A liposome drug delivery system targeting neutrophil PD-L1, its preparation method and application

Abstract

The present invention provides a liposome drug delivery system targeting neutrophil PD-L1, its preparation method, and application, belonging to the field of biomedicine. This liposome drug delivery system targeting neutrophil PD-L1 has the following characteristics: a neutrophil-specific antibody or polypeptide, a liposome, and a PD-L1-specific siRNA, wherein the neutrophil-specific antibody or polypeptide is covalently bound to the liposome surface, and the PD-L1-specific siRNA is encapsulated within the liposome. Experimental results have shown that this liposome drug delivery system targeting neutrophil PD-L1 can be delivered to neutrophils, exerting cellular regulatory effects and intervening in sepsis, acute respiratory distress syndrome, and multiple organ dysfunction syndrome.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a liposome drug delivery system targeting neutrophil PD-L1, a preparation method thereof, and applications thereof. Background Art

[0002] Neutrophils are the most numerous white blood cells in the human peripheral blood and serve as the body's first line of defense against invading pathogens. When an infection occurs, neutrophils are mobilized from the bone marrow into the peripheral blood. Neutrophils in the peripheral blood can cross capillary walls through processes such as rolling, adhesion, and migration, entering the site of infection, identifying pathogens, and killing them through phagocytosis and respiratory burst. However, under certain conditions, such as sepsis, acute respiratory distress syndrome, and multiple organ dysfunction syndrome (MODS), neutrophils may experience a degree of dysfunction. For example, in sepsis, neutrophils exhibit dysfunction in migration and phagocytosis, significantly reducing their ability to eliminate pathogens and rendering them ineffective in their bactericidal effects. In these cases, neutrophils are unable to precisely migrate to the site of infection and instead migrate non-directionally to areas such as the lungs, liver, kidneys, and brain, releasing large numbers of extracellular traps that damage these vital organs and ultimately lead to MODS. PD-L1 is a key molecule in this neutrophil dysfunction. Although various anti-PD-L1 antibodies or peptides are currently available for in vivo blocking of PD-L1, they induce various other side effects due to the lack of cell specificity.

[0003] Currently, in the field of tumor treatment, relevant scholars have covalently bound monoclonal antibodies to liposomes encapsulating therapeutic drugs. Monoclonal antibodies can accurately identify molecules such as VEGF / PD1 / PDL1 on the surface of tumor cells (CN 106727331 B), and precisely deliver liposome-encapsulated drugs to tumor cells. However, it is not clear whether similar technology can be used to deliver liposomes encapsulating PD-L1 siRNA to neutrophils to exert cell regulatory effects and intervene in sepsis, acute respiratory distress syndrome, and multiple organ dysfunction syndrome. Summary of the Invention

[0004] The present invention is made to solve the above problems, and its purpose is to provide a liposome drug delivery system targeting neutrophil PD-L1, and its preparation method and application.

[0005] The present invention provides a liposome drug delivery system targeting neutrophil PD-L1, which has the following characteristics: neutrophil-specific antibodies or polypeptides, liposomes, and PD-L1-specific siRNA, wherein the neutrophil-specific antibodies or polypeptides are covalently bound to the surface of the liposomes, and the PD-L1-specific siRNA is encapsulated in the liposomes.

[0006] The liposome drug delivery system targeting neutrophil PD-L1 provided by the present invention may also have the following characteristics: wherein the neutrophil-specific antibody is a monoclonal antibody against human CD15 or a monoclonal antibody against mouse Ly6G, and the neutrophil-specific polypeptide is a polypeptide that specifically recognizes neutrophil surface molecules.

[0007] The liposome drug delivery system targeting neutrophil PD-L1 provided by the present invention may also have the following characteristics: wherein the neutrophil-specific polypeptide is MUB40, and its amino acid sequence is CTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIF.

[0008] The liposome drug delivery system targeting neutrophil PD-L1 provided by the present invention may also have the following characteristics: wherein the liposome is any one or more of soybean lecithin (SPC), cholesterol, (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), and phospholipid-polyethylene glycol 2000.

