Co-release liposomes for regulating systemic and local immune responses and preparation method thereof

Abstract

The present invention belongs to the technical field of pharmaceutical preparations, specifically to co-delivery liposomes for regulating systemic and local immune responses and their preparation methods. The co-delivery liposomes of the present invention use calcium phosphate as the core to encapsulate two drugs: one is a small molecule drug that regulates the systemic immune response, and the other is a cytokine that regulates the local immune response of the tumor. By regulating the systemic and local immune responses of the tumor host, the infiltrating T cells in the immune desert tumor are increased, thereby improving the anti-tumor effect of immunotherapy. The small molecule drug is preferably cannabidiol, and the cytokine is preferably tumor necrosis factor LIGHT protein. The co-delivery liposomes constructed by the present invention are suitable for immune desert tumors. In situ transplant tumor model experiments have shown that it helps to improve the T cell immune response in patients and improve the response rate of tumor patients to ICIs. The co-delivery liposomes can significantly enhance the anti-tumor efficacy of ICI.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a co-release liposome capable of regulating systemic and local immune responses and a preparation method thereof. Background Art

[0002] Cancer is one of the most serious diseases that threatens human health and disrupts family and social harmony. Currently, the main clinical treatments for cancer include surgery, radiotherapy, and chemotherapy. While these have improved the survival of cancer patients to some extent, their survival remains short and their effectiveness remains limited. In recent years, with the advancement of immunology and life sciences, cancer immunotherapy has become an emerging treatment option, the fourth most important approach after surgery, radiotherapy, and chemotherapy.

[0003] Immunotherapy mainly includes immune checkpoint inhibitors (ICIs), tumor vaccines, chimeric antigen receptor T cell therapy, oncolytic viruses and cytokine therapy. Among them, ICIs are the most important research progress in the field of tumor immunotherapy in the past decade. The most representative immune checkpoint molecules are cytotoxic T-lymphocyte associated antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1). The corresponding anti-CTLA-4 antibodies and anti-PD-1 antibodies have achieved good therapeutic effects in some tumors. However, in most tumors, the response rate of ICIs is still less than 20%, and the response rate in "immune desert" tumors is even less than 10%. Therefore, the efficacy of ICIs alone is limited, and it is necessary to seek treatment options to improve the response rate of ICIs based on the characteristics of the tumor.

[0004] Unlike traditional therapies, ICIs primarily target immune cells rather than tumor cells and rely primarily on the body's pre-existing anti-tumor T cell immune response. However, most "immune desert" tumors lack T cell infiltration, which reduces the tumor's response rate to ICIs. The lack of effector T cell infiltration in "immune desert" tumors is mainly due to two reasons: systemic immunosuppression and local immunosuppression. First, cancer is a systemic disease. As the tumor grows chronically, it often induces abnormal changes in the function and composition of systemic immunity, leading to a decrease in systemic dendritic cells (DCs), a significant increase in granulocyte-monocyte progenitor cells, and a significant decrease in circulating T cells. Second, the tumor microenvironment (TME) is highly immunosuppressive, which can induce dysfunction of antigen-presenting cells (APCs) within the tumor. For example, tumor-associated macrophages (TAMs) and DCs within the TME induce immunosuppression and fail to perform antigen presentation, inhibiting local immune responses and hindering the generation of tumor-specific T cells. Both of these factors contribute to a reduction in the production of specific T cells in "immune desert" tumors. Furthermore, due to the rapid growth of tumors and abnormal vascular function, a large number of newly formed vascular endothelial cells lack functional adhesion factors such as ICAM-1 and VCAM-1, creating a physical barrier to T cell infiltration, preventing circulating T cells from successfully infiltrating the tumor parenchyma. Furthermore, due to immunosuppression in the TME, chemokines that recruit T cells, such as CXCL9, CXCL10, CCL21, and CXCL13, are at low levels, further reducing the tumor's recruitment of peripheral T cells. In summary, for "immune desert" tumors, systemic immunosuppression and local tumor immunosuppression jointly contribute to the lack of T cells within the tumor. Therefore, it is necessary to find new therapeutic drugs to enhance the host's systemic immunity while simultaneously improving the level of local immune response within the tumor, increasing the production of specific T cells in the host, and thereby improving the response rate of "immune desert" tumors to ICIs.

[0005] Cannabidiol (CBD) is a non-hallucinogenic component of cannabis. It was first isolated from cannabis by researchers in 1940, and its chemical structure was subsequently identified in 1963. CBD has shown potential clinical application value in the fields of neuropsychiatric diseases, cardiovascular and tumor diseases. Although many studies have focused on the application of CBD in anti-tumor in recent years, most of the current research focuses on the regulation and killing of tumor cells by CBD, and there are few reports on the role of CBD in regulating the immune system. The present invention uses CBD to enhance systemic immunity. Considering that CBD has poor water solubility and its in vivo bioavailability is only about 10%, the present invention constructs CBD into a phosphorylated prodrug CP. CP can effectively increase the number of T cells in tumor-bearing mice in vivo. When used in combination with the pro-inflammatory cytokine LIGHT, it can effectively increase T cell infiltration in "immune desert-type" tumors and improve the efficacy of ICIs. The new effects of CBD and its prodrug discovered in the present invention are applied to the adjuvant treatment of "immune desert" tumors, which can clinically help improve the T cell immune response in patients and improve the response rate of tumor patients to ICIs, and have great clinical translation value. Summary of the Invention

[0006] The present invention aims to provide a co-delivery system for a phosphorylated prodrug that enhances systemic immunity and a cytokine that improves the local immune microenvironment, thereby enhancing the responsiveness of immune-desert tumors to ICIs. The co-delivery of the phosphorylated prodrug and plasmid using calcium phosphate liposome technology aims to increase effector T cell infiltration in immune-desert tumors. Specifically, the present invention provides a co-delivery liposome that modulates both systemic and local immune responses and a method for its preparation.

[0007] The co-delivery liposomes for regulating systemic and local immune responses provided by the present invention have calcium phosphate as the core and are loaded with two drugs; one of the drugs is a small molecule drug for regulating systemic immune responses, and the other drug is a cytokine for regulating local immune responses of tumors. By regulating the systemic and local immune responses of the tumor host, the T cells infiltrating in immune desert tumors are increased, thereby improving the anti-tumor effect of immunotherapy.

