Lung-targeted nanoemulsion preparation for co-delivery of antitumor drugs and antibacterial drugs, preparation method and application

Abstract

The invention provides a lung-targeted nanoemulsion preparation for co-delivery of an antitumor drug and an antibacterial drug, a preparation method and an application, relates to the technical field of medical biology, and provides a new strategy of an in-situ vaccine activated by utilizing antitumor and antibacterial synergistic immunity for the first time. Accurate delivery of anti-tumor drugs and antibacterial drugs in lungs and tumor parts is achieved through the nanoemulsion modified by the ACE2 targeting material, meanwhile, immune response of the tumor parts and the whole body is effectively activated through a space-time coupled'antigen-adjuvant co-delivery 'strategy, and good anti-tumor and antibacterial effects are achieved. The new strategy of'in-situ vaccine 'provides a new thought for the treatment of bacterial symbiotic lung cancer, and is expected to promote the development of precise treatment of lung cancer.

Description

Technical Field

[0001] The embodiments of the present invention relate to the field of medical biotechnology, and in particular to a lung-targeted nanoemulsion preparation for co-delivering anti-tumor drugs and antibacterial drugs, a preparation method, and applications. Background Art

[0002] Recent advances in the field of tumor microbiome research have provided new insights into the thorny issue of lung cancer. Studies have shown that tumor commensal microbiota can play a role in promoting cancer in various solid tumors, including breast and colorectal cancer, through a three-pronged regulatory network: tumor-specific colonization, metabolic intervention, and immune microenvironment remodeling. As a respiratory mucosal organ that is continuously exposed to the outside world, lung tissue is susceptible to colonization by diverse microorganisms, paving the way for the relationship between lung cancer development and the microbiome. Existing evidence suggests that the commensal microbiota in lung cancer tissue differs from that in normal lung tissue, with significantly higher abundances of Firmicutes and Proteobacteria. These commensal microbiota can drive tumor progression through multiple mechanisms. For example, lung cancer commensal microbiota can not only promote tumor cell proliferation by activating lung-resident γδT cells, but also induce the growth of myeloid-derived suppressor cells, thereby inhibiting cytotoxic T lymphocyte activity and promoting tumor progression. They can even mediate anti-tumor drug resistance, hindering the effectiveness of lung cancer treatment. Therefore, in-depth exploration of the relationship between commensal microbiota and lung cancer progression and the development of targeted intervention strategies to eliminate tumors and their commensal microbiota may provide new insights into cancer treatment.

[0003] Currently, the strategy of using antibiotics to intervene in tumor commensal flora to assist chemotherapy has made initial progress. For example, related studies have suggested that oral ampicillin can reduce the load of Staphylococcus epidermidis in breast cancer, and its efficacy can be significantly improved when used in combination with paclitaxel. Related studies have also significantly reduced breast cancer lung metastasis by combining oral or injectable multiple antibiotics. However, systemic antibiotic treatment has inherent defects such as poor organ specificity and large off-target side effects. For example, it may affect macrophage function and destroy the homeostasis of intestinal flora. Long-term use may induce compensatory immunosuppression and thus weaken the anti-tumor efficacy, and even increase the risk of other diseases such as inflammatory bowel disease, neurological diseases, and obesity.

[0004] Currently, local antimicrobial intervention strategies are gaining increasing attention. Studies have targeted the lung microbiome with aerosolized vancomycin or neomycin, significantly reducing lung metastases in mice with B16F10 melanoma and enhancing the chemotherapeutic activity of dacarbazine against advanced B16F10 metastases. Although local intervention strategies have shown positive effects in some cases, they are not applicable to all organs. Therefore, a new treatment strategy is urgently needed. Summary of the Invention

[0005] Embodiments of the present invention provide lung-targeted nanoemulsion formulations, preparation methods, and applications for the co-delivery of antitumor and antibacterial drugs, providing a novel "in situ vaccine" strategy. This novel "in situ vaccine" strategy involves delivering an antitumor drug with immunogenic cell death (ICD)-inducing ability to kill tumors and release endogenous tumor antigens in situ. This strategy also involves the co-delivery of an antibacterial drug to eliminate tumor-associated bacteria and release endogenous bacterial adjuvants in situ. Furthermore, the in situ-generated tumor antigens and bacterial adjuvants are used to activate the body's immune response.

[0006] In a first aspect, the present invention provides a lung-targeted nanoemulsion formulation for co-delivering antitumor drugs and antibacterial drugs, wherein the antitumor drugs include at least one antitumor drug with an ICD effect; and the antibacterial drugs include at least one antibiotic or antimicrobial peptide.

[0007] Optionally, the anti-tumor drugs with ICD effect include but are not limited to: cyclophosphamide, docetaxel, doxorubicin, mitoxantrone, paclitaxel and crizotinib; the antibiotics or antimicrobial peptides include but are not limited to: gentamicin, polymyxin E, moxifloxacin and synthetic antimicrobial anti-biofilm peptide SAAP-148 (amino acid sequence: LKRVWKRVFKLLKRYWRQLKKPVR).

[0008] Optionally, the nanoemulsion preparation further comprises: an aqueous phase, an oil phase and an emulsifier, wherein the oil phase is any pharmaceutically acceptable oil, and the emulsifier is selected from one or more combinations of anionic surfactants, nonionic surfactants and zwitterionic surfactants.

[0009] Optionally, the nanoemulsion is externally modified with DSPE-PEG 2000 -ACE2 targeting peptides AYQ, GQ, or AY.

[0010] Optionally, when the antitumor drug is crizotinib and the antibacterial drug is SAAP-148, the nanoemulsion preparation is an oil-in-water nanoemulsion system, which uses the oil phase to encapsulate hydrophobic crizotinib and adds an anionic surfactant to adsorb positively charged SAAP-148 at the oil-water interface.

[0011] Optionally, the anionic surfactant includes but is not limited to carboxylates, sulfates, sulfonates, phosphates, phosphatidic acid, chenodeoxycholic acid, chenodeoxycholic acid sodium salt, bile acid, bovine or sheep bile, dehydrocholic acid, deoxycholic acid, deoxycholic acid, deoxycholic acid methyl ester, digoxin, N,N-dimethyldodecylamine N-oxide, sodium laurate, sodium glycocholate, hydrated glycocholic acid, monohydrated glycocholic acid, 3-sulfate glycocholic acid disodium salt, ethyl glycocholate, sodium N-lauroyl sarcosine, N-lauroyl sarcosine solution, N-lauroyl sarcosine solution, lithium lauryl sulfate, Lugol Solution, Type 4 Niaproof, Sodium 1-Octanesulfonate, Sodium 1-Butanesulfonate, Sodium 1-Decanesulfonate, Sodium 1-Decanesulfonate, Sodium 1-Dodecanesulfonate, Sodium 1-Heptanesulfonate Anhydrous, Sodium 1-Heptanesulfonate Anhydrous, Sodium 1-Nonanesulfonate, Sodium 1-Propanesulfonate, Sodium 2-Bromoethanesulfonate, Sodium Cholate Hydrate, Sodium Cholate, Sodium Deoxycholate, Sodium Lauryl Sulfate, Sodium Hexanesulfonate Anhydrous, Sodium Octyl Sulfate, Sodium Pentanesulfonate Anhydrous, Sodium Taurocholate, Sodium Taurodeoxycholate, Sodium Taurodeoxycholate Hydrate, Sodium Taurocholate, Taurocholic Acid 3-Sulfate Disodium Salt, Sodium Taurocholate, Lauryl sulfate, ursodeoxycholic acid, salts of C6-C30 fatty acids and aromatic acids, semi-synthetic derivatives thereof, and combinations thereof.

