Multifunctional nano-preparation for reversing tumor immune microenvironment and preparation method and application thereof

Abstract

The application discloses a multifunctional nano preparation for reversing tumor immune microenvironment and a preparation method and application thereof, and belongs to the technical field of biological medicine. The multifunctional nano preparation is composed of a carrier formed by a composite lipid material, a hydrophobic prodrug molecule and cholesterol and siRNA loaded in the carrier. The molar ratio of siRNA, the hydrophobic prodrug molecule, cholesterol and the composite lipid material is 0.8-2.5:0.1-5:1.5-7:10, wherein the molar number of siRNA is calculated according to the molar number of its base. The composite lipid material comprises an ionizable cationic lipid, a phospholipid and an iRGD peptide modified phospholipid-polyethylene glycol. The hydrophobic prodrug molecule is obtained by coupling 7-ethyl-10-hydroxy camptothecin and cholesterol through a disulfide bond. The multifunctional nano preparation can successfully realize the conversion of a "cold tumor" into a "hot tumor" and significantly improve the curative effect of a tumor vaccine, thereby providing a new strategy and scheme for clinical cancer treatment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a multifunctional nano preparation for reversing tumor immune microenvironment and a preparation method and application thereof. BACKGROUND

[0002] The mRNA vaccine induces the body to produce specific immune responses by introducing mRNA sequences encoding specific antigens into the body, thereby achieving the purpose of preventing or treating diseases. Nowadays, mRNA vaccines have gradually become a powerful weapon against various infectious diseases, immunodeficiency diseases and cancers. However, in clinical experiments, a single vaccine treatment method is often difficult to achieve efficient inhibition of tumors, and mRNA vaccine therapy is often combined with chemotherapy drugs, antibody drugs and other treatment methods. This is mainly due to the presence of various immune suppression mechanisms in the tumor microenvironment, such as excessive expression of immune checkpoints, high infiltration of immunosuppressive cells, and complex extracellular matrix components, which together induce the inactivation of cytotoxic T lymphocytes. How to transform the difficult-to-treat "cold" tumor into a "hot" tumor that is more susceptible to immune attack has become a key problem that needs to be solved in the current field of tumor immunotherapy.

[0003] The Chinese patent document with publication number CN119074930A discloses the application of a oncolytic poxvirus and an immune checkpoint PD-L1 / TDO2 inhibitor in the preparation of an anti-tumor drug. The oncolytic poxvirus rVV-CCL5 has good anti-tumor effect, and after being combined with the immune checkpoint PD-L1 inhibitor and the immune checkpoint TDO2, it can convert "cold tumors" into "hot tumors" and has stronger anti-tumor effect. However, whether it is some existing chemotherapy drugs, or the above-mentioned oncolytic poxvirus, or the immune checkpoint antibody drug, there may be a problem of inducing systemic adverse reactions after a large amount of drug administration, thereby limiting the efficacy and application range.

[0004] Stimulus-responsive tumor treatment products can only "unloading" when the tumor environment is unique stimulation, such as the disclosure of the Chinese patent document No. CN116063245A discloses a central degradable mRNA liposome nanoparticle and its preparation method and application, the invention introduces a stimulus-responsive group into the amino core structure of the ionizable lipid to obtain a central degradable ionizable lipid, and then the central degradable ionizable lipid, phospholipid, cholesterol and polyethylene glycol lipid four components are mixed to load mRNA at a specific molar ratio, thereby improving the endosome escape efficiency of the liposome nanoparticle in cells, and the transfection efficiency is high. By changing the amino core structure, the hydrophobic tail and the ratio of the four components, the mRNA can be targeted to deliver to the liver, spleen, lung, secondary lymphatic system, blood and tumor of mice. The invention is to modify the carrier to achieve stimulus responsiveness, if the prodrug design is carried out, the prodrug molecule itself responds to release the antitumor drug when it is subjected to the unique stimulation of the tumor environment, which is another new strategy to improve drug utilization and reduce side effects. SUMMARY

[0005] The present application provides a multifunctional nanometer preparation for reversing the tumor immune microenvironment, which co-loads SN38 prodrug and siRNA, and has tumor targeting property; low-dose SN38 prodrug can not only inhibit tumor cell proliferation, but also mediate immunogenic death (ICD) to reverse the immune microenvironment together with immune checkpoint blockade (ICB) realized by gene therapy; the multifunctional nanometer preparation can successfully realize the transformation of "cold tumor" to "hot tumor", significantly improve the efficacy of tumor vaccine, and provide new strategies and schemes for clinical cancer treatment.