[0009] The liposome drug delivery system targeting neutrophil PD-L1 provided by the present invention may also have the following characteristics: the nucleotide sequence of the positive chain of the PD-L1 specific siRNA is GAGGUAAUCUGGACAAACATT, and the nucleotide sequence of the antisense chain is UGUUUGUCCAGAUUACCUCTT.

[0010] The liposome drug delivery system targeting neutrophil PD-L1 provided by the present invention may also have the following characteristics: wherein the mass ratio of the neutrophil-specific antibody or polypeptide, the liposome, and the PD-L1-specific siRNA is 3:17:1.

[0011] The present invention also provides a method for preparing a liposome drug delivery system targeting neutrophil PD-L1, which is used to prepare the above-mentioned liposome drug delivery system targeting neutrophil PD-L1, characterized in that it comprises the following steps:

[0012] Step 1, dissolving SPC, DOTAP, cholesterol, and neutrophil-specific antibodies or peptides in an organic solvent to obtain a lipid solution;

[0013] In step 2, the PD-L1-specific siRNA was dissolved in a citric acid buffer solution containing 25% ethanol, and then slowly added to the lipid solution. After mixing, the mixture was incubated for 15-25 minutes. The mixture was treated with ultrasound and a liposome extruder, and then dialyzed using a nanodialysis device to obtain a liposome delivery system targeting neutrophil PD-L1.

[0014] The preparation method of the liposome drug delivery system targeting neutrophil PD-L1 provided by the present invention may also have the following characteristics: wherein, in step 1, if a neutrophil-specific antibody is used, the neutrophil-specific antibody needs to be reacted with the PEGylated lipid DSPE-PEG-2K-NHS under weak alkaline conditions at room temperature for 5-6 hours, and then dialyzed to obtain the DSPE-PEG-2K-antibody, and then the DSPE-PEG-2K-antibody, SPC, DOTAP and cholesterol are dissolved in 3 mL of anhydrous ethanol at a mass ratio of 3:10:5:2 to obtain a lipid solution. In step 1, if a neutrophil-specific polypeptide is used, SPC, DOTAP and cholesterol are first dissolved together in 2 mL of anhydrous ethanol at a mass ratio of 3:10:5:2, and then the neutrophil-specific polypeptide is dissolved in 1 mL of hexafluoroisopropanol, and then mixed to obtain a lipid solution.

[0015] In the preparation method of the liposome drug delivery system targeting neutrophil PD-L1 provided by the present invention, it can also have the following characteristics: wherein, the citric acid buffer is 50 mM citrate, pH 4, the filter membrane used in the liposome extruder is a 100 nm filter membrane, and the nanodialysis device uses a polycarbonate membrane with a pore size of 30 nm.

[0016] The present invention also provides the use of a liposome drug delivery system targeting neutrophil PD-L1 in the treatment of neutrophil PD-L1-related diseases, including sepsis, acute respiratory distress syndrome, and multiple organ dysfunction syndrome.

[0017] Functions and effects of the invention

[0018] According to the present invention, a liposome drug delivery system targeting neutrophil PD-L1, its preparation method and application, the liposome drug delivery system includes a neutrophil-specific antibody or polypeptide, liposomes and PD-L1-specific siRNA, wherein the neutrophil-specific antibody or polypeptide is covalently bound to the surface of the liposome, and the PD-L1-specific siRNA is encapsulated within the liposome. Experiments have found that the liposome drug delivery system can be delivered to neutrophils to exert a cell regulatory effect and intervene in sepsis, acute respiratory distress syndrome and multiple organ dysfunction syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is the UV absorption spectrum of the polypeptide-coupled liposomes encapsulating PD-L1 siRNA in Example 3 of the present invention;

[0020] Figure 2 is the fluorescence spectrum of the polypeptide-coupled liposomes encapsulating PD-L1 siRNA in Example 3 of the present invention;

[0021] Figure 3 is a scanning electron micrograph of the polypeptide-coupled liposomes encapsulating PD-L1 siRNA in Example 3 of the present invention;

[0022] Figure 4 is the UV absorption spectrum of the antibody-coupled liposomes encapsulating PD-L1 siRNA in Example 4 of the present invention;