[0008] The small molecule drug for regulating systemic immune response is selected from natural products with immunoenhancing effects, and the cytokines for regulating local immune response of tumors are selected from inflammatory cytokines or T cell chemokines.

[0009] Furthermore, the small molecule drug for regulating systemic immune response is preferably cannabidiol (CBD); the hydroxyl group of cannabidiol is used to phosphorylate it to prepare a phosphorylated prodrug CP, and the complexation property of CP with calcium ions is used to encapsulate CP into the calcium phosphate core.

[0010] Furthermore, the cytokine that regulates local immune response to tumors is preferably the 14th member of the tumor necrosis factor superfamily (TNFSF14), also known as LIGHT protein. A plasmid encoding the LIGHT protein (pLIGHT) is used to encapsulate it in a calcium phosphate core using electrostatic interaction.

[0011] Furthermore, the co-delivery liposome particle size is 20-80 nm, the drug loading amount of the plasmid encoding the LIGHT protein is 0.1-0.5%, the drug loading amount of the phosphorylated prodrug CP is 1-2%, and the mass ratio of the encapsulated plasmid to CP is 1:2-1:20.

[0012] Furthermore, the extracellular segment sequence encoding the LIGHT protein includes three parts: a signal peptide Flt3l sequence, an isoleucine zipper ILZ sequence and a LIGHT extracellular segment sequence.

[0013] Furthermore, the calcium phosphate core adopts the amphiphilic phospholipid 1,2-oleoylphosphatidic acid (DOPA) dispersant, the polar phosphate head of which is complexed with the core at the oil-water interface, and the hydrophobic tail is distributed in the oil phase, forming a hydrophobic layer with stabilizing function on the surface of the core.

[0014] The outer layer of the calcium phosphate core is coated with phospholipids, and the phospholipids are (2,3-dioleoyl-propyl)-trimethylamine (DOTAP), methoxy-terminated polyethylene glycol 2000 - Distearoylphosphatidylethanolamine (mPEG 2000 -DSPE) and targeting peptide modified with polyethylene glycol 2000 - Distearoylphosphatidylethanolamine. Polyethylene glycol increases the long circulation of nanoparticles.

[0015] The co-delivery liposome is modified with a tumor targeting peptide.

[0016] The tumor targeting peptide is selected from apolipoprotein A, apolipoprotein B, apolipoprotein E and their corresponding synthetic polypeptides.

[0017] The tumor targeting peptide is selected from apolipoprotein E polypeptide (ApoE), the polypeptide sequence is CWG-(LRKLRKRLLR)2-NH2, and is bound to maleimide-terminated polyethylene glycol. 2000 - Distearoylphosphatidylethanolamine (Mal-PEG 2000 -DSPE) to obtain ApoE polypeptide modified with polyethylene glycol 2000 - Distearoylphosphatidylethanolamine (ApoE-PEG 2000 -DSPE), which is convenient for modification to the surface of lipid calcium phosphate nanoparticles.

[0018] The present invention also provides a method for preparing the co-delivery liposome, which comprises the following specific steps:

[0019] (1) preparing calcium phosphate liposome cores loaded with cytokines (such as LIGHT protein particles) and small molecule drugs by reverse microemulsion method; specifically, adding an oil-in-water emulsion containing compressed cytokines (such as LIGHT protein particles) to an oil-in-water emulsion containing phosphorylated small molecule drugs, and after sufficient reaction to form a core, centrifugation and washing are performed;

[0020] (2) Calcium phosphate nanomaterials are mixed with liposome materials, and calcium phosphate liposomes are prepared by a thin film dispersion method; specifically, the calcium phosphate core containing cytokines (such as LIGHT protein particles) and phosphorylated small molecule drugs is dispersed with the lipid material in a thin film, and then an asymmetric lipid layer is coated on the calcium phosphate core by a hydration method.

[0021] Furthermore, the specific process of step (1) is as follows:

[0022] Two 20 mL aliquots of the oil phase were stirred at room temperature for 15-30 minutes. To one aliquot, serving as the calcium phase, 200-300 μL of 2.5 M CaCl₂ and 60-600 μg of pLIGHT were added and stirred at room temperature for 15-20 minutes. 100-300 μL of a 12.5 mM CP solution and 100-300 μL of a 12.5 mM Na₂HPO₄ solution were mixed thoroughly and added to the other aliquot of the oil phase, serving as the phosphorus phase, and stirred for 15-20 minutes. The two phases were combined, and 100-300 μL of a chloroform solution of DOPA was added and stirred for 45-90 minutes. After stirring, anhydrous ethanol was added to break the emulsion, and the mixture was centrifuged at 12,500 g for 20 minutes at 4°C. The precipitate was washed twice with anhydrous ethanol and resuspended in chloroform to obtain the calcium phosphate nanoparticle cores, which were stored at -20°C until use.

[0023] The specific process of step (2) is:

[0024] The calcium phosphate nanoparticle core obtained in step (1) is mixed with DOTAP and mPEG 2000 -DSPE, cholesterol, and ApoE-PEG 2000 -DSPE was dispersed in chloroform and formed into a film by rotary evaporation. The film was hydrated with 5% glucose or PBS and ultrasonically dispersed to obtain the co-release liposome ApoE-pLIGHT@CaCP.

[0025] The co-delivery liposomes can be used alone or in combination with T cell-based immunotherapy to treat immune desert tumors.

[0026] The co-release technology used in the present invention is mainly calcium phosphate liposome technology, which prolongs the circulation time of the preparation in the body by incorporating PEGylated lipids into the outer layer, and at the same time increases the transfection efficiency of the plasmid with the help of positively charged DOTAP.

[0027] The present invention uses tumor targeting peptides to increase the aggregation of co-delivered liposomes at the tumor site. Preferably, ApoE polypeptide is used. ApoE polypeptide is covalently linked to Mal-PEG 2000 -DSPE end, which has good tumor targeting ability.

