Engineered taxus chinensis exosome as well as preparation method and application thereof

Abstract

The invention discloses an engineered taxus chinensis exosome as well as a preparation method and application thereof, and relates to the technical field of biological medicines. According to the invention, firstly, the taxus chinensis-derived medicinal plant exosome is successfully separated, and secondly, the taxus chinensis exosome is further subjected to engineering modification, including loading of a chemotherapeutic drug CDDP and increasing of RGP-KDEL peptide modification. Preferably, the engineered taxus chinensis exosome subjected to targeted modification is loaded into the microneedle and is used for local precise drug delivery, the systemic toxicity of the drug is reduced to the maximum extent, multi-level targeted drug delivery is achieved, and then the anti-cancer curative effect is enhanced. The engineered taxus chinensis exosome constructed by the invention has the advantages of simple preparation method, obvious anti-tumor growth effect, obvious anti-tumor targeting property, good biocompatibility, high delivery efficiency and the like, can be used as a novel nano-drug for tumor treatment, and provides a new strategy for efficient transdermal drug delivery.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a preparation method and application of an engineered Taxus exosome microneedle transdermal patch (RGP-KDEL-TC@CDDP) for targeted treatment of breast cancer. Background Art

[0002] Triple-negative breast cancer (TNBC) is a highly aggressive subtype of breast cancer that lacks estrogen receptors, progesterone receptors, and human epidermal growth factor receptors. Due to the lack of targeted treatment options, combined with the significant toxicity of existing chemical drugs and the development of chemotherapy resistance, the clinical treatment of TNBC is particularly challenging. Cisplatin (CDDP), as a broad-spectrum anti-tumor chemotherapy drug, causes DNA damage in cancer cells by cross-linking with DNA and is also commonly used in the treatment of breast cancer as an endoplasmic reticulum-targeted drug. However, its anti-cancer effect is limited, and there are bottlenecks such as low solubility and large toxic side effects, so a single drug cannot achieve the expected anti-tumor effect. Currently, the combination of chemotherapy drugs and nano-delivery materials has been widely regarded as a potential alternative therapy to traditional single chemotherapy. Although the reported nano-materials have been proven to be effective in anti-tumor, they always face obvious obstacles such as low biocompatibility, non-degradability in vivo, and limited drug delivery efficiency. Therefore, the development of a new generation of nano-delivery materials is an urgent problem to be solved.

[0003] Taxus plants, as a medicinal plant, are widely used in the treatment of cancer. Medicinal components in Taxus such as paclitaxel have been proven to have good clinical anti-cancer treatment effects. In addition, studies have shown that exosomes derived from plants have advantages such as high biocompatibility, strong stability, and improved drug bioavailability, and can be used as a new type of safe drug carrier and an effective carrier for loading CDDP chemical drugs. However, due to its low targeting efficiency to animal cells and the limitation of cell uptake by its negative surface charge, the anti-cancer efficacy is greatly restricted. In response to this, engineered exosomes constructed by targeting, due to their characteristics such as precise targeting, high efficiency, and wide applicability, show absolute advantages in targeted tumor treatment and are also the focus of current research on precision targeted anti-tumor drugs. However, currently, the delivery methods of targeted anti-breast cancer drugs in clinical practice are mainly oral and intravenous injection. Although targeted drugs can directly enter the bloodstream to enhance the bioavailability of drugs, the toxicity and side effects caused by the absorption and accumulation of anti-cancer components in normal organs and blood greatly limit their application.

[0004] Transdermal drug delivery refers to the absorption of drugs through the skin into the blood circulation to exert systemic or local therapeutic effects. Compared with traditional drug delivery methods (such as oral administration and injection), transdermal drug delivery has the characteristics of avoiding the first-pass effect, reducing side effects, improving compliance, and long-acting sustained release, showing unique advantages in the treatment of breast cancer. As an emerging transdermal drug delivery system, microneedles can form micron-sized pores in the epidermis and dermis of the skin by penetrating the stratum corneum of the skin without damaging the nerves and blood vessels in the dermis. Anticancer drugs enter the skin through the pores, which can greatly improve the transdermal penetration efficiency and bioavailability of drugs, attracting extensive attention to targeted anti-breast cancer drug delivery and treatment. Summary of the Invention

[0005] In view of the deficiencies of the prior art, the present invention provides an engineered Taxus exosome microneedle transdermal patch, its preparation method and application.

