Curcumin-containing nano-drug for treating cancer, preparation method therefor, and use thereof

Abstract

A curcumin-containing nano-drug for treating cancer, a preparation method therefor, and use thereof. The nano-drug comprises curcumin and an extracellular vesicle expressing an anti-cancer protein TRAIL, or the nano-drug comprises curcumin and a tumor-antigen binding polypeptide-modified extracellular vesicle expressing an anti-cancer protein TRAIL. The curcumin is loaded into the extracellular vesicle for combined use, such that an extremely strong synergistic effect can be generated; the anti-cancer activity of the EV-T can be significantly enhanced by means of polypeptide modification; the composite nano-drug obtained by loading the curcumin into the extracellular vesicle can solve the problem of insolubility of curcumin, thereby further improving the anti-cancer effect.

Description

A nanomedicine containing curcumin for treating cancer, and its preparation method and application

[0001] This application claims priority to a Chinese patent application filed with the Patent Office of China on March 7, 2024, with application number 202410258359.2 and invention name “A nanomedicine containing curcumin for treating cancer, its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application belongs to the field of bio-nano preparations and oncology application technology, and specifically relates to a nano drug containing curcumin for treating cancer, and its preparation method and application. Background Art

[0003] Curcumin (CUR), a polyphenolic compound extracted from turmeric, has promising anticancer potential. Curcumin can modulate the expression of apoptotic factors, growth factors, and inflammatory proteins, exhibiting both anticancer and anti-inflammatory activities. It can be used alone or in combination with other anticancer drugs for cancer treatment. However, the clinical application of curcumin is limited by significant issues, including its low antitumor activity. Therefore, there is an urgent need to develop new formulations to overcome these issues and achieve better therapeutic effects.

[0004] Summary of the Invention

[0005] The purpose of this application is to provide a nanomedicine containing curcumin for the treatment of cancer, as well as its preparation method and application. The nanomedicine described in this application is highly effective and safe, with high anti-tumor activity and good biosafety, providing new ideas and strategies for conquering cancer, a major disease.

[0006] The present application provides a nanomedicine for treating cancer containing curcumin, wherein the nanomedicine comprises curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL, or the nanomedicine comprises curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL modified with a tumor antigen binding polypeptide.

[0007] Preferably, the tumor antigen-binding polypeptide modification includes RGD polypeptide modification.

[0008] Preferably, the RGD polypeptide modification is performed using an RGD-CP05 fusion peptide, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] Preferably, in the nanomedicine, curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL are simply mixed; or curcumin is loaded into extracellular vesicles expressing the anti-cancer protein TRAIL; or curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL modified with a tumor antigen binding polypeptide are simply mixed; or curcumin is loaded into extracellular vesicles expressing the anti-cancer protein TRAIL modified with a tumor antigen binding polypeptide.

[0010] Preferably, the cells used for producing the extracellular vesicles include mesenchymal stem cells, bone marrow stem cells, embryonic stem cells, umbilical cord stem cells, induced pluripotent stem cells, tumor cells, tumor stem cells, immune cells or fibroblasts.

[0011] Preferably, the dosage form of the nanomedicine includes a nanoformulation; in the nanoformulation, the particle size distribution of the extracellular vesicles is 60 to 120 nm.

[0012] The present application also provides the use of extracellular vesicles expressing the anti-cancer protein TRAIL modified with a tumor antigen binding polypeptide as a nano-delivery platform for targeted delivery of anti-cancer drugs in the preparation of anti-tumor nano-medicines; the anti-cancer drugs include curcumin.

[0013] Preferably, when curcumin is loaded into extracellular vesicles expressing the anti-cancer protein TRAIL, the method for preparing the nanomedicine comprises the following steps:

[0014] Extracellular vesicles expressing the anticancer protein TRAIL were mixed with curcumin and placed on ice for sonication to obtain EV-T-CUR;

[0015] When curcumin is loaded into extracellular vesicles modified with tumor antigen binding polypeptides and expressing the anti-cancer protein TRAIL, the preparation method of the nanomedicine includes the following steps:

[0016] Extracellular vesicles expressing the anti-cancer protein TRAIL modified with tumor antigen binding peptides were mixed with curcumin and placed on ice for ultrasonic treatment to obtain RGD@EV-T-CUR.

[0017] Preferably, the method for preparing extracellular vesicles modified with tumor antigen binding polypeptides and expressing the anti-cancer protein TRAIL comprises the following steps:

[0018] Tumor antigen binding peptides were synthesized, and the tumor antigen binding peptides were mixed with extracellular vesicles expressing the anti-cancer protein TRAIL, incubated, ultracentrifuged, and the precipitate was collected to obtain RGD@EV-T.

