A tumor energy depletion nanoassembly and its preparation method and application

Abstract

The present invention discloses a tumor energy depletion nanoassembly and its preparation method and application, which belongs to the technical field of new excipients and new dosage forms for combined treatment of pharmaceutical preparations. The tumor energy depletion nanoassembly is co-assembled by a mitochondrial oxidative phosphorylation inhibitor and a glycolysis inhibitor through intermolecular forces, and modified with a PEG modifier; the molar ratio of the mitochondrial oxidative phosphorylation inhibitor and the glycolysis inhibitor is 10:1 to 1:10, and the mass ratio of the sum of the mitochondrial oxidative phosphorylation inhibitor and the glycolysis inhibitor to the PEG modifier is 10:90 to 90:10. The tumor energy depletion nanoassembly of the present invention has a high drug loading capacity, good stability, and low toxic and side effects, providing a new strategy for the assembly of homologous synergistic drug nanoparticles, and providing an effective nano-platform for the development of carrier-free hybrid nanoassemblies and energy depletion-driven combined tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of new excipients and new dosage forms for combined treatment of pharmaceutical preparations, and particularly relates to a tumor energy-depleting nanoassembly, a preparation method and an application thereof. Background Art

[0002] Malignant tumors pose a serious threat to human health worldwide. Given the suboptimal efficacy and severe toxicity of existing treatments, such as conventional chemotherapy, the development of new, effective, and safe approaches has been a top priority in clinical cancer therapy. In recent years, tumor cell metabolism has been increasingly recognized as a promising strategy for cancer treatment. Abnormal tumor metabolism is a key characteristic of tumor cells and is closely associated with tumor development and progression. Given the significant metabolic differences between normal and tumor cells, therapies that disrupt tumor energy metabolism have attracted widespread attention as an emerging approach to tumor starvation therapy. Although anaerobic glycolysis plays a crucial role in tumor energy supply, oxidative phosphorylation is not lost even in anaerobic glycolysis-dependent cancer cells. In contrast, most cancer cells contain metabolically active mitochondria, a significant proportion of which are highly dependent on oxidative phosphorylation for energy. In particular, in most tumor cells, mitochondrial oxidative phosphorylation is immediately restored upon blockade of anaerobic glycolysis. Furthermore, anaerobic glycolysis is actually a reluctant alternative for tumor cell survival, rather than a highly efficient mode of energy supply. Tumor cells rely heavily on mitochondrial oxidative phosphorylation to maintain high proliferation rates. Therefore, tumor energy depletion therapy is largely influenced by the existing mutual compensation mechanism of the dual energy supply mode in tumor cells.

[0003] As previously mentioned, two major metabolic pathways provide energy for tumor cells: anaerobic glycolysis and mitochondrial oxidative phosphorylation, which are highly complementary. Therefore, inhibiting or disrupting only one energy metabolic pathway has proven ineffective in cancer starvation therapy. Furthermore, the metabolic heterogeneity of tumors significantly hinders energy depletion-driven therapies. To address these challenges, there is growing interest in dual glycolysis / mitochondrial blockade strategies for complete energy depletion of tumor cells. However, due to the significant differences in the physicochemical properties of different drugs, the effective co-delivery of two energy blockers remains a challenge, inevitably leading to difficulties in drug co-loading, dose ratio adjustment, and simultaneous in vivo delivery. Furthermore, while a variety of small molecule metabolic inhibitors have been developed, they suffer from poor inhibitory selectivity and pose a significant risk of off-target toxicity to normal tissues. Precisely because of the potential for off-target metabolic inhibition, dual glycolysis / mitochondrial blockade significantly increases the risk of multidrug toxicity associated with two or more metabolic inhibitors, which remains one of the greatest challenges in cancer combination therapy. In summary, systemic tumor energy depletion-driven therapies face multiple challenges in terms of glycolysis / mitochondrial closed-loop blockade, simultaneous drug delivery, and good therapeutic efficacy and safety. We urgently need to rationally develop new strategies to address these challenges.

[0004] With the widespread application of biomedical nanotechnology, the simultaneous loading of multiple drugs into a single nanocarrier is becoming increasingly challenging. Currently, most co-delivered nanodrugs are prepared by non-covalently encapsulating drugs into organic or inorganic nanocarriers. Conventional co-delivered nanodrugs suffer from shortcomings such as low co-loading efficiency, inconvenient dosage adjustment, mismatched pharmacokinetic behavior, and suboptimal synergistic effects. These issues can be attributed to differences in the affinity between the nanocarrier material and the different drug molecules. On the one hand, poor nanocarrier-drug affinity often leads to low drug loading efficiency and premature drug leakage. On the other hand, while excessive intermolecular affinity between the carrier and the drug facilitates drug encapsulation, it inevitably results in delayed drug release. In the past decade, carrier-free nanodrugs co-assembled with small molecule drugs or prodrugs have demonstrated numerous advantages over traditional co-delivery nanocarriers, including ease of preparation, high drug co-loading efficiency, matched pharmacokinetics, efficient tumor-specific accumulation, and synchronized drug release. Notably, the formation of carrier-free hybrid nanoassemblies driven by intermolecular interactions imparts excellent synchronized drug delivery properties. That is, the intermolecular interactions between the two drugs drive their co-assembly into hybrid nanoassemblies, while under certain tumor stimuli, the disappearance of intermolecular forces promotes the disintegration of the nanostructure and site-specific drug release. However, despite the many advantages of carrier-free hybrid nanoassemblies, the problem of multi-drug superposition toxicity has not been well solved in combination cancer therapy.

[0005] In recent years, carrier-free nanoparticle delivery systems based on pure drug co-assembly have shown promising prospects in drug delivery. This is particularly true for certain anticancer drugs that can be co-assembled into stable nanoparticles without the need for carrier materials. The construction of hybrid nanoassemblies containing multiple drug molecules holds even greater potential for combination therapy. The development of tumor-depleting nanoassemblies with controlled drug release is a critical and pressing research topic. Summary of the Invention

[0006] To address the technical problems of the prior art, the present invention provides a tumor energy-depleting nanoassembly, its preparation method, and its application. This invention addresses the problems of JQ1's poor water solubility, poor assembly ability, low drug loading due to encapsulation in polymers, drug leakage, and excipient-related toxicity. It provides a novel oxidative phosphorylation inhibitor with mitochondrial targeting capability co-assembled with the glycolysis inhibitor JQ1, thereby achieving the technical benefits of high drug loading, good stability, and low toxic side effects. This addresses the problem of unsatisfactory therapeutic effects of single drugs and enhances the energy-depletion-induced tumor eradication effect. The present invention aims to synthesize co-assembled nanoparticles of a novel oxidative phosphorylation inhibitor TPP-AA with mitochondrial targeting capability and the glycolysis inhibitor JQ1, or nanoparticles assembled from a novel oxidative phosphorylation inhibitor TPP-AA with mitochondrial targeting capability, the glycolysis inhibitor JQ1, and a PEG modifier. The present invention achieves energy depletion through the dual inhibition of novel mitochondrial oxidative phosphorylation and glycolysis, thereby inducing tumor treatment.