[0006] First, the present invention successfully prepares and isolates exosomes derived from the medicinal plant Taxus for the first time, and proves that it can be used for tumor treatment by delivering functional nucleic acid molecules to regulate related pathways to inhibit the proliferation, migration, invasion and apoptosis of breast cancer cells. Secondly, the engineered Taxus exosomes in the present invention can target tumor cells and further target endoplasmic reticulum organelles at the same time. At the same time, the engineered Taxus exosome microneedle patch in the present invention can be used for local precise drug delivery, minimizing the systemic toxicity of drugs, achieving multi-level targeted drug delivery and sustained release of the contained anti-cancer active ingredients, and then exerting anti-tumor efficacy. Finally, the engineered Taxus exosome microneedle transdermal patch in the present invention has the advantages of simple preparation method, significant tumor-targeting effect, good biocompatibility, high delivery efficiency, etc. The present invention will provide new reference significance and value for the further development of various engineered medicinal plant exosomes and their application in tumor treatment.

[0007] To achieve the above object, the technical solution adopted by the present invention when aiming to solve technical problems is as follows:

[0008] The present invention first provides a preparation method of engineered Taxus exosomes, which includes the following steps:

[0009] 1) Mix the fresh active part of Taxus with phosphate buffer solution and squeeze juice to obtain a slurry containing Taxus exosomes;

[0010] 2) Use a sieve to filter out the residue in the slurry obtained in step 1) and collect the juice, and the juice is centrifuged by differential centrifugation to obtain a precipitate containing Taxus exosomes (TC-Exos);

[0011] 3) Resuspend the precipitate obtained in step 2) with PBS and purify to obtain Taxus exosomes;

[0012] 4) The Taxus exosomes obtained in step 3) were combined with CDDP by ultrasonic-assisted method to obtain drug-loaded Taxus exosomes (TC@CDDP) loaded with CDDP;

[0013] 5) The drug-loaded Taxus exosomes loaded with CDDP obtained in step 4) were functionalized with RGP and KDEL respectively. The amino acid sequence of the RGP is shown in SEQ ID No.1, and the amino acid sequence of KDEL is shown in SEQ ID No.2, to obtain engineered modified Taxus exosomes (RGP-KDEL-TC@CDDP)

[0014] According to the preferred embodiment of the present invention, the differential centrifugation method in step 2) is as follows: The filtered juice was centrifuged at 1000×g for 5 - 20 min, 4000×g - 10000×g for 20 - 60 min, and 10000×g for 60 - 120 min in sequence at 4°C to obtain the supernatant; then the supernatant was centrifuged at 100000×g - 150000×g for 60 - 150 min at 4°C to obtain the precipitate containing Taxus exosomes (TC-Exos).

[0015] According to the preferred embodiment of the present invention, the purification in step 3) is by filtration through a 0.22 μm membrane filter.

[0016] According to the preferred embodiment of the present invention, the ultrasonic-assisted method in step 4) is as follows: After resuspending the Taxus exosomes with PBS, the chemotherapeutic drug CDDP was added at 1 - 2 times the mass of the exosomes, and ultrasonic-assisted treatment was carried out for 5 - 10 cycles. Each cycle included 1 - 10 s of pulse and 2 - 3 s of pause, and after each cycle, ice-cooling treatment was carried out for 1 - 5 min; after ultrasonic treatment, incubation was carried out at 37°C for 30 - 120 min to restore the exosome membrane activity, and then it was centrifuged at 100000×g - 150000×g at 4°C for 60 - 150 min to obtain drug-loaded Taxus exosomes (TC@CDDP) loaded with CDDP chemotherapeutic drug.

[0017] According to the preferred embodiment of the present invention, step 5) is as follows: After resuspending the drug-loaded Taxus exosomes with PBS, RGP was added at 0.1 - 1 times the mass of the exosomes and KDEL was added at 0.01 - 1 times the mass of the exosomes, and shaking was carried out on a shaker at 37°C at 100 - 300 rpm for 30 - 120 min, and then it was added to a 10 - 100KD ultrafiltration tube and centrifuged at 1000 - 5000 rpm for 10 - 30 min to obtain targeted modified engineered Taxus exosomes.

[0018] The present invention also provides the engineered Taxus exosomes prepared by the preparation method described above.

[0019] The present invention further provides a microneedle transdermal patch based on the engineered Taxus exosomes, and its preparation method is as follows: Using the shaker incubation method, the RGP-KDEL-TC@CDDP is carried in the porous microneedles to prepare an engineered Taxus exosomes microneedle transdermal patch (RGP-KDEL-TC@CDDP-MN).

