Nano-composite for targeted induction of mitochondrial autophagy as well as preparation method and application of nano-composite

Abstract

The invention relates to a nano compound for targeted induction of mitochondrial autophagy as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The invention provides a nano-composite for targeted induction of mitochondrial autophagy. The nano-composite comprises a mitochondrial autophagy inducer and siPD-L1, wherein the surface of the mitochondrial autophagy inducer is provided with a Meo-PEG-b-PDPA block copolymer. According to the invention, the mitochondrial autophagy inducer is matched with siPD-L1, and the Meo-PEG-b-PDPA block copolymer is added for coating to obtain the nano-composite for targeted induction of mitochondrial autophagy, so that the nano-composite can break through a series of physiological barriers, is targeted and positioned to a tumor site, penetrates tumor tissues and cell membranes, and can be used for inducing mitochondrial autophagy. And a mitochondrial autophagy inducer and siPD-L1 are effectively released in cytoplasm, so that excessive mitochondrial autophagy and PD-L1 silencing of tumor cells are induced, and the anti-tumor effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular, to a nanocomplex for targeted induction of mitophagy, a preparation method thereof, and applications thereof. Background Art

[0002] In recent years, immunotherapy has been widely used in the treatment of refractory solid tumors. In particular, immune checkpoint blockade therapies targeting cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death 1 (PD-1), and its ligand PD-L1 have significantly changed the current cancer treatment landscape. However, treatment responses are only seen in a small fraction of cancer patients, and the vast majority of patients do not gain survival benefits from immune blockade therapies. This outcome is mainly due to the immunosuppressive tumor microenvironment (TME), which can induce the exhaustion of tumor-infiltrating immune cells, especially CD8 + T cells. To address this issue, therapies are combined with other treatment modalities (such as chemotherapy) to enhance anti-tumor immunity by remodeling the immunosuppressive TME. However, clinical observations have shown that due to the high termination rate caused by severe adverse events, most cancer patients do not obtain durable benefits from this combination therapy. Therefore, there is an unmet need to develop new treatment strategies to improve anti-tumor immunity and expand the scope of cancer immunotherapy. Summary of the Invention

[0003] An object of the present invention is to overcome the deficiencies of the prior art and provide a nanocomplex for targeted induction of mitophagy, a preparation method thereof, and applications thereof, which can improve tumor immunity and expand cancer immunotherapy.

[0004] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0005] In a first aspect, the present invention provides a nanocomplex for targeted induction of mitophagy, the nanocomplex comprising a mitophagy inducer with a Meo-PEG-b-PDPA block copolymer on its surface and siPD-L1;

[0006] The ratio of the mitophagy inducer to siPD-L1 is mitophagy inducer: siPD-L1 = (0.01 - 0.10) mg: 1 nmol;

[0007] The ratio of siPD-L1 to the Meo-PEG-b-PDPA block copolymer is siPD-L1: Meo-PEG-b-PDPA block copolymer = 1 nmol: (1 - 5) mg.

[0008] Mitophagy is a conserved cellular process that maintains mitochondrial stability and health by eliminating dysfunctional or senescent mitochondria. Mitophagy plays a double-edged role in regulating cell fate. Moderate mitophagy, as a protective mechanism, can eliminate damaged mitochondria to promote cell survival, while excessive mitophagy can induce the loss of mitochondrial function, leading to cell death. Based on the important role of mitophagy in regulating cell fate and the immune microenvironment, targeted induction of mitophagy in tumor cells may be a novel and promising strategy to simultaneously induce tumor cell death and reshape the immunosuppressive TME to enhance cancer immunotherapy.

[0009] The RNA interference (RNAi) technology, which has developed rapidly in recent years, has achieved ideal results in the field of tumor treatment. For target genes, siRNA has better target specificity and selectivity, can silence the mRNA of the target gene to inhibit its translation, and does not affect the expression of other genes in cancer cells. The biggest challenge in the clinical translation of RNAi technology is how to develop low-toxic and highly efficient nucleic acid carriers to help siRNA drugs break through a series of physiological barriers, target tumor cells, penetrate tumor tissues and tumor cell membranes, efficiently escape from endosomes, and effectively release siRNA in the cytoplasm, etc.