[0007] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0008] In the first aspect, the present invention provides a tumor energy depletion nanoassembly, which is co-assembled by an oxidative phosphorylation inhibitor and a glycolysis inhibitor with mitochondrial targeting ability through intermolecular forces and modified with a PEG modifier; the molar ratio of the oxidative phosphorylation inhibitor and the glycolysis inhibitor is 10:1 to 1:10, and the mass ratio of the sum of the oxidative phosphorylation inhibitor and the glycolysis inhibitor to the PEG modifier is 10:90 to 90:10.

[0009] Furthermore, the intermolecular forces include π-π stacking, hydrophobic interaction and hydrogen bonding.

[0010] Furthermore, the mitochondrial oxidative phosphorylation inhibitor includes a mitochondrial electron transport chain complex I inhibitor, a mitochondrial targeted oxidant based on triphenylphosphine, or a combination of triphenylphosphine and unsaturated fatty acids.

[0011] Furthermore, the unsaturated fatty acids in the combination of triphenylphosphine (TPP) and unsaturated fatty acids include at least one of oleic acid, linoleic acid, linolenic acid, ω-3 polyunsaturated fatty acids, ω-6 polyunsaturated fatty acids, arachidonic acid (AA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and various branched-chain fatty acids.

[0012] Furthermore, the unsaturated fatty acid in the mitochondrial oxidative phosphorylation inhibitor is preferably a combination of TPP and AA (TPP-AA).

[0013] Furthermore, the glycolysis inhibitor includes at least one of a GLUTs inhibitor, an HKII inhibitor, a PDKII inhibitor, a PK inhibitor, an LDH inhibitor, JQ1, lonidamine, 2-deoxyglucose, trametinib or vemurafenib.

[0014] Furthermore, the glycolysis inhibitor is preferably JQ1.

[0015] Furthermore, the PEG modifier includes one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG, and PE-PEG, and the molecular weight of PEG is 200-20000.

[0016] Furthermore, the PEG modifier is preferably DSPE-PEG 2K or DSPE-SS-PEG 2K .

[0017] Furthermore, the molar ratio of TPP-AA to JQ1 is 1:5 to 5:1.

[0018] Furthermore, the molar ratio of TPP-AA to JQ1 is preferably 1:3.

[0019] In a second aspect, the present invention provides a method for preparing a tumor energy-depleting nanoassembly, comprising the following steps:

[0020] The mitochondrial oxidative phosphorylation inhibitor and the glycolysis inhibitor are dissolved in organic solvents respectively, mixed under stirring, and the mixed solution is slowly added dropwise to water to spontaneously form uniform co-assembled nanoparticles; the organic solvent of the PEG modifier is added dropwise to the co-assembled nanoparticles under stirring, and the organic solvent is removed to obtain the nanoparticles.

[0021] Further, the stirring speed is 200-2000 rpm

[0022] Furthermore, the organic solvent is one of ethanol, tetrahydrofuran, and dimethyl sulfoxide, or a combination of any two thereof.

[0023] Furthermore, the solvent is preferably tetrahydrofuran.

[0024] Furthermore, the method for removing the organic solvent includes rotary evaporation or dialysis.

[0025] Furthermore, in the preparation method, the method for removing the organic solvent can be solvent evaporation, ultrafiltration and membrane permeation.

[0026] In a third aspect, the present invention provides a tumor energy-depleting nanoassembly prepared by the above method.

[0027] In a fourth aspect, the present invention provides the use of tumor energy-depleting nanoassemblies in the preparation of drug delivery systems.

[0028] Fifthly, the present invention provides the use of tumor energy depletion nanoassemblies in the preparation of anti-tumor drugs.

[0029] In a sixth aspect, the present invention provides the use of tumor energy-depleting nanoassemblies in the preparation of an application system for injection, oral administration or local administration.

[0030] The present invention has the following beneficial effects compared to the prior art:

[0031] The present invention prepares a co-assembled nanoparticle composed of a mitochondrial oxidative phosphorylation inhibitor (preferably TPP-AA) and a glycolysis inhibitor (preferably JQ1), which is modified with a PEG modifier to produce a bidirectional closed-loop energy depletion-induced tumor eradication nanoassembly. TPP-AA can target and effectively destroy mitochondria, resulting in inhibition of the mitochondrial oxidative phosphorylation pathway, thereby enhancing anti-tumor efficacy.

[0032] The co-assembled nanoparticles of the mitochondrial oxidative phosphorylation inhibitor and the glycolysis inhibitor of the present invention achieve technical effects such as high drug loading, good stability, and low toxic side effects, meeting the urgent clinical demand for high-efficiency and low-toxic preparations, providing a new strategy for the assembly of homologous synergistic drug nanoparticles, and providing an effective nano-platform for the development of carrier-free hybrid nanoassemblies and energy depletion-driven combined tumor therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 4 is the high performance liquid chromatogram of Example 1 of the present invention.

[0034] Figure 2 The mass spectrum and NMR diagram of Example 1 of the present invention are shown.

[0035] Figure 3 This is a diagram of the 4T1 cytotoxicity of Example 2 of the present invention.

[0036] Figure 4This is the L02 cytotoxicity graph of Example 2 of the present invention.

[0037] Figure 5 This is a diagram of mitochondrial lipid peroxidation in Example 2 of the present invention.

[0038] Figure 6 This is a diagram of the mitochondrial membrane potential of Example 2 of the present invention.

[0039] Figure 7 The particle size distribution and transmission electron microscopy image of the nanoparticles prepared in Example 3 of the present invention.

[0040] Figure 8 Zeta potential diagram of nanoparticles prepared in Example 3 of the present invention.

[0041] Figure 9 This is a PBS (pH 7.4) stability diagram of non-nanoparticles prepared in Example 4 of the present invention.

[0042] Figure 10 This is the PBS (pH 7.4) stability diagram of TAJP nanoparticles and TAJS nanoparticles prepared in Example 4 of the present invention.