[0020] According to a preferred embodiment of the present invention, the incubation method is: adding 100 - 1000 μg of RGP-KDEL-TC@CDDP onto the porous microneedles (MN), shaking at 100 - 300 rpm under a normal temperature shaker for 10 - 120 min, and then sucking off the excess exosomes to prepare an engineered Taxus exosomes microneedle transdermal patch (RGP-KDEL-TC@CDDP-MN).

[0021] The present invention also provides the application of the engineered Taxus exosomes or the engineered Taxus exosomes microneedle transdermal patch in the preparation of anti-tumor drugs. Further preferably, the tumor is breast cancer.

[0022] The reagents and raw materials used in the present invention are all commercially available.

[0023] The advantages of the present invention are as follows:

[0024] 1) The engineered Taxus exosomes microneedle transdermal patch for targeted treatment of breast cancer provided by the present invention is inexpensive, easily available, safe, low-toxic, and has high biocompatibility, and can overcome the problems of high cost, poor efficacy, easy drug resistance, large toxicity and adverse reactions, and low absorption and delivery efficiency of anti-tumor drugs.

[0025] 2) The active substances in the engineered Taxus exosomes microneedle transdermal patch for targeted treatment of breast cancer provided by the present invention can significantly reduce MAPK in tumor cells and up-regulate the expression of proteins in the TNF pathway and efficiently activate the endoplasmic reticulum stress response, inhibit tumor cell proliferation, migration, invasion and efficiently induce tumor cell apoptosis, thereby exerting the anti-tumor growth and metastasis efficacy.

[0026] 3) The engineered Taxus exosomes microneedle transdermal patch for targeted treatment of breast cancer provided by the present invention can be used as a novel drug delivery means, can efficiently target tumor cells and organelles, while reducing systemic toxicity, can also be efficiently taken up by animal cells and deliver and slowly release the active substances contained therein, cross-regulate the expression of genes and proteins in mammalian cells to exert efficacy, and provides a new strategy for cross-kingdom medical treatment of animals and plants, effectively solving the problems of possible toxicity, large toxic side effects and low bioavailability of the drug itself. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will be further described below with reference to the accompanying drawings:

[0028] Figure 1 Particle size distribution diagrams of (a) TC@Exos; (b) TC@CDDP and (c) RGP-KDEL-TC@CDDP obtained in an embodiment of the present invention;

[0029] Figure 2 Transmission electron microscope (TEM) images (scale bar: 100 nm) of (a) TC@Exos; (b) TC@CDDP and (c) RGP-KDEL-TC@CDDP obtained in an embodiment of the present invention;

[0030] Figure 3 Flow cytometry analysis diagrams of the cellular uptake of TC@Exos, TC@CDDP and RGP-KDEL-TC@CDDP by 4T1 at different time points in an embodiment of the present invention;

[0031] Figure 4 Confocal laser scanning microscopy (CLSM) analysis diagrams of the co-localization of 4T1 with TC@Exos, TC@CDDP and RGP-KDEL-TC@CDDP in the endoplasmic reticulum (scale bar: 100 μm) in an embodiment of the present invention;

[0032] Figure 5 Effects of different concentrations of TC@Exos, TC@CDDP and RGP-KDEL-TC@CDDP on the proliferation of 4T1 cells in an embodiment of the present invention;

[0033] Figure 6 Effects of TC@Exos, TC@CDDP and RGP-KDEL-TC@CDDP on the migration level of 4T1 cells (scale bar: 400 μm) in an embodiment of the present invention;

[0034] Figure 7 Effects of TC@Exos, TC@CDDP and RGP-KDEL-TC@CDDP on the invasion level of 4T1 cells (scale bar: 100 μm) in an embodiment of the present invention;

[0035] Figure 8 Effects of TC@Exos, TC@CDDP and RGP-KDEL-TC@CDDP on the apoptosis level of 4T1 cells in an embodiment of the present invention;

[0036] Figure 9 Effects of TC@Exos, TC@CDDP and RGP-KDEL-TC@CDDP on the ROS level of 4T1 cells in an embodiment of the present invention;

[0037] Figure 10 Western blot expression analysis diagrams of the effects of TC@Exos on the MAPK and TNF pathway proteins in 4T1 cells in an embodiment of the present invention;

[0038] Figure 11 Efficacy diagrams of TC@Exos-MN, TC@CDDP-MN, and RGP-KDEL-TC@CDDP-MN in anti-tumor and anti-angiogenesis in vivo in one embodiment of the present invention (scale bar: 100 μm);

[0039] Figure 12 Anti-tumor mechanism diagram of TC@Exos-MN, TC@CDDP-MN, and RGP-KDEL-TC@CDDP-MN in vivo in one embodiment of the present invention;

[0040] Figure 13 Biosafety diagrams of TC@Exos-MN, TC@CDDP-MN, and RGP-KDEL-TC@CDDP-MN in vivo in one embodiment of the present invention (scale bar: 100 μm). Detailed implementation manners

[0041] The present invention will be further described below by way of examples.

