Biomimetic nanodrugs for mitochondrial dysfunction, their manufacturing methods, and applications.

Abstract

This invention provides biomimetic nanopharmaceuticals for mitochondrial dysfunction, methods for producing the same, and applications. [Solution] The biomimetic nanodrug comprises an RGD-engineered exosome as a carrier, decalinium chloride modified on the surface of the carrier, and oxaliplatin encapsulated inside the carrier. The biomimetic nanodrug of the present invention (OXA@Exo-RD) protects cargoes that induce mitochondrial dysfunction in the blood circulation process, sequentially increasing the accumulation of mitochondria in the cargoes by targeting them. Cargo 1 (DQA) induces oxidative stress, triggering mitochondrial-mediated apoptosis and mitochondrial dysfunction, while cargo 2 (OXA) disrupts mitochondrial DNA and inhibits the initiation of DNA repair, thereby improving chemotherapy resistance. Both cargoes synergistically overcome CRC drug resistance and inhibit its migration.

Description

[Technical Field]

[0001] This invention relates to biomimetic nanopharmaceuticals for mitochondrial dysfunction, methods for producing the same, and applications, and belongs to the field of biomimetic nanopharmaceuticals. [Background technology]

[0002] Colorectal cancer (CRC) is one of the most common malignancies, and both its incidence and mortality rates are on the rise, posing a serious threat to human health and life. Oxaliplatin (OXA) is the first-line chemotherapy drug for patients with advanced CRC. It targets and binds to DNA, inhibiting the helix breakdown of the DNA double helix, thereby inhibiting DNA replication and exerting an antitumor effect. While OXA has a promising outlook for application, its drug resistance and poor responses due to nonspecific distribution significantly limit its therapeutic effect. More importantly, tumor metastasis is another obstacle to the effective treatment of CRC and can lead to treatment failure.

[0003] DNA damage repair mechanisms reduce the effectiveness of chemotherapy by repairing interstrand crosslinks between platinum-based drugs and DNA, which is one of the main causes of chemotherapy resistance. Due to the overexpression of genes correlated with DNA damage repair in drug-resistant tumor cells, there is a strong need for effective measures to inhibit the initiation of DNA repair and restore sensitivity to platinum-based drugs in drug-resistant tumor cells. Currently, several inhibitors correlated with DNA repair, such as DNA-pk inhibitors, PARP inhibitors, and MGMT inhibitors, are used in combination with platinum-based drugs to improve the efficacy of antitumor therapy. Furthermore, studies have shown that mitochondria do not possess DNA damage repair function.

[0004] Therefore, the technical problem to be solved in this field is how to deliver platinum to mitochondria rather than the cell nucleus in order to avoid chemotherapy resistance and / or inhibit tumor metastasis. [Overview of the Initiative]

[0005] To improve the above-mentioned problems, the present invention provides a biomimetic nanodrug comprising an RGD-engineered exosome (Exo-R) as a carrier, decalinium chloride (DQA) modified on the surface of the carrier, and oxaliplatin (OXA) encapsulated inside the carrier.

[0006] According to embodiments of the present invention, the RGD engineered exosome (Exo-R) refers to an exosome to which the RGD peptide is bound.

[0007] According to embodiments of the present invention, the RGD peptide is selected from peptides containing arginine-glycine-aspartic acid (Arg-Gly-Asp, RGD), such as cRGD (valine-arginine-glycine-aspartic acid-glutamic acid cyclic peptide).

[0008] According to embodiments of the present invention, the exosome, also called an exosome, is a small membrane vesicle secreted by a cell. The diameter of the exosome may be about 20 nm to about 200 nm, for example, about 30 nm to about 200 nm.

[0009] According to embodiments of the present invention, the surface of the carrier is modified with decalinium chloride (DQA), and preferably, the decalinium chloride (DQA) is inserted into the surface of the carrier.

[0010] According to embodiments of the present invention, the content of decalinium chloride (DQA) on the carrier surface is 5 to 10 wt%, preferably 6 to 9 wt%, for example, 6 wt%, 7 wt%, 8 wt%, 9 wt%, and an example thereof is 7.3 wt%.

[0011] According to embodiments of the present invention, the content of oxaliplatin (OXA) within the carrier is 5 to 10 wt%, preferably 5 to 8 wt%, for example, 5 wt%, 6 wt%, 7 wt%, or 8 wt%, and an example thereof is 5.8 wt%.