[0023] Figure 5 is the fluorescence spectrum of the antibody-coupled liposomes encapsulating PD-L1 siRNA in Example 4 of the present invention;

[0024] Figure 6 is a scanning electron micrograph of the antibody-coupled liposomes encapsulating PD-L1 siRNA in Example 4 of the present invention;

[0025] Figure 7 This is the in vitro intervention result of the antibody or polypeptide coupled to the PD-L1 siRNA-encapsulated liposome in Example 5 of the present invention;

[0026] Figure 8 This is the in vitro intervention result of the antibody or polypeptide coupled to the PD-L1 siRNA-encapsulated liposome in Example 5 of the present invention;

[0027] Figure 9 This is the in vitro intervention result of the antibody or polypeptide coupled to the PD-L1 siRNA-encapsulated liposome in Example 5 of the present invention;

[0028] Figure 10 This is the in vitro intervention result of the antibody or polypeptide coupled to the PD-L1 siRNA-encapsulated liposome in Example 5 of the present invention. DETAILED DESCRIPTION

[0029] In order to make the technical means, creative features, objectives and effects achieved by the present invention easier to understand, the following examples and accompanying drawings specifically illustrate the liposome drug delivery system targeting neutrophil PD-L1 of the present invention, its preparation method and application.

[0030] Example 1: Preparation of Peptide-coupled Liposomes Encapsulating PD-L1 siRNA

[0031] Example 1 provides a liposome drug delivery system targeting neutrophil PD-L1 and a preparation method thereof. The preparation method comprises the following steps:

[0032] Step 1: Dissolve soybean phosphatidylcholine (SPC), (2,3-dioleoyl-propyl)-trimethylammonium chloride (DOTAP), and cholesterol in 2 mL of anhydrous ethanol at a ratio of 50%:25%:10% by weight to obtain a first mixed solution with a final lipid concentration of 10 mg / mL. Then, dissolve the neutrophil-specific peptide in 1 mL of hexafluoroisopropanol to obtain a second mixed solution. The first and second mixed solutions are transferred to an eggplant-shaped flask and mixed to obtain a peptide-lipid solution. In this peptide-lipid solution, the mass ratio of neutrophil-specific peptide to lipid is 15%:85%. The neutrophil-specific peptide is MUB40, and its amino acid sequence is Ac-CTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIF-amide, where "Ac-" indicates acetylation and "-amide" indicates amidation.

[0033] In step 2, the PD-L1-specific siRNA was dissolved in 1 mL of citric acid buffer (50 mM citrate, pH 4) containing 25% ethanol and then slowly added to the peptide-lipid solution prepared in step 1. After mixing, the solution was incubated for 20 minutes. The solution was then sonicated and treated with a liposome extruder (100 nm filter membrane). The solution was then dialyzed using a nanodialysis device (polycarbonate membrane, 30 nm pore size) to obtain peptide-conjugated PD-L1 siRNA-encapsulated liposomes, a liposome delivery system targeting neutrophil PD-L1. The nucleotide sequence of the sense strand of the PD-L1-specific siRNA was GAGGUAAUCUGGACAAACATT, and the nucleotide sequence of the antisense strand was UGUUUGUCCAGAUUACCUCTT. The mass ratio of the neutrophil-specific peptide, lipid, and PD-L1-specific siRNA in the resulting peptide-conjugated PD-L1 siRNA-encapsulated liposomes was 3:17:1.

[0034] A certain amount of freeze-drying protective agent is added to the liposome drug delivery system targeting neutrophil PD-L1, and the system is freeze-dried and stored for future use.

[0035] Example 2: Preparation of antibody-coupled liposomes encapsulating PD-L1-siRNA

[0036] Example 2 provides a liposome drug delivery system targeting neutrophil PD-L1 and a preparation method thereof. The preparation method comprises the following steps:

[0037] In step 1, a PEGylated lipid (DSPE-PEG-2K-NHS) and an antibody were reacted under weakly alkaline conditions (pH 8) at room temperature for 6 hours, followed by purification by dialysis to obtain the DSPE-PEG-2K-antibody. The antibody was either a monoclonal antibody against human CD15 or a monoclonal antibody against mouse Ly6G; in this example, the anti-mouse Ly6G monoclonal antibody was used. Next, SPC, DOTAP, DSPE-PEG-2K-antibody, and cholesterol were dissolved in 3 mL of anhydrous ethanol at a ratio of 50%:25%:15%:10% by weight. The solution was transferred to an eggplant flask to obtain an antibody-lipid solution with a final concentration of 10 mg / mL.

