Acid-sensitive liposome for co-regulating acidic tumor microenvironment and immune checkpoint as well as preparation method and application of acid-sensitive liposome

Abstract

The invention provides an acid-sensitive liposome for co-regulating an acidic tumor microenvironment and an immune checkpoint as well as a preparation method and application of the acid-sensitive liposome. The acid-sensitive liposome comprises a carrier formed by assembling a membrane material, an MCT inhibitor loaded in a hydrophobic layer of the carrier, and a polyethyleneimine adsorbed PD-1 / PD-L1 inhibitor compound loaded in a hydrophilic inner cavity of the carrier, the membrane material comprises dimethyl dioctadecyl ammonium salt, cholesterol succinic acid monoester and distearoyl phosphatidyl ethanolamine-polyethylene glycol. The liposome provided by the invention has excellent acid sensitivity, and releases an MCT inhibitor and a PD-1 / PD-L1 inhibitor in an acid environment; lactic acid excretion is inhibited, tumor cell acidosis is caused, and the pH of the tumor microenvironment is up-regulated; the interaction of immune checkpoints is blocked, and the T cell mediated anti-tumor immune response is amplified; the immunosuppression microenvironment is synergistically improved, and tumor immune combined therapy is realized.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor pharmaceuticals, and particularly relates to an acid-sensitive liposome that co-regulates an acidic tumor microenvironment and immune checkpoints, and a preparation method and application thereof. Background Art

[0002] Programmed death ligand-1 (PD-L1) is overexpressed on the surface of tumor cells, which can be recognized and bound by programmed death protein-1 (PD-1) on the surface of T cells, transmitting inhibitory signals to T cells, inhibiting T cell activation and proliferation, inducing T cell apoptosis, so that the body cannot produce an effective immune response, ultimately resulting in immune escape. PD-1 / PD-L1 inhibitors can block the binding of T cells to tumor cells, restore T cell activity, induce a normal immune response, and ultimately kill and eliminate tumor cells. However, most PD-1 / PD-L1 inhibitors are macromolecular drugs with poor in vivo stability. After intravenous injection into the blood circulation, some will be destroyed by proteases in the blood, and the amount reaching the target site is small, resulting in a weak induced immune effect.

[0003] Due to hypoxia and abnormal proliferation of tumors, tumor cells obtain energy through a large amount of oxygen-independent glycolysis. This glycolysis causes tumor cells to produce a large amount of lactic acid, as well as excessive protons and carbon dioxide, which together lead to a weakly acidic tumor microenvironment (TME). The acidic environment of TME inhibits T cell activation and recruits immunosuppressive cells (such as tumor-associated macrophages, regulatory cells, and myeloid-derived suppressor cells), thereby reducing the immune response induced by T cells.

[0004] Based on the above two effects, the effective response rate of patients to PD-1 / PD-L1 inhibitors is relatively low. Therefore, there is an urgent need to develop effective delivery technologies to improve the targeting and stability of drugs, and at the same time regulate the acidic tumor microenvironment to achieve an efficient tumor treatment effect. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide an acid-sensitive liposome that co-regulates an acidic tumor microenvironment and immune checkpoints, and a preparation method and application thereof. The liposome provided by the present invention co-encapsulates an MCT inhibitor and a PD-1 / PD-L1 inhibitor, has excellent acid sensitivity, can be stably delivered to the tumor site, release drugs in the acidic tumor microenvironment, and synergistically achieve tumor immune combination therapy.

[0006] To achieve the purpose of this invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides an acid-sensitive liposome that co-regulates the acidic tumor microenvironment and immune checkpoints. The acid-sensitive liposome includes a carrier formed by assembling membrane materials, an MCT inhibitor loaded in the hydrophobic layer of the carrier, and a PD-1 / PD-L1 inhibitor complex adsorbed by polyethyleneimine loaded in the hydrophilic inner cavity of the carrier;

[0008] The membrane materials include dimethyldioctadecylammonium salt, cholesterol succinate monoesters, and distearoylphosphatidylethanolamine-polyethylene glycol.

[0009] In the present invention, dimethyldioctadecylammonium salt, cholesterol succinate monoesters, and distearoylphosphatidylethanolamine-polyethylene glycol form a liposome with a phospholipid bilayer structure. The MCT inhibitor is loaded in the hydrophobic layer of the liposome, and the polyethyleneimine (PEI) adsorbed PD-1 / PD-L1 inhibitor is loaded in the hydrophilic inner cavity of the liposome.

[0010] Among them, cholesterol succinate monoesters (CHEMS) has pH sensitivity and is protonated in an acidic environment, resulting in changes in the liposome membrane structure and releasing the encapsulated drug; dimethyldioctadecylammonium salt carries a positive charge and participates in the formation of the phospholipid bilayer of the liposome; distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG) can extend the circulation time of the liposome in the blood, reduce the chance of being recognized and cleared by the immune system, improve the stability of the liposome, and prevent particle aggregation.

[0011] The liposome provided by the present invention has excellent acid sensitivity and can be stably delivered to the tumor site, releasing the MCT inhibitor and the PD-1 / PD-L1 inhibitor in the acidic tumor microenvironment. The MCT inhibitor can inhibit the efflux of lactic acid, causing lactic acid to accumulate in tumor cells, leading to tumor cell acidosis, and at the same time up-regulating the pH value of the tumor microenvironment; the PD-1 / PD-L1 inhibitor can block the interaction of the immune checkpoint PD-1 / PD-L1, amplifying the T cell-mediated anti-tumor immune response. The two work together to improve the immunosuppressive microenvironment and achieve tumor immunotherapy in combination.

[0012] Preferably, the MCT inhibitor includes any one or a combination of at least two of quercetin, diclofenac, AZD3965, or phloretin.