[0012] The present invention further provides a method for manufacturing the above biomimetic nano-drug, and the method includes the following steps.

[0013] (1) Manufacturing RGD-engineered exosomes modified with decalinium chloride, i.e., Exo-RD, and (2) Encapsulating oxaliplatin into the above Exo-RD to obtain OXA@Exo-RD.

[0014] According to an embodiment of the present invention, the manufacturing method further includes the step of manufacturing or preparing RGD-engineered exosomes Exo-R.

[0015] According to an embodiment of the present invention, in the above step (1), DSPE-PEG 2000 -DQA, PBS buffer solution and Exo-R are mixed to obtain Exo-RD.

[0016] According to an embodiment of the present invention, step (1) includes the following.

[0017] (1a) Mixing DSPE-PEG 2000 -DQA with PBS buffer solution to obtain a mixed solution, (1b) Mixing Exo-R with the mixed solution in step (1a).

[0018] Preferably, the above Exo-R is added to the mixed solution in step (1a) in the form of its suspension. For example, the suspension of the above Exo-R is a PBS suspension of Exo-R.

[0019] According to an embodiment of the present invention, in step (1a), the temperature for mixing DSPE-PEG 2000 -DQA with PBS buffer solution is 30 - 70 °C, and / or the mixing time is 10 - 30 min.

[0020] According to an embodiment of the present invention, in step (1b), the mixing temperature is 30 - 50 °C, and / or the mixing time is 1 - 4 h.

[0021] According to embodiments of the present invention, in step (1b), the concentration of the suspension of Exo-R is 100 to 300 μg / mL, preferably 150 to 200 μg / mL, for example 150 μg / mL, 160 μg / mL, 170 μg / mL, 180 μg / mL, 190 μg / mL, or 200 μg / mL.

[0022] According to embodiments of the present invention, in step (1a), DSPE-PEG in the above mixture 2000 -The concentration of DQA is 20 to 100 μg / mL, preferably 20 to 60 μg / mL, for example 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, or 60 μg / mL.

[0023] According to embodiments of the present invention, in step (1b), Exo-R surface protein and DSPE-PEG 2000 -The mass ratio with DQA is 5:1 to 1:15, for example, 5:1, 1:1, 1:5, 1:10, or 1:15.

[0024] According to embodiments of the present invention, in step (2), the mass ratio of Exo-RD surface protein to oxaliplatin is 10:1 to 1:10, for example, 10:1, 8:1, 6:1, 5:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:5, 1:6, 1:8, or 1:10.

[0025] According to embodiments of the present invention, step (2) specifically involves employing an electroporation method (e.g., an X-Porator H1 electroporation system) to encapsulate oxaliplatin (OXA) in the Exo-RD.

[0026] According to embodiments of the present invention, the above DSPE-PEG 2000 -DQA is manufactured using methods known in this field, for example, DSPE-PEG 2000 -COO-NHS is reacted with decalinium chloride to produce DSPE-PEG. 2000- Obtain -DQA. As an example, the above DSPE-PEG 2000 - The manufacturing method of -DQA includes the following steps.

[0027] (S1) 1,2-distearoyl phosphatidylethanolamine-polyethylene glycol 2000 -carboxy (DSPE-PEG 2000 -COOH), N-hydroxysuccinimide (NHS), carbodiimide hydrochloride (EDC-HCl) and triethylamine are mixed and reacted to obtain DSPE-PEG 2000 -COO-NHS, (S2) React DSPE-PEG 2000 -COO-NHS with decalinium chloride (DQA) to obtain the above DSPE-PEG 2000 -DQA.

[0028] Preferably, step (S1) is carried out in the presence of an organic solvent. For example, the above organic solvent is chloroform.

[0029] Preferably, step (S2) is carried out in the presence of an organic solvent. For example, the above organic solvent is DMSO.

[0030] The present invention further provides a pharmaceutical composition containing the above biomimetic nanodrug.

[0031] The present invention further provides the application of the above biomimetic nanodrug in the treatment of cancer.

[0032] The present invention also further provides a method for treating cancer, which includes administering a therapeutically effective amount of the above biomimetic nanodrug to a patient who needs it.<​​​​​​According to embodiments of the present invention, the cancer may be colon cancer and / or rectal cancer, for example, colorectal cancer.