[0042] The mouse breast cancer cells (4T1) used in this example were cultured in a complete medium containing 10% fetal bovine serum, penicillin (50 U / mL), and streptomycin (50 U / mL), and placed in an environment of 37 °C and 5% CO2.

[0043] Example 1 Extraction of Taxus exosomes by ultracentrifugation and purification using a membrane filter

[0044] Take fresh Taxus branches and leaves, wash them with deionized water, and extract the juice with PBS buffer in a juicer. After filtering the residue with a filter screen, the juice was centrifuged successively at 4 °C and 1000×g for 10 min, 4000×g for 20 min, and 10000×g for 60 min using a tabletop refrigerated centrifuge to remove large particles and cell debris. Then the supernatant was centrifuged at 4 °C and 150000×g for 120 min using a floor ultracentrifuge, and the bottom precipitate was resuspended with an appropriate amount of PBS and filtered through a 0.22 μm membrane filter to obtain Taxus exosomes (TC-Exos). The particle size and potential of the exosomes were measured using a Malvern particle size analyzer, the morphology of the exosomes was photographed using a transmission electron microscope, and the protein concentration of the exosomes was quantified using a BCA kit. As Figure 1 shown, the particle size of the obtained Taxus exosomes was 152.63 nm. As Figure 2 shown, Taxus exosomes presented a cup-shaped structure under the transmission electron microscope, which conforms to the classical structural characteristics of exosomes.

[0045] Example 2 Preparation of engineered modified Taxus exosomes by ultrasonic-assisted method and incubation method

[0046] Taxol exosomes and the chemotherapeutic drug CDDP were added at a mass ratio of 1:1 (W:W). The ultrasonic treatment cycle was set to 6 cycles, each cycle consisting of a 4 s pulse and a 2 s pause, and after each cycle, it was cooled on ice for 2 min. After the ultrasonic treatment was completed, it was incubated at 37 °C for 60 min to restore the exosome membrane activity. Subsequently, it was centrifuged at 4 °C at 150,000×g for 90 min to obtain drug-loaded Taxol exosomes (TC@CDDP) of the CDDP chemotherapeutic drug. Subsequently, TC@CDDP was added at 1-fold the mass of the exosomes to RGP (RWrNMGGGGIVRRADRAAVP) and at 0.05-fold the mass of the exosomes to KDEL (Lys-Asp-Glu-Leu) respectively, and shaken at 100 rpm on a shaker at 37 °C for 60 min. Subsequently, it was added to a 100 KD ultrafiltration tube and centrifuged at 3000 rpm for 15 min to obtain a precipitate of engineered modified Taxol exosomes loaded with CDDP (RGP-KDEL-TC@CDDP). The bottom precipitate was resuspended with a small amount of PBS, and the particle size and potential of the exosomes were measured using a Malvern particle size analyzer, and the morphology of the exosomes was photographed using a transmission electron microscope. As Figure 1 shown, the particle sizes of the obtained TC@CDDP and RGP-KDEL-TC@CDDP were 163.50 and 181.07 nm respectively. As Figure 2 shown, TC@CDDP and RGP-KDEL-TC@CDDP presented a cup-and-saucer structure under the transmission electron microscope. After being engineered modified, there was no significant change in the structure of Taxol exosomes, but compared with unmodified Taxol exosomes, the exosome membrane surface of TC@CDDP and RGP-KDEL-TC@CDDP was rough, and there was a more obvious phenomenon of membrane surface thickening and depression in RGP-KDEL-TC@CDDP.

[0047] Example 3 Characterization and Qualitative and Quantitative Analysis of Inclusions in Taxol Exosomes

[0048] Isolation and analysis of nucleic acids contained in Taxol exosomes: Total RNA of Taxol exosomes was isolated and purified using Trizol reagent, and nucleic acids in TC-Exos, including mRNA and small RNAs (miRNA, IncRNA, circle RNA), were isolated and analyzed through procedures of lysis, labeling, amplification, paired-end sequencing, and sequence mapping.