[0043] Figure 11 Stability diagram of TAJP nanoparticles and TAJS nanoparticles prepared in Example 4 of the present invention in PBS (pH 7.4) containing 10% FBS.

[0044] Figure 12 This is a graph showing the storage stability of TAJP nanoparticles and TAJS nanoparticles prepared in Example 4 of the present invention at 4°C.

[0045] Figure 13 This is the molecular docking diagram of TPP-AA and JQ1 according to embodiment 5 of the present invention.

[0046] Figure 14 This is the force destruction of the non-PEGylated nanoparticles of Example 5 of the present invention in PBS (pH 7.4) containing sodium chloride, sodium lauryl sulfate and urea.

[0047] Figure 15 Graph showing the stability of TAJP nanoparticles and TAJS nanoparticles of Example 6 of the present invention in PBS (pH 7.4) containing 0 mM and 20 mM DTT.

[0048] Figure 16 The cumulative release of JQ1 from the TAJP nanoparticles and TAJS nanoparticles of Example 6 of the present invention in PBS (pH 7.4) containing 0 mM and 20 mM DTT.

[0049] Figure 17These are confocal microscope photographs of the cellular uptake of the Cy7 solution, TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles according to Example 7 of the present invention at 0.5 hours and 2 hours.

[0050] Figure 18 Flow cytometric quantitative graphs of the Cy7 solution, TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles of Example 7 of the present invention at 0.5 hours and 2 hours.

[0051] Figure 19 These are the cytotoxicity results of TPP-AA solution (a), JQ1 solution (b), TAJ solution (c), TAJP nanoparticles (d) and TAJS nanoparticles (e) in Example 8 of the present invention on 4T1 cells.

[0052] Figure 20 These are the cytotoxicity results of TPP-AA solution (a), JQ1 solution (b), TAJ solution (c), TAJP nanoparticles (d) and TAJS nanoparticles (e) in Example 8 of the present invention on CT26 cells.

[0053] Figure 21 These are the apoptosis results of 4T1 cells by the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) of Example 9 of the present invention.

[0054] Figure 22 These are qualitative graphs of the reactive oxygen species levels in 4T1 cells after treatment with the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) of Example 10 of the present invention.

[0055] Figure 23 Quantitative graph of the reactive oxygen species level in 4T1 cells after treatment with the control group, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles of Example 10 of the present invention.

[0056] Figure 24 The mitochondrial lipid peroxidation levels of 4T1 cells after being treated with the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) of Example 11 of the present invention.

[0057] Figure 25 This is a qualitative graph of the changes in mitochondrial membrane potential of 4T1 cells after being treated with the control group, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles and TAJS nanoparticles of Example 12 of the present invention.

[0058] Figure 26 This is a quantitative graph of the changes in mitochondrial membrane potential of 4T1 cells after treatment with the control group, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles and TAJS nanoparticles of Example 12 of the present invention.

[0059] Figure 27 These are the cell protein blotting results of the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) of Example 13 of the present invention.

[0060] Figures 28-30 Graphs showing changes in lactate, lactate dehydrogenase (LDH), and ATP levels in the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e), and TAJS nanoparticles (f) of Example 14 of the present invention.

[0061] Figure 31 1 is a graph showing the blood concentration-time curves of the Cy7 solution, TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles of Example 15 of the present invention.

[0062] Figure 32 This is the in vivo imaging of mice administered with Cy7 solution, TAJP-Cy7 nanoparticles, and TAJS-Cy7 nanoparticles in Example 16 of the present invention.

[0063] Figure 33 These are fluorescence imaging images of isolated mouse organs after administration of Cy7 solution, TAJP-Cy7 nanoparticles, and TAJS-Cy7 nanoparticles in Example 16 of the present invention.

[0064] Figure 34 This is a fluorescence quantitative graph of isolated organs of mice administered with Cy7 solution, TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles in Example 16 of the present invention.

[0065] Figure 35 This is a graph showing the mouse tumor growth curves of the anti-tumor experiment in the 4T1 tumor model using the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) of Example 17 of the present invention.

[0066] Figure 36 Statistical graphs of the tumor-bearing rates of mice in the anti-tumor experiment of Example 17 of the present invention, including the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) in the 4T1 tumor model.

[0067] Figure 37 These are in vitro tumor images of the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) in the 4T1 tumor model of Example 17 of the present invention.

[0068] Figure 38 These are H&E and TUNEL staining images of mouse in vitro tumors from the anti-tumor experiment of the control group, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles and TAJS nanoparticles in the 4T1 tumor model of Example 17 of the present invention.

[0069] Figure 39 These are histopathological sections of the control group, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles in the 4T1 tumor model of Example 17 of the present invention.

[0070] Figure 40 The results of liver and kidney function analysis of the control group, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles and TAJS nanoparticles in the 4T1 tumor model of Example 17 of the present invention are as follows:

[0071] Figure 41 This is a graph showing the changes in mouse body weight in the anti-tumor experiment of the control group, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles and TAJS nanoparticles in the 4T1 tumor model according to Example 17 of the present invention.

[0072] Figure 42 This is a graph of the survival rate of mice in the safety experiment of Example 17 of the present invention, wherein A1: TAJSol 10 mg / kg, B1: TAJPNAs 10 mg / kg, C1: TAJSNAs 10 mg / kg, A2: TAJSol 30 mg / kg, B2: TAJPNAs 30 mg / kg, C2: TAJSNAs 30 mg / kg, A3: TAJSol 50 mg / kg, B3: TAJPNAs 50 mg / kg, C3: TAJSNAs 50 mg / kg.

[0073] Figure 43This is a graph showing the changes in mouse body weight in the safety experiment of Example 17 of the present invention, wherein A1: TAJ Sol 10 mg / kg, B1: TAJPNAs 10 mg / kg, C1: TAJSNAs 10 mg / kg, A2: TAJSol 30 mg / kg, B2: TAJPNAs 30 mg / kg, C2: TAJSNAs 30 mg / kg, A3: TAJSol 50 mg / kg, B3: TAJPNAs 50 mg / kg, C3: TAJSNAs 50 mg / kg.

[0074] Figure 44 This is a graph showing the changes in c-Myc levels in the anti-tumor experiment of the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) in the 4T1 tumor model according to Example 17 of the present invention.

[0075] Figures 45-47 This is a graph showing changes in lactate, LDH, and ATP levels in the anti-tumor experiment of the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e), and TAJS nanoparticles (f) in the 4T1 tumor model according to Example 17 of the present invention.

