CN118903047A - Atomization inhalation PS-SR nanoparticle targeting macrophages, and preparation method and application thereof - Google Patents

Abstract

The invention provides a macrophage-targeted atomized inhalation PS-SR nanoparticle and a preparation method and application thereof, belonging to the technical field of biological medicine. The PS-SR nanoparticle provided by the invention comprises SHP099, R848 and PS liposome. PS-SR nanoparticles can target macrophages by PS liposome and aerosol inhalation, delivering R848 and SHP099 into macrophages. R848 polarizes M2 type macrophages into M1 type, and releases pro-inflammatory cytokines to realize anti-tumor effect; SHP099 can inhibit downstream protein SHP2 of CD47-SIRP alpha in macrophage to block tumor cell escape in macrophage, so as to enhance tumor cell eliminating ability of macrophage. The activated macrophages present T cells with antigens, so that T cell immunity of organisms is effectively activated, cancer cells at tumor positions are further killed, and efficient inhibition of metastasis is realized.

Description

PS-SR nanoparticles for macrophage targeting by aerosol inhalation and preparation method and application thereof

Technical Field

The present invention belongs to the technical field of biomedicine, and in particular relates to PS-SR nanoparticles targeted for macrophages by atomization inhalation, and a preparation method and application thereof.

Background Art

Tumor-associated macrophages account for more than 50% of the tumor as a whole and are an important type of immune cell in the tumor. Tumor-associated macrophages have two phenotypes with different functional states, M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes. M1 macrophages can phagocytize tumor cells to inhibit tumor growth and recurrence, while M2 macrophages are just the opposite, and their main function is to promote tumor cell growth and invasion. Resiquimod (R848) is an agonist of Toll-like receptors 7 and 8 (TLR7/TLR8), an immune response regulator with TLR7/8 agonist activity, inducing upregulation of cytokines such as TNF-α, IL-12 and NO. It has been reported that R848 can polarize pro-tumor macrophages into anti-tumor macrophages for immunotherapy. However, the administration of free R848 can cause nonspecific systemic inflammatory responses, which is greatly limited in clinical application. Therefore, new strategies are needed to more accurately regulate the activity of R848.

After CD47 binds to SIRPα, it transmits a "don't eat me" signal to macrophages, thereby inhibiting the phagocytosis of tumor cells by macrophages, allowing tumor cells to escape immune surveillance. SHP099 is an orally active SHP2 inhibitor that can effectively block the downstream protein SHP2 of CD47-SIRPα in tumor cells and macrophages, blocking the escape of tumor cells in macrophages, and enhancing the ability of macrophages to clear tumor cells.

Phosphatidylserine (PS), also known as serine phospholipids and diacylglycerol phosphoserine, is a ubiquitous phospholipid that is usually located in the inner layer of the cell membrane. It is one of the cell membrane components and is related to a series of membrane functions. When cells undergo apoptosis, PS is expressed externally on the cell membrane. Macrophages recognize PS expressed by apoptotic cells and then remove them through efferocytosis.

Therefore, it is essential to develop a nanoformulation that can effectively target macrophages, deliver R848 and SHP099 into macrophages, and reduce adverse reactions of the body while taking advantage of the characteristics of macrophages that recognize and eliminate cells expressing PS. Currently, intravenous administration is a common means of anti-tumor treatment, but intravenous injection often brings a lot of inconvenience and pain to patients, and usually requires professionals to perform intravenous injection operations, which causes patients to go to the hospital frequently for treatment, thus affecting patient compliance. Therefore, it is of great significance to explore a more convenient, effective and patient-acceptable treatment method. It brings hope for improving the treatment effect and prognosis of cancer patients.

Summary of the invention

In view of the above-mentioned deficiencies, the present invention provides a PS-SR nanoparticle for aerosol inhalation targeting macrophages, and a preparation method and application thereof. The PS-SR nanoparticles provided by the present invention include SHP099, R848 and PS liposomes. The PS-SR nanoparticles provided by the present invention can target macrophages by aerosol inhalation, and deliver R848 and SHP099 into macrophages. R848 polarizes M2 macrophages to M1 type, releases proinflammatory cytokines to achieve anti-tumor effects; SHP099 blocks tumor cells from escaping in macrophages by inhibiting SHP2, a downstream protein of CD47-SIRPα in macrophages, thereby enhancing the tumor cell clearance ability of macrophages. Activated macrophages present antigens to T cells, effectively activating the body's T cell immunity, further killing cancer cells at the tumor site, and achieving efficient inhibition of metastatic tumors.

