Nano-drug for co-delivery of evodiamine and Ppa as well as preparation method and application of nano-drug

Abstract

The invention discloses a nano-drug for co-delivery of evodiamine and Ppa as well as a preparation method and application of the nano-drug, and a pharmaceutical composition is a TPGS (at) EVO / Ppa nano-drug and comprises evodiamine (EVO), pyropheophorbide-A (Ppa) and a drug carrier vitamin E polyethylene glycol succinate (TPGS). The nano-drug has a synergistic anti-tumor effect, further enhances the immunotherapy effect, has a remarkable killing effect on tumor cells under laser irradiation, and can effectively inhibit tumor growth.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of pharmaceutical preparations, in particular to a nano-drug co-delivering evodiamine and Ppa, a preparation method and application thereof. BACKGROUND

[0002] In recent years, immunotherapy has attracted extensive attention in the treatment of triple-negative breast cancer (TNBC). Clinical research results show that chemotherapy combined with immune checkpoint therapy depending on the PD-1 / PD-L1 pathway has good therapeutic effect on TNBC, but due to the high level of immunosuppressive tumor microenvironment (TME) and low level of CD8 + T cell infiltration, only a small number of patients are sensitive to immunotherapy. Therefore, it is necessary to explore effective combination therapy to reduce the level of immunosuppressive TME and promote tumor infiltrating lymphocytes, such as chemo-immunotherapy or photodynamic-immunotherapy, which can synergistically enhance the immunotherapeutic effect of TNBC with immune checkpoint inhibitor therapy. The activation of immunotherapy requires the release of damage-associated molecular patterns (DAMPs), the capture of antigens by dendritic cells (DCs), the antigen presentation and maturation of DCs, and the production of killer T cells. To enhance immunotherapy, numerous strategies are being explored. Nanodrugs show great potential in improving tumor immunotherapy. For example, PDT not only directly kills tumor cells under laser irradiation using photosensitizers, but also induces immunogenic cell death (ICD) of tumor cells and promotes the release of DAMPs such as adenosine triphosphate (ATP), calreticulin (CRT), high mobility group protein B1 (HMGB1) from tumor cells, thereby stimulating the maturation of DCs. However, due to the low level of CD8 + T lymphocyte infiltration in tumor tissue, the ability of PDT to stimulate immunity alone is not enough to completely eliminate tumor metastasis. It has been reported that natural products have potential immunomodulatory effects. Therefore, co-delivery of natural products and photosensitizer molecules is of great significance to enhance the immunotherapeutic effect of TNBC.

[0003] Evodiamine (EVO) is a natural alkaloid extracted from Evodia rutaecarpa, which has significant anti-tumor activity and various pharmacological effects. It is reported that the anti-tumor effect of EVO is mainly related to inducing apoptosis of cancer cells, inhibiting proliferation of cancer cells, and affecting cancer cell cycle and migration. However, due to its poor water solubility and low bioavailability, its clinical application is greatly limited. Nanodrug delivery system has the advantages of increasing drug solubility, prolonging drug retention time in vivo, improving drug targeting ability through enhanced permeability and retention (EPR) effect, and reducing systemic toxicity. To achieve photodynamic-chemical-immune combination therapy for TNBC, co-delivery of Ppa and EVO is essential. SUMMARY

[0004] The present application aims to provide a nano-drug for co-delivery of evodiamine and Ppa and a preparation method thereof. The present application aims to design and solve the key scientific problems of nano-drugs with high loading efficiency and drug stability and realize the synergistic enhancement of triple negative breast cancer (TNBC) immunotherapy effect. The intermolecular π-π weak interaction strategy is adopted to design the nano-drug (TPGS@EVO / Ppa) based on vitamin E polyethylene glycol succinate (TPGS) carrier to realize the efficient co-delivery of natural product evodiamine and photosensitizer pyropheophorbide-A, and realize the synergistic treatment effect of TNBC photodynamic-chemotherapy-immune combined with immune checkpoint inhibitors.

[0005] To achieve the object of the present application, the following embodiments are provided.

[0006] In an embodiment, the present application provides a nano-drug for co-delivery of evodiamine and Ppa, which comprises evodiamine (EVO) and pyropheophorbide-A (Ppa) and a drug carrier vitamin E polyethylene glycol succinate (TPGS), referred to as TPGS@EVO / Ppa nano-drug.

[0007] In some embodiments, the TPGS@EVO / Ppa nano-drug of the present application, the weight ratio of evodiamine (EVO) to pyropheophorbide-A (Ppa) is (1-4):1, preferably, the weight ratio of EVO to Ppa is 2±0.1.

[0008] In some embodiments, the TPGS@EVO / Ppa nano-drug of the present application, the mass ratio of TPGS to the drug (EVO+Ppa total weight) is 2:1 to 4:1, preferably 4:1.

