Pharmaceutical composition of paclitaxel dimer prodrug nanoparticles and traditional Chinese medicine compound and application of pharmaceutical composition

Abstract

The invention relates to the technical field of biological medicine, and discloses a pharmaceutical composition of paclitaxel dimer prodrug nanoparticles and a traditional Chinese medicine compound and application of the pharmaceutical composition, and the pharmaceutical composition is composed of Qiyuansanlong prescription freeze-dried powder and paclitaxel dimer prodrug nanoparticles; wherein the paclitaxel dimer prodrug nanoparticles comprise one of NQO1 response type PTX dimer prodrug nanoparticles (PTX-Q-PTX NPs) or GSH (glutathione) response type PTX dimer prodrug nanoparticles (PTX-SeSe-PTXNPs), and the paclitaxel dimer prodrug nanoparticles comprise one of NQO1 response type PTX dimer prodrug nanoparticles (PTX-Q-PTX NPs) and GSH response type PTX dimer prodrug nanoparticles (PTX-SeSe-PTXNPs). According to the pharmaceutical composition disclosed by the invention, a traditional Chinese and western medicine synergistic treatment system is innovatively constructed, and the advantage of multi-dimensional synergistic interaction is shown. Firstly, an NQO1 / GSH double-response type intelligent release system is adopted, and a biomarker highly expressed by tumor tissue can be specifically responded to realize precise drug release, so that the PTX concentration in tumors is improved compared with that of a traditional dosage form, and meanwhile, the drug circulation time is prolonged. Secondly, the active ingredients in the Qiyuansanlong prescription freeze-dried powder can significantly down-regulate the PD-L1 expression level, synergistically enhance CD4 + T and CD8 + T cell infiltration, and form chemical-immune dual killing effects.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and in particular to a pharmaceutical composition of paclitaxel dimer prodrug nanoparticles and a traditional Chinese medicine compound, and its application. Background Art

[0002] Lung cancer has become the most common cancer globally due to its high incidence and mortality, posing a great threat to human health. The reasons are immunosuppression and inefficient drug treatment. The efficient treatment of lung cancer has become a major challenge that the global medical community urgently needs to overcome.

[0003] For decades, chemotherapy has always been the main means of treating lung cancer. Among them, as the core drug in the first-line clinical chemotherapy regimen, paclitaxel (PTX) has an irreplaceable position in the systemic treatment of non-small cell lung cancer (NSCLC). However, long-term clinical applications have found that although the traditional formulations of PTX can effectively inhibit tumor growth and proliferation, the polyoxyethylene castor oil and ethanol carriers used in it are prone to cause allergic reactions, and there are problems such as uneven distribution in tumor tissues and obvious toxic and side effects on normal tissues. To overcome these defects, although the subsequent developed PTX nanoformulations avoid carrier-related toxicities and improve the targeting of drugs in lung cancer tissues, they still face technical bottlenecks such as low drug loading, poor stability, easy recrystallization and drug leakage during storage. More seriously, studies have found that PTX formulations can induce the upregulation of the expression of the tumor cell PD-L1 protein, resulting in the formation of an immunosuppressive microenvironment during the treatment process, seriously affecting the treatment effect. Therefore, developing a new type of PTX formulation with high efficiency and low toxicity has become an urgent need for the current treatment of lung cancer.

[0004] Small molecule prodrug self-assembled nanoparticles that combine nanotechnology and prodrug strategies are considered an effective method to solve the chemotherapy dilemma. They can not only passively target tumor tissues through the EPR effect, increase the concentration of drugs in tumors, thereby reducing the toxic and side effects of PTX. More importantly, in the tumor-specific microenvironment, stimuli-responsive prodrugs can achieve precise drug release and tumor-specific killing. It is reported that the bond angle of the disulfide bond is close to 90°, which can improve the structural toughness of the original drug and is widely used in the design of self-assembled original drugs. It constructs a stimuli-responsive carrier using disulfide bonds (good structural toughness) and diselenide bonds (strong redox sensitivity), which can synergistically deliver drugs, improve water solubility, optimize pharmacokinetics, and achieve precise drug release in the tumor microenvironment. However, studies have shown that these drugs can induce the upregulation of PD-L1 expression, promote the formation of an immunosuppressive state in the tumor microenvironment, and thus significantly reduce the final efficacy of long-term treatment.

[0005] Qi-Yu-San-Long formula (Q), an effective traditional Chinese medicine compound for the clinical treatment of lung cancer, has been proven to significantly relieve the clinical symptoms of advanced lung cancer patients, improve the quality of life and prolong the survival period. Under the guidance of traditional Chinese medicine theory, Qi-Yu-San-Long formula is a compound preparation scientifically formulated with ten traditional Chinese medicines. Its formula includes: the monarch drugs Astragalus membranaceus and Polygonatum odoratum to strengthen the healthy qi and consolidate the root; the ministerial drugs Gecko and Pheretima aspergillum to dredge collaterals and disperse nodules; the adjuvant drugs Solanum nigrum, Hedyotis diffusa, and Coix lacryma-jobi to clear heat and detoxify; and the compatibility of Euphorbia helioscopia and Curcuma zedoaria to enhance the effect of promoting blood circulation and dissipating nodules. Previous experimental studies have shown that the antitumor effect of Qi-Yu-San-Long formula is related to the regulation of PI3K / Akt / mTOR and Wnt / β-catenin signaling pathways. At the same time, it can significantly reduce the PD-L1 mRNA level and PD-L1 protein level in tumor tissues, and enhance Th1 immune responses by mediating the PD-1 signaling pathway, regulate the tumor immune microenvironment, inhibit tumor cell immune escape, and have a certain anti-tumor immune regulatory effect on tumor-bearing mice.

[0006] NQO1 (Quinone Oxidoreductase 1) is a two-electron oxidoreductase that catalyzes and reduces a variety of compounds, including quinone nitroaromatic compounds, imidazoles, and iron ions. Compared with normal tissues, NQO1 is highly expressed in most solid tumors (such as lung cancer, brain cancer, gastric cancer, and colon cancer), making this enzyme an attractive endogenous activator in tumor-specific drug delivery systems. At the same time, NQO1 has a "dual" effect in cancer biology. On the one hand, it provides multiple layers of protection for normal cells against carcinogenesis; on the other hand, overexpression in cancer cells can promote the proliferation, metastasis, and drug resistance of cancer cells. However, the cell death mediated by NQO1 and its potential role in combating drug-resistant cancers remain unclear.

[0007] Existing lung cancer chemotherapy drugs (such as paclitaxel, PTX) have problems such as poor water solubility, low bioavailability, and significant systemic toxic side effects, and cannot overcome the interference of high concentrations of glutathione (GSH) and reactive oxygen species (H2O2) in the tumor microenvironment (TME) on drug release. In addition, although PTX can induce immunogenic cell death (ICD), it activates the PD-L1 immune checkpoint, leading to immune escape, while existing PD-L1 inhibitors are costly and lack sufficient targeting. Although the traditional Chinese medicine Qi-Yu-San-Long formula (Q) has an immune regulatory effect, its anti-tumor specificity is insufficient. Therefore, there is an urgent need to develop a new strategy for the treatment of lung cancer with high efficiency, low toxicity, and both chemical and immune synergistic effects. Summary of the Invention

[0008] The object of the present invention is to provide a pharmaceutical composition of a paclitaxel dimer prodrug nanoparticle and a traditional Chinese medicine compound and its application, specifically relating to a chemo-immunotherapy system based on the combined application of a paclitaxel (PTX) dimer nano-prodrug and a traditional Chinese medicine compound (Qi-Yu-San-Long-Fang, Q) for the synergistic chemo-immunotherapy of lung cancer. To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a pharmaceutical composition of a paclitaxel dimer prodrug nanoparticle and a traditional Chinese medicine compound, which is composed of the freeze-dried powder of Qiyu Sanlong Formula and the paclitaxel dimer prodrug nanoparticle; wherein,

[0010] The paclitaxel dimer prodrug nanoparticle includes one of the quinone oxidoreductase 1 (NQO1)-responsive paclitaxel (PTX) dimer prodrug nanoparticle PTX-Q-PTXNPs or the glutathione (GSH)-responsive paclitaxel (PTX) dimer prodrug nanoparticle PTX-SeSe-PTXNPs.

[0011] Further, the concentrations of PTX-Q-PTX NPs and their control nanoparticles are set equally with the PTX concentration at 5 - 80000 nM, preferably 50 - 40000 nM, more preferably 500 - 20000 nM, and the QYSLD concentration correspondingly is 8 - 22 mg / mL, preferably 10 - 20 mg / mL, more preferably 12 - 18 mg / mL.

[0012] Further, the concentrations of PTX-SeSe-PTX NPs and their control nanoparticles are set equally with the PTX concentration at 0.1 - 8000 nM, preferably 0.2 - 2000 nM, more preferably 0.25 - 500 nM, and the QYSLD concentration correspondingly is 8 - 22 mg / mL, preferably 8 - 20 mg / mL, more preferably 8 - 14 mg / mL.

[0013] Further, the preparation method of the dimer prodrug nanoparticle PTX-Q-PTX NPs is as follows:

[0014] Dissolve the dimer prodrug PTX-Q-PTX and DSPE-PEG 2K in a mixed solvent of absolute ethanol and acetone, and slowly drop it into deionized water under stirring conditions, and then remove the organic solvent by rotary evaporation under reduced pressure at room temperature to obtain the dimer prodrug nanoparticle PTX-Q-PTXNPs.

[0015] Further, the molar ratio of PTX-Q-PTX to DSPE-PEG 2K is 1:2 - 3; preferably 1:2;

[0016] The volume ratio of absolute ethanol to acetone is 2 - 3:3; preferably 2:3.

[0017] Furthermore, the preparation method of the dimer prodrug nanoparticles PTX-SeSe-PTX NPs is as follows:

[0018] Dissolve the dimer prodrug PTX-SeSe-PTX and DSPE-PEG 2K in a mixed solvent of absolute ethanol and tetrahydrofuran. Under stirring conditions, slowly drop the solution into deionized water, and then remove the organic solvents by rotary evaporation under reduced pressure at room temperature to obtain the dimer prodrug nanoparticles PTX-SeSe-PTX NPs.

[0019] Furthermore, the molar ratio of PTX-SeSe-PTX to DSPE-PEG 2K is 4:1 - 3, preferably 4:1;

[0020] The volume ratio of absolute ethanol to tetrahydrofuran is 5 - 7:1; preferably 7:1.