[0035] According to embodiments of the present invention, the tumor may be a colon tumor and / or a rectal tumor, for example, a colorectal tumor. [Effects of the Invention]

[0036] The inventors have surprisingly found that the biomimetic nanodrug (OXA@Exo-RD) provided by the present invention sequentially targets drug-resistant colorectal cancer cells and mitochondria, thereby increasing the accumulation of the mitochondrial function inhibitors dequalinium chloride (DQA) and oxaliplatin (OXA). This induces mitochondrial-mediated apoptosis and mitochondrial dysfunction, synergistically improving the drug resistance problem in colorectal cancer and inhibiting cancer cell metastasis. Furthermore, the biomimetic nanodrug of the present invention can protect the "cargo" (i.e., DQA and OXA) that induces mitochondrial dysfunction during the blood circulation process. Of these, cargo 1 (DQA) induces oxidative stress and triggers mitochondrial-mediated apoptosis and mitochondrial dysfunction, while cargo 2 (OXA) disrupts mitochondrial DNA and prevents the initiation of DNA repair, thereby improving chemotherapy resistance.

[0037] Furthermore, the biomimetic nanodrugs of the present invention have advantages such as good biocompatibility, high stability, long circulation time, and low immunogenicity, and can significantly avoid the problem of adverse reactions due to nonspecific distribution. [Brief explanation of the drawing]

[0038] [Figure 1] This is a manufacturing flowchart for OXA@Exo-RD of the present invention. [Figure 2] These are transmission electron microscope characterization diagrams of Exo in Comparative Example 1 and OXA@Exo-RD nanoparticles in Example 1. [Figure 3]This chart shows the results of the anticancer activity tests of OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD 24 hours after treatment with HCT116 and HCT116 / OXA cells. [Figure 4] This chart shows the in vivo anti-cancer results for PBS, OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD. [Figure 5] This diagram shows the anti-metastatic results for PBS, OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD. [Modes for carrying out the invention]

[0039] The technical aspects of the present invention will be described in more detail below, in accordance with specific embodiments. The embodiments described below are merely illustrative and interpretable to illustrate the present invention, and should not be interpreted as limiting the scope of the claims. Any technology realized based on the above-described aspects of the present invention falls within the scope of the claims according to the present invention.

[0040] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or may be manufactured by known methods.

[0041] DSPE-PEG 2000 -cRGD and DSPE-PEG 2000 -COOH was purchased from Xi'an Ruixi Biotechnology Co., Ltd., DQA was purchased from Sigma-Aldrich, oxaliplatin, N-hydroxysuccinimide (NHS), and carbodiimide hydrochloride (EDC) were purchased from Malcolm Chemical Reagents Co., Ltd., and 4-6 week old female BALB / c nude mice for animal experiments were obtained from Vital River (Beijing).

[0042] Example 1: Manufacturing of OXA@Exo-RD Figure 1 is a flowchart of the manufacturing process for OXA@Exo-RD according to the present invention. As shown in Figure 1, first, Exo-R is purified from the culture supernatant, then it is conjugated with DQA by a post-insertion treatment method to obtain Exo-RD, and finally, OXA is encapsulated in Exo-RD by electroporation, thereby sequentially forming the target and mitochondrial dysfunction drug delivery system OXA@Exo-RD. The specific manufacturing method is as follows.

[0043] 1.1 DSPE-PEG 2000 - DQA manufacturing 50 mg of 1,2-distearoylphosphatidylethanolamine-polyethylene glycol 2000 -carboxy(DSPE-PEG) 2000 Dissolve 2 mg of N-hydroxysuccinimide (NHS) and 6.8 mg of carbodiimide hydrochloride (EDC-HCl) in 4 mL of chloroform, then add 3 drops of triethylamine, and allow the reaction mixture to react at room temperature in the dark for 3 hours to prepare DSPE-PEG. 2000 - Obtain COO-NHS

[0044] Next, 9.2 mg of decalinium chloride (DQA) dissolved in DMSO (4 mL) is administered via DSPE-PEG. 2000 Add -COO-NHS and stir for 1 hour. Remove chloroform by rotary evaporation, then add 20 mL of deionized water. Dialyze the crude product in deionized water for 72 hours using a 3000 Da MWCO regenerated cellulose dialysis tube to remove uncoupled reactants, and freeze-dry to obtain a dry powder.

