Multifunctional nano preparation for reversing tumor immune microenvironment as well as preparation method and application of multifunctional nano preparation

Abstract

The invention discloses a multifunctional nano preparation for reversing a 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, hydrophobic prodrug molecules and cholesterol and siRNA loaded on the carrier; the molar ratio of the siRNA to the hydrophobic prodrug molecule to the cholesterol to the composite lipid material is (0.8-2.5): (0.1-5): (1.5-7): 10, and the mole number of the siRNA is calculated according to the mole number of basic groups of the siRNA; the composite lipid material is prepared from ionizable cationic lipid, phospholipid and iRGD peptide modified phospholipid-polyethylene glycol, the hydrophobic prodrug molecule is obtained by coupling 7-ethyl-10-hydroxycamptothecin and cholesterol through a disulfide bond. The multifunctional nano preparation can successfully realize conversion of'cold tumor 'into'hot tumor', remarkably improves the curative effect of tumor vaccines, and provides a new strategy and scheme for clinical cancer treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a multifunctional nano preparation for reversing tumor immune microenvironment, and a preparation method and application thereof. Background Art

[0002] mRNA vaccines induce 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 trials, single vaccine treatments are often difficult to achieve efficient suppression of tumors, and mRNA vaccine therapy is often used in combination with chemotherapy drugs, antibody drugs and other treatments. This is mainly attributed to the presence of multiple immunosuppressive mechanisms in the tumor microenvironment, such as overexpression of immune checkpoints, high infiltration of immunosuppressive cells, and complex extracellular matrix components, which jointly induce the inactivation of cytotoxic T lymphocytes. How to transform difficult-to-treat "cold" tumors into "hot" tumors that are more susceptible to immune attacks has become a key issue that needs to be urgently addressed in the current field of tumor immunotherapy.

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

[0004] Stimulus-responsive tumor treatment products can "unload" only when there is a unique stimulus in the tumor environment. For example, the Chinese patent document with publication number 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 mixes the central degradable ionizable lipid, phospholipid, cholesterol, and polyethylene glycol lipid four components in a specific molar ratio to load mRNA, thereby improving the intracellular endosomal escape efficiency of the liposome nanoparticles, and the transfection efficiency is high. By changing the amino core structure, hydrophobic tail and the ratio of the four components, it is possible to achieve targeted delivery of mRNA to the mouse liver, spleen, lungs, secondary lymphatic system, blood and tumors. The invention is to modify the carrier to achieve stimulus responsiveness. If a prodrug is designed, the prodrug molecule itself responds to release anti-tumor drugs when it is stimulated by the unique stimulation of the tumor environment, which is another new strategy to improve drug utilization and reduce side effects. Summary of the invention

[0005] The present invention provides a multifunctional nanoformulation for reversing the tumor immune microenvironment. The multifunctional nanoformulation co-loads SN38 prodrug and siRNA and has tumor targeting. A low dose of SN38 prodrug can not only inhibit the proliferation of tumor cells to a certain extent, but the immunogenic death (ICD) mediated by SN38 prodrug can also reverse the immune microenvironment together with the immune checkpoint blockade (ICB) achieved by gene therapy. The multifunctional nanoformulation can successfully realize the transformation of "cold tumor" into "hot tumor", significantly improve the efficacy of tumor vaccines, and provide new strategies and solutions for clinical cancer treatment.

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

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

[0008] The composite lipid material comprises 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-hydroxycamptothecin and cholesterol through a disulfide bond.

[0010] Furthermore, 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 degree of polymerization of the polyethylene glycol part is 500-5000.

