A double-drug co-loaded liposome and a preparation method and application thereof

Abstract

The present application relates to a kind of double drug co-loading liposome and its preparation method and application, belong to the field of pharmaceutical preparation.DOX is a hydrophilic drug, mainly by pH gradient drug loading method is loaded in the water phase in liposome;R837 is as a kind of hydrophobic immunoadjuvant, mainly by thin film dispersion method is loaded on the phospholipid bilayer of liposome;The double drug co-loading liposome provided in the present application can be gathered in tumor microenvironment (TME) by enhanced permeability and retention (EPR) effect, change M2TAMs in it into M1TAMs, activate adaptive immune response, and improve immunosuppressive TME.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of pharmaceutical preparations, and particularly relates to a double-drug co-loaded liposome as well as a preparation method and application thereof. BACKGROUND

[0002] Cancer immunotherapy is a revolutionary treatment after surgical intervention, chemotherapy and radiotherapy, aiming to fight cancer by activating, restoring or enhancing the patient's systemic anti-tumor immune response. This method greatly improves the survival of many patients and has great potential for wide application. However, in clinical practice, due to insufficient tumor antigenicity and immunosuppressive microenvironment, the effect of immunotherapy on some patients is limited, resulting in considerable variability in treatment response and insufficient effectiveness against metastasis and recurrence. Therefore, it is urgent to develop more effective combination therapy strategies to address these challenges.

[0003] Recent studies have shown that the classic anthracycline broad-spectrum chemotherapy drug doxorubicin (DOX) can induce immunogenic cell death (ICD) by forming damage-associated molecular patterns (DAMPs), which can activate adaptive immune responses, promote the maturation of dendritic cells (DCs), enhance the killing ability of specific effector T cells against tumors, improve the immunogenicity of tumors, and ultimately promote the effectiveness of immunotherapy.

[0004] However, intravenous injection of DOX solution can cause severe systemic toxicity. In order to solve this problem, various studies have focused on formulating DOX into liposomes. Liposomes are nanometer particles mainly composed of phospholipids, which can deliver both hydrophilic and hydrophobic agents, and have good biocompatibility. Preparing liposomal DOX can significantly reduce drug-induced side effects, prolong circulation time, and improve unfavorable biodistribution, thereby exerting stronger antitumor effects and inducing greater tumor antigenicity.

[0005] In addition to insufficient tumor cell antigenicity, another factor limiting the effectiveness of immunotherapy is the presence of an immunosuppressive tumor microenvironment (TME). Among them, tumor-associated macrophages (TAMs) are the most important immunosuppressive cells in the tumor microenvironment, playing a key regulatory role in tumor occurrence and development. Upon exposure to various stimuli, TAMs can polarize into two different types: immune-stimulating M1 phenotype and immune-suppressive M2 phenotype. M1 TAMs can activate anti-tumor immune responses by releasing pro-inflammatory cytokines. In contrast, M2 TAMs promote tumor growth, metastasis and recurrence through various mechanisms.

[0006] Imiquimod (R837) is one of the FDA-approved hydrophobic Toll-like receptor 7 (TLR7) agonists for cancer immunotherapy. It has been proven to have good safety and tolerability. R837 can specifically bind to TLR7, effectively repolarize M2 TAMs that promote tumor growth into anti-tumor M1 TAMs, activate adaptive immune responses, and improve immunosuppressive TME.

[0007] Most of the prior art separately prepares liposomes for immunotherapy drugs and immunoadjuvants, which leads to inconvenient drug use. If the immunotherapy drug and the immunoadjuvant can be co-loaded in one drug, the synergistic effect of the two can be utilized to promote antitumor immunotherapy, which will be of great significance. SUMMARY

[0008] To solve the above-mentioned technical problems, the present application designs DOX / R837 co-loaded liposomes (DOX / R837@CL). The liposomes can deliver immunomodulators to the TME by using the EPR effect. The released R837 can repolarize M2 TAMs into M1 TAMs and regulate the immunosuppressive TME. At the same time, the released DOX can trigger ICD, enhance tumor antigenicity, and synergistically promote antitumor immunotherapy.