[0049] Isolation and analysis of lipids contained in Taxol exosomes: Lipids were extracted using the MTBE method. Briefly, the sample (100 μL) was spiked with an internal lipid standard, then separated by reversed-phase liquid chromatography, detected for positive and negative ions using electrospray ionization, and finally peak extraction and identification were performed on lipid molecules and internal standard lipid molecules contained in Taxol exosomes. The results showed that TC-Exos contained various lipid small molecules such as triglycerides, diglycerides, phosphatidylethanolamine, and phosphatidylcholine.

[0050] Analysis of the drug loading capacity of Taxus exosomes loaded with CDDP: The drug loading of the loaded CDDP was determined by ultraviolet spectrophotometry. The results showed that the maximum drug loading of Taxus exosomes was 0.25 μg / μg Exos.

[0051] Example 4 Cellular uptake of Taxus exosomes and engineered Taxus exosomes by 4T1 at different time points

[0052] First, mix 25 μL of DMSO with DiO dye to form a stock solution of 1 mg / mL. When in use, dilute the stock solution 100 - 200 times, add it to the exosome solution to prepare a working solution of 5 - 10 μM and incubate for 30 min, then ultracentrifuge at 150,000 g for 1 h to remove free dye and complete exosome labeling. Second, take a 6-well plate, collect 4T1 cells in the logarithmic growth phase, and gently pipette them into a single-cell suspension. Seed the cells into the 6-well plate at a density of 2×10 5 / well. Gently shake to evenly distribute the cells in the wells. Place the culture plate in a cell culture incubator at 37°C with 5% CO2. After overnight attachment, add the labeled exosomes and incubate for 0, 3, 6, 9, 18, and 24 h respectively. Finally, remove the culture medium, wash with PBS, add trypsin for digestion and centrifuge, and analyze the exosome uptake by flow cytometry. As Figure 3 shown, within 24 h, 4T1 cells significantly increased their uptake ability of RGP-KDEL-TC@CDDP, and the uptake rate at 24 h was as high as 97.7%, which was 1.1 times that of Ar-Exos.

[0053] Example 5 Analysis diagram of the co-localization of endoplasmic reticulum of 4T1 with Taxus exosomes and engineered Taxus exosomes by CLSM

[0054] Take a confocal dish, collect 4T1 cells in the logarithmic growth phase, and gently pipette them into a single-cell suspension. Seed the cells into the confocal dish at a density of 1×10 5 / well. Gently shake to evenly distribute the cells in the dish. Place the culture plate in a cell culture incubator at 37°C with 5% CO2. After overnight attachment, discard the original culture medium, add complete culture medium containing 500 μg / mL TC-Exos, TC@CDDP, and RGP-KDEL-TC@CDDP, and set up control wells at the same time, and continue to place them in the incubator for 12 h. Use an ER fluorescence probe detection kit, according to the manufacturer's instructions, with an excitation wavelength of 587 nm and an emission wavelength of 615 nm, and take fluorescence photos before and after treatment. As Figure 4As shown, the fluorescence intensity of RGP-KDEL-TC@CDDP and the endoplasmic reticulum increased significantly, indicating that the engineered Taxus exosomes could significantly enhance the co-localization ability with the endoplasmic reticulum.

[0055] Example 6 Construction and Structural Characterization of RGP-KDEL-TC@CDDP-MN

[0056] Commercially available porous microneedles (MN) can be used. In this example, a 10×10 uniform porous MN array with a height of 600 nm, a base diameter of 380 nm, and a porosity of 40.87% was selected for the preparation of the microneedle transdermal patch of the present invention.

[0057] Using the incubation method, 400 μg of the prepared TC-Exos, TC@CDDP, and RGP-KDEL-TC@CDDP were added onto three groups of porous microneedles (MN) respectively, and shaken at 100 rpm for 30 min under a normal temperature shaker. Subsequently, the excess exosomes were aspirated to prepare the single Taxus exosome microneedle transdermal patch (TC-Exos-MN), the Taxus microneedle transdermal patch loaded with the chemotherapeutic drug CDDP (TC@CDDP), and the engineered Taxus exosome microneedle transdermal patch (RGP-KDEL-TC@CDDP-MN). Subsequently, the morphology of RGP-KDEL-TC@CDDP-MN in the final system was observed under an optical microscope and a SEM scanning electron microscope, and then elemental scanning was carried out to characterize the loading situation of the engineered Taxus exosomes. Finally, the intact skin of the mouse back was used to simulate the release of the overall drug at different time points on a small transdermal instrument for characterization. As Figure 5 shown, compared with the blank microneedle MN, the drug-loaded microneedle of RGP-KDEL-TC@CDDP-MN presented a pale yellow translucent appearance, with a drug loading of 380.95 μg / patch and an encapsulation efficiency of 20.51%; SEM imaging showed that RGP-KDEL-TC@CDDP completely occupied the pores, showing a flaky morphology. In addition, the drug release test further demonstrated that the RGP-KDEL-TC@CDDP-MN microneedle transdermal patch had the efficacy of sustained release of the contained drug.