[0019] Preferably, the ultrasonic treatment conditions include: ultrasonic treatment for 10 to 60 seconds, pause for 10 to 60 seconds, 5 to 12 cycles, temperature of 15 to 30° C., and ultrasonic power of 10 to 30%.

[0020] The present application provides a nanomedicine for treating cancer containing curcumin. The nanomedicine described in the present application includes curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL, or the nanomedicine includes curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL modified with a tumor antigen binding polypeptide. The drug components can be simply mixed and used in combination, or they can be prepared into a composite loaded drug. By combining curcumin to sensitize the TRAIL response of cancer cells, the common TRAIL resistance of cancer cells can be overcome. Specifically, the combination of CUR with EV-T or RGD@EV-T can produce a very strong synergistic effect and obtain an anti-cancer activity significantly higher than that of a single drug. When combined with CUR, RGD@EV-T showed a higher cancer cell growth inhibition rate than EV-T, indicating that RGD modification significantly enhanced the anti-cancer activity of EV-T. In addition, the combination of CUR with EV-T or RGD@EV-T has no effect on normal cells, and the combination of the composite drugs in the present application has good safety.

[0021] When curcumin is present in an extracellular vesicle expressing the anticancer protein TRAIL or an extracellular vesicle expressing the anticancer protein TRAIL modified by a tumor antigen binding polypeptide in a loaded manner, the application obtains a novel composite nanomedicine. Nanoparticles come from cells, specifically extracellular vesicles, and CUR has a higher EV encapsulation rate, which can solve the problem of curcumin's poor solubility. The application first prepares extracellular vesicles EV-T expressing the anticancer protein TRAIL, and then binds the tumor-targeting polypeptide RGD to the EV-T membrane surface through the mediation of the CP05 anchor peptide to obtain RGD-modified EV-T, i.e., RGD@EV-T (in the present application, EV-T is engineered to carry membrane-surface-bound RGD to obtain tumor-targeted modified EV-T, i.e., RGD@EV-T, which can significantly enhance the tumor targeting of EV-T), and then CUR is loaded to prepare a targeted-modified, composite-carrying TRAIL and CUR novel targeted curcumin nanomedicine RGD@EV-T-CUR. By omitting the RGD modification process of the tumor-targeting peptide, a novel targeted curcumin nanoparticle, EV-T-CUR, was obtained, which is a composite carrier of TRAIL and CUR. CUR and TRAIL, co-delivered via EV, exhibit a high degree of synergy, effectively and specifically killing a wide range of cancer cells. RGD modification further significantly enhances the tumor targeting of EV-T-CUR, demonstrating potent anti-tumor activity in both cell and animal tumor models, and exhibiting good safety. This application creates a novel, highly effective and safe tumor-targeted curcumin nanoparticle formulation with excellent potential for development into a broad-spectrum targeted anticancer nanoparticle. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 is a transmission electron microscope photo of EV-T;

[0024] Figure 2 shows the results of quantitative detection of TRAIL content in EV, EV-T and RGD@EV-T using an ELISA kit;

[0025] Figure 3 shows the results of a DiR fluorescently labeled EV-T and RGD@EV-T cellular uptake test using renal cancer cells Caki-1; wherein, a is a diagram of cellular uptake of the DiR-labeled preparation, b is a diagram of quantitative analysis of cellular uptake, c is a diagram of biodistribution of the DiR-labeled preparation, and d is a diagram of quantitative analysis of the biodistribution of the preparation;

[0026] FIG4 is a graph showing the expression level of TRAIL in EV, EV-T and RGD@EV-T detected by immunoblotting (WB);

[0027] Figure 5 is a graph showing the detection peaks and standard curves of curcumin CUR detected by HPLC; wherein a is a graph showing the detection peaks of CUR, b is a graph showing the standard curve for detection, and c is a graph showing the loading rate of CUR;

[0028] Figure 6 shows the cytotoxicity test results of EV-T, RGD@EV-T, and RGD@EV on cancer cell lines and normal cell lines; among them, a is a dose experiment diagram of the effect of EV-T on cell proliferation, b is a dose experiment diagram of the effect of RGD@EV-T on cell proliferation, and c is a dose experiment diagram of the effect of RGD@EV on cell proliferation;

[0029] Figure 7 shows the cytotoxicity test results of different concentrations of CUR combined with EV-T or RGD@EV-T on cancer cell lines and normal cell lines; among them, a is the cytotoxicity test result of curcumin CUR on 4 cancer cells and 2 normal cells; bg are the effects of CUR alone or in combination with EV-T or RGD@EV-T on the proliferation of A549 (b), M231 (c), Hela (d), Caki-1 (e), HK-2 (f) and MSC (g) cells;

[0030] Figure 8 shows the growth inhibition (CCK-8 assay) and apoptosis induction (Annexin V / PI staining and flow cytometry analysis) and cytoskeleton effects (F-actin fluorescence labeling) of Caki-1 cancer cells treated with various reagents (CUR, EV-T, RGD@EV-T, EV-T-CUR, and RGD@EV-T-CUR). Figure a shows the cell proliferation and activity assay, figure b shows the cell apoptosis assay, and figure c shows the comparison of cytoskeleton changes after 24 hours of treatment with various reagents.