[0012] Optionally, the aqueous phase components of the nanoemulsion formulation include: SAAP-148, water, phospholipids and sodium deoxycholate; the ratio of SAAP-148, water, phospholipids and sodium deoxycholate is: 1-4 mg: 1-4 mL: 10-15 mg: 1.5-2.5 mg; the oil phase components of the nanoemulsion formulation include: crizotinib, soybean oil and MCT, and the ratio of crizotinib, soybean oil and MCT is: 1-4 mg: 10-15 mg: 10-15 mg. Preferably, the ratio of SAAP-148, water, phospholipids and sodium deoxycholate is: 2 mg: 2 mL: 10 mg: 1.5 mg; the oil phase components of the nanoemulsion formulation include: crizotinib, soybean oil and MCT, and the ratio of crizotinib, soybean oil and MCT is: 2 mg: 15 mg: 15 mg.

[0013] Optionally, the phospholipids include: soybean lecithin S 100 , phospholipids PL 100 , egg yolk lecithin E 80 , soy lecithin S 75 and other medically acceptable phospholipids.

[0014] Preferably, the phospholipids include: soybean lecithin S 100 and / or egg yolk lecithin E 80 .

[0015] The second aspect of the present invention provides a method for preparing the lung-targeted nanoemulsion formulation for co-delivering anti-tumor drugs and antibacterial drugs according to the first aspect, the method comprising:

[0016] (1) Preparation of the aqueous phase: Sterile water for injection or a hydrophilic drug dissolved in sterile water for injection is added to the phospholipids, and the mixture is emulsified by vortexing and heating in a 37°C water bath. A surfactant is then added and further vortexed. The hydrophilic drug is positively charged SAAP-148.

[0017] (2) Preparation of the oil phase: Soybean oil and MCT are added to the hydrophobic drug, and the oil phase containing the hydrophobic drug is dissolved with a small amount of dichloromethane, and the oil phase is fully dissolved by water bath ultrasound. The hydrophobic drug is crizotinib;

[0018] (3) Mixed ultrasound: the oil phase was dropped into the water phase while vortexing, and then ultrasonicated with an ice bath probe at 180 W for 5 s and 5 s for 10 min. Finally, the dichloromethane was removed by rotary evaporation to obtain a nanoemulsion formulation that co-delivered antitumor drugs and antibacterial drugs;

[0019] (4) Modification of DSPE-PEG on the outside of the nanoemulsion by post-insertion method 2000 -ACE2 targeting peptide to obtain a lung-targeted nanoemulsion formulation for co-delivery of anti-tumor drugs and antibacterial drugs.

[0020] Alternatively, DSPE-PEG is modified on the outside of the nanoemulsion using a post-insertion method. 2000 -ACE2 targeting peptides include:

[0021] Take 1 mL of the prepared nanoemulsion preparation, add 1 mg of the prepared lyophilized powder of the targeting material, gently shake and incubate in a 37°C water bath for 2 minutes. The pH should be neutral. During this period, the hydrophobic end of the amphiphilic targeting material can be inserted into the oil phase, and the hydrophilic end faces the water phase.

[0022] Optionally, the steps of preparing the freeze-dried powder of the targeting material include:

[0023] (1) Exposure of thiol groups in the peptide: Weigh 6 mg of peptide AYQ, add 500 μL of DMSO to dissolve it, and add 1 mL of PBS containing a final concentration of 5 mM TCEP dropwise while stirring. Mix under magnetic stirring for 1 h to fully expose the thiol groups in the peptide. This is referred to as the first system. At this time, the solution pH should be neutral.

[0024] (2) Reaction: Weigh 15 mg of DSPE-PEG2000-Mal and dissolve it completely in 1 mL of PBS by heating, water bath sonication, etc. Slowly add this solution dropwise to the first system and react under magnetic stirring at 25°C in the dark for 12 h. During this time, the sulfhydryl group and the maleimide will react at this neutral pH to form an irreversible, stable thioether bond.

[0025] (3) Dialysis: After the reaction is completed, the reaction solution is dialyzed using a dialysis bag with a molecular weight cutoff of 2000 Da for 48 hours to remove the organic solvent and unreacted components in the reaction system. The water is changed every 3 hours during this period.

[0026] (4) Freeze-drying: After dialysis, the product was freeze-dried to obtain a white fluffy substance, referred to as DSPE-PEG. 2000 -AYQ, sealed and stored in a -20℃ refrigerator.

[0027] A third aspect of the embodiments of the present invention provides the use of the lung-targeted nanoemulsion formulation for co-delivering anti-tumor drugs and antibacterial drugs described in the first aspect in the preparation of a bacterial symbiotic tumor treatment preparation, wherein the anti-tumor drug induces immunogenic cell death to kill the tumor, releases endogenous tumor antigens, and converts dying tumor cells into in situ antigens; and the antibacterial drug is co-delivered to eliminate tumor symbiotic bacteria and release endogenous bacterial adjuvants.

[0028] Optionally, the nanoemulsion preparation modified with DSPE-PEG2000-ACE2 targeting peptide AYQ, GQ or AY has lung targeting and tumor targeting capabilities.

[0029] Optionally, the bacterial symbiotic tumor is an in situ bacterial symbiotic lung cancer, and the preparation is used to inhibit tumor growth, reduce bacterial load, delay the course of the disease, and clear bacteria inside and outside the lungs and tumor cells; or, the bacterial symbiotic tumor is a lung metastatic bacterial symbiotic lung cancer, and the preparation is used to reduce the number and area of ​​lung metastases, and the preparation is also used to reduce bacterial content.