[0021] Furthermore, the preparation method of the dimer prodrug PTX-Q-PTX includes the following steps:

[0022] (1) Add methanesulfonic acid to 2,5-dimethylhydroquinone. Evacuate and protect with nitrogen, heat until the solid compound dissolves, then slowly drop 3,3-dimethylmethyl acrylate into it. After heating and stirring the reaction, stop. After the reaction system cools to room temperature, extract and wash with organic solvents in turn, remove water, concentrate under reduced pressure until a solid precipitates, and finally filter to obtain the bis-lactone chromanone derivative;

[0023] (2) Dissolve the bis-lactone chromanone derivative in tetrahydrofuran, then add lithium hydroxide solution to it. Stir the reaction under open conditions at room temperature, and monitor the conversion rate of the reaction by thin layer chromatography. When the conversion is complete, first adjust the pH of the reaction mixture to 3 - 4, then extract and wash with organic solvents in turn, remove water, and dry under reduced pressure to obtain the intermediate compound quinone dipropionic acid QDPA;

[0024] (3) Dissolve PTX in dichloromethane, then add QDPA, EDCI, and DMAP in turn. Stir the reaction under N2 protection at room temperature. Add EDCI and DMAP again and continue the reaction until it ends. The reaction solution is eluted and separated and purified with a mixed organic solvent to obtain the dimer prodrug PTX-Q-PTX.

[0025] Further, the molar ratio of 2,5 - dimethyl - p - benzenediol, methanesulfonic acid and methyl 3,3 - dimethylacrylate is 2 - 4:3 - 6:3 - 6, preferably 2:3:3; the molar ratio of the dilactone chromanone derivative to lithium hydroxide is 2 - 5:3 - 5, preferably 2:3; the molar ratio of PTX, QDPA, EDCI and DMAP is 3 - 5:2 - 3:10 - 15:1 - 2, preferably 3:2:10:1.

[0026] Further, the preparation method of the dimer prodrug PTX - SeSe - PTX includes the following steps:

[0027] (1) Add selenium powder into a reaction vessel filled with water, dropwise add sodium borohydride dissolved in water under ice bath. After the reaction solution becomes clear, add another portion of selenium powder, heat under reflux, then add bromopropionic acid solution into the above system, react at room temperature overnight. Protect the whole process with N2 until the reaction ends, adjust the pH of the reaction solution, extract with organic solvents in turn, recrystallize and dry to obtain the intermediate product 3,3'-diselenodipropionic acid;

[0028] (2) Dissolve PTX in dichloromethane, then add diselenodipropionic acid, EDCI and DMAP in turn, stir and react at room temperature under N2 protection. Add EDCI and DMAP again and continue the reaction until the end. The reaction solution is eluted and separated and purified with a mixed organic solvent to obtain the dimer prodrug PTX - SeSe - PTX.

[0029] Further, the molar ratio of selenium powder, sodium borohydride and bromopropionic acid is 1 - 2:2 - 4:1 - 3, preferably 1:2:1; the molar ratio of PTX, diselenodipropionic acid, EDCI and DMAP is 3 - 5:4 - 5:10 - 15:1 - 2, preferably 3:4:10:1.

[0030] The present invention also provides an application of a pharmaceutical composition of a paclitaxel dimer prodrug nanoparticle and a traditional Chinese medicine compound in the preparation of an anti - lung cancer drug.

[0031] The technical effects and advantages of the present invention:

[0032] First, the present invention synthesizes a paclitaxel dimer prodrug PQPNPs bridged by quinodipropionic acid (QDPA), and uses the paclitaxel dimer prodrug PCPNPs bridged by a non - sensitive carbon bond as a control. The NQO1 - responsive paclitaxel dimer prodrug and non - sensitive paclitaxel dimer prodrug nanodelivery systems (PQPNPs / PCP NPs) are constructed by the nanoprecipitation method, and combined with Qiyu Sanlong Formula for tumor chemo - immunotherapy ( Figure 1 ). This combination therapy has the following advantages:

[0033] (1) High drug loading, long circulation, and tumor tissue targeting: The dimer prodrug itself serves as a carrier of the nano-prodrug, with high drug loading capacity. Meanwhile, DSPE-PEG 2K can increase the circulation time of the nanoparticles, and then passively target tumor tissues through the enhanced permeability and retention (EPR) effect.

[0034] (2) NQO1-triggered precise and rapid drug release: High expression of NQO1 in tumors oxidizes the quinone structure in QDPA to a hydroxyl group. The hydroxyl group attacks the carbonyl group intramolecularly to form a lactone ring, and the ester bond breaks to rapidly release PTX, increasing the accumulation of PTX in cells and exerting a chemotherapeutic effect.

[0035] (3) Qiyu Sanlong formula regulates the tumor immune microenvironment: By downregulating the expression of PD-1 / PD-L1, it remodels the tumor suppressive microenvironment and then synergistically exerts tumor chemo-immunotherapy with PTX.

[0036] Second, the present invention synthesized selenium-selenium (Se-Se), sulfur-sulfur (S-S) bond-linked paclitaxel dimer prodrugs diPTX-2Se and diPTX-2S, and used the paclitaxel dimer prodrug diPTX-2C linked by a non-sensitive carbon bond as a control. The nano-precipitation method was used to construct GSH / H2O2-responsive paclitaxel dimer prodrug and non-sensitive paclitaxel dimer prodrug nano-delivery systems (diPTX-2Se NPs / diPTX-2S NPs / diPTX-2C NPs), combined with Qiyu Sanlong formula, for tumor chemo-immunotherapy ( Figure 1 ). This combined therapy has the following advantages:

[0037] (1) Redox-responsive prodrug design: Paclitaxel dimer prodrugs were constructed through selenium-selenium (Se-Se), sulfur-sulfur (S-S) or carbon chain (C-C) linkage bonds. Among them, the Se-Se bond has the optimal redox sensitivity and self-assembly stability, and can be rapidly cleaved under high GSH / H2O2 conditions in the tumor microenvironment (TME) to achieve tumor-specific PTX release and reduce normal cell damage.

[0038] (2) Self-assembled nano-delivery system: The prodrug was modified with DSPE-PEG 2K to form nanoparticles (NPs) with uniform particle size (about 109 - 133 nm) and negative surface charge, which passively target tumors through the enhanced permeability and retention (EPR) effect, prolong the blood circulation time, and improve the drug loading rate (>98%).

[0039] (3) Synergistic immune regulation of traditional Chinese and Western medicine: Combining oral administration of traditional Chinese medicine Q significantly downregulates the expression of PD-L1 in tumor tissues, and at the same time promotes CD4 + / CD8 +T cell infiltration and polarization of M2 macrophages into M1 macrophages reshape the immunosuppressive microenvironment, forming a positive feedback loop of "killing-immune activation" with chemotherapy, enhancing the ICD effect of PTX, providing an efficient, low-toxicity, and highly synergistic integrated solution for lung cancer treatment, applicable to different tumor burdens (small tumors / large tumors) and dose requirements (low dose / high dose) scenarios, and possessing important clinical translational value.

[0040] (4) High-efficiency targeting and precise release: The diselenide prodrug has a high release efficiency, and NPs specifically release PTX at the tumor site. Its cytotoxicity is 10 times higher in lung cancer cells (LLC) than in normal cells (3T3) (IC50: 0.249 nM). Q reduces the expression of PD-L1, and Q does not affect the targeting of NPs, reducing chemotherapy-induced immunosuppression.

[0041] (5) Synergistic anti-tumor and immune activation: Q combined with diPTX-2Se NPs significantly inhibits tumor growth (the tumor volume in the low-dose model decreases by 80%), and through downregulating PD-L1, increasing the proportion of CD4 + T and CD8 + T cell proportions, and promoting the polarization of M2 to M1 macrophages, achieving synergistic enhancement of chemotherapy and immunity.

[0042] (6) Broad-spectrum and safety: Nanoparticles are enriched in tumors through the EPR effect, reducing toxicity to normal tissues. They show significant efficacy in both small tumor models at low doses (5 mg / kg PTX) and large tumor models at high doses (10 mg / kg PTX), and do not cause weight loss or organ toxicity, verifying the universality and safety of the solution.

[0043] Other features and advantages of the present invention will be described in the subsequent specification, and in part, will become apparent from the specification or be understood by implementing the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the structures pointed out in the specification and the drawings. Brief Description of the Drawings

[0044] To more clearly illustrate the technical solutions in the embodiments of the present invention or in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following-described drawings are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0045] Figure 1 It is a mechanism diagram of the application of the drug composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound of the present invention in the treatment of lung cancer;

[0046] Figure 21H-NMR spectrum of the paclitaxel dimer prodrug PQP in Example 1 of the present invention; 1 H-NMR spectrum;

[0047] Figure 3 TOF-MS spectrum of the paclitaxel dimer prodrug PQP in Example 1 of the present invention;

[0048] Figure 4 High performance liquid chromatography (HPLC) chromatogram of the paclitaxel dimer prodrug PQP in Example 1 of the present invention;

[0049] Figure 5 1H-NMR spectrum of the paclitaxel dimer prodrug PCP in Example 1 of the present invention; 1 H-NMR spectrum;

[0050] Figure 6 TOF-MS spectrum of the paclitaxel dimer prodrug PCP in Example 1 of the present invention;

[0051] Figure 7 High performance liquid chromatography (HPLC) chromatogram of the paclitaxel dimer prodrug PCP in Example 1 of the present invention;

[0052] Figure 8 1H-NMR spectrum of the paclitaxel dimer prodrug 2diPTX-2Se in Example 1 of the present invention; 1 H-NMR spectrum;

[0053] Figure 9 TOF-MS spectrum of the paclitaxel dimer prodrug 2diPTX-2Se in Example 1 of the present invention;

[0054] Figure 10 High performance liquid chromatography (HPLC) chromatogram of the paclitaxel dimer prodrug 2diPTX-2Se in Example 1 of the present invention;

[0055] Figure 11 1H-NMR spectrum of the paclitaxel dimer prodrug 2diPTX-2S in Example 1 of the present invention; 1 H-NMR spectrum;

[0056] Figure 12 TOF-MS spectrum of the paclitaxel dimer prodrug 2diPTX-2S in Example 1 of the present invention;

[0057] Figure 13 High performance liquid chromatography (HPLC) chromatogram of the paclitaxel dimer prodrug 2diPTX-2S in Example 1 of the present invention;

[0058] Figure 14 1H-NMR spectrum of the paclitaxel dimer prodrug 2diPTX-2C in Example 1 of the present invention; 1 H-NMR spectrum;

[0059] Figure 15TOF-MS spectrum of paclitaxel dimer prodrug 2diPTX-2C in Example 1 of the present invention;

[0060] Figure 16 High performance liquid chromatography of paclitaxel dimer prodrug 2diPTX-2C in Example 1 of the present invention;

[0061] Figure 17 Characterization results and self-assembly mechanism investigation results of PQPNPs and PCPNPs in Example 2 of the present invention; wherein, (A) and (B) are the hydrodynamic diameters of PQPNPs and PCPNPs respectively; (C) is the ζ-potential of PQPNPs and PCPNPs; (D) and (E) are the TEM images of PQPNPs and PCPNPs respectively; (F) and (G) are the changes in hydrodynamic diameter and PDI of PQPNPs and PCP NPs incubated with 10% FBS PBS (pH 7.4) solution and deionized water for different times;

[0062] Figure 18 Characterization results and self-assembly mechanism investigation results of diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs in Example 2 of the present invention; wherein, (A), (B) and (C) are the hydrodynamic diameters and TEM images of diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs respectively; (D) is the ζ-potential of diPTX-2Se NPs, diPTX-2S NPs and diPTX-2CNPs; (E), (F) and (G) are the TEM images of diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs respectively, and (H), (I) and (J) are the changes in size and PDI of diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs after treatment with Water, NaCl, Urea and SDS for 24h;

[0063] Figure 19 Stability investigation results of PQPNPs and PCPNPs in Example 3 of the present invention; wherein, (A) is the change in hydrodynamic diameter of PQPNPs and PCPNPs incubated with 10% FBS PBS (pH 7.4) solution for different times; (B) is the change in hydrodynamic diameter of PQPNPs and PCPNPs incubated with deionized water for different times;

[0064] Figure 20This is the result graph of the stability investigation of diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in Example 3 of the present invention; among them, (A) shows the change in hydrodynamic diameter of diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs incubated with 10% FBS PBS (pH 7.4) solution for different times; (B) shows the change in hydrodynamic diameter of diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs incubated with deionized water for different times.