[0009] In some embodiments, the TPGS@EVO / Ppa nano-drug of the present application has a nanoparticle size of 135-146.5 nm.

[0010] In another embodiment, the present application provides a preparation method of the above-mentioned TPGS@EVO / Ppa nano-drug, comprising the following steps:

[0011] 1) dissolving EVO in an organic solvent to obtain an EVO solution;

[0012] 2) dissolving Ppa in an organic solvent to obtain a Ppa solution;

[0013] 3) dissolving TPGS in an organic solvent to obtain a TPGS solution;

[0014] 4) mixing the solutions of steps 1), 2) and 3), adding deionized water, stirring or ultrasonic treatment to prepare the TPGS@EVO / Ppa nano-drug.

[0015] In some embodiments, the present application provides a preparation method of the above-mentioned TPGS@EVO / Ppa nanomedicine, comprising dissolving EVO, Ppa and TPGS in an organic solvent, stirring, dissolving, mixing completely, adding deionized water, and continuing to stir to obtain the TPGS@EVO / Ppa nanomedicine.

[0016] In some embodiments, the preferred organic solvent is DMSO.

[0017] In some embodiments, the present application also provides a pharmaceutical composition comprising the TPGS@EVO / Ppa nanomedicine and a pharmaceutical excipient.

[0018] In some embodiments, the present application also provides the use of the TPGS@EVO / Ppa nanomedicine in the preparation of an anti-tumor drug.

[0019] In some embodiments, the application described above, the tumor is breast cancer, hepatocellular carcinoma, glioblastoma, ovarian cancer or lung cancer, more preferably triple negative breast cancer (TNBC).

[0020] Studies have shown that pyropheophorbide-a (Ppa) is an excellent porphyrin photosensitizer, which can improve the self-assembly ability with other small molecules through π-π weak interaction, and achieve good photodynamic immunotherapy effect. However, although EVO and Ppa both have good anti-tumor effect, the low bioavailability and poor solubility of the two bring great challenges to drug delivery. Vitamin E polyethylene glycol succinate (TPGS) is a water-soluble derivative synthesized by the reaction of succinic acid ester carboxyl of vitamin E with polyethylene glycol hydroxyl, which has both lipophilicity of vitamin E and hydrophilicity of polyethylene glycol long chain. In this study, TPGS was used as a drug carrier to co-deliver EVO and Ppa to prepare a nanomedicine (TPGS@EVO / Ppa, hereinafter referred to as "TEP"), and combined with an immune checkpoint inhibitor to achieve efficient photodynamic-chemical-immune combined therapy for TNBC. Experimental results show that the TEP nanomedicine has excellent endocytosis efficiency and ability to kill cancer cells.

[0021] The TEP nanomedicine designed in this study has the following advantages:

[0022] (1) EVO and Ppa molecules can improve the drug loading efficiency and stability of the drug through π-π weak interaction force, solving the problem of hydrophobicity and low bioavailability;

[0023] (2) Under photodynamic therapy, the TEP nanomedicine can kill primary tumor cells, achieve ICD effect of tumor cells, promote the release of DAMPs, and thus activate the acquired immunity of breast cancer;

[0024] (3) EVO not only can kill breast cancer cells, but also can further enhance the killing effect of killer T cells. In addition, subsequent immunotherapy with a-PD-L1 can further enhance systemic anti-tumor immunity. By designing the above nano-drug, the synergistic effect of photodynamic-chemical-immune combined therapy is achieved in the treatment of TNBC.

[0025] In summary, the present application aims at the technical problems that evodiamine (EVO) and pyropheophorbide-A (Ppa) have poor water solubility and low bioavailability, which greatly limits their clinical application. By using nano-drug loading technology, the solubility of the drug is increased, the in vivo retention time of the drug is prolonged, and the targeting ability of the drug is improved and the systemic toxicity is reduced through enhanced permeability and retention effect. Therefore, the present application designs and prepares nano-drug TPGS@EVO / Ppa (referred to as TEP) to co-deliver EVO and Ppa, and further synergistically enhances the immunotherapy effect. Experiments show that TEP has a significant killing effect on tumor cells under laser irradiation, and can effectively inhibit tumor growth. In addition, TEP promotes the release of damage-associated molecular patterns such as calreticulin, high mobility group protein B1 and adenosine triphosphate, thereby stimulating the maturation of dendritic cells and activating adaptive immunity, which significantly increases the proportion of killer T cells in the tumor, showing excellent photodynamic-chemotherapy-immune combined synergistic treatment effect. TEP as an innovative nano-drug provides a new strategy for preclinical treatment of tumors, especially triple-negative breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 1 is a schematic diagram of the preparation of TPGS@EVO / Ppa nano-drug and the mechanism of killing cancer cells and enhancing immunotherapy effect, wherein A is a flow chart of the preparation of nano-drug (TPGS@EVO / Ppa); B is a schematic diagram of the mechanism of killing cancer cells and enhancing immunotherapy effect of nano-drug.