[0045] 1.2 Manufacturing of OXA@Exo-RD First, RGD-engineered exosomes (Exo-R) are obtained from the cell culture supernatant using gradient centrifugation. Specifically, hUMSCs are cultured for 24 hours in DMEM containing FBS with 10% exosomes removed and 1% penicillin-streptomycin. The culture medium is then DSPE-PEG. 2000Replace with DMEM containing cRGD (100 μg / mL) and continue culturing for 24 hours. When the cells reach 80-90% confluence, collect the medium using a pipette. Obtain Exo-R by gradient ultracentrifugation of the obtained supernatant. Resuspend the obtained Exo-R in PBS and store at -80°C for further analysis. All centrifugation should be performed at 4°C.

[0046] Next, DQA is incorporated into Exo-R using a post-insertion method to produce Exo-RD. Specifically, 40 μg / mL DSPE-PEG is used. 2000 Solution A is obtained by heating DQA in PBS buffer at 60°C for 15 minutes. Next, a 200 μg / mL Exo-R suspension is mixed with Solution A at 40°C for 3 hours. After cooling the crude product to room temperature, exosomes modified with both cRGD and DQA (Exo-RD) are obtained. Finally, 20 μg / mL oxaliplatin (OXA) is encapsulated in 200 μg / mL Exo-RD to obtain OXA@Exo-RD. Specifically, purified Exo-RD (or Exo-RD in PBS solution) (200 μg / mL) and oxaliplatin (or oxaliplatin in PBS solution) (20 μg / mL) are mixed, electroporated at 200 V and 100 μF using an X-Porator H1 electroporation system, and then the prepared OXA@Exo-RD is collected by centrifugation and stored at 4°C for later use.

[0047] In OXA@Exo-RD, the content of decalinium chloride (DQA) on the surface of the Exo-RD carrier is 7.3 wt%, and the content of oxaliplatin (OXA) inside the Exo-RD carrier is 5.8 wt%.

[0048] Comparative Example 1: Manufacturing of OXA@Exo The manufacturing method for OXA@Exo is as follows:

[0049] First, exosomes (Exo) are obtained from the cell culture supernatant using gradient centrifugation. Specifically, hUMSCs are cultured in DMEM containing FBS with 10% exosomes removed and 1% penicillin-streptomycin. When the cells reach 80-90% confluence, the medium is collected using a pipette. The obtained supernatant is obtained by gradient ultracentrifugation. The obtained Exo is resuspended in PBS and stored at -80°C for further analysis. All centrifugation is performed at 4°C.

[0050] Next, oxaliplatin (OXA) is encapsulated in Exo to obtain OXA@Exo. Specifically, purified Exo (200 μg / mL) and oxaliplatin (20 μg / mL) are mixed, electroporated at 200 V and 100 μF using an X-Porator H1 electroporation system, and the prepared OXA@Exo is collected by centrifugation and stored at 4°C for later use.

[0051] Comparative Example 2: Manufacturing of OXA@Exo-R The manufacturing method for OXA@Exo-R is as follows:

[0052] The manufacturing of Exo-R is the same as in Example 1.

[0053] Next, oxaliplatin (OXA) is encapsulated in Exo-R to obtain OXA@Exo-R. Specifically, purified Exo-R (200 μg / mL) and oxaliplatin (20 μg / mL) are mixed, electroporation is performed using an X-Porator H1 electroporation system at 200 V and 100 μF, and the prepared OXA@Exo-R is collected by centrifugation and stored at 4°C for later use.

[0054] Comparative Example 3: Manufacturing of OXA@Exo-D The manufacturing method for OXA@Exo-D is as follows:

[0055] DSPE-PEG 2000 -The manufacturing of DQA is the same as in Example 1.

[0056] Exosomes (Exo) were obtained from the cell culture supernatant using gradient centrifugation, and the production of Exo was the same as in Comparative Example 1.

[0057] Next, DQA is incorporated into Exo using a post-insertion method to produce Exo-D. Specifically, 40 μg / mL DSPE-PEG is used. 2000 -DQA is dissolved in PBS buffer and heated at 60°C for 15 minutes to obtain solution A. Next, a 200 μg / mL Exo suspension is mixed with solution A at 40°C for 3 hours. After the crude product is cooled to room temperature, exosomes (Exo-D) that are also modified with DQA are obtained.