[0030] This study proposes a novel "in situ vaccine" strategy for the first time, leveraging synergistic anti-tumor and antibacterial immune activation. Using a nanoemulsion modified with an ACE2-targeting material, the team achieved precise delivery of anti-tumor and antibacterial drugs to the lungs and tumor sites. Simultaneously, through a spatiotemporally coupled "antigen-adjuvant co-delivery" strategy, the team effectively activated the immune response at the tumor site and throughout the body, achieving robust anti-tumor and antibacterial effects. This novel "in situ vaccine" strategy offers new insights into the treatment of bacterially symbiotic lung cancer and is expected to advance the development of precision medicine for lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0032] Figure 1A schematic diagram showing the working principle of a lung-targeted nanoemulsion formulation for co-delivering anti-tumor drugs and antibacterial drugs provided by an embodiment of the present invention is shown;

[0033] Figure 2 The dose-effect curves and IC values ​​of each anti-tumor drug on killing tumor cells in vitro are shown. 50 value;

[0034] Figure 3 The ability of each anti-tumor drug to induce ICD of tumor cells in vitro is shown;

[0035] Figure 4 The OD values ​​of different bacteria after treatment with different concentrations of antibiotics are shown. 600 Visualization of values;

[0036] Figure 5 The fluorescence intensity of eGFP in the supernatant after each antimicrobial drug acts on eGFP-E.coli is shown;

[0037] Figure 6 The ratio of macrophage polarization to M1 and M2 phenotypes induced by each antimicrobial drug is shown;

[0038] Figure 7 Shows the synthetic verification results of ACE2 targeting materials;

[0039] Figure 8 The fluorescence distribution of DiD nanoemulsions modified with three ACE2 targeting materials in lung tissue is shown;

[0040] Figure 9 The changing trends of average particle size, PDI, and Zeta potential of nanoemulsions when stored at 4°C are shown;

[0041] Figure 10 The cellular uptake of the nanoemulsion is shown;

[0042] Figure 11 The cellular uptake mechanism of nanoemulsion is shown;

[0043] Figure 12 The tumor sphere penetration ability of the nanoemulsion is shown;

[0044] Figure 13 The dose-effect curve and IC of nanoemulsion and active ingredients in killing lung cancer cells are shown. 50 value;

[0045] Figure 14 Shown are the changes in tumor sphere viability after nanoemulsion treatment;

[0046] Figure 15 The visualization results of MFI values ​​after treating different bacteria with different concentrations of antimicrobial drugs are shown;

[0047] Figure 16 The ability of nanoemulsion to eliminate commensal bacteria from tumor cells was demonstrated;

[0048] Figure 17 The ability of nanoemulsion to eliminate commensal bacteria in tumor spheres was shown;

[0049] Figure 18 The safety of the nanoemulsion and active ingredients on cells in vitro was demonstrated;

[0050] Figure 19 Demonstrated proof of concept for nanoemulsion-induced production of tumor antigens and bacterial adjuvants;

[0051] Figure 20 The distribution of nanoemulsion in various tissues at different time points after intravenous injection is shown;

[0052] Figure 21 The distribution of intravenously injected nanoemulsion in the lung and in orthotopic lung tumors is shown;

[0053] Figure 22 Shows the results of IVIS bioluminescence detection during the treatment of LLC-Luc orthotopic bacterial symbiosis in lung cancer mice;

[0054] Figure 23 Shown are the survival curves and median survival time of mice with LLC-Luc orthotopic bacterial symbiosis after treatment;

[0055] Figure 24 Shows the lung tissue status of mice with LLC-Luc orthotopic bacterial symbiosis after treatment;

[0056] Figure 25 Shows LLC-GFP-Luc orthotopic bacterial symbiosis after treatment of lung cancer mice with GFP + The proportion of cells in the lungs;

[0057] Figure 26 Shows the anti-tumor effect of LLC-Luc after treatment of lung cancer mice with orthotopic bacterial symbiosis;

[0058] Figure 27 Shows the antibacterial effect of TC1 after treatment of lung-metastatic bacterial commensal lung cancer mice;

[0059] Figure 28 The ICD index in the tumor tissue lysate after treatment is shown;

[0060] Figure 29 The infiltration of immune cells with anti-tumor effects at the tumor site after treatment is shown;

[0061] Figure 30 The proportion of Tregs in the tumor site after treatment is shown;

[0062] Figure 31 The typing of macrophages in the tumor site after treatment is shown;

[0063] Figure 32 The levels of cytokines in the tumor site after treatment are shown;

[0064] Figure 33 shows the maturation of DCs in mediastinal lymph nodes after treatment;

[0065] Figure 34 shows the activation of T cells in the spleen after treatment;

[0066] Figure 35 The figure shows the secretion of cytokines by T cells in the spleen after treatment after stimulation with tumor antigens;

[0067] Figure 36 Shown is the abundance of effector memory T cells in the spleen after treatment. DETAILED DESCRIPTION

[0068] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0069] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in this field can be used. The reagents and other instruments used, if the manufacturers are not specified, are all conventional reagents that can be purchased on the market.

[0070] The present invention proposes a new strategy of "in situ vaccine", which is to kill tumors by delivering anti-tumor drugs with ICD induction ability, releasing endogenous tumor antigens in situ; at the same time, co-deliver antibacterial drugs to eliminate tumor symbiotic bacteria, and release endogenous bacterial adjuvants in situ; and then use the tumor antigens and bacterial adjuvants produced in situ to activate the body's immune response (such as Figure 1 , which shows a schematic diagram of the working principle of the lung-targeted nanoemulsion formulation for co-delivering anti-tumor drugs and antibacterial drugs provided by an embodiment of the present invention). Specifically, the key points of this strategy are as follows:

[0071] (1) Anti-tumor drugs release endogenous tumor antigens in situ. Some anti-tumor drugs have been shown to have ICD-inducing ability, which can induce dying tumor cells to release endogenous tumor antigens, convert them into in situ vaccines, and stimulate anti-tumor immune responses. During the ICD process, tumor cells die under external stimuli and transform from a non-immunogenic to an immunogenic state, during which a series of damage-associated molecular patterns (DAMPs) are released, thereby activating the body's anti-tumor immune response. The characteristic mechanisms of this process include: promoting the release of ATP molecules by cells during the apoptotic blistering phase or necrosis phase through autophagy, which acts as a chemokine to attract dendritic cells (DCs) through purinergic receptors; triggering the exposure of calreticulin (CALR) on the cell surface through the endoplasmic reticulum stress response, which acts as an "eat me" signal to promote DCs to phagocytose dead cell fragments carrying tumor antigens; in addition, DAMPs such as high-mobility group protein 1 (HMGB-1) and heat shock proteins (HSP70, HSP90) are also released during the ICD process. These molecules initiate immune responses by binding to pattern recognition receptors (PRRs) on the surface of DCs.

[0072] (2) Antimicrobial drugs release endogenous bacterial adjuvants in situ. Although the ICD process induced by antitumor drugs can convert dying tumor cells into in situ vaccines, the intensity of the antitumor immune response triggered by this is usually limited by problems such as antigen presentation efficiency and insufficient co-stimulatory signals. The introduction of immune adjuvants can enhance this immune response to a certain extent. It is worth noting that in the environment of bacterial tumor symbiosis, the death of commensal bacteria caused by antimicrobial drugs can release a large number of microbial-associated molecular patterns (MAMPs) in situ, such as lipopolysaccharide, peptidoglycan, bacterial DNA, etc. These MAMPs released in situ can act as endogenous adjuvants, activate myeloid cells through pathways such as TLR2 / 4 / 9, and synergize with DAMPs to promote DCs maturation and presentation of tumor antigens. Therefore, by eliminating the tumor commensal flora, it can be transformed from a cancer-promoting "accomplice" to an endogenous adjuvant "factory", thereby enhancing the immune effect of ICD treatment.