[0013] Preferably, the PD-1 / PD-L1 inhibitor includes any one or a combination of at least two of pembrolizumab, toripalimab, pembrolizumab, nivolumab, pidilizumab, cemiplimab, spartalizumab, AMP-224, MEDI0680, atezolizumab, avelumab, durvalumab, or MDX-1105.

[0014] The acid-sensitive liposomes provided by the present invention have good stability and high drug-loading capacity for drugs with different physical and chemical properties, solving the problem that it is difficult to formulate drugs due to poor solubility.

[0015] Preferably, the dimethyldioctadecylammonium salt includes dimethyldioctadecylammonium chloride and / or dimethyldioctadecylammonium bromide.

[0016] Preferably, the molecular weight of polyethylene glycol in the distearoyl phosphatidylethanolamine-polyethylene glycol is 1000-6000, and can be, for example, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, etc.

[0017] Other specific point values within the above numerical ranges can be selected and will not be elaborated here one by one.

[0018] In a second aspect, the present invention provides a method for preparing the acid-sensitive liposomes as described in the first aspect, and the preparation method includes:

[0019] (1) Dissolve the dimethyldioctadecylammonium salt, cholesterol succinate monoesters, distearoyl phosphatidylethanolamine-polyethylene glycol, and MCT inhibitor in an organic solvent, mix, and remove the organic solvent by rotary evaporation under reduced pressure;

[0020] (2) Add the PD-1 / PD-L1 inhibitor complex adsorbed by polyethyleneimine, hydrate with phosphate buffer, sonicate, and repeatedly extrude through a polycarbonate membrane to obtain the acid-sensitive liposomes.

[0021] The preparation process of the acid-sensitive liposomes provided by the present invention is simple. By combining the MCT inhibitor and the PD-1 / PD-L1 inhibitor, it jointly regulates the acidic tumor microenvironment, inhibits immune checkpoints, amplifies the anti-tumor immune response, and realizes tumor immune combination therapy.

[0022] Preferably, the molar ratio of the dimethyldioctadecylammonium salt, cholesterol succinate monoesters, and distearoyl phosphatidylethanolamine-polyethylene glycol is (6.5-9):10:(0.025-0.4).

[0023] The specific point values in (6.5-9) can be 6.5, 6.7, 6.9, 7, 7.2, 7.4, 7.5, 7.6, 7.8, 8, 8.2, 8.4, 8.5, 8.6, 8.8, 9, etc.; the specific point values in (0.025-0.4) can be 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, etc.

[0024] Preferably, the molar ratio of the dimethyldioctadecylammonium salt, cholesterol succinate monoesters, and distearoylphosphatidylethanolamine-polyethylene glycol is (7.5-8.5):10:(0.05-0.1).

[0025] Specific point values in (7.5-8.5) can be 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, etc.; specific point values in (0.05-0.1) can be 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, etc.

[0026] Preferably, the mass ratio of the MCT inhibitor to the total mass of the dimethyldioctadecylammonium salt, cholesterol succinate monoesters, and distearoylphosphatidylethanolamine-polyethylene glycol is 1:(10-100).

[0027] Specific point values in (10-100) can be 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, etc.

[0028] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0029] The acid-sensitive liposomes provided by the present invention can regulate the acid sensitivity of liposomes by controlling the dosage of film materials.

[0030] Preferably, the organic solvent includes any one or a combination of at least two of chloroform, methanol, dichloromethane, or ethanol.

[0031] Preferably, the temperature of the rotary evaporation under reduced pressure is 30-60 °C, for example, it can be 30 °C, 32 °C, 34 °C, 35 °C, 36 °C, 38 °C, 40 °C, 42 °C, 44 °C, 45 °C, 46 °C, 48 °C, 50 °C, 52 °C, 54 °C, 55 °C, 56 °C, 58 °C, 60 °C, etc.; the time is 20-80 min, for example, it can be 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, etc.

[0032] Preferably, the mass ratio of the PD-1 / PD-L1 inhibitor to the total mass of the dimethyldioctadecylammonium salt, cholesterol succinate monoesters, and distearoylphosphatidylethanolamine-polyethylene glycol is 1:(10-30).

[0033] Specific point values in (10-30) can be 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, etc.

[0034] Preferably, the pH of the phosphate buffer is 7-9, and can be, for example, 7, 7.2, 7.4, 7.5, 7.6, 7.8, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, etc.

[0035] Preferably, the temperature for hydration is 30-60°C, and can be, for example, 30°C, 32°C, 34°C, 35°C, 36°C, 38°C, 40°C, 42°C, 44°C, 45°C, 46°C, 48°C, 50°C, 52°C, 54°C, 55°C, 56°C, 58°C, 60°C, etc.; the time is 10-30 min, and can be, for example, 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, etc.

[0036] Preferably, the temperature for ultrasonic treatment is 30-50°C, and can be, for example, 30°C, 32°C, 34°C, 36°C, 38°C, 40°C, 42°C, 44°C, 46°C, 48°C, 50°C, etc.; the time is 4-10 min, and can be, for example, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, etc.

[0037] Preferably, the pore size of the polycarbonate membrane is 100-600 nm, and can be, for example, 100 nm, 150 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, 600 nm, etc.

[0038] Preferably, the number of times of repeated extrusion is 10-50 times, and can be, for example, 10 times, 15 times, 20 times, 25 times, 30 times, 35 times, 40 times, 45 times, 50 times, etc.

[0039] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0040] Preferably, the method for preparing the polyethyleneimine-adsorbed PD-1 / PD-L1 inhibitor complex includes: dissolving polyethyleneimine and the PD-1 / PD-L1 inhibitor in a phosphate buffer, mixing, and ultrafiltering to obtain the polyethyleneimine-adsorbed PD-1 / PD-L1 inhibitor complex.

