Cationic immune liposome dry powder inhalant as well as preparation method and application thereof

Abstract

The invention discloses a cationic immune liposome dry powder inhaler as well as a preparation method and application thereof, and belongs to the technical field of pharmaceutical preparations. The cationic immune liposome dry powder inhaler disclosed by the invention is prepared from siRNA (small interfering Ribonucleic Acid) used for KRAS mutation, (2, 3-dioleyloxypropyl)-trimethyl ammonium chloride, dipalmitic acid phosphatidylcholine, cholesterol, 1, 2-distearoyl-SN-glycerol-3-phosphorylethanolamine-N-maleimide-polyethylene glycol 2000, palbolizumab and a freeze-drying protective agent. The composition is prepared through a freeze-drying process. The preparation method of the dry powder inhaler comprises the following steps: preparing the cationic liposome carrying the siRNA, preparing the cationic immune liposome coupled with the antibody, and freeze-drying the dry powder inhaler. Compared with a traditional administration mode, the inhalant has the advantages of being simple in preparation process, good in drug stability, high in targeting performance, obvious in-vivo distribution advantage and the like, and a new strategy is provided for treatment of the non-small cell lung cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of pharmaceutical preparations, and in particular relates to a cationic immunoliposome dry powder inhaler and a preparation method and application thereof. Background Art

[0002] Lung cancer is the most common type of cancer and one of the most deadly cancers worldwide. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases. The Kirsten Rat Sarcoma Viral Oncogene Homolog (KRAS) is a key member of the RAS protein family and one of the most commonly mutated oncogenes in non-small cell lung cancer. However, due to the unusually smooth surface of the KRAS protein and the lack of a hydrophobic pocket for drug binding, the development of KRAS-targeted drugs faces significant challenges. In 2022, the FDA announced the approval of Adagrasib (MRTX849), an oral KRAS G12C inhibitor developed by Mirati Therapeutics, for the treatment of patients with KRAS G12C-mutated non-small cell lung cancer (NSCLC) who have received at least one prior systemic therapy. This is the second KRAS G12C inhibitor to be marketed globally, following Lumakras (sotorasib). Although these KRAS-targeted drugs have demonstrated significant efficacy in monotherapy, acquired drug resistance and adverse reactions such as gastrointestinal reactions and hepatotoxicity have also emerged.

[0003] Small interfering RNA (siRNA) is an important tool in targeted therapy and precision medicine, precisely inhibiting the expression of disease-related genes by utilizing sequence-specific RNA interference mechanisms. However, issues such as short in vivo half-life, low delivery efficiency, and potential safety issues severely limit the clinical application of siRNA. Numerous studies have shown that KRAS mutations not only affect tumor development but also accelerate tumor progression and promote immune escape by altering the tumor microenvironment. Pembrolizumab is a monoclonal antibody targeting programmed death protein-1 (PD-1). Combining KRAS inhibitors with PD-1 / PD-L1 inhibitors can improve the tumor microenvironment of KRAS-mutant NSCLC, activate T lymphocytes, and enhance their anti-tumor activity, thereby effectively fighting tumors. The combined strategy of gene silencing and immunotherapy has great potential in the treatment of KRAS-mutant lung cancer, and combined administration will have a synergistic effect in killing tumor cells. In the existing technology, both siRNA and pembrolizumab are administered via intravenous injection, which results in poor drug bioavailability, poor in vivo stability, and inability to achieve synergistic co-delivery to lung tumors. Therefore, it is necessary to develop a new drug co-delivery system for lung tumors. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the inventors have provided a cationic immunoliposome dry powder inhaler, its preparation method, and application. Pembrolizumab is coupled to cationic liposomes loaded with siRNA targeting KRAS mutations. The resulting dry powder inhaler is freeze-dried and prepared for pulmonary inhalation. This method features a simple preparation process, high drug loading, and excellent formulation properties. This cationic immunoliposome dry powder inhaler not only overcomes the limitations of traditional intravenous administration, but also effectively improves the in vivo stability of both siRNA and pembrolizumab, increasing the targeted accumulation of the combined drug in lung tumors. It also simultaneously exerts a synergistic therapeutic effect of gene silencing and immunotherapy, providing a new therapeutic approach for the treatment of non-small cell lung cancer.