[0073] (3) Co-delivery of antitumor drugs and antibacterial drugs. Based on this, the "co-delivery of antitumor drugs and antibacterial drugs" in the context of bacterial symbiotic tumors can form a spatiotemporally coupled "antigen-adjuvant co-delivery" while simultaneously achieving antitumor and antibacterial effects, thereby jointly activating a more powerful immune response in the body.

[0074] In response to this "in situ vaccine" strategy, on this basis, the embodiments of the present invention have carried out formulation design and optimization. By systematically screening potential ICD-inducing drugs and commonly used antibacterial drugs, and combining their differences in physical and chemical properties, the co-delivery of the two types of drugs was achieved using water-in-oil nanoemulsion. In addition, studies have shown that the lung tissue of clinical lung cancer patients has significantly increased levels of ACE2 receptors compared to normal patients, and tumor tissue also contains a certain level of ACE2 receptors. Therefore, the embodiments of the present invention also introduce ACE2 targeting materials, which are modified into the outer layer of the nanoemulsion by post-insertion, in order to improve the lung and tumor targeting ability and tumor penetration ability of the formulation in vivo.

[0075] The present invention presents an in vitro study of the formulation. Through a series of cytological experiments, the key role of ACE2-targeting material modification in optimizing cellular uptake, promoting nanoemulsion penetration in 3D tumor spheres, enhancing the tumor spheroid killing effect, and enhancing the ability to clear commensal bacteria was revealed. The safety of the nanoemulsion on normal cells was also evaluated, and the concept of the new "in situ vaccine" strategy proposed in the present invention was verified through a bone marrow-derived dendritic cell (BMDC) maturation experiment.

[0076] The present invention presents in vivo studies of the formulation. First, the formulation's accumulation in the lung and lung tumors was investigated, providing a basis for precise targeted delivery. The formulation's anti-tumor and antibacterial effects were then evaluated in both in situ and lung metastatic bacterial symbiotic lung cancer models, further exploring the in vivo immunological mechanisms of the formulation's therapeutic effects.

[0077] Example 1 Design and preparation of lung-targeted nanoemulsion

[0078] The present invention innovatively proposes a co-delivery strategy, which is to deliver anti-tumor drugs with immunogenic cell death (ICD) inducing ability to kill tumors and produce endogenous tumor antigens, and at the same time co-deliver antibacterial drugs to eliminate tumor symbiotic bacteria and release endogenous bacterial adjuvants, and then use the endogenous tumor antigens and endogenous bacterial adjuvants generated by the co-delivery strategy to jointly activate the body's immune response.

[0079] The present invention first identifies anti-tumor drugs with potential ICD-inducing effects, as well as antibacterial drugs widely used in clinical practice, and comprehensively evaluates and screens them using multiple in vitro indicators. On this basis, a formulation strategy for co-delivering anti-tumor and antibacterial drugs using an oil-in-water nanoemulsion system is further proposed. Given the significant differences in the physicochemical properties of the two types of drugs, the oil and water phases are used to encapsulate the anti-tumor and antibacterial drugs, respectively, and the optimal formulation is obtained through a series of optimization screening. At the same time, the present invention also introduces existing ACE2-targeting materials and modifies them into the outer layer of the nanoemulsion through post-insertion to improve the formulation's lung and tumor targeting capabilities and tumor penetration in vivo.

[0080] 1. Experimental methods:

[0081] 1.1 Selection of antitumor drugs

[0082] 1.1.1 Investigation of the ability of antitumor drugs to kill tumor cells in vitro

[0083] The CCK-8 method was used to detect tumor cell viability, and then the in vitro killing ability of various anti-tumor drugs on tumor cells was investigated.

[0084] 1.1.2 Investigation of HMGB-1 Release Levels Induced by Antitumor Drugs in Tumor Cells in Vitro

[0085] The present invention uses an HMGB-1 quantitative detection ELISA kit to detect the HMGB-1 concentration in the cell culture supernatant. The kit adopts the double antibody sandwich ELISA principle, and the kit is used according to the instructions.

[0086] 1.1.3 Investigation of ATP release levels induced by antitumor drugs in vitro

[0087] The present invention uses an enhanced ATP detection kit to measure ATP concentration in cell culture supernatants. The kit works on the principle that firefly luciferase requires ATP to provide energy to catalyze luciferin to produce fluorescence. The kit was used according to the instructions in the manufacturer's instructions.

[0088] 1.2 Selection of antimicrobial drugs

[0089] 1.2.1 Investigation of the antibacterial ability of antimicrobial drugs in vitro

[0090] The present invention determines the minimum inhibitory concentration (MIC) of each antibacterial drug against different bacteria by microplate absorbance method, thereby examining its in vitro antibacterial ability.

[0091] 1.2.2 Investigation of the ability of antimicrobial drugs to release bacterial cytoplasmic components

[0092] In order to preliminarily investigate the ability of various antimicrobial drugs to release adjuvant substances after sterilization, Escherichia coli expressing eGFP was used as a model strain and eGFP was used as a representative of cytoplasmic components. The release of bacterial cytoplasmic components was indirectly reflected by examining the fluorescence intensity of eGFP in the bacterial supernatant after the action of antimicrobial drugs.

[0093] 1.2.3 Investigation of the immunomodulatory ability of antimicrobial drugs

[0094] Under continuous stimulation by macrophage colony-stimulating factor (M-CSF), bone marrow hematopoietic stem cells activate the cell surface CSF1R receptor, initiating PI 3K / Akt and MAPK signaling pathways, driving the differentiation of mononuclear progenitor cells into mature macrophages. Based on this principle, cells were extracted from mouse bone marrow and differentiated into bone marrow-derived macrophages (BMDMs) through M-CSF stimulation. The extraction and culture methods followed established methods in the relevant art. Cells were seeded in 24-well plates at a density of approximately 1.5e6 cells per well using R10 medium and incubated in a 37°C, 5% CO2 incubator for approximately 4 hours. After stabilization, various antimicrobial agents were added. An LPS group was also included as a positive control. After 24 hours of culture, the cells were pipetted into centrifuge tubes and centrifuged at 3000 rpm for 4 minutes. The cell pellets were collected and stained by flow cytometry. After staining, M1 and M2 macrophages were phenotypically distinguished and their ratios were calculated using a flow cytometer. The anti-mouse flow cytometry antibodies used include: CD11bFITC, F4 / 80-PE, CD86-APC, CD206-APC-eFluor780.

[0095] 1.3 Preparation and formulation optimization of nanoemulsion

[0096] The basic preparation method of oil-in-water nanoemulsion is as follows:

[0097] (1) Preparation of aqueous phase: Sterile water for injection or hydrophilic drugs dissolved in it are added to phospholipids, vortexed and heated in a 37°C water bath to help emulsification, and then surfactants are added for further vortexing.