[0027] Figure 2Figures for characterization, release, ROS generation of TPGS@EVO / Ppa nanodrugs of Example 2, wherein A-C are the nanodrug particle size distribution and polydispersity index (PDI) measured in water by dynamic light scattering (DLS): (A) TE, (B) TP, (C) TEP; D-F are the transmission electron microscopy images of the nanodrugs: (D) TE, (E) TP, (F) TEP; G is the potential (zeta potential) of the nanodrugs (TE, TP, TEP) in water; H-I are the changes in particle size (H) and PDI (I) of the nanodrugs in 7-day storage stability test; J is the in vitro 96-hour drug release curve of TEP; K is the change curve of the UV-Vis absorption spectrum of DPBF (singlet oxygen indicator fluorescent probe) in the wavelength range of 300-500 nm under laser irradiation at different times (0-120 seconds) for TEP+DPBF+laser group; L is the change rate of UV-Vis absorption spectrum of DPBF at 422 nm under different experimental conditions, all images are 200 nm scale, n=3.

[0028] Figure 3 Figures for the uptake of nanodrugs by 4T1 cells, light-induced ROS generation and in vitro cytotoxicity of Example 3, wherein A is the uptake of nanodrugs (5.0 pg / mL) by 4T1 cells at different time points (0.5 h and 2.0 h) (CLSM images), scale is 50 pm, n=3; B is the ability of different drugs to generate ROS in 4T1 cells, scale is 150 pm, n=3; C is the statistical analysis of flow cytometry for the quantification of ROS generated by different drugs in 4T1 cells; D is the survival rate of 4T1 cells after treatment with different concentrations of drugs for 48 hours; E is the statistical analysis of the apoptosis rate of cells after treatment with different drugs for 24 hours; F is the live / dead cell staining images of 4T1 cells after different treatments, scale is 200 pm, n=3; G is the apoptosis of 4T1 cells after treatment with different drugs for 24 hours analyzed by flow cytometry. (Ns means no statistical significance, * means p<0.05, ** means p<0.01, *** means p<0.001, **** means p<0.0001).

[0029] Figure 4Figure for evaluating results of in vitro induction of ICD and maturation of BMDCs of Example 4, wherein A is an immunofluorescence image of calreticulin exposed on the cell surface observed by CLSM; B is a statistical analysis of the average fluorescence intensity of CRT; C is an immunofluorescence image of drug-induced release of high mobility group box 1 protein B1 observed by CLSM; D is a statistical analysis of the average fluorescence intensity of HMGB1; E is a quantitative evaluation of adenosine triphosphate (ATP) release of 4T1 cells under different treatments; F is a schematic diagram of a co-culture model of mouse BMDCs and 4T1 cells; G is a flow cytometry result showing the maturation state (CD80+CD86+cell population) of DCs after co-culture; H is a statistical analysis of the proportion of mature DCs in different groups, all images have a scale of 50 μm, n = 3. (Ns represents no statistical significance, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001). DETAILED DESCRIPTION

[0030] The following examples are provided to further illustrate the present application. However, the following examples are provided only for the purpose of further illustrating the present application and are not intended to limit the present application. Those skilled in the art will understand that equivalent substitutions, or corresponding modifications, made to the content of the present application, still fall within the scope of protection of the present application.

[0031] In the following examples, the preparation route of TPGS@EVO / Ppa (hereinafter referred to as “TEP”) nanomedicine and the schematic diagram of the anticancer mechanism are as shown in Figure 1

[0032] Example 1 Synthesis and screening of nanomedicine TEP

[0033] Preparation method: Take 133.3 μL of EVO solution (10 mg / mL, DMSO), 66.7 μL of Ppa solution (10 mg / mL, DMSO), and 200.0 μL of TPGS solution (40 mg / mL, DMSO) and mix thoroughly, then add 2.6 mL of deionized water under probe sonication. After ice bath ultrasonic treatment for 3 min, TPGS@EVO / Ppa (TEP) nanomedicine solution is obtained.

[0034] Adjust the mass ratio of TPGS to drug (EVO+Ppa) to 2:1 and 4:1, and adjust the mass ratio of EVO to Ppa to 1:1, 2:1, 3:1 and 4:1, see Table 1 for specific dosages. Investigate the effect of the carrier on the nanomedicine preparation, and evaluate the effect of drug ratio on the characteristics of the nanomedicine, such as particle size, zeta potential, and stability in aqueous solution.

[0035] Table 1. Effect of different dosing ratios on the characteristics of nanomedicine TEP ​

[0036]

[0037] Preparation of comparative nanodrugs (TE and TP) :

[0038] Method for preparing TE (TPGS@EVO) : Take 133.3 μL of EVO solution (10 mg / mL, DMSO) and 200.0 μL of TPGS solution (40 mg / mL, DMSO) and mix thoroughly, and then add 2.667 mL of deionized water under the condition of probe ultrasonic. After ultrasonic treatment under ice bath condition for 3 min, TPGS@EVO (TE) nanodrug solution is obtained.

[0039] Method for preparing TP (TPGS@ / Ppa) : Take 66.7 μL of Ppa solution (10 mg / mL, DMSO) and 200.0 μL of TPGS solution (40 mg / mL, DMSO) and mix thoroughly, and then add 2.7333 mL of deionized water under the condition of probe ultrasonic. After ultrasonic treatment under ice bath condition for 3 min, TPGS@ / Ppa (TP) nanodrug solution is obtained.

[0040] The prepared TE nanodrug and TP nanodrug are used for subsequent comparison experiments of properties and activities of TEP.

[0041] Example 2 Nanodrug characterization

[0042] The TEP nanodrug used in the experiment of this example is the TEP nanodrug prepared in Example 1 with the feeding amount of experimental serial number 3 (under the condition of this mass ratio: TPGS: EVO: Ppa = 4: 0.67: 0.33) (named as TEP3).

[0043] Characterization of nanodrug: Transmission electron microscope and dynamic light scattering instrument are used to study the particle size, polydispersity index (PDI), zeta potential and morphology of the nanodrug.

[0044] UV-Vis absorption spectrum is measured by UV-Vis spectrophotometer;

[0045] EVO standard curve is prepared by high performance liquid chromatography;

[0046] Storage stability investigation: The TEP nanodrug is stored in aqueous solution, and its stability is investigated at room temperature for 7 days. Instability refers to unstable particle size or leakage of encapsulated drug, etc.

[0047] The encapsulation efficiency (EE%) and drug loading efficiency (LE%) of EVO in TE and TEP3 nanodrug are determined, and the encapsulation efficiency (EE%) of EVO in TE and TEP nanodrug is determined after dialysis in PBS aqueous solution for 12 hours.

[0048] The chromatographic conditions used in this study include: a reversed-phase chromatographic column (Phenomenx Luna 5U C18 100a, 250 x 4.6 mm, 5 μm), a mobile phase of a mixture of methanol and water (75:25, v / v), a flow rate of 1 mL / min, a column temperature of 35 °C, a detection wavelength of 225 nm, and an injection volume of 10 μL.

[0049] Finally, the EE% of EVO in different nanomedicines was calculated according to the amount of free EVO in the dialysate, and the formula is as follows:

[0050] EE% = 1 - (amount of unencapsulated EVO) / (amount of EVO administered) x 100%

[0051] The Ppa standard curve was prepared using a multifunctional enzyme marker (Spectra Max i 3x, Molecular Devices, USA) (excitation: 490 nm, emission: 670 nm), and the EE% of Ppa in TP and TEP3 nanomedicines was determined, and the formula is as follows:

[0052] EE% = 1 - (amount of unencapsulated Ppa) / (amount of Ppa administered) x 100%

[0053] The calculated EE% is shown in Table 2.

[0054] Table 2. EE% of EVO or Ppa in different nanomedicines

[0055] EVO (EE %) Ppa (EE %) EVO (LE %) Ppa (LE %) TE 92.1±0.67 -- 13.3±0.08 -- TP -- 99.7±0.01 -- 7.7±0.01 TEP 92.4±0.22 99.7±0.04 12.4±0.02 6.7±0.01

[0056] Evaluation of EVO release from TEP nanomedicines

[0057] The EVO drug release behavior (in terms of EVO) of TEP3 at different pH values was studied. The TEP solution was added to a dialysis bag (3000 Da) and sealed. Then the dialysis bag was immersed in 30.0 mL of PBS (containing 30% absolute ethanol, pH 5.0 or 7.4) and placed on a constant temperature shaker at 37 °C, 120 rpm. At predetermined time points, 3.0 mL of release medium was removed and replaced with fresh PBS. Finally, the drug concentration was determined by HPLC and the cumulative release curve of EVO was plotted.

[0058] Evaluation of the effect of nanomedicine TEP on ROS production

[0059] Indirect detection of reactive oxygen species (ROS) by DPBF probe: in the presence of ROS, the ultraviolet absorption of DPBF decreases due to oxidation. To 1 mL of TEP3 nanomedicine solution (100 μg / mL), 50 μL of DPBF solution (1 mM, DMF) was added, mixed thoroughly, and then a 660 nm laser (0.3 W / cm2 ) irradiation, and the UV absorption spectrum of the mixed solution was tested at different time points. At the same time, the UV absorption changes of DPBF under laser irradiation or the mixed solution of DPBF and TEP in the dark were tested under the same conditions.