[0028] The present invention adopts the reverse microemulsion method combined with the thin film dispersion method to prepare the co-release liposomes encapsulating two drugs, and mixes the tumor targeting peptide material ApoE-PEG into the lipid material. 2000 -DSPE, as a preferred embodiment, the encapsulation efficiency of LIGHT plasmid is 51.7%, the drug loading is 0.21%, the encapsulation efficiency of CP is 53.4%, the drug loading is 1.45%, and the mass ratio of LIGHT plasmid to CP encapsulated in the liposome is 1:7. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The synthesis route and mass spectrometric characterization of cannabidiol prodrugs. (A) Schematic diagram of the synthesis route of phosphorylated cannabidiol (CP), where CBD stands for cannabidiol, POCl3 stands for phosphorus oxychloride, THF stands for tetrahydrofuran, TEA stands for triethylamine, and rt stands for room temperature; (B) Mass spectrum in positive ion mode; (C) Mass spectrum in negative ion mode.

[0030] Figure 2 CP NMR hydrogen spectrum ( 1 H-NMR).

[0031] Figure 3 CP NMR carbon spectrum ( 13 C-NMR).

[0032] Figure 4 Schematic diagram of the preparation process of co-delivery liposomes.

[0033] Figure 5 The figure shows the formulation characterization of the co-delivery liposome. (A) Schematic diagram of the LIGHT plasmid encoding LIGHT; (B)

[0034] Particle size distribution, polydispersity index (PDI) and surface potential of ApoE-pLIGHT@CaCP, and appearance photo of the preparation; (C) Transmission electron microscopy image of ApoE-pLIGHT@CaCP, scale bar 50 nm; (D) Fluorescence microscopy image of GL261 cells transfected with EGFP-ApoE-pLIGHT@CaCP in vitro, using the commercially available Hieff TransTM Liposomal Transfection Reagent as a positive control, scale bar 100 μm; (E) GL261 cells were transfected with ApoE-pLIGHT@CaCP in vitro, using the commercially available Hieff TransTM Liposomal Transfection Reagent as a positive control, and ELISA was used to detect the secretion of the cytokine LIGHT by the transfected cells. Data are expressed as mean ± SD (n = 5), ns indicates no significant difference; (F) Secretion level of LIGHT protein after ApoE-pLIGHT@CaCP transfection in vivo, data are expressed as mean ± SD (n = 3), **p < 0.01.

[0035] Figure 6 CP is converted to the prodrug cannabidiol. (A) Schematic diagram of the prodrug conversion scheme; (B) HPLC analysis results.

[0036] Figure 7 Validation of ApoE-pLIGHT@CaCP for brain targeting in vitro and in vivo. (A) Schematic diagram of the in vitro Transwell assay; (B) Quantitative in vitro Transwell assay results, data are presented as mean ± SD (n = 3), **p < 0.001; (C) In vitro imaging of healthy C57BL / 6 mouse brains; (D) Semi-quantitative in vitro brain imaging data, data are presented as mean ± SD (n = 3), *p < 0.05; (E) In vivo imaging of C57BL / 6 mice bearing orthotopic gliomas; (F) Semi-quantitative in vivo imaging data of C57BL / 6 mice bearing orthotopic gliomas, data are presented as mean ± SD (n = 3), *p < 0.05, **p < 0.01, ****p < 0.001. (G) In vitro imaging of glioma-bearing brains; (H) Semi-quantitative in vitro imaging data of glioma-bearing brains, data are presented as mean ± SD (n = 3), *p < 0.05.

[0037] Figure 8ApoE-pLIGHT@CaCP promotes antigen-presenting cell activation. (A) Semi-quantitative data of ApoE-pLIGHT@CaCP on the uptake of coumarin 6-labeled nanoparticles by BV2 cells, data are expressed as mean ± SD (n = 6), **p < 0.001; (B) Semi-quantitative data of ApoE-pLIGHT@CaCP on the uptake of coumarin 6-labeled nanoparticles by RAW264.7 cells, data are expressed as mean ± SD (n = 6), **p < 0.001; (C) Semi-quantitative data of ApoE-pLIGHT@CaCP on the uptake of coumarin 6-labeled nanoparticles by BMDC cells, data are expressed as mean ± SD (n = 3), **p < 0.01; (D)

[0038] Effect of ApoE-pLIGHT@CaCP on BMDC cell maturation. Data are expressed as mean ± SD (n = 3). ***p < 0.001. (E) Effect of ApoE-pLIGHT@CaCP on OVA antigen presentation by BMDC cells. Data are expressed as mean ± SD (n = 3). ***p < 0.001.

[0039] Figure 9 The in vivo anti-glioma and immunomodulatory effects of ApoE-CaCP. (A) Schematic diagram and design of administration of different doses of ApoE-CaCP; (B) Tumor bioluminescence images of tumor-bearing mice; (C) Semi-quantitative bioluminescence images, data are expressed as mean ± SD (n = 5), ns, no significant difference; (D) Spleen anatomical photos of mice in each group; (E) Spleen index of tumor-bearing mice in each group, data are expressed as mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001; (F) Number of splenocytes in the spleen of tumor-bearing mice in each group, data are expressed as mean ± SD (n = 3), *p < 0.05, **p < 0.01, ** *p<0.001; (G) Photographs of thymus anatomy of tumor-bearing mice in each group; (H) Thymus index of tumor-bearing mice in each group, data are expressed as mean ± SD (n=5), **p<0.01, ***p<0.001; (I) Thymocyte number in the thymus of tumor-bearing mice in each group, data are expressed as mean ± SD (n=3), *p<0.05, ***p<0.001; (J) T cell number in the thymus of tumor-bearing mice in each group, data are expressed as mean ± SD (n=3), *p<0.05, **p<0.01, ***p<0.001; (K) CD8 + and CD4 + The number of T cells, data are expressed as mean ± SD (n = 3), *p < 0.05, **p < 0.01; (L) CD8 + and CD4+ Initial T cell numbers, data are expressed as mean ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001; (M) CD8 + and CD4 + The number of T cells is expressed as mean ± SD (n = 3), *p < 0.05; (N) CD8 + and CD4 + The number of initial T cells is shown as mean ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

[0040] Figure 10 The effect of ApoE-CaCP on T cell infiltration in glioma. (A) CD8 + The changes in the proportion of T cells are expressed as mean ± SD (n = 3), ns, no significant difference; (B) The CD4 + The changes in T cell ratios are expressed as mean ± SD (n = 3), ns, no significant difference.