[0058] Example 7 Survival Rates of Tumor Cell Models Specifically Inhibited by Taxus Exosomes and Engineered Taxus Exosomes

[0059] The CCK-8 kit was used to detect the proliferation activity of cells. 4T1 cells were seeded in 96-well plates at a cell density of 5000 cells / well and incubated overnight in an incubator at 37°C and 5% CO2. After the cells adhered to the wall, the medium was changed, and 500, 800, 1000, 1500, and 2000 μg / mL exosomes and engineered modified exosome solutions diluted with the culture medium were respectively given, and blank wells and control wells were set at the same time. After administration, the cells were continuously incubated in the incubator for 24 h, then 10 μL of CCK-8 solution diluted with fresh serum-free culture medium was changed, and after continuous incubation for 1 h, the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader. As Figure 5 shown, the proliferation rate of 4T1 cells decreased with the increase in the administration concentration of Taxus exosomes and engineered modified Taxus exosomes, indicating that the treatment with Taxus exosomes and engineered modified Taxus exosomes effectively inhibited the growth and survival of tumor cells.

[0060] Example 8 Specific inhibition of migration, invasion and pro-apoptotic ability of engineered modified Taxus exosomes on 4T1

[0061] Take a 24-well plate, and seed 4T1 at a quantity of 1×10 5 / well into the 24-well plate, and the amount of cell suspension added is 1 mL per well. Place the culture plate in a cell incubator at 37°C and 5% CO2 and culture it routinely overnight. The next day, discard the original culture medium, and quickly make a longitudinal scratch in the cell layer with a sterile 200 μL pipette tip to form a scratch with a uniform width, and establish a wound model of cultured cells. Add serum-free culture medium containing 500 μg / mL TC-Exos, TC@CDDP, and RGP-KDEL-TC@CDDP to each well, observe and photograph the width of the wound at 0 h after the scratch under an optical microscope, and take samples and photograph at 6, 12, and 24 h to observe the wound healing situation of each group and measure it with relevant drawing software.

[0062] Take a 24-well plate, add 80 μL of Matrigel to each well and culture it in a cell incubator for 30 min until it solidifies. Seed 4T1 cells at a quantity of 5×10 4 / well into the 24-well plate, and the amount of cell suspension added is 400 μL per well. Set up a blank culture medium group, 500 μg / mL TC-Exos, TC@CDDP, and RGP-KDEL-TC@CDDP groups. After culturing the culture plate in a cell incubator at 37°C and 5% CO2 routinely for 24 h, wash it twice with PBS, fix it with 4% paraformaldehyde for 15 min, stain it with 0.1% crystal violet for 5 min, wash it twice with PBS, observe and photograph the invasion cells of each group under an optical microscope, and analyze it with Image J1.8.0 image processing software.

[0063] Take a 6-well plate, and seed 4T1 cells at a quantity of 1×105 / well were inoculated in a 6-well plate, and 2 mL of cell suspension was added to each well. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for routine culture overnight. The next day, the old culture medium was discarded, 500 μg / mL TC-Exos, TC@CDDP and RGP-KDEL-TC@CDDP were added to the treatment group, and blank culture medium was added to the blank group, and the plates were placed in the incubator for 24 hours. The number of apoptotic cells in different groups was qualitatively and quantitatively analyzed using an apoptosis kit.

[0064] Take a 6-well plate and plate 4T1 cells at a concentration of 1×10 5 The number of cells / well was inoculated in a 6-well plate, and the amount of cell suspension added was 2 mL per well. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for routine culture overnight. The next day, the old culture medium was discarded, 500 μg / mL TC-Exos, TC@CDDP and RGP-KDEL-TC@CDDP were added to the treatment group, and blank culture medium was added to the blank group, and the cells were placed in the incubator for another 24 hours. The cells in each group were treated with a ROS kit, and the ROS fluorescence intensity of different groups was detected under a flow cytometer.

[0065] like Figure 6 , 7 As shown in Figures 8 and 9, in the blank group, the migration rate and cell invasion number of 4T1 cells were higher, and cell apoptosis was lower, while RGP-KDEL-TC@CDDP at a concentration of 500 μg / mL could significantly inhibit the migration, invasion, apoptosis and ROS production of 4T1 cells, exerting an anti-tumor effect.