[0010] In the present invention, a mitophagy inducer is combined with siPD-L1 and coated with a Meo-PEG-b-PDPA block copolymer to form a nano-complex for targeted induction of mitophagy, which enables the nano-complex to break through a series of physiological barriers, target and localize to the tumor site, penetrate tumor tissues and cell membranes, efficiently escape from endosomes, and effectively release the mitophagy inducer and siPD-L1 in the cytoplasm, thereby inducing excessive mitophagy and PD-L1 silencing in tumor cells, reversing the tumor immune microenvironment, improving the therapeutic effect of immune checkpoint blockade, and thus achieving anti-tumor efficacy.

[0011] As a preferred embodiment of the nano-complex of the present invention, the ratio of the mitophagy inducer to siPD-L1 is mitophagy inducer:siPD-L1 = (0.05 - 0.08) mg:1 nmol.

[0012] As a preferred embodiment of the nano-complex of the present invention, the ratio of the mitophagy inducer to siPD-L1 is mitophagy inducer:siPD-L1 = 0.08 mg:1 nmol.

[0013] The ratio of siPD-L1 to the Meo-PEG-b-PDPA block copolymer is siPD-L1:Meo-PEG-b-PDPA block copolymer = 1 nmol:(1.5 - 4) mg.

[0014] The ratio of the siPD-L1 to the Meo-PEG-b-PDPA block copolymer is siPD-L1: Meo-PEG-b-PDPA block copolymer = 1 nmol: 2 mg.

[0015] As a preferred embodiment of the nanocomplex of the present invention, the mitophagy inducer includes at least one of carbonyl cyanide, fisetin, and resveratrol.

[0016] As a preferred embodiment of the nanocomplex of the present invention, the carbonyl cyanide includes 3-chlorophenylhydrazone and / or carbonyl cyanide 4-trifluoromethoxyphenylhydrazone.

[0017] As a preferred embodiment of the nanocomplex of the present invention, the mitophagy inducer is 3-chlorophenylhydrazone.

[0018] As a preferred embodiment of the nanocomplex of the present invention, the siPD-L1 is double-stranded RNA, and the nucleotide sequence of the siPD-L1 is shown in SEQ ID NO.1-2 and / or SEQ ID NO.3-4.

[0019] In a second aspect, the present invention provides a method for preparing the above-mentioned nanocomplex, and the nanocomplex is prepared by a nanoprecipitation method.

[0020] As a preferred embodiment of the preparation method of the present invention, the nanoprecipitation method includes the following operations:

[0021] S1. Mix the mitophagy inducer with siPD-L1 to obtain a core.

[0022] S2. Mix the core obtained in step S1 with the Meo-PEG-b-PDPA block copolymer, add water at 700-1100 rpm, ultrafilter, and wash to obtain a nanocomplex for targeting and inducing mitophagy.

[0023] As a preferred embodiment of the preparation method of the present invention, in step S1, the solvent of the mitophagy inducer is N,N-dimethylformamide, and the solvent of the siPD-L1 is water.

[0024] As a preferred embodiment of the preparation method of the present invention, in step S2, the ultrafiltration is through an ultrafiltration membrane with a molecular weight cut-off of 900-110 kDa.

[0025] As a preferred embodiment of the preparation method of the present invention, in step S2, the ultrafiltration is through an ultrafiltration membrane with a molecular weight cut-off of 100 kDa.

[0026] As a preferred embodiment of the preparation method of the present invention, in step S2, the washing is carried out 3 - 4 times with a phosphate buffer solution.

[0027] As a preferred embodiment of the preparation method of the present invention, in step S2, the washing is carried out 3 times with a phosphate buffer solution.

[0028] In the third aspect, the present invention provides the application of the above-mentioned nanocomposite in the preparation of anti-tumor drugs.

[0029] The present invention has confirmed through in vitro and in vivo experiments that CCCP can induce mitochondrial autophagy in tumor cells to inhibit the transcriptional expression of the immunosuppressive chemokine CCL2 in tumor cells, thereby inhibiting the entry of immunosuppressive monocytes into the tumor immune microenvironment and enhancing the immune checkpoint blockade therapy for tumors.

[0030] In the fourth aspect, the present invention provides an anti-tumor drug, comprising the above-mentioned nanocomposite and pharmaceutically acceptable excipients.