[0041] Figure 11 Figure 3. In vivo anti-glioma effect of ApoE-pLIGHT@CaCP. (A) Schematic diagram and design of the administration of each formulation; (B) Tumor bioluminescence images of tumor-bearing mice in each treatment group; (C) Semi-quantitative tumor bioluminescence images, data are expressed as mean ± SD (n = 5), ns, no significant difference; (D) Survival curves of tumor-bearing mice treated with each formulation (n = 6); (E) Body weight curves of tumor-bearing mice treated with each formulation (n = 6).

[0042] Figure 12 ApoE-pLIGHT@CaCP regulates local and systemic immune responses. (A) Immunofluorescence staining of high endothelial venules in tumors, MECA79 + CD31 +Positive areas are high endothelial venules; (B) Ratio of high endothelial venule-positive areas, data are expressed as mean ± SD (n = 6), *p < 0.05, ***p < 0.001; (C) Immunofluorescence staining of B lymphocytes and T lymphocytes in tumors; (D) Semi-quantitative image of T lymphocytes in tumors, data are expressed as mean ± SD (n = 6), *p < 0.05, ***p < 0.001; (E) Flow cytometry analysis of the proportion of various immune cells in the tumor microenvironment , data are expressed as mean ± SD (n = 3), *p < 0.05, **p < 0.01, ***p < 0.001; (F) ELISA analysis of intratumoral lymphocyte chemotactic factors and immunosuppressive cytokines, data are expressed as mean values; (G) Spleen index and thymus index of tumor-bearing mice in each group, data are expressed as mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001; (H) CD8 + and CD4 + The proportion of T cells, data are expressed as mean ± SD (n = 5), *p < 0.05, **p < 0.01, ***p < 0.001; (I) CD8 + and CD4 + T cell ratio and tumor-specific CD8 + T cell ratio, *p<0.05, **p<0.01, ***p<0.001; (J) CD80+CD86+ DC cell ratio in lymph nodes of tumor-bearing mice, and CD8 + and CD4 + T cell proportion, **p<0.01, ***p<0.001.

[0043] Figure 13 ApoE-pLIGHT@CaCP synergizes with αPD-1 to treat orthotopic gliomas. (A) Schematic diagram and design of each formulation; (B) Tumor bioluminescence images of tumor-bearing mice in each treatment group; (C) Semi-quantitative tumor bioluminescence images, data are expressed as mean ± SD (n = 6), ns, no significant difference; (D) Survival curves of tumor-bearing mice treated with each formulation (n = 6); (E) Body weight curves of tumor-bearing mice treated with each formulation (n = 6); (F) HE staining and Ki-67 immunohistochemical staining of tumors; (G) Schematic diagram of secondary tumor cell inoculation challenge; (H) Survival curves of mice after secondary tumor cell inoculation challenge. DETAILED DESCRIPTION

[0044] The present invention will be further described below by way of specific embodiments in conjunction with the accompanying drawings. In the specific embodiments, many details are added to facilitate a better understanding of the present invention. However, those skilled in the art will readily appreciate that some of the features may be omitted in different circumstances, or may be replaced by other elements, materials, or methods.

[0045] Example 1: Synthesis of cannabidiol prodrug.

[0046] Since cannabidiol has poor water solubility and low oral bioavailability, in order to improve the bioavailability and administration of cannabidiol, a cannabidiol prodrug was first synthesized by phosphorylating the hydroxyl group of cannabidiol.

[0047] Synthesis: 600 μM cannabidiol was dissolved in 10 mL of anhydrous tetrahydrofuran (THF), and 500 μL of triethylamine (TEA) was added. 180 μL of phosphorus oxychloride (POCl3) was dispersed in 10 mL of anhydrous THF and then added dropwise to the THF solution of cannabidiol. The reaction was carried out at 0°C for 30 minutes. After completion, the reaction was continued at room temperature under nitrogen for 6 hours. 20 mL of triple-distilled water was added, and the THF was removed by rotary evaporation. 5% NaOH was added to adjust the pH to 10.0, and unreacted cannabidiol was removed by extraction with ethyl acetate. Finally, 6 M concentrated hydrochloric acid was added to the extracted aqueous solution to precipitate phosphorylated cannabidiol (CP). CP was dissolved in anhydrous ethanol, and the excess sodium chloride was removed. The anhydrous ethanol was then removed by rotary evaporation. The purified CP was dissolved in a mixture of water and tert-butyl alcohol and lyophilized to obtain a powdered CP. The product was characterized by mass spectrometry and nuclear magnetic resonance.

[0048] The results show that: Figure 1 As shown in (B) and (C), the [M+H] + and [M+Na] + Peak, [MH] detected in negative ion mode - Peak. Figure 2 The H-MR spectrum shown in the figure is consistent with the chemical shifts of the hydrogen peaks after analysis and CP: δ = 6.91 (s, 2H), 5.11 (s, 1H), 4.45 (s, 1H), 4.39 (s, 1H), 3.87 (d, J = 12.1 Hz, 2H), 2.91-2.82 (m, 1H), 2.48-2.40 (t, 2H), 2.20 (s, 1H), 1.60 (s, 6H), 1.52 (s, 2H), 1.31 (s, 4H), 1.26-1.18 (m, 2H), 0.87 (t, J = 6.9 Hz, 3H). Figure 3The carbon magnetic resonance spectrum shown in the figure shows that the chemical shifts of each carbon peak are consistent with the CP after analysis: δ = 147.91, 140.29, 130.78, 124.24, 122.20, 114.48, 110.50, 45.14, 44.25, 36.05, 34.73, 30.83, 30.20, 29.65, 28.67, 28.59, 23.30, 21.79, 18.55, 13.75, 8.71.

[0049] In summary, this scheme successfully synthesized the prodrug phosphorylated cannabidiol CP.

[0050] Example 2: Preparation and characterization of co-delivery liposomes encapsulating cannabidiol prodrug CP and plasmid pLIGHT.

[0051] After synthesizing the cannabidiol prodrug CP, in order to achieve systemic immune regulation and local immune regulation of brain tumors, we chose intravenous injection as the administration method. At the same time, in order to increase the aggregation of CP in brain tumors, CP was encapsulated in calcium phosphate nanoparticles and the nanoparticles were modified with the brain-targeting peptide ApoE, so as to achieve the purpose of systemic and local immune regulation, and at the same time solve the problem of low bioavailability of cannabidiol.

[0052] Preparation of calcium phosphate liposomes containing CP alone: Two 20 mL portions of oil phase (cyclohexane:CO-520 = 70:30, v / v) were stirred at room temperature for 15 minutes. One portion served as the calcium phase, to which 300 μL of 2.5 M CaCl₂ was added, and the mixture was stirred at room temperature for 20 minutes. 200 μL of 12.5 mM CP solution and 100 μL of 12.5 mM Na₂HPO₄ solution were mixed and added to the other portion of the oil phase, serving as the phosphorus phase, and stirred for 20 minutes. The two phases were mixed, and 300 μL of DOPA chloroform solution was added, and the mixture was stirred for 45 minutes. After stirring, anhydrous ethanol was added to break the emulsion, and the mixture was centrifuged at 12,500 g for 20 minutes at 4°C. The precipitate was washed twice with anhydrous ethanol and finally resuspended in chloroform. The resulting core was mixed with DOTAP and mPEG₂. 2000 -DSPE, cholesterol, and ApoE-PEG 2000 -DSPE was dispersed in chloroform (where the ApoE polypeptide enhances brain targeting), rotary evaporated to form a film, hydrated with 5% glucose, and ultrasonically dispersed to yield calcium phosphate liposomes, ApoE-CaCP. Dynamic light scattering was used to characterize the particle size and potential of the ApoE-CaCP and to measure the CP encapsulation efficiency.

[0053] To prepare calcium phosphate liposomes ApoE-pLIGHT@CaCP co-delivering CP and plasmid pLIGHT, two 20 mL oil phases (cyclohexane:CO-520 = 70:30, v / v) were stirred at room temperature for 15 minutes. In one of the two solutions, 180 μg of pLIGHT was mixed with 300 μL of 2.5 M CaCl₂, followed by 25 μL of 8 mg / mL cyclooctapeptide-encapsulated plasmid, and stirred at room temperature for 20 minutes. 200 μL of 12.5 mM CP solution and 100 μL of 12.5 mM Na₂HPO₄ solution were mixed and added to the other oil phase, serving as the phosphate phase, and stirred for 20 minutes. The remaining steps were repeated as above to prepare the co-delivery liposomes ApoE-pLIGHT@CaCP. The encapsulation efficiency of CP and pDNA was measured.

[0054] In vitro: GL261 cells in the logarithmic growth phase were seeded in confocal microscopy (CFM) culture dishes. When the cells reached 70% confluency, they were treated with Hieff TransTM Liposomal Transfection Reagent and EGFP-ApoE-pLIGHT@CaCP for 6 hours. After washing three times with PBS, the cells were cultured in serum-free medium (Opti-MEM) for an additional 24 hours. The cells were photographed, EGFP expression was observed by confocal microscopy, and LIGHT protein expression was detected by ELISA.

[0055] In vivo: An orthotopic GL261 glioblastoma model was established. 30w GL261 cells were inoculated on day 0. On day 10, ApoE-pLIGHT@CaP or ApoE-pLIGHT@CaCP were injected twice every two days. The CP dose was 10mg / kg, and the pDNA dose was 30μg / mouse. Twenty-four hours after the second injection, serum, heart, liver, spleen, lung, kidney, brain, thymus, and tumor tissues were dissected and isolated. LIGHT protein expression in these tissues and organs was assessed by ELISA.

[0056] The results showed that the preparation process of co-delivery liposomes is shown in the following figure: Figure 4 As shown in Figure 2, the particle size of ApoE-CaCP is 34.3±5.0nm, the polydispersity coefficient is 0.262±0.011, the zeta potential is 15.8±1.7mv, and the CP encapsulation efficiency is 52.08±3.62%. Figure 5 A. The particle size distribution and formulation appearance of ApoE-pLIGHT@CaCP are shown in Figure 5 As shown in B, the particle size is 35.9±1.5nm, the polydispersity coefficient is 0.250±0.021, and the zeta potential is 13.1±2.1mv; transmission electron microscopy is shown in Figure 5As shown in Figure C, round spherical particles were observed. The CP encapsulation efficiency was 53.39±5.46%, and the pDNA encapsulation efficiency was 51.7±4.8%.

[0057] like Figure 5 As shown in D, after transfection of the ApoE-pLIGHT@CaCP group, the fluorescence intensity of EGFP was comparable to that of commercially available transfection preparations. Figure 5 The results of ELISA test in E are consistent. Figure 5 As shown in Figure F, compared with calcium phosphate liposomes encapsulating pLIGHT alone, co-delivery liposomes can significantly enhance its protein expression level in tumors.

[0058] Example 3: Transformation of prodrug-loaded CP on cells in vitro.

[0059] In order to verify that the synthesized CP can be converted into the original drug cannabidiol in cells, ApoE-pLIGHT@CaCP was incubated with cells to detect whether the CP in the cells can be converted into the original drug cannabidiol.

[0060] GL261 and DC2.4 cells were seeded in 6-well plates and allowed to adhere overnight in complete culture medium. The culture medium was then replaced with serum-free culture medium containing ApoE-pLIGHT@CaCP (CP concentration was 50 μg / mL). After incubation for 4 h, the culture medium and cells were collected, the cells were disrupted by ultrasound, and the suspension was lyophilized. The lyophilized product was dissolved in 500 μL of methanol and centrifuged at 14,500 rpm to remove insoluble matter. The supernatant was detected by high-performance liquid chromatography.

[0061] The results show that: Figure 6 As shown, after incubation of ApoE-pLIGHT@CaCP with GL261, DC2.4, RAW264.7, and BV2 cells for 4 hours, cannabidiol was detected in the cells. In summary, CP can be converted into the original drug cannabidiol within cells.

[0062] Example 4: Brain targeting evaluation of ApoE-pLIGHT@CaCP.

[0063] bEnd.3 cells in the logarithmic growth phase were seeded in a 24-well Transwell chamber at a density of 20,000 cells per well. When the transendothelial resistance reached 200 Ω·cm 2DiI-labeled pLIGHT@CaCP and ApoE-pLIGHT@CaCP were placed in Traswell chambers and cultured at 37°C for 4 hours. The fluorescence intensity of DiI was measured and the transendothelial efficiency of the nanoparticles was calculated. To verify the transendothelial effect mediated by ApoE, a competitive assay was performed in which 100 μg / mL of ApoE peptide was pre-incubated for 1 hour before the addition of ApoE-pLIGHT@CaCP.