[0098] (2) Preparation of the oil phase: Soybean oil and MCT were added to the hydrophobic drug, and then a small amount of dichloromethane was used to dissolve the oil phase containing the hydrophobic drug, and the drug was fully dissolved by water bath ultrasound.

[0099] (3) Mixed ultrasound: the oil phase was dropped into the water phase while vortexing, and then ultrasonicated with an ice bath probe at 180 W for 5 s and 5 s for 10 min. Finally, the dichloromethane was removed by rotary evaporation to obtain the nanoemulsion (denoted as NE).

[0100] According to the formulation screening conditions listed in Tables 2 and 3, the composition ratios of the aqueous phase and the oil phase in the nanoemulsion formulations were screened respectively. The precipitation, average particle size (Z-Average Size), polydispersity index (Polydispersity Index, PDI), and zeta potential (ζ) were used as evaluation indicators. Finally, the nanoemulsion formulation that could form an emulsion and had a small PDI was determined and used to prepare a nanoemulsion containing CZT and SAAP-148 (denoted as CS NE).

[0101] 1.4 Selection and synthesis of nanoemulsion modified materials

[0102] 1.4.1 Investigation of the Effect of Lung Cancer Modeling on ACE2 Receptor Content in Various Tissues

[0103] An orthotopic lung cancer mouse model was established, and a non-modeling control group (naive) was established simultaneously. On day 12 after modeling, mice were euthanized, and single-cell suspensions of the heart, liver, spleen, lung, kidney, and tumor were prepared. Anti-ACE2 primary antibody was diluted 1:200 by volume in antibody diluent, and the appropriate number of cells was resuspended in this solution. The cells were mixed and incubated at 4°C for 1 hour. Staining was then terminated by adding 1 mL of PBS, and the cell pellet, collected by centrifugation, was resuspended and washed again in PBS. After centrifugation, the supernatant was discarded, and the cells were resuspended in Cy3-labeled secondary antibody, mixed, and incubated at 4°C for 40 minutes. Staining was then terminated by adding 1 mL of PBS, and the cell pellet, collected by centrifugation, was resuspended and washed again in PBS. Finally, the cells were resuspended in 200-300 μL of PBS, depending on the cell density, and filtered through a 70 μm mesh. The Cy3 positivity rate was determined by flow cytometry.

[0104] 1.4.2 Synthesis and Verification of ACE2-Targeting Materials

[0105] The reaction between the sulfhydryl group of cysteine ​​(Cysteine, C) and maleimide (Maleimide, Mal) in the ACE2 receptor affinity peptide was used to react with DSPE-PEG 2000 -Mal coupling. Taking the peptide AYQ as an example, the specific synthesis method is as follows:

[0106] (1) Exposure of thiol groups in the polypeptide: Weigh 6 mg of the polypeptide AYQ and dissolve it in 500 μL of DMSO. Add 1 mL of PBS containing a final concentration of 5 mM TCEP dropwise while stirring. Mix under magnetic stirring for 1 h to fully expose the thiol groups in the polypeptide. This is referred to as System 1. At this point, the pH of the solution should be neutral.

[0107] (2) Reaction: Weigh 15 mg of DSPE-PEG 2000 -Mal was completely dissolved in 1 mL of PBS by heating, water bath sonication, etc. This solution was slowly added dropwise to System 1 and allowed to react at 25°C under magnetic stirring in the dark for 12 hours. During this time, the sulfhydryl group and maleimide reacted at this neutral pH to form an irreversible, stable thioether bond.

[0108] (3) Dialysis: After the reaction, the reaction solution was dialyzed for 48 h using a dialysis bag with a molecular weight cutoff of 2000 Da to remove the organic solvent and unreacted components in the reaction system. The water was changed every 3 h during the period.

[0109] (4) Freeze-drying: After dialysis, the product was freeze-dried to obtain a white fluffy substance, referred to as DSPE-PEG. 2000-AYQ, sealed and stored in a -20℃ refrigerator.

[0110] To determine whether DSPE-PEG was successfully prepared 2000 -Mal, the product was dissolved in deuterated DMSO and analyzed by nuclear magnetic resonance spectroscopy ( 1 H NMR) to detect characteristic peaks, and a free thiol detection kit (DTNB method) to detect incompletely reacted free thiol groups.

[0111] 1.4.3 Preparation of DiD-loaded Nanoemulsion

[0112] Referring to the above nanoemulsion preparation method, the dichloromethane in the oil phase is replaced with a DiD stock solution prepared with dichloromethane to prepare a DiD-loaded nanoemulsion.

[0113] 1.4.4 Modification of Nanoemulsion

[0114] DSPE-PEG was modified on the outside of the nanoemulsion using the post-insertion method. 2000 -ACE2 targeting peptide (denoted as NA) or DSPE-PEG 2000 (denoted as NP), the specific method is as follows:

[0115] Take 1 mL of the prepared nanoemulsion and add 1 mg of the prepared freeze-dried powder (denoted as NA-AYQ, NA-AY, NA-GQ) or DSPE-PEG 2000 (denoted as NP), gently shake and incubate in a 37°C water bath for 2 minutes. The pH should be neutral at this time. During this period, the hydrophobic end of the amphiphilic material can be inserted into the oil phase, and the hydrophilic end faces the water phase.

[0116] 1.4.5 Investigation of lung tissue distribution after intravenous injection of nanoemulsion

[0117] Based on the prepared DiD-loaded nanoemulsions, NP, NA-AYQ, NA-AY, and NA-GQ were prepared. 100 μL of each preparation was administered to C57BL / 6 mice via tail vein injection, for a final DiD dosage of 1.5 μg per mouse. Ten hours after administration, the mice were euthanized, and lung tissue was removed, rinsed with saline, and dried with filter paper. DiD fluorescence signals in the lung tissues were imaged using IVIS and semi-quantitatively analyzed.

[0118] 1.5 Characterization of Nanoemulsion

[0119] 1.5.1 Investigation of the Encapsulation Efficiency and Drug Loading of Crizotinib in Nanoemulsion

[0120] CS NE, CS NA or CS NP were prepared as described above. The encapsulation efficiency and drug loading of CZT in the nanoemulsion were determined by ultrafiltration combined with high performance liquid chromatography (HPLC).

[0121] 1.5.2 Investigation of the adsorption rate of SAAP-148 nanoemulsion

[0122] The adsorption rate of SAAP-148 by nanoemulsion was determined by ultrafiltration combined with HPLC.

[0123] 1.5.3 Investigation of the Storage Stability of Nanoemulsion

[0124] The prepared CS NE, CS NA, or CS NP was sealed and stored at 4°C. The hydrated particle size, particle size distribution, and zeta potential of each preparation were monitored every two days using a Malvern particle size analyzer. Note that the preparation was diluted 10-fold with sterile water for injection before testing.