[0031] As a preferred embodiment of the anti-tumor drug of the present invention, the dosage form of the anti-tumor drug includes at least one of solid dosage forms, semi-solid dosage forms, liquid dosage forms, gas dosage forms, injection dosage forms and special dosage forms, and the special dosage forms include but are not limited to transdermal patches, implants, suppositories, etc.

[0032] As a preferred embodiment of the anti-tumor drug of the present invention, the types of tumors targeted by the anti-tumor drug include but are not limited to at least one of breast cancer, lung cancer, gastric cancer, liver cancer, pancreatic cancer, colorectal cancer and prostate cancer.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] The present invention combines a mitochondrial autophagy inducer with siPD-L1 and coats it with a Meo-PEG-b-PDPA block copolymer to form a nanocomposite that targets and induces mitochondrial autophagy. The nanocomposite can break through a series of physiological barriers, target and localize to the tumor site, penetrate tumor tissues and cell membranes, efficiently escape from endosomes, and effectively release the mitochondrial autophagy inducer and siPD-L1 in the cytoplasm. Furthermore, it can induce excessive mitochondrial autophagy and PD-L1 silencing in tumor cells, reverse the tumor immune microenvironment, improve the therapeutic effect of immune checkpoint blockade, and thus achieve anti-tumor efficacy. Description of the Drawings

[0035] Figure 1 It is the particle size distribution diagram of the nanocomposite obtained in Example 1 of Effect Example 1 of the present invention;

[0036] Figure 2 It is the transmission electron microscope image of the nanocomposite obtained in Example 1 of Effect Example 1 of the present invention;

[0037] Figure 3 Release curves of CCCP (A) and siPD-L1 (B) of NPs (CCCP / siPD-L1) in Example 1 of the effects of the present invention under different pH environments;

[0038] Figure 4 Effects of different treatments on the expression levels of PD-L1 mRNA (A) and protein (B) in MDA-MB-231 cells in Example 2 of the effects of the present invention;

[0039] Figure 5 Localization of NPs (CCCP / siPD-L1) in MDA-MB-231 cells treated differently in Example 2 of the effects of the present invention;

[0040] Figure 6 Effects of different treatments on the expression levels of mitophagy-related proteins in MDA-MB-231 cells in Example 3 of the effects of the present invention;

[0041] Figure 7 Effects of different treatments on mitochondria and lysosomes in MDA-MB-231 cells in Example 3 of the effects of the present invention;

[0042] Figure 8 Effects of different treatments on the ATP content (A), proliferation rate (B), and clone number (C) of MDA-MB-231 cells in Example 3 of the effects of the present invention;

[0043] Figure 9 Effects of different treatments on the clone number of MDA-MB-231 cells in Example 3 of the effects of the present invention;

[0044] Figure 10 Effects of different treatments on the differential gene expression levels of MDA-MB-231 cells in Example 4 of the effects of the present invention;

[0045] Figure 11 Effects of different treatments on the expression levels of CCL2 mRNA (A) and protein (B) and extracellular concentration (C) of MDA-MB-231 cells in Example 4 of the effects of the present invention;

[0046] Figure 12 Effects of cells treated with NPs (CCCP / siPD-L1) on the recruitment of immunosuppressive monocytes in Example 4 of the effects of the present invention;

[0047] Figure 13 Effects of different treatments on tumors in 4T1 orthotopic tumor-bearing mice in Example 5 of the effects of the present invention;

[0048] Figure 14Effects of different treatments on tumor volume (A) and weight (B) of 4T1 orthotopic tumor-bearing mice in Example 5 of the present invention;

[0049] Figure 15 Effects of different treatments on tumor metastasis in lung metastasis model mice in Example 5 of the present invention;

[0050] In the above figures, those marked with "*" indicate significant differences between the two groups (p < 0.05), those marked with "**" indicate significant differences between the two groups (p < 0.01), those marked with "***" indicate significant differences between the two groups (p < 0.001), and those marked with "****" indicate significant differences between the two groups (p < 0.0001);

[0051] PBS is PBS treatment, Free CCCP is treatment with uncoated CCCP, Control NPs is treatment with NPs (G0-C14 / siPD-L1), NPs (CCCP / siCTL) is treatment with NPs (CCCP / siCTL), and NPs (CCCP / siPD-L1) is NPs (CCCP / siPD-L1). Detailed implementation manners

[0052] To better illustrate the purpose, technical solution and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0053] Other materials, reagents, etc. used in the examples, comparative examples and effect examples can be obtained from commercial channels without special instructions.