[0058] Finally, oxaliplatin (OXA) is encapsulated in Exo-D to obtain OXA@Exo-D. Specifically, purified Exo-D (200 μg / mL) and oxaliplatin (20 μg / mL) are mixed, electroporated at 200 V and 100 μF using an X-Porator H1 electroporation system, and the prepared OXA@Exo-D is collected by centrifugation and stored at 4°C for later use.

[0059] Test Example 1 Figure 2 shows the transmission electron microscope characterization of Exo in Comparative Example 1 and OXA@Exo-RD nanoparticles in Example 1. As shown in Figure 2, TEM observation revealed that both Exo and OXA@Exo-RD were disk-shaped, which is a typical morphology for Exo and indicates that the extraction of Exo was successful and that its integrity was maintained after post-modification and drug encapsulation.

[0060] Test Example 2 The cytotoxicity of OXA, OXA@Exo (Comparative Example 1), OXA@Exo-R (Comparative Example 2), OXA@Exo-D (Comparative Example 3), and OXA@Exo-RD (Example 1) in OXA-sensitive and drug-resistant HCT116 cells was evaluated using the CCK-8 experiment.

[0061] The specific experimental procedure is as follows: First, HCT116 and HCT116 / OXA cells are placed in a 96-well plate (1 × 10⁶). 4The samples were inoculated into wells and incubated overnight. Next, OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD, each containing 0.01–20 μg / mL of Pt, were added to the culture plates and incubated for 24 hours. 10 μL of CCK-8 solution was added to each well and incubated for 2 hours, after which the absorbance at 450 nm was recorded using a microplate reader. After serial dilutions of the different drugs, IC was calculated using dose-response curves. 50 I calculated it.

[0062] The results of the analysis of the anticancer activity of OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD 24 hours after treatment with HCT116 and HCT116 / OXA cells are shown in Figure 3. Figure 3 shows that OXA@Exo-RD showed stronger cytotoxicity against HCT116 / OXA cells compared to OXA, and IC 50 The decrease in the index (0.87 μg / mL vs 8.09 μg / mL) also confirms this.

[0063] Furthermore, the ICs for HCT116 / OXA and HCT116 50 The (drug resistance factor) ratio decreased from 3.50 for OXA to 1.21 for OXA@Exo-RD, indicating that OXA@Exo-RD can reduce drug resistance in OXA.

[0064] Test Example 3 Considering that OXA@Exo-RD showed significant inhibitory effects against OXA-resistant HCT116 cells in the in vitro experiment of Test Example 2, this test example further evaluated the in vivo antitumor therapeutic effect of OXA@Exo-RD in a subcutaneous tumor-carrying model of colorectal cancer resistant to OXA.

[0065] BALB / c nude mice carrying HCT116 / OXA tumors were randomly divided into 6 groups (n=6), and PBS, OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD were administered intravenously every other day. Using OXA as the baseline, the equivalent doses of OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD were each 2 mg / kg, for a total of 5 doses.

[0066] The antitumor effect was reflected by monitoring the change in tumor volume over 21 days in different treatment groups. Figure 4 shows the in vivo anticancer results for PBS, OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD, where V represents the tumor volume at different treatment days and V0 represents the initial tumor volume. Figure 4 shows that OXA@Exo-RD showed the best growth inhibitory effect against subcutaneous OXA-resistant CRCs and resulted in the smallest tumor volume.

[0067] Test Example 4 Liver metastases are a leading cause of death in colorectal cancer patients. Given the crucial role of mitochondria in cancer metastasis, this study further investigated the anti-metastatic effects of OXA@Exo-RD in an HCT116 / OXA metastasis model.

[0068] In constructing a liver metastasis model, the mouse spleen was ligated and divided into two parts, and the vascular pedicles of each part were complete. 2 × 10⁶ in 50 μL of DMEM culture medium 6 Individual HCT116 / OXA cells were injected into the distal portion of the spleen. The injection site was gently massaged with a cotton swab for 5 minutes to prevent the spillage of tumor cells and to promote their migration to the liver. Five minutes after inoculation, when the tumor cells entered the portal vein, half of the spleen containing the injected cells was removed to mimic the resection of a primary tumor.

[0069] On day 1 after splenic injection, mice were randomly divided into 6 groups (n=3 mice per group). PBS, OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD were administered intravenously every other day, with an equivalent dose of 2 mg / kg for OXA, administered a total of 5 times. The equivalent doses of OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD were all 2 mg / kg, relative to OXA. Mouse body weight and activity levels were recorded throughout the administration period.