[0009] The technical solution adopted by the present application is: a double-drug co-loaded liposome, wherein the double-drug co-loaded liposome is prepared by loading an immunotherapy drug in the water phase of the liposome and loading an immunoadjuvant on the phospholipid bilayer of the liposome.

[0010] Further, the immunotherapy drug is a hydrophilic immunotherapy drug.

[0011] Further, the hydrophilic immunotherapy drug is doxorubicin (DOX).

[0012] Further, the immunoadjuvant is a hydrophobic immunoadjuvant.

[0013] Further, the hydrophobic immunoadjuvant is imiquimod (R837).

[0014] The preparation method of the double-drug co-loaded liposome comprises the following steps:

[0015] 1) Take phospholipid (S100), cholesterol (CH) and immunoadjuvant in a container, dissolve them with chloroform, and then remove the chloroform by rotary evaporation to form a lipid film; then add a 60-70℃ citric acid buffer solution, hydrate for 20-30min; then use a probe ultrasonic instrument to ultrasonically treat the obtained mixture for 8-10min, and then filter the obtained product through a polycarbonate membrane to remove large particles to obtain the liposome;

[0016] 2) mixing the sodium phosphate solution with the liposome obtained in step 1), then adding the hydrochloric acid solution of the immunotherapeutic drug, treating at 60-70 DEG C for 20-30 min, passing through a cation exchange resin, to obtain the double-drug-loaded liposome.

[0017] Further, in step 1), the mass ratio of phospholipid: cholesterol: immunoadjuvant is 9-11: 3-5: 1.

[0018] Further, in step 2), the mass ratio of phospholipid: immunotherapeutic drug is 9-11: 1.

[0019] Further, in step 2), the concentration of the sodium phosphate solution is 0.4-0.6 M.

[0020] The double-drug-loaded liposome provided by the application is used for preparing a cancer immunotherapy drug.

[0021] The application has the following beneficial effects:

[0022] 1) The double-drug-loaded liposome DOX / R837@CL provided by the application is a hydrophilic immunotherapeutic drug, which is mainly loaded in the water phase of the liposome by a pH gradient drug loading method; R837 is a hydrophobic immunoadjuvant, which is mainly loaded on the phospholipid bilayer of the liposome by a film dispersion method. The double-drug-loaded liposome can be gathered in the tumor microenvironment (TME) through the enhanced permeability and retention (EPR) effect, convert M2 TAMs in the double-drug-loaded liposome into M1 TAMs, activate adaptive immune response, and improve the immunosuppressive TME.

[0023] 2) The double-drug-loaded liposome DOX / R837@CL provided by the application can deliver the immunotherapeutic drug and the immunoadjuvant to the immune microenvironment (TME) through the EPR effect, activate adaptive immune response, and improve the immunosuppressive TME. The released R837 can repolarize the M2 TAMs promoting tumor growth into the M1 TAMs resisting tumor, activate adaptive immune response, and improve the immunosuppressive TME. Meanwhile, the released DOX can trigger ICD, enhance tumor antigenicity, and synergistically promote the anti-tumor immunotherapy.

[0024] 3) The application can simultaneously deliver two drugs and improve the drug loading efficiency.

[0025] 4) The method of the application is expected to combine the nanodrug delivery system with immunotherapy, and promote the development of cancer treatment. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is the UV full scan spectrum of DOX-S, DOX / R837-S, DOX@CL and DOX / R837@CL.

[0027] Figure 2 Figure 6 is a transmission electron micrograph of DOX / R837@CL.

[0028] Figure 3 Figure 7 is an in vitro release curve of DOX in DOX / R837@CL.

[0029] Figure 4 Figure 8 is an in vitro release curve of R837 in DOX / R837@CL. DETAILED DESCRIPTION

[0030] Example 1 DOX and R837 dual drug-loaded liposome (DOX / R837@CL)

[0031] (I) Preparation method as follows:

[0032] 1. Preparation of R837@CL

[0033] 1) Take 100 mg S100, 40 mg CH and 10 mg R837 in a round-bottom flask, add 5 mL of chloroform to dissolve, and use a rotary evaporator to remove the solvent chloroform to form a thin lipid film.

[0034] 2) Add 5 mL of preheated citric acid buffer (200 mmol / L) at 65°C, and hydrate for 20 min.

[0035] 3) Use a probe sonicator to ultrasonicate the resulting mixture for 8 min (200 W x 2 min, 400 W x 6 min).

[0036] 4) Filter the product obtained in step 3) through polycarbonate membranes of different pore sizes (0.8, 0.45 and 0.22 μm) in sequence to remove large particles, to obtain liposomes, labeled as R837@CL.

[0037] 2. Preparation of DOX@CL

[0038] 1) Take 100 mg S100, 40 mg CH in a round-bottom flask, add 5 mL of chloroform to dissolve, and use a rotary evaporator to remove the solvent chloroform to form a thin lipid film.

[0039] 2) Add 5 mL of preheated citric acid buffer (200 mmol / L) at 65°C, and hydrate for 20 min.

[0040] 3) Use a probe sonicator to ultrasonicate the resulting mixture for 8 min (200 W x 2 min, 400 W x 6 min).

[0041] 4) The product obtained in step 3) was filtered through polycarbonate membranes (0.8, 0.45 and 0.22 μm) in turn to remove large particles to obtain blank liposomes.

[0042] 5) 1 mL of the blank liposomes was taken, 400 μL of a sodium phosphate solution (0.5 M) was added to make the pH of the outer water phase 7.0 to obtain pH gradient liposomes.

[0043] 6) The pH gradient liposomes were mixed with 500 μL of a DOX-HCl solution (4 mg / mL), and stirred in a water bath at 60°C for 20 min.

[0044] 7) The un-loaded DOX-HCl was removed by using a cation exchange resin to prepare DOX liposomes, which were marked as DOX@CL.

[0045] 3. Preparation of DOX / R837@CL

[0046] 1) 1 mL of R837@CL was taken, 400 μL of a sodium phosphate solution (0.5 M) was added to make the pH of the outer water phase 7.0 to obtain pH gradient liposomes.

[0047] 2) The pH gradient liposomes were mixed with 500 μL of a DOX-HCl solution (4 mg / mL), and stirred in a water bath at 60°C for 20 min.

[0048] 3) The un-loaded DOX-HCl was removed by using a cation exchange resin to prepare DOX and R837 co-loaded liposomes, which were marked as DOX / R837@CL.

[0049] (B) Detection and characterization

[0050] 1. Determination of particle size and Zeta potential

[0051] An appropriate amount of R837@CL and DOX / R837@CL samples were diluted with sterilized water for injection and transferred into a sample cell, and the particle size and polydispersity index of the samples were determined at a wavelength of 632.8 nm by using a Nano-zs 90 dynamic light scattering particle size instrument. Each sample was operated in parallel for 3 times, and the particle size, Zeta potential and PDI value of the sample were obtained, respectively. The results are shown in Table 1.

[0052] Table 1. Characterization of liposomes

[0053]

[0054] The data are expressed as mean ± standard deviation (n = 3)

[0055] As shown in Table 1, the particle size of the prepared DOX / R837@CL was about 100 nm, and the particle size distribution was relatively uniform. The Zeta potential was about -20 mV.

[0056] 2. Determination of detection wavelength of DOX and R837

[0057] A certain amount of DOX-S, DOX / R837-S, DOX@CL and DOX / R837@CL (20.0 μg / mL) was subjected to full wavelength scanning at 200-800 nm, and the results were as shown in Figure 1 .

[0058] It can be seen from the above table that DOX has a strong absorption at 495 nm, and the membrane material and DOX do not interfere with the detection at 245 nm, so the detection wavelength of R837 is determined to be 245 nm. Figure 1

[0059] 3. Determination of encapsulation efficiency (EE%) of DOX and R837

[0060] Two portions of 0.1 mL each of the prepared liposomes R837@CL and DOX / R837@CL were precisely transferred, one portion was transferred to a 10 mL volumetric flask, 1.2 mL of sterile water for injection was added, diluted to the mark with the demulsifier methanol, mixed well, and the total drug absorbance A0 was determined at a wavelength of 495 nm. The other portion was placed at the top of a dextran gel column and centrifuged at 2000 rpm for 4 minutes, then 400 μL of sterile water for injection was added to the top of the column and centrifuged at 2000 rpm for 4 minutes, this operation was repeated for 3 times, the eluate was combined and transferred to a 10 mL volumetric flask, diluted to the mark with the demulsifier methanol, mixed well, and the absorbance values at 245 nm (R837) and 495 nm (DOX) were measured by UV / visible spectrophotometer. The encapsulated drug absorbance A1 was measured, and the encapsulation efficiency EE% of DOX and R837 was calculated according to the following formula, and the results were as shown in Table 1.