[0125] 2. Experimental Results and Discussion

[0126] 2.1 Selection of antitumor drugs

[0127] In this study, LLC and TC1 were used as representative lung cancer cells. Seven anti-tumor drugs were selected through literature research. These drugs were selected by examining their tumor cell killing ability and ICD-inducing ability. These included six chemotherapy drugs generally believed to have an ICD effect: cyclophosphamide (CYC), docetaxel (DOC), doxorubicin (DOX), mitoxantrone (MTN), paclitaxel (TAX), and crizotinib (CZT), a small molecule targeted drug recently reported to have a strong ICD effect on lung cancer.

[0128] 2.1.1 Antitumor drug killing ability on tumor cells in vitro

[0129] The ability to kill tumor cells is an important criterion for selecting anti-tumor drugs. The selected drugs need to show strong killing effects on lung cancer cells. The present invention uses CCK-8 method to investigate the killing ability of seven anti-tumor drugs against LLC and TC1, and the dose-effect curves and corresponding IC 50 The results are as follows Figure 2 The results showed that, in addition to cyclophosphamide and docetaxel, crizotinib, doxorubicin, mitoxantrone, and paclitaxel all had good killing effects on the two tumor cell lines in the laboratory and could be used as alternative anti-tumor drugs in the embodiments of the present invention.

[0130] 2.1.2 Ability of antitumor drugs to induce ICD in tumor cells in vitro

[0131] The inducibility of ICD is another important indicator for selecting anti-tumor drugs in the present invention. ICD refers to the process in which tumor cells change from a non-immunogenic to an immunogenic state when they die under external stimulation, during which a series of DAMPs are released, thereby activating the body's anti-tumor immune response. The characteristic mechanisms of this process include: on the one hand, the autophagy reaction prompts cells to release ATP molecules in the blistering or necrotic phase, which acts as a chemokine to attract dendritic cells (DCs) through purinergic receptors; on the other hand, the endoplasmic reticulum stress response triggers CALR exposure on the cell surface, which acts as an "eat me" signal to promote DCs to phagocytose dead cell fragments carrying tumor antigens. In addition, other DAMPs such as HMGB-1 and heat shock proteins (HSP70, HSP90) are also released during the ICD process. These molecules bind to PRRs on the surface of DCs, triggering cascade cellular responses, and ultimately activating innate immune responses and adaptive immune responses, forming a systemic anti-tumor immune effect.

[0132] For seven anti-tumor drugs, two concentrations of 6.25 μM and 3.13 μM were selected for testing. The concentration of culture supernatant was detected using HMGB-1 and ATP as representative DAMPs indicators. The measured cell supernatant concentration was normalized to Z-Score and displayed in Figure 3 middle, Figure 3 The ability of various anti-cancer drugs to induce ICD in tumor cells in vitro is shown. The high and low triangle gradients represent a high concentration of 6.25 μM and a low concentration of 3.13 μM, respectively. HMGB-1 and ATP concentrations were normalized using a Z-score, reflecting the standard deviation relative to the mean. Positive values ​​(red) indicate concentrations above the average, while negative values ​​(blue) indicate concentrations below the average. Larger absolute values ​​indicate higher correlation levels. Among the seven selected anti-cancer drugs, CZT exhibits the strongest ICD-inducing ability against these two tumor cell lines, followed by TAX.

[0133] Considering the tumor cell killing ability and ICD induction ability, CZT was subsequently selected as the preferred anti-tumor drug in the examples of the present invention.

[0134] 2.2 Selection of antimicrobial drugs

[0135] In this study, four antimicrobial agents with different mechanisms of action were selected through literature research. These agents were selected through in vitro testing of their antibacterial abilities, immunomodulatory abilities, and ability to release bacterial adjuvant components. Gentamicin, colistin, and moxifloxacin were selected as representative antibiotics, while SAAP-148, reported in the literature, was selected as a representative antimicrobial peptide.

[0136] 2.2.1 In vitro antibacterial activity of antimicrobial drugs

[0137] Antimicrobial activity is the fundamental basis for selecting antimicrobial drugs, and the selected antimicrobial drugs need to have good inhibitory effects on a variety of bacterial strains. The present invention selects Gram-positive (G+) bacteria Staphylococcus aureus (S. aureus) and Gram-negative (G-) bacteria Escherichia coli (E. coli) as representatives of common facultative anaerobic bacteria, and Gram-positive bacteria Lactobacillus iners (L. iners) as a representative of anaerobic bacteria. The antimicrobial activity is measured by examining the MIC of each antimicrobial drug. According to literature reports, Lactobacillus iners can frequently exchange genes with the host or other pathogens during proliferation, and can quickly develop resistance to a variety of antibiotics, so it is necessary to focus on its inhibitory ability.

[0138] Figure 4 OD after different bacteria were treated with different concentrations of antibiotics 600 Visualization of the values, which shows the in vitro antibacterial ability of each antibacterial drug, where the darker the blue, the OD 600 The larger the value, the greater the number of bacteria. The red check mark indicates the MIC value, which is the minimum concentration point that completely inhibits bacterial growth. 600 It is usually positively correlated with bacterial concentration and can be used to measure bacterial concentration and reflect bacterial growth. MIC is usually the minimum concentration that can inhibit bacterial growth, that is, maintain OD 600 The minimum concentration that does not increase the activity is indicated in Figure 4 by the concentration corresponding to the "red checkmark." The MIC results indicate that polymyxin E's antibacterial activity is limited to Gram-negative bacteria, with poor inhibition against Gram-positive bacteria, S. aureus and L. iners. While gentamicin exhibits some inhibitory activity against E. coli and S. aureus, it lacks antibacterial activity against Lactobacillus iners. Moxifloxacin and SAAP-148, due to their broad-spectrum antibacterial activity, exhibited good inhibitory effects against all three bacteria and are therefore suitable candidates for use in the present invention.

[0139] 2.2.2 Ability of antimicrobial drugs to release bacterial cytoplasmic components

[0140] Since the sites of action and mechanisms of action of various antibacterial drugs are different (as shown in Table 1), the bacterial components that can be released as adjuvant components during the sterilization process are also different. In view of the design of the present invention, the present invention hopes to select antimicrobial peptides that can maximize the release of bacterial adjuvant components for subsequent immune effects together with tumor antigens produced by the ICD effect of antitumor drugs. In the present invention, by detecting the eGFP fluorescence intensity of the cytoplasmic component eGFP released into the supernatant after each drug acts on eGFP-E. coli, the release of bacterial cytoplasmic components was preliminarily investigated.