[0006] The specific technical solutions adopted are as follows:

[0007] A multifunctional nanometer preparation for reversing the tumor immune microenvironment is composed of a carrier formed by a complex lipid material, a hydrophobic prodrug molecule and cholesterol, and siRNA loaded therein; the molar ratio of siRNA, hydrophobic prodrug molecule, cholesterol and complex lipid material is 0.8-2.5:0.1-5:1.5-7:10, and further 1.2-2:2-4:3-5:10, wherein the molar number of siRNA is calculated based on the molar number of its base;

[0008] The complex lipid material includes ionizable cationic lipid, phospholipid and iRGD peptide modified phospholipid-polyethylene glycol; the molar ratio of ionizable cationic lipid, phospholipid and iRGD peptide modified phospholipid-polyethylene glycol is 20-75:2.5-15:0.5-5;

[0009] The hydrophobic prodrug molecule is obtained by coupling 7-ethyl-10-hydroxy camptothecin and cholesterol through a disulfide bond.

[0010] Further, the ionizable cationic lipid is selected from SM102, Dlin-MC3-DMA, cKK-E12 or ALC-0315; the phospholipid is selected from DSPC or DOPE; the iRGD peptide modified phospholipid-polyethylene glycol is selected from DSPE-PEG-iRGD or DMG-PEG-iRGD, wherein the polyethylene glycol moiety has a degree of polymerization of 500-5000.

[0011] Specifically, the preparation method of the hydrophobic prodrug molecule is as follows:

[0012] S01, after the hydroxyl group on the benzene ring of 7-ethyl-10-hydroxycamptothecin is protected, the protected 7-ethyl-10-hydroxycamptothecin is mixed with triphosgene BTC and 4-dimethylaminopyridine DMAP in an organic solvent, and then stirred under anhydrous and anaerobic conditions in the dark, and then 2,2'-dithiodiethanol is added to obtain a first intermediate;

[0013] S02, after the first intermediate is mixed with triphosgene BTC and 4-dimethylaminopyridine DMAP in an organic solvent and stirred under anhydrous and anaerobic conditions in the dark, cholesterol is added to obtain a second intermediate, and then the second intermediate is deprotected to obtain the hydrophobic prodrug molecule.

[0014] Preferably, in step S01, the molar ratio of the protected 7-ethyl-10-hydroxycamptothecin, triphosgene BTC, 4-dimethylaminopyridine DMAP and 2,2'-dithiodiethanol is 1:0.2-0.8:1.2-4.5:2-10, and further preferably 1:0.4:3:5;

[0015] Preferably, in step S02, the molar ratio of the first intermediate, triphosgene BTC, 4-dimethylaminopyridine DMAP and cholesterol is 1:0.2-0.8:1.2-4.5:1-5, and further preferably 1:0.37:3.2:1.2.

[0016] Preferably, di-tert-butyl dicarbonate, tert-butyl dimethyl silyl ether, tert-butyl diphenyl silyl ether or benzyl ether is used to protect the hydroxyl group on the benzene ring of 7-ethyl-10-hydroxycamptothecin; the organic solvent in steps S01 and S02 is selected from dichloromethane, tetrahydrofuran, N,N-dimethylformamide or dimethyl sulfoxide.

[0017] Preferably, the siRNA is siPD-L1; the sequence of the sense strand of siPD-L1 is 5'-GCGUUUACUGCUGCAUAAUTT-3' (SEQ ID NO. 1), and the sequence of the antisense strand is 5'-AUUAUGCAGCAGUAAACGCTT-3' (SEQ ID NO. 2).