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

[0012] S01 protects the hydroxyl group on the benzene ring of 7-ethyl-10-hydroxycamptothecin, mixes it with triphosgene BTC and 4-dimethylaminopyridine DMAP in an organic solvent in anhydrous and oxygen-free conditions and avoids light, and then adds 2,2'-dithiodiethanol to react to obtain a first intermediate;

[0013] S02: The first intermediate is mixed with triphosgene BTC and 4-dimethylaminopyridine DMAP in an organic solvent in anhydrous and oxygen-free state and protected from light, and then cholesterol is added to react to obtain a second intermediate. The second intermediate is deprotected to obtain the hydrophobic prodrug molecule.

[0014] Preferably, in step S01, the molar ratio of 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, more 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 more preferably 1:0.37:3.2:1.2.

[0016] Preferably, the hydroxyl group on the benzene ring of 7-ethyl-10-hydroxycamptothecin is protected by di-tert-butyl dicarbonate, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether or benzyl ether; and the organic solvent in step S01 and step S02 is selected from dichloromethane, tetrahydrofuran, N,N-dimethylformamide or dimethyl sulfoxide.

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

[0018] Preferably, the multifunctional nanoparticle 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 present invention also provides a method for preparing the multifunctional nanoformulation, comprising first preparing a mixed solution of a composite lipid material and cholesterol, and mixing the mixed solution with an organic solution of a hydrophobic prodrug molecule to obtain an oil phase solution; then preparing an aqueous phase solution containing siRNA, mixing the oil phase solution with the aqueous phase solution, and dialyzing to obtain the multifunctional nanoformulation.

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

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

[0022] The present invention also provides the use of the multifunctional nano preparation in preparing tumor treatment products.

[0023] The present invention also provides a tumor treatment product, comprising the multifunctional nano preparation and 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 present invention has the following beneficial effects:

[0025] The present invention develops a multifunctional nanoformulation for co-delivering SN38 prodrug and siPD-L1 based on the LNP carrier platform, and the multifunctional nanoformulation can improve the tumor immunosuppressive microenvironment to improve the efficacy of the vaccine. 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 death (ICD). The damage-associated molecular patterns (DAMPs) and immunogenic substances released by the ICD process will promote the maturation of local antigen-presenting cells (APCs). On the one hand, mature APCs can migrate to lymph nodes for antigen presentation, and on the other hand, they can release immune factors to promote the maturation of other local immune cells in the tumor, thereby achieving the purpose of improving the immunosuppressive microenvironment. The introduction of siPD-L1 into nanocarriers effectively solves the problem of SN38 inducing upregulation of tumor-intrinsic immune checkpoint molecules (PD-L1). The results of in vivo anti-tumor experiments showed that the combined tumor inhibition effect of the multifunctional nanopreparation and mRNA vaccine was significantly better than that of the single treatment group. The inhibition rate in the B16F10-OVA subcutaneous tumor model reached 93.5% (vaccine 45.8%), and the inhibition rate in the 4T1 subcutaneous tumor model could reach 56.9% (vaccine 30.8%). This was attributed to the improvement of the immunosuppressive microenvironment by the multifunctional nanopreparation, which promoted the infiltration and activity of immune cells activated by the vaccine, thereby achieving efficient killing of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 For SN38-ss-Chol 13 C NMR spectrum.

[0027] Figure 2 Schematic diagram of the preparation of the multifunctional nanoformulation RSLNP / siPD-L1.

[0028] Figure 3 A in the figure is a response release process diagram after SN38-ss-Chol and GSH are co-incubated; B is a fluorescence spectrum diagram of SN38-ss-Chol and GSH (10mM) incubated for different time periods.

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

[0030] Figure 5 A is a flow cytometer graph after co-incubation of cells with different vectors labeled with Cy5-siRNA; B is an in vivo imaging image of tumor-bearing mice after injection of different nanoformulations via tail vein (DIR labeling); C is an in vitro imaging image of liver and tumor tissue 24 hours after injection; D is a comparative analysis of the relative fluorescence intensity of the liver and tumor.