[0065] Figure 21 This is the result graph of the in vitro release behavior of PQPNPs and PCPNPs in Example 4 of the present invention; among them, (A) is the in vitro release curve of PQPNPs and PCPNPs in a release medium containing 30 mM Na2S2O4; (B) is the in vitro release curve of PQPNPs and PCPNPs in a release medium containing 60 mM Na2S2O4; (C) and (E) are the appearance morphologies of PQPNPs before and after adding 60 mM Na2S2O4, respectively; (D) and (F) are the appearance morphologies of PCPNPs before and after adding 60 mM Na2S2O4, respectively.

[0066] Figure 22 This is the result graph of the in vitro release behavior of diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in Example 4 of the present invention; among them, (A), (B), and (C) are the drug releases induced by diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs determined by HPLC in a release medium containing 10 mM DTT, respectively; (D) is the corresponding release rate; (E), (F), and (G) are the drug releases induced by diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs determined by HPLC in a release medium containing 10 mM H2O2, respectively; (H) is the corresponding release rate; (I) and (J) are the release rates of PTX in diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in the presence of 10 mM DTT and H2O2, respectively.

[0067] Figure 23 This is the result graph of the cytotoxicity of Q combined with PQPNPs and PCPNPs in Example 6 of the present invention; among them, (A), (B), and (C) are the cell viabilities (n = 6) of Taxol, PQPNPs, PCPNPs, QYSLD, PQPNPs&Q, and PCP NPs&Q co-incubated with LLC cells, A549 cells, and L929 cells for 48 h determined by the CCK-8 method, respectively.

[0068] Figure 24 This is the graph showing the results of flow cytometry for the cellular uptake of Q combined with PQPNPs and PCPNPs in Example 6 of the present invention. Among them, (A), (B), and (C) are the average fluorescence intensities (n = 3) of LLC cells, A549 cells, and L929 cells incubated with C-6, C-6&Q, PQPNPs / C6, PCPNPs / C6, PQPNPs / C6&Q, and PCPNPs / C6&Q, respectively, as quantitatively detected by flow cytometry (FCM).

[0069] Figure 25 This is the graph showing the results of confocal laser scanning microscopy for the cellular uptake of Q combined with PQPNPs and PCPNPs in Example 6 of the present invention. Among them, (A), (B), and (C) are the CLSM images of LLC cells, A549 cells, and L929 cells cultured with C-6, C-6&Q, PQPNPs / C6, PCPNPs / C6, PQPNPs / C6&Q, and PCPNPs / C6&Q for 0.5 h and 2 h, respectively.

[0070] Figure 26 This is the graph showing the cytotoxicity results of Q combined with diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in Example 6 of the present invention. Among them, (A) and (B) are the cell viabilities (n = 6) of Taxol, diPTX-2Se NPs, diPTX-2S NPs, diPTX-2C NPs, Q, diPTX-2Se NPs&Q, diPTX-2S NPs&Q, and diPTX-2C NPs&Q co-incubated with LLC cells and 3T3 cells for 48 h as determined by the CCK-8 method.

[0071] Figure 27 This is the graph showing the results of flow cytometry for the cellular uptake of Q combined with diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in Example 6 of the present invention. Among them, (A) and (B) are the average fluorescence intensities (n = 3) of LLC cells and 3T3 cells incubated with C-6, diPTX-2Se / C6 NPs, diPTX-2S / C6 NPs, diPTX-2C / C6 NPs, diPTX-2Se / C6 NPs&Q, diPTX-2S / C6 NPs&Q, and diPTX-2C / C6 NPs&Q, respectively, as quantitatively detected by flow cytometry (FCM).

[0072] Figure 28This is the figure of the cellular uptake results of laser confocal detection of Q combined with diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in Example 6 of the present invention; among them, (A) and (B) are the CLSM images of LLC cells and 3T3 cells cultured with C-6, diPTX-2Se / C6 NPs, diPTX-2S / C6 NPs, diPTX-2C / C6 NPs, diPTX-2Se / C6 NPs&Q, diPTX-2S / C6 NPs&Q, and diPTX-2C / C6 NPs&Q for 0.5 h and 2 h respectively.

[0073] Figure 29 This is the figure of the pharmacokinetic study results of Q combined with PQPNPs and PCPNPs in Example 7 of the present invention.

[0074] Figure 30 This is the figure of the pharmacokinetic study results of Q combined with diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in Example 7 of the present invention.

[0075] Figure 31 This is the figure of the biodistribution study results of Q combined with PQPNPs and PCPNPs in Example 8 of the present invention.

[0076] Figure 32 This is the figure of the semi-quantitative analysis of the fluorescence intensity of the in vivo tumor tissues and ex vivo organs at 4 h and 24 h after treatment with Q combined with PQPNPs and PCPNPs in Example 8 of the present invention; among them, (A) is for 4 h; (B) is for 24 h.

[0077] Figure 33 This is the figure of the biodistribution study results of Q combined with diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in Example 8 of the present invention.

[0078] Figure 34 This is the figure of the semi-quantitative analysis of the fluorescence intensity of the in vivo tumor tissues and ex vivo organs at 4 h and 24 h after treatment with Q combined with diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs in Example 8 of the present invention; among them, (A) is for 4 h; (B) is for 24 h.

[0079] Figure 35 This is the dosing schedule diagram of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention.

[0080] Figure 36This is the graph of the tumor volume change in each group of mice and the graph of the body weight change curve of mice after 15 days of different treatments with Q combined with PQPNPs and PCPNPs in Example 9 of the present invention; among them, (A) is the graph of volume change; (B) is the graph of body weight change curve.

[0081] Figure 37 This is the ex vivo tumor photo of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention.

[0082] Figure 38 This is the tumor weight graph of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention.

[0083] Figure 39 This is the H&E and TUNEL staining graph of the tumor sections of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention.

[0084] Figure 40 This is the graph showing the expression of PD-L1 protein in the tumor tissues of each group detected by western blot of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention.

[0085] Figure 41 This is the quantitative analysis graph of the expression of PD-L1 protein in the tumor tissues of each group detected by western blot of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention.

[0086] Figure 42 This is the detection of the expression of PD-L1 protein in LLC cells of each group by western blot of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention.

[0087] Figure 43 This is the detection of the expression of PD-L1 protein in LLC cells of each group by western blot of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention.

[0088] Figure 44 This is the detection of CD4 + T, CD8 + T cells and quantitative analysis graph by flow cytometry of Q combined with PQPNPs and PCPNPs in tumor tissues in Example 9 of the present invention; among them, (A) is the cell graph; (B) and (C) are the quantitative analysis graphs.

[0089] Figure 45 This is the detection of M1 and M2 cells and quantitative analysis graph by flow cytometry of Q combined with PQPNPs and PCPNPs in tumor tissues in Example 9 of the present invention; among them, (A) is the cell graph; (B) and (C) are the quantitative analysis graphs.

[0090] Figure 46 H&E staining images of major organs (heart, liver, spleen, lung, kidney) of mice in each group of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention;

[0091] Figure 47 Analysis charts of blood routine, liver and kidney functions of mice in each group of Q combined with PQPNPs and PCPNPs in Example 9 of the present invention; among them, (A) is the blood routine analysis chart; (B) is the liver and kidney function analysis chart;

[0092] Figure 48 High-dose anti-tumor effect and biosafety of Q combined with diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs in Example 10 of the present invention; among them, (A) is the high-dose anti-tumor efficacy experimental protocol; (B) is the tumor volume change of mice in each group after 19 days of different treatments (n = 5); (C) is the tumor weight; (D) is the ex vivo tumor picture (n = 5); (E) is the body weight change curve of mice (n = 5); (F) is the detection of PD-L1 protein expression in LLC tumor tissues of each group by Western blot; (G) is the corresponding quantitative analysis (n = 3); (H) is H&E and TUNEL staining of tumor sections; (I) is the immunofluorescence staining images of CD4 and CD8 of tumor sections, scale bar: 100 μm, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001;

[0093] Figure 49 Low-dose small tumor efficacy study of Q combined with diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs in Example 10 of the present invention; among them, (A) is the low-dose small tumor efficacy experimental protocol; (B) is the tumor volume change of mice in each group after 14 days of different treatments (n = 5); (C) is the tumor weight; (D) is the ex vivo tumor picture (n = 5); (E) is the body weight change curve of mice (n = 5); (F) is the detection of PD-L1 protein expression in LLC tumor tissues of each group by Western blot; (G) is the corresponding quantitative analysis; (H) and (I) are the detection of CD4 + T, CD8 + T cells and the corresponding quantitative analysis; (L) and (M) are M2 cells and the corresponding quantitative analysis; (J) and (K) are M1 cells and the corresponding quantitative analysis (n = 3). *p < 0.05, **p < 0.01, ****p < 0.0001;

[0094] Figure 50This is the high-dose large tumor efficacy study of Q combined with diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs in Example 10 of the present invention; among them, (A) is the experimental protocol for high-dose large tumor efficacy; (B) is the change in tumor volume of mice after 12 days of different treatments (n = 5); (C) is the tumor weight and (D) is the ex vivo tumor pictures (n = 5); (E) is the body weight change curve of mice (n = 5); (F) is the detection of the expression of PD-L1 protein in LLC tumor tissues of each group by Western blot; (G) is the corresponding quantitative analysis; (H) and (I) are the detection of CD4 + T, CD8 + T cells and the corresponding quantitative analysis; (L) and (M) are M2 cells and the corresponding quantitative analysis respectively; (J) and (K) are M1 cells and the corresponding quantitative analysis respectively (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Detailed implementation manners

[0095] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0096] The flowcharts shown in the accompanying drawings are only illustrative and do not necessarily include all the steps. For example, some steps can be decomposed, and some steps can be combined or partially combined. Therefore, the actual execution order may be changed according to the actual situation.