[0018] Preferably, the multifunctional nano-preparation has a particle size of 80-220 nm, an encapsulation rate of 70%-100%, and a drug loading rate of 5%-30%. The particle size is small and uniform, and the encapsulation rate and drug loading rate are high.

[0019] The application further provides a preparation method of the multifunctional nano-preparation. First, a mixed solution of composite lipid material and cholesterol is prepared, and is mixed with an organic solution of hydrophobic prodrug molecules to obtain an oil phase solution; then, an aqueous phase solution containing siRNA is prepared, and the oil phase solution is mixed with the aqueous phase solution, followed by dialysis to obtain the multifunctional nano-preparation.

[0020] Further, in the mixed solution of composite lipid material and cholesterol, the solvent is an alcohol substance; in the organic solution of hydrophobic prodrug molecules, the solvent is an alcohol substance and dimethyl sulfoxide; and the solvent of the aqueous phase solution is a buffer solution, preferably a citric acid-citrate buffer solution, further preferably having a pH of 4 and a concentration of 50 mM.

[0021] Further, the volume ratio of the oil phase solution to the aqueous phase solution is 2-5:1.

[0022] The application further provides an application of the multifunctional nano-preparation in the preparation of a tumor treatment product.

[0023] The application further provides a tumor treatment product comprising the multifunctional nano-preparation and an mRNA vaccine. The multifunctional nano-preparation can improve the tumor immunosuppressive microenvironment to improve the efficacy of the vaccine.

[0024] Compared with the prior art, the application has the following beneficial effects:

[0025] The present application develops a multifunctional nanoformulation co-delivering SN38 prodrug and siPD-L1 based on LNP carrier platform, which can improve the tumor immunosuppressive microenvironment to improve the efficacy of the vaccine. The SN38 prodrug can achieve responsive release of SN38 in the presence of GSH, and the targeted delivery of SN38 at the tumor site can inhibit tumor growth and induce immunogenic cell death (ICD). The damage-associated molecular patterns (DAMPs) and immunogenic substances released during the ICD process will promote the maturation of local antigen-presenting cells (APCs). Mature APCs can migrate to lymph nodes for antigen presentation on one hand, and release immune factors to promote the maturation of other immune cells in the tumor microenvironment on the other hand, thereby achieving the purpose of improving the immunosuppressive microenvironment. The introduction of siPD-L1 into the nano-carrier effectively solves the problem of up-regulation of the tumor's inherent immune checkpoint molecule (PD-L1) induced by SN38. The results of in vivo anti-tumor experiments show that the combination of multifunctional nanoformulation and mRNA vaccine has significantly better tumor inhibition effect than single treatment group, with an inhibition rate of 93.5% (vaccine 45.8%) in the B16F10-OVA subcutaneous tumor model, and an inhibition rate of 56.9% (vaccine 30.8%) in the 4T1 subcutaneous tumor model, which is attributed to the improvement of the immunosuppressive microenvironment by the multifunctional nanoformulation, which promotes the infiltration and activity of immune cells activated by the vaccine, thereby achieving high-efficiency killing effect on tumor cells. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Figure 1 is a preparation schematic diagram of SN38-ss-Chol. 13 Figure 2 is a 1H NMR spectrum of SN38-ss-Chol.

[0027] Figure 2 Figure 3 is a preparation schematic diagram of multifunctional nanoformulation RSLNP / siPD-L1.

[0028] Figure 3 Figure 4A is a responsive release process diagram of SN38-ss-Chol after co-incubation with GSH; Figure 4B is a fluorescence spectrum diagram of SN38-ss-Chol after co-incubation with GSH (10 mM) for different time.

[0029] Figure 4 Figure 5 is a UV absorption diagram of different materials.

[0030] Figure 5 Figure 6A is a flow diagram of Cy5-siRNA-labeled different carriers after co-incubation with cells; Figure 6B is a live imaging diagram of tumor-bearing mice after tail vein injection of different nanoformulations (DIR labeling); Figure 6C is an ex vivo imaging diagram of liver and tumor tissues 24 h after injection; Figure 6D is a relative fluorescence intensity comparison analysis diagram of liver and tumor.