[0031] Figure 6A is a schematic diagram of the establishment of the mouse B16F10-OVA subcutaneous tumor model and the treatment plan; B is a graph showing the changes in mouse body weight; C is a graph showing the growth curve of tumor volume; D is a graph showing the average tumor mass; E is a graph showing the summary of tumor inhibition rate; and F is a graph showing 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 cytometric image of mature dendritic cells in tumor-draining lymph nodes; C is the CD11c + MHC-II + Quantitative analysis diagram; D is CD11c in tumor-draining lymph nodes + CD86 + Quantitative analysis of mature dendritic cells; E is a schematic diagram of RSLNP / siPD-L1 improving the tumor immunosuppressive microenvironment.

[0033] Figure 8 A in the figure is the CD8 + T cell immunofluorescence staining; B is CD3 + CD8 + Representative flow cytometry images of T cells; C is M1 macrophages (F4 / 80 + CD80 + ) representative flow cytometry images; D and E are the relative multiple comparisons of IFN-γ and TNF-α in tumor tissues; F, G, H and I are the relative multiple comparisons of CD3 + CD8 + T cells, CD3 + CD4 + Quantitative analysis of T cells, CD3-NK1.1+ cells and M1 macrophages in tumors; J is the M1 (F4 / 80 + CD80 + ) and M2(F4 / 80 + CD206 + ) ratio diagram; K is spleen CD3 + CD8 + CD62L-CD44 in T cells + Quantitative analysis of effector memory T cells. DETAILED DESCRIPTION

[0034] In order to make the purpose, features and advantages of the present invention more obvious and easy to understand, the following is a detailed description through specific embodiments. In the following description, many specific details are set forth in order to fully understand the present invention. However, the present invention can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below. The technical features in each embodiment of the present invention can be combined accordingly without conflicting with each other.

[0035] The operating methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or according to the conditions recommended by the manufacturer. The contents not described in detail in this specification belong to the prior art known to professionals in the field. The experimental materials used in the following examples, unless otherwise specified, can be purchased from conventional biochemical reagent companies.

[0036] In the following examples, siRNA is selected from siPD-L1; the sense strand sequence of siPD-L1 is 5`-GCGUUUACUGCUGCAUAAUTT-3`, and the antisense strand sequence is 5`-AUUAUGCAGCAGUAAACGCTT-3`. mOVA vaccine can be synthesized according to the prior art or purchased.

[0037] Example 1 Synthesis of Hydrophobic Prodrug Molecule SN38-ss-Chol

[0038] The synthetic route of the hydrophobic prodrug molecule SN38-ss-Chol is as follows:

[0039]

[0040] 1.96 g of 7-ethyl-10-hydroxycamptothecin SN38 (5 mmol) was suspended in 200 mL of anhydrous dichloromethane, and 1.42 g of di-tert-butyl dicarbonate (Boc 2 0, 6.5 mmol) and 10 mL of anhydrous pyridine, stirred at room temperature overnight to obtain a clear solution, washed with hydrochloric acid solution (0.5 mol / L) and sodium bicarbonate solution (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] Take 476mg SN38-Boc (1mmol) and dissolve it in 15mL anhydrous dichloromethane. Add 118.4mg triphosgene (BTC, 0.4mmol) under ice bath conditions, then add 366mg 4-dimethylaminopyridine (DMAP, 3mmol), stir for 30 minutes under anhydrous, oxygen-free and light-proof conditions, then transfer to room temperature and continue stirring for 1-2 hours. Then add 770mg 2,2'-dithiodiethanol (5mmol) and continue stirring to react overnight. The product is 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] Take 336mg SN38(Boc)-ss-OH (0.5mmol) and dissolve it in 10mL anhydrous dichloromethane. Add 55mg BTC (0.185mmol) under ice bath conditions, then add 195mg DMAP (1.60mmol), stir for 30 minutes under anhydrous, oxygen-free and light-proof conditions, then transfer to room temperature and continue stirring for 1-2 hours. Then add 232mg cholesterol (0.6mmol), continue to stir and react overnight in the dark. Purify by column chromatography to obtain pure SN38(Boc)-ss-Chol.