[0076] Figure 48 Statistical graphs of the tumor-bearing rates of mice in the anti-tumor experiment of Example 17 of the present invention, including the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) in the CT26 tumor model.

[0077] Figure 49 This is a graph showing the mouse tumor growth curves of the anti-tumor experiment in the CT26 tumor model using the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) of Example 17 of the present invention.

[0078] Figure 50 These are in vitro tumor images of the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) in the CT26 tumor model of Example 17 of the present invention.

[0079] Figure 51 This is a graph showing the changes in mouse body weight in the anti-tumor experiment in the CT26 tumor model using the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) of Example 17 of the present invention.

[0080] Figure 52 These are histopathological sections of the control group, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles in the CT26 tumor model of Example 17 of the present invention.

[0081] Figure 53 These are the results of liver and kidney function analysis of the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e) and TAJS nanoparticles (f) in the CT26 tumor model of Example 17 of the present invention.

[0082] Figure 54 Graph showing changes in lactate dehydrogenase (A), lactate (B), and ATP (C) levels in the anti-tumor experiment of the control group (a), TPP-AA solution (b), JQ1 solution (c), TAJ solution (d), TAJP nanoparticles (e), and TAJS nanoparticles (f) in the CT26 tumor model according to Example 17 of the present invention. DETAILED DESCRIPTION

[0083] The present invention is described in detail below with reference to the embodiments, but the embodiments of the present invention are not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments obtained without creative work all fall within the scope of protection of the present invention.

[0084] Example 1: Synthesis of a novel oxidative phosphorylation inhibitor TPP-AA with mitochondrial targeting function

[0085] Triphenylphosphine (TPP, 30 mmol) and Na2CO3 (50 mmol) were dissolved in 50 mL of dry acetonitrile. Then, 30 mmol of 3-bromo-1-propanol was added and the mixture was heated at 85°C under nitrogen reflux for 7 hours for condensation. After filtering out the Na2CO3, TPP-OH was recrystallized from glacial ether. TPP-AA was then coupled via esterification. TPP-OH (3 mmol), arachidonic acid AA (3 mmol), DMAP (0.6 mmol), EDCI (6 mmol), and HOBT (3 mmol) were added to 25 mL of dichloromethane and stirred at 37°C for 12 hours. TPP-AA was purified by preparative liquid chromatography. The product was characterized by HPLC, MS, and NMR.

[0086] The results are as follows Figure 1 Figure 2As shown, the purity of the isolated and synthesized TPP-AA is 99.8%. The molecular weight of TPP-AA was determined by MS and was 607.36957, which is consistent with the actual molecular weight. The hydrogen of TPP-AA was analyzed by nuclear magnetic resonance spectroscopy, confirming the successful synthesis of TPP-AA.

[0087] Example 2: Cytotoxicity study and mechanism of TPP-AA

[0088] The cytotoxicity of TPP-OH, AA and TPP-AA was compared by cytotoxicity assay. 4T1 cells (mouse breast cancer cells) were cultured at 2×10 3 Cells / well were seeded in a 96-well plate at a density of 1×10 cells / well for 12 hours, and then 4T1 cells were treated with TPP-OH solution (TPP-OHSol), AA solution (AASol) and TPP-AA solution (TPP-AASol) for 48 hours. After incubation for 48 hours, 5 mg / mL MTT (25 μL / well) was added and incubated at 37°C for 4 hours, and then the formazan was dissolved with DMSO (200 μL / well). Finally, the ultraviolet (UV) absorbance at 490 nm was measured using a multifunctional microplate reader (Thermo Scientific, USA). Using the same method, a cytotoxicity experiment was used to compare the cytotoxicity of TPP-OHSol, AASol and TPP-AASol on L02 cells (normal human liver cells). In order to further verify the difference in cytotoxicity between TPP-AASol and AASol, we performed measurements of mitochondrial lipid peroxidation and changes in mitochondrial membrane potential. Briefly, 4T1 cells were seeded at a density of 1×10 5 Cells were seeded at a density of 100 μg / well in 35 mm glass-bottomed culture dishes and incubated for 12 hours. The medium was then replaced with AASol (10 μM, 50 μM) and TPP-AASol (10 μM, 50 μM) and incubated for 12 hours. Cells were then treated as indicated to measure mitochondrial lipid peroxidation and mitochondrial membrane potential and observed using confocal laser scanning microscopy (CLSM).

[0089] The results are as follows Figure 3 As shown in the results, TPP-AA is much more cytotoxic to 4T1 cells than AA and TPP-OH. More importantly, TPP-AA not only exhibits strong cytotoxicity to 4T1 cells, but also has negligible cytotoxicity to normal cell lines (L02 cells) within the same concentration range, showing good therapeutic selectivity ( Figure 4This selective cytotoxicity should be attributed to the huge difference in mitochondrial membrane potential between normal cells and tumor cells, which enables it to be used as a new type of oxidative phosphorylation inhibitor with good safety in cancer starvation therapy. Based on our previous hypothesis, we studied the effects of different concentrations of TPP-AA and AA on 4T1 cells in terms of mitochondrial lipid peroxidation and mitochondrial membrane potential. Figure 5 As shown in the results, compared with AA, TPP-AA had a higher level of mitochondrial lipid peroxidation in a concentration-dependent manner. In addition, as the concentration increased, the mitochondrial membrane potential of cells treated with TPP-AA decreased sharply, while the mitochondrial membrane potential of cells treated with AA did not change significantly ( Figure 6 These results suggest that TPP-AA can effectively target mitochondria and induce mitochondrial damage. This may be because AA is prone to lipid peroxidation in the highly oxidative mitochondrial environment, leading to the accumulation of lipid peroxides in mitochondria and mitochondrial membranes, further amplifying oxidative stress.

[0090] Example 3: Screening of the synergistic dosage ratio of TPP-AA and JQ1

[0091] TPP-AA and JQ1 were dissolved in tetrahydrofuran (THF) at varying molar ratios to obtain a 5 mg / mL drug-containing solution. Under stirring, 200 μL of this solution was slowly added dropwise to 2 mL of deionized water. TPP-AA and JQ1 spontaneously formed uniform nanoparticles. The organic solvent was then removed from the nanoformulation by rotary evaporation at 30°C, yielding a solvent-free nanoformulation (TAJNPs).