[0005] The present invention specifically provides a cationic immunoliposome dry powder inhaler, which comprises: siRNA for KRAS mutation, (2,3-dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol, 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000, pembrolizumab and a lyoprotectant; the cationic immunoliposome dry powder inhaler is prepared by a freeze-drying process.

[0006] Preferably, the siRNA sequence for KRAS mutation in the cationic immunoliposome dry powder inhaler is: sense chain: 5′-CAGCUAAUUCAGAAUCAUU-3′; antisense chain: 5′-AAUGAUUCUGAAUUAGCUG-3′.

[0007] The preparation method of the cationic immunoliposome dry powder inhaler comprises three processes: preparing cationic liposomes loaded with siRNA, preparing antibody-coupled cationic immunoliposomes, and freeze-drying to prepare the dry powder inhaler.

[0008] Preferably, the siRNA-loaded cationic liposomes are prepared by the following method: (2,3-dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000 in a molar ratio of 1:1:0.5:0.06 are dissolved in anhydrous ethanol, the organic solvent is removed under reduced pressure by a rotary evaporator, an aqueous solution of siRNA for KRAS mutation is added for hydration, the particle size is homogenized by an ultrasonic cell disruptor, and the particles are granulated with a microporous filter membrane to obtain siRNA-loaded cationic liposomes.

[0009] Preferably, the antibody-coupled cationic immunoliposomes are prepared by the following method: pembrolizumab is diluted with PBS, and a thiol reagent is added at a molar ratio of antibody to Traut's reagent of 1:20, and the mixture is reacted at room temperature in the dark for 1 hour; after the reaction, the free reagent is removed by ultrafiltration and centrifugation to obtain the thiol-modified antibody; the modified antibody is mixed with the siRNA-loaded cationic liposomes, incubated with stirring in the dark for 12 hours, and purified by ultrafiltration to obtain the cationic immunoliposomes.

[0010] Preferably, the dry powder inhaler is prepared by the following method: pretreating the sample by slow freezing, mixing the lyoprotectant and liposomes at a volume ratio of 0.5:1, performing a freeze-drying process, and maintaining the drying stage for 12 hours to obtain a cationic immunoliposome dry powder inhaler.

[0011] Preferably, the lyoprotectant used in the dry powder inhaler is a composition of 5% by mass mannitol and 5% by mass tert-butanol.

[0012] The cationic immunoliposome dry powder inhaler is used to treat KRAS gene mutant non-small cell lung cancer.

[0013] The cationic immunoliposome dry powder inhaler is used for pulmonary inhalation administration.

[0014] Compared with the prior art, the present invention has the following advantages:

[0015] (1) A cationic immunoliposome dry powder inhaler was prepared to address the problems of KRAS drug shortage and unstable siRNA drug delivery, and to synergistically target the PD-1 receptor to achieve a combined treatment strategy of gene silencing and immunotherapy.

[0016] (2) Targeted co-delivery of siRNA and pembrolizumab can be achieved through the pulmonary administration route, which can accurately co-deliver to the lung lesion area and achieve drug accumulation and release at the tumor site, thereby effectively inhibiting tumor cell migration and inducing its apoptosis.

[0017] (3) A method for preparing a cationic immunoliposome dry powder inhaler is provided, which has a simple preparation process, a high drug loading rate, a mild freeze-drying process, and good drug stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 :Agarose gel electrophoresis of siRNA cationic liposomes

[0019] Figure 2 :Transmission electron microscopy images of cationic immunoliposomes

[0020] Figure 3 :Scanning electron micrograph of cationic immunoliposome dry powder inhaler

[0021] Figure 4 :Cytotoxicity of cationic immunoliposome dry powder inhaler

[0022] Figure 5 :Western blot analysis of cationic immunoliposome dry powder inhaler

[0023] Figure 6 :Comparison of tumor size treated with cationic immunoliposome dry powder inhalation

[0024] Figure 7 :Bio distribution of cationic immunoliposome dry powder inhaler DETAILED DESCRIPTION

[0025] The present invention will be further described below with reference to the examples and experimental data. It should be understood that the examples of the present invention are provided for illustration only and are not intended to limit the present invention. Therefore, simple modifications to the present invention based on the method of the present invention fall within the scope of the present invention.