To achieve the above purpose, the technical solution adopted by the present invention is as follows:

In one aspect, the present invention provides a PS-SR nanoparticle for aerosol inhalation, wherein the PS-SR nanoparticle comprises SHP099, R848 and PS liposomes, and the PS liposomes comprise brain PS, DSPC and cholesterol;

The structural formula of SHP099 is shown in Formula I, and the structural formula of R848 is shown in Formula II;

Specifically, the concentration ratio of SHP099, R848 and PS liposome is 2-4:4-6:3-3.5.

Preferably, the concentration ratio of SHP099, R848 and PS liposome is 3:5:3.25.

Specifically, the concentration ratio of brain PS, DSPC and cholesterol in the PS liposome is 4-6:3-5:0.5-2.

Preferably, the concentration ratio of brain PS, DSPC and cholesterol in the PS liposome is 5:4:1.

Specifically, the particle size of the PS liposome is ≤100 nm.

Specifically, the preparation method of the PS liposome is:

S1. Brain PS, DSPC and cholesterol were added to chloroform solution, stirred for 5 min, and evaporated under reduced pressure to form a PS lipid film;

S2. The PS lipid membrane is hydrated and purified to form PS liposomes.

Preferably, the PS-SR nanoparticles are atomized by a nebulizer.

Specifically, the preparation method of the PS-SR nanoparticles comprises:

S1. Evenly mix an organic solvent containing SHP099 and an organic solvent containing R848 to obtain a mixed solution;

S2, adding PS liposomes to the mixed solution, evaporating under reduced pressure to form a PS-SR lipid membrane;

S3. The PS-SR lipid film is hydrated and purified to form PS-SR nanoparticles.

Specifically, the input volumes of the organic solvent containing SHP099 and the organic solvent containing R848 in step S1 are 1:1-2.

Preferably, in step S1, the input volume of the organic solvent containing SHP099 and the organic solvent containing R848 is 1:1.5.

Specifically, the organic solvent containing SHP099 in step S1 contains 5-15 mg/mL SHP099.

Preferably, the organic solvent containing SHP099 in step S1 contains 10 mg/mL SHP099.

Specifically, the organic solvent containing R848 in step S2 contains 5-15 mg/mL R848.

Preferably, the organic solvent containing R848 in step S2 contains 10 mg/mL R848.

Preferably, the organic solvent in step S1 includes methanol.

Specifically, step S1 includes mixing the SHP099 methanol solution and the R848 methanol solution evenly, adding the mixture into a round-bottom flask containing chloroform, and stirring.

Preferably, the volume ratio of the mixed solution of the SHP099 methanol solution and the R848 methanol solution to chloroform is 1:8.

Specifically, the input volume ratio of the PS liposomes described in step S2 to the mixed solution is 7:20.

Specifically, the purpose of evaporating under reduced pressure in step S2 is to evaporate the organic solvent.

Specifically, the solution used for hydration in step S3 includes but is not limited to PBS, physiological saline or purified water.

Preferably, the solution used for hydration in step S3 is PBS.

Specifically, the hydration in step S3 includes the following steps: (1) adding PBS and ultrasonically hydrating in a water bath for 5 minutes; (2) ultrasonically hydrating at 70° C. for another 2 minutes using a 20W probe.

Specifically, the purification described in step S3 includes filtration and dialysis.

Preferably, the filter membrane used for the filtration is a 0.45 μm filter membrane.

Preferably, the purpose of the filtration is to remove free lipid aggregates.

Preferably, the dialysis process is as follows: encapsulating the PS-SR in a dialysis bag with a molecular cutoff of 500, and placing the bag in a dialysis cylinder containing ddH ₂ O for 4 hours.

Preferably, the purpose of the dialysis is to remove unentrapped free drugs.

In certain embodiments, the PS-SR concentration can be adjusted by adjusting the input amount of SHP099, R848, PS liposomes or the added amount of PBS.

In another aspect, the present invention provides a method for preparing the above-mentioned PS-SR nanoparticles, the preparation method comprising:

S1. Evenly mix an organic solvent containing SHP099 and an organic solvent containing R848 to obtain a mixed solution;

S2, adding PS liposomes to the mixed solution, evaporating under reduced pressure to form a PS-SR lipid membrane;

S3. The PS-SR lipid film is hydrated and purified to form PS-SR nanoparticles.