[0092] In addition, the MTT assay was used to evaluate the synergistic effect of TPP-AA and JQ1. Briefly, 4T1 cells were plated at 2×10 3 4T1 cells were seeded in 96-well plates at a density of 10 cells / well for 12 hours. The cells were then treated with various concentrations of TPP-AA solution, JQ1 solution, or a mixture of TPP-AA and JQ1 at molar ratios of 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, and 5:1. After 48 hours, the cells were incubated with 5 mg / mL MTT (25 μL / well) at 37°C for 4 hours. The formazan produced by the cells was then solubilized with DMSO (200 μL / well). Finally, UV absorbance at 490 nm was measured using a microplate reader. Cell viability and the synergy index (CI) were calculated to evaluate the synergistic effect of TPP-AA and JQ1.

[0093] The particle size, particle size distribution and synergistic index of TPP-AA and JQ1 of the prepared nanoformulation were tested, and the results are shown in Table 1.

[0094] Table 1. Particle size, particle size distribution, and synergistic index of TAJ nanoparticles and TPP-AA and JQ1

[0095]

[0096] As shown in Table 1, the nanoparticles ranged in size from 80 to 170 nm, with a synergistic index of 0.33 to 1.07. A CI value <1 indicates a synergistic effect, a CI value = 1 indicates an additive effect, and a CI value > 1 indicates an antagonistic effect. This indicates that TPP-AA and JQ1 exhibit a synergistic effect, except when the molar ratio of TPP-AA to JQ1 is 2:1. When the TPP-AA:JQ1 ratio is 1:3, the TAJ nanoparticles are more evenly distributed, and the synergistic effect between TPP-AA and JQ1 is greater. A TPP-AA:JQ1 ratio of 1:3 is initially preferred.

[0097] (1) Preparation of non-PEGylated TAJ nanoparticles: 0.31 mg TPP-AA and 0.69 mg (3 times the molar amount) of JQ1 were accurately weighed and dissolved in 200 μL of tetrahydrofuran. The solution was slowly added dropwise to 2 mL of deionized water under stirring to spontaneously form uniform TAJ nanoparticles. The organic solvent in the nanoformulation was then removed by rotary evaporation at 30°C to obtain a nanocolloidal solution free of any organic solvent.

[0098] (2) DSPE-PEG 2K and DSPE-SS-PEG 2K Preparation method of modified TAJP nanoparticles and TAJS nanoparticles: accurately weigh 0.31 mg TPP-AA and 0.69 mg (3 times the molar amount) of JQ1, and dissolve them in 200 μL tetrahydrofuran; accurately weigh 0.85 mg DSPE-PEG 2K and DSPE-SS-PEG 2K , dissolve in tetrahydrofuran to a 10 mg / mL stock solution. Mix 200 μL of TAJ solution with 85 μL of DSPE-PEG 2K or DSPE-SS-PEG 2K After mixing thoroughly, the solution was slowly added dropwise to 2 mL of deionized water with stirring to obtain uniform TAJP and TAJS nanoparticles. The organic solvent in the nanoformulation was then removed by rotary evaporation at 30°C to obtain a solvent-free nanocolloidal solution. The particle size, size distribution, and zeta potential of the prepared TAJP and TAJS nanoparticles were determined by dynamic light scattering.

[0099] The particle size and Zeta potential of the TAJ nanoparticles prepared in Example 3 were measured by a Malvern particle size analyzer. The results showed that the particle size was about 80 nm and the Zeta potential was about 20 mV. The particle size and morphology of the TAJP nanoparticles and TAJS nanoparticles prepared in Example 3 were measured by a Malvern particle size analyzer and a transmission electron microscope. The results are as follows: Figure 7 and Figure 8 The transmission electron microscopy image shows that the nanoparticles are uniform spherical, with a particle size of about 100nm and a Zeta potential of about -20mV.

[0100] Example 4: Colloidal stability test of nanoparticles

[0101] 1 mL of the TAJP nanoparticles and TAJS nanoparticles (0.5 mg / mL) prepared in Example 3 was taken out and added to 10 mL of PBS (pH 7.4) and PBS (pH 7.4) containing 10% FBS, and incubated in a shaker at 37°C for 12 hours, and the particle size change was measured by dynamic light scattering at predetermined time points. In addition, TAJP nanoparticles and TAJS nanoparticles (0.5 mg / mL) were placed at 4°C for 7 days. The stability of the nanoparticles was evaluated by measuring the particle size change of the nanoparticles using dynamic light scattering. The results are shown in FIG. Figure 9 As shown in Figure 2, non-PEG-modified FA nanoparticles are unstable in PBS. In contrast, FAP nanoparticles and FAS nanoparticles have better colloidal stability in PBS, and the particle size does not change significantly within 12 hours ( Figure 10 ).like Figure 11 As shown in Figure 2, FAP nanoparticles and FAS nanoparticles have good colloidal stability in PBS containing 10% FBS. Figure 12 As shown in the figure, PEG-modified FAP nanoparticles and FAS nanoparticles have good storage stability. PEG-modified FAP nanoparticles and FAS nanoparticles are preferred.

[0102] Example 5: Analysis of the assembly mechanism of TPP-AA and JQ1

[0103] Computer simulations were used to explore the assembly mechanism of TPP-AA and JQ1. Molecular docking calculations were performed using the Vina program on the Yinfu cloud computing platform. Energy minimization of the compounds TPP-AA and JQ1 was performed under the MMFF94 force field to obtain a 3D structure, forming a stable nanoassembly. Semi-flexible docking was performed using the AutoDockVina program, and sodium chloride, sodium dodecyl sulfate, and urea were used for force disruption. The results are shown in Figure 2. Figure 13 and Figure 14 As shown, there are multiple forces between TPP-AA and JQ1 molecules, such as π-π stacking, hydrophobic forces and hydrogen bonding, which make great contributions to the co-assembly of TPP-AA and JQ1.

[0104] Example 6: Study on reduction-triggered disassembly of nanoassemblies

[0105] As mentioned above, good colloidal stability is crucial for the in vivo delivery of nanoassemblies, but rapid release of drugs in tumors is also a necessary prerequisite for exerting antitumor activity. 2K Modified on the surface of the nanoassembly, reduction-responsive nanoparticles (TAJSNAs) were obtained. In order to study the disintegration behavior of the reduction-triggered nanoparticles, the stability of the nanoparticles (TAJPNAs and TAJSNAs) under reduction stimulation was first evaluated using PBS (pH 7.4) containing DTT (20mM). TAJPNAs and TAJSNAs (0.5mg / mL) were incubated at 37°C in PBS (pH 7.4) containing 0mM and 20mM DTT, respectively. The particle size of NAs at different time points was measured using a Malvern particle size analyzer. Figure 15 As shown in the figure, under the action of 20mM DTT, the particle size of TAJSNAs increased significantly. In contrast, under the same conditions, DTT had no effect on TAJPNAs. The results showed that DSPE-SS-PEG 2K The modified TAJSNAs have the property of dissociating from reducing substances.