[0141] Table 1 Mechanism of action of various antibacterial drugs

[0142]

[0143] The results are as follows Figure 5 As shown, Figure 5 The fluorescence intensity of eGFP in the supernatant after each antimicrobial drug was applied to eGFP-E. coli is shown. It can be seen that the eGFP fluorescence intensity in the bacterial supernatant was the highest after SAAP-148 treatment. This is likely due to SAAP-148's efficient destruction of the bacterial membrane structure, allowing the contents to be released. The eGFP intensity in the gentamicin-treated group was the lowest, suggesting that it was not conducive to the release of bacterial adjuvant components. This may be because gentamicin blocks bacterial protein synthesis, thus potentially negatively affecting eGFP and potential adjuvant components in the cytoplasm. In addition, moxifloxacin and polymyxin performed well in terms of eGFP release, but were inferior to SAAP-148. The potential reasons for this deserve further exploration. Therefore, based on its ability to release bacterial cytoplasmic components, SAAP-148 may be a more ideal antimicrobial drug in the present invention.

[0144] 2.2.3 Antimicrobial Immunomodulatory Ability

[0145] Cationic amphiphilic antimicrobial peptides are an important component of the body's innate immune defense. In addition to their antimicrobial ability, they are also believed to be able to regulate innate immune responses. LL-37 is the only antimicrobial peptide belonging to the Cathelicidins family found in the human body. It has been shown to have broad-spectrum antimicrobial activity and immunomodulatory ability. It can promote the polarization of macrophages to the M1 pro-inflammatory phenotype. In addition, it promotes the phagocytic ability of DCs and upregulates the expression of co-stimulatory molecules, thereby promoting T cell activation. It can also specifically promote extracellular cGAMP to activate the STING signaling pathway and thus promote host immune responses. The alternative antimicrobial drug selected in the present invention, SAAP-148, is modified from the antimicrobial peptide LL-37 and may therefore have potential immunomodulatory effects. In the present invention, the ratio of inducing macrophages to produce M1 phenotype and M2 phenotype is used as a representative indicator to measure the immunomodulatory ability of each antimicrobial drug. The results are as follows. Figure 6 As shown, Figure 6 The figures show the ratio of macrophage polarization to the M1 and M2 phenotypes induced by each antimicrobial drug. This indicates that SAAP-148 can induce a higher proportion of macrophage polarization to the M1 proinflammatory phenotype, while other antitumor drugs cannot. Therefore, from the perspective of immunomodulatory ability, SAAP-148 may be a more ideal antimicrobial drug for use in the present invention.

[0146] Taking into account the in vitro antibacterial ability, the ability to release bacterial cytoplasmic components, and the immunomodulatory ability, the present invention subsequently selected the antimicrobial peptide SAAP-148 as the preferred antibacterial drug.

[0147] 2.3 Preparation and formulation optimization of nanoemulsion

[0148] Based on the above results, the final anti-tumor drug selected was CZT. Because it is a highly hydrophobic drug, it can be entrapped in the inner oil phase of the oil-in-water nanoemulsion system through hydrophobic interactions.

[0149] Based on these comprehensive considerations, the antimicrobial agent ultimately selected was SAAP-148. Because it is a positively charged, water-soluble peptide, the design involved first dissolving it in the external aqueous phase and then adding the anionic surfactant, sodium deoxycholate, to the external aqueous phase. This allowed SAAP-148 to adsorb to the oil-water interface through positive and negative electrical forces, while also minimizing the cytotoxicity associated with the formulation's strong positive charge.

[0150] After determining the selected drugs, the present invention optimizes the nanoemulsion formulation for co-loading the two drugs as follows.

[0151] First, because the external aqueous phase involves the positively charged SAAP-148 and the anionic surfactant sodium deoxycholate, the present invention requires optimizing the ratio of these two components to ensure that the entire system does not precipitate due to the strong positive and negative interactions. Based on the screening schemes and results shown in Table 2, schemes C, E, and F, which did not cause precipitation, were selected for the next step of screening.

[0152] Table 2 Screening scheme and results of the dosage of nanoemulsion aqueous phase

[0153]

[0154] Next, based on experience with nanoemulsion preparation in related research, the soybean oil and MCT ratio in the oil phase was fixed at 1:1, and two dosages were selected. The oil phase dosage was further screened based on the C, E, and F aqueous phase ratios determined in the previous step. The specific protocols and results are shown in Table 3. Particle size results showed little difference among the six protocols; the PDI of the nanoemulsions prepared by Protocols 1 and 2 was more ideal, with Protocol 1 being the optimal. The potentials of the nanoemulsions prepared by each protocol varied significantly. Retaining a positive charge favors antibacterial efficacy, but excessively high positive charge can lead to strong cytotoxicity. Considering that the outer surface of the nanoemulsion will undergo further modification to reduce the potential, we believe that all potentials are within acceptable ranges. Overall, Protocol 1, which exhibited the lowest PDI, was selected for subsequent experiments.

[0155] Table 3 Screening scheme and results for the amount of oil phase after the nanoemulsion fixed the water phase ratio

[0156]

[0157] 2.4 Selection and synthesis of nanoemulsion modified materials

[0158] 2.4.1 Effect of lung cancer modeling on ACE2 receptor content in various tissues

[0159] According to relevant studies, the level of ACE2 receptors in the lung tissue of clinical lung cancer patients is increased. Accordingly, the present invention compared the levels of ACE2 receptors in various organs of healthy mice and mice with orthotopic lung cancer. The results are consistent with those reported in the literature, showing that the level of ACE2 receptors in the lung tissue of mice with orthotopic lung cancer is significantly increased, while there is no significant difference in other organs. In addition, a certain level of ACE2 receptors is also contained in the tumor tissue of mice with lung cancer. This conclusion suggests that the use of ACE2 targeting peptides for nanoemulsion modification may enhance the targeting and retention ability of the preparation for the lungs, or enhance the binding and penetration ability of the preparation for tumor tissue. Therefore, its use in the treatment of lung cancer has a certain rationality.

[0160] 2.4.2 Synthesis and Verification of ACE2-Targeting Materials

[0161] The synthesis of ACE2 targeting materials was carried out by DSPE-PEG 2000 -Mal's maleimide reacts with the sulfhydryl group on the ACE2 targeting peptide. Taking any ACE2 targeting peptide AYQ as an example, Figure 7 Demonstrated its synthesis as DSPEPEG 2000 -AYQ verification results, which show the synthesis verification results of ACE2 targeting materials. Among them, part (A) shows DSPE-PEG 2000 -AYQ and its synthetic raw materials 1 H NMR results; Part (B) shows DS PE-PEG 2000 Comparison of free thiol content between -AYQ and AYQ. 1 HNMR can be seen ( Figure 7 -(A)), DSPE-PEG 2000 -AYQ and DSPE-PEG 2000 -Mal has a PEG resonance peak at about 3.5ppm, indicating that they all have long PEG chains; DSPE-PEG 2000 -Mal has a characteristic peak of maleimide group at 6.5-7.0ppm, while DSPE-PEG 2000 -AYQ, the peak disappeared, indicating that the maleimide group was completely consumed during the synthesis process; in addition, the synthetic product showed a resonance peak between 2.5-3.5ppm that completely overlapped with the two synthetic raw materials, further indicating that DSPE-PEG 2000 -AYQ was successfully synthesized. In addition, the free thiol content results ( Figure 7 -(B)) shows that the raw material peptide AYQ contains a certain level of free thiol groups, which disappears in the synthetic product, again proving that DSPE-PEG 2000 -AYQ coupling was successful.