[0031] Figure 9 shows a comparison of the therapeutic effects of various therapeutic agents tested in a nude mouse subcutaneous renal cancer model; wherein, a is a tumor growth curve, b is a representative tumor image at the end of the experiment, c is a tumor weight comparison image at the end of the experiment, d is a TUNEL apoptosis staining image of animal organ tissues, and e is a quantitative analysis image of TUNEL staining. DETAILED DESCRIPTION

[0032] The present application provides a nanomedicine for treating cancer containing curcumin, which comprises curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL (tumor necrosis factor-related apoptosis-inducing ligand (TNF-related apoptosis-inducing ligand, TRAIL)) (cells can secrete and release EVs expressing membrane-bound TRAIL, named EV-T), or the nanomedicine comprises curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL modified with a tumor antigen binding polypeptide (i.e., tumor-targeted).

[0033] This application first discovered that curcumin and EV-T have a high degree of synergistic apoptosis-inducing effect, especially that they can specifically and efficiently kill a variety of cancer cells, with broad-spectrum anti-cancer activity, while having no effect on normal cells. In addition, it was found that curcumin can be loaded into EV-T, perfectly solving its problem of poor water solubility. Tumor-targeted modification of EV-T to carry tumor-targeting peptides bound to the membrane surface can obtain tumor-targeted modification, significantly enhancing the tumor targeting of EV-T.

[0034] The present application does not specifically limit the preparation method of extracellular vesicles (EV-T) expressing the anticancer protein TRAIL, and conventional methods can be used for preparation, such as references (Cytotherapy 2015, 17(7):885-896; Journal of Extracellular Vesicles, 2017, 6(1), 1265291). Curcumin loaded into extracellular vesicles EV synergizes with the anticancer factor TRAIL expressed on the surface of EV, and has efficient, broad-spectrum and specific anticancer activity. In the present application, the tumor antigen binding polypeptide modification preferably includes RGD polypeptide modification. In the present application, the RGD polypeptide modification is preferably performed using RGD-CP05 fusion peptide, and the amino acid sequence of the RGD-CP05 fusion peptide is shown in SEQ ID NO.1: RGDCRHSQMTVTSRL. The present application does not specifically limit the source of the RGD-CP05 fusion peptide, and it can be synthesized by a company. The RGD-CP05 fusion peptide described in the present application is preferably synthesized by Hefei Sener Biotechnology Co., Ltd.

[0035] In the present application, in the nanomedicine, curcumin and extracellular vesicles expressing the anticancer protein TRAIL are simply mixed (i.e., mixed and combined); or curcumin is loaded into the extracellular vesicles expressing the anticancer protein TRAIL; or curcumin and extracellular vesicles expressing the anticancer protein TRAIL modified with a tumor antigen binding polypeptide are simply mixed (i.e., mixed and combined); or curcumin is loaded into the extracellular vesicles expressing the anticancer protein TRAIL modified with a tumor antigen binding polypeptide. In the present application, curcumin and extracellular vesicles expressing the anticancer protein TRAIL or extracellular vesicles expressing the anticancer protein TRAIL modified with a tumor antigen binding polypeptide are used in combination to produce a very strong synergistic effect, obtaining an anticancer activity significantly higher than that of a single drug. And after modification with a tumor antigen binding polypeptide, a higher cancer cell growth inhibition rate is shown, indicating that the modification with the tumor antigen binding polypeptide significantly enhances the anticancer activity. In addition, the drug combination has no effect on normal cells, and the mixed combination scheme of the present application has good safety. CUR and TRAIL co-delivered via EV have a high degree of synergistic effect and can kill a wide range of cancer cells with high efficiency and specificity. RGD modification further significantly enhances the tumor targeting of EV-T-CUR. Most preferably, this application loads curcumin (CUR) into RGD@EV-T to prepare a new targeted nanoformulation RGD@EV-T-CUR for the composite co-delivery of CUR and TRAIL. It has shown efficient anti-tumor activity in both cell models and animal tumor models, and has good safety. This new formulation can potentially be developed into a targeted nanomedicine for the treatment of cancer and has good application prospects.

[0036] In the present application, the cells used for the production of extracellular vesicles preferably include mesenchymal stem cells (MSCs), bone marrow stem cells, embryonic stem cells, umbilical cord stem cells, induced pluripotent stem cells, tumor cells, tumor stem cells, immune cells or fibroblasts. Most preferably, they are mesenchymal stem cells derived from human umbilical cord.