[0031] Figure 6A is the establishment of mouse B16F10-OVA subcutaneous tumor model and treatment scheme diagram; B is the mouse weight change curve; C is the tumor volume growth curve; D is the average tumor weight graph; E is the tumor inhibition rate summary graph; F is the H&E staining and TUNEL staining of tumor tissue.

[0032] Figure 7 A is the immunofluorescence staining of CRT and HMGB1 in tumor tissue; B is the representative flow cytometry of mature dendritic cells in tumor draining lymph nodes; C is the representative flow cytometry of CD11c + MHC-II + quantitative analysis graph; D is the quantitative analysis graph of CD11c + CD86 + mature dendritic cells in tumor draining lymph nodes; E is the schematic diagram of RSLNP / siPD-L1 improving tumor immunosuppressive microenvironment.

[0033] Figure 8 A is the CD8 + T cell immunofluorescence staining of tumor tissue in each group of mice after treatment; B is the CD3 + CD8 + T cell representative flow cytometry of tumor tissue; C is the representative flow cytometry of M1 macrophages (F4 / 80 + CD80 + ) in tumor tissue; D and E are the relative fold comparison graphs of IFN-γ and TNF-α in tumor tissue, respectively; F, G, H and I are the quantitative analysis graphs of CD3 + CD8 + T cells, CD3 + CD4 + cells in tumor tissue; J is the ratio graph of M1 (F4 / 80 + CD80 + ) and M2 (F4 / 80 + CD206 + ) in tumor tissue; K is the CD62L-CD44 + CD8 + T cells in spleen; L is the CD62L-CD44 + effector memory T cell quantitative analysis graph. DETAILED DESCRIPTION

[0034] To make the objectives, features, and advantages of this invention more apparent and understandable, a detailed description is provided below through specific embodiments. Many specific details are set forth in the following description to provide a thorough understanding of the invention. However, the invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below. Technical features in various embodiments of the invention can be combined appropriately without mutual conflict.

[0035] Unless otherwise specified, the operating methods in the following examples are generally performed under conventional conditions or as recommended by the manufacturer. Contents not described in detail in this specification are prior art known to those skilled in the art. Unless otherwise specified, the experimental materials used in the examples below can be purchased from conventional biochemical reagent companies.

[0036] In the following examples, the siRNA used is siPD-L1; the sense strand sequence of siPD-L1 is 5`-GCGUUUACUGCUGCAUAAUTT-3`, and the antisense strand sequence is 5`-AUUAUGCAGCAGUAAACGCTT-3`. The mOVA vaccine can be synthesized or purchased according to existing technology records.

[0037] Example 1: Synthesis of the hydrophobic prodrug molecule SN38-ss-Chol

[0038] The synthetic route for the hydrophobic prodrug molecule SN38-ss-Chol is shown below:

[0039]

[0040] 1.96 g of 7-ethyl-10-hydroxycamptothecin SN38 (5 mmol) was suspended in 200 mL of anhydrous dichloromethane. 1.42 g of di-tert-butyl dicarbonate (Boc₂O, 6.5 mmol) and 10 mL of anhydrous pyridine were added, and the mixture was stirred overnight at room temperature to obtain a clear solution. The solution was washed with hydrochloric acid (0.5 mol / L) and sodium bicarbonate (1.0 mol / L), respectively. The organic phase was dried over anhydrous sodium sulfate and distilled under reduced pressure to obtain the SN38-Boc product.

[0041] To 476 mg of SN38-Boc (1 mmol) dissolved in 15 mL of anhydrous dichloromethane, 118.4 mg of triphosgene (BTC, 0.4 mmol) was added under ice bath conditions, followed by 366 mg of 4-dimethylaminopyridine (DMAP, 3 mmol), and stirred for 30 minutes under anhydrous and oxygen-free conditions in the dark, then transferred to room temperature and continued to stir for 1-2 hours. 770 mg of 2,2'-dithiodiethanol (5 mmol) was added and the reaction was continued overnight. The product was washed with water, dried under reduced pressure, and purified by silica gel chromatography (ethyl acetate / n-hexane: 3 / 1) to obtain the pure product SN38(Boc)-ss-OH.