[0162] 2.4.3 Selection of ACE2-targeting materials

[0163] In related studies, based on the amino acid sequence of the ACE2 receptor and the novel coronavirus S protein receptor binding domain, rational design combined with computer simulation optimization screening was used to obtain nine peptides with potential binding to the ACE2 receptor (called ACE2 targeting peptides), and in vitro experiments demonstrated that seven of them had high affinity for the ACE2 receptor. In the present invention, in order to achieve a better lung targeting effect, three peptides with the highest affinity for the ACE2 receptor and containing cysteine ​​were selected for selection. The peptide sequences are shown in Table 4.

[0164] Table 4 Abbreviations and amino acid sequences of three ACE2 targeting peptides

[0165] Peptide abbreviation Amino acid sequence AY AFNCYFLLQSYGFQPTNGIGY GQ GVEGFNCYFPLQSYGFQPTNGIGYQ AYQ AFNCYFLLQSYGFQPTYGIGYQ

[0166] The lipophilic fluorescent dye DiD was used instead of the encapsulated drug to investigate the effects of three DSPEPEG 2000 -ACE2 targeting peptide modified nanoemulsion (NA) fluorescence distribution in lung tissue, and modified DSPE-PEG 2000 -Mal nanoemulsion (NP) was used as a control to preliminarily determine whether the modification of the three ACE2 targeting peptides could improve the lung targeting ability of the preparation, and the most appropriate modification material was selected accordingly. Figure 8 The fluorescence distribution of three ACE2-targeting material-modified DiD nanoemulsions in lung tissue is shown, where the IVIS imaging results 10 hours after tail vein injection are shown in (A); the fluorescence semi-quantitative statistical results of the IVIS imaging results are shown in (B). The IVIS imaging results show that ( Figure 8 -(A)), the modifications of the three ACE2 targeting peptides can improve the lung targeting ability of the preparation, although there is no significant difference in the semi-quantitative results ( Figure 8 -(B)), but the present invention selected AYQ with slightly higher fluorescence intensity for modification of nanoemulsion in subsequent experiments.

[0167] 2.5 Characterization of Nanoemulsion

[0168] 2.5.1 Encapsulation Efficiency and Drug Loading of Nanoemulsion

[0169] The results in Table 5 show that the three nanoemulsions achieved an encapsulation efficiency of approximately 90% for CZT and a drug loading of approximately 3%. The adsorption rate of SAAP-148 was less than 70% for the unmodified nanoemulsion, while the two modified nanoemulsions achieved an adsorption rate of nearly 90%. This phenomenon is speculated to be due to the additional structural modifications that further encapsulate the external aqueous phase, making it more difficult for the antimicrobial peptide adsorbed at the oil-water interface to escape from the entire system.

[0170] Table 5 Encapsulation efficiency, adsorption rate and drug loading of nanoemulsion

[0171] CZT encapsulation efficiency (%) CZT drug loading (%) SAAP adsorption rate (%) CS NE 89.14±3.45 3.20±0.17 67.14±5.24 CS NP 90.45±2.74 2.98±0.84 87.64±3.98 CS NA 92.14±2.05 3.04±0.26 89.56±3.01

[0172] 2.5.2 Storage stability of nanoemulsions

[0173] Figure 9 The results show that Figure 9 The data show the trends in average particle size, PDI, and zeta potential of the nanoemulsions when stored at 4°C. The three nanoemulsions exhibited good storage stability. They remained stable at 4°C for at least 10 days, with no significant fluctuations in average particle size, PDI, or zeta potential.

[0174] In the examples of the present invention, during the drug screening process, CZT was ultimately selected as the anti-tumor drug component by comprehensively evaluating the tumor cell killing ability and ICD-inducing ability of different anti-tumor drugs. Simultaneously, in-depth investigations were conducted on the in vitro antibacterial activity, bacterial cytoplasmic component release, and immunomodulatory capacity of various antibacterial drugs, ultimately selecting the antimicrobial peptide SAAP-148 as the antibacterial drug component. Through this multi-dimensional indicator evaluation, the present invention effectively screened candidate drugs that exhibited advantages in both killing effects and immune activation, ensuring the integration of the drug efficacy of the subsequent nanoemulsion.

[0175] In terms of formulation optimization, the present invention addresses the issue of differing water solubility between the two drugs by employing an oil-in-water nanoemulsion system. By adjusting the ratio of the antimicrobial peptide to the anionic surfactant in the external aqueous phase, a stable aqueous phase is achieved. Furthermore, by varying the amount of the internal oil phase, the particle size and potential distribution are optimized, providing a nanoemulsion formulation with excellent physicochemical properties for subsequent biological evaluation.

[0176] In addition, to achieve lung-targeted delivery, the present invention also synthesized ACE2 targeting peptide materials. The ACE2 targeting peptide was successfully coupled to DSP E-PEG through chemical reaction. 2000 The distribution of the modified nanoemulsion in the lungs was preliminarily evaluated using DiD as a fluorescent tracer. The results showed that modification of the ACE2-targeting peptide, especially AYQ modification, effectively enhanced the nanoemulsion's lung accumulation ability, providing a feasible technical solution for achieving precise drug delivery.

[0177] Finally, it was verified that the morphology, particle size distribution, potential, encapsulation efficiency, drug loading and storage stability of the nanoemulsion all met the expected requirements, and the preparation showed a good balance between stability and functionality.

[0178] In summary, the research of this invention, from drug efficacy screening to nanoformulation construction and then to the modification of targeting materials, has successfully achieved the dual needs of anti-tumor and antibacterial, co-delivery of drugs of different properties and precise targeting of the lungs, providing a solid pharmaceutical foundation for subsequent research.

[0179] Example 2 In vitro cytological study of lung-targeted nanoemulsion

[0180] 1. Experimental Methods

[0181] 1.1 Construction of an in vitro bacteria-tumor symbiotic system

[0182] The method for constructing the in vitro bacteria-tumor cell symbiotic system in the present invention is mainly based on the methods reported in related studies, and is adjusted in combination with actual conditions.

[0183] 1.2 Construction of an in vitro bacteria-tumorsphere symbiotic system

[0184] The method for constructing the in vitro bacteria-tumor sphere symbiotic system in the present invention mainly refers to the method reported by relevant research institutes and is adjusted in combination with actual conditions.

[0185] 1.3 Extraction and culture of mouse bone marrow-derived dendritic cells

[0186] Bone marrow hematopoietic stem cells and mononuclear progenitor cells, under the continuous stimulation of granulocyte-macrophage colony-stimulating factor (GM-CSF), activate the cell surface CSF2R receptor, start the JAK-STAT and MAPK signaling pathways, and drive the cells to differentiate into the dendritic cell lineage. Based on this principle, the present invention extracts cells from mouse bone marrