses the "nuclear import" mechanism of the engineered stem cells. The fusion protein actively and efficiently transports the chemotherapeutic drugs in the culture medium into the nucleus and enriches them. When the exosomes biogenesis in the intracellular membrane system (such as multivesicular bodies), these nuclear-enriched drugs are naturally wrapped into the exosomes. This "biological drug loading" method based on the physiological process of the cells has high drug loading efficiency and maximizes the integrity and function of the exosomes. Intelligent responsive drug release, reducing systemic toxicity: the system is loaded with a prodrug that is responsive to the tumor microenvironment (TME). The prodrug is in an inactive state during circulation, which can effectively reduce the toxicity to normal tissues. When the delivery system is enriched in tumor tissue, the high expression of MMP-2 / 9 enzymes in the tumor microenvironment can specifically cleave the connecting peptide in the prodrug, allowing it to rapidly release active chemotherapeutic drugs at the lesion site. This "intelligent" drug release mode realizes the precise activation of drugs, significantly improves the therapeutic window, and reduces the toxic side effects. Synergistic therapeutic effect: the system delivers conventional chemotherapeutic drugs and prodrugs that can be activated by TME, which can produce a synergistic effect. The conventional drug provides rapid killing, while the prodrug is continuously activated in the TME, which can prolong the action time and overcome part of the drug resistance problem, thereby achieving more complete and persistent killing effect on tumors. DETAILED DESCRIPTION

[0018] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below.

[0019] The present application provides a tumor-targeting therapy intelligent delivery system and method based on stem cell exosomes, comprising: engineered exosomes secreted by engineered stem cells, the engineered stem cells overexpressing a fusion protein of a cell-penetrating peptide-nuclear localization signal peptide; the engineered exosomes containing a chemotherapeutic drug and a prodrug responsive to a tumor microenvironment.

[0020] Further, the cell-penetrating peptide is a TAT peptide, and the nuclear localization signal peptide is an SV40 large T antigen NLS peptide.

[0021] Further, the chemotherapy drug is selected from one or more of doxorubicin, paclitaxel or cisplatin.

[0022] Further, the prodrug with tumor microenvironment responsiveness is a compound in which a chemotherapy drug is connected with a quenching group or a shielding group by a peptide chain cleavable by matrix metalloproteinase.

[0023] The tumor-targeted therapy intelligent delivery method based on stem cell exosomes comprises the following steps: S1: constructing a gene vector encoding a transmembrane peptide-nuclear localization signal peptide fusion protein, transfecting the gene vector into stem cells, and screening to obtain engineered stem cells stably expressing the fusion protein; S2: culturing the engineered stem cells in a culture medium containing a chemotherapy drug and a prodrug with tumor microenvironment responsiveness; S3: collecting the supernatant of the cultured cells, isolating and purifying the supernatant to obtain engineered exosomes loaded with the chemotherapy drug and the prodrug.

[0024] Further, in step S2, the chemotherapy drug and the prodrug are added to the culture medium simultaneously or sequentially.

[0025] Further, in step S3, the isolation and purification is performed by differential ultracentrifugation.

[0026] Further, the tumor-targeted therapy intelligent delivery system and method based on stem cell exosomes are used in the preparation of a drug for treating tumors.

[0027] A pharmaceutical composition comprising the intelligent delivery system and a pharmaceutically acceptable carrier.

[0028] Further, the tumor is breast cancer, liver cancer or brain glioma.