[0097] The terms "first", "second", etc. in the specification and claims of the present application and the above accompanying drawings are used to distinguish similar objects and do not necessarily need to be used to describe a specific order or sequence. It should be understood that such data can be interchanged under appropriate circumstances so that the embodiments of the present application described here can be implemented in an order other than those illustrated or described here.

[0098] In addition, the terms "comprising" and "having" and any variations thereof are intended to cover non-exclusive inclusion. For example, a process, method, system, product or device comprising a series of steps or sub-modules does not necessarily have to be limited to those clearly listed steps or sub-modules, but may include other steps or sub-modules that are not clearly listed or are inherent to these processes, methods, products or devices.

[0099] Example 1: Synthesis and characterization of paclitaxel dimer prodrug

[0100] (1) Synthesis and Structural Characterization of NQO1-Responsive PTX Dimer Prodrug PTX-Q-PTX (PQP):

[0101] Synthesis of 2,5-Dimethylhydroquinone: Using 2,5-dimethyl-p-benzoquinone (6.99 mmol) as the raw material, it was reacted with an aqueous solution of sodium dithionite (50.95 mmol) in diethyl ether under nitrogen protection for 4 h. After extraction with diethyl ether (2×35 mL), washing with saturated NaCl, and drying with anhydrous Na2SO4, a pale yellow solid product (6.89 mmol, yield 98.5%) was obtained.

[0102] Preparation of dilactone chromanone derivative: 2,5-Dimethylhydroquinone (6.89 mmol) was dissolved in methanesulfonic acid (8.6 mL), heated and dissolved at 70 °C under nitrogen protection, and 3,3-dimethyl-methyl acrylate (1.9 mL) was slowly added dropwise, and the reaction was carried out for 48 h. After extraction with ethyl acetate (2×), washing with water / saturated NaHCO3, and drying and concentration, white crystals were precipitated (1.86 mmol, yield 27.0%).

[0103] Synthesis of quinone dipropionic acid (QDPA): The dilactone chromanone derivative (0.83 mmol) was dissolved in THF (15 mL), 1 mol / L LiOH solution (30 mL) was added, and the mixture was stirred at room temperature for 1 h until TLC showed complete conversion. The pH was adjusted to 3-4 with dilute hydrochloric acid, and after extraction with ethyl acetate (3×) and washing with saturated NaCl, yellow solid QDPA (0.44 mmol, yield 53.2%) was obtained after drying.

[0104] Coupling and Purification of PQP: PTX (1.8 mmol) and QDPA (1.1 mmol) were used as raw materials, and EDCI / DMAP was used as a catalyst in CH2Cl2. The raw materials were added in two stages (0 h and 1 h), and the reaction was carried out at room temperature for 24 h. After purification by silica gel column chromatography (CH2Cl2 / ethyl acetate = 2:1), the NQO1-responsive PTX dimer prodrug PTX-Q-PTX was obtained. The structure was confirmed by 1H NMR spectroscopy ( Figure 2 ) and high-resolution mass spectrometry ( Figure 3 ). The purity of the paclitaxel prodrug was detected by high-performance liquid chromatography. The detection wavelength was 227 nm, the mobile phase was 70% acetonitrile and 30% aqueous phase, and the column temperature was 30 °C. The detection results showed that the purity of PTX-Q-PTX > 96%, which could meet the requirements of subsequent experiments ( Figure 4 ).

[0105] The synthesis route of PTX-Q-PTX is as follows:

[0106]

[0107]

[0108] (2) Synthesis and structural characterization of non-sensitive PTX dimer prodrug PTX-C-PTX (PCP):

[0109] PTX (1.366 g, 1.6 mmol) and sebacic acid (143 mg, 0.89 mmol) were reacted in CH2Cl2 with EDCI (added in two portions, total 5.2 mmol) and DMAP (total 0.36 mmol) as catalysts under N2 protection at room temperature (the first stage for 1 h and the second stage for 24 h), and the conversion was monitored by TLC. After the reaction was completed, it was extracted with EtOAc (3 × 100 mL), concentrated by rotary evaporation, and purified by preparative liquid chromatography (redissolved with chromatographic grade acetonitrile) to obtain PTX-C-PTX. The structure was confirmed by 1 H NMR( Figure 5 ) and HRMS( Figure 6 ). The purity of the paclitaxel prodrug was detected by high performance liquid chromatography with a detection wavelength of 227 nm, a mobile phase of 70% acetonitrile and 30% aqueous phase, and a column temperature of 30 °C. The detection results showed that the purity of PTX-C-PTX > 96%, which could meet the requirements of subsequent experiments ( Figure 7 ).

[0110] The synthetic route of PTX-C-PTX is as follows:

[0111]

[0112] (3) Synthesis and structural characterization of GSH-responsive PTX dimer prodrug PTX-SeSe-PTX (2diPTX-2Se):

[0113] Synthesis of 3,3'-diselenodipropionic acid: Selenium powder (80 mmol) and sodium borohydride (80 mmol) were reacted under ice bath protection with N2, then heated to 105 °C and refluxed for 1 h, and bromopropionic acid (80 mmol) was added and reacted at room temperature overnight. The pH was adjusted to 3 - 4 with dilute hydrochloric acid, and the precipitate was extracted with ethyl acetate and recrystallized to obtain the product.

[0114] Coupling and purification of PTX-SeSe-PTX: PTX (1.8 mmol) and diselenodipropionic acid (0.8 mmol) were catalyzed in two stages (0 h and 1 h) with EDCI (total 8.7 mmol) and DMAP (total 0.75 mmol) in CH2Cl2, and reacted at room temperature for 24 h under N2 protection. It was purified by silica gel column chromatography (CH2Cl2 / ethyl acetate = 2:1) to obtain the target product. Structural characterization: 1 H NMR( Figure 8 ) and HRMS( Figure 9) Confirmation. The purity of the paclitaxel prodrug was detected by high performance liquid chromatography at a detection wavelength of 227 nm. The mobile phase was 70% acetonitrile and 30% aqueous phase, and the column temperature was 30 °C. The detection results showed that the purity of PTX-SeSe-PTX was > 98%, which could meet the requirements of subsequent experiments ( Figure 10 ).

[0115] The synthesis route of PTX-SeSe-PTX is as follows:

[0116]

[0117] (4) Synthesis and structural characterization of the GSH-responsive PTX dimer prodrug PTX-SS-PTX (2diPTX-2S):

[0118] PTX (1.8 mmol) and dithiodipropionic acid (0.8 mmol) were catalyzed in two stages (0 h and 1 h) with EDCI (total amount 8.7 mmol) and DMAP (total amount 0.75 mmol) in CH2Cl2, and reacted at room temperature for 24 h under N2 protection. Purification: Separated and purified by silica gel column chromatography (CH2Cl2 / ethyl acetate = 2:1) to obtain the target product. Structure confirmation: 1 H NMR( Figure 11 ) and HRMS( Figure 12 ) were used to verify the structure of PTX-SS-PTX. The purity of the paclitaxel prodrug was detected by high performance liquid chromatography at a detection wavelength of 227 nm. The mobile phase was 70% acetonitrile and 30% aqueous phase, and the column temperature was 30 °C. The detection results showed that the purity of PTX-SS-PTX was > 98%, which could meet the requirements of subsequent experiments ( Figure 13 ).

[0119] The synthesis route of PTX-SS-PTX is as follows:

[0120]

[0121] (5) Synthesis and structural characterization of the non-responsive PTX dimer prodrug PTX-CC-PTX (2diPTX-2C):

[0122] PTX (1.8 mmol) and adipic acid (0.8 mmol) were catalyzed in two stages (0 h and 1 h) with EDCI (total amount 8.7 mmol) and DMAP (total amount 0.75 mmol) in CH2Cl2, and reacted at room temperature for 25 h under N2 protection. Purification: Separated and purified by silica gel column chromatography (CH2Cl2 / ethyl acetate = 2:1) to obtain the target product. Structure confirmation: 1 H NMR( Figure 14 ) and HRMS( Figure 15)Verify the PTX-CC-PTX structure. The purity of the paclitaxel prodrug was detected by high performance liquid chromatography at a detection wavelength of 227 nm, with a mobile phase of 70% acetonitrile and 30% aqueous phase, and a column temperature of 30 °C. The detection results showed that the purity of PTX-CC-PTX > 98%, which could meet the requirements of subsequent experiments. Figure 16 )

[0123] The synthesis route of PTX-CC-PTX is as follows:

[0124]

[0125] Example 2: Preparation and characterization of nanoparticles;

[0126] (1) Preparation of PQPNPs and their control nanoparticles: Based on the one-step nanoprecipitation method established by the team before, 4 mg of PQP / PCP and 8 mg of DSPE-PEG 2K were dissolved in a mixed solvent of 1 mL of absolute ethanol and acetone. Under stirring conditions, the above mixed solution was slowly dropped into 4 mL of deionized water. In the mixed solvent system, it spontaneously assembled into uniform nanoparticles. After removing the organic solvents by rotary evaporation under reduced pressure at room temperature, PQPNPs and PCPNPs were obtained. Neither of the two self-assembled nanoparticles showed turbidity, stratification or precipitation. They showed obvious light blue opalescence. When the two nanopreparations were irradiated with laser, obvious Tyndall effects were observed, indicating that the drug formed nanostructures in the solution.

[0127] Characterization of nanoparticles: After diluting the PQP NPs and PCP NPs solutions 20 times with deionized water, the particle size and Zeta potential of the diluted nanoparticles were measured using a Malvern Zetasizer particle size analyzer. Figure 17 A-C); At the same time, the diluted nanoparticle solution was dropped onto a copper mesh covered with a carbon film, allowed to dry naturally, and negatively stained with 2% phosphotungstic acid. The appearance morphology was observed using a transmission electron microscope (TEM). Figure 17 D-E). From the results, it can be seen that the above two kinds of nanoparticles have a uniform particle size distribution and a small particle size. The TEM pictures also show that the nanoparticles have a uniform spherical morphology, which is beneficial to enhancing the accumulation of nanoparticles at the tumor site, while the negative Zeta potential can help the nanoparticles avoid being captured by the liver and prolong the circulation time of the drug in the body.