[0043] 314 mg of SN38(Boc)-ss-Chol was dissolved in 4 mL of dichloromethane, and 1 mL of trifluoroacetic acid was added. The mixture was stirred at room temperature in the dark for 1 hour. The crude product was washed with water, dried and distilled under reduced pressure. The crude product was then purified by column chromatography (dichloromethane / methanol: 100 / 1.2) to obtain the pure hydrophobic prodrug molecule SN38-ss-Chol. 13 C NMR spectrum Figure 1 As shown, 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 a preset ratio to obtain an oil phase solution. siPD-L1 (sense chain sequence is 5`-GCGUUUACUGCUGCAUAAUTT-3`, antisense chain sequence is 5`-AUUAUGCAGCAGUAAACGCTT-3`) was dissolved in 50mM citric acid-citrate buffer (pH=4) to obtain an aqueous phase solution. The oil phase solution and the aqueous phase solution were mixed at a volume ratio of 3:1 using a syringe pump (LONGER). The mixture was dialyzed with PBS (molecular weight = 14 kDa, Biosharp) at 4°C for more than 4 hours to obtain the multifunctional nanoformulation RSLNP / siPD-L1; RSLNP / siPD-L1 with various SN38-ss-Chol doping ratios was obtained by adjusting the concentration of SN38-ss-Chol. Experiments have shown that the RSLNP / siPD-L1 prepared with a molar ratio of siRNA, SN38-ss-Chol, cholesterol and composite lipid material of 1.63:2.96:3.34:10 (wherein the molar number of siPD-L1 is calculated based on the molar number of its base) has the best effect. Cell and animal experiments were further performed using the control product prepared under the relevant parameters. The preparation schematic diagram of the multifunctional nanoformulation RSLNP / siPD-L1 is shown in FIG. Figure 2 shown.

[0046] Example 3

[0047] The response release function of SN38-ss-Chol prepared in Example 1 was verified using a fluorescence spectrophotometer. Figure 3 As shown in A and B in Figure 1, the fluorescence intensity of the prodrug SN38-ss-Chol is significantly lower than that of SN38, which may be caused by intermolecular electron transfer. After incubation with GSH, the characteristic peak of SN38 gradually increased due to the cleavage of the disulfide bond, proving that the prodrug molecule SN38-ss-Chol can achieve responsive release of SN38 in the presence of GSH for subsequent use.

[0048] The encapsulation of SN38-ss-Chol was further verified by UV spectrophotometer. The results showed that the main characteristic peaks of Chol-ss-SN38 (386nm and 368nm) also appeared in RSLNP / siRNA (ie: RSLNP / siPD-L1) ( Figure 4) RLNP / siRNA did not load SN38-ss-Chol, which proved that SN38-ss-Chol was successfully encapsulated in LNP. By plotting 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 (the particle size was 188.39 ± 2.83 nm at this time). At the same time, the encapsulation efficiency of siRNA (siPD-L1) was detected by Nanodrop to be 94.1%. The effective encapsulation and loading of the drug provided guarantee for the further application of the nanoplatform.

[0049] Example 4

[0050] Explore the tumor cell targeting delivery ability of the RSLNP / siPD-L1 prepared in Example 2; Figure 5 In A, the flow cytometry results of cell uptake after co-incubating different carriers with cells for 6 h are shown. The fluorescence values of the RLNP and RSLNP groups modified with iRGD peptide are higher than those of the LNP group (prepared with DSPE-PEG2000), which may be attributed to the high expression of integrin receptors on tumor cells promoting the uptake of LNP modified with iRGD peptide. Figure 5 In B, the in vivo imaging diagrams (DIR probe labeled) of 4T1 tumor-bearing mice after intravenous injection of different nanocarriers are shown. By observing the fluorescence distribution of the nanodrugs at 8 h and 24 h, it is obvious that the enrichment of the carrier RSLNP modified with iRGD peptide (iRGD peptide-LNP / SN38-ss-Chol) in the tumor site is improved compared with SLNP (LNP / SN38-ss-Chol); further dissect the liver and tumor tissues to obtain and perform ex vivo imaging analysis. The results show that the enrichment of RSLNP in the liver is decreased compared with SLNP, but the enrichment in the tumor tissue is increased ( Figure 5 In C and D). The results show that the introduction of iRGD peptide will be beneficial to the tumor-targeted enrichment of nanodrugs to improve the drug utilization rate.