Specifically, the input volumes of the organic solvent containing SHP099 and the organic solvent containing R848 in step S1 are 1:1-2.

Preferably, in step S1, the input volume of the organic solvent containing SHP099 and the organic solvent containing R848 is 1:1.5.

Specifically, the organic solvent containing SHP099 in step S1 contains 5-15 mg/mL SHP099.

Preferably, the organic solvent containing SHP099 in step S1 contains 10 mg/mL SHP099.

Specifically, the organic solvent containing R848 in step S2 contains 5-15 mg/mL R848.

Preferably, the organic solvent containing R848 in step S2 contains 10 mg/mL R848.

Preferably, the organic solvent in step S1 includes methanol.

Specifically, step S1 includes mixing the SHP099 methanol solution and the R848 methanol solution evenly, adding the mixture into a round-bottom flask containing chloroform, and stirring.

Preferably, the volume ratio of the mixed solution of the SHP099 methanol solution and the R848 methanol solution to chloroform is 1:8.

Specifically, the input volume ratio of the PS liposomes described in step S2 to the mixed solution is 7:20.

Specifically, the purpose of evaporating under reduced pressure in step S2 is to evaporate the organic solvent.

Specifically, the solution used for hydration in step S3 includes but is not limited to PBS, physiological saline or purified water.

Preferably, the solution used for hydration in step S3 is PBS.

Specifically, the hydration in step S3 includes the following steps: (1) adding PBS and ultrasonically hydrating in a water bath for 5 minutes; (2) ultrasonically hydrating at 70° C. for another 2 minutes using a 20W probe.

Specifically, the purification described in step S3 includes filtration and dialysis.

Preferably, the filter membrane used for the filtration is a 0.45 μm filter membrane.

Preferably, the purpose of the filtration is to remove free lipid aggregates.

Preferably, the dialysis process is as follows: encapsulating the PS-SR in a dialysis bag with a molecular cutoff of 500, and placing the bag in a dialysis cylinder containing ddH ₂ O for 4 hours.

Preferably, the purpose of the dialysis is to remove unentrapped free drugs.

In certain embodiments, the PS-SR concentration can be adjusted by adjusting the input amount of SHP099, R848, PS liposomes or the added amount of PBS.

In another aspect, the present invention provides use of the PS-SR nanoparticles in the preparation of anti-tumor drugs.

Specifically, the dosage form of the drug includes solution, suspension or aerosol.

Specifically, the PS-SR nanoparticles target macrophages and deliver R848 and SHP099 into macrophages to achieve anti-tumor effects.

Specifically, the tumor includes metastatic tumor.

Preferably, the tumor comprises lung cancer metastasis, breast cancer metastasis, colorectal cancer metastasis, gastric cancer metastasis, liver cancer metastasis, pancreatic cancer metastasis or prostate cancer metastasis.

Further preferably, the tumor is a lung metastasis of breast cancer.

In yet another aspect, the present invention provides a pharmaceutical composition comprising the PS-SR nanoparticles.

The beneficial effects of the present invention are:

The PS-SR nanoparticles provided by the present invention can target macrophages through atomization inhalation, and deliver R848 and SHP099 into macrophages. R848 polarizes M2 macrophages to M1 type, releases pro-inflammatory cytokines to achieve anti-tumor effects; SHP099 inhibits the downstream protein SHP2 of CD47-SIRPα in macrophages, blocks the escape of tumor cells in macrophages, and enhances the tumor cell clearance ability of macrophages. Activated macrophages present antigens to T cells, effectively activating the body's T cell immunity, further killing cancer cells in the tumor site, and achieving efficient inhibition of metastatic tumors.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the characterization of the nanodrug PS-SR; A in the figure is the particle size PDI diagram of PS; B is the particle size PDI diagram of PS-SR; C is TEM; D is the storage stability of PS-SR.

Figure 2 is a high performance liquid chromatogram of R848.

FIG3 is a high performance liquid chromatogram of SHP099.

FIG4 is a high performance liquid chromatogram of PS-SR.

Figure 5 is a cell uptake experiment diagram; A in the figure shows the uptake of cy5.5-labeled PS-SR by different cells; B shows the uptake of PS-SR by three cells at different concentrations after incubation for 4 hours; C shows the uptake of PS-SR by three cells at a concentration of 3 mg/L for different time periods.