[0064] Healthy 6-8 week old C57BL / 6 mice were injected with DiR-labeled pLIGHT@CaCP and ApoE-pLIGHT@CaCP via the tail vein. The injection dose of DiR was 1 mg / kg. 24 hours after injection, the brains were dissected and imaged in vitro and semi-quantitatively analyzed.

[0065] An orthotopic GL261 glioblastoma model was established in C57BL / 6 mice. On the 10th day after tumor implantation, DiR-labeled pLIGHT@CaCP and ApoE-pLIGHT@CaCP were injected into the tail vein at a dose of 1 mg / kg DiR. In vivo imaging was performed 1, 2, 4, 8, 12, and 24 hours after injection. After 24 hours of in vivo imaging, the ex vivo brain was dissected for imaging and semi-quantitative analysis.

[0066] The results show that: Figure 7 A Transwell test was performed as shown in Figure 2. Figure 7 As shown in B, under the action of ApoE polypeptide, the ability of calcium phosphate liposomes to cross the BBB can be significantly increased. Figure 7 As shown in C and D, ApoE can also significantly increase the brain targeting of calcium phosphate liposomes in healthy mice. Figure 7 As shown in Figures EH, ApoE peptide significantly enhanced the in vivo targeting of calcium phosphate liposomes to gliomas in vivo using in vivo and ex vivo imaging. In summary, ApoE peptide has good brain targeting and can enhance the targeting of the formulation to gliomas.

[0067] Example 5: Activation effect of ApoE-pLIGHT@CaCP on antigen-presenting cells.

[0068] In vitro experiments demonstrated that ApoE-pLIGHT@CaCP can be successfully converted into the original drug cannabidiol within cells. Therefore, the following study further investigated the activation effect of ApoE-pLIGHT@CaCP on antigen-presenting cells.

[0069] Phagocytic ability assessment: 5000 RAW264.7 and BV2 cells were seeded in 96-well plates respectively and cultured overnight in complete medium. The culture medium was replaced with DMEM or medium containing ApoE-CaCP, ApoE-pLIGHT@CaP and ApoE-pLIGHT@CaCP (CP = 4.0 μg / mL, pLIGHT = 0.58 μg / mL) and cultured for 24 hours. The supernatant was then discarded and coumarin 6-labeled mPEG-PLA nanoparticles diluted in serum-free medium were added and incubated for another 2 hours. After completion, the supernatant was discarded and the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 15 minutes, washed 3 times with PBS, stained with Hoechst for 10 minutes, and washed 3 times with PBS. The high-content drug screening system was used to detect the coumarin 6 fluorescence intensity of each group of cells to evaluate the effect of cannabidiol on the phagocytic ability of various RAW264.7 and BV2 cells. For BMDCs cells, 5×10 5 The cells were seeded at a density of 100 nanoparticles per well in a 12-well plate. After incubation for 24 hours with the above-mentioned preparation, the medium was replaced with serum-free medium containing coumarin-6-labeled mPEG-PLA nanoparticles and incubated for another 2 hours. The cells were washed three times with PBS and stained with anti-mouse eFluor-CD11c antibody. The fluorescence intensity of coumarin-6 was measured by flow cytometry.

[0070] Evaluation of cell activation and antigen presentation: BMDCs were seeded in 12-well plates and stained with DMEM or culture medium containing ApoE-CaCP, ApoE-pLIGHT@CaP, and ApoE-pLIGHT@CaCP (CP = 4.0 μg / mL, pLIGHT = 0.58 μg / mL). After incubation for 24 h, the culture medium was replaced with 100 μg / mL OVA and incubated for 12 h. The cells were collected, washed three times with PBS, and stained with anti-mouse eFluor-CD11c, anti-mouse APC-CD86, and FITC-CD80 to assess the degree of DC cell activation; anti-mouse APC-SIINFEKL-MHC-I and anti-mouse eFluor-CD11c antibodies were added for staining. After washing three times with PBS, antigen presentation was detected by flow cytometry.

[0071] The results showed that the phagocytic ability test results are shown in the attached Figure 8 AC, after incubation with ApoE-CaCP and ApoE-pLIGHT@CaCP, the phagocytic ability of each antigen presenting cell to nanoparticles was significantly enhanced. Figure 8 As shown in D, after incubation of ApoE-CaCP and ApoE-pLIGHT@CaCP, CD80 + CD86 +The positive cells increased significantly, indicating that ApoE-CaCP and ApoE-pLIGHT@CaCP can significantly promote the maturation of DC cells. Figure 8 As shown in E, after incubation with ApoE-CaCP and ApoE-pLIGHT@CaCP, the presentation of OVA antigen by DC cells was significantly increased, indicating that ApoE-pLIGHT@CaCP can enhance the cross-presentation of OVA antigen by DC.

[0072] In summary, phosphorylated cannabidiol can also promote the activation of antigen-presenting cells.

[0073] Example 6(1): In vivo anti-glioma and immunomodulatory effects of ApoE-CaCP.

[0074] A GL261 orthotopic glioblastoma model was established to investigate the antitumor efficacy and immunomodulatory effects of ApoE-CaCP, and a preliminary dose screening was performed. Dosing began on day 6 after orthotopic glioma inoculation and was repeated five times. Tumor progression was monitored using bioluminescence. After dosing, mice were weighed, and peripheral blood, brain tumors, spleens, and thymuses were collected. The spleens and thymuses were weighed, and the spleen and thymus indices were calculated. Flow cytometry was used to analyze changes in immune cells in the peripheral blood and tumors.

[0075] The results show that: Figure 9 As shown in Figures ac, there was no significant anti-tumor effect at each ApoE-CaCP dosage, indicating that the anti-glioma effect of ApoE-CaCP alone was weak. Figure 9 As shown in Figures 9d, 9e, 9g and 9h, glioma caused a significant decrease in the spleen index and thymus index of tumor-bearing mice, while ApoE-CaCP significantly increased the spleen index and thymus index of tumor-bearing mice. Figure 9 As shown in Figures 9f and 9i, the number of cells in the spleen and thymus increased significantly after ApoE-CaCP treatment. Figure 9 As shown in j, ApoE-CaCP significantly increased the number of T cells in the thymus. Analysis of spleen and peripheral blood revealed that Figure 9 As shown in k and 9m, CD8 + and CD4 + The number of T lymphocytes increased significantly. Figure 9 As shown in l and 9m, the initial CD8 + and CD4 + T lymphocytes also increased significantly, which was consistent with the trend of changes in thymic index and intrathymic T cells, suggesting that ApoE-CaCP treatment increased thymic output cells. Figure 10 As shown, ApoE-CaCP has an effect on tumor-infiltrating CD8 + and CD4 +There was no significant effect on T cells.