[0041] In the present invention, polyethyleneimine (PEI) is a cationic polymer containing a large number of amino groups, which can adsorb the PD-1 / PD-L1 inhibitor to more stably encapsulate it in the hydrophilic inner cavity of the liposome; meanwhile, it can be used for the antibody after stable release to prevent it from denaturing or degrading in the complex in vivo environment.

[0042] Preferably, the molar ratio of the polyethyleneimine to the PD-1 / PD-L1 inhibitor is (1-10):1.

[0043] The specific point values in (1-10) can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.

[0044] Preferably, the pH of the phosphate buffer solution is 7-9, and can be, for example, 7, 7.2, 7.4, 7.5, 7.6, 7.8, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9, etc.

[0045] Other specific point values within the above numerical ranges can be selected and will not be elaborated one by one here.

[0046] In a third aspect, the present invention provides the use of the acid-sensitive liposome as described in the first aspect in the preparation of a tumor immunotherapy drug.

[0047] Compared with the prior art, the present invention has the following beneficial effects:

[0048] The liposome provided by the present invention has excellent acid sensitivity, can be stably delivered to the tumor site, and releases the MCT inhibitor and the PD-1 / PD-L1 inhibitor in the acidic tumor microenvironment. The MCT inhibitor can inhibit the efflux of lactic acid, causing lactic acid to accumulate in tumor cells, leading to tumor cell acidosis, and at the same time upregulating the pH value of the tumor microenvironment; the PD-1 / PD-L1 inhibitor can block the interaction of the immune checkpoint PD-1 / PD-L1, amplifying the T cell-mediated anti-tumor immune response. The two work together to improve the immunosuppressive microenvironment and achieve tumor immunotherapy combination. Description of the Drawings

[0049] Figure 1 Transmission electron microscope image of the acid-sensitive liposome PEI-Atezo@LP-Que prepared in Example 3 in a phosphate buffer solution at pH 7.4;

[0050] Figure 2 Transmission electron microscope image of the acid-sensitive liposome PEI-Atezo@LP-Que prepared in Example 3 in a phosphate buffer solution at pH 6.5;

[0051] Figure 3 Particle size distribution diagram of the acid-sensitive liposome PEI-Atezo@LP-Que prepared in Example 3;

[0052] Figure 4 Zeta potential distribution diagram of the acid-sensitive liposome PEI-Atezo@LP-Que prepared in Example 3;

[0053] Figure 5 Particle size and PDI distribution diagrams of the acid-sensitive liposomes PEI-Atezo@LP-Que prepared in Example 3 within 7 days in pH 7.4 phosphate buffer;

[0054] Figure 6 Zeta potential distribution diagram of the acid-sensitive liposomes PEI-Atezo@LP-Que prepared in Example 3 within 7 days in pH 7.4 phosphate buffer;

[0055] Figure 7 Cumulative release curve of quercetin of the acid-sensitive liposomes PEI-Atezo@LP-Que prepared in Example 3 within 72 h in pH 7.4 and pH 6.5 phosphate buffers;

[0056] Figure 8 Kill effect diagrams of free drug and drug-loaded acid-sensitive liposomes on A375 tumor cells for 48 h;

[0057] Figure 9 Uptake diagrams of the acid-sensitive liposomes PEI-Atezo@LP-Que prepared in Example 3 on A375 tumor cells for 0.5 h, 4 h, 8 h, and 24 h under different pH conditions;

[0058] Figure 10 Flow cytometry detection diagram of the acid-sensitive liposomes PEI-Atezo@LP-Que prepared in Example 3 on A375 tumor cells for 24 h;

[0059] Figure 11 Flow cytometry detection diagram of apoptosis induction of free drug and drug-loaded acid-sensitive liposomes on A375 tumor cells for 24 h;

[0060] Figure 12 Diagrams of changes in extracellular fluid lactic acid content and pH value of free drug and drug-loaded acid-sensitive liposomes on A375 tumor cells for 12 h. Detailed implementation manners

[0061] To further elaborate on the technical means and effects adopted by the present invention, the following further illustrates the technical solutions of the present invention in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0062] For those not specifying specific techniques or conditions in the examples, they shall be in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product specifications. For reagents or instruments not specifying the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0063] Quercetin and polyethyleneimine (PEI, 10 kDa) used in the following examples were purchased from Shanghai Macklin Biochemical Co., Ltd., Atezolizumab was purchased from Selleck, dimethyldioctadecylammonium chloride (DODAC) and cholesteryl succinate (CHEMS) were purchased from Sigma-Aldrich, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was purchased from AVT Shanghai Pharmaceutical Technology Co., Ltd.

[0064] Effect of Membrane Material Dosage on Acid Sensitivity of Liposomes in Example 1

[0065] (1) Dimethyldioctadecylammonium salt and cholesteryl succinate monoesters

[0066] Dimethyldioctadecylammonium chloride (DODAC) and cholesteryl succinate (CHEMS) with different molar ratios (6.5:10, 7:10, 7.5:10, 8:10, 8.5:10) were dissolved in 10 mL of chloroform. After being fully mixed and dissolved, the organic solvent was removed by rotary evaporation under reduced pressure at 40 °C and 100 rpm for 30 min, and a uniform liposome film was formed on the bottle wall; 3 mL of phosphate buffer (pH = 8) was used for hydration at 45 °C for 15 min and ultrasonic treatment for 5 min; the obtained mixture was successively passed through 400 nm and 200 nm polycarbonate membranes and extruded repeatedly 20 times respectively to obtain drug-free blank liposomes (abbreviated as LP1), and their particle sizes were measured and recorded as Linitial; 3 mL of phosphate buffer with different pH values (5.0, 5.5, 6.0, 6.5, 7.0, 7.4) was added to the above liposomes respectively, and they were incubated in a water bath at 37 °C for 30 min, and their particle sizes were measured again and recorded as L5.0 - L7.4 respectively.