Figure 6 is a polarization experiment diagram of macrophages; A and E in the figure are the expression of Arg-1, a marker protein of the M2 type; B and F are the expression of CD206, a marker protein of the M2 type; C and G are the expression of CD80, a marker protein of the M1 type; D and H are the expression of iNOS, a marker protein of the M1 type.

Figure 7 shows the cell phagocytosis experiment and cytotoxicity experiment; A in the figure is the phagocytic ability of macrophages for tumor cells detected by flow cytometry; B is the F4/80 ⁺ GFP ⁺ double positive ratio; C is the phagocytic ability of macrophages for tumor cells detected by CLSM; D is the NO expression level in the cell supernatant detected by Elisa; E is the TNF-α expression level in the cell supernatant detected by Elisa; F is the live and dead cell staining picture; G is the macrophage viability.

FIG8 is a diagram of a mouse fluorescence imaging experiment.

Figure 9 is a diagram of a tumor inhibition experiment; A in the figure is H&E staining; B is Ki67 and TUNEL staining.

Figure 10 is an immune cell analysis; A-H in the figure are immune cell analysis performed by flow cytometry; I is a CD4 fluorescent staining section of lung tumor tissue; J is a CD8 fluorescent staining section of lung tumor tissue; left in the figure represents the left lung of the mouse, and right represents the right lung of the mouse.

FIG11 shows the lung tissues of mice taken after treatment and H&E staining.

Figure 12 is an in vivo biosafety analysis; A in the figure is H&E staining of the heart, liver, spleen, lungs and kidneys; B is the change in mouse body weight; and C is a blood sample biochemical analysis.

DETAILED DESCRIPTION

The present invention will be further described in detail below by way of examples. The following examples are only a part of the present invention and are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are not specifically stated, and the materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

Example 1 Preparation of PS-SR

1. Preparation

(1) Preparation of PS-SR

100 μL of SHP099 methanol solution (10 mg/mL) and 150 μL of R848 methanol solution (10 mg/mL) were mixed and added to a round-bottom flask containing 2 mL of chloroform, stirred at 600 rpm for 2 minutes, 70 μL of liposomes (20 mM, brain PS: DSPC: cholesterol = 5: 4: 1, CHCl ₃ solution) were added to the flask, stirred for 5 minutes, and then chloroform and methanol were evaporated under reduced pressure to form a lipid film. Drug-loaded liposomes PS-SR were formed by adding 1 ml of PBS (0.1 M, pH = 7.4) and hydrating under water bath sonication for 5 minutes, and then rehydrating at 70°C, 20W probe sonication for 2 minutes. The resulting NPs were filtered with a 0.45 μm membrane to remove free lipid aggregates, dialyzed for 4 hours to remove unentrapped free drugs, and stored at 4°C. The purified drug-loaded liposome PS-SR contained 0.4 mg/mL SHP099, 0.667 mg/mL R848, and 0.433 mg/mL PS liposomes, and the concentration of the prepared PS-SR was 1.5 mg/mL.

(2) PS preparation

70 μL of PS liposomes (20 mM, brain PS: DSPC: cholesterol = 5: 4: 1, CHCl ₃ solution) were added to the flask, stirred for 5 minutes, and then chloroform was evaporated under reduced pressure to form a lipid film. Empty liposome PS was obtained by adding 1 ml of PBS (0.1 M, pH = 7.4) and hydrating for 5 minutes under water bath sonication, and then rehydrating for 2 minutes at 70°C and 20W probe sonication. The resulting NPs were filtered with a 0.45 μm membrane to remove free lipid aggregates, and dialyzed for 4 hours to remove, and stored at 4°C.

(3) PS-SR atomization

Aerosol (nebulized PS-SR) was generated by a nebulizer connected to the animal box through a tube. Unanesthetized mice were placed in a sealed box and exposed to the aerosol at an air flow rate of level 1 for 10 minutes each time, during which 1 mL of the solution in the nebulizer was nebulized.

2. Characterization and storage stability

The particle sizes of PS and PS-SR were detected by DLS, and the results are shown in Figure 1A and Figure 1B. The particle size and PDI of PS were 83.04 nm and 0.264, respectively, and the particle size and PDI of PS-SR were 114.6 nm and 0.176, respectively. TEM images show that PS and PS-SR can be assembled into spherical nanoparticles with good dispersion, and the atomized PS-SR still maintains the morphology of spherical nanoparticles (Figure 1C). In addition, in order to evaluate the stability of PS-SR, DLS was used to monitor the changes in the particle size and distribution of PS-SR within six days. As expected, PS-SR maintained good stability within six days (Figure 1D).

3. Encapsulation rate

HPLC verification showed that the absorption peaks of R848 and SHP099 appeared in PS-SR (Figure 4), indicating that PS-SR successfully encapsulated R848 and SHP099. The encapsulation efficiency of R848 in the PS-SR prepared in this experiment was 44.4±8%, and the encapsulation efficiency of SHP099 was 40±8%.

Example 2 In vitro cell experiment

1. Cellular uptake experiment

In order to evaluate the targeting of PS to macrophages, different concentrations of PS-SR were incubated with mouse breast cancer cells 4T1, mouse embryonic fibroblasts 3T3, and mouse alveolar macrophages MH-S. The specific process was as follows: 4T1, 3T3, and MH-S cells were co-cultured with cy5.5-labeled PS-SR (1, 3, 5 mg/L) for 2, 4, and 8 hours, and then the cells were washed three times with PBS and stained with the nuclear dye Hoechst 33342 for 15 minutes.

Fluorescence confocal microscopy (CLSM) was used to observe the uptake of cy5.5-labeled PS-SR by different cells such as mouse breast cancer cells 4T1, mouse embryonic fibroblasts 3T3, and mouse alveolar macrophages MH-S (Figure 5A).

PS-SR was incubated with the three cells at different concentrations and for different times. It can be observed that the uptake of PS-SR by the three cells was time-dependent and concentration-dependent. Moreover, among the three cell lines, MH-S had the highest uptake of PS-SR regardless of time gradient or concentration gradient (B in Figure 5). The flow cytometry results also reached the same conclusion (C in Figure 5), indicating that PS successfully mediated the targeting performance of nanoparticles to macrophages, providing a powerful platform for PS to successfully deliver R848 and SHP099 to macrophages.

2. Polarization of macrophages

R848 can polarize tumor-promoting M2 macrophages into anti-tumor M1 macrophages, and M1 and M2 macrophages have different surface marker proteins. In order to observe the changes in marker proteins on the surface of macrophages after treatment with different drugs, immunofluorescence staining was performed on the treated MH-S cells, and the expression of different proteins was observed under CLSM. The specific process is:

LPS or IL-4 (50 ng/mL) was incubated with MH-S cells for 48 h and then replaced with SHP099 (3 mg/L), R848 (5 mg/L), SHP1099 (3 g/L) + R848 (5 mg/L), or PS-SR (11.158 mg/L) for 48 h. The cells were washed three times with PBS and then fixed with 4% paraformaldehyde for 15 min and exposed to 0.1% Triton for 5 min. 10% goat serum was then added to seal the cells for 1 h. Next, the cells were cultured with monoclonal antibodies anti-iNOS (1:500), anti-CD80 (1:200), and anti-Arg-1 (1:200). The next day, the cells were stained with goat anti-rabbit IgG (H+L) labeled with Alexa Fluor 488 (1:500) for 1 h and re-stained with DAPI for 15 min. Afterwards, green fluorescence was detected and counted using CLSM.

As shown in Figure 6, the expression of Arg-1 and CD206, the marker proteins of M2 on macrophages, was reduced to a certain extent in the groups treated with R848 (A in Figure 6 and B in Figure 6), and in all drug-treated groups (except the IL-4 and LPS-treated groups), PS-SR downregulated these two proteins the most. At the same time, CD80 and iNOS, the marker proteins of M1 on macrophages, were also upregulated in the groups containing R848. Not surprisingly, the upregulation effect was the most obvious in the PS-SR group, showing strong green fluorescence. However, SHP099 had little effect on the polarization of macrophages. The fluorescence statistics reached a consistent conclusion, which showed that R848 could successfully polarize M2 macrophages into M1 macrophages, laying a successful foundation for the anti-tumor effect of PS-SR in polarizing macrophages in vivo.