[0026] 1. Preparation of siRNA-loaded cationic liposomes

[0027] Example 1

[0028] (2,3-Dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol, and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000 (at a molar ratio of 1:1:0.5:0.06) were dissolved in 2 mL of anhydrous ethanol and transferred to a round-bottom flask. The organic solvent was removed using a rotary evaporator in a constant-temperature water bath to form a uniform lipid film. Subsequently, 3 mL of an aqueous solution containing 400 μg of siRNA was added, and the mixture was hydrated at the same temperature. The mixture was then ultrasonically crushed and sieved to obtain siRNA-loaded cationic liposomes.

[0029] Example 2

[0030] (2,3-Dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol, and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000 (at a molar ratio of 1:3:0.5:0.06) were dissolved in 2 mL of anhydrous ethanol and transferred to a round-bottom flask. The organic solvent was removed using a rotary evaporator in a constant-temperature water bath to form a uniform lipid film. Subsequently, 3 mL of an aqueous solution containing 400 μg of siRNA was added, and the mixture was hydrated at the same temperature. The mixture was then ultrasonically crushed and sieved to obtain siRNA-loaded cationic liposomes.

[0031] Example 3

[0032] (2,3-Dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol, and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000 at a molar ratio of 1:5:0.5:0.06 were dissolved in 2 mL of anhydrous ethanol and transferred to a round-bottom flask. The organic solvent was removed using a rotary evaporator in a constant-temperature water bath to form a uniform lipid film. Subsequently, 3 mL of an aqueous solution containing 400 μg of siRNA was added, and the mixture was hydrated at the same temperature. The mixture was ultrasonically crushed and sieved to obtain siRNA-loaded cationic liposomes.

[0033] Example 4

[0034] (2,3-Dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol, and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000 at a molar ratio of 1:1:1:0.06 were dissolved in 2 mL of anhydrous ethanol and transferred to a round-bottom flask. The organic solvent was removed by rotary evaporation in a constant-temperature water bath to form a uniform lipid film. Subsequently, 3 mL of an aqueous solution containing 400 μg of siRNA was added, and the mixture was hydrated at the same temperature. The mixture was ultrasonically crushed and sieved to obtain siRNA-loaded cationic liposomes.

[0035] Example 5

[0036] (2,3-Dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol, and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000 (at a molar ratio of 1:1:2:0.06) were dissolved in 2 mL of anhydrous ethanol and transferred to a round-bottom flask. The organic solvent was removed using a rotary evaporator in a constant-temperature water bath to form a uniform lipid film. Subsequently, 3 mL of an aqueous solution containing 400 μg of siRNA was added, and the mixture was hydrated at the same temperature. The mixture was then ultrasonically crushed and sieved to obtain siRNA-loaded cationic liposomes.

[0037] Verification Example

[0038] The solutions obtained in Examples 1-5 were filtered through a 0.45 μm microporous filter membrane, and the particle size, polydispersity index, and zeta potential were monitored using a laser particle size analyzer. The binding ability of lipids with different N / P ratios to siRNA was investigated by agarose gel electrophoresis.

[0039] Table 1. Particle size, polydispersity index and potential of various examples and comparative examples

[0040] Example Particle size (nm) Polydispersity Index (PDI) Potential (mV) Example 1 93.61 0.30 16.29 Example 2 150.96 0.21 24.04 Example 3 193.11 0.14 50.46 Example 4 107.73 0.26 41.95 Example 5 157.95 0.21 32.66

[0041] The results in Table 1 show that the particle size of the siRNA-loaded cationic liposomes obtained in the present invention is less than 200 nm and the zeta potential is positive. Figure 1 As shown, the siRNA-loaded cationic liposomes prepared by the present invention have high siRNA delivery efficiency and loading efficiency.

[0042] 2. Preparation of Antibody-Conjugated Cationic Immunoliposomes

[0043] Pembrolizumab was diluted to 0.5 mg / mL in PBS, and the thiol reagent was added at a molar ratio of 1:20. The reaction was allowed to proceed in the dark at room temperature for 1 hour. After completion of the reaction, free reagent was removed by ultrafiltration and centrifugation to obtain the thiolated antibody. The modified antibody was mixed with siRNA-loaded cationic liposomes, incubated with stirring in the dark for 12 hours, and unconjugated antibody was purified by ultrafiltration to prepare antibody-conjugated cationic immunoliposomes. The morphology of the antibody-conjugated cationic immunoliposomes was directly observed using transmission electron microscopy (TEM).

[0044] The results are as follows Figure 2 The antibody-coupled cationic immunoliposomes are spherical or quasi-spherical in shape under a transmission electron microscope and are evenly dispersed.

[0045] 3. Freeze-drying to prepare dry powder inhaler

[0046] Example 1

[0047] The antibody-coupled cationic immunoliposomes were pretreated by slow freezing at -80°C for 24 h. While keeping other freeze-drying process parameters consistent, 5% by mass mannitol and 5% by mass tert-butanol were used as freeze-drying protectants and freeze-dried with the above liposome solution at a volume ratio of 0.5:1 to obtain a dry powder inhaler.

[0048] Example 2

[0049] The antibody-coupled cationic immunoliposomes were pretreated by slow freezing at -80°C for 24 h. While keeping other freeze-drying process parameters consistent, 5% by mass mannitol and 10% by mass tert-butanol were used as freeze-drying protectants and freeze-dried with the above liposome solution at a volume ratio of 0.5:1 to obtain a dry powder inhaler.

[0050] Example 3

[0051] The antibody-coupled cationic immunoliposomes were pretreated by slow freezing at -80°C for 24 h. While keeping other freeze-drying process parameters consistent, 5% by mass mannitol and 15% by mass tert-butanol were used as freeze-drying protectants and freeze-dried with the above liposome solution at a volume ratio of 0.5:1 to obtain a dry powder inhaler.

[0052] Example 4

[0053] The antibody-coupled cationic immunoliposomes were pretreated by slow freezing at -80°C for 24 h. While keeping other freeze-drying process parameters consistent, 5% by mass mannitol and 5% by mass tert-butanol were used as freeze-drying protectants and freeze-dried with the above liposome solution at a volume ratio of 0.5:1 to obtain a dry powder inhaler.

[0054] Verification Example

[0055] A small amount of the dry powder inhaler obtained in Examples 1-4 was weighed and observed for appearance and morphology. The mixture was then reconstituted with deionized water to determine dispersibility. The mixture was filtered through a 0.45 μm microporous membrane and the particle size and zeta potential were monitored using a laser particle size analyzer. The dry powder inhaler obtained in Example 1 was uniformly dispersed in the conductive adhesive. After being treated with gold sputtering for 200 s, the multi-scale morphology was observed using a scanning electron microscope (SEM) at an accelerating voltage of 10.0 kV (magnification 500-10,000×).

[0056] Table 2. Appearance, particle size, potential and redispersibility of various examples and comparative examples

[0057] Example Appearance Particle size (nm) Potential (mV) Redispersibility Example 1 Fluffy, light 133.05 2.68 +++ Example 2 Fluffy, light 134.95 16.90 +++ Example 3 Fluffy, light 135.81 28.17 +++ Example 4 Fluffy, light 151.76 3.36 +++

[0058] Note: +++: completely dissolved within 30 seconds; ++: dissolved within 1-3 minutes; +: dissolved within more than 5 minutes or not dissolved

[0059] The results in Table 2 show that the cationic immunoliposome dry powder inhalers obtained in the present invention are fluffy and light, have good redispersibility, have a particle size of less than 200 nm after redispersion, and have positive zeta potentials. Figure 3 The results show that the dry powder inhaler prepared by the present invention has an appearance of a needle-shaped crystal structure with a uniform shape and a large specific surface area.

[0060] 4. In vitro antitumor activity assay of cationic immunoliposome dry powder inhalation

[0061] A549 cells in the logarithmic growth phase were collected and 5×10 3100 μL of cell suspension was seeded into each well of a 96-well plate and incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours. The old culture medium was removed, and 100 μL of drug-containing culture medium (for the siRNA group and the cationic immunoliposome dry powder inhaler reconstituted group) was added to the dosing wells to achieve final drug concentrations of 0.625, 1.25, 2.5, 5, and 10 μg / mL. Control wells and a zeroing well were also set. The 96-well plate was incubated in a 37°C and 5% CO2 incubator for 24 and 48 hours. 20 μL of 5 mg / mL MTT reagent was added to each well and incubated in a 37°C and 5% CO2 incubator for 4 hours. The solution in the wells was aspirated, and 100 μL of dimethyl sulfoxide solution was added. The absorbance value (OD value) in each well of the 96-well plate was measured by a microplate reader at 570 nm, and the effects of naked siRNA and cationic immunoliposome dry powder inhalation on the viability of A549 lung cancer cells were compared.

[0062] The results are as follows Figure 4 As shown, compared with naked siRNA, the use of cationic immunoliposome dry powder inhalation can more significantly inhibit the proliferation of A549 cells (*p≤0.05, **p≤0.01, ***p≤0.001, ****p≤0.0001).

[0063] 5. Degradation of KRAS protein by cationic immunoliposome dry powder inhalation

[0064] A549 cells were plated at 3 × 10 5 A549 cells were seeded into six-well plates at a density of 100 cells / well and cultured overnight at 37°C to allow attachment. After incubation for 48 hours, the cells were treated with naked siRNA solution and cationic immunoliposome dry powder inhaler reconstitution solution, respectively. 100 μL of RIPA lysis buffer was added to each well and lysed for 2-4 minutes. Cells were then transferred to a 1.5 mL centrifuge tube using a cell scraper and lysed for another 30 minutes. Centrifuge at 4°C and 12,000 rpm for 10 minutes. The supernatant was the total protein solution. All procedures were performed on ice.

[0065] Determination of protein concentration: The protein concentration was determined using the BCA method.

[0066] SDS-PAGE electrophoresis: Prepare 15% separating gel and 5% stacking gel, add TEMED and mix thoroughly before pouring the gel. Insert a comb and gently remove it after the stacking gel solidifies. Add 5 μL of protein sample to each well and 3 μL of protein marker to an adjacent well as a reference. Set the electrophoresis conditions to 90 V for the separating gel and 150 V for the stacking gel. Run the gel until the bromophenol blue migrates to approximately 1 cm from the bottom of the gel.

[0067] Transfer: Use a 0.45 μm pore size polyvinylidene fluoride (PVDF) membrane, activated with methanol for 2 minutes before use. Place the separation gel in the transfer solution. Place a sponge pad, three pieces of filter paper, separation gel, three pieces of filter paper, and sponge pad in the order of black plate at the bottom and white plate at the top. Once secured, place the membrane in an electroporation tank. Set the transfer condition to a constant current of 300 mA for 30 minutes.

[0068] Immunofluorescence reaction: Immerse the PVDF membrane in TBST solution, add 5% skim milk powder, and block at room temperature on a shaker for 30 minutes. Subsequently, incubate the membrane in a primary antibody working solution diluted 1:1000 at 4°C overnight. Prepare a secondary antibody working solution using horseradish peroxidase-conjugated goat anti-rabbit antibody at a dilution of 1:5000 and incubate the PVDF membrane at room temperature for 30 minutes. Finally, mix equal volumes of ECL luminescent solution A and solution B. After blotting the liquid from the membrane, immerse the membrane in the mixed luminescent solution for 1 minute and then develop the membrane in a chemiluminescence imager.

[0069] The results are as follows Figure 5 As shown, compared with the naked siRNA solution control group (control), the expression of KRAS protein in A549 cells treated with the reconstituted solution of cationic immunoliposome dry powder inhaler was significantly downregulated.

[0070] 6. In vivo Antitumor Activity Determination of Cationic Immunoliposome Dry Powder Inhalation

[0071] Nude mice bearing orthotopic non-small cell lung cancer were randomly divided into three groups: a model group (no medication), a lung-administered siRNA solution, and a cationic immunoliposome dry powder inhaler. 100 nM siRNA was administered to each mouse every other day for 14 days. Euthanasia was performed 48 hours after the last dose, and the lungs were fully excised, rinsed with PBS, and immediately subjected to gross morphological observation and photography.