[0106] In addition, the in vitro drug release behavior of JQ1 in a release medium containing DTT (20 mM) was further evaluated. The in vitro drug release behavior of TAJP nanoparticles and TAJS nanoparticles was evaluated by an in vitro dialysis method. PBS (pH 7.4) containing 10% tetrahydrofuran was used as the release medium. 0 mM and 20 mM DTT were added thereto, 1 mL of TAJP nanoparticles and TAJS nanoparticles (equivalent to 0.345 mg of JQ1) was added to the dialysis membrane, and 30 mL of release medium was placed in a conical flask. The flask was placed in a shaker at 37 ° C. At the preset time points (0 hours, 0.5 hours, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours), 200 μL of release medium was taken out and the same volume of medium was added to the conical flask. The cumulative release of JQ1 was measured using high performance liquid chromatography. As Figure 16 As shown, TAJPNAs and TAJSNAs slowly released JQ1 in the blank release medium (0mM DTT), while TAJSNAs showed DTT-triggered drug release characteristics. In the presence of 20mM DTT, about 60% of JQ1 was released within 24h. In contrast, non-sensitive TAJPNAs showed slow drug release behavior. TAJSNAs without PEG shell had poor colloidal stability and accelerated drug release. These results indicate that DSPE-SS-PEG 2K The modified nanoparticles can achieve reduction-selective drug release. The reduction-sensitive drug release pattern of TAJSNAs will help to produce strong cytotoxicity against tumor cells by rapidly triggering drug release from tumor cells.

[0107] Example 7: Cellular Uptake of Nanoparticles

[0108] Confocal microscopy and flow cytometry were used to qualitatively and quantitatively determine the uptake of TAJP nanoparticles and TAJS nanoparticles prepared in Example 3 in 4T1 cells (mouse breast cancer cells). 4 Cells were seeded onto 24-well plates at a density of cells / well and incubated in an incubator for 12 hours to allow them to adhere. After the cells adhered, Cy7 solution, TAJP-Cy7 nanoparticles, and TAJS-Cy7 nanoparticles were added. The concentration of Cy7 was 10 μM. After incubation at 37°C for 0.5 hours and 2 hours, the cells were washed and fixed. Finally, confocal microscopy was used to analyze the cellular uptake of various agents. The experimental results are shown in the figure. Figure 17 The quantitative operation was performed by plating 4T1 cells at 2×10 5 Cells were seeded onto 12-well plates at a density of cells / well and incubated in an incubator for 12 hours to allow them to adhere. After the cells adhered, Cy7 solution, TAJP-Cy7 nanoparticles, and TAJS-Cy7 nanoparticles were added. The concentration of Cy7 was 10 μM. After incubation at 37°C for 0.5 hours and 2 hours, the cells were washed and digested and collected. Finally, the cellular uptake of various preparations was analyzed by flow cytometry. The experimental results are shown in the figure. Figure 18 shown.

[0109] The above experimental results show that cellular uptake exhibits time-dependent uptake, and cells treated with TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles have higher intracellular fluorescence intensity than cells treated with Cy7 solution. Therefore, the prepared TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles have higher cellular uptake efficiency than Cy7 solution.

[0110] Example 8: Cytotoxicity of Nanoparticles

[0111] The cytotoxicity of TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles and TAJS nanoparticles on mouse breast cancer (4T1) cells and colon cancer (CT26) cells was investigated by MTT assay. 4 After the cells are adhered to the wall, they are cultured with a medium containing TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles and TAJS nanoparticles, with 200 μL per well. The control group is cultured with a medium without drug solution. After 48 hours, the 96-well plate is removed and 5 mg mL of the suspension is added to each well. -1Incubate in an incubator for 4 hours with 25 μL of MTT solution, then shake the plate. Place the 96-well plate upside down on filter paper to thoroughly absorb any remaining liquid. Add 200 μL of DMSO to each well and shake on a shaker for 10 minutes to dissolve the blue-purple crystals. Measure the absorbance of each well at 490 nm using a microplate reader after zeroing.

[0112] Cytotoxicity results such as Figure 19-20 As shown, TAJ solution and TAJP nanoparticles exhibited synergistic effects in 4T1 and CT26 cells, resulting in more significant cell growth inhibition compared to free drug alone. TAJS nanoparticles exhibited the most potent antitumor activity in vitro, attributed to their efficient cellular uptake and tumor-sensitive intracellular drug release.

[0113] Example 9: Apoptosis

[0114] Apoptosis is one of the basic characteristics of cells and plays a very important role in all aspects of the body. AnnexinV-FITC Apoptosis Detection Kit detects cell apoptosis. FITC-AnnexinV binds to apoptotic cells and emits green fluorescence, distinguishing apoptotic cells from normal cells. Propidium iodide (PI) is a nucleic acid dye. FITC-AnnexinV is paired with PI to distinguish between early and late apoptotic cells. Simply plate 4T1 cells at 2×10 5 Cells were seeded at a density of 10 μM / well in 6-well plates and cultured for 12 hours. The medium was then replaced with equal concentrations of JQ1 (10 μM) in TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles. The cells were then incubated for 24 hours and treated according to the instructions of the Annexin V-FITC Apoptosis Detection Kit. Flow cytometry was used to detect cell apoptosis using Annexin V and PI double staining.

[0115] like Figure 21 As shown in Figure 3, apoptosis assays also confirmed that the TAJS nanoparticle group had the highest degree of apoptosis (Q2 + Q3 = 52.2), further indicating that TPP-AA and JQ1 energy depletion have a strong synergistic effect in driving apoptosis. These results validate our hypothesis that the nanoassembly can effectively eliminate tumor cells through dual glycolysis / mitochondrial blockade of energy depletion.

[0116] Example 10: Reactive oxygen species (ROS) detection

[0117] The DCFH-DA probe detects intracellular ROS levels. In fact, DCFH-DA is a general indicator of oxidative stress and does not show fluorescence, but intracellular esterases and ROS can oxidize non-fluorescent DCFH-DA to DCFH and fluorescent DCF. First, 4T1 cells were cultured at 5×10 4Cells were seeded in 24-well plates at a density of 100 μg / well for 12 h. 4T1 cells were then treated with TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles at the same concentration of JQ1 (10 μM). After incubation with 4T1 cells for 4 h, DCFH-DA (10 μM) was incubated for 30 min, and DCF fluorescence was detected using an inverted microscope. In addition, 4T1 cells were treated and collected using the same method. The cells were resuspended in PBS at pH 7.4, and the fluorescence of intracellular ROS was detected by flow cytometry.

