Double-drug liposome co-carrying oxymatrine and STING agonist prodrug as well as preparation method and application of double-drug liposome

Abstract

The invention relates to a double-drug liposome co-carrying oxymatrine and an STING agonist prodrug as well as a preparation method and application of the double-drug liposome. The double-drug liposome comprises a drug carrier and oxymatrine, wherein the drug carrier consists of a membrane material; the oxymatrine is loaded in a hydrophilic inner cavity of the carrier; the membrane material is prepared from distearoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine-polyethylene glycol and a cholesterol-STING agonist prodrug conjugate; the cholesterol-STING agonist prodrug conjugate is a conjugate formed by coupling an STING agonist prodrug with cholesterol through a chemical bond. According to the invention, the traditional Chinese medicine active component oxymatrine and the STING agonist prodrug are creatively integrated into the same liposome, so that synergistic controlled release and mechanism complementation can be realized; wherein the STING prodrug is embedded into the carrier in a chemical bond coupling manner, so that drug leakage caused by physical entrapment is avoided, the acute toxicity of the free STING agonist is remarkably reduced, and meanwhile, the innate immune activation capability of the free STING agonist is enhanced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to a double-drug liposome co-loading matrine and a STING agonist prodrug, a preparation method and application thereof. BACKGROUND

[0002] Tumor immunotherapy is an important direction in the field of anti-tumor, which recognizes and eliminates tumor cells by activating or reconstructing the function of the body's immune system. However, the immune "cold" tumors represented by esophageal cancer and colon cancer have poor response to single immunotherapy due to their immunosuppressive tumor microenvironment and lack of tumor-infiltrating lymphocytes, and new strategies for synergistic treatment that can effectively reverse the immunosuppressive microenvironment are urgently needed. Chemotherapy-induced immunogenic cell death (ICD) provides a possibility for transforming "cold" tumors into "hot" tumors, but this method still often faces problems such as insufficient depth of immune activation and significant side effects.

[0003] Natural products matrine and STING agonists show unique immunomodulatory potential. Matrine has certain anti-tumor and immunomodulatory activity, but has problems such as poor water solubility, fast in vivo metabolism, and low bioavailability, which leads to unstable single-drug efficacy, especially in immune "cold" tumors. On the other hand, although STING agonists can strongly activate innate immunity and induce the production of type I interferons, their free forms usually have high systemic toxicity, poor in vivo stability, and are prone to trigger systemic immune storm, and clinical intratumoral injection is often required, which greatly limits its application range and treatment effect.

[0004] Therefore, how to develop an intelligent delivery system that can achieve efficient co-delivery of the two, improve pharmacokinetics, and realize targeted immune microenvironment regulation is a technical problem to be solved in the field. SUMMARY

[0005] In view of the deficiencies of the prior art, the purpose of the present application is to provide a double-drug liposome co-loading matrine and a STING agonist prodrug, a preparation method and application thereof.

[0006] To achieve the purpose of the present application, the following technical solutions are adopted:

[0007] In a first aspect, the present application provides a double-drug liposome co-loading matrine and a STING agonist prodrug, which comprises a drug carrier composed of a membrane material and matrine loaded in the hydrophilic inner cavity of the carrier; the membrane material comprises distearoyl phosphatidylcholine, distearoyl phosphatidyl ethanolamine-polyethylene glycol and cholesterol-STING agonist prodrug conjugate; the cholesterol-STING agonist prodrug conjugate is a conjugate formed by coupling the STING agonist prodrug and cholesterol through a chemical bond.

[0008] The application creatively integrates the active ingredient of traditional Chinese medicine, oxymatrine, and the STING agonist prodrug in the same liposome to achieve synergistic controlled release and mechanism complementation; wherein the STING prodrug is embedded in the carrier through chemical coupling, which not only avoids drug leakage caused by physical packaging, but also significantly reduces the acute toxicity of free STING agonists, while enhancing the innate immune activation ability.