[0054] 3-chlorophenylhydrazone (hereinafter referred to as CCCP) was purchased from MedChemExpress, with the product number HY-100941. A CCCP solution with a concentration of 8 mg / mL (the solvent is N,N-dimethylformamide, DMF) was prepared before preparing the nanocomposite.

[0055] Methoxy-polyethylene glycol-b-poly(2-(diisopropylamino)ethyl methacrylate) (hereinafter referred to as Meo-PEG-b-PDPA block copolymer) was purchased from Suzhou Juling High Polymer Technology Co., Ltd.

[0056] The cationic lipid compound alkyl-modified polyamidoamine dendrimer (hereinafter referred to as G0-C14) was purchased from Suzhou Juling High Polymer Technology Co., Ltd.

[0057] siPD-L1 is a small interfering RNA targeting the PD-L1 gene. In the following Examples, Comparative Examples and Efficacy Examples, siPD-L1 labeled with Cy5 is denoted as Cy5-siPD-L1, which was prepared by commissioning a biological company. The nucleotide sequences of human siPD-L1 are shown in SEQ ID NO.1-2, and the nucleotide sequences of murine siPD-L1 are shown in SEQ ID NO.3-4. In the following Examples, Comparative Examples and Efficacy Examples, whether the siPD-L1 is human or murine can be selected according to the experimental subject, and it is formulated into a 0.1 nmol / μL siPD-L1 solution (the solvent is enzyme-free ultrapure water) before preparing the nanocomplex.

[0058] siCTL is a control small interfering RNA of siPD-L1 without biological activity. The siCTL of the present invention was prepared by commissioning a biological company. The nucleotide sequence of siCTL is shown in SEQ ID NO.5-6, and it is formulated into a 0.1 nmol / μL siCTL solution (the solvent is enzyme-free ultrapure water) before preparing the nanocomplex.

[0059] The "dT" at the 3' end of the nucleotide sequences of the above siRNAs all represents deoxythymidine (T).

[0060] MDA-MB-231 cells are a human-derived breast cancer cell line, and 4T1 cells are a mouse-derived breast cancer cell line. The cell culture conditions are 37 °C and 5% CO2.

[0061] The qRT-PCR primer sequences mentioned in the following Efficacy Examples are as follows (5'-3'):

[0062] PD-L1(human)-F: TGC CGA CTA CAA GCG AAT TAC TG;

[0063] PD-L1(human)-R: CTG CTT GTC CAG ATG ACT TCG G;

[0064] PD-L1(mouse)-F: TGC GGA CTA CAA GCG AAT CAC G;

[0065] PD-L1(mouse)-R: CTC AGC TTC TGG ATA ACC CTC G;

[0066] GAPDH(human)-F: GTC TCC TCT GAC TTC AAC AGC G;

[0067] GAPDH(human)-R: ACC ACC CTG TTG CTG TAG CCA A;

[0068] GAPDH (mouse)-F: CAT CAC TGC CAC CCA GAA GAC TG;

[0069] GAPDH (mouse)-R: ATG CCA GTG AGC TTC CCG TTC AG;

[0070] CCL2 (human)-F: AGA ATC ACC AGC AGC AAG TGT CC;

[0071] CCL2 (human)-R: TCC TGA ACC CAC TTC TGC TTG G;

[0072] CCL2 (mouse)-F: GCT ACA AGA GGA TCA CCA GCA G;

[0073] CCL2 (mouse)-R: GTC TGG ACC CAT TCC TTC TTG G;

[0074] CXCL3 (human)-F: TTC ACC TCA AGA ACA TCC AAA GTG;

[0075] CXCL3 (human)-R: TTC TTC CCA TTC TTG AGT GTG GC;

[0076] IL-1β (human)-F: CCA CAG ACC TTC CAG GAG AAT G;

[0077] IL-1β (human)-R: GTG CAG TTC AGT GAT CGT ACAGG;