[0037] In the present application, the dosage form of the nanomedicine preferably includes a nanoformulation; in the nanoformulation, the particle size distribution of the extracellular vesicles is preferably 60 to 120 nm, and the average particle size is preferably 72.77 nm.

[0038] This application also provides the use of extracellular vesicles (EVs) modified with tumor antigen-binding polypeptides and expressing the anticancer protein TRAIL as a nano-delivery platform for the targeted delivery of anticancer drugs, preferably including curcumin, in the preparation of anti-tumor nanomedicines. Curcumin loaded into EVs synergizes with the anticancer factor TRAIL expressed on the surface of the EVs modified with tumor antigen-binding polypeptides, resulting in highly effective, tumor-targeted, broad-spectrum, and specific anticancer activity.

[0039] In the present application, when curcumin is loaded into extracellular vesicles expressing the anticancer protein TRAIL, the preparation method of the nanomedicine comprises the following steps: mixing the extracellular vesicles expressing the anticancer protein TRAIL with curcumin, placing them on ice for ultrasonic treatment, and obtaining EV-T-CUR. The present application does not specifically limit the preparation method of extracellular vesicles (EV-T) expressing the anticancer protein TRAIL, and conventional methods can be used for preparation, such as references (Cytotherapy 2015, 17 (7): 885-896; Journal of Extracellular Vesicles, 2017, 6 (1), 1265291). Specifically, the present application preferably first constructs a lentiviral vector expressing TRAIL, prepares a genetically engineered modified virus, establishes a cell line with high TRAIL expression by lentiviral transfection, cultures the cells to obtain a culture supernatant containing EV-T, and purifies EV-T by ultracentrifugation. The preparation of the engineered modified virus in the present application is not limited to lentivirus, and preferably retrovirus or adenovirus can also be used for preparation. In the present application, the conditions for the ultrasonic treatment preferably include: ultrasonic treatment for 10 to 60 seconds, pause for 10 to 60 seconds, and 5 to 12 cycles; temperature of 15 to 30°C, and ultrasonic power set to 10 to 30%, more preferably include: ultrasonic treatment for 35 seconds, pause for 35 seconds, and 6 cycles; temperature of 25°C, and ultrasonic power of 25%.

[0040] When curcumin is loaded into the extracellular vesicles expressing the anti-cancer protein TRAIL modified by the tumor antigen binding polypeptide, the preparation method of the nanomedicine includes the following steps: the extracellular vesicles expressing the anti-cancer protein TRAIL modified by the tumor antigen binding polypeptide (RGD@EV-T) are mixed with curcumin, placed on ice for ultrasonic treatment, and RGD@EV-T-CUR (targeted polypeptide-modified composite extracellular vesicle nanoparticles co-carrying curcumin and anti-cancer factor TRAIL). The present application first prepares the extracellular vesicles expressing the anti-cancer protein TRAIL modified by the tumor antigen binding polypeptide, including the following steps: synthesizing the tumor antigen binding polypeptide, mixing the tumor antigen binding polypeptide and the extracellular vesicles expressing the anti-cancer protein TRAIL, incubating, ultracentrifuging, and taking the precipitate to obtain RGD@EV-T. The present application does not specifically limit the source of the tumor antigen binding polypeptide, and the polypeptide product synthesized by the company can be used.

[0041] In the present application, the mixing mass ratio of the tumor antigen binding polypeptide and the extracellular vesicles expressing the anti-cancer protein TRAIL is preferably (0.8-1.1): (0.95-1.05), more preferably 1: 1. After mixing, the present application is preferably incubated with shaking at 4°C for 6 hours to promote the binding of the polypeptide to EV-T, specifically by inserting the CP05 anchor peptide into the EV-T membrane to complete the surface modification. In the present application, the ultracentrifugation conditions are preferably 110,000-120,000g, 1.5h, 4°C, to remove free unbound polypeptides, and the precipitate is RGD-targeted modified EV-T, i.e., RGD@EV-T.

[0042] After obtaining the extracellular vesicles expressing the anti-cancer protein TRAIL modified with the tumor antigen binding polypeptide, the present application preferably resuspends them and mixes them with a curcumin solution (dissolved in DMSO, 10mM stock solution), places them on ice for ultrasonic treatment to load curcumin, and obtains RGD@EV-T-CUR. The ultrasonic treatment described in the present application preferably uses an ultrasonic cell disruptor, and the model of the ultrasonic cell disruptor is preferably: Sonics VC505. In the present application, the conditions of the ultrasonic treatment preferably include: ultrasonication for 10 to 60 seconds, pause for 10 to 60 seconds, and 5 to 12 cycles; temperature of 15 to 30°C, and ultrasonic power set to 10 to 30%, more preferably including: ultrasonication for 35 seconds, pause for 35 seconds, and 6 cycles; temperature of 25°C, and ultrasonic power set to 25%.