[0009] Preferably, the STING agonist prodrug comprises any one or a combination of at least two of DMXAA, NVS-STG2, and MSA-2.

[0010] Preferably, the molecular weight of polyethylene glycol in the distearoyl phosphatidyl ethanolamine-polyethylene glycol is 1000-6000 (for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, etc.).

[0011] Preferably, the mass ratio of the distearoyl phosphatidyl choline, the distearoyl phosphatidyl ethanolamine-polyethylene glycol, and the cholesteryl-STING agonist prodrug conjugate is (2-10):1:(1-6), wherein the specific value of 2-10 can be selected from 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and the specific value of 1-6 can be selected from 1, 2, 3, 4, 5, 6, etc.

[0012] Preferably, the mass ratio of the STING agonist prodrug to cholesteryl is 1:(1-5) (for example, 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, etc.).

[0013] Preferably, the mass ratio of oxymatrine to the drug carrier is 1:(2-8) (for example, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, etc.).

[0014] Preferably, the cholesteryl-STING agonist prodrug conjugate is a conjugate formed by esterification of the STING agonist prodrug and cholesteryl.

[0015] In a second aspect, the application provides a preparation method of the double-drug liposome according to the first aspect, which comprises:

[0016] (1) dissolving the cholesteryl-STING agonist prodrug conjugate, distearoyl phosphatidyl choline, and distearoyl phosphatidyl ethanolamine-polyethylene glycol in an organic solvent, removing the organic solvent by rotary evaporation under reduced pressure, and drying;

[0017] (2) Mix with a hydrating agent for hydration, sonicate, heat to phase transition temperature, and repeatedly extrude with a polycarbonate film to obtain a drug carrier;

[0018] (3) Adjust the pH of the aqueous phase outside the drug carrier to 6.8-7.2 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, etc.), mix with oxymatrine, incubate, and obtain dual-drug liposomes.

[0019] Preferably, the organic solvent in step (1) includes any one or a combination of at least two of chloroform, methanol, dichloromethane or ethanol.

[0020] Preferably, the temperature of the reduced pressure rotary evaporation in step (1) is 30-60℃ (e.g., 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, 60℃, etc.), and the rotation speed is 50-100 rpm (e.g., 50 rpm, 55 rpm, 60 rpm, 65 rpm, 70 rpm, 75 rpm, 80 rpm, 85 rpm, 90 rpm, 95 rpm, 100 rpm, etc.).

[0021] Preferably, the hydration agent in step (2) includes any one or a combination of at least two of sodium citrate buffer, phosphate buffer, sodium chloride buffer, and potassium chloride buffer.

[0022] Preferably, the ultrasound time in step (2) is 8-15 min (e.g., 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.), and the temperature is 0-5℃ (e.g., 0℃, 1℃, 2℃, 3℃, 4℃, 5℃, etc.).

[0023] Preferably, the pore size of the polycarbonate membrane in step (2) is 100-600 nm (e.g., 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, etc.).

[0024] Preferably, the number of times the extrusion is repeated in step (2) is 10-60 times (e.g., 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, 55 times, 60 times, etc.).

[0025] Preferably, the reagent used to adjust the pH of the aqueous phase outside the drug carrier in step (3) includes any one or a combination of at least two of NaOH solution, Tris alkali solution, and sodium carbonate solution.

[0026] Preferably, the incubation time in step (3) is 8-20 min (e.g., 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, 20 min, etc.), and the temperature is 40-60℃ (e.g., 40℃, 45℃, 50℃, 55℃, 60℃, etc.).

[0027] All other specific point values ​​not listed above within the numerical ranges mentioned above can be selected and are all within the protection scope of this invention. For the sake of brevity, they will not be described in detail here.