[0067] The test results are shown in Table 1. When the molar ratio of DODAC to CHEMS is 8.5:10, the particle size change of liposomes in phosphate buffer at pH 6.5 is the largest, the liposome structure is inverted, and it has the best acid sensitivity.

[0068] Table 1

[0069]

[0070] (2) Distearoylphosphatidylethanolamine - polyethylene glycol

[0071] To enhance the passive targeting effect of liposomes, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000) was added to the membrane material.

[0072] Dissolve DODAC, CHEMS, and DSPE-PEG2000 with different molar ratios (8.5:10:0.025, 8.5:10:0.05, 8.5:10:0.1, 8.5:10:0.2, 8.5:10:0.4) in 10 mL of chloroform. After fully mixing and dissolving, remove the organic solvent by rotary evaporation under reduced pressure at 40 °C and 100 rpm for 30 min to form a uniform lipid film on the bottle wall. Hydrate with 3 mL of phosphate buffer (pH = 8) at 45 °C for 15 min and sonicate for 5 min. The resulting mixture is successively passed through 400 nm and 200 nm polycarbonate membranes and extruded 20 times repeatedly to obtain drug-free blank liposomes (abbreviated as LP2), and measure their particle size, denoted as Linitial. Add the above liposomes to 3 mL of phosphate buffer with different pH values (5.0, 5.5, 6.0, 6.5, 7.0, 7.4) respectively, incubate in a 37 °C water bath for 30 min, and measure their particle size again, denoted as L5.0 - L7.4 respectively.

[0073] The test results are shown in Table 2. As the content of DSPE-PEG increases, the change in the particle size of liposomes in phosphate buffer at pH 6.5 decreases. DSPE-PEG affects the structural inversion of liposomes and their acid sensitivity. When the molar ratio of DODAC, CHEMS, and DSPE-PEG is 8.5:10:0.05, the liposomes have the optimal targeting effect and appropriate acid sensitivity, which is beneficial for drug delivery.

[0074] Table 2

[0075]

[0076] Example 2 Effects of Polyethyleneimine Adsorption of PD-1 / PD-L1 Inhibitor on the Particle Size, Zeta Potential, and Encapsulation Efficiency of Nanoparticles

[0077] Dissolve polyethyleneimine (PEI, 10 kDa) and atezolizumab (abbreviated as Atezo) with different molar ratios (1:1, 5:1, 10:1) in 3 mL of phosphate buffer (pH = 8). After fully mixing and dissolving, vortex for 1 min, and ultrafiltrate and centrifuge at 4 °C and 3000 xg for 30 min to remove free substances, obtaining polyethyleneimine-adsorbed atezolizumab (abbreviated as PEI-Atezo).

[0078] The test results are shown in Table 3. As the amount of PEI input increases, the particle size, zeta potential, and Atezo encapsulation efficiency of the nanoparticles gradually increase. When the molar ratio of PEI to Atezo is 5:1, the particle size is smaller and the Atezo encapsulation efficiency is the largest.

[0079] Table 3

[0080]

[0081] Example 3

[0082] Dissolve 4.98 mg of DODAC, 4.86 mg of CHEMS and 140 μg of DSPE-PEG2000 (molar ratio 8.5:10:0.05) in 10 mL of chloroform. Add 100 μL of a 1 mg / mL quercetin (abbreviated as Que) solution. After thorough mixing and dissolution, remove the organic solvent by rotary evaporation under reduced pressure at 40 °C and 100 rpm for 30 min to form a uniform liposome film on the bottle wall. Add the PEI-Atezo complex (containing 0.67 mg of Atezo, molar ratio of PEI to Atezo is 5:1), and hydrate at 45 °C for 15 min and sonicate for 5 min with 3 mL of phosphate buffer (pH = 8). The resulting mixture is successively passed through 400 nm and 200 nm polycarbonate membranes and extruded repeatedly 20 times each to obtain acid-sensitive liposomes loaded with PEI-Atezo and Que (abbreviated as PEI-Atezo@LP-Que).

[0083] Example 4

[0084] Dissolve 4.98 mg of DODAC, 4.86 mg of CHEMS and 140 μg of DSPE-PEG2000 (molar ratio 8.5:10:0.05) in 10 mL of methanol. Add 120 μL of a 1 mg / mL diclofenac solution. After thorough mixing and dissolution, remove the organic solvent by rotary evaporation under reduced pressure at 42 °C and 120 rpm for 50 min to form a uniform liposome film on the bottle wall. Add the PEI-nivolumab complex (containing 0.80 mg of nivolumab, molar ratio of PEI to nivolumab is 5:1), and hydrate at 47 °C for 30 min and sonicate for 4 min with 3 mL of phosphate buffer (pH = 7.5). The resulting mixture is successively passed through 400 nm and 200 nm polycarbonate membranes and extruded repeatedly 30 times each to obtain acid-sensitive liposomes loaded with PEI-nivolumab and diclofenac.

[0085] Example 5

[0086] Dissolve 4.98 mg of DODAC, 4.86 mg of CHEMS, and 140 μg of DSPE-PEG2000 (molar ratio 8.5:10:0.05) in 10 mL of dichloromethane. Add 150 μL of a 1 mg / mL solution of AZD3965, and after thorough mixing and dissolution, remove the organic solvent by rotary evaporation under reduced pressure at 50 °C and 80 rpm for 20 min to form a uniform lipid film on the bottle wall. Add the PEI-pembrolizumab complex (containing 0.99 mg of pembrolizumab, with a molar ratio of PEI to pembrolizumab of 5:1), and hydrate at 55 °C for 10 min and sonicate for 8 min with 3 mL of phosphate buffer (pH = 8.5). The resulting mixture is successively passed through 400 nm and 200 nm polycarbonate membranes and extruded 15 times each to obtain acid-sensitive liposomes loaded with PEI-pembrolizumab and AZD3965.