[0038] In step 2, the PD-L1-specific siRNA was dissolved in 1 mL of citric acid buffer (50 mM citrate, pH 4) containing 25% ethanol and then slowly added to the antibody-lipid solution prepared in step 1. After mixing, the solution was incubated for 20 minutes. The solution was then sonicated and treated with a liposome extruder (100 nm filter membrane). The solution was then dialyzed using a nanodialysis device (polycarbonate membrane, 30 nm pore size) to obtain antibody-conjugated PD-L1 siRNA-encapsulated liposomes, a liposome delivery system targeting neutrophil PD-L1. The nucleotide sequence of the sense strand of the PD-L1-specific siRNA was GAGGUAAUCUGGACAAACATT, and the nucleotide sequence of the antisense strand was UGUUUGUCCAGAUUACCUCTT. The mass ratio of antibody, lipid, and PD-L1-specific siRNA in the resulting antibody-conjugated PD-L1 siRNA-encapsulated liposomes was 3:17:1.

[0039] A certain amount of freeze-drying protective agent is added to the liposome drug delivery system targeting neutrophil PD-L1, and the system is freeze-dried and stored for future use.

[0040] Example 3: Characterization of Peptide-Conjugated Liposome-Encapsulated PD-L1 siRNA

[0041] The peptide-coupled PD-L1 siRNA-encapsulated liposomes prepared in Example 1 were characterized by measuring particle size, potential, drug loading, encapsulation efficiency, UV absorption, fluorescence spectrum, and microstructure (electron microscopy). The results are as follows:

[0042] (1) Particle size and potential:

[0043] Table 1: Particle size and potential of peptide-coupled PD-L1 siRNA liposomes

[0044]

[0045] (2) Drug loading and encapsulation efficiency

[0046] After testing, the drug loading capacity of the peptide-coupled PD-L1 siRNA liposomes was 4.4%, and the encapsulation efficiency was 89%.

[0047] (3) Ultraviolet absorption

[0048] Figure 1 This is the ultraviolet absorption spectrum of the polypeptide-coupled PD-L1 siRNA-encapsulated liposomes in Example 3 of the present invention.

[0049] (4) Fluorescence spectrum

[0050] Figure 2 This is the fluorescence spectrum of the polypeptide-coupled PD-L1 siRNA-encapsulated liposomes in Example 3 of the present invention.

[0051] (5) Scanning electron microscopy

[0052] Figure 3 This is a scanning electron micrograph of the polypeptide-coupled PD-L1 siRNA-encapsulated liposome in Example 3 of the present invention.

[0053] Example 4: Characterization of Antibody-Conjugated Liposome-Encapsulated PD-L1 siRNA

[0054] The antibody-coupled PD-L1 siRNA-encapsulated liposomes prepared in Example 2 were characterized by measuring particle size, potential, drug loading, encapsulation efficiency, UV absorption, fluorescence spectrum, and microstructure (electron microscopy). The specific results are as follows:

[0055] (1) Particle size and potential:

[0056] Table 2: Particle size and potential of antibody-coupled PD-L1 siRNA-encapsulated liposomes

[0057]

[0058] (2) Drug loading and encapsulation efficiency

[0059] After testing, the drug loading capacity of antibody-coupled PD-L1 siRNA liposomes was 4.3%, and the encapsulation efficiency was 87%.

[0060] (3) Ultraviolet absorption

[0061] Figure 4 This is the UV absorption spectrum of the antibody-coupled PD-L1 siRNA-encapsulated liposomes in Example 4 of the present invention.

[0062] (4) Fluorescence spectrum

[0063] Figure 5 This is the fluorescence spectrum of the antibody-coupled PD-L1 siRNA-encapsulated liposomes in Example 4 of the present invention.

[0064] (5) Scanning electron microscopy

[0065] Figure 6 This is a scanning electron micrograph of the antibody-coupled PD-L1 siRNA-encapsulated liposomes in Example 4 of the present invention.