[0028] Thirdly, the present invention provides the use of the dual-drug liposome as described in the first aspect in the preparation of products for tumor immunotherapy.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] Oxymatrine possesses certain anti-tumor and immunomodulatory potential, while STING agonists exhibit strong immune-activating capabilities. This invention creatively integrates the active ingredient oxymatrine from traditional Chinese medicine with a STING agonist prodrug within the same liposome (LipoDO), achieving synergistic controlled release and complementary mechanisms. The STING prodrug is chemically coupled into the carrier, avoiding drug leakage caused by physical encapsulation, significantly reducing the acute toxicity of free STING agonists, and enhancing their innate immune-activating capabilities, resulting in significantly superior anti-tumor effects compared to single-drug or traditional co-administration methods. The liposome platform constructed in this invention exhibits excellent structural scalability and can be widely applied to the combined delivery of natural products and various immunomodulators, providing a new and feasible technical pathway for the precise regulation of complex tumor microenvironments and nanoimmunotherapy. Attached Figure Description

[0031] Figure 1 This is a synthetic route diagram of the cholesterol-DMXAA conjugate in this invention;

[0032] Figure 2 This is the 1H NMR spectrum characterization of the cholesterol-DMXAA conjugate in this invention;

[0033] Figure 3 These are TEM morphology images and structural schematic diagrams of LipoDO in this invention. In the figure, a is the TEM morphology image of the dual-drug liposome, and b is the structural schematic diagram of the dual-drug liposome.

[0034] Figure 4 This is a schematic diagram of the uptake of LipoDO by immune cells in this invention;

[0035] Figure 5This is a biodistribution map of LipoDO in mice in this invention. In the figure, a is the fluorescence signal map of LipoDO in mice, b is the distribution of LipoDO in various tissues, and c is the enrichment analysis of LipoDO in mouse tumor sites.

[0036] Figure 6 The figures show the tumor-suppressing effects of different drug treatment groups in a colon cancer model. Figure a shows the tumor anatomy of each group, figure b shows the tumor growth curve, and figure c shows the weight change of mice in each group. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be considered as specific limitations thereof.

[0038] Preparation Example 1

[0039] This embodiment provides a cholesterol-DMXAA conjugate, prepared by the following method:

[0040] (1) Accurately weigh 154.7 mg of cholesterol, 56.5 mg of DMXAA (a prodrug of the STING agonist), 2.44 mg of 4-dimethylaminopyridine (DMAP), and 45.5 mg of N,N'-dicyclohexylcarbodiimide (DCC). Dissolve the reactants completely in 50 mL of dichloromethane and stir magnetically at 25 °C for 3 h. Remove the organic solvent by rotary evaporation at 40 °C and 100 rpm to obtain a crude reaction mixture. Redissolve the reaction mixture in 10 mL of methanol for later use.

[0041] (2) Pack a silica gel column using a wet method, activate the column with a mobile phase (methanol:dichloromethane = 1:10), and purify the target product by column chromatography. The crude reaction mixture obtained in step (1) is eluted with a methanol-dichloromethane system in the same ratio as the mobile phase.

[0042] During elution, thin-layer chromatography (TLC) was used to monitor the eluting components. Fractional collection began when a new product spot appeared on the TLC and continued until the spot essentially disappeared, thus determining the elution range of the target product. All eluents containing the target product were combined and the solvent was removed by rotary evaporation at 40°C and 100 rpm to obtain a preliminarily purified mixed product.

[0043] (3) The mixed product was redissolved in the mobile phase solvent and separated again by silica gel column chromatography. Each eluted fraction was analyzed by TLC and compared with the spot positions of the raw material and intermediate. The fractions with Rf values ​​different from those of the raw material and presenting a single new spot were collected, combined, and the solvent was evaporated in a water bath at 40°C and 100 rpm to obtain the cholesterol-DMXAA conjugate.