[0066] Case 9: Taxus exosomes inhibit the expression of MAPK and TNF pathway proteins in tumor cells

[0067] Take a 6-well plate and plate 4T1 cells at a concentration of 2 × 10 5 / well were inoculated into 6-well plates, and 2mL of cell suspension was added to each well. The culture plate was placed in a cell culture incubator at 37°C and 5% CO2 for routine culture overnight. The next day, the original culture medium was discarded, and 500μg / mL TC-Exos culture medium was added to each well of the treatment group, and the cells were cultured in the incubator for another 24h. Western blot was used to perform qualitative and quantitative analysis of cells in different groups, and Image J 1.8.0 image processing software was used to analyze the optical density values ​​of the bands to determine the effect of TC-Exos on the expression of MAPK and TNF pathway proteins in tumor cells. Figure 10 As shown in the figure, TC-Exos significantly downregulated the expression of MAPK and upregulated the expression of TNF pathway proteins, indicating that yew exosomes can exert anti-breast cancer effects by inhibiting MAPK and activating TNF pathway.

[0068] Evaluation of the anti-tumor and anti-angiogenic effects of Taxus exosomes and modified Taxus exosomes on a subcutaneous breast cancer mouse model and analysis of related mechanisms

[0069] The experimental animals were female BALB / c mice aged 6 - 8 weeks. A 0.1 mL suspension of 1×10 7 / mL 4T1 cells was inoculated into the right axilla to establish a subcutaneous transplanted breast cancer model. When the average tumor volume reached 50 - 100 mm 3 , the tumor-bearing mice were randomly divided into 4 experimental groups, with 6 mice in each group. PBS, 2.5 mg / kg CDDP-IV, CDDP-MN (2.5 mg / kg CDDP), 10 mg / kg TC-Exos-MN, TC@CDDP-MN (2.5 mg / kg CDDP), and RGP-KDEL-TC@CDDP-MN (2.5 mg / kg CDDP) solutions were administered every 2 days. The body weight and tumor volume of the mice were monitored every other day. On the 14th day, the mice were sacrificed, and the tumors of each group were collected for qualitative and quantitative analysis. Western blot assays were used to determine the expression of proteins related to the MAPK, TNF pathways, apoptotic proteins, and metastasis proteins. Immunofluorescence staining was used to analyze the expression of angiogenesis-related proteins VEGF and CD31. At the same time, an Elisa kit was used to detect and verify the expression of serum factor-related indicators related to immunity. ImageJ 1.8.0 was used for image processing. As Figure 11 、 12 shown, the tumors in the control group increased rapidly within 12 days, while the exosome group significantly delayed the tumor growth trend, reduced the expression of Ki67, VEGF, and CD31, and the effect of RGP-KDEL-TC@CDDP-MN was the best. At the same time, RGP-KDEL-TC@CDDP-MN could significantly inhibit the expression of MAPK pathway proteins and promote the expression of TNF pathway proteins, inhibit the expression of tumor metastasis proteins MMP2 and MMP9, enhance the expression of apoptotic proteins Cleaved Capase3, Cleaved Capase 9, and endoplasmic reticulum stress mediator Capase12 proteins, effectively activate the intracellular ROS response, and then induce the intracellular endoplasmic reticulum stress response, regulate immunity and reduce the inflammatory response, achieving the functions of inhibiting breast cancer growth and promoting cancer cell apoptosis.

[0070] Case 11 In vivo biosafety evaluation of an engineered Taxus exosome microneedle transdermal patch for targeted treatment of breast cancer

[0071] The experiment was terminated 14 days after the administration of the drugs respectively. Whole blood was collected from the orbits. One part was placed in a negative pressure anticoagulation tube for the detection and analysis of blood routine indexes of mice in each drug administration group at the end of the experiment. The other part was placed in an ordinary centrifuge tube and left to stand in a 4°C refrigerator. After stratification, it was left at room temperature for several minutes, and then the serum was separated and collected under the conditions of 3000 rpm for 15 minutes in a refrigerated centrifuge. The blood biochemical indexes were detected and analyzed on an automatic biochemical analyzer. Finally, the mice were sacrificed by cervical dislocation, and the hearts, livers, spleens, lungs, kidneys and brain-kidneys of the mice in each group were taken and fixed in 10% neutral formalin. Then the above-mentioned organs were paraffin-embedded, sliced with a microtome and stained with hematoxylin-eosin to evaluate the safety. As Figure 13 shown, CDDP-IV significantly reduced the levels of ALT and AST, while significantly increased the levels of UREA and CREA. The other indexes had no significant changes, indicating that CDDP-IV caused a certain degree of liver and kidney toxicity, while CDDP-MN effectively alleviated the occurrence of liver and kidney toxicity. In addition, the final system RGP-KDEL-TC@CDDP-MN had no obvious pathological changes in the main organs and had no adverse effects on liver and kidney functions. The above results indicate that RGP-KDEL-TC@CDDP-MN has good biosafety, can effectively reduce the problems of the drug itself with large toxicity and side effects, and can be used as a new type of nano-formulation targeted drug.