[0029] The beneficial effects of the present application are as follows: double active targeting, improving tumor enrichment efficiency: the present application genetically engineers stem cells to secrete exosome membranes displaying a transmembrane peptide-nuclear localization signal peptide fusion protein. The fusion protein not only imparts exosomes with efficient transmembrane ability, but its nuclear localization signal (NLS) characteristics can also guide exosomes to further enrich in the nucleus after entering tumor cells, achieving double active targeting of "tissue targeting (tumor)" and "organelle targeting (nucleus)", greatly improving the concentration of drugs at the action site. "Nuclear drug loading" mechanism, efficient and safe drug loading: traditional methods directly load drugs on exosomes, which can easily damage exosomes. The present application innovatively uses the "nuclear import" mechanism of engineered stem cells. The fusion protein actively and efficiently transports chemotherapeutic drugs in the culture medium into the nucleus and enriches them. When exosomes biogenesis in the intracellular membrane system (such as multivesicular bodies), these nuclear-enriched drugs are naturally encapsulated into exosomes. This "biological drug loading" method based on the physiological process of cells has high drug loading efficiency and maximizes the integrity and function of exosomes. Intelligent responsive drug release, reducing systemic toxicity: the system encapsulates a prodrug with tumor microenvironment (TME) responsiveness. The prodrug is in an inactive state during circulation, which can effectively reduce the toxicity to normal tissues. When the delivery system is enriched in tumor tissue, the high expression of MMP-2 / 9 enzymes in the tumor microenvironment can specifically cleave the connecting peptide in the prodrug, allowing it to rapidly release active chemotherapeutic drugs at the lesion site. This "intelligent" drug release mode achieves precise activation of drugs, significantly improves the therapeutic window, and reduces toxic side effects. Synergistic therapeutic effect: the system delivers conventional chemotherapeutic drugs and TME-activatable prodrugs together, which can produce a synergistic effect. Conventional drugs provide rapid killing, while prodrugs are continuously activated in the TME, which can extend the action time and overcome some drug resistance problems, thereby achieving more complete and persistent killing effect on tumors.

[0030] Example 1: Preparation of the intelligent delivery system Construction of engineered stem cells: Synthesize the DNA sequence encoding the TAT-SV40 NLS fusion protein and clone it into a lentiviral expression vector. After preparing lentiviral particles, infect human mesenchymal stem cells (hMSCs). Through puromycin screening and Western Blot verification, obtain engineered hMSCs stably overexpressing TAT-NLS fusion protein.

[0031] Preparation of drug-loaded exosomes: engineered hMSCs were seeded in culture flasks, and when the cell density reached 80%, the complete medium containing 100 nM doxorubicin (Dox) and 50 μM MMP-9-responsive doxorubicin prodrug (Dox-MMP-substrate) was replaced, and the culture was continued for 24 hours. The prodrug Dox-MMP-substrate is a peptide chain (GPLGVRG) that can be cleaved by MMP-9 to connect Dox with a fluorescence quenching group.

[0032] Isolation and purification of exosomes: The cell supernatant was collected, and the cells and debris were removed by centrifugation at 300 g, 2000 g, and 10000 g, respectively. Finally, the engineered exosomes loaded with Dox and Dox-MMP-substrate (denoted as TNL-Exo@Dox / ProDox) were obtained by ultracentrifugation at 110,000 g for 70 minutes. The obtained exosomes were identified by transmission electron microscopy and nanoparticle tracking analysis (NTA) and were in a typical cup-shaped morphology with a particle size mainly concentrated around 100 nm.

[0033] Example 2: In vitro tumor cell killing experiment To verify the targeting and therapeutic effect of the delivery system of the present application, in vitro experiments were performed.

[0034] Experimental grouping: Four groups were set up: ① PBS control group; ② free Dox group; ③ native exosome-loaded Dox group (Native-Exo@Dox); ④ TNL-Exo@Dox / ProDox group of the present application.

[0035] Experimental method: Human breast cancer MDA-MB-231 cells with high expression of MMP-9 were selected. The cells were seeded in 96-well plates and treated with the above four groups of drugs, respectively. After 48 hours, the cell survival rate was detected by CCK-8 method.

[0036] Results: Compared with the PBS group, the free Dox group showed certain cytotoxicity. The inhibitory effect of the Native-Exo@Dox group was better than that of the free Dox, which proved the advantage of exosome delivery. The TNL-Exo@Dox / ProDox group of the present application showed the strongest tumor cell inhibition effect, and the cell survival rate was significantly lower than that of the other groups (p<0.01). This indicates that the dual targeting ability of TNL-Exo and the MMP-9-responsive drug release produce a synergistic killing effect.