[0061] EE% = A1 / A0 x 100%

[0062] As can be seen from Table 1, the encapsulation efficiency of DOX in liposome DOX / R837@CL reached 89.140 ± 3.299%, and the encapsulation efficiency of R837 reached 60.817 ± 2.842%, which can meet the subsequent in vitro and in vivo experiments.

[0063] 4. Transmission electron microscopy (TEM) test

[0064] The morphology and particle size distribution of liposome DOX / R837@CL were observed by transmission electron microscopy. A small amount of diluted sample was added to a 230 mesh copper mesh, stained with 2% phosphotungstic acid solution, the excess liquid was absorbed and naturally air dried, and placed under a transmission electron microscope for photography, and the results were as shown in Figure 2 .

[0065] As can be seen from the above table, the morphology of the liposome is spherical, and the particle size is uniform, which meets the requirements of the subsequent experiments. Figure 2 ​As shown, the prepared liposome DOX / R837@CL all had double-layer spherical vesicular structure, uniform size, and basically consistent with the data measured by the particle size instrument.

[0066] 5. Investigation of in vitro release behavior

[0067] Each 1.0 mL of DOX solution (DOX-S), DOX / R837 mixed solution (DOX / R837-S), DOX ordinary liposome (DOX@CL) and DOX / R837 co-loaded liposome (DOX / R837@CL) was precisely pipetted into a dialysis bag (10 kDa molecular weight cut-off), clamped at both ends and placed in 100 mL PBS release medium, and stirred at 100 rpm at 37℃ in the dark. 1.0 mL of dialysate was collected at 0.5, 1, 2, 4, 6, 12 and 24 h and supplemented with an equal amount of fresh release medium. The dialysate was filtered with a 0.22 μm microporous filter, and the concentrations of DOX and R837 in the filtrate were detected by ultraviolet-visible spectrophotometer, and the cumulative release amount of the drug was calculated. The results are shown in Figure 3 and Figure 4

[0068] As shown in Figure 3 and Figure 4 It can be seen that within 24 h, the cumulative release rates of R837 and DOX in different liposome preparations were less than 40% and 20%, respectively.​

Claims

1. A dual-drug co-loaded liposome, characterized in that, The dual-drug co-loaded liposomes are prepared by encapsulating immunotherapy drugs in the aqueous phase of liposomes and loading immune adjuvants onto the phospholipid bilayer of liposomes. The preparation method of the dual-drug co-loaded liposomes includes the following steps: 1) Take phospholipids, cholesterol and immune adjuvants in a container, dissolve them in chloroform, remove the chloroform by rotary evaporation to form a lipid film; then add citrate buffer at 60-70 ℃ and hydrate for 20-30 min; then use a probe sonicator to sonicate the resulting mixture for 8-10 min, and filter the resulting product through a polycarbonate membrane to remove large particles to obtain liposomes; 2) Mix the sodium phosphate solution with the liposomes obtained in step 1), then add the hydrochloric acid solution of the immunotherapy drug, treat at 60-70 °C for 20-30 min, and then pass through a cation exchange resin to obtain dual-drug co-loaded liposomes; The immunotherapy drug is the hydrophilic immunotherapy drug doxorubicin, and the immune adjuvant is the hydrophobic immune adjuvant imiquimod.

2. The dual-drug co-loaded liposome according to claim 1, characterized in that, In step 1), the ratio of phospholipids:cholesterol:immune adjuvant by mass is 9-11:3-5:

1.

3. The dual-drug co-loaded liposome according to claim 1, characterized in that, In step 2), the ratio of phospholipids to immunotherapy drugs by mass is 9-11:

1.

4. The dual-drug co-loaded liposome according to claim 1, characterized in that, In step 2), the concentration of sodium phosphate solution is 0.4-0.6 M.

5. The use of the dual-drug co-loaded liposomes according to claim 1 in the preparation of antitumor immunotherapy drugs.

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