[0060] The above investigation and experimental results are shown in Figure 2 . Figure 2 , A-C are the nanoparticle size distribution and polydispersity index (PDI) of the nanodrugs measured by dynamic light scattering (DLS) in water: (A) TE, (B) TP, (C) TEP; D-F are the transmission electron microscopy images of the nanodrugs: (D) TE, (E) TP, (F) TEP; G is the potential (Zeta potential) of the nanodrugs (TE, TP, TEP) in water; H-I are the changes in particle size (H) and PDI (I) of the nanodrugs in the 7-day storage stability test; J is the in vitro 96-hour drug release curve of TEP; K is the UV-visible absorption spectrum change curve of DPBF (singlet oxygen indicating fluorescent probe) in the wavelength range of 300-500 nm under laser irradiation at different times (0-120 seconds) in the TEP+DPBF+laser group; L is the change rate of the UV-visible absorption spectrum of DPBF at 422 nm under different experimental conditions. All images have a scale of 200 nm, n=3.

[0061] The successful preparation of the TEP nanodrug of the present application is proved by Figure 2 , Table 1, and Table 2. The nanodrug TPGS@EVO / Ppa (TEP) was prepared by loading EVO and Ppa under ultrasonic conditions through hydrophobic interaction and π-π stacking interaction using TPGS. The prescription feed ratio was screened by single factor experiment design (Table 1). When the proportion of EVO was larger, the drug loading was unstable, drug precipitation occurred, and it could not be further applied (TPGS: EVO: Ppa = 4:0.75:0.25). Previous experiments have shown that EVO requires a higher drug concentration than Ppa to produce effective cytotoxicity on tumor cells. Therefore, increasing the proportion of EVO in the nanodrug preparation helps to improve the efficacy of combination therapy, so the optimal feed ratio of EVO and Ppa was determined (TPGS: EVO: Ppa = 4:0.67:0.33). TEP exhibited good physicochemical properties, including suitable particle size, Zeta potential, polydispersity index (PDI), and stability. Single-drug-loaded nanodrugs TPGS@EVO (TE) and TPGS@Ppa (TP) were also prepared using the same method. Dynamic light scattering (DLS) results showed that the particle sizes of the three nanodrugs (TE, TP, TEP) were 188.6±3.3 nm, 92.9±3.1 nm, and 145.0±0.7 nm, respectively Figure 2A-C). Transmission electron microscopy (TEM) results showed that the morphology of the three nanodrugs (TE, TP, TEP3) were close to spherical, uniformly distributed, and no obvious aggregation phenomenon Figure 2 D-F), which was similar to the DLS measurement results. TEP had a smaller particle size than TE, which was beneficial to the better passive endocytosis of the nanodrug, and demonstrated the advantage of co-delivery of drugs. All the nanodrugs showed negative Zeta potential Figure 2 G), TEP had a lower potential than TE, which was more conducive to prolonging its blood circulation time in vivo. The PDI of the nanodrug was less than 0.3, indicating that the nanodrug had good size distribution. In addition, the stability test results showed that TEP remained good stability within 7 days, and its particle size and PDI did not change significantly Figure 1 H-I). The standard curve of EVO was established by high performance liquid chromatography (HPLC): y = 138.44x + 17.4, R 2 = 0.9999; at the same time, the multifunctional enzyme marker was used to generate the standard curve of Ppa: y = 3443619x + 104314, R 2 = 0.9989. Both curves showed excellent linear relationship, which was convenient for accurate calculation of the drug loading capacity (DLC) of each nanodrug by dialysis method. As shown in Table 2, TPGS showed significant drug loading capacity for EVO and Ppa, and the encapsulation efficiency was more than 90%. The loading efficiency of TPGS for EVO and Ppa was 12.4% (EVO) and 6.7% (Ppa), respectively.

[0062] The drug release study results further confirmed that the drug was released rapidly within 12 hours, and then the release rate slowed down. Finally, about 50% of EVO was released from the nanodrug within 96 hours. In addition, the laser had no significant effect on drug release in the experiment Figure 2 J). To verify its photodynamic therapy ability, the ability of the nanodrug to produce reactive oxygen species (ROS) was explored using the DPBF probe. The results showed that the ultraviolet absorption intensity of DPBF at 422 nm in the TEP + laser + DPBF group decreased with the increase of irradiation time Figure 2 K). These results showed that TEP could produce a large amount of ROS within 2 minutes under 660 nm laser irradiation Figure 2 L).

[0063] Example 3 Cell Uptake and Light-induced ROS Generation and Toxicity Experiments

[0064] The TEP nanodrug used in the experiment of this example was the TEP nanodrug prepared in Example 1 with the experimental serial number 3 (under the condition of this mass ratio: TPGS: EVO: Ppa = 4: 0.67: 0.33) (named TEP3).