[0072] The above-described embodiments merely represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. A preparation method of engineered Taxus chinensis exosomes, characterized in that, It includes the following steps: 1) Mix the active part of fresh medicinal Taxus chinensis with phosphate buffer solution for juicing to obtain a slurry containing Taxus exosomes; 2) Use a sieve to filter out the residues in the slurry obtained in step 1) and collect the juice. The juice is centrifuged by differential centrifugation to obtain a precipitate containing Taxus exosomes (TC-Exos); 3) Resuspend the precipitate obtained in step 2) with PBS and purify to obtain Taxus exosomes; 4) Use the ultrasonic-assisted method to co-load CDDP on the Taxus exosomes obtained in step 3) to obtain drug-loaded Taxus exosomes loaded with CDDP (TC@CDDP); 5) Functionalize the drug-loaded Taxus exosomes loaded with CDDP obtained in step 4) with RGP and KDEL respectively. The amino acid sequence of the RGP is shown in SEQ ID No.1, and the amino acid sequence of the KDEL is shown in SEQ ID No.2 to obtain engineered modified Taxus exosomes (RGP-KDEL-TC@CDDP).

2. The method according to claim 1, wherein The differential centrifugation method described in step 2) is as follows: Centrifuge the filtered juice at 4°C successively at 1000×g for 5 - 20 min, at 4000×g - 10000×g for 20 - 60 min, and at 10000×g for 60 - 120 min to obtain the supernatant; then centrifuge the supernatant at 4°C at 100000×g - 150000×g for 60 - 150 min to obtain a precipitate containing Taxus exosomes (TC-Exos).

3. The method according to claim 1, characterized in that The purification described in step 3) is filtration through a 0.22 μm membrane filter.

4. The method according to claim 1, wherein The ultrasonic-assisted method described in step 4) is as follows: After resuspending the Taxus exosomes with PBS, add the chemotherapeutic drug CDDP according to 1 - 2 times the mass of the exosomes, and perform ultrasonic-assisted treatment for 5 - 10 cycles. Each cycle includes 1 - 10 s of pulses and 2 - 3 s of pauses, and after each cycle, cool it on ice for 1 - 5 min; after ultrasonic treatment, incubate it at 37°C for 30 - 120 min to restore the exosome membrane activity, and then centrifuge it at 4°C at 100000×g - 150000×g for 60 - 150 min to obtain drug-loaded Taxus exosomes loaded with CDDP chemotherapy drug (TC@CDDP).

5. The method according to claim 1, characterized in that, Step 5) is as follows: After resuspending the drug-loaded Taxus exosomes with PBS, add RGP according to 0.1 - 1 times the mass of the exosomes and add KDEL according to 0.01 - 1 times the mass of the exosomes, shake it at 37°C on a shaker at 100 - 300 rpm for 30 - 120 min, and then add it to a 10 - 100KD ultrafiltration tube and centrifuge it at 1000 - 5000 rpm for 10 - 30 min to obtain targeted modified engineered Taxus exosomes.

6. Engineered Taxus exosomes prepared by the preparation method described in any one of claims 1 - 5.

7. A microneedle transdermal patch based on the engineered Taxus chinensis exosomes described in claim 6, characterized in that, The preparation method thereof is as follows: Adopt the shaker incubation method to carry RGP-KDEL-TC@CDDP in porous microneedles to prepare an engineered Taxus exosome microneedle transdermal patch (RGP-KDEL-TC@CDDP-MN).

8. The engineered Taxus exosome microneedle transdermal patch according to claim 7, wherein The incubation method is as follows: Add 100 - 1000 μg of RGP-KDEL-TC@CDDP onto the porous microneedles (MN), shake at 100 - 300 rpm for 10 - 120 min under a normal temperature shaker, and then aspirate the excess exosomes to prepare the engineered Taxus exosome microneedle transdermal patch (RGP-KDEL-TC@CDDP-MN).

9. Use of the engineered Taxus exosomes according to claim 6 or the microneedle transdermal patch of the engineered Taxus exosomes according to claim 8 in the preparation of antitumor drugs.

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

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