[0087] Test Example 1

[0088] The encapsulation efficiency and drug loading of the acid-sensitive liposomes prepared in Test Examples 3-5 were tested. The test results are shown in Table 4. The MCT inhibitor is embedded in the hydrophobic layer of the liposome through hydrophobic interaction, with an encapsulation efficiency of over 85%. The PD-1 / PD-L1 inhibitor is adsorbed by PEI and then encapsulated in the hydrophilic inner cavity of the liposome, with an encapsulation efficiency of over 80%. The ratio of the two drugs in the liposome meets the range of 1:5 - 1:10, which can meet the subsequent application requirements.

[0089] Table 4

[0090]

[0091] Test Example 2

[0092] (1) Test sample: Acid-sensitive liposomes (PEI-Atezo@LP-Que) prepared in Example 3;

[0093] (2) Test content:

[0094] ① Observe its morphological characteristics using a transmission electron microscope (TEM); the test results are as Figure 1 and Figure 2 shown. The acid-sensitive liposomes are nearly spherical and uniformly distributed in the physiological environment (pH = 7.4) ( Figure 1 ), and a layered structure can be faintly seen at the spherical boundary, which is a characteristic of liposomes. The average particle size is about 200 nm under the electron microscope; the particle size of the acid-sensitive liposomes increases and the morphology becomes irregular with ruptured edge structures in the acidic environment (pH = 6.5) ( Figure 2 ).

[0095] ② The particle size distribution and surface Zeta potential were measured using a dynamic light scattering particle size analyzer (DLS) at a measurement temperature of 25 °C, and each sample was measured 3 times; the test results are as Figure 3 and Figure 4 shown. The hydrated particle size ( Figure 3 ) of the acid-sensitive liposomes was 214.83 ± 3.0 nm, and the polydispersity index PDI was 0.15 ± 0.022, indicating a uniform particle size distribution; its surface potential ( Figure 4 ) was -24.1 ± 0.53 mV, with a negatively charged surface, which is beneficial for improving stability and half-life in blood circulation;

[0096] ③ The particle size and potential changes of the acid-sensitive liposomes in pH 7.4 phosphate buffer were measured continuously for 7 days to investigate their stability; the test results are as Figure 5 and Figure 6 shown. In pH 7.4 phosphate buffer, the particle size and potential of the acid-sensitive liposomes did not change significantly within 7 days, and the polydispersity index PDI did not exceed 0.4, with good dispersibility, indicating high stability within 7 days.

[0097] Test Example 3

[0098] (1) Test samples: The acid-sensitive liposomes (PEI-Atezo@LP-Que) prepared in Example 3, with free quercetin (Que) as the control group;

[0099] (2) Test content: The dynamic membrane dialysis method was used for testing. The test samples were placed in dialysis bags (MWCO, 1000 Da), with 20 mL of phosphate buffer at different pH values (7.4, 6.5) as the external phase, and shaken in a constant temperature water bath at 37 °C with an oscillation speed of 100 rpm; at preset time points of 0.5, 1, 2, 4, 8, 12, 24, 36, 48, 72 h, 2 mL of the external phase was taken out, and the dialysis external phase was replaced with 20 mL of the same release medium to ensure that the dialysis medium was always in a sink state. The experiment was repeated three times; the concentration of Que in the dialysis fluid was measured using an ultraviolet spectrophotometer, and the release curve was plotted;

[0100] (3) Test results: As Figure 7As shown, the release of Que has an obvious pH dependence. At 48 h, the cumulative release rate of Que in the liposome group in pH 6.5 phosphate buffer was about 70%, which was significantly higher than that in pH 7.4 phosphate buffer. This may be due to the encapsulation of the acid-sensitive shell. The normal physiological environment limits the leakage of the drug, indicating that the nanoparticles have a certain stability under normal physiological conditions and there will be no obvious drug release at non-target sites. However, in an acidic environment, the conformation of the acid-sensitive material changes, causing the structure of the liposome to change and release the drug. Moreover, compared with free Que, the release efficiency of Que in the liposome is significantly slowed down, showing a sustained-release effect, which is beneficial to prolonging the circulation half-life of the drug in vivo, reducing the toxicity to normal tissues, and enhancing the anti-tumor effect. The above results indicate that PEI-Atezo@LP-Que has good stability in the normal body fluid environment, can ensure less drug release, achieve effective drug delivery to tumor cells, and can respond to release drugs under acidic conditions, showing acid sensitivity.

[0101] Test Example 4

[0102] (1) Test samples: ① Free atezolizumab (Atezo); ② Free quercetin (Que); ③ PEI-Atezo prepared in Example 2 (molar ratio 5:1); ④ During the preparation of Example 3, only "PEI-Atezo" was replaced with an equal amount of "PEI" and quercetin was not added, and the rest of the steps remained unchanged, obtaining acid-sensitive liposomes loaded with PEI (abbreviated as PEI@LP); ⑤ During the preparation of Example 3, only PEI-Atezo was not added, and the rest of the steps remained unchanged, obtaining acid-sensitive liposomes loaded with quercetin (abbreviated as LP-Que); ⑥ During the preparation of Example 3, only "PEI-Atezo" was replaced with an equal amount of "Atezo", and the rest of the steps remained unchanged, obtaining acid-sensitive liposomes loaded with unmodified atezolizumab and quercetin (abbreviated as Atezo@LP-Que); ⑦ Acid-sensitive liposomes prepared in Example 3 (PEI-Atezo@LP-Que);