3. Cell phagocytosis and cytotoxicity experiments

SHP2 protein is a signal protein downstream of macrophages mediated by the engagement of CD47 protein on tumor cells and SIRPα on macrophages. It is responsible for the activity of actin involved in phagocytosis downstream of CD47-SIRPα signal. Inhibition of SHP2 protein can block the escape pathway of CD47-SIRPα mediated by tumor cells and macrophages. After treating macrophages MH-S with different drugs, the phagocytic ability of macrophages for tumor cells was detected by flow cytometry and CLSM. The specific process is as follows:

PS-SR (11.158 mg/L) was cultured with MH-S cells for 48 hours. Next, 4T1-GFP cells were added for 2 hours (macrophages: 4T1-GEP cells = 3:1). After washing with PBS, the cells were fixed with 4% paraformaldehyde for 15 minutes, then permeabilized with 0.1% Triton and replaced with 10% goat serum for 1 hour. Monoclonal antibody anti-CD80 (1:500) was added and incubated overnight at 4°C, and goat anti-rabbit IgG (h+L) labeled with Alexa Fluor 647 (1:500). After incubation with SHP099 (100 mg/L), R848 (166.7 mg/L), SHP099 and R848 (166.7 mg/L), or PS-SR (371.9 mg/L) for 6 h, MH-S cells were stained with fluorescent monoclonal antibody F4/80 (bound to PB450) and studied by flow cytometry.

As can be seen from Figure 7A, in all groups containing SHP099, the phagocytic ability of macrophages for tumor cells increased. R848 also slightly increased the phagocytic ratio of macrophages for tumor cells due to its polarization effect, and the PS-SR treatment group had the highest double positive ratio (Figure 7B), suggesting that under the effective delivery of PS-SR, SHP099 more effectively increased the inhibition of SHP2 protein, thereby increasing the phagocytic ability of macrophages. The 3D image displayed by the confocal results more intuitively gives the effect of macrophages engulfing tumor cells (Figure 7C).

In addition, in order to detect the level of pro-inflammatory cytokines secreted by macrophages after drug treatment, we used Elisa kits to detect the amount of NO and TNF-α in the cell supernatant after drug treatment. As shown in Figure 7D and Figure 7E, the amount of NO and TNF-α secreted by macrophages in the positive control group increased sharply after LPS treatment, and the levels of these two cytokines in the group containing R848 also increased to a certain extent. Compared with the IL-4 treatment group, the cytokine secretion in the group containing R848 was more than twice that of the IL-4 treatment group, which provided data support for the subsequent verification of macrophages killing tumor cells through pro-inflammatory cytokines by live and dead cell staining (Figure 7F). Regardless of SHP099, R848 or PS, we did not observe its direct killing effect on macrophages (Figure 7G), which further indicates that PS-SR achieves anti-tumor effects by transforming macrophages rather than targeting tumor cells.

Example 3 Animal Experiment

1. Mouse fluorescence imaging

10 ⁶ 4T1 cells were injected into the tail vein of 6-week-old female Balb/c mice to establish a mouse breast cancer lung metastasis model. 14 days later, the mice were subjected to aerosol inhalation of PS-SR (labeled with cy5.5) using a nebulizer, with an inhalation volume of 1 mL. Fluorescence imaging of the main organs of the mice was performed 1 hour, 24 hours, and 48 hours after the end of the inhalation to observe the accumulation of PS-SR in the lungs.

As can be seen from A in Figure 8, the retention time of cy5.5-labeled PS-SR in the lungs of mice is as long as 48 hours. The lungs show the strongest red fluorescence at 1 hour, and a certain amount of PS-SR remains in the lungs after 48 hours. Regardless of the time point, the red fluorescence in the lungs is the strongest, which reflects the advantages of aerosol therapy. Nanomedicines can be effectively delivered to lung tumors through the autonomous breathing of mice, greatly reducing the toxic side effects caused by the retention of nanomedicines in other organs. The mouse lungs were observed under CLSM and their fluorescence was counted, and consistent conclusions were obtained (B and C in Figure 8).

In addition, in order to investigate the uptake of PS-SR by lung macrophages, mouse lungs were taken for immunofluorescence staining to observe the co-localization of PS-SR and macrophages. As can be seen from D in Figure 8, whether it is 24 hours or 48 hours, the red fluorescence of cy5.5-labeled PS-SR and the green fluorescence of macrophage labeling are highly overlapped, showing a distinct yellow color, which indicates that the nanomedicine with PS as the carrier successfully achieves the targeting of alveolar macrophages, providing a basis for the drug in it to play a role in macrophages.