[0042] To 336 mg of SN38(Boc)-ss-OH (0.5 mmol) dissolved in 10 mL of anhydrous dichloromethane, 55 mg of BTC (0.185 mmol) was added under ice bath conditions, followed by 195 mg of DMAP (1.60 mmol), and stirred for 30 minutes under anhydrous and oxygen-free conditions in the dark, then transferred to room temperature and continued to stir for 1-2 hours. 232 mg of cholesterol (0.6 mmol) was added and the reaction was continued overnight under stirring in the dark. The pure product SN38(Boc)-ss-Chol was obtained by column chromatography purification.

[0043] To 314 mg of SN38(Boc)-ss-Chol dissolved in 4 mL of dichloromethane, 1 mL of trifluoroacetic acid was added, and the reaction was stirred at room temperature in the dark for 1 hour. The crude product was obtained by washing with water, drying, and distillation under reduced pressure, and then purified by column chromatography (dichloromethane / methanol: 100 / 1.2) to obtain the pure product of the hydrophobic prodrug molecule SN38-ss-Chol, with a purity of 95% and a yield of 70%. 13 The C NMR spectrum is shown in Figure 1 and the corresponding results confirmed the successful synthesis of SN38-ss-Chol.

[0044] Example 2

[0045] Cationic liposome SM102, distearoylphosphatidylcholine DSPC, cholesterol and DSPE-PEG2000-iRGD were dissolved in the ethanol phase to obtain a lipid solution, wherein the molar ratio of SM102, DSPC and DSPE-PEG2000-iRGD was 50:10:1.5; SN38-ss Chol of different concentrations was dissolved in a mixed solution of DMSO and ethanol (v / v: 1 / 3), and then mixed with the lipid solution according to the preset ratio to obtain an oil phase solution. siPD-L1 (the sequence of the sense strand is 5'-GCGUUUACUGCUGCAUAAUTT-3', and the sequence of the antisense strand is 5'-AUUAUGCAGCAGUAAACGCTT-3') was dissolved in 50 mM citric acid-citrate buffer (pH = 4) to obtain an aqueous phase solution. The oil phase solution and the aqueous phase solution were mixed in a volume ratio of 3:1 using a syringe pump (LONGER). The mixture was dialyzed against PBS (molecular weight = 14 kDa, Biosharp) at 4°C for more than 4 hours to obtain the multifunctional nano-preparation RSLNP / siPD-L1; by adjusting the concentration of SN38-ss-Chol, RSLNP / siPD-L1 with different SN38-ss-Chol doping ratios was obtained. Experiments showed that the RSLNP / siPD-L1 prepared by using the molar ratio of siRNA, SN38-ss-Chol, cholesterol and complex lipid material as 1.63:2.96:3.34:10 (wherein the number of moles of siPD-L1 is calculated based on the number of moles of its base; ) had the best effect. Further cell and animal experiments were carried out using the RSLNP / siPD-L1 prepared therefrom or a control product prepared under related parameters, and a preparation schematic of the multifunctional nano-preparation RSLNP / siPD-L1 is shown in Figure 2 .

[0046] Example 3

[0047] The response release function of SN38-ss-Chol prepared in Example 1 was verified by using a fluorescence spectrophotometer. As shown in A and B in Figure 3 , the fluorescence intensity of the prodrug SN38-ss-Chol was greatly reduced compared with that of SN38, which may be caused by intermolecular electron transfer. After co-incubation with GSH, the characteristic peak of SN38 gradually increased due to the breaking of the disulfide bond, which proved that the prodrug molecule SN38-ss-Chol could realize the responsive release of SN38 in the presence of GSH for subsequent use.