[0103] (2) Test content: The test samples were diluted with medium before use. For each group, the corresponding Atezol concentrations were set at 0.5, 1, 2, 4, 8, 16 μg / mL, and the corresponding Que concentrations were set at 0.0625, 0.125, 0.25, 0.5, 1, 2 μg / mL; The A375 cells were resuspended and counted, and according to 5×10 3Cells were inoculated into a 96-well cell culture plate at a density of [number] cells / well. After the cells adhered to the wall, IFN-γ (10 ng / ml) was added for stimulation and culture for 24 h. At the same time, Jurkat cells were pre-activated with PHA at a final concentration of 1 μg / ml and PMA at 50 ng / ml and incubated for 24 h. The activated Jurkat cells were inoculated into IFN-γ-pretreated A375 cells at a target-effector ratio of 1:10, and the corresponding concentrations of the test samples were added according to the experimental groups. At the same time, parallel experiments of the control group were carried out. After co-culture for 48 h, the supernatant was discarded, and the cells were washed repeatedly with sterile PBS 3 times to remove the remaining Jurkat cells. The inhibitory effect on tumor cells was detected by the MTT method. After adding 20 μL of MTT solution, the cells were returned to the incubator for incubation for 4 hours. Then 150 μL of DMSO solution was added, and the mixture was shaken on a shaker for 10 min, and the OD value was measured at a wavelength of 570 nm to calculate the cell survival rate;

[0104] (3) Test results: As Figure 8 shown, the killing effect of Que or Atezo alone on tumor cells was limited, and the inhibitory effect was not obvious. Compared with free Que and Atezo, all liposome formulation groups showed stronger inhibitory effects. Among them, Atezo@LP-Que and PEI-Atezo@LP-Que contained both Que and Atezo and showed strong tumor cell toxicity. On this basis, the adsorption effect of PEI in PEI-Atezo@LP-Que could delay the release of the monoclonal antibody, prolong the action time of the monoclonal antibody in vivo, enhance the killing effect on tumor cells, and further improve the anti-tumor immune effect.

[0105] Test Example 5

[0106] (1) Test samples: In the acid-sensitive liposomes (PEI-Atezo@LP-Que) prepared in Example 3, Nile red was used to replace Que, and Atezo was labeled with FITC;

[0107] (2) Test content: Use sterile forceps to pick up the pre-treated cell slides and place them in a 6-well plate. Gently press the slides to make them close to the bottom of the well. Take A375 cells in the logarithmic growth phase with a fusion state of about 80%, digest them with trypsin, add culture medium and blow them into a uniform single-cell suspension. Count the cells with a cell counting plate. According to a density of 2×10 5 cells / well, spread them in the 6-well plate and shake the culture plate to disperse the cells evenly. Place them in an incubator at 37°C and 5% CO 2Cultured in an incubator at 37°C with 5% CO₂. After the cells adhered to the wall, IFN-γ (10 ng / ml) was added for stimulation and cultured for 24 h. The culture medium was discarded, and 2 mL of test samples with pH 7.4 or 6.5 were added respectively, and the culture was continued for 0.5, 4, 8, and 24 h. After the incubation ended, the culture medium was discarded, and the cells were rinsed 3 times with PBS. 2 mL of 4% paraformaldehyde solution was gently added dropwise to each well of the plate and fixed at room temperature for 20 min. Then the fixing solution was discarded, and the cells were rinsed 3 times with PBS. 0.1 mL of DAPI dye was added to each well for nuclear staining, and the cells were incubated at room temperature in the dark for 5 min. After the staining ended, the dye was aspirated and discarded, and the cells were rinsed 3 times with PBS. After sealing the coverslip with nail polish, the uptake of the drug by A375 cells was observed under a confocal microscope;

[0108] (3) Test results: As Figure 9 shown, the blue in the figure is the nucleus labeled by DAPI, the red is the cell membrane and cytoplasm labeled by Nile red, and the green is the cell membrane labeled by FITC. The Merge figure is the superimposed figure of blue, red, and green fluorescence. The results showed that with the extension of the incubation time, different degrees of red and green could be observed under the confocal microscope, indicating that the uptake of the preparation by A375 cells under pH 7.4 and pH 6.5 conditions gradually increased. And at the same time, the red and green fluorescence was stronger under pH 6.5 conditions, indicating that more PEI-Atezo@LP-Que was taken up under acidic conditions.

[0109] Test Example 6

[0110] (1) Test sample: In the acid-sensitive liposome (PEI-Atezo@LP-Que) prepared in Example 3, Nile red was used to replace Que, and Atezo was labeled with FITC;

[0111] (2) Test content: Take A375 cells in the logarithmic growth phase with a confluence of about 80%, digest them with trypsin, add the culture medium and blow them into a uniform single-cell suspension. Count the cells with a cell counting plate, and seed them in a 6-well plate at a density of 2×10 5 cells / well. Shake the culture plate to disperse the cells evenly, and place them in an incubator at 37°C with 5% CO 2 ₂. After the cells adhered to the wall, IFN-γ (10 ng / ml) was added for stimulation and cultured for 24 h. The culture medium was discarded, and 2 mL of test samples with pH 7.4 and 6.5 were added respectively, and a control group (PBS) was set, and the culture was continued for 24 h. After the incubation ended, the culture medium was discarded, and the cells were rinsed 2 times with PBS. 100 μL of trypsin was added to each well for digestion. After adding an appropriate amount of culture medium to terminate the digestion, the cells were centrifuged at 4°C and 1000 rpm for 5 min. The supernatant was discarded, and the cells were washed 2 times with PBS and then resuspended in 500 μL of PBS and stored on ice. The uptake of the drug by A375 cells was detected by flow cytometry;