[0065] Cellular uptake experiments:

[0066] The uptake of nanomedicine by 4T1 cells was analyzed by confocal laser scanning microscopy (CLSM, LSM880, Zeiss, Germany) and flow cytometry (FCM). 5 The density of cells / well was seeded in a 12-well plate containing a cell slide, incubated at 37°C for 24 hours, and then treated with TEP3 for 0.5, 2, and 4 hours, respectively. After treatment, the cells were fixed with 4% paraformaldehyde for 15 minutes, stained with DAPI (1.0 μg / mL) for 20 minutes, and washed 3 times. The cell slide was removed and placed on a slide and sealed with an anti-fluorescence quencher. Finally, CLSM was used to observe and capture fluorescence images. For flow cytometric analysis, 4T1 cells were collected after 0.5h and 2.0h of drug treatment, fixed with 4% paraformaldehyde, and then resuspended in PBS. Finally, the fluorescence intensity was detected by flow cytometry (Fortessa, BD, US).

[0067] Light-induced ROS generation:

[0068] The light-induced ROS production of 4T1 cells was analyzed by inverted fluorescence microscopy and flow cytometry. 4T1 cells in the logarithmic growth phase were plated at 1×10 5 The cells were seeded at a density of 100 cells / well in a 12-well plate. 4T1 cells were then cultured at 37°C and 5% CO2 for 24 hours. The cells were divided into control group, EVO group, TE group, TEP3 group, TP+L group, and TEP3+L group. The culture medium was discarded and the cells were washed once with PBS. Each group was treated with 1 mL of drug-containing (corresponding drug to each group) culture medium (equivalent to EVO 3.0 μg / mL) for 2 hours. The laser (L) treatment group was irradiated with laser (660 nm, 0.3 W / cm 2 , 3min), and then the cells were treated according to the instructions of the ROS detection kit. Fluorescence images of the cells were observed using an inverted fluorescence microscope (DMi8, Leica, Germany). In addition, the ability of different groups of nanomedicines (control group, EVO, TE, TEP3, TP+L, TEP3+L) to produce ROS at the cellular level was quantitatively verified by flow cytometry. The treated cells were collected, stained with 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), and the fluorescence intensity was measured by FCM.

[0069] In vitro cytotoxicity:

[0070] The cytotoxicity of different drug preparations on 4T1 cells was evaluated by CCK-8 assay. 44T1 cells were seeded in 96-well plates and cultured for 24 h. The cells were treated with different concentrations of EVO, TE, TP and TEP3, with or without laser irradiation (660 nm, 0.3 W / cm 2 , 2 min). After 48 h of treatment, 10 μL CCK-8 was added to each well and incubated at 37 °C for 1 h. Finally, the absorbance at 450 nm of each well was measured by a microplate reader to calculate the cell survival rate.

[0071] Cell survival rate was evaluated by live / dead cell staining. 4T1 cells were seeded in 12-well plates and treated with EVO, TE, TP and TEP3 after 24 h. The laser treatment group was given laser irradiation (660 nm, 0.3 W / cm 2 , 3 min) after 2 h of endocytosis. After 24 h of treatment, the culture medium was discarded and the cells were washed with PBS, and then PBS containing Calcein-AM and PI was added. After 20 min of incubation in the dark, the cells were observed using a fluorescence microscope.

[0072] Flow cytometry analysis of apoptosis:

[0073] 4T1 cells were seeded in 6-well plates and cultured for 24 h. Then the cells were treated with EVO, TE, TP and TEP3, with or without laser irradiation (660 nm, 0.3 W / cm 2 , 5 min). After 24 h of treatment, the cells were collected and washed with PBS, and then stained with Annexin V-FITC / PI to detect apoptosis by flow cytometry.

[0074] The results of the above experiments are shown in Figure 3 , where A is the uptake of the nanodrugs (5.0 μg / mL) by 4T1 cells at different time points (0.5 h and 2.0 h) (CLSM images), the scale bar is 50 μm, n = 3; B is the ability of different drugs to generate ROS in 4T1 cells, the scale bar is 150 μm, n = 3; C is the statistical analysis of the quantitative results of flow cytometry for the generation of ROS in 4T1 cells by different drugs; D is the survival rate of 4T1 cells after treatment with different concentrations of drugs for 48 h; E is the statistical analysis of the apoptosis rate of cells after treatment with different drugs for 24 h; F is the live / dead cell staining image of 4T1 cells after different treatments, the scale bar is 200 μm, n = 3; G is the apoptosis of 4T1 cells after treatment with different drugs for 24 h analyzed by flow cytometry. (Ns indicates no statistical significance, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001).