[0066] Example 5: Therapeutic effect of antibody or peptide-coupled PD-L1 siRNA liposomes for sepsis

[0067] Animals: Male C57BL / 6J mice, 8-10 weeks old

[0068] Model: Cecal ligation and puncture (CLP) sepsis mouse model. Under sevoflurane anesthesia, the skin, subcutaneous tissue, and peritoneum were incised along the midline of the mouse abdomen to expose the cecum. The distal 60% of the cecum was ligated with silk suture. The cecum was pierced with a 21G suture, and a small amount of intestinal contents was squeezed out before the cecum was reinserted. The peritoneum and skin were sutured layer by layer, and tramadol was administered for analgesia.

[0069] Grouping: The mice were divided into sham operation group, CLP group, si-NC-LNP, si-PD-L1-LNP, si-NC-polypeptide-LNP, si-PD-L1-polypeptide-LNP (i.e., the polypeptide-coupled PD-L1 siRNA-encapsulated liposomes prepared in Example 1), si-NC-anti-Ly6G-LNP, and si-PD-L1-anti-Ly6G-LNP (i.e., the antibody-coupled PD-L1 siRNA-encapsulated liposomes prepared in Example 2). Each group was treated with PBS or the corresponding liposomes via the tail vein.

[0070] result:

[0071] 1. In vitro intervention efficiency

[0072] Mouse bone marrow neutrophils were isolated, stimulated with LPS and IFN-γ, and given corresponding liposomes for intervention. PD-L1 expression was detected 21 hours later.

[0073] Figure 7 This is the in vitro intervention result of the antibody or polypeptide coupled PD-L1 siRNA-encapsulated liposome in Example 5 of the present invention.

[0074] like Figure 7 As shown in the figure, the results showed that PD-L1 expression was significantly increased after LPS / IFN-γ stimulation, while PD-L1 expression was significantly decreased after intervention with peptide-coupled PD-L1 siRNA liposomes and antibody-coupled PD-L1 siRNA liposomes.

[0075] 2. In vivo intervention efficiency

[0076] The corresponding PBS or liposome drugs were injected into the tail vein of CLP mice, and blood was collected by cardiac puncture 24 hours later to detect the expression level of PD-L1.

[0077] Figure 8 This is the in vitro intervention result of the antibody or polypeptide coupled PD-L1 siRNA-encapsulated liposome in Example 5 of the present invention.

[0078] like Figure 8 As shown, the results showed that the expression of PD-L1 in neutrophils and monocytes of CLP mice was significantly increased, while the expression of PD-L1 in neutrophils was significantly decreased after treatment with peptide- or antibody-coupled liposomes, while the expression of PD-L1 in monocytes was not affected, suggesting that peptide- or antibody-coupled PD-L1 siRNA liposomes can specifically knock down the expression of PD-L1 in neutrophils.

[0079] 3. Therapeutic effects on sepsis survival rate, systemic inflammation and immune cell apoptosis

[0080] Figure 9 This is the in vitro intervention result of the antibody or polypeptide coupled PD-L1 siRNA-encapsulated liposome in Example 5 of the present invention.

[0081] like Figure 9 As shown in the results, after liposome drug treatment, the 7-day survival rate of septic mice was significantly improved, the levels of peripheral blood inflammatory factors IL-1β, IL-6 and TNF-α were significantly reduced, and the apoptosis of spleen and thymocytes was significantly alleviated, suggesting that peptide- or antibody-coupled PD-L1 siRNA liposomes can improve the survival rate of septic mice and reduce systemic inflammation and immune cell apoptosis.

[0082] 4. Effects on neutrophil distribution in septic mice

[0083] Figure 10 This is the in vitro intervention result of the antibody or polypeptide coupled PD-L1 siRNA-encapsulated liposome in Example 5 of the present invention.

[0084] like Figure 10 As shown in the results, after liposome drug treatment, the number of neutrophils in the peritoneal cavity of septic mice increased significantly, while the number of neutrophils in organs such as the heart, lungs, liver, and kidneys decreased significantly, suggesting that peptide- or antibody-coupled PD-L1 siRNA liposomes can correct the dysfunction of neutrophil migration and cause them to accumulate at the site of infection rather than non-specific organs such as the heart, lungs, liver, and kidneys.