[0043] After sonication, the present application preferably performs incubation to restore the integrity of the EV membrane, thereby obtaining a targeted modified nanoformulation RGD@EV-T-CUR that carries curcumin and the anticancer factor TRAIL. In the present application, the incubation conditions are preferably 30-40°C for 0.5-2.5 hours, more preferably 37°C on a shaker for 1 hour.

[0044] This application modified EV-derived cells through genetic engineering, and then isolated and purified EVs expressing TRAIL (EV-T). The tumor targeting ability of EV-T was improved through RGD polypeptide modification. EV was used as a nanocarrier to deliver curcumin and TRAIL in combination, fully leveraging the synergistic effect of the two and overcoming the TRAIL tolerance of cancer cells. The results showed that the targeted modified composite drug-loaded EV-T (RGD@EV-T-CUR) had specific and broad-spectrum cancer cell killing activity, as well as efficient therapeutic effects in animal tumor models and good safety.

[0045] This application uses the tumor-targeting peptide RGD to modify EV-T and load curcumin to prepare a targeted composite drug-loaded nanoformulation RGD@EV-T-CUR, which can significantly increase the enrichment effect of curcumin and TRAIL in tumor delivery and has a very significant synergistic effect of combined drugs; this application can solve the problems of low water solubility, lack of targeting and low bioavailability of curcumin; by combining curcumin to sensitize cancer cells, the common cancer cell resistance to TRAIL can be overcome; after the targeted composite drug-loaded nanoformulation of this application enters the blood through intravenous administration, it can be highly enriched in tumor tissue, induce cancer cell apoptosis, significantly inhibit tumor growth, and have good therapeutic safety. The targeted composite drug-loaded nanoformulation of this application can achieve multi-target combined precision treatment of drug-resistant cancers based on the co-delivery of biomolecules and chemical drugs by extracellular vesicles, providing potentially more efficient and safer innovative targeted nanomedicines for cancer treatment.

[0046] To further illustrate the present application, a nanomedicine containing curcumin for treating cancer, its preparation method, and application provided by the present application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present application.

[0047] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0048] Example 1

[0049] TRAIL-expressing extracellular vesicles (EV-T) were surface-modified with RGD.

[0050] As reported in the literature (Cytotherapy 2015, 17(7):885-896; Journal of Extracellular Vesicles, 2017, 6(1), 1265291), human umbilical cord-derived mesenchymal stem cells (UC-MSCs) at passage P3 were transfected with a lentivirus expressing TRAIL to establish the TRAIL-gene-modified cell line MSCTRAIL. Parental MSC cells and MSCTRAIL cells were cultured, and the culture supernatants were collected. EVs and EV-Ts were separated and purified by ultracentrifugation as reported in the literature. Briefly, the culture supernatant was first centrifuged at low speed to remove cells and cell debris and clarify the supernatant; then, it was filtered using a 0.22 μm filter membrane to remove EVs larger than 220 nm, and then the supernatant containing EVs was concentrated 5-10 times using a 100 kDa ultrafiltration column (4°C, 30 min, 3000 g). Finally, EVs and EV-T were precipitated by ultracentrifugation at 4°C (1.5 h, 120,000 g). The EV and EV-T precipitates were then resuspended in PBS solution, and their protein content was quantified. The samples were aliquoted and frozen at -80°C for later use.

[0051] The morphology and particle size of the prepared EVs and EV-T were observed by transmission electron microscopy (Figure 1), and a double-membrane vesicle structure was detected with a particle size distribution of 60-120 nm, which are typical cell-derived exosome nanoparticles. A TRAIL-specific ELISA kit was used to measure the TRAIL content carried by EVs, EV-T and RGD@EV-T (preparation method see below) (Figure 2). It can be seen that both EV-T and RGD@EV-T significantly expressed TRAIL protein, with an expression level of 98.5±2.1pgTRAIL / μg EVs, indicating that RGD modification did not significantly affect the level of TRAIL protein carried by EV-T, and at the same time, EVs did not have obvious TRAIL expression.

[0052] EV-Ts surface RGD modification is achieved by binding the anchor peptide CP05 to the EV surface protein CD63. First, a commercially available fusion peptide of RGD and CP05, RGD-CP05, was synthesized with the sequence RGDCRHSQMTVTSRL (SEQ ID NO. 1) and a purity of ≥95%. To modify the surface of EV-Ts, EV-Ts (10 μg) were incubated with the RGD-CP05 peptide (10 μg) at 4°C for 6 hours with shaking to promote the binding of the peptide to EV-T. The resulting RGD-modified nanoformulation was named RGD@EV-T. Unbound peptide was removed by ultracentrifugation (120,000 g, 1.5 hours, 4°C). The purified RGD@EV-T precipitate was finally resuspended in PBS, aliquoted, and stored at -80°C until further use.

[0053] To test the modification of EV-T by the RGD-CP05 peptide, RGD-CP05 peptide labeled with rhodamine B (RhoB), namely RGDRhoB-CP05, was conjugated to EV-T for modification. The resulting RGDRhoB@EV-T was first fluorescently labeled with DiR and then added to Caki-1 renal cancer cells for observation of EV cellular uptake. The results are shown in Figure 3, which shows the results of the DiR fluorescently labeled EV-T and RGD@EV-T cellular uptake assay using Caki-1 renal cancer cells to evaluate the effect of RGD-targeted modification on enhancing EV-T tumor targeting. As shown in Figure 3a, RhoB and DiR labels co-localize to EV-T that has been taken up by cells, indicating successful modification of EV-T by RGD. To verify the enhanced tumor targeting effect of RGD modification on EV-T, PBS control, EV-T, and RGD@EV-T were fluorescently labeled with DiR. Unlabeled dye was then removed by ultracentrifugation. The tumor targeting properties of the formulations were then observed by uptake into Caki-1 cells combined with confocal microscopy, as well as in animal tumor models, intravenous infusion, and in vivo imaging. As shown in Figure 3(b) and Figures 3(c) and 3(d), respectively, RGD modification significantly enhanced both tumor cell and tumor tissue targeting of EV-T.

[0054] Western blotting (WB) was performed on the prepared EVs, EV-T, and RGD@EV-T to detect the expression of extracellular vesicle markers TSG101 and TRAIL. As shown in Figure 4, all three preparations expressed TSG101, demonstrating clear extracellular vesicle characteristics. EVs derived from the parental MSC cells did not express TRAIL, but both EV-T and RGD@EV-T expressed TRAIL, indicating that RGD surface modification did not affect the ability of EV-T to carry the anticancer protein TRAIL.

[0055] Example 2

[0056] Curcumin (CUR) was loaded into EV-T and RGD@EV-T prepared in Example 1.

[0057] Commercially available CUR (HY-N0005, MedChemExpress, Shanghai) was dissolved in DMSO to prepare a 10 mM stock solution. High-performance liquid chromatography (HPLC) was used to quantify the CUR concentration in the solution using a C18 column and a detection wavelength of 420 nm. A CUR standard curve was established, and the test series concentrations were 0.0 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25.0 μg / mL, 50.0 μg / mL, and 100 μg / mL.

[0058] Low-frequency ultrasound-mediated loading was used to encapsulate CUR into EV-T and RGD@EV-T, respectively, to prepare composite nanodrugs EV-T-CUR and RGD@EV-T-CUR. Specifically, 2 μl of a 10 mM CUR solution was added to 1 mL of a 20 μg / mL EV-T or RGD@EV-T solution (containing 2.0 ng / mL TRAIL), mixed thoroughly, and loaded with CUR using ultrasound or co-incubation. The drug loading parameters on a Sonics VC505 were as follows: 35 seconds of ultrasound, 35 seconds of pause, 25% power, 25°C temperature, and 6 cycles. After ultrasonic treatment, the samples were placed on a shaker at 37°C for 1 hour to allow the EV-T to restore its membrane structure. Finally, the samples were ultracentrifuged (120,000 g, 1.5 hours, 4°C) to remove free, unloaded CUR from the solution (precipitate). Purified EV-T-CUR and RGD@EV-T-CUR precipitates were obtained, which were then resuspended in PBS and observed by transmission electron microscopy as described in Example 1 to confirm that the integrity of the EV-T nanovesicles was not compromised after ultrasonic loading with CUR. For the co-incubation loading method, CUR was incubated with the EV-T / RGD@EV-T mixed solution at 4°C for 1 hour, and the resulting EV-T-CUR and RGD@EV-T-CUR were purified by ultracentrifugation. To measure drug loading efficiency, the loaded and purified nanoformulations were added to RIPA lysis buffer to release the CUR encapsulated in the EVs. The CUR content was then determined by HPLC, and the corresponding CUR loading efficiency was calculated. The results showed that the detection peak time of CUR was 5.91 min (a in Figure 5), and its standard regression curve was y = 0.1515x-1.034 (R 2 =0.9958) (b in FIG5 ), the drug loading rate of CUR by ultrasonic loading method was 53.4±1.02%, which was significantly better than the loading rate of 31.5% by mixed loading method (c in FIG5 ).

[0059] Example 3

[0060] The in vitro anticancer activity of the nanoformulation was detected by CCK-8 assay.