[0051] Example 5

[0052] Construct a B16F10-OVA melanoma tumor-bearing mouse model to explore its in vivo tumor suppression effect and the improvement effect on the efficacy of mRNA tumor vaccine. The treatment plan is as Figure 6As 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; first, B16F10-OVA tumor cells were subcutaneously inoculated 6 days in advance, and the drug treatment was started on the 0th day. On the 11th day, the mice were euthanized and the tumors and organs were dissected for analysis, and the tumor size and mouse weight changes were monitored throughout the process.

[0053] Depend on Figure 6 As shown in B, there was no significant drop in the weight of mice in each group. However, the weight of mice in the free CPT-11 treatment group was slightly lower than that in the other nanoformulation groups. Groups G2, G3, and G4 all had the effect of delaying tumor growth compared with the PBS group ( Figure 6 In the G5 group, siPD-L1 was introduced into RSLNP, which further delayed tumor growth and the inhibition rate was as high as 73.2% ( Figure 6 D and E in the figure). The G6 group treated with the vaccine alone also showed an anti-tumor effect (inhibition rate of 45.8%), but this effect did not completely eradicate the tumor, which may be attributed to the tumor immunosuppressive microenvironment limiting its therapeutic effect. The G7 combined treatment group achieved the most ideal results, with an inhibition rate of up to 93.5%, which was significantly better than single therapy. The above results prove that a lower dose of RSLNP / siPD-L1 can enhance the therapeutic effect of mRNA vaccine, thereby achieving a highly effective inhibitory effect on melanoma.

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

[0055] Example 6

[0056] Calreticulin (CRT) exposure and high-mobility group protein B1 (HMGB1) release are the main indicators of ICD, so the ICD status of tumor tissues was evaluated by immunofluorescence staining. Figure 7 As shown in A, fluorescence of CRT (green) and HMGB1 (red) can be observed in groups G2-G5, and the brightness of group G5 is significantly stronger than that of group G2, indicating that RSLNP / siPD-L1 efficiently mediates tumor ICD. The brightest and most widely distributed fluorescence in the field of view of group G7 further confirms that the triple therapy of chemotherapy, immune blockade and vaccine produces the most powerful ICD effect.

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

[0058] CD8 in tumor tissues of mice in each group after treatment + T cell immunofluorescence staining Figure 8 As shown in A, CD3 + CD8 + T cells and M1 macrophages (F4 / 80 + CD80 + Representative flow cytometry images of Figure 8 As shown in B and C in Figure 8, and as shown in D and E in Figure 8, the secretion of immune factors in the G7 combined treatment group was the highest compared with the single vaccine treatment group, which indicates that after treatment, the pro-inflammatory factors in the tumor are also in an active secretion state, and the immunosuppressive microenvironment is gradually improved. As shown in FH in Figure 8, the CD3 + CD8 + The proportion of T cells showed the highest level and was significantly better than that of the single treatment group, indicating that RSNLNP / siPD-L1 did improve the tumor microenvironment and promote CD8 + T infiltration, also in CD3 + CD4 + The same trend was also shown in the evaluation results of T cells and NK cells.