[0085] Those skilled in the art will appreciate that the present invention is not limited to the foregoing embodiments. The foregoing embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A liposome drug delivery system targeting neutrophil PD-L1, characterized in that: include: Neutrophil-specific antibodies or peptides, liposomes, and PD-L1-specific siRNA, Among them, neutrophil-specific antibodies or peptides are covalently bound to the surface of liposomes, and PD-L1-specific siRNA is encapsulated in the liposomes. The neutrophil-specific antibody is a monoclonal antibody against human CD15 or a monoclonal antibody against mouse Ly6G. The neutrophil-specific polypeptide is a polypeptide that specifically recognizes neutrophil surface molecules. The neutrophil-specific polypeptide is MUB40, and its amino acid sequence is CTAEGIKKFEGDGYELFKDNFPAGEKFDNDDTNDQFYTVIF. The nucleotide sequence of the sense strand of the PD-L1 specific siRNA is GAGGUAAUCUGGACAAACATT, and the nucleotide sequence of the antisense strand is UGUUUGUCCAGAUUACCUCTT.

2. The liposome drug delivery system targeting neutrophil PD-L1 according to claim 1, characterized in that: in, The liposomes are any one or more of soybean lecithin SPC, cholesterol, (2,3-dioleoyl-propyl)-trimethylammonium chloride DOTAP, and phospholipid-polyethylene glycol 2000.

3. The liposome drug delivery system targeting neutrophil PD-L1 according to claim 1, characterized in that: in, The mass ratio of neutrophil-specific antibody or peptide, liposome, and PD-L1-specific siRNA is 3:17:

1.

4. A method for preparing a liposome drug delivery system targeting neutrophil PD-L1, for preparing the liposome drug delivery system targeting neutrophil PD-L1 according to any one of claims 1 to 3, characterized in that: The following steps are involved: Step 1, dissolving soybean lecithin SPC, (2,3-dioleoyl-propyl)-trimethylammonium chloride DOTAP, cholesterol, and neutrophil-specific antibody or polypeptide in an organic solvent to obtain a lipid solution; In step 2, the PD-L1-specific siRNA is dissolved in a citric acid buffer solution containing 25% ethanol, and then slowly added to the lipid solution. After mixing, the mixture is incubated for 15-25 minutes. The mixture is treated with ultrasound and a liposome extruder, and then dialyzed using a nanodialysis device to obtain the neutrophil PD-L1-targeting liposome delivery system.

5. The method for preparing the liposome drug delivery system targeting neutrophil PD-L1 according to claim 4, characterized in that: in, In step 1, if a neutrophil-specific antibody is used, the neutrophil-specific antibody needs to be reacted with the PEGylated lipid DSPE-PEG-2K-NHS under weak alkaline conditions at room temperature for 5-6 hours, and then dialyzed to obtain the DSPE-PEG-2K-antibody. The DSPE-PEG-2K-antibody, the SPC, the DOTAP, and the cholesterol are dissolved in 3 mL of anhydrous ethanol in a mass ratio of 3:10:5:2 to obtain the lipid solution. In step 1, if a neutrophil-specific polypeptide is used, the SPC, the DOTAP, and the cholesterol are first dissolved in 2 mL of anhydrous ethanol at a ratio of 3:10:5:2, and then the neutrophil-specific polypeptide is dissolved in 1 mL of hexafluoroisopropanol, and then mixed to obtain the lipid solution.

6. The method for preparing the liposome drug delivery system targeting neutrophil PD-L1 according to claim 4, characterized in that: in, The citric acid buffer is 50 mM citrate, pH 4, The filter membrane used in the liposome extruder is a 100 nm filter membrane. The nanodialysis device uses a polycarbonate membrane with a pore size of 30 nm.

7. Use of the neutrophil PD-L1 targeting liposome drug delivery system according to any one of claims 1 to 3 in the treatment of neutrophil PD-L1 related diseases, wherein the neutrophil PD-L1 related disease is sepsis.

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