[0044] The synthetic route of cholesterol-DMXAA conjugates is as follows: Figure 1 As shown. After separating the cholesterol-DMXAA conjugate, it was redissolved in deuterated chloroform (CDCl3), and the H-spectrum was measured using a BRUKER 400MHz liquid chromatography-nuclear magnetic resonance spectra. Figure 2 As shown, the analysis of the spectrum and comparison of the chemical shifts of the product, the chemical shifts, intensities, splitting fractions and coupling constants of the peaks, the number of nuclei and their chemical environment are in line with expectations, indicating that the cholesterol-DMXAA conjugate was successfully synthesized.

[0045] Example 1

[0046] This embodiment provides a dual-drug liposome, LipoDO, prepared by the following method:

[0047] (1) Distearylphosphatidylcholine DSPC (66.1 mg), distearylphosphatidylethanolamine-polyethylene glycol 2000 DSPE-PEG2000 (11.7 mg), and cholesterol-DMXAA conjugate (22.2 mg) were fully dissolved in 50 mL of dichloromethane. The organic solvent was evaporated under reduced pressure at 40 °C and 80 rpm in a water bath. The flask was then placed in a freeze dryer overnight to allow it to dry completely.

[0048] (2) Add 3 mL of sodium citrate buffer solution to hydrate the lipid membrane. The suspension is sonicated with a probe for 10 min under ice bath conditions, with a 2 s pause every 2 s of sonication. Place the solution in a 60℃ metal bath to reach the phase transition temperature. Extrude granules using 400 nm, 200 nm, and 100 nm polycarbonate membranes respectively. Extrude each type of membrane at least 11 times.

[0049] (3) Adjust the pH outside the capsule to 7.0 with 1 mM sodium carbonate solution, add 10 mg / mL oxymatrine solution to the alkaline empty liposomes, the drug-liposome mass ratio is 1:2.5, incubate in a metal bath at 50℃ for 10 min, remove the unloaded free drug by ultrafiltration centrifugation, and obtain LipoDO.

[0050] The morphology of the synthesized LipoDO was characterized, and the results are as follows: Figure 3As shown in the figure, a is a transmission electron microscope (TEM) image of LipoDO, showing that LipoDO has uniform particle size and uniform particle size distribution, and is a vesicle structure of single or multilayer phospholipids; b is a schematic diagram of the structure of LipoDO.

[0051] Comparative Example 1

[0052] This comparative example provides an oxidized matrine liposome, LipoOMT, prepared by the following method:

[0053] (1) Distearylphosphatidylcholine DSPC (66.1 mg), distearylphosphatidylethanolamine-polyethylene glycol 2000 DSPE-PEG2000 (11.7 mg), and cholesterol (22.2 mg) were fully dissolved in 50 mL of dichloromethane. The organic solvent was evaporated under reduced pressure at 40 °C and 80 rpm in a water bath. The flask was then placed in a freeze dryer overnight to allow it to dry completely.

[0054] (2) Add 3 mL of sodium citrate buffer solution to hydrate the lipid membrane. The suspension is sonicated with a probe for 10 min under ice bath conditions, with a 2 s pause every 2 s of sonication. Place the solution in a 60℃ metal bath to reach the phase transition temperature. Extrude granules using 400 nm, 200 nm, and 100 nm polycarbonate membranes respectively. Extrude each type of membrane at least 11 times.

[0055] (3) Adjust the pH outside the capsule to 7.0 with 1 mM sodium carbonate solution, add 10 mg / mL oxymatrine solution to the alkaline empty liposomes, the drug-liposome mass ratio is 1:2.5, incubate in a metal bath at 50℃ for 10 min, remove the unloaded free drug by ultrafiltration centrifugation, and obtain LipoOMT.

[0056] Test Example 1

[0057] This test case examines the uptake of LipoDO by primary dendritic cells (BMDC) and primary macrophages (BMDM) derived from mouse bone marrow.

[0058] The BMDCs used in the experiment were extracted using the conventional bone marrow induction method in this field (obtained by inducing and culturing mouse bone marrow cells with GM-CSF, and the cell purity was ≥90% as determined by flow cytometry); BMDMs were extracted using the conventional peritoneal lavage method in this field (collected by centrifugation after intraperitoneal injection of sterile PBS, and the cell viability was verified as ≥95% by trypan blue staining).

[0059] The processing and presentation functions of antigen-presenting cells are key steps in inducing an immune response. This test case examined the uptake capacity of BMDCs for LipoDO. During liposome synthesis, replacing DSPE-PEG2000 with DSPE-PEG2000-Cy5 successfully synthesized Cy5-labeled liposomes LipoDO-Cy5, which were a light blue solution. 5 × 10 5 BMDC and BMDM cells were seeded in 6-well plates. After cell adhesion, the cells were incubated with Cy5-labeled LipoDO at a concentration of 5 μM. After 24 h, the cells were washed twice with PBS, collected, and then analyzed using an Attune NxT flow cytometer.

[0060] The results are as follows Figure 4 As shown in the figure. The results show that both BMDC and BMDM have good uptake capacity for LipoDO-Cy5, indicating that LipoDO can be well uptaken by BMDC and BMDM.

[0061] Test Example 2

[0062] This test case examines the distribution of LipoDO in mice.

[0063] The in vivo processes of liposomes affect their delivery efficacy. When injected into the bloodstream via the tail vein, they first accumulate at the tumor site through osmosis and retention effects, then penetrate into the tumor tissue. After endocytosis, the drug is released intracellularly to exert its therapeutic effect. Polyethylene glycol increases the systemic stability of liposomes, resulting in a long-circulation effect.

[0064] The CT-26 cell line in logarithmic growth phase was divided into groups of 5 × 10⁻⁶ cells. 5 To establish a colon cancer mouse model, 100 μL / 100 μg ... 3 Three mice were set up as parallel control groups. They were injected with LipoDO labeled with Cy5 (Cy5 concentration of 5 μM, injection volume of 200 μL) via tail vein. In vivo imaging of the mice was performed at 3 h, 12 h, and 24 h. After imaging at 24 h, the mice were sacrificed and their major organs and tumor tissues were dissected.

[0065] The results are as follows Figure 5 As shown in the figure, a) is the fluorescence signal of LipoDO in mice, b) is the distribution of LipoDO in various tissues, and c) is the enrichment analysis of LipoDO in mouse tumor sites. Imaging results show that LipoDO has good enrichment in tumor sites, and Cy5 fluorescence signals can still be detected in tumor sites 24 h after administration.

[0066] Test Example 3

[0067] This test case examines the in vivo antitumor effect of LipoDO.

[0068] The CT-26 cell line in logarithmic growth phase was divided into groups of 5 × 10⁻⁶ cells. 5 To establish a colon cancer mouse model, 100 μL / 100 μg ... 3 Mice were randomly divided into 5 groups of 5 mice each: (1) PBS group; (2) DMXAA group (23 mg / kg); (3) DMXAA + OMT mixed group (100 mg / kg); (4) LipoDO group (30 mg / kg); and (5) LipoDO (30 mg / kg) + oxaliplatin (OXP) (5 mg / kg) combination group. Samples from each group were injected into the model mice via the tail vein (in the LipoDO + OXP combination group, LipoDO was injected into the mice via the tail vein, and OXP was injected into the mice via the peritoneum). Tumor size was monitored every two days. The tumor size in the PBS group was increased to 1000 mm². 3 The experiment was terminated when the tumor reached a certain size, and the tumor was dissected and photographed.

[0069] The results are as follows Figure 6 As shown in the figure, a represents the tumor anatomy of each group, and b represents the tumor growth curve. The results show that, compared with the PBS group, the LipoDO group has a better tumor inhibition effect, while the DMXAA group and the DMXAA+OMT mixed group also have certain anti-tumor effects. The combination of LipoDO and oxaliplatin has an excellent tumor inhibition effect, indicating that the combination of the two drugs can effectively regulate the immune microenvironment, improve its responsiveness to immune checkpoint inhibitors, and thus exert a synergistic effect and enhance anti-tumor activity. c represents the weight change of mice in each group, indicating that the samples in each group have no harmful effects on mouse growth and have good biocompatibility.

[0070] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. A dual drug liposome co-loading oxymatrine and a STING agonist prodrug, characterized in that, The double-drug liposome comprises a drug carrier composed of a membrane material and oxymatrine loaded in the hydrophilic inner cavity of the carrier; The membrane material comprises distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol and cholesteryl-STING agonist prodrug conjugate; The cholesteryl-STING agonist prodrug conjugate is a conjugate formed by coupling the STING agonist prodrug and cholesteryl through a chemical bond.

2. The dual drug liposome of claim 1, wherein, The STING agonist prodrug comprises any one of DMXAA, c-di-GMP, cGAMP, ADU-S100 or a combination of at least two thereof.

3. The dual drug liposome according to claim 1 or 2, characterized in that, The molecular weight of polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol is 1000-6000.

4. The dual drug liposome according to any one of claims 1-3, wherein, The mass ratio of the distearoylphosphatidylcholine, distearoylphosphatidylethanolamine-polyethylene glycol and cholesteryl-STING agonist prodrug conjugate is (2-10):1:(1-6); Preferably, the mass ratio of the STING agonist prodrug and cholesteryl is 1:(1-5); Preferably, the mass ratio of the oxymatrine and drug carrier is 1:(2-8).

5. The dual drug liposome according to any one of claims 1-4, wherein, The cholesteryl-STING agonist prodrug conjugate is a conjugate formed by coupling the STING agonist prodrug and cholesteryl through esterification.

6. The method for preparing dual-drug liposomes according to any one of claims 1-5, characterized in that, The preparation method comprises: (1) dissolving the cholesteryl-STING agonist prodrug conjugate, distearoylphosphatidylcholine and distearoylphosphatidylethanolamine-polyethylene glycol in an organic solvent, removing the organic solvent by rotary evaporation under reduced pressure and drying; (2) mixing with a hydrating agent to hydrate, ultrasonic, heating to the phase transition temperature, repeatedly extruding with a polycarbonate membrane to obtain a drug carrier; (3) adjusting the pH of the external aqueous phase of the drug carrier to 6.8-7.2, mixing with oxymatrine, incubating to obtain a double-drug liposome.

7. The method of claim 6, wherein the liposome is a double drug liposome. The organic solvent in step (1) comprises any one of trichloromethane, methanol, dichloromethane or ethanol or a combination of at least two thereof; Preferably, the temperature of the rotary evaporation under reduced pressure in step (1) is 30-60℃ and the rotation speed is 50-100 rpm.

8. The method of claim 6 or 7, wherein the liposome is a double drug liposome. The hydrating agent in step (2) comprises any one of sodium citrate buffer, phosphate buffer, sodium chloride buffer, potassium chloride buffer or a combination of at least two thereof; Preferably, the ultrasonic time in step (2) is 8-15 min and the temperature is 0-5℃; Preferably, the pore size of the polycarbonate membrane in step (2) is 100-600 nm; Preferably, the number of repeated extrusions in step (2) is 10-60 times.

9. The method for preparing dual-drug liposomes according to any one of claims 6-8, characterized in that, The reagent for adjusting the pH of the external aqueous phase of the drug carrier in step (3) comprises any one of NaOH solution, Tris alkali solution, sodium carbonate solution or a combination of at least two thereof; Preferably, the incubation time in step (3) is 8-20 min and the temperature is 40-60℃.

10. Use of the double-drug liposome according to any one of claims 1-5 in the preparation of a product for tumor immunotherapy.

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