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

[0060] For tumor treatment, long-term immunity is crucial for tumor recurrence and metastasis, and the immune memory effect caused by different treatment groups was further explored. Figure 8 As shown in K, CD62L in G6 group - CD44 + T cells (central memory T cells) in CD8 +The higher proportion of T cells indicates that tumor vaccines can activate the body's immune memory at a high level. At the same time, the immune memory effect of the G7 group was further enhanced, which will be more conducive to improving the long-term effect of tumor immunotherapy.

[0061] The embodiments described above provide a detailed description of the technical solutions of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, supplements or similar substitutions made within the scope of the principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A multifunctional nanoformulation for reversing the tumor immune microenvironment, characterized in that: The invention is composed of a carrier formed by a composite lipid material, a hydrophobic prodrug molecule and cholesterol and a loaded siRNA; the molar ratio of siRNA, hydrophobic prodrug molecule, cholesterol and composite lipid material is 0.8-2.5:0.1-5:1.5-7:10, wherein the molar number of siRNA is calculated based on the molar number of its bases; The composite lipid material comprises 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; The hydrophobic prodrug molecule is obtained by coupling 7-ethyl-10-hydroxycamptothecin and cholesterol through a disulfide bond.

2. The multifunctional nanoformulation according to claim 1, characterized in that: The ionizable cationic lipids include SM102, Dlin-MC3-DMA, cKK-E12 or ALC-0315; the phospholipids include DSPC or DOPE; the iRGD peptide-modified phospholipid-polyethylene glycol includes DSPE-PEG-iRGD or DMG-PEG-iRGD, wherein the degree of polymerization of the polyethylene glycol part is 500-5000.

3. The multifunctional nanoformulation according to claim 1, characterized in that: The preparation method of the hydrophobic prodrug molecule is: S01 protects the hydroxyl group on the benzene ring of 7-ethyl-10-hydroxycamptothecin, mixes it with triphosgene BTC and 4-dimethylaminopyridine DMAP in an organic solvent in anhydrous and oxygen-free conditions and avoids light, and then adds 2,2'-dithiodiethanol to react to obtain a first intermediate; S02: The first intermediate is mixed with triphosgene BTC and 4-dimethylaminopyridine DMAP in an organic solvent in anhydrous and oxygen-free state and protected from light, and then cholesterol is added to react to obtain a second intermediate. The second intermediate is deprotected to obtain the hydrophobic prodrug molecule.

4. The multifunctional nanoformulation according to claim 3, characterized in that: In step S01, the molar ratio of 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; 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.

5. The multifunctional nanoformulation according to claim 3, characterized in that: In step S01, the hydroxyl group on the benzene ring of 7-ethyl-10-hydroxycamptothecin is protected by di-tert-butyl dicarbonate, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether or benzyl ether; the organic solvent in step S01 and step S02 is selected from dichloromethane, tetrahydrofuran, N,N-dimethylformamide or dimethyl sulfoxide.

6. The multifunctional nanoformulation according to claim 1, characterized in that: The siRNA is siPD-L1.

7. The method for preparing the multifunctional nanoformulation according to any one of claims 1 to 6, characterized in that: First, a mixed solution of a composite lipid material and cholesterol is prepared, and the mixed solution is mixed with an organic solution of a hydrophobic prodrug molecule to obtain an oil phase solution; then an aqueous phase solution containing siRNA is prepared, and the oil phase solution and the aqueous phase solution are mixed and dialyzed to obtain the multifunctional nano preparation.

8. The method for preparing the multifunctional nanoformulation according to claim 7, characterized in that: In the mixed solution of the composite lipid material and cholesterol, the solvent is an alcohol substance; in the organic solution of the hydrophobic prodrug molecule, the solvent is an alcohol substance and dimethyl sulfoxide; and the solvent of the aqueous phase solution is a buffer solution.

9. Use of the multifunctional nanoformulation according to any one of claims 1 to 6 in the preparation of tumor treatment products.

10. A tumor treatment product, characterized in that: It comprises the multifunctional nanoformulation and mRNA vaccine described in any one of claims 1-6.

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