[0112] (3) Test results: As Figure 10 shown, the uptake of PEI-Atezo@LP-Que under the condition of pH 6.5 was significantly higher than that under pH 7.4, and the results were statistically significant, which was consistent with the results of the confocal experiment. Analyzing the reasons, the liposome contains an acid-sensitive film material, cholesterol monosuccinate. Under normal conditions, it remains stable and can be partially taken up by cells. Under acidic conditions, it can undergo structural inversion, causing the drugs in the lipid bilayer to leak out and be taken up by cells. At the same time, the inner core PEI-Atezo complex is exposed. In a proton environment, Atezolizumab in the PEI-Atezo complex is released and binds to the PD-L1 receptor on the surface of melanoma cells A375. The results indicate that the PEI-Atezo@LP-Que nanoparticles have good acid responsiveness.

[0113] Test Example 7

[0114] (1) Test samples: ① Free Atezolizumab (Atezo); ② Free quercetin (Que); ③ PEI-Atezo prepared in Example 2 (molar ratio of 5:1); ④ During the preparation of Example 3, only "PEI-Atezo" was replaced with an equal amount of "PEI", and quercetin was not added, and the rest of the steps remained unchanged, resulting in acid-sensitive liposomes loaded with PEI (abbreviated as PEI@LP); ⑤ During the preparation of Example 3, only PEI-Atezo was not added, and the rest of the steps remained unchanged, resulting in acid-sensitive liposomes loaded with quercetin (abbreviated as LP-Que); ⑥ During the preparation of Example 3, only "PEI-Atezo" was replaced with an equal amount of "Atezo", and the rest of the steps remained unchanged, resulting in acid-sensitive liposomes loaded with unmodified Atezolizumab and quercetin (abbreviated as Atezo@LP-Que); ⑦ Acid-sensitive liposomes prepared in Example 3 (PEI-Atezo@LP-Que);

[0115] (2) Test content: Take A375 cells in the logarithmic growth phase with a confluence state of about 80%, digest them with trypsin, add medium and blow them into a uniform single-cell suspension. Count with a cell counting plate, adjust the concentration to 1×10 5 cells / mL, seed 6-well plates at a density of 2×10 5 cells / well, 2 mL per well, and place them in an incubator at 37°C and 5% CO 2 . After the cells adhere, add IFN-γ (10 ng / ml) and stimulate for 24 h; discard the medium, add the test samples respectively. The concentration of Atezo in each group is 16 μg / mL, and the concentration of Que is 2 μg / mL, and a blank group is set; at 37°C and 5% CO 2Cultivate in an incubator for 24 hours, collect the old culture medium, add PBS and wash twice. Add 100 μL of trypsin to each well for digestion, add the old culture medium to terminate digestion, collect the cell suspension in a centrifuge tube, centrifuge at 4 °C and 1000 g for 5 min, resuspend with 1 mL of PBS and count. Take 50,000 - 100,000 cells, centrifuge at 4 °C and 1000 rpm for 5 min, discard the supernatant. According to the kit instructions, add 195 μL of Annexin-FITC binding solution, gently resuspend the cells, then add 5 μL of Annexin-FITC and 10 μL of propidium iodide (PI) staining solution respectively, mix well, place on ice and incubate in the dark for 15 min, and detect with a flow cytometer.

[0116] (3) Test results: As Figure 11 shown, the effect of inducing apoptosis of tumor cells by free drug alone is weak, while Atezo@LP-Que can slowly release the drug for anti-tumor treatment. Atezo and Que alone basically do not induce apoptosis and have weak effects. The apoptosis rates of the PEI-Atezo group (9.31%), LP-Que group (37.17%), PEI@LP group (26.09%), Atezo@LP-Que group (45.71%), and PEI-Atezo@LP-Que group (47.23%) are all obvious. Compared with single PEI-Atezo or LP-Que, PEI-Atezo@LP-Que significantly enhances the apoptosis and necrosis of A375 cells, showing the synergistic effect of Atezo and Que in promoting the apoptosis of A375 cells.

[0117] Test Example 8

[0118] (1) Test samples: ① Free atezolizumab (Atezo); ② Free quercetin (Que); ③ PEI-Atezo prepared in Example 2 (molar ratio 5:1); ④ In the preparation process of Example 3, only replace "PEI-Atezo" with an equal amount of "PEI" and do not add quercetin, and keep the rest of the steps unchanged, to obtain acid-sensitive liposomes loaded with PEI (abbreviated as PEI@LP); ⑤ In the preparation process of Example 3, only do not add PEI-Atezo, and keep the rest of the steps unchanged, to obtain acid-sensitive liposomes loaded with quercetin (abbreviated as LP-Que); ⑥ In the preparation process of Example 3, only replace "PEI-Atezo" with an equal amount of "Atezo", and keep the rest of the steps unchanged, to obtain acid-sensitive liposomes loaded with unmodified atezolizumab and quercetin (abbreviated as Atezo@LP-Que); ⑦ Acid-sensitive liposomes (PEI-Atezo@LP-Que) prepared in Example 3;

[0119] (2) Test content: Take A375 cells in logarithmic growth phase with a fusion state of about 80%, digest them with trypsin, add medium and blow them into a uniform single-cell suspension. Count the cells with a cell counting plate and adjust the concentration to 1×10 5 cells / mL. Seed a 6-well plate at a density of 2×10 5 cells / well, 2 mL per well, and place it in an incubator at 37°C and 5% CO 2 for 24 h; discard the medium, add the test samples respectively, with the Que concentration of 4 μg / mL in each group, and set up a blank group; after culturing for 12 h, take the supernatant, centrifuge at 4°C and 10,000 rpm for 5 min, take the supernatant, and detect the pH value of the extracellular fluid with a pH detector.