[0048] The entrapment of SN38-ss-Chol was further verified by using a UV spectrophotometer. The results showed that the main characteristic peaks (386 nm and 368 nm) of Chol-ss-SN38 also appeared in RSLNP / siRNA (i.e. RSLNP / siPD-L1) Figure 4), RLNP / siRNA unloaded SN38-ss-Chol, which proved that SN38-ss-Chol was successfully encapsulated in LNP. By drawing its standard curve and calculating, the encapsulation efficiency was 97.1% and the drug loading rate was 13.5% under the condition of 18% doping ratio (at this time the particle size was 188.39 ± 2.83 nm). At the same time, the encapsulation efficiency of siRNA (siPD-L1) was 94.1% by Nanodrop detection. The effective encapsulation and loading of drugs will provide guarantee for the further application of nano-preparation.

[0049] Example 4

[0050] The tumor cell targeting delivery ability of RSLNP / siPD-L1 prepared in Example 2 was explored. Figure 5 A in FIG. 6 is the flow cytometry detection of cell uptake results after different carriers were co-incubated with cells for 6h, the fluorescence values of iRGD peptide targeted RLNP and RSLNP groups were higher than that of LNP group (prepared by DSPE-PEG2000), which may be attributed to the high expression of integrin receptor on tumor cells promoting the uptake of iRGD peptide modified LNP. Figure 5 B in FIG. 6 is the in vivo imaging diagram (DIR probe labeling) of 4T1 tumor-bearing mice after intravenous injection of different nano-carriers. By observing the fluorescence distribution of nano-drugs at 8h and 24h, it can be seen that the enrichment of iRGD peptide modified carrier RSLNP (iRGD peptide-LNP / SN38-ss-Chol) in tumor site is improved compared with SLNP (LNP / SN38-ss-Chol). Further dissection of liver and tumor tissues and ex vivo imaging analysis showed that the enrichment of RSLNP in liver was reduced compared with SLNP, but the enrichment of tumor tissue was improved (C and D in FIG. 6). The results showed that the introduction of iRGD peptide would be beneficial to the tumor targeting enrichment of nano-drugs to improve drug utilization. Figure 5

[0051] Example 5

[0052] B16F10-OVA melanoma-bearing mouse model was constructed to explore its in vivo tumor inhibition effect and the improvement of mRNA tumor vaccine efficacy. The treatment scheme was as follows Figure 6 ​As shown in A, G1 is the PBS buffer control group, G2 is the CPT-11 chemotherapy drug group, G3 is the SLNP (LNP / SN38-ss-Chol) group, G4 is the RSLNP (iRGD peptide-LNP / SN38-ss-Chol) group, G5 is the RSLNP / siPD-L1 (iRGD peptide-LNP / SN38-ss-Chol / siPD-L1) group, G6 is the mRNA vaccine group, and G7 is the RSLNP / siPD-L1 and mRNA vaccine combination group. Six days prior to treatment, B16F10-OVA tumor cells were subcutaneously injected. Treatment began on day 0, and on day 11, mice underwent euthanasia to dissect the tumors and organs for analysis. Tumor size and mouse weight changes were monitored throughout the process.

[0053] Depend on Figure 6 As shown in B, there was no significant and sudden decrease in the body weight of mice in any group. However, the body weight of mice in the free CPT-11 treatment group was slightly lower than that of mice in other nanoparticle formulation groups. Compared with the PBS group, groups G2, G3, and G4 all showed the effect of delaying tumor growth. Figure 6 In the G4 group (C), the difference in efficacy between the G4 group and the PBS group was more significant. In the G5 group, the introduction of siPD-L1 into RSLNP further delayed tumor growth with an inhibition rate as high as 73.2% (C). Figure 6 (D and E in the text). The G6 group, treated with the vaccine alone, also demonstrated tumor-suppressive activity (inhibition rate 45.8%), but this effect did not completely eradicate the tumor, possibly due to the tumor immunosuppressive microenvironment limiting its therapeutic efficacy. The G7 combination therapy group achieved the most ideal results, with an inhibition rate as high as 93.5%, significantly superior to single therapy. These results demonstrate that lower doses of RSLNP / siPD-L1 enhance the therapeutic effect of mRNA vaccines, thereby achieving highly effective inhibition of melanoma.