[0075] Figure 3The results showed that the TEP nanomedicine of the present invention has excellent uptake, light-induced ROS generation and in vitro cell killing effects on 4T1 cells. In this experiment, the cellular uptake ability of TEP3 was evaluated by incubating 4T1 cells for different time periods (0.5 or 2.0 hours). The results showed that the red fluorescence intensity on 4T1 cells gradually increased over time ( Figure 3 A). DCFH-DA was used as a probe to detect reactive oxygen species (ROS) in vitro. Observation under an inverted fluorescence microscope revealed that the TEP+laser group showed strong green fluorescence, indicating that a large amount of ROS was produced. At the same time, the TP+laser group also showed significant fluorescence. In contrast, the other groups (including the TEP3 group without laser irradiation) did not show significant green fluorescence ( Figure 3 B). In addition, FCM was used to quantitatively analyze the ROS production in each group. The results showed that the ROS fluorescence intensity in the TEP+laser group was 37% higher than that in the TP+laser group, which proved that the co-delivery of EVO and Ppa was beneficial to synergistically enhance the production of ROS ( Figure 3 C). The CCK-8 method was used to evaluate the killing ability of nanomedicine on 4T1 tumor cells within 48 hours. The results showed that the laser-irradiated nanomedicine (TP or TEP) group exhibited significant cytotoxic effects on 4T1 cells at low concentrations (EVO: 0.38μg / mL, Ppa: 0.19μg / mL), indicating that photodynamic therapy (PDT) can effectively induce 4T1 cell death. More importantly, the killing effect of the TEP+laser group was significantly better than that of the TP+laser group, which was attributed to the significant advantages of the combined treatment of EVO and Ppa ( Figure 3 D). Live / dead cell staining experiments further elucidated the killing efficiency of nanomedicines on 4T1 cells after different treatments. After staining with calcein-AM (green) and propidium iodide (PI, red), the TP+laser group and the TEP+laser group emitted obvious red fluorescence, indicating that the cells died. In contrast, although the other treatment groups did not show significant cytotoxicity, they significantly inhibited the proliferation of 4T1 cells, and the cell density in the fluorescence images was lower than that of the control group. In addition, although both the TP+laser group and the TEP+laser group showed significant killing ability, the cell density in the TEP+laser group was significantly lower than that in the TP+laser group ( Figure 3 F) Detect cell apoptosis rate by FCM. Figure 3 As shown in E and 3G, the non-laser treatment group had limited effect, with apoptosis rates of approximately 45.3±2.1%, 27.6±4.0%, and 23.1±3.0% in the TEP3, TE, and EVO groups, respectively. In contrast, the apoptosis rates in the laser treatment group were 70.3±8.6% in the TP+laser group and as high as 74.6±1.8% in the TEP+laser group, indicating that chemotherapy combined with PDT has a significant advantage in anti-tumor efficacy.

[0076] Example 4 Inducing ICD and DC in vitro

[0077] The TEP nanomedicine used in the experiment of this example is the TEP nanomedicine prepared in Example 1 with the feeding amount of experimental serial number 3 (under the condition of this mass ratio: TPGS: EVO: Ppa = 4: 0.67: 0.33) (named as TEP3).

[0078] Induction of immunogenic cell death (ICD)

[0079] 4T1 cells were seeded in 12-well plates containing cell slides at a density of 1 x 10 5 Cells were seeded in 12-well plates containing cell slides at a density of 1 x 10 2 After treatment, cells were fixed with 4% paraformaldehyde, then incubated with CRT and HMGB1 -related antibodies, respectively, and stained with DAPI (1.0 μg / mL). The cell slides were taken out and placed on glass slides, and then sealed with an anti-fluorescence quencher. Finally, observation and analysis were performed by CLSM. At the same time, a small amount of culture supernatant was collected 2 hours after laser irradiation, and ATP analysis was performed by an ATP detection kit.

[0080] Induction of DC (dendritic cell) maturation in vitro: Excess tissue of mouse hind limbs was removed, and the bone marrow in the bone marrow cavity was washed thoroughly with 1640 medium, and bone marrow-derived dendritic cells (BMDCs) were collected by filtration and centrifugation, then cultured in 1640 medium containing GM-CSF (20 ng / mL) for 1 week for standby use. 4T1 cells were seeded in the upper chamber of a Transwell chamber, treated with EVO, TE, TP and TEP, respectively, with or without laser irradiation (660 nm, 0.3 W / cm 2 , 3 min), and then co-cultured with BMDCs placed in a 24-well plate for 24 hours. Finally, DCs were collected, stained with anti-CD11c, anti-CD80 and anti-CD86 antibodies, and analyzed by FCM.