[0076] In conclusion, ApoE-CaCP can enhance the systemic immunity of glioma-bearing mice, with the 10 mg / kg dose being the most significant, but it has little effect on the CD8 + and CD4 + There was no significant effect on T cells, which may be the reason for its weak anti-tumor efficacy.

[0077] Example 6(2): Anti-glioma effect of ApoE-pLIGHT@CaCP.

[0078] Given that ApoE-CaCP can enhance systemic immunity but has limited effect on increasing the number of local tumor-infiltrating T cells, we will now combine CP with the T cell-recruiting cytokine LIGHT to investigate its anti-tumor efficacy. A GL261 orthotopic glioblastoma model was established to investigate the anti-tumor efficacy of ApoE-pLIGHT@CaCP. The dosing regimen is as shown in the attached figure. Figure 11 Figure A. Bioluminescence was used to monitor tumor progression and evaluate the inhibitory effect of ApoE-pLIGHT@CaCP on glioma. Survival and body weight of tumor-bearing mice were also monitored to further assess the antitumor efficacy.

[0079] The results show that: Figure 11 As shown in Figure BE, after 5 doses, compared with the control group and the single-drug treatment group, ApoE-pLIGHT@CaCP significantly inhibited the growth of in situ glioma, prolonged the survival of tumor-bearing mice, and effectively alleviated the loss of body weight of tumor-bearing mice, indicating that CP combined with cytokine LIGHT can significantly inhibit the growth of in situ glioma.

[0080] Example 7: In vivo immune regulatory effect of ApoE-pLIGHT@CaCP.

[0081] ApoE-pLIGHT@CaCP significantly inhibited the growth of GL261 orthotopic gliomas in vivo. Therefore, we further investigated its effects on local and systemic tumor immunity. Six days after orthotopic GL261 tumor inoculation, and one day after five consecutive dosings, brains were dissected and fixed with 4% paraformaldehyde for 48 hours. Dehydrated and precipitated in 15% and then 30% sucrose solutions, embedded in OCT, and cryosectioned for immunofluorescence staining. Following dosing, brain tumors were isolated, filtered through a 70μm mesh to obtain single-cell suspensions. After three washes with PBS, cells were distributed and stained with antibodies for flow cytometry. Finally, cells were fixed with 4% paraformaldehyde and analyzed by flow cytometry. The mice were weighed, and brain tumors, spleens, thymuses, draining lymph nodes, and peripheral blood were collected. The levels of CCL21, CXCL13, IL-10, and TGF-β cytokines in the tumors were detected by ELISA. The spleen and thymus were weighed, and the spleen index and thymus index were calculated. The CD8 + and CD4 + T lymphocytes, analysis of CD8 in peripheral blood + and CD4 + T lymphocytes and tumor-specific CD8 + T lymphocytes, analysis of CD80 in lymph nodes + CD86 + Positive DC cells and CD8 + and CD4 + T lymphocytes.

[0082] The results show that: Figure 12 As shown in A and B, after ApoE-pLIGHT@CaCP treatment, high endothelial venules (MECA39) were formed in the tumor under the action of LIGHT cytokines, and the infiltrating T cells in the tumor increased significantly ( Figure 12 C and D). The proportion of DCs and activated DCs in tumors increased significantly, and CD8 + T, CD4 + T, tumor-specific CD8 + The proportion of T cells and B lymphocytes increased significantly, while the proportion of immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2 phenotype macrophages decreased significantly ( Figure 12 E). Consistent with the trend of changes in immune cells within the tumor, chemokines CCL21 and CXCL13, which recruit T and B lymphocytes within the tumor, were significantly downregulated, while immunosuppressive cytokines IL-10 and TGF-β were significantly downregulated ( Figure 12 F). In addition, after ApoE-pLIGHT@CaCP treatment, the spleen index and thymus index of tumor-bearing mice increased significantly ( Figure 12 G), CD8 +T and CD4 + The proportion of T lymphocytes increased significantly ( Figure 12 H), the same trend was also observed in lymph nodes and peripheral blood ( Figure 12 I and J), tumor-specific CD8 + The proportion of T cells increased significantly, indicating an enhanced specific immune response to tumors ( Figure 12 I), and the increase in the proportion of activated DC cells in the lymph nodes further verified the enhancement of the immune response.

[0083] In summary, ApoE-pLIGHT@CaCP administration significantly enhanced the local and systemic immune responses to tumors in tumor-bearing mice.

[0084] Example 8: ApoE-pLIGHT@CaCP combined with αPD-1 antibody to treat glioblastoma.

[0085] ApoE-pLIGHT@CaCP significantly enhanced local and systemic immune responses in mice bearing orthotopic gliomas, significantly increasing the number of T cells infiltrating the tumor. Therefore, the efficacy of combining ApoE-pLIGHT@CaCP with ICB was investigated to provide clinical treatment options and practical application value.

[0086] The drug was administered on the 6th day after in situ GL261 tumor implantation. The detailed dosing regimen and time points for each group are shown in the attached Figure 13 As shown in Figure A, the dose of 5% glucose was 10 mL / kg, the dose of TMZ was 20 mg / kg, the dose of CP was 10 mg / kg, the dose of LIGHT plasmid was 1.5 mg / kg, and the dose of αPD-1 antibody was 5 mg / kg. Bioluminescence was used to monitor tumor progression, and the improvement in survival of tumor-bearing mice with the combined therapy strategy was evaluated. Changes in body weight of tumor-bearing mice were also examined. After the final administration, mice were autopsied for HE staining and Ki-67 immunohistochemistry. For long-term surviving mice in the combined therapy group, GL261 cells were re-inoculated into the contralateral brain to assess immune memory. Untreated healthy mice served as a control group, and the survival of the inoculated mice was observed.