[0037] Example 3: Evaluation of in vivo anti-tumor effect A MDA-MB-231 breast cancer tumor-bearing nude mouse model was established, and when the tumor volume reached about 100 mm³, the mice were randomly divided into four groups (same as in Example 2), and the corresponding preparations were injected through the tail vein twice a week.

[0038] In vivo imaging: At different time points after drug administration, the distribution of exosomes (labeled with DIR dye) in vivo was observed by a small animal in vivo imaging system. The results showed that the fluorescence signal intensity of the TNL-Exo group at the tumor site was much higher than that of the Native-Exo group, and reached a peak at 24 hours, proving its excellent tumor targeting ability.

[0039] Therapeutic effect: The tumor volume and mouse weight were measured regularly during the treatment. The results showed that the tumor growth inhibition rate of the TNL-Exo@Dox / ProDox group was the highest, and the tumors of some mice completely regressed. At the same time, the body weight of the mice in this group remained stable, and no obvious toxic reaction occurred. While the free Dox group had a certain tumor inhibition effect, the body weight of the mice decreased obviously, showing systemic toxicity.

[0040] Conclusion: The intelligent delivery system of the present application shows high efficiency and safety in the treatment of tumors in animals, which is significantly better than traditional chemotherapy and ordinary exosome delivery systems.

[0041] The above description of disclosed embodiments enables those skilled in the art to carry out or use the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tumor-targeted therapy intelligent delivery system based on stem cell exosomes, characterized in that, include: Engineered exosomes secreted by engineered stem cells, wherein the engineered stem cells overexpress a membrane-penetrating peptide-nuclear localization signal peptide fusion protein; The engineered exosomes contain chemotherapy drugs and prodrugs that are responsive to the tumor microenvironment.

2. The intelligent delivery system for tumor-targeted therapy based on stem cell exosomes according to claim 1, characterized in that, The membrane-penetrating peptide is a TAT peptide, and the nuclear localization signal peptide is the SV40 large T antigen NLS peptide.

3. The intelligent delivery system for tumor-targeted therapy based on stem cell exosomes according to claim 1, characterized in that, The chemotherapy drug is selected from one or more of doxorubicin, paclitaxel, or cisplatin.

4. The intelligent delivery system for tumor-targeted therapy based on stem cell exosomes according to claim 1, characterized in that, The prodrug with tumor microenvironment responsiveness is a compound formed by linking a chemotherapeutic drug with a quenching group or a shielding group via a matrix metalloproteinase-cleavable peptide chain.

5. A smart delivery method for tumor-targeted therapy based on stem cell exosomes, characterized in that, Includes the following steps: S1: Construct a gene vector encoding a membrane-penetrating peptide-nuclear localization signal peptide fusion protein, transfect it into stem cells, and screen to obtain engineered stem cells that express the protein stably; S2: Culture the engineered stem cells in a culture medium containing chemotherapeutic drugs and prodrugs that are responsive to the tumor microenvironment; S3: Collect the cell supernatant after culture, separate and purify it to obtain engineered exosomes loaded with chemotherapy drugs and prodrugs.

6. The intelligent delivery method for tumor-targeted therapy based on stem cell exosomes according to claim 5, characterized in that, In step S2, the chemotherapy drug and the prodrug are added to the culture medium simultaneously or sequentially.

7. The intelligent delivery method for tumor-targeted therapy based on stem cell exosomes according to claim 5, characterized in that, In step S3, the separation and purification are performed using differential ultracentrifugation.

8. A pharmaceutical composition, characterized in that, It includes the intelligent delivery system as described in any one of claims 1-4 and a pharmaceutically acceptable carrier.

9. The use of the intelligent delivery system as described in any one of claims 1-4 or the pharmaceutical composition as described in claim 8 in the preparation of a medicament for treating tumors.

10. The application according to claim 9, characterized in that, The tumor is breast cancer, liver cancer, or glioma.