[0061] To test the anticancer activity of EV-T, RGD@EV-T, and RGD@EV alone or in combination with CUR, this application selected six cell lines for CCK-8 assays to test their effects on cell proliferation and activity. The cell lines used included one renal cancer cell line (Caki-1), one lung cancer cell line (A549), one breast cancer cell line (M231), one cervical cancer cell line (Hela), and two normal cell lines (human mesenchymal stem cells (MSCs) and human renal tubular epithelial cells (HK-2).

[0062] The results, as shown in Figure 6, show that both EV-T and RGD@EV-T exhibited significant growth inhibition against cancer cells, with a dose-dependent effect. In contrast, RGD@EV lacked significant anticancer activity, indicating that the activity was primarily conferred by EV-carried TRAIL, rather than by EV itself or RGD modification. As shown in Figure 7, compared with the PBS control (Ctrl), CUR alone exhibited a modest growth inhibitory effect on tumor cells. When combined with EV-T or RGD@EV-T (carrying 2 ng / mL TRAIL), CUR exhibited a strong synergistic effect, achieving significantly higher anticancer activity than either single-agent treatment. When combined with CUR, RGD@EV-T exhibited a higher cancer cell growth inhibition rate than EV-T, indicating that RGD modification significantly enhanced the anticancer activity of EV-T. Notably, the synergistic cytotoxicity of CUR and EV-T or RGD@EV-T was specific to cancer cells, with no effect on normal cells, demonstrating the favorable safety profile of this combination therapy.

[0063] Example 4

[0064] Detection of the specific cancer cell apoptosis-inducing effect of nanoformulations.

[0065] After confirming that CUR and EV-T synergistically inhibit cancer cell proliferation, this application next studied the effects of CUR-encapsulated EV-T and RGD@EV-T (EV-T-CUR and RGD@EV-T-CUR, containing 20 μM CUR and 2.0 ng / mL TRAIL, respectively) on cancer cell proliferation, apoptosis and cytoskeleton. The detection methods used were CCK-8 assay, FITC-Annexin-V / PI staining combined with flow cytometry analysis, and FITC-phalloidin labeling of actin fibers (F-actin).

[0066] The results, as shown in Figure 8a and b, show that treatment with 20 μM CUR and 2.0 ng / mL EV-T-carried TRAIL alone had limited effects on inhibiting Caki-1 cell proliferation and inducing apoptosis. In contrast, treatment with EV-T-CUR and RGD@EV-T-CUR produced significant synergistic effects, with RGD@EV-T and RGD@EV-T-CUR being more effective than EV-T and EV-T-CUR, respectively. Cytoskeleton markers (Figure 8c) further confirmed that both EV-T-CUR and RGD@EV-T-CUR treatment induced significant cytoskeletal disruption in cancer cells, indicating significant apoptosis, with the latter being particularly effective.

[0067] Example 5

[0068] Observation of the targeted killing effect of composite nanoformulations jointly delivering CUR and TRAIL on animal tumor models.

[0069] BALB / c female nude mice aged 4 to 5 weeks were purchased from the market and raised in an SPF-grade environment until they reached a weight of approximately 20 g. Mice in good condition were selected for subcutaneous tumor model establishment. Caki-1 cells were inoculated at a cell count of 3 million per mouse for model establishment. Two weeks after Caki-1 implantation, when the tumor grew to 200 mm 3 Treatment was initiated by intratumoral injection of control (PBS), RGD@EV-T, CUR, and RGD@EV-T-CUR, respectively. A very low dose of CUR (1.0 mg / kg, relative to the mouse dose) was encapsulated into RGD@EV-T to prepare a composite RGD@EV-T-CUR formulation. Three intratumoral injections were performed, with an interval of 48 hours between injections.

[0070] The dose of RGD@EV-T-CUR per injection was 60 μg / animal (3.0 mg / kg, containing 0.2 μg / kg TRAIL and 1.0 mg / kg CUR), the dose of RGD@EV-T was 40 μg / animal (2.0 mg / kg, containing 0.2 μg / kg TRAIL), and the dose of CUR per injection was 20 μg (1.0 mg / kg). The animal condition was monitored regularly and tumor growth was measured. The results are shown in Figures 9a to 9c. Based on the tumor growth curve, tumor morphology, and weight after the end of the experiment, the combined treatment with RGD@EV-T-CUR almost completely inhibited tumor growth compared with the control; in contrast, either RGD@EV-T or CUR alone showed only limited inhibitory effects. In addition, the safety of the RGD@EV-T-CUR combination treatment was evaluated using the tissue cell apoptosis detection technique TUNEL. The results are shown in Figures 9d to 9e. Compared with the control mice, the combined treatment did not significantly induce cell apoptosis in the heart, lung, liver, kidney and spleen tissues of the animals, revealing the good safety of this treatment.