[0087] The results show that: Figure 13 As shown in B and C, each drug-treated group significantly inhibited the growth of in situ glioma, among which ApoE-pLIGHT@CaCP+αPD-1 had the strongest inhibitory effect on tumors and significantly prolonged the survival time of tumor-bearing mice ( Figure 13 D) During the administration period, the body weight of the tumor-bearing mice in the ApoE-pLIGHT@CaCP+αPD-1 group did not decrease significantly, and the body weight of the tumor-bearing mice increased steadily after the administration. Figure 13As shown in F, HE staining results showed that the tumor in the 5% glucose group grew vigorously, while the brain tumor growth activity of mice in the ApoE-pLIGHT@CaCP+αPD-1 group was the weakest. Ki-67 staining also showed that the proliferation of tumors in the ApoE-pLIGHT@CaCP+αPD-1 group was the weakest. Figure 13 D shows that 5 mice in the ApoE-pLIGHT@CaCP+αPD-1 group survived for a long time, and the secondary tumor challenge ( Figure 13 G) The experiments showed that these five mice could tolerate a second inoculation of tumors, indicating that the treatment induced the formation of immune memory.

Claims

1. A co-delivery liposome for regulating systemic and local immune responses, characterized in that: The core of the drug is calcium phosphate, which contains two drugs: one is a small molecule drug that regulates the systemic immune response, and the other is a cytokine that regulates the local immune response of the tumor. By regulating the systemic and local immune responses of the tumor host, it increases the infiltration of T cells in immune-desert tumors and improves the anti-tumor effect of immunotherapy. The small molecule drug that regulates the systemic immune response is cannabidiol, and the cytokine that regulates the local immune response of tumors is the 14th member of the tumor necrosis factor superfamily, also known as LIGHT protein.

2. The co-release liposome according to claim 1, wherein The small molecule drug adopts a phosphorylated cannabidiol prodrug; the LIGHT protein adopts a plasmid form, and the plasmid is a sequence that can encode the extracellular segment of the LIGHT protein.

3. The co-release liposome according to claim 2, wherein The particle size is 20~80 nm, the drug loading capacity of the plasmid pLIGHT encoding the LIGHT protein is 0.1~0.5%, the drug loading capacity of the phosphorylated prodrug CP is 1~2%, and the mass ratio of the encapsulated plasmid to CP is 1:2~1:

20.

4. The co-delivery liposome according to claim 2, characterized in that The coding sequence of the LIGHT protein extracellular segment includes three parts: a signal peptide Flt3l sequence, an isoleucine zipper ILZ sequence and a LIGHT extracellular segment sequence.

5. The co-delivery liposome according to claim 2, characterized in that The calcium phosphate core uses the amphiphilic phospholipid 1,2-oleoylphosphatidic acid as a dispersant. The polar phosphate head of the calcium phosphate core is complexed with the core at the oil-water interface, and the hydrophobic tail is distributed in the oil phase, forming a hydrophobic layer with a stabilizing function on the surface of the core. The calcium phosphate core is coated with phospholipids, wherein the phospholipids are (2,3-dioleoyl-propyl)-trimethylamine DOTAP, methoxy-terminated polyethylene glycol 2000 -Distearoylphosphatidylethanolamine mPEG 2000 -DSPE and targeting peptide modified with polyethylene glycol 2000 -Distearoylphosphatidylethanolamine.

6. The co-delivery liposome according to claim 5, characterized in that The co-delivery liposome is modified with a tumor targeting peptide; the tumor targeting peptide is selected from apolipoprotein A, apolipoprotein B, and apolipoprotein E.

7. The co-delivery liposome according to claim 6, characterized in that The tumor targeting peptide is selected from the apolipoprotein E polypeptide ApoE, the polypeptide sequence is CWG-(LRKLRKRLLR)2-NH2, and is capped with maleimide-terminated polyethylene glycol. 2000 - Distearoylphosphatidylethanolamine linked to obtain ApoE polypeptide modified with polyethylene glycol 2000 -Distearoylphosphatidylethanolamine ApoE-PEG 2000 -DSPE, which is convenient for modification to the surface of lipid calcium phosphate liposomes.

8. A method for preparing the co-delivery liposome according to any one of claims 1 to 7, characterized in that: The specific steps are: (1) Calcium phosphate liposome cores loaded with cytokines and small molecule drugs are prepared by the reverse microemulsion method; specifically, an oil-in-water emulsion containing compressed cytokines is added to an oil-in-water emulsion containing phosphorylated small molecule drugs, and after sufficient reaction to form a core, the core is centrifuged and washed; (2) Mixing calcium phosphate nanomaterials with liposome materials and preparing calcium phosphate liposomes using a thin film dispersion method; Specifically, the calcium phosphate core containing cytokines and phosphorylated small molecule drugs is dispersed in a thin film with lipid materials, and then an asymmetric lipid layer is coated on the calcium phosphate core through a hydration method.

9. The preparation method according to claim 8, characterized in that: The specific process of step (1) is as follows: Stir two 20 mL portions of oil phase at room temperature for 15-30 min. Add 200-300 μL of 2.5 MCaCl2 and 60-600 μg of pLIGHT to one portion as the calcium phase and stir at room temperature for 15-20 min. Mix 100-300 μL of 12.5 mM CP solution and 100-300 μL of 12.5 mM Na2HPO4 solution and add to the other portion of oil phase as the phosphorus phase and stir for 15-20 min. The two phases were mixed, and 100-300 μL of DOPA chloroform solution was added and stirred for 45-90 min. After stirring, anhydrous ethanol was added to break the emulsion, and the mixture was centrifuged at 12,500 g for 20 min at 4°C. The precipitate was washed twice with anhydrous ethanol and finally resuspended in chloroform to form the calcium phosphate nanoparticle core. The mixture was stored at -20°C until use. The specific process of step (2) is as follows: The calcium phosphate nanoparticle core obtained in step (1) was mixed with DOTAP and mPEG 2000 -DSPE, cholesterol, and ApoE-PEG 2000 -DSPE was dispersed in chloroform and rotary evaporated to form a film. The film was hydrated with 5% glucose or PBS and ultrasonically dispersed to obtain the co-delivery liposome ApoE-pLIGHT@CaCP.

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