[0081] The experimental results are shown in Figure 4Evaluation of ICD induction and BMDC maturation in vitro. In the figure, A is the immunofluorescence image of calreticulin exposed on the cell surface observed by CLSM; B is the statistical analysis of the average fluorescence intensity of CRT; C is the immunofluorescence image of drug-induced high mobility group box 1 release observed by CLSM; D is the statistical analysis of the average fluorescence intensity of HMGB1; E is the quantitative evaluation of adenosine triphosphate (ATP) release of 4T1 cells under different treatments; F is a schematic diagram of the co-culture model of mouse BMDCs and 4T1 cells; G is the flow cytometry result showing the maturation state of DCs after co-culture (CD80+CD86+ cell population); H is the statistical analysis of the proportion of mature DCs in different groups. All images have a scale of 50 μm, n = 3. (Ns represents no statistical significance, * represents p < 0.05, ** represents p < 0.01, *** represents p < 0.001, **** represents p < 0.0001).

[0082] Figure 4 The results show that the TEP nanodrug of the present application can induce immunogenic death of tumor cells and stimulate maturation of dendritic cells in vitro. In the process of ICD of tumors, cells release a large amount of DAMPs, which can activate immune cells and stimulate anti-tumor immune response. Using CLSM, it can be clearly observed that the CRT signal (green) in the photodynamic therapy (PDT) treatment group (TP + laser and TEP + laser). The CRT positive rate of the TP + laser group is 16.2 times that of the control group, and that of the TEP + laser group is 17.85 times that of the control group, which indicates that the synergistic effect of EVO and Ppa under laser irradiation may increase the exposure of CRT Figure 4 A-B). HMGB1 is a non-histone chromatin-binding protein that is released into the extracellular space when ICD occurs. As shown in Figure 4 C-D, in the Ppa-mediated PDT treatment group (TP + laser and TEP + laser), HMGB1 shows significant efflux (green), and the fluorescence intensity of HMGB1 in the TEP + laser group is only 20.1% of that in the control group, which is much lower than that in the control group. In contrast, the nanodrug group without laser irradiation shows strong green fluorescence and is co-localized with DAPI staining, indicating that there is no significant efflux of HMGB1. In addition, ATP is another DAMP that can be released from the cytoplasm due to cell damage during ICD. Compared with the control group, the ATP concentration in the TP + laser group and the TEP + laser group increased by 1.5 times and 5.4 times, respectively Figure 4 E). The co-culture experiment was used to study the stimulating effect of 4T1 cells after ICD on the maturation of dendritic cells (DCs) Figure 4F). Flow cytometry analysis showed that the maturity of DCs (CD11c+CD80+CD86+) in the TEP+ laser group was 2.9 times higher than that in the control group. This result confirmed that the ICD effect of 4T1 cells can induce DCs maturation.

Claims

1. A nanomedicine for co-delivering evodiamine and Ppa, comprising evodiamine (EVO), pyropheophorbide-A (Ppa) and a drug carrier vitamin E polyethylene glycol succinate (TPGS).

2. The nanomedicine according to claim 1, wherein the weight ratio of evodiamine (EVO) to pyropheophorbide-A (Ppa) is (1-4):1, and preferably the weight ratio of EVO to Ppa is 2±0.

1.

3. The nanomedicine according to claim 1, wherein the mass ratio of TPGS to the drug is 2:1 to 4:1, preferably 4:1, and the mass of the drug is the total weight of EVO and Ppa.

4. The nanomedicine according to any one of claims 1 to 3, wherein the nanoparticle size is 135-146.5 nm.

5. A method for preparing the nanomedicine according to any one of claims 1 to 3, comprising the following steps: 1) dissolving evodiamine (EVO) in an organic solvent to obtain an EVO solution; 2) dissolving pyropheophorbide-A (Ppa) in an organic solvent to obtain a Ppa solution; 3) dissolving TPGS in an organic solvent to obtain a TPGS solution; 4) The solutions of steps 1), 2) and 3) are mixed, deionized water is added, and the mixture is stirred or ultrasonically treated to prepare TPGS@EVO / Ppa nanomedicine.

6. A method for preparing the nanodrug according to any one of claims 1-3, comprising dissolving EVO, Ppa and TPGS in an organic solvent, stirring, dissolving and mixing completely, adding deionized water, and continuing to stir to obtain TPGS@EVO / Ppa nanodrug.

7. The preparation method according to claim 5 or 6, wherein the organic solvent is dimethyl sulfoxide (DMSO).

8. A pharmaceutical composition comprising the nanodrug according to any one of claims 1 to 3 and pharmaceutical excipients.

9. Use of the nanomedicine according to any one of claims 1 to 3 in the preparation of anti-tumor drugs.

10. The use according to claim 9, wherein the tumor is breast cancer, hepatocellular carcinoma, glioblastoma, ovarian cancer or lung cancer.

Images