[0071] In summary, mesenchymal stem cells were first genetically modified to secrete extracellular vesicles (EV-T) expressing the anti-cancer factor TRAIL. After isolating and purifying the EV-T, the EV-T was modified with the tumor-targeting peptide RGD and then loaded with the synergistic therapeutic drug CUR, achieving the co-delivery of CUR and TRAIL based on a targeted modified composite nanoformulation. Compared with the treatment of EV-T and CUR alone, the use of RGD-modified EV-T to deliver CUR can significantly improve the inhibitory effect on tumor growth, effectively and synergistically kill multiple cancer cells (breast cancer, lung cancer, cervical cancer, and kidney cancer), and has good cancer specificity and therapeutic safety. It can be seen that the use of targeted modified EVs to carry curcumin and TRAIL can achieve combined targeted treatment of multiple cancers based on the EV nanocarrier delivery system, while having high therapeutic efficiency and good safety.

[0072] Although the above embodiment provides a detailed description of the present application, it is only a part of the embodiments of the present application, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creative work, and these embodiments all fall within the scope of protection of the present application.

Claims

1. A nanomedicine for treating cancer containing curcumin, characterized in that: The nano drug comprises curcumin and extracellular vesicles expressing the anticancer protein TRAIL, or the nano drug comprises curcumin and extracellular vesicles expressing the anticancer protein TRAIL modified by a tumor antigen binding polypeptide.

2. The nanomedicine according to claim 1, characterized in that The tumor antigen binding polypeptide modification includes RGD polypeptide modification.

3. The nanomedicine according to claim 2, characterized in that The RGD polypeptide modification is performed using an RGD-CP05 fusion peptide, the amino acid sequence of which is shown in SEQ ID NO.

1.

4. The nanomedicine according to claim 1, characterized in that In the nanomedicine, curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL are simply mixed; or curcumin is loaded into the extracellular vesicles expressing the anti-cancer protein TRAIL; or curcumin and extracellular vesicles expressing the anti-cancer protein TRAIL modified with a tumor antigen binding polypeptide are simply mixed; or curcumin is loaded into the extracellular vesicles expressing the anti-cancer protein TRAIL modified with a tumor antigen binding polypeptide.

5. The nanomedicine according to claim 1, 2 or 4, characterized in that The cells used for producing the extracellular vesicles include mesenchymal stem cells, bone marrow stem cells, embryonic stem cells, umbilical cord stem cells, induced pluripotent stem cells, tumor cells, tumor stem cells, immune cells or fibroblasts.

6. The nanomedicine according to claim 1, characterized in that In the nanomedicine, the particle size distribution of the extracellular vesicles is 60-120 nm.

7. Use of extracellular vesicles expressing the anti-cancer protein TRAIL modified with tumor antigen binding polypeptides as a nano-delivery platform for targeted delivery of anti-cancer drugs in the preparation of anti-tumor nano-medicines; the anti-cancer drugs include curcumin.

8. The method for preparing the nanomedicine according to any one of claims 1 to 6, characterized in that: When curcumin is loaded into extracellular vesicles expressing the anti-cancer protein TRAIL, the preparation method of the nanomedicine includes the following steps: Extracellular vesicles expressing the anticancer protein TRAIL were mixed with curcumin and placed on ice for sonication to obtain EV-T-CUR; When curcumin is loaded into extracellular vesicles modified with tumor antigen binding polypeptides and expressing the anti-cancer protein TRAIL, the preparation method of the nanomedicine includes the following steps: Extracellular vesicles expressing the anti-cancer protein TRAIL modified with tumor antigen binding peptides were mixed with curcumin and placed on ice for ultrasonic treatment to obtain RGD@EV-T-CUR.

9. The preparation method according to claim 8, characterized in that The method for preparing extracellular vesicles modified with tumor antigen binding polypeptides and expressing the anti-cancer protein TRAIL comprises the following steps: Tumor antigen binding peptides were synthesized, and the tumor antigen binding peptides were mixed with extracellular vesicles expressing the anti-cancer protein TRAIL, incubated, ultracentrifuged, and the precipitate was collected to obtain RGD@EV-T.

10. The preparation method according to claim 9, characterized in that The mass ratio of the mixture of the tumor antigen binding polypeptide and the extracellular vesicles expressing the anti-cancer protein TRAIL is (0.8-1.1):(0.95-1.05).

11. The preparation method according to claim 8, characterized in that The ultrasonic treatment conditions include: ultrasonic treatment for 10 to 60 seconds, pause for 10 to 60 seconds, 5 to 12 cycles, temperature of 15 to 30° C., and ultrasonic power of 10 to 30%.

12. The preparation method according to claim 8, characterized in that After the ultrasonic treatment, incubation is also included.

13. A method for treating cancer, characterized in that: The method comprises the following steps: administering the nano drug according to any one of claims 1 to 6 or the nano drug prepared by the preparation method according to any one of claims 8 to 10 intravenously.

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