[0070] On day 21, mice were killed, liver metastases were counted, and the level of liver metastasis from colorectal cancer was assessed. The livers were harvested after treatment, fixed with 4% paraformaldehyde, and embedded in paraffin. The paraffin-embedded tumors were cut into 4 μm sections, stained with hematoxylin-eosin (H&E), and then subjected to histopathological analysis.

[0071] Figure 5 shows the anti-metastasis results for PBS, OXA, OXA@Exo, OXA@Exo-R, OXA@Exo-D, and OXA@Exo-RD, among which, A is a representative image of the liver after different treatments. B is the number of liver metastases in each group at the end of the study. C shows representative H&E stained images of liver tissue from each group.

[0072] As shown in Figures 5A and 5C, mice treated with PBS showed diffuse metastatic lesions in their livers and had the highest tumor burden based on liver weight, while OXA monotherapy had very little effect on liver metastatic tumors. As shown in Figure 5B, the number of liver metastases was lowest after OXA@Exo-RD treatment. Compared to the PBS group, the metastasis inhibition rates for the OXA, OXA@Exo, and OXA@Exo-RD groups were 31.9%, 51.2%, and 66.3%, respectively. Furthermore, OXA@Exo-D and OXA@Exo-RD increased the metastasis inhibition rates to 80.7% and 86.6%, respectively, which are approximately 1.5 and 1.7 times higher than the OXA group, demonstrating that OXA@Exo-RD has superior anti-metastatic ability.

[0073] The above examples illustrate specific embodiments of the present invention. However, the claims of this application are not limited to the above embodiments. Any modifications, equivalent substitutions, improvements, etc., made by those skilled in the art, without departing from the spirit and principles of the present invention, are also included within the scope of the claims of this application.

Claims

1. The present invention is characterized by comprising RGD engineered exosomes as a carrier, decalinium chloride modified on the surface of the carrier, and oxaliplatin encapsulated inside the carrier. Biomimetic nanodrugs.

2. The surface of the carrier is modified with decalinium chloride. Preferably, the decalinium chloride is inserted into the surface of the carrier, The biomimetic nanodrug according to claim 1.

3. The content of the decalinium chloride on the carrier surface is 5 to 10 wt%, Preferably, the content of oxaliplatin within the carrier is 5 to 10 wt%, The biomimetic nanodrug according to claim 1.

4. A method for producing a biomimetic nanodrug according to any one of claims 1 to 3, (1) To produce RGD-engineered exosomes modified with decalinium chloride, i.e., Exo-RD, and (2) The method is characterized by comprising encapsulating oxaliplatin in the Exo-RD to obtain OXA@Exo-RD. method.

5. Step (1) above is DSPE-PEG 2000 - This includes mixing DQA, PBS buffer, and Exo-R to obtain Exo-RD, Preferably, step (1) is (1a) DSPE-PEG 2000 - Mix DQA with PBS buffer to obtain a mixture. (1b) Mixing Exo-R with the mixture from step (1a), Preferably, in step (1b), the concentration of the Exo-R suspension is 100 to 300 μg / mL. Preferably, in step (1a), the DSPE-PEG of the mixed liquid 2000 -The concentration of DQA is characterized by being 20 to 100 μg / mL. The method according to claim 4.

6. In step (1b), Exo-R surface protein and DSPE-PEG 2000 -The mass ratio with DQA is 5:1 to 1:

15. Preferably, in step (2), the mass ratio of Exo-RD surface protein to oxaliplatin is 10:1 to 1:

10. The method according to claim 4.

7. Step (2) specifically involves employing electroporation to encapsulate oxaliplatin in Exo-RD. The method according to claim 4.

8. A biomimetic nanodrug comprising the biomimetic nanodrug described in any one of claims 1 to 3, Pharmaceutical composition.

9. A biomimetic nanodrug according to any one of claims 1 to 3 in the treatment of cancer, application.

10. A method for treating cancer, comprising administering a therapeutically effective amount of a biomimetic nanodrug according to any one of claims 1 to 3 to a patient in need thereof. Preferably, a method for reducing tumor growth, characterized by administering a therapeutically effective amount of a biomimetic nanodrug according to any one of claims 1 to 3 to a patient in need thereof. Preferably, the cancer is colon cancer and / or rectal cancer, for example, colorectal cancer. Preferably, the tumor is characterized by being a colon tumor and / or rectal tumor, for example, a colorectal tumor. method.

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