[0078] CCL20 (human)-F: AAG TTG TCT GTG TGC GCA AAT CC;

[0079] CCL20 (human)-R: CCATTC CAG AAA AGC CAC AGT TTT;

[0080] CXCL2 (human)-F: GGC AGA AAG CTT GTC TCA ACC C;

[0081] CXCL2 (human)-R: CTC CTT CAG GAA CAG CCA CCA A;

[0082] CSF2 (human)-F: GGA GCATGT GAATGC CAT CCA G;

[0083] CSF2 (human)-R: CTG GAG GTC AAACAT TTC TGA GAT;

[0084] IL-6 (human)-F: AGA CAG CCA CTC ACC TCT TCA G;

[0085] IL-6 (human)-R: TTC TGC CAG TGC CTC TTT GCT G.

[0086] All the kits mentioned in the following effect examples are commercially available. For example, lysosome, mitochondrion, and nucleus staining are completed by fluorescence probe staining (40741ES, 40738ES, and 40728ES03 purchased from Yeasen Biotechnology Co., Ltd.), and the ATP detection kit can use HY-K0314 purchased from MedChemExpress (MCE).

[0087] Examples 1 - 3

[0088] Examples 1 - 3 respectively provide a nanocomposite for targeting and inducing mitophagy and a preparation method thereof. The components and their dosages of the nanocomposite are shown in Table 1. The preparation method includes the following steps:

[0089] (1) Mix the DMF solution of the mitophagy inducer with the aqueous solution of siPD-L1 to obtain the core.

[0090] (2) Mix the core obtained in step (1) with the Meo-PEG-b-PDPA block copolymer, add 5 mL of deionized water at 1000 rpm, then perform ultrafiltration (the cut-off molecular weight of the ultrafiltration membrane is 100 kDa), wash 3 times with PBS to obtain the nanocomposite for targeting and inducing mitophagy. Resuspend the nanocomposite in deionized water and adjust the concentration of siRNA in the nanocomposite to 1 nmol / mL.

[0091] Table 1 Components and Dosages of Different Nanocomposites

[0092]

[0093] Comparative Example 1

[0094] Comparative Example 1 provided a nanocomposite for targeted induction of mitophagy and its preparation method. The components and dosages of the nanocomposite were similar to those in Example 1, except that siPD-L1 was replaced with siCTL, and the other components and dosages remained unchanged. The preparation method was the same as that in Example 1, and this material was named NPs(CCCP / siCTL).

[0095] Comparative Example 2

[0096] Comparative Example 2 provided a nanocomposite for targeted induction of mitophagy and its preparation method. The components and dosages of the nanocomposite were similar to those in Example 1, except that CCCP was replaced with G0-C14, and the other components and dosages remained unchanged. The preparation method was the same as that in Example 1, and this material was named Control NPs.

[0097] Effect Example 1

[0098] The nanocomposites obtained in Examples 1-3 were characterized, and the specific scheme was as follows:

[0099] 1. The average particle size, Zeta potential, and polydispersity index (PdI) of the nanocomposites obtained in Examples 1-3 were measured by dynamic light scattering method, and the results are shown in Table 2 and Figure 1 .

[0100] Table 2 Measured values of average particle size, Zeta potential, and PdI of different nanocomposites

[0101] Group Average Particle Size (nm) Zeta Potential (mV) PdI Example 1 78 8.9 0.10 Example 2 75 7.2 0.15 Example 3 72 8.1 0.19

[0102] As shown in Table 2, the method of the present invention can successfully prepare nanocomposites with an average particle size of 72-78 nm. Based on parameters such as reagent dosage and the size of the nanocomposite, the nanocomposite in Example 1 was selected as the optimal product, and the nanocomposite in Example 1 was named NPs(CCCP / siPD-L1).

[0103] 2. 10 μL of NPs(CCCP / siPD-L1) was dropped onto a copper grid and left to stand for 5 min, then the excess sample was removed by filter paper. Then, 10 μL of 2% v / v uranyl acetate was dropped for negative staining for 5 min. After removing the excess sample with filter paper, it was air-dried overnight in the dark and observed using a transmission electron microscope. As Figure 2 shown, due to the above treatment, the particle size of NPs(CCCP / siPD-L1) decreased but still showed a uniform spherical shape.