2. Tumor inhibition experiment

In order to explore the in vivo tumor-suppressing effect of PS-SR, the anti-tumor effect of PS-SR in the Balb/c mouse model carrying 4T1 was further evaluated. The lung metastasis model was established by injecting 1×10 ⁶ 4T1 cells into the tail vein of mice, and the mice were divided into the model (Blank) group, SHP099 group, R848 group, SHP099+R848 group, and PS-SR group. Six days after modeling, different drugs were given respectively. After the experiment, the lung tissues of the mice were taken for H&E staining, ki67 and tunel staining.

Blank group: inhaled an equal amount of PBS;

SHP099 group: 1 mL SHP099 (7.5 mg/kg) was administered by inhalation;

R848 group: 1 mL R848 (12.5 mg/kg) was administered by inhalation;

SHP099+R848 group: 1 mL SHP099 (7.5 mg/kg) and R848 (12.5 mg/kg) were administered by inhalation

PS-SR group: 1 mL PS-SR (28.14 mg/kg) was administered by inhalation. The average weight of mice was 20 g. In the 28.14 mg/kg drug inhaled, the total amount of SHP099 was 150 μg and the total amount of R848 was 250 μg. 224 μl of the original prepared PS-SR nanoparticles were taken and fixed to 1 ml for inhalation.

As can be seen from A in Figure 9, in the group without drug treatment, that is, the Blank group, the mouse liver and lungs showed the most tumor cells, while the groups treated with SHP099, R848 or a combination of the two reduced the number of tumors in the liver and lungs to a certain extent, and PS-SR had the strongest tumor inhibition effect in the liver and lungs. At the same time, the white pulp and red pulp of the spleen in the PS-SR group tended to be orderly, reflecting that PS-SR normalizes body functions by activating the immune system. The mouse lungs were taken for ki67 and tunel staining (B in Figure 9). Corresponding to the H&E results, PS-SR showed the lowest cell proliferation index and the strongest tumor cell killing ability. This further reflects the superior therapeutic effect of PS-SR by transforming macrophages to enhance their anti-tumor ability.

3. Immune cell analysis

To study the in vivo immune effects, mice treated with different drugs were euthanized, and cells from lymph nodes and lung metastases were extracted for immune cell analysis using flow cytometry.

In lymphoid tissues, the number of cytotoxic T lymphocytes (CD3 ⁺ CD8 ⁺ ) in the SHP099, R848, SHP099+R848, and PS-SR groups increased significantly compared with Blank, with the most significant increase in PS-SR (A in Figure 10). After treatment in the PS-SR group, helper T lymphocytes (CD3 ⁺ CD4 ⁺ ) and cytotoxic T cells (CD3 ⁺ CD8 ⁺ ) in lung metastases significantly expanded (B in Figure 10 and C in Figure 10), indicating that T cell-related immunotherapy was activated after PS-SR treatment, causing a strong immune response. In addition, due to the expansion of cytotoxic T cells, PS-SR also greatly reduced the percentage of Treg cells (D in Figure 10).

Because R848 activates antigen-presenting cells, the number of CD11c ⁺ CD80 ⁺ CD86 ⁺ dendritic cells in tumor tissues was further detected (E in Figure 10). PS-SR greatly increased the maturation of dendritic cells, and the maturation of dendritic cells provided a strong support for the successful activation of T cell immunity.

In addition, the proportion of macrophages in lung metastases was also detected. As can be seen from Figure 10 FH, the proportion of F4/80 ⁺ CD206 ⁺ macrophages in the group treated with SHP099 decreased, while all groups containing R848 significantly downregulated the number of F4/80 ⁺ CD206 ⁺ macrophages, suggesting that the polarization effect of PS-SR in vivo successfully transformed tumor-promoting macrophages into anti-tumor macrophages.

4. Immune cell analysis

In order to confirm that PS-SR exerts its anti-tumor effect by acting on macrophages, 1×10 ⁶ 4T1 cells were injected into the tail vein of mice to construct a lung metastasis model. The mice were pre-treated with aerosolized clodronate liposomes (200μg), intraperitoneal injection of CD4 neutralizing antibodies (400μg) and CD8 neutralizing antibodies (400μg) to deplete antigen-presenting cells, CD4 ⁺ T cells and CD8 ⁺ T cells in the mice, and then given PS-SR (28.14mg/kg) for treatment. After the treatment, the lungs of the mice were taken for H&E staining to observe the number of lung tumors.