[0054] like Figure 6 As shown in F, compared with the single treatment group, the combined treatment group had significantly increased levels of apoptosis and tissue necrosis, which is consistent with the tumor suppression results.

[0055] Example 6

[0056] Calreticulin (CRT) exposure and high-mobility group box 1 (HMGB1) release are key indicators of ICD (intracytoplasmic reticulum), therefore, immunofluorescence staining is used to assess the ICD status of tumor tissues. Figure 7 As shown in Figure A, CRT (green) and HMGB1 (red) fluorescence were observed in groups G2-G5, with group G5 showing significantly stronger fluorescence than group G2, indicating that RSLNP / siPD-L1 efficiently mediated tumor ICD. Group G7 exhibited the brightest and most widely distributed fluorescence in the field of view, further confirming that the combined effects of chemotherapy, immune blockade, and vaccine therapy produced the most powerful ICD effect.

[0057] Immune cells were extracted from mouse lymph nodes, spleen, and tumors for flow cytometry analysis. The results are as follows: Figure 7 As shown in Figure BD, the proportion of mature DCs in group G5 was significantly higher than that in group G1. This is attributed to the release of local tumor immunogenic substances mediated by RSLNP / siPD-L1, which promoted DC maturation and lymph node drainage. The proportion of mature DCs was the highest in group G7, indicating that the combination therapy brought the activation state of antigen-presenting cells (APCs) in TDLNs to its peak, which will be beneficial to the activation of cytotoxic T lymphocytes. A schematic diagram of RSLNP / siPD-L1 improving the tumor immunosuppressive microenvironment is shown in Figure E.

[0058] CD8+ in tumor tissues of mice after treatment in each group + T-cell immunofluorescence staining image as shown below Figure 8 As shown in A, CD3 in tumor tissue + CD8 + T cells and M1 macrophages (F4 / 80) + CD80 + Representative flow cytometry images are as follows: Figure 8 As shown in B and C in Figure 8, and D and E in Figure 8, the G7 combination therapy group showed the highest level of immune factor secretion, as did the single vaccine therapy group. This indicates that after treatment, pro-inflammatory factors within the tumor were also actively secreted, and the immunosuppressive microenvironment was gradually improved. As shown in F and H in Figure 8, the G7 combination therapy group showed the highest level of intratumoral CD3. + CD8 + The proportion of T cells was the highest and significantly better than in the single-treatment group, indicating that RSNLNP / siPD-L1 indeed improved the tumor microenvironment and thus promoted CD8. + T infiltration, also in CD3 + CD4 + The same trend was also observed in the assessment results of T cells and NK cells.

[0059] The M1 / M2 ratio of macrophages is often used as an indicator of tumor immune status and clinical prognosis. For example... Figure 8 As shown in I and J, both the M1 macrophages with tumor-suppressing properties and the M1 / M2 ratio showed that the G7 group was the best.

[0060] For cancer treatment, long-acting immunotherapy is crucial against tumor recurrence and metastasis, and further research has explored the immune memory effects induced by different treatment groups. For example... Figure 8 As shown in K, G6 group CD62L - CD44 + T cells (central memory T cells) in CD8 +The proportion of T is high, indicating that the tumor vaccine can activate the immune memory of the body at a high level. At the same time, the immune memory effect of the G7 group is further strengthened, which will be more conducive to improving the long-acting effect of tumor immunotherapy.

[0061] The above-described embodiments have described the technical solutions of the present application in detail, and it should be understood that the above-described is only a specific embodiment of the present application and is not used to limit the present application. Any modification, supplement or similar way of substitution made within the principle range of the present application shall be included in the protection scope of the present application.