[0128] Investigation of the self-assembly mechanism of nanoparticles: Taking the particle size change as an index, the self-assembly mechanisms of PQPNPs and PCPNPs were investigated. The specific operations were as follows. 1 mL of the prepared nanopreparation was mixed evenly with 9 mL of deionized water, NaCl solution, urea solution, and Triton X-100 solution. Among them, NaCl, urea, and Triton X-100 inhibited the intermolecular ionic bond, hydrogen bond, and hydrophobic interaction, respectively. After incubation at room temperature for 12 h, the changes in the particle size and polydispersity index (PDI) of PQPNPs and PCPNPs were measured. From Figure 17 the results of F-G, it can be seen that the particle size changes of PQPNPs and PCPNPs were not obvious after incubation with deionized water, NaCl solution, and urea solution, while the particle size and PDI of the two kinds of nanoparticles changed significantly after incubation with Triton X-100. This may be because Triton X-100, as a surfactant, destroyed the original intermolecular hydrophobic force of the nanoparticles. Therefore, the hydrophobic interaction may be the most important mechanism driving the self-assembly of nanoparticles.

[0129] (2) Preparation of diPTX-2Se NPs and their comparative nanoparticles: A certain proportion of PTX-SeSe-PTX, PTX-SS-PTX, and PTX-CC-PTX were dissolved in 2K 1 mL of a mixed solvent of absolute ethanol and tetrahydrofuran. Under stirring conditions, the above mixed solution was slowly dropped into 4 mL of deionized water. In the mixed solvent system, uniform nanoparticles were spontaneously assembled. After removing ethanol by rotary evaporation under reduced pressure at room temperature, diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2CNPs were obtained. The three self-assembled nanoparticle systems all remained clear and transparent, and no stratification or precipitation was observed. Under natural light, the system showed a characteristic light blue opalescence. When irradiated with a 650 nm laser beam, both nanopreparations showed a significant Tyndall phenomenon. Combining their colloidal solution characteristics, it was confirmed that the drug molecules formed a stable nano-scale colloidal structure through self-assembly.

[0130] Characterization of nanoparticles: The solutions of prodrug self-assembled diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs were diluted 20 times and 50 times with deionized water, respectively. After dilution, the particle size and Zeta potential ( Figure 18 A-D) of the diluted nanoparticles were measured with a Malvern Zetasizer particle size analyzer; at the same time, the diluted nanoparticle solution was dropped onto a copper mesh covered with a carbon film, allowed to dry naturally, and negatively stained with 2% phosphotungstic acid. The appearance morphology was observed by TEM ( Figure 18E-G). Experimental data showed that all three nanostructures had a monodisperse particle size distribution, and their hydrodynamic diameters were in the nanoscale range. High-resolution transmission electron microscopy (HR-TEM) characterization showed that the particles had a regular spherical morphology. This homogenized nano-size property promoted the selective accumulation in tumor tissues through the enhanced permeability and retention effect (EPR effect). At the same time, the negatively charged property on the material surface could significantly reduce the recognition and uptake by the reticuloendothelial system (RES), thus effectively prolonging the plasma half-life of the drug.

[0131] Investigation of the self-assembly mechanism of nanoparticles: Taking the particle size change as an index, the self-assembly mechanisms of diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs were investigated. The specific operation was as follows. 1 mL of the prepared nanopreparation was mixed evenly with 9 mL of deionized water, NaCl solution, urea solution and sodium dodecyl sulfate (SDS) solution. Among them, NaCl, urea and SDS inhibited the intermolecular ionic bonds, hydrogen bonds and hydrophobic interactions respectively. After incubation at room temperature for 12 h, the particle size and polydispersity index (PDI) changes of PQPNPs and PCPNPs were measured. From Figure 18 The results of H-J, it could be seen that the particle size changes of diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs were not obvious after incubation with deionized water, NaCl solution and urea solution, while the particle size and PDI of the two kinds of nanoparticles changed significantly after incubation with SDS. This selective response indicated that the surfactant destroyed the hydrophobic association in the nanostructure through competitive binding, confirming that hydrophobic interaction was the key driving force to maintain the self-assembled conformation of this nano-system.

[0132] Example 3: Investigation of the stability of nanoparticles;

[0133] (1) Investigation of the stability of PQPNPs and their comparative nanoparticles: Taking the particle size change as the evaluation index, the plasma stability ( Figure 19 A) and long-term stability ( Figure 19 B) of PQPNPs and PCPNPs were investigated. The specific operation was as follows: In a 37 °C constant temperature oscillator (speed of 100 rpm), PQPNPs and PCPNPs were incubated in PBS (pH 7.4) with 10% fetal bovine serum for 48 h, and the particle size of the prodrug nanoparticles was measured at 0, 2, 4, 6, 8, 12, 24 and 48 h respectively. In addition, the long-term storage stability of the self-assembled nanoparticles of the small molecule prodrug stored at 4 °C for 1 month was also investigated. The experimental results showed that the plasma stability and long-term stability of PQPNPs and PCPNPs were good, which might be related to the relatively large absolute value of the Zeta potential of the prodrug nanoparticles and the existence of a PEG hydration layer on the surface, inhibiting the adhesion between nanoparticles, thus improving the stability.

[0134] (2) Investigation on the stability of diPTX-2Se NPs and their control nanoparticles: Taking the particle size change as the evaluation index, the plasma stability ( Figure 20 A) and long-term stability ( Figure 20 B) of the prodrug self-assembled diPTX-2Se nanoparticles, diPTX-2S and diPTX-2C nanoparticles were investigated. The results showed that the hydrodynamic diameters of diPTX-2Se NPs, diPTX-2S NPs and diPTX-2C NPs were almost unchanged, indicating good stability.

[0135] Example 4: In vitro release behavior of paclitaxel prodrug self-assembled nanoparticles;

[0136] (1) In vitro release behavior of PQPNPs and their control nanoparticles: The drug release of PQPNPs and PCPNPs in the release media containing 60 mM Na2S2O4 and 30 mM Na2S2O4 was investigated. The specific steps were as follows: An appropriate amount of the prepared PQPNPs / PCPNPs was added to 30 mL of PBS solution (containing 30% absolute ethanol, V / V) with a pH of 7.4 containing 60 mM Na2S2O4 and 30 mM Na2S2O4, respectively. The whole system was placed in a constant temperature oscillator (37 °C, 100 rpm). Samples of 100 μL were taken at 0, 0.5, 1, 3, 6, 9, 12, 24, 36, and 48 h, and the concentration of the free drug in them was determined by HPLC. At the same time, TEM was used to observe the morphological changes of PQPNPs and PCPNPs before and after the addition of 60 mM Na2S2O4. The following is the release mechanism of PQPNPs under the action of NQO1:

[0137]

[0138] From Figure 21 the results of A - B, it can be seen that in the release medium containing 30 mM Na2S2O4, about 80% of the free drug was released from PQP NPs within 48 h, while in the release medium containing 60 mM Na2S2O4, about 100% of the free drug was released within 48 h, and the release rate was uniform without obvious burst release phenomenon; while in the two different concentration release media, almost no free drug was released from PCPNPs within 48 h, which reflected the good NQO1 enzyme response ability of PQPNPs. The mechanism is that under the action of NQO1, the hydroxyl group attacks the carbonyl group intramolecularly to form a lactone ring, and at the same time, the ester bond breaks to rapidly release PTX, increasing the accumulation of PTX in the cells. Figure 21 The TEM images of C - F also confirmed this point.

[0139] (2) In vitro release behavior of diPTX-2Se NPs and their control NPs: Using PBS (pH 7.4) containing 30% ethanol (v / v) as the release medium, the release of the prodrug self-assembled diPTX-2Se NPs, diPTX-2S and diPTX-2C NPs was investigated in the absence or presence of 1 mM, 5 mM and 10 mM DTT (simulating the in vivo GSH microenvironment), 10 mM H2O2 (simulating the tumor oxidative response microenvironment), and PTX. The following are the release mechanisms of diPTX-2Se NPs and their control NPs under the action of H2O2 and DTT:

[0140]

[0141] From Figure 22 The results from A-J show that in the presence of DTT, diPTX-2Se NPs were almost completely degraded within 96 h, diPTX-2S NPs were cleaved within 6 h, while DTT could hardly destroy diPTX-2C NPs, indicating that diPTX-2Se NPs and diPTX-2S NPs have better reductive reactivity. The PTX release curve shows that although diPTX-2S NPs have a faster degradation ability, diPTX-2Se NPs have the highest PTX release rate after 96 h. By HPLC detection, the elution peak intensities of diPTX-2Se NPs and diPTX-2S NPs gradually decreased at 13 min and 11.9 min respectively, while a new peak of PTX appeared around 4 min and the peak intensity increased gradually with time. In contrast, under the same treatment conditions, the HPLC peak of diPTX-2C NPs hardly changed, which confirmed the non-reactivity of the carbon chain. At the same time, diPTX-2Se NPs released PTX slightly faster than diPTX-2S NPs and finally reached the same release amount.

[0142] Example 5: Preparation of freeze-dried Qiyu Sanlong formula;

[0143] First step: Add 30 g of Astragalus membranaceus, 20 g each of Solanum nigrum, Hedyotis diffusa, and Coix lacryma-jobi, 10 g each of Polygonatum odoratum, Rhizoma zedoariae, and Fritillaria thunbergii, and 6 g each of Gekko swinhonis, Pheretima aspergillum, and Euphorbia helioscopia to 10 times the amount of distilled water and soak for 1 h. Boil it vigorously and then simmer gently for 90 min. Filter the extract, add 1.07 L of distilled water to the residue, boil it vigorously, and simmer gently for 40 min, then filter the extract. Mix and concentrate the two extracts in vacuo at 50 °C to 2 g / mL to obtain the Qiyu Sanlong formula solution.

[0144] Step 2: Filter and concentrate the Qiyusanlong Fang solution, then mix and dispense it into sterile containers to ensure the uniformity of the medicinal liquid in each container. After gradient cooling and freezing, place it in a freeze dryer for vacuum freeze-drying. Collect the freeze-dried powder and store it sealed at -20°C. The mother liquor of the Qiyusanlong Fang freeze-dried powder solution is prepared with high-glucose DMEM medium at a concentration of 50 mg / mL, filtered and sterilized with a 0.22-μm microporous membrane, and diluted to the required concentration with high-glucose DMEM medium before use.

[0145] Example 6: Cytotoxicity and cellular uptake experiments of Qiyusanlong Fang combined with paclitaxel prodrug nanoparticles;

[0146] (1) Cytotoxicity and cellular uptake experiments of PQPNPs and their control nanoparticles:

[0147] This study is based on the uniformly stable nanopharmaceuticals prepared in the early stage, and further evaluates their in vitro anti-tumor activities. Three cell models, L929 (low NQO1 normal cells), A549 (high NQO1 human lung cancer), and LLC (high NQO1 mouse lung cancer), are selected. The cytotoxicities of Taxol, PQPNPs, PCPNPs, Q, and their combined preparations (PQP / PCPNPs&Q) are detected by the CCK-8 method. Among them, the concentrations of PQPNPs and PCPNPs are set equally to the PTX concentration at 5, 50, 500, 5000, 10000, 20000, 40000, 80000 nM, and the corresponding concentrations of QYSLD are set at 8, 10, 12, 14, 16, 18, 20, 22 mg / mL. Specific method: Seed 2×10 3 cells / well in a 96-well plate. After overnight adherence, give drug-containing treatments (using drug-free medium as a control). After 48 h, add CCK-8 reagent and measure the OD value at 450 nm.

[0148] As Figure 23As shown in A - C, Taxol (paclitaxel solution) showed rapid and strong cytotoxicity in L929, A549, and LLC cells, and its effect was significantly stronger than that of the PQP / PCPNPs group that required drug slow release. It was speculated that because the solution could directly diffuse into the cells, while the nanoparticles needed to undergo a release process. The anti - tumor activity of PQP NPs was better than that of PCP NPs (the IC50 values of LLC / A549 were 3839 / 3746 nM vs. 45601 / 18660 nM respectively), indicating that the non - responsiveness of the carbon chain in PCP NPs led to low drug release efficiency. In addition, PQP NPs had weak toxicity to normal cells L929 (IC50 = 28107 nM), with a significant difference in activity from tumor cells (about 7 - fold), suggesting that the low NQO1 expression in L929 cells restricted the cleavage of the prodrug ester bond, verifying its safety advantage. It was worth noting that Q alone had high killing power against tumor cells (the IC50 values of LLC / A549 were 18.13 / 19.69 mg / mL), and the anti - tumor effect was further enhanced after combination with PQP / PCPNPs, indicating the synergistic potential between Q and paclitaxel prodrug nanoparticles.

[0149] Using coumarin - 6 (C - 6) as a fluorescent probe, C - 6 - labeled PQPNPs, PCPNPs, and their combination with Q group (containing Q at the IC50 concentration) were prepared respectively, and cell uptake was evaluated by flow cytometry (FCM) and confocal laser scanning microscopy (CLSM). The specific procedure was as follows: LLC / A549 / L929 cells were seeded into 6 - well plates (for FCM analysis) or 12 - well plates (containing cell slides, for CLSM observation) at 5×105 cells / well or 1×105 cells / well respectively. After culturing for 24 h, C - 6 - labeled nanoparticles (the final concentration in the FCM group was 500 ng / mL, and in the CLSM group was 200 ng / mL) and the combination group (containing Q) were added, and incubated for 0.5 h and 2 h respectively. After terminating the uptake, the cells were washed. Sample processing and detection: In the FCM group, the cells were collected by trypsin digestion and centrifugation, and then the fluorescence intensity was directly analyzed; in the CLSM group, the cells were fixed with 4% paraformaldehyde and stained with DAPI for nuclei, and the intracellular distribution of C - 6 (green fluorescence) and its co - localization with the nucleus were observed to quantitatively and visually compare the uptake efficiency of PQP / PCPNPs and the synergistic effect of Q.

[0150] From the flow cytometry results ( Figure 24 A - C) and the pictures taken by confocal laser scanning microscopy ( Figure 25As can be seen from (A-C), the cellular uptake of nanoparticles is higher than that of coumarin-6 solution, and it is time-dependent. This is because the nanoparticles enter the cells through the endocytosis pathway, while the small molecules in the solution enter through the diffusion pathway. Therefore, the nanoparticles modified with DSPE-PEG can effectively increase the drug uptake efficiency. It is worth noting that Q hardly affects the uptake of NPs, indicating good compatibility between the two.

[0151] (2) Cytotoxicity and cellular uptake experiments of diPTX-2Se NPs and their control nanoparticles:

[0152] In this study, the cytotoxicity and synergistic effects of paclitaxel (PTX), paclitaxel prodrug nanoparticles modified with selenium / sulfur / carbon bonds (diPTX-2Se / 2S / 2C NPs), and their combination with Q on mouse lung cancer cells (LLC) and mouse embryonic fibroblasts (3T3) were evaluated by the CCK-8 method. Among them, the concentrations of diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs were set equally to those of PTX at 1, 2, 8, 31.25, 125, 500, 2000, 8000 nM, and the corresponding concentrations of QYSLD were set at 8, 10, 12, 14, 16, 18, 20, 22 mg / mL.

[0153] As Figure 26 As shown in A-B, Qiyusanlong formula and all PTX prodrug nanoparticles exhibited concentration-dependent anti-cancer activities. Compared with normal cells (3T3), the cytotoxicity of diPTX-2Se NPs, diPTX-2S NPs, and diPTX-2C NPs against tumor cells (LLC) was significantly increased, which was due to the lower redox level in 3T3 cells and less release of PTX from the prodrug. Compared with diPTX-2S NPs and diPTX-2C NPs, diPTX-2Se NPs had the lowest IC50 (0.249 nM) against LLC cells. It is worth noting that after Q was combined with the three kinds of nanoparticles, the IC50 values were significantly reduced. These results indicate that the combination of Q and PTX prodrug nanoparticles has a more powerful anti-tumor effect.

[0154] Using coumarin-6 (C-6) as a fluorescent probe, C-6-labeled diPTX-2Se NPs, diPTX-2S NPs, diPTX-2C NPs, and their combination with Q group (containing Q at the IC50 concentration) were prepared respectively, and cellular uptake was evaluated by flow cytometry (FCM) and confocal laser scanning microscopy (CLSM).

[0155] From flow cytometry ( Figure 27 A-B) and confocal laser scanning microscopy ( Figure 28) From the results, we can see that the green fluorescence of the nanoparticles was weak at 0.5 h and increased significantly with time at 2 h, indicating that the nanoparticles can be effectively internalized by LLC and 3T3 cells. Compared with the nanoparticles, the green fluorescence of the C-6 solution preparation was almost invisible, because the nanoparticles entered the cell interior through the endocytosis pathway, while the small molecules in the solution preparation passed through the diffusion pathway. Therefore, the nanoparticles modified with DSPE-PEG2K effectively increased the drug absorption efficiency. DiPTX-2Se / C6 NPs, diPTX-2S / C6 NPs, and diPTX-2C / C6 NPs showed almost the same green fluorescence, and the addition of Qiyusanlong formula had little effect on the uptake of the nanoparticles, indicating good compatibility between the two.

[0156] Example 7: Pharmacokinetic experiment of Qiyusanlong formula combined with paclitaxel prodrug nanoparticles;

[0157] (1) The pharmacokinetics and tissue distribution characteristics of PQPNPs, PCPNPs, and their combination with Q were evaluated through an SD rat model (male, 180 - 220 g, n = 3 / group). Figure 29 ) The experiment was divided into 5 groups: DiR-labeled nanoparticles (PQP / DiRNPs, PCP / DiRNPs) and the combination group (NPs + Q) were administered by tail vein injection (DiR dose 2 mg / kg), and the combination group was simultaneously gavaged with a high dose of Q (80.48 g / kg, selected based on the optimal tumor suppression dose in the previous period). Blood samples were collected from the ophthalmic vein at preset time points (0.083 - 24 h). After anticoagulation with heparin, centrifugation, and methanol precipitation of the blood samples, the DiR fluorescence intensity (Ex / Em = 748 / 780 nm) was detected using an enzyme-linked immunosorbent assay (ELISA) reader. As shown in the figure, compared with free DiR, the area under the blood drug concentration curve (AUC 0-24 h) of DiR-labeled NPs was larger, indicating that NPs can prolong the blood circulation of the prodrug.

[0158] (2) As Figure 30 shown, the in vivo metabolic behavior of DiR-labeled paclitaxel prodrug nanoparticles (diPTX-2Se / 2S / 2C NPs) and their combination with Qiyusanlong formula (Q) was evaluated through a male SD rat model (7 groups, n = 3 / group): In the experimental groups, DiR-Sol or DiR-labeled nanoparticles (equivalent DiR 2 mg / kg) were administered by tail vein injection, and the combination group was simultaneously gavaged with a high dose of Q (80.48 g / kg). Blood samples were collected from the orbit multiple times within 0.083 - 24 h. After anticoagulation with heparin, centrifugation, and methanol precipitation, the DiR fluorescence intensity (Ex / Em = 748 / 780 nm) in the plasma was detected. From the above results, we can see that compared with free DiR-Sol, the paclitaxel prodrug nanoparticles can prolong the blood circulation of the prodrug, and the addition of Qiyusanlong formula has basically no effect on the pharmacokinetic behavior of the paclitaxel prodrug nanoparticles in vivo.

[0159] Example 8: Tissue distribution experiment of Qiyu Sanlong formula combined with paclitaxel prodrug nanoparticles;

[0160] (1) Tissue distribution experiment of PQPNPs and their control nanoparticles: As Figure 31 - 32 shown, based on the C57BL / 6J mouse LLC subcutaneous tumor model (tumor volume ~ 300 mm 3 ), the in vivo distribution of PQP / DiRNPs, PCP / DiRNPs and their combination with Qiyu Sanlong formula (Q) was compared by DiR fluorescence labeling: The experimental groups were injected with DiR-Sol, PQP / DiRNPs or PCP / DiRNPs (DiR dose 2 mg / kg) via the tail vein, and the combined group was simultaneously intragastrically administered Q (80.48 g / kg). At 4 h and 24 h after administration, the heart, liver, spleen, lung, kidney and tumor tissues were taken, and the fluorescence distribution was analyzed by an in vivo imaging system (Ex / Em = 748 / 780 nm). From Figure 31 - 32 the results, it can be seen that Dir was mainly distributed in the liver and lungs and hardly distributed in the tumor tissue. In contrast, the fluorescence intensity of the Dir-labeled nanoparticles in the tumor tissue increased significantly, and the near-infrared fluorescence of the excised major organs and tumor tissue further confirmed the effective accumulation and retention of NPs at the tumor site, which was caused by the long-circulation effect produced by PEG modification and the passive tumor targeting effect mediated by the EPR effect of the nanoparticles. In addition, the influence of the addition of Q on the pharmacokinetics and biodistribution of NPs was negligible.

[0161] (2) Tissue distribution experiment of diPTX-2Se NPs and their control nanoparticles: Based on the C57BL / 6J mouse LLC subcutaneous tumor model (tumor volume ~ 300 mm 3 ), the in vivo distribution characteristics of selenium / sulfur / carbon bond-modified paclitaxel prodrug nanoparticles (diPTX-2Se / 2S / 2C NPs) and their combination with Qiyu Sanlong formula (Q) were compared by DiR fluorescence labeling. From Figure 33 - 34 the results, it can be seen that the pharmacokinetic distribution study showed that the free DiR molecule (DiR-Sol) was mainly taken up efficiently by the liver and lung tissues after administration, while the accumulation amount at the tumor site was only at the background level. In sharp contrast, the accumulation efficiency of the DiR-labeled nanoparticle formulation group in the tumor tissue was significantly increased to 15.6 times, and the ex vivo near-infrared imaging quantitative analysis further verified the tumor specificity. It should be noted that after the combined application of Qiyu Sanlong formula, there was no significant difference between groups confirmed by one-way ANOVA, indicating that the in vivo distribution behavior of the traditional compound components and the nanoparticle delivery system showed pharmacokinetic independence.

[0162] Example 9: In vivo antitumor effect and safety evaluation of Qiyu Sanlong formula combined with PQPNPs

[0163] (1) Antitumor effect in vivo

[0164] We first established a mouse lung cancer model by subcutaneously injecting LLC cells into the back of C57BL / 6J mice ( Figure 35 ). When the tumor volume reached 100 - 150 mm 3 , drug administration began. The mice were randomly divided into 7 groups (N = 5) ( Figure 36 ). The PQPNPs, PCPNPs, PBS, Taxol, PQPNPs&Q, PCPNPs&Q, and Q groups were given an intravenous injection of NPs equivalent to 10 mg / kg (drug weight / body weight) of PTX to the mice, and the gavage dose of Q was 80.48 g / kg. At the same time, the tumor volume and body weight were recorded. On the 15th day, the mice were sacrificed, and whole blood, serum, heart, liver, spleen, lung, kidney, and tumor specimens of each group of mice were taken.

[0165] As can be seen from the figure, different NPs groups before and after combination with Q and Q alone showed different degrees of antitumor ability. In contrast, the tumor in the Saline group grew rapidly. It is worth noting that compared with PQPNPs or PCPNPs alone, the addition of Q significantly reduced the tumor volume, and after the addition of Q, the antitumor ability of PQPNPs was very close to that of the PTX group, which fully demonstrated the role of Q. Ex vivo tumor pictures ( Figure 37 ) and weight measurement ( Figure 38 ) are shown in the figure. Compared with other treatment groups, the tumor size obtained by PTX was the smallest and the tumor weight was the lightest, followed by PQP NPs&Q. This was consistent with the tumor volume of the live mice. In addition, hematoxylin and eosin (H&E) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining analysis showed ( Figure 39 ) that compared with other treatment groups, PTX and PQPNPs&Q showed more obvious cell necrosis and apoptosis.

[0166] (2) Detection of PD-L1 protein expression in mouse tumor tissues

[0167] We detected PD-L1 in tumor tissues by Western blot. Take 100 mg of tumor tissues stored at -80 °C, add 1 mL of RIPA buffer, and grind the tissues under ice bath conditions. Put the ground solution into a centrifuge and centrifuge at a centrifugal force of 12,000 xg for 15 min. Take the supernatant, add loading buffer to prepare protein samples, and heat in a water bath at 95 °C for 5 min to denature the proteins. Prepare a 10% sodium sulfate gel, add protein samples, and perform electrophoresis under the conditions of 80 V (concentrating gel) and 120 V (separating gel). Wait for the proteins to be fully separated, and transfer the samples to a 0.45 μm PVDF membrane at 4 °C, 200 mA for 2 h. Immerse the PVDF membrane in 5% skim milk powder and block at room temperature for 2 h. After blocking, transfer the membrane to the primary antibody diluted with 5% BSA and incubate overnight at 4 °C. The next day, incubate the membrane in the secondary antibody for 1 h and develop using ECL chemiluminescence method. Use Image J software to calculate the gray value of the bands ( Figure 40 ).

[0168] The results are as Figure 41 shown. Compared with the Saline group, PQPNPs, PCPNPs, and Taxol can all up-regulate the expression of PD-L1 in tumor tissues. Due to the EPR effect of NPs and the release effect of nanoparticles, more PTX is released from PQPNPs in tumor tissues than from PCPNPs, so the up-regulation is more obvious. Q can also down-regulate PD-L1 in tumor tissues. Moreover, compared with the NPs group, the addition of Q can also significantly down-regulate PD-L1 in tumor tissues to enhance the anti-tumor immune response.

[0169] (3) Detection of PD-L1 protein expression in LLC cells

[0170] We detected PD-L1 in LLC cells of different administration groups by Western blot. We selected mouse Lewis lung cancer (LLC) cells as a tumor cell model with high expression of NQO1. The results are as Figure 42 - 43 shown. Compared with the Saline group, PQPNPs, PCPNPs, and Taxol can all up-regulate the expression of PD-L1 in LLC cells. However, due to the high expression of NQO1 in tumor cells, more PTX is released from PQPNPs in LLC cells, so the up-regulation is more obvious. Q can down-regulate PD-L1 in tumor tissues. It should be noted that compared with the NPs group, the addition of Q can significantly down-regulate PD-L1 in LLC cells.

[0171] (4) Detection of the expression of CD4 + T, CD8 + T, M1, and M2 cells in tumor tissues

[0172] We used flow cytometry to detect the expression of CD4 + T, CD8 + T, M1, and M2 cells in the tumor tissues of mice after drug administration. The mice in each pharmacodynamic group were sacrificed after drug administration, and the tumors were removed. The tumors were placed in a sieve, ground, and the cells were collected. The cells were resuspended in 2 mL of red blood cell lysis buffer and lysed at room temperature for 2 - 3 min. Immediately, 10 mL of PBS was added, and the mixture was centrifuged to discard the supernatant. The cells were resuspended in cell staining buffer, filtered through a sieve again, counted, and the cell concentration was adjusted to 1×10 7 cells / mL. 5 μL of fluorescently labeled antibodies (anti-FITC-CD4, anti-APC-CD8, anti-APC-CD80, anti-PE-CD206) were added to the cells in sequence, and the mixture was incubated in the dark for 30 min. The cells were resuspended in 2 mL of cell staining buffer, centrifuged, and the supernatant was discarded. The cells were resuspended in 0.2 mL of cell staining buffer and detected and analyzed using a flow cytometer.

[0173] The results were as Figure 44 - 45 shown. Compared with NPs alone, NPs combined with Q significantly upregulated the proportions of CD4 + T and CD8 + T cells in the tumor tissues. We further examined the expression of M1 and M2 macrophages in the tumor tissues. The results showed that whether it was Q alone or the group of NPs combined with Q, both significantly downregulated the expression of M2 and upregulated the expression of M1 macrophages, promoted the polarization of M2 to M1 macrophages, and reshaped the tumor immunosuppressive microenvironment, thus demonstrating a strong anti-tumor effect.

[0174] (5) Safety evaluation

[0175] The biological safety of different treatment groups was evaluated by indicators such as the body weight of mice, H&E staining of major organs (heart, liver, spleen, kidney), blood routine, liver and kidney functions.

[0176] As Figure 46 - 47 seen, no obvious physiological morphological changes or tissue damage were observed in the H&E staining of major organs in each group. Compared with mice treated with normal saline, there were no significant changes in the blood routine parameters and liver and kidney function indicators of mice treated with the drug. This demonstrated that our drug has good biological safety.

[0177] Example 10: In vivo pharmacodynamic experiment and biological safety evaluation of Qiyu Sanlong formula combined with diPTX-2Se NPs;

[0178] (1) High-dose anti-tumor effect and biological safety:

[0179] Male C57BL / 6J mice (20 - 25 g) were obtained and raised under specified conditions. An animal model of subcutaneous transplanted tumor of LLC cells was constructed ( Figure 48 A). When the tumor volume reached approximately 150 mm 3 , the tumor-bearing mice were randomly divided into 9 groups (n = 5) ( Figure 48 B - C): Saline, Taxol, Q, diPTX-2C NPs, diPTX-2C NPs&Q, diPTX-2S NPs, diPTX-2S NPs&Q, diPTX-2Se NPs, diPTX-2Se NPs&Q. Before treatment, the mice were marked, weighed, and measured ( Figure 48 D - E). The equivalent dose of PTX injected via the tail vein was 10 mg / kg, and the equivalent dose of Q administered by gavage was 80.48 g / kg. The body weight and tumor volume of the mice were measured daily. On the 19th day, the mice were sacrificed, and the tumors were excised to visually evaluate tumor inhibition. To study the safety of the preparations, potential liver and kidney toxicity and blood routine were examined. The main organs (heart, liver, spleen, lung, kidney) and tumors were collected, fixed in 4% paraformaldehyde solution, then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) to evaluate the potential toxicity of the main organs and the degree of apoptosis of cancer cells. TUNEL, CD4, and CD8 detection kits were used to detect the apoptosis of cells in the tumor tissue and the expression of CD4 + T, CD8 + T cell expression according to the instructions provided by the manufacturer, and Western blotting was used to detect the expression of PD-L1 in the tumor tissue of each treatment group.

[0180] The experiments showed ( Figure 48 F - I) that Qiyusanlong formula (Q) combined with selenium-bond-responsive paclitaxel prodrug nanoparticles (diPTX-2Se NPs) exhibited the best anti-tumor effect: the tumor volume / weight decreased by 68% compared with the saline group, and H&E and TUNEL staining showed the largest tumor necrosis area and apoptosis index; mechanistically, diPTX-2Se NPs targeted the release of paclitaxel through the EPR effect. Although it upregulated the expression of tumor PD-L1, Q significantly downregulated PD-L1 to block immune escape and promoted CD4 + / CD8 + T cell infiltration, achieving "chemotherapy-immunotherapy" synergistic enhancement, while diPTX-2S / 2C NPs had inferior efficacy due to insufficient responsiveness or stability. This study revealed the dual advantages of redox-responsive nano-drug delivery combined with traditional Chinese medicine compound in precise tumor suppression and immune microenvironment regulation, providing a new strategy for chemotherapy resistance and immunosuppression problems.

[0181] (2) Low-dose small tumor efficacy experiment:

[0182] Male C57BL / 6J mice (20 - 25 g) were obtained and raised under specified conditions. An animal model of subcutaneous transplanted tumor of LLC cells was constructed ( Figure 49 A). When the tumor volume reached approximately 100 mm 3 , the tumor-bearing mice were randomly divided into 5 groups (n = 5) ( Figure 49 B - C): Saline, Taxol, Q, diPTX-2Se NPs, diPTX-2Se NPs&Q. Before treatment, the mice were marked, weighed, and measured ( Figure 49 D - E). The equivalent dose of PTX injected via the tail vein was 5 mg / kg, and the equivalent dose of Q administered by gavage was 80.48 g / kg. The body weight and tumor volume of the mice were measured daily. On the 14th day, the mice were sacrificed and the tumors were excised to visually evaluate tumor inhibition. Western blotting was used to detect the expression of PD-L1 in the tumor tissues of each treatment group ( Figure 49 F - G), and flow cytometry was used to detect the expression of CD4 + T, CD8 + T cells and M2, M1 macrophages in the tumor tissues of each treatment group.

[0183] In the low-dose small-tumor experiment of Qiyusanlong formula combined with paclitaxel prodrug nanoparticles, it can be seen that ( Figure 49 H - M), the tumor volume of each treatment group was ranked as follows: Saline > Q > Taxol > diPTX-2Se NPs > diPTX-2Se NPs&Q, which was consistent with the in vitro tumor pictures and weight measurements. Compared with other treatment groups, diPTX-2Se NPs&Q showed excellent anti-tumor efficacy and did not cause weight loss. Inspired by the previous results of CD4 and CD8 immunofluorescence staining, we used flow cytometry to detect the expression of CD4 + T, CD8 + T cells in the tumor tissues. Compared with Q or diPTX-2Se NPs, the diPTX-2Se NPs&Q treatment group significantly up-regulated the proportion of CD4 + T, CD8 + T cells in the tumor tissues. We further investigated the expression of M1 and M2 macrophages in the tumor tissues. diPTX-2Se NPs&Q significantly down-regulated the expression of M2 and up-regulated the expression of M1 macrophages, promoted the polarization of M2 to M1 macrophages, and reshaped the tumor immunosuppressive microenvironment, thus showing a strong anti-tumor effect.

[0184] (3) High-dose large-tumor efficacy experiment:

[0185] Male C57BL / 6J mice (20 - 25 g) were obtained and raised under specified conditions. An animal model of subcutaneous transplanted tumor of LLC cells was constructed ( Figure 50 A). When the tumor volume reached approximately 300 mm 3 , the tumor-bearing mice were randomly divided into 5 groups (n = 5) ( Figure 50 B - C): Saline, Taxol, Q, diPTX-2Se NPs, diPTX-2Se NPs&Q. Before treatment, the mice were marked, weighed, and measured ( Figure 50 D - E). The equivalent dose of PTX injected via the tail vein was 10 mg / kg, and the equivalent dose of Q administered by gavage was 80.48 g / kg. The body weight and tumor volume of the mice were measured daily. On the 14th day, the mice were sacrificed, and the tumors were excised to visually evaluate tumor inhibition. Western blotting was used to detect the expression of PD-L1 in the tumor tissues of each treatment group ( Figure 50 F - G), and flow cytometry was used to detect the expression of CD4 + T, CD8 + T cells, and M2 and M1 macrophages in the tumor tissues of each treatment group.

[0186] From the above research results, we can see that ( Figure 50 H - M), diPTX-2Se NPs&Q had the best anti-tumor effect. The in vitro tumor pictures and weight measurements showed the same results, and there was no significant change in the body weight of the mice in each group. Flow cytometry was used to detect the expression of CD4 + T, CD8 + T cells, M1, and M2 macrophages in the tumor tissues. The results also showed that the proportions of CD4 + T and CD8 + T cells in the diPTX-2Se NPs&Q treatment group increased significantly, promoting the polarization of M2 to M1 macrophages. In summary, the diPTX-2Se NPs&Q treatment group could down-regulate the expression of PD-L1 on the basis of chemotherapy, induce the proliferation of T cells (CD4 + T cells and CD8 + T cells), promote the polarization of M2 to M1 macrophages, and reshape the tumor immunosuppressive microenvironment, thereby exerting a synergistic anti-tumor effect of chemoimmunotherapy.

[0187] In this study, a novel chemo-immunotherapy system was constructed by integrating the immunomodulatory function of Qiyu Sanlong formula (Q) with a redox-responsive paclitaxel prodrug nanodelivery system. Based on the characteristics of the tumor microenvironment, an NQO1 enzyme-responsive PTX-Q-PTX dimer prodrug (PQP system) and a redox-responsive PTX-SeSe-PTX / PTX-SS-PTX dimer prodrug (diPTX-2Se / S NPs) were designed respectively, and non-responsive controls (PTX-C-PTX and PTX-CC-PTX) were prepared. Experiments confirmed that both types of nanosystems possessed excellent colloidal stability (PDI < 0.2) and microenvironment-specific drug release capabilities: PQP achieved precise release of PTX in NQO1-overexpressing tumor cells, while diPTX-2Se / S NPs were selectively activated through a GSH / H2O2-responsive disulfide / diselenide bond cleavage mechanism. In a Lewis lung cancer model, Q demonstrated multi-dimensional immune remodeling functions, including significantly enhancing the infiltration rate of CD4 + / CD8 + T cells, inducing M2→M1 macrophage polarization, and downregulating the expression of PD-L1. The combination treatment group showed significant synergistic effects in both small and large tumor models, and its mechanism of action was attributed to: ① the EPR effect of the nanocarrier and intelligent drug release achieved chemosensitization; ② the traditional Chinese medicine components reversed the immunosuppressive microenvironment by regulating immune checkpoints and immune cell reprogramming. This study provided an innovative paradigm for the integrated traditional Chinese and Western medicine treatment of tumors, and confirmed the dual advantages of nanodrugs and traditional Chinese medicine compounds in terms of pharmacokinetic independence and therapeutic synergy.

[0188] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A pharmaceutical composition of a paclitaxel dimer prodrug nanoparticle and a traditional Chinese medicine compound, characterized in that, The pharmaceutical composition is composed of freeze-dried Qiyu Sanlong formula and paclitaxel dimer prodrug nanoparticles; among them, the paclitaxel dimer prodrug nanoparticles include one of quinone oxidoreductase 1 (NQO1)-responsive paclitaxel (PTX) dimer prodrug nanoparticles PTX-Q-PTX NPs or glutathione (GSH)-responsive paclitaxel (PTX) dimer prodrug nanoparticles PTX-SeSe-PTX NPs.

2. The pharmaceutical composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound according to claim 1, characterized in that The preparation method of the dimer prodrug nanoparticles PTX-Q-PTX NPs is as follows: Dissolve the dimer prodrug PTX-Q-PTX and DSPE-PEG 2K in a mixed solvent of absolute ethanol and acetone. Under stirring conditions, slowly add it dropwise to deionized water, and then remove the organic solvent by rotary evaporation under reduced pressure at room temperature to obtain the dimer prodrug nanoparticles PTX-Q-PTX NPs.

3. The pharmaceutical composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound according to claim 2, wherein The molar ratio of the PTX-Q-PTX to DSPE-PEG 2K is 1:2-3; The volume ratio of the absolute ethanol and acetone is 2-3:

3.

4. The pharmaceutical composition of paclitaxel dimer prodrug nanoparticles and traditional Chinese medicine compound according to claim 1, wherein, The preparation method of the dimer prodrug nanoparticles PTX-SeSe-PTX NPs is as follows: The dimeric prodrug PTX-SeSe-PTX and DSPE-PEG 2K were dissolved in a mixed solvent of absolute ethanol and tetrahydrofuran. Under stirring conditions, it was slowly added dropwise to deionized water, and then the organic solvents were removed by rotary evaporation under reduced pressure at room temperature to obtain the dimeric prodrug nanoparticles PTX-SeSe-PTX NPs.

5. The pharmaceutical composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound according to claim 4, characterized in that The PTX-SeSe-PTX and DSPE-PEG 2K have a molar ratio of 4:1 - 3; The volume ratio of the absolute ethanol and tetrahydrofuran is 5-7:

1.

6. The pharmaceutical composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound according to claim 2, wherein, The preparation method of the dimer prodrug PTX-Q-PTX includes the following steps: (1) Methanesulfonic acid is added to 2,5-dimethylhydroquinone, vacuum is pumped and protected by nitrogen, heated until the solid compound dissolves, and then methyl 3,3-dimethylacrylate is slowly dropped into it. After heating and stirring the reaction, it is stopped. After the reaction system is cooled to room temperature, it is successively extracted and washed with organic solvents, dehydrated, concentrated under reduced pressure until a solid precipitates, and finally filtered to obtain the bislactone chromanone derivative; (2) The bislactone chromanone derivative is dissolved in tetrahydrofuran, and then lithium hydroxide solution is added thereto. The reaction is stirred under open conditions at room temperature, and the conversion rate of the reaction is monitored by thin layer chromatography. When the conversion is complete, the reaction mixture is first adjusted to pH 3 to 4, and then successively extracted and washed with organic solvents, dehydrated, and dried under reduced pressure to obtain the intermediate compound quinone dipropionic acid QDPA; (3) PTX is dissolved in dichloromethane, and then QDPA, EDCI and DMAP are successively added. The reaction is stirred at room temperature under N2 protection. EDCI and DMAP are added again, and the reaction continues until the end. The reaction solution is eluted and separated and purified with a mixed organic solvent to obtain the dimer prodrug PTX-Q-PTX.

7. The pharmaceutical composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound according to claim 6, wherein, The molar ratio of 2,5-dimethylhydroquinone, methanesulfonic acid and methyl 3,3-dimethylacrylate is 2-4:3-6:3-6; the molar ratio of the bislactone chromanone derivative to lithium hydroxide is 2-5:3-5; the molar ratio of PTX, QDPA, EDCI and DMAP is 3-5:2-3:10-15:1-2.

8. The pharmaceutical composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound according to claim 4, characterized in that The preparation method of the dimer prodrug PTX-SeSe-PTX includes the following steps: (1) Selenium powder is added to a reaction vessel containing water, and sodium borohydride dissolved in water is added dropwise under ice bath. After the reaction solution becomes clear, another portion of selenium powder is added, and the mixture is heated under reflux. Then the bromopropionic acid solution is added to the above system, and the reaction is carried out overnight at room temperature. The whole process is protected by N2 until the reaction ends. The pH of the reaction solution is adjusted, and it is successively extracted, recrystallized and dried with organic solvents to obtain the intermediate product 3,3'-diselenodipropionic acid; (2) Dissolve PTX in dichloromethane, then sequentially add diselenodipropionic acid, EDCI, and DMAP, stir and react at room temperature under N₂ protection. Add EDCI and DMAP again and continue the reaction until completion. The reaction solution is eluted, separated, and purified with a mixed organic solvent to obtain the dimer prodrug PTX-SeSe-PTX.

9. The pharmaceutical composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound according to claim 8, wherein, The molar ratio of the selenium powder, sodium borohydride, and bromopropionic acid is 1 - 2: 2 - 4: 1 - 3; the molar ratio of PTX, diselenodipropionic acid, EDCI, and DMAP is 3 - 5: 4 - 5: 10 - 15: 1 - 2.

10. Use of the pharmaceutical composition of the paclitaxel dimer prodrug nanoparticles and the traditional Chinese medicine compound according to any one of claims 1 - 9 in the preparation of an anti-lung cancer drug.

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