As can be seen from Figure 11, the Blank group and the CD8 neutralizing antibody + PS-SR group and the clodronate liposome + PS-SR group have comparable therapeutic effects, with almost no tumor inhibition effect. The CD4 neutralizing antibody + PS-SR group has significantly fewer tumor nodules than the Blank group, indicating that the anti-tumor effect of PS-SR mainly relies on the killing of macrophages in the mouse body and the subsequent activated CD8 ⁺ T cells, with CD4 ⁺ T cells accounting for a small part of the effect. The PS-SR group that has not undergone any pre-treatment showed the best anti-tumor efficiency. The experimental results successfully confirmed the design of the present invention: that is, it is effective and reliable to activate the body's immune system by acting on macrophages in the body to fight against tumors.

5. In vivo biosafety analysis

The mice were divided into a blank control group, a SHP099 group, a R848 group, and a PS-SR group, and the drugs were administered according to the groups.

Blank group: inhaled an equal amount of PBS;

SHP099 group: 1 mL SHP099 (7.5 mg/kg) was administered by inhalation;

R848 group: 1 mL R848 (12.5 mg/kg) was administered by inhalation;

PS-SR group: 1 mL PS-SR (28.14 mg/kg) was administered by inhalation.

After the last administration, the heart, liver, spleen, lung and kidney of the mice were collected for H&E staining to investigate the biosafety of PS-SR in vivo.

The results are shown in A in Figure 12. The SHP099, R848 and PS-SR groups did not cause obvious damage to the organs and the morphology of each tissue was normal. In addition, the changes in the weight of the mice were monitored during the treatment (B in Figure 12). After drug treatment in each group, the weight of the mice fluctuated slightly, all within the acceptable range of variation (10%-15%). Finally, we extracted blood samples from the mice for biochemical analysis (C in Figure 12), and the biochemical indicators of each group were basically kept within the normal range. These results show that the systemic toxicity of the therapeutic drug after inhalation treatment is negligible.

The above detailed description is a specific description of one of the feasible embodiments of the present invention, and the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims (10)

1. An aerosolized inhaled PS-SR nanoparticle, wherein the PS-SR nanoparticle comprises SHP099, R848, and PS liposomes, and wherein the PS liposomes comprise brain PS, DSPC, and cholesterol;

The structural formula of the SHP099 is shown in a formula I, and the structural formula of the R848 is shown in a formula II;

2. The PS-SR nanoparticle according to claim 1, wherein the concentration ratio of SHP099, R848 and PS liposome is 2-4:4-6:3-3.5.

3. The PS-SR nanoparticle according to claim 1, wherein the PS liposome has a concentration ratio of brain PS, DSPC and cholesterol of 4-6:3-5:0.5-2.

4. The PS-SR nanoparticle according to claim 1, wherein the PS liposome is prepared by the following steps:

S1, adding brain PS, DSPC and cholesterol into chloroform solution, stirring, and evaporating under reduced pressure to form PS lipid film;

S2, purifying the PS lipid membrane after hydration to form PS liposome.

5. The method for preparing PS-SR nanoparticle according to any one of claims 1 to 4, comprising:

S1, uniformly mixing an organic solvent containing SHP099 and an organic solvent containing R848 to obtain a mixed solution;

s2, adding PS liposome into the mixed solution, and evaporating under reduced pressure to form a PS-SR lipid film;

S3, purifying the PS-SR lipid membrane after hydration to form PS-SR nanoparticles.

6. Use of PS-SR nanoparticles according to any one of claims 1-4 for the preparation of an antitumor drug.

7. The use according to claim 6, wherein the dosage form of the medicament comprises a solution, suspension or aerosol.

8. The use of claim 6, wherein the PS-SR nanoparticle targets macrophages, delivering R848 and SHP099 into macrophages for anti-tumor effect.

9. The use according to claim 6, wherein the tumor comprises a lung cancer metastasis, a breast cancer metastasis, a colorectal cancer metastasis, a gastric cancer metastasis, a liver cancer metastasis, a pancreatic cancer metastasis or a prostate cancer metastasis.

10. A pharmaceutical composition comprising the PS-SR nanoparticle according to any one of claims 1-4.