[0104] 3. Determination of the encapsulation efficiency of the nanocomposite obtained in Example 1 by dialysis: The nanocomposite labeled with Cy5 (hereinafter referred to as NPs(CCCP / Cy5-siPD-L1)) was prepared according to the method of Example 1, dispersed in 1 mL of PBS, transferred to a dialysis bag with a molecular weight cut-off of 100 kDa, and the dialysis bag was placed in PBS with pH = 7.4 or pH = 6.0. At predetermined time intervals, 5 μL of the nanoparticle solution was taken out and mixed with 100 μL of DMSO, the Cy5 fluorescence intensity was detected, and the CCCP absorption signal at a wavelength of 370 nm was detected using an ultraviolet spectrometer, and the cumulative release rates of siPD-L1 and CCCP were calculated. The results are shown in Figure 3 , and the cumulative release rate calculation formula is as follows:

[0105] Cumulative release rate (%) = (M t / M ∞ ) × 100

[0106] In the formula, M t is the siRNA or CCCP released from the nanoparticles at the specified time point; M ∞ is the total amount of siRNA or CCCP loaded in the nanoparticles.

[0107] As Figure 3 shown, the release amounts of CCCP and siRNA are higher in an environment with pH = 6.0, and the TME is an environment closer to pH = 6.0, that is, the NPs(CCCP / siPD-L1) of the present invention can release the core (CCCP and siPD-L1) more efficiently in the TME environment.

[0108] Effect Example 2

[0109] To evaluate the function of NPs(CCCP / siPD-L1) in silencing PD-L1 and inducing autophagy in vitro, NPs(CCCP / siPD-L1) was co-cultured with cells. The specific protocol is as follows:

[0110] 1. Seed MDA-MB-231 cells at 5 × 10 per well 4Cells were seeded in 6-well plates at a density of [cell density], with each well containing 2 mL of DMEM (containing 10% v / v fetal bovine serum). After incubation for 24 h, NPs (CCCP / Cy5-siPD-L1) were added to the cells at doses of 0, 10, 30, or 50 nM siRNA (i.e., the final concentration of siPD-L1 in each well was 0, 10, 30, or 50 nM). NPs (CCCP / siCTL) were used as the control group (the final concentration of siCTL was 50 nM). After culturing for 72 h, total RNA and proteins were extracted from the cells in each treatment group. The mRNA and protein expression levels of PD-L1 and GAPDH were detected by qRT-PCR and Western blotting, respectively. The expression of PD-L1 in the treatment group of NPs (CCCP / Cy5-siPD-L1) with 50 nM siPD-L1 was observed by laser confocal microscopy. Each treatment had at least 3 replicates. The above results are shown in Figures 4 - 5 .

[0111] As Figure 4 shown, when the concentration of siPD-L1 in NPs (CCCP / Cy5-siPD-L1) was 10 - 50 nM, it could effectively reduce the mRNA and protein expression levels of PD-L1 in MDA-MB-231, and the silencing effect of PD-L1 was dose-dependent on the siRNA dose.

[0112] As Figure 5 shown, after treatment with NPs (CCCP / Cy5-siPD-L1), blue fluorescence emitted by Cy5 labeling was observed in MDA-MB-231 cells, indicating that the nanocomplex NPs (CCCP / siPD-L1) of the present invention could enter tumor cells to release siPD-L1 to interfere with the expression of PD-L1 in tumor cells.

[0113] 2. MDA-MB-231 cells were co-cultured with NPs (CCCP / siPD-L1), NPs (CCCP / siCTL), or Control NPs with a siRNA concentration of 50 nM according to the method of Experiment 1 above. After incubation for 72 h, the cells were washed with PBS, and total proteins were extracted from the cells in each treatment group for Western blotting to detect the protein expression levels of autophagy substrates p62, cytoplasmic LC3-I, membrane LC3-II, PINK1, Parkin, PD-L1, and GAPDH. The ATP content in the total proteins was measured according to the instructions of the kit, and the mitochondria and lysosomes in the cells were localized using a staining kit. The results are shown in Figures 6 - 7 .

[0114] The MDA-MB-231 cells were co-cultured with NPs (CCCP / siPD-L1), NPs (CCCP / siCTL) or Control NPs with an siRNA concentration of 50 nM according to the above co-culture method. After 24 h of culture, they were washed with PBS, and the cell proliferation rate was detected by the AlamarBlue method. The results are shown in Figure 8 .

[0115] The MDA-MB-231 cells were co-cultured with NPs (CCCP / siPD-L1), NPs (CCCP / siCTL) or Control NPs with an siRNA concentration of 50 nM according to the above method. After incubation for 1 week, at least 3 parallels were selected for each treatment to add crystal violet, and the cell growth was observed under an MVX10 MacroView dissecting microscope equipped with an Olympus DP80 camera. The results are shown in Figures 8 - 9 .

[0116] As Figures 6 - 9 shown, the expression level of p62 decreased, the ratio of LC3-II / LC3-I increased, the expression levels of PINK1, Parkin and PD-L1 increased, and the ATP concentration, proliferation rate and clone number decreased, indicating that mitophagy was successfully induced, and excessive mitophagy phagocytosed more healthy mitochondria, resulting in a decrease in the ability of cells to synthesize ATP, thereby inhibiting the proliferation ability of tumor cells.

[0117] In summary, NPs (CCCP / siPD-L1) can silence the expression of PD-L1 in cells, combined with CCCP to induce mitophagy, and can effectively inhibit the proliferation of breast cancer (BCa) cells.

[0118] Effect Example 3

[0119] To further study the effect of NPs (CCCP / Cy5-siPD-L1) on the tumor microenvironment, the total cellular RNA was extracted from MDA-MB-231 cells treated with NPs (CCCP / Cy5-siPD-L1) (siRNA concentration of 50 nM) after 48 h of culture for transcriptome sequencing. Using Control NPs as the control group, KEGG enrichment analysis of differentially expressed genes was performed, and the common genes (CCL2, CXCL3, IL-1β, CCL20, CXCL2, CSF2 and IL-6) of the top 3 signaling pathways with the highest scores were selected for qRT-PCR. The results are shown in Figure 10 .

[0120] As Figure 10 shown, among the numerous common genes, the expression level of the CCL2 gene was significantly down-regulated. Therefore, the CCL2 gene was selected as a marker gene for research.

[0121] The MDA-MB-231 cells were co-cultured with NPs (CCCP / siPD-L1), NPs (CCCP / siCTL) or Control NPs with an siRNA concentration of 50 nM according to the method of Experiment 1 in Effect Example 2. After incubation for 48 h, the RNA and total protein in the cell supernatant were extracted for qRT-PCR and Western blotting to detect the level of CCL2 secreted by the cells, and the recruitment of immunosuppressive monocytes was detected by flow cytometry. The results are shown in Figures 11 - 12 .

[0122] As Figures 11 - 12 shown, the transcriptional level of CCL2 in MDA-MB-231 cells treated with NPs (CCCP / siPD-L1) was inhibited, resulting in a decrease in its protein level and extracellular release, and at the same time, it was also able to inhibit the recruitment of immunosuppressive monocytes.

[0123] Effect Example 5

[0124] To evaluate whether the nanocomposite also has good antitumor efficacy in vivo, a tumor-bearing mouse model was constructed and the nanocomposite was injected for experiments. The specific protocol is as follows:

[0125] 1. Mouse-derived 4T1 cells were injected orthotopically into nude mice. When the tumor volume reached 100 mm 3 , the drugs were injected according to the following groups at intervals of 1 day for 3 times. The tumor size was recorded at 1 and 2 weeks after the last injection. The results are shown in Figures 13 - 14 and Table 3. The treatments for each group are as follows:

[0126] (1) 200 μL PBS;

[0127] (2) 200 μL CCCP DMF solution (8 mg / mL);

[0128] (3) 200 μL Control NPs (siCTL concentration of 1 nM);

[0129] (4) 200 μL NPs (CCCP / siCTL) (siCTL concentration of 1 nM);

[0130] (5) 200 μL NPs (CCCP / siPD-L1) (siPD-L1 concentration of 1 nM).

[0131] Table 3 Statistical results of tumor volume and weight of orthotopic tumor-bearing mice treated with different treatments (14 days after injection, x±SD)

[0132]

[0133] As shown in Table 3 andFigures 13 - 14 As shown, the tumor volume and weight of mice treated with NPs (CCCP / siPD-L1) were the smallest, and the combined effect of CCCP or siPD-L1 alone could not achieve the efficacy of their combination, indicating that the nanocomposite of the present invention is based on the combination of CCCP and siPD-L1 to synergistically enhance the anti-tumor efficacy.

[0134] 2. A 4T1 orthotopic tumor-bearing mouse model was constructed according to the method of Experiment 1 above. A metastasis model was constructed by injecting 4T1 cells expressing lusifer via the tail vein. After the tumor volume could be identified in the in vivo imaging system, drugs were injected according to the grouping in Experiment 1. The injection frequency and dose were the same as those in Experiment 1. The tumor metastasis was observed using the in vivo imaging system on days 0, 7, and 14 after the 3rd injection, and the average radiance was calculated. The results are shown in Table 4 and Figure 15 .

[0135] Table 4 Statistical results of the average radiance of mice with lung metastatic tumors treated with different treatments (14 days after injection, x±SD)

[0136]

[0137] As shown in Table 4 and Figure 15 shown, the lung metastasis of mice treated with NPs (CCCP / siPD-L1) was significantly improved, and the combined effect of CCCP or siPD-L1 alone could not achieve the efficacy of their combination, indicating that the nanocomposite of the present invention is based on the combination of CCCP and siPD-L1 and then coated with Meo-PEG-b-PDPA block copolymer to synergistically enhance the anti-tumor efficacy.

[0138] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A nanocomplex for targeted induction of mitophagy, characterized in that, The nanocomposite comprises a mitophagy inducer with Meo-PEG-b-PDPA block copolymer on its surface and siPD-L1; The ratio of the mitophagy inducer to siPD-L1 is mitophagy inducer: siPD-L1 = (0.01 - 0.10) mg: 1 nmol; The ratio of siPD-L1 to Meo-PEG-b-PDPA block copolymer is siPD-L1: Meo-PEG-b-PDPA block copolymer = 1 nmol: (1 - 5) mg.

2. The nanocomposite according to claim 1, characterized in that, The ratio of the mitophagy inducer to siPD-L1 is mitophagy inducer: siPD-L1 = (0.05 - 0.08) mg: 1 nmol; The ratio of siPD-L1 to Meo-PEG-b-PDPA block copolymer is siPD-L1: Meo-PEG-b-PDPA block copolymer = 1 nmol: (1.5 - 4) mg.

3. The nano - composite according to claim 1, wherein, The mitophagy inducer includes at least one of carbonyl cyanide, fisetin, and resveratrol.

4. The nanocomposite according to claim 3, characterized in that, The carbonyl cyanide includes 3-chlorophenylhydrazone and / or carbonyl cyanide 4-trifluoromethoxyphenylhydrazone.

5. The nanocomposite according to claim 1, wherein The siPD-L1 is double-stranded RNA, and the nucleotide sequence of the siPD-L1 is as shown in SEQ ID NO.1 - 2 and / or SEQ ID NO.3 - 4.

6. The preparation method of the nano - composite as described in any one of claims 1 - 5, characterized in that, The nanocomposite is prepared by the nanoprecipitation method.

7. The preparation method according to claim 6, characterized in that, The nanoprecipitation method includes the following operations: S1. Mix the mitophagy inducer and siPD-L1 to obtain a core; S2. Mix the core obtained in step S1 with Meo-PEG-b-PDPA block copolymer, add water at 700 - 1100 rpm, ultrafilter, and wash to obtain a nanocomposite for targeted induction of mitophagy.

8. The preparation method according to claim 7, characterized in that, Includes at least one of the following (Ⅰ) - (Ⅱ): (Ⅰ) In step S1, the solvent of the mitophagy inducer is N,N-dimethylformamide, and the solvent of the siPD-L1 is water; (Ⅱ) In step S2, the ultrafiltration is through an ultrafiltration membrane with a molecular weight cut-off of 900 - 110 kDa; (Ⅲ) In step S2, the washing is performed 3 - 4 times with phosphate buffer solution.

9. Use of the nanocomposite according to any one of claims 1 - 5 in the preparation of an anti-tumor drug.

10. An anti-tumor drug, characterized in that, Includes the nanocomposite according to any one of claims 1 - 5 and pharmaceutically acceptable excipients.

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