[0118] like Figure 22-23 As shown in the results, TPP-AA solution can promote the production of intracellular ROS to a certain extent, and the combined treatment group TAJ solution, TAJP nanoparticles and TAJS nanoparticles caused higher ROS levels. Among them, TAJS nanoparticles caused the most significant ROS production.

[0119] Example 11: Mitochondrial lipid peroxidation detection

[0120] MitoPeDPP was used as a fluorescent dye to detect intracellular lipid peroxides. Briefly, 4T1 cells were plated at 1×10 5 Cells were seeded at a density of 10 μM / well in 35 mm glass-bottomed culture dishes and incubated for 12 hours. The medium was then replaced with equal concentrations of JQ1 (10 μM) in TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles, and incubated for 12 hours. Afterwards, the cells were treated as directed by MitoPeDPP and observed by CLSM.

[0121] like Figure 24 As shown, both TPP-AA solution and JQ1 solution promoted the production of mitochondrial lipid peroxides to some extent. Furthermore, based on the synergistic effect of TPP-AA and JQ1, the combined treatment groups TAJ solution, TAJP nanoparticles, and TAJS nanoparticles induced higher levels of mitochondrial lipid peroxides. Among them, TAJS nanoparticles led to the greatest accumulation of mitochondrial lipid peroxides.

[0122] Example 12: Mitochondrial membrane potential measurement

[0123] JC-1 is used as a fluorescent probe to detect early cell apoptosis. When the mitochondrial membrane potential is high, JC-1 can accumulate in the mitochondrial matrix and produce red fluorescence. When the mitochondrial membrane potential is low, JC-1 produces green fluorescence as a monomer. The change of JC-1 fluorescence from red to green can easily detect the decrease in cell membrane potential and can be used as an indicator of early apoptosis. Briefly, 4T1 cells were cultured at 2×10 5Cells were seeded at a density of 10 μM per well in 35 mm glass-bottomed cell culture dishes and incubated for 12 hours. The culture medium was then replaced with equal concentrations of JQ1 (10 μM) in TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles, and incubated for 12 hours. Subsequently, the cells were treated with JC-1 according to the instructions of the mitochondrial membrane potential assay kit and observed by CLSM. In addition, 4T1 cells were treated using the same method and processed according to the instructions of the JC-1 mitochondrial membrane potential assay kit. JC-1 intracellular fluorescence was detected by flow cytometry.

[0124] like Figures 25-26 As shown, both TPP-AA solution and JQ1 solution reduced mitochondrial membrane potential to some extent. Combined treatment with TAJ solution, TAJP nanoparticles, and TAJS nanoparticles significantly reduced mitochondrial membrane potential. TAJS nanoparticles led to the most significant reduction in mitochondrial membrane potential.

[0125] Example 13: Western blotting

[0126] Western blot analysis was used to detect c-Myc expression in 4T1 cells. 4T1 cells were treated with blank medium, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles containing equal concentrations of JQ1 (10 μM). Total cellular protein concentration was quantified using the BCA assay. Equal amounts of protein samples were subjected to SDS-PAGE electrophoresis and electrophoretic transfer. The membranes were then blocked with 5% skim milk for 1 hour. The PVDF membranes were then incubated with rabbit monoclonal antibodies against c-Myc and GAPDH, respectively, overnight at 4°C. The PVDF membranes were incubated with secondary antibodies at 37°C for 1 hour. After incubation, ECL Western Blotting Substrate was added to visualize protein bands.

[0127] like Figure 27 As shown in the results, the JQ1-containing groups significantly downregulated the expression of c-Myc. In addition, TAJS nanoparticles showed a clear advantage in downregulating the expression of c-Myc.

[0128] Example 14: Lactate, lactate dehydrogenase (LDH) and ATP detection

[0129] 4T1 cells were cultured at 4 × 10 6 Cells were seeded at a density of 100 cells / well in 6-well plates and cultured for 12 hours before being treated with blank culture medium, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles, and TAJS nanoparticles, respectively. Following treatment, 4T1 cells were washed and collected. Intracellular lactate, lactate dehydrogenase, and ATP levels were measured according to the manufacturer's instructions.

[0130] like Figures 28-29 As shown, reduction-sensitive nanoparticles (TAJS nanoparticles) can significantly reduce LDH content and inhibit lactate efflux, which is attributed to JQ1-mediated glycolysis inhibition. The experimental results show that compared with other groups, TAJS nanoparticles have a significant advantage in reducing ATP levels ( Figure 30 TAJS nanoparticles with excellent cellular uptake efficiency and cytotoxicity caused the greatest energy consumption. Obviously, the combination of TPP-AA and JQ1 can successfully achieve dual blockade energy and has strong cytotoxicity.

[0131] Example 15: Pharmacokinetic Study of Nanoparticles

[0132] SD rats weighing between 180-220 g were randomly divided into groups and fasted for 12 h before administration, with free access to water. Cy7 solution and TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles prepared according to Example 3 (both at 2 mg / kg of Cy7) were injected intravenously. Blood was collected from the eye sockets at the specified time points and plasma was separated. Cy7 was then extracted by protein precipitation method, and the pharmacokinetic behavior of each preparation was finally detected using an enzyme marker (excitation 750 nm, emission 773 nm). The experimental results are shown in Figure 2. Figure 31 As shown in the figure, due to its short half-life, Cy7 solution is quickly metabolized and cleared. Compared with the solution, TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles have significantly prolonged circulation time and significantly increased the AUC of Cy7, providing a good foundation for the accumulation of the drug in tumors in vivo.

[0133] Example 16: Tissue distribution experiment of nanoparticles

[0134] 4T1 cell suspension was inoculated subcutaneously on the ventral side of BALB / c mice. When the tumor volume reached 300 mm 3 At 4, 6, 8, 12, and 24 hours after administration, mice were anesthetized and subjected to in vivo imaging analysis. Figure 32 shown.

[0135] The time point with the highest accumulation in each group was selected for fluorescence intensity analysis of isolated tissues and organs. Figures 33-34 shown.

[0136] The above results showed that compared with Cy7 solution, the fluorescence intensity of TAJP-Cy7 nanoparticles and TAJS-Cy7 nanoparticles in tumor tissue was significantly increased, and reached maximum accumulation at 8 hours.