[0072] The results are as follows Figure 6 As shown, compared with the model group and siRNA solution, the lung tumor volume of the cationic immunoliposome dry powder inhalation group was the smallest, proving that the cationic immunoliposome dry powder inhalation prepared by the present invention can effectively inhibit the growth of lung tumors and has the best tumor inhibition effect.

[0073] 7. In vivo distribution experiment of cationic immunoliposome dry powder inhalation

[0074] Nude mice in good condition with an orthotopic lung cancer model were divided into groups of three and received both tail vein and lung injections of a cationic immunoliposome dry powder inhaler loaded with the fluorescent dye DiR. Each nude mouse was sacrificed 72 hours after administration, and its major organs were removed and placed in a Boluten in vivo fluorescence imaging system to observe the distribution of DiR in these major organs.

[0075] The results are as follows Figure 7 As shown, compared with tail vein administration, the fluorescence signal at the tumor site of tumor-bearing nude mice administered with DiR-loaded cationic immunoliposome dry powder inhalation via the lung remained stable and the decay rate was slow, indicating that the cationic immunoliposome dry powder inhalation prepared by the present invention can significantly prolong the retention time of the drug in lung tumor tissue.

Claims

1. A cationic immunoliposome dry powder inhaler, characterized in that: The cationic immunoliposome dry powder inhaler comprises: siRNA for KRAS mutation, (2,3-dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol, 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000, pembrolizumab and a lyophilization protectant; the cationic immunoliposome dry powder inhaler is prepared by a freeze-drying process.

2. The cationic immunoliposome dry powder inhaler according to claim 1, characterized in that The preparation method of the cationic immunoliposome dry powder inhaler comprises three processes: preparing cationic liposomes loaded with siRNA, preparing antibody-coupled cationic immunoliposomes, and freeze-drying to prepare the dry powder inhaler.

3. The cationic immunoliposome dry powder inhaler according to claim 2, characterized in that The siRNA-loaded cationic liposomes are prepared by the following method: (2,3-dioleyloxypropyl)-trimethylammonium chloride, dipalmitoylphosphatidylcholine, cholesterol, and 1,2-distearoyl-SN-glycero-3-phosphoethanolamine-N-maleimide-polyethylene glycol 2000 in a molar ratio of 1:1:0.5:0.06 are dissolved in anhydrous ethanol, the organic solvent is removed under reduced pressure on a rotary evaporator, an siRNA aqueous solution for KRAS mutation is added for hydration, the particle size is homogenized using an ultrasonic cell disruptor, and the particles are granulated with a microporous filter to obtain the siRNA-loaded cationic liposomes.

4. The cationic immunoliposome dry powder inhaler according to claim 2, characterized in that The antibody-coupled cationic immunoliposomes were prepared by the following method: pembrolizumab was diluted with PBS, and a thiol reagent was added at a molar ratio of antibody to Traut's reagent of 1:20, and the reaction was carried out at room temperature in the dark for 1 hour; after the reaction, the free reagent was removed by ultrafiltration and centrifugation to obtain the thiol-modified antibody; the modified antibody was mixed with the siRNA-loaded cationic liposomes, incubated with stirring in the dark for 12 hours, and purified by ultrafiltration to obtain the cationic immunoliposomes.

5. The cationic immunoliposome dry powder inhaler according to claim 2, characterized in that The dry powder inhaler is prepared by the following method: pretreating the sample by slow freezing, mixing the freeze-drying protectant and liposomes at a volume ratio of 0.5:1, performing a freeze-drying process, and maintaining the drying stage for 12 hours to obtain a cationic immunoliposome dry powder inhaler.

6. The cationic immunoliposome dry powder inhaler according to claim 5, characterized in that The freeze-drying protective agent used in the dry powder inhaler is a composition of 5% by mass of mannitol and 5% by mass of tert-butyl alcohol.

7. The cationic immunoliposome dry powder inhaler according to claim 1, characterized in that The cationic immunoliposome dry powder inhaler is used to treat KRAS gene mutant non-small cell lung cancer.

8. The cationic immunoliposome dry powder inhaler according to claim 1, characterized in that The cationic immunoliposome dry powder inhaler is used for pulmonary inhalation administration.

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