Claims

1. A multifunctional nanoformulation for reversing the tumor immune microenvironment, characterized in that, The carrier is formed by a complex lipid material, a hydrophobic prodrug molecule and cholesterol, and the siRNA loaded by the carrier; the molar ratio of the siRNA, the hydrophobic prodrug molecule, the cholesterol and the complex lipid material is 1.63:2.96:3.34:10, wherein the molar number of the siRNA is calculated based on the molar number of its base; The complex lipid material comprises an ionizable cationic lipid, a phospholipid and an iRGD peptide modified phospholipid-polyethylene glycol; the molar ratio of the ionizable cationic lipid, the phospholipid and the iRGD peptide modified phospholipid-polyethylene glycol is 20-75:2.5-15:0.5-5; the ionizable cationic lipid is selected from SM102, Dlin-MC3-DMA, cKK-E12 or ALC-0315; the phospholipid is selected from DSPC or DOPE; the iRGD peptide modified phospholipid-polyethylene glycol is selected from DSPE-PEG-iRGD or DMG-PEG-iRGD, wherein the polymerization degree of the polyethylene glycol part is 500-5000; The hydrophobic prodrug molecule is obtained by coupling 7-ethyl-10-hydroxy camptothecin and cholesterol through a disulfide bond; The siRNA is siPD-L1.

2. The multifunctional nanoformulation according to claim 1, wherein, The preparation method of the hydrophobic prodrug molecule is as follows: S01, after protecting the hydroxyl on the benzene ring of 7-ethyl-10-hydroxy camptothecin, the protected 7-ethyl-10-hydroxy camptothecin, triphosgene BTC and 4-dimethylaminopyridine DMAP are mixed in an organic solvent, and then stirred under anhydrous and anaerobic conditions in the dark, and then 2,2'-dithiodiethanol is added to obtain a first intermediate; S02, after the first intermediate, triphosgene BTC and 4-dimethylaminopyridine DMAP are mixed in an organic solvent, and then stirred under anhydrous and anaerobic conditions in the dark, and then cholesterol is added to obtain a second intermediate, and then the second intermediate is deprotected to obtain the hydrophobic prodrug molecule.

3. The multifunctional nano-preparation according to claim 2, characterized in that, In step S01, the molar ratio of the protected 7-ethyl-10-hydroxy camptothecin, triphosgene BTC, 4-dimethylaminopyridine DMAP and 2,2'-dithiodiethanol is 1:0.2-0.8:1.2-4.5:2-10; In step S02, the molar ratio of the first intermediate, triphosgene BTC, 4-dimethylaminopyridine DMAP and cholesterol is 1:0.2-0.8:1.2-4.5:1-5.

4. The multifunctional nanoformulation as claimed in claim 2, wherein, In step S01, di-tert-butyl dicarbonate, tert-butyl dimethyl silyl ether, tert-butyl diphenyl silyl ether or benzyl ether is used to protect the hydroxyl on the benzene ring of 7-ethyl-10-hydroxy camptothecin; the organic solvent in step S01 and step S02 is selected from dichloromethane, tetrahydrofuran, N,N-dimethylformamide or dimethyl sulfoxide.

5. The method of preparing multifunctional nanoformulation as claimed in any one of claims 1 to 4, wherein, First, a mixed solution of the complex lipid material and cholesterol is prepared, which is mixed with an organic solution of the hydrophobic prodrug molecule to obtain an oil phase solution; then an aqueous phase solution containing siRNA is prepared, and the oil phase solution is mixed with the aqueous phase solution, and then dialyzed to obtain the multifunctional nano-preparation.

6. The method of claim 5, wherein the multifunctional nanoformulation is prepared by, The solvent in the mixture of the composite lipid material and cholesterol is an alcohol; the solvent in the organic solution of the hydrophobic prodrug molecule is an alcohol and dimethyl sulfoxide; and the solvent in the aqueous solution is a buffer.

7. Use of the multifunctional nano-preparation according to any one of claims 1 to 4 for the preparation of a medicament for the treatment of tumors, characterized in that, The tumor is melanoma.

8. A medicament for treating a tumor, characterized by comprising the compound according to claim 1. The tumor is melanoma. The tumor is melanoma.

Images