[0137] Example 17: In vivo anti-tumor experiment of nanoparticles

[0138] 4T1 cell suspension (10 7 cells / 100 μL) were inoculated subcutaneously on the ventral side of female mice. 3 The mice were randomly divided into groups of 5 each and were given PBS, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles prepared in Example 3, and TAJS nanoparticles, respectively. The drugs were administered once every other day for 5 consecutive times, and the total dose of TPP-AA and JQ1 was 30 mg / kg. The survival status of the mice was observed every day, their body weight was weighed, and the tumor volume was measured. After the last administration, the mice were killed after an interval of 1 day, and the organs and tumors were obtained for further analysis and evaluation. The main organs (heart, liver, spleen, lungs, and kidneys) were collected and fixed with 4% tissue fixative for H&E staining.

[0139] In addition, the safety of the nanoparticles was tested using the same method. In short, mice were injected with TAJ solution, TAJP nanoparticles and TAJS nanoparticles (total concentrations of 10, 30 and 50 mg / kg). The safety of the treatment was evaluated by the weight changes and survival rates of the mice during treatment. In addition, a CT26 breast tumor-bearing BALB / c mouse model was established, and its in vivo anti-tumor effect was detected using the same method. PBS, TPP-AA solution, JQ1 solution, TAJ solution, TAJP nanoparticles prepared in Example 3 and TAJS nanoparticles (15 mg / kg, JQ1 equivalent) were intravenously administered to model mice. The anti-tumor effect was verified by the evaluation of various indicators.

[0140] Tumor growth curve, tumor-bearing rate and in vitro tumor photos after treatment of 4T1 tumor model mice are shown in the figure. Figures 35-37 As shown in the figure, at the administered dose, there was no significant difference between the TPP-AA solution group and the PBS group, while the anti-tumor activity of the TAJ solution group was more significant than that of the JQ1 solution group, indicating that TPP-AA and JQ1 have a strong synergistic anti-tumor effect. In addition, TAJS nanoparticles with tumor-specific drug release properties showed better closed-loop energy depletion-driven tumor eradication effect than insensitive TAJS nanoparticles. Figure 38 As shown in Figure 2, H&E and TUNEL staining also showed that TAJS nanoparticles have a strong tumor killing effect. A preliminary study was conducted on their therapeutic safety. Figures 39-40 As shown in the figure, after multiple treatments, no obvious abnormalities were found in the H&E stained sections of the heart, liver, spleen, lung, and kidney, as well as in the liver and kidney blood indicators. During the entire treatment process, the change in body weight was negligible ( Figure 41 To further investigate the safety of nanoparticles, the survival of mice at different doses was determined. Figures 42-43As shown in Figure 3, when the dose was 50 mg / kg, the survival rate of mice in the TAJ solution group was greatly reduced after multiple administrations, while the TAJP nanoparticle group and the TAJS nanoparticle group still survived. In summary, these results indicate that the dose we selected for mouse administration is safe and effective. Subsequently, we conducted in vivo mechanism verification, such as Figure 44 As shown in Figure 3, TAJS nanoparticles significantly reduced the level of c-Myc in tumor tissues, which was consistent with the results of in vitro experiments. Figures 45-47 As shown, TAJS nanoparticles could significantly reduce LDH content, inhibit lactate efflux and decrease ATP level.

[0141] In addition, we further explored the therapeutic effects and mechanisms of nano-depleting agents on CT26 tumor-bearing mice. Figures 48-50 As shown, the combined treatment groups (TAJ solution, TAJP nanoparticles, and TAJS nanoparticles) all showed good anti-tumor effects compared with the control group. As expected, TAJS nanoparticles had the best tumor killing ability, while the changes in mouse body weight were negligible ( Figure 51 At the same time, no obvious abnormalities were found in the liver and kidney function indicators and H&E staining results of major organs after treatment, indicating that its in vivo therapeutic effect is good and its safety is high ( Figures 52-53 ). We then evaluated the anti-tumor mechanism in CT26 tumor-bearing mice. Figure 54 The results are consistent with the above results ( Figures 45-47 ).

[0142] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A tumor energy depletion nanoassembly, characterized in that: The tumor energy depletion nanoassembly is co-assembled by a mitochondrial oxidative phosphorylation inhibitor and a glycolysis inhibitor through intermolecular forces and modified with a PEG modifier; the molar ratio of the mitochondrial oxidative phosphorylation inhibitor to the glycolysis inhibitor is 10:1 to 1:10, and the mass ratio of the sum of the mitochondrial oxidative phosphorylation inhibitor and the glycolysis inhibitor to the PEG modifier is 10:90 to 90:10; the mitochondrial oxidative phosphorylation inhibitor is a conjugate of triphenylphosphine and an unsaturated fatty acid; The unsaturated fatty acid in the combination of triphenylphosphine and unsaturated fatty acid is arachidonic acid; The PEG modifier is DSPE-PEG 2K or DSPE-SS-PEG 2K ; The glycolysis inhibitor is JQ1.

2. The tumor energy-depleting nanoassembly according to claim 1, wherein The intermolecular forces include π-π stacking, hydrophobic interaction and hydrogen bonding.

3. The method for preparing the tumor energy-depleting nanoassembly according to any one of claims 1 to 2, characterized in that: The steps include: The mitochondrial oxidative phosphorylation inhibitor and the glycolysis inhibitor are dissolved in organic solvents respectively, mixed under stirring, and the mixed solution is slowly added dropwise to water to spontaneously form uniform co-assembled nanoparticles; the organic solvent of the PEG modifier is added dropwise to the co-assembled nanoparticles under stirring, and the organic solvent is removed to obtain the nanoparticles.

4. The preparation method according to claim 3, wherein The organic solvent is one of ethanol, tetrahydrofuran, and dimethyl sulfoxide, or a combination of any two thereof.

5. Use of the tumor energy-depleting nanoassembly according to any one of claims 1 to 2 or the tumor energy-depleting nanoassembly prepared by the preparation method according to claim 3 or 4 in the preparation of a drug delivery system.

6. Use of the tumor energy-depleting nanoassembly according to any one of claims 1 to 2 or the tumor energy-depleting nanoassembly prepared by the preparation method according to claim 3 or 4 in the preparation of anti-tumor drugs.

7. Use of the tumor energy-depleting nanoassembly according to any one of claims 1 to 2 or the tumor energy-depleting nanoassembly prepared by the preparation method according to claim 3 or 4 in the preparation of an injection, oral administration or local administration system.

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