[0120] (3) Test results: As Figure 12 shown, compared with the blank control group, the lactic acid content in the medium of the Que group, LP-Que group, Atezo@LP-Que group, and PEI-Atezo@LP-Que group decreased significantly, indicating that Que can effectively inhibit the lactic acid efflux in tumor cells. The lactic acid detection results are consistent with the changes in the pH value of the culture medium. Compared with the blank control group, the pH of the groups containing Que increased, showing a certain degree of relief from weak acidity to neutrality, regulating the acidic tumor microenvironment. The results show that the liposome preparation provided by the present invention can further slow-release the drug, has a stronger effect of inhibiting lactic acid efflux, and regulates the tumor acidic microenvironment.

[0121] The applicant declares that the present invention uses the above embodiments to illustrate an acid-sensitive liposome for co-regulating the acidic tumor microenvironment and immune checkpoint, its preparation method and application, but the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent substitution of each raw material of the products of the present invention, the addition of auxiliary components, and the selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0122] 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 technical concept scope of the present invention, various simple modifications can be made to the technical solutions of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0123] In addition, it should be noted that in the various specific technical features described in the above specific embodiments, they can be combined in any suitable manner without conflict. To avoid unnecessary repetition, the present invention will not separately describe various possible combination methods.

Claims

1. An acid-sensitive liposome for co-regulating acidic tumor microenvironment and immune checkpoints, characterized in that: The acid-sensitive liposome includes a carrier formed by assembling membrane materials, an MCT inhibitor loaded in a hydrophobic layer of the carrier, and a polyethyleneimine-adsorbed PD-1 / PD-L1 inhibitor complex loaded in a hydrophilic inner cavity of the carrier; The membrane material comprises dimethyl dioctadecyl ammonium salt, cholesterol succinate monoester and distearoyl phosphatidylethanolamine-polyethylene glycol.

2. The acid-sensitive liposome according to claim 1, characterized in that The MCT inhibitor includes any one of quercetin, diclofenac, AZD3965 or phloretin or a combination of at least two thereof; Preferably, the PD-1 / PD-L1 inhibitor includes any one of pembrolizumab, toripalizumab, pembrolizumab, nivolumab, pidilizumab, cemiprilimab, spartalizumab, AMP-224, MEDI0680, atezolizumab, avelumab, durvalumab or MDX-1105, or a combination of at least two thereof.

3. The acid-sensitive liposome according to claim 1, characterized in that The dimethyldioctadecylammonium salt includes dimethyldioctadecylammonium chloride and / or dimethyldioctadecylammonium bromide; Preferably, the molecular weight of the polyethylene glycol in the distearoylphosphatidylethanolamine-polyethylene glycol is 1000-6000.

4. The method for preparing the acid-sensitive liposome according to any one of claims 1 to 3, characterized in that: The preparation method comprises: (1) dissolving dimethyl dioctadecyl ammonium salt, cholesterol succinate monoester, distearoyl phosphatidylethanolamine-polyethylene glycol and MCT inhibitor in an organic solvent, mixing, and removing the organic solvent by vacuum rotary evaporation; (2) adding the PD-1 / PD-L1 inhibitor complex adsorbed by polyethyleneimine, hydrating with phosphate buffer, ultrasonicating, and repeatedly extruding through a polycarbonate membrane to obtain the acid-sensitive liposome.

5. The preparation method according to claim 4, characterized in that: The molar ratio of the dimethyl dioctadecyl ammonium salt, cholesterol succinate and distearoyl phosphatidylethanolamine-polyethylene glycol is (6.5-9):10:(0.025-0.4); Preferably, the molar ratio of the dimethyl dioctadecyl ammonium salt, cholesterol succinate and distearoyl phosphatidylethanolamine-polyethylene glycol is (7.5-8.5):10:(0.05-0.1); Preferably, the ratio of the mass of the MCT inhibitor to the total mass of dimethyl dioctadecyl ammonium salt, cholesterol succinate and distearoylphosphatidylethanolamine-polyethylene glycol is 1:(10-100).

6. The preparation method according to claim 4, characterized in that: The organic solvent includes any one of chloroform, methanol, dichloromethane or ethanol, or a combination of at least two thereof; Preferably, the temperature of the vacuum rotary evaporation is 30-60°C and the time is 20-80 min.

7. The preparation method according to claim 4, characterized in that: The ratio of the mass of the PD-1 / PD-L1 inhibitor to the total mass of dimethyl dioctadecyl ammonium salt, cholesterol succinate and distearoylphosphatidylethanolamine-polyethylene glycol is 1:(10-30); Preferably, the pH of the phosphate buffer is 7-9; Preferably, the hydration temperature is 30-60°C and the time is 10-30min; Preferably, the temperature of the ultrasound is 30-50°C and the time is 4-10min; Preferably, the pore size of the polycarbonate membrane is 100-600 nm; Preferably, the number of repeated extrusions is 10-50 times.

8. The preparation method according to claim 4, characterized in that: The preparation method of the polyethyleneimine-adsorbed PD-1 / PD-L1 inhibitor complex comprises: dissolving polyethyleneimine and a PD-1 / PD-L1 inhibitor in a phosphate buffer, mixing, and ultrafiltration to obtain the polyethyleneimine-adsorbed PD-1 / PD-L1 inhibitor complex.

9. The preparation method according to claim 8, characterized in that: The molar ratio of the polyethyleneimine to the PD-1 / PD-L1 inhibitor is (1-10):1; Preferably, the pH of the phosphate buffer is 7-9.

10. Use of the acid-sensitive liposome according to any one of claims 1 to 3 in the preparation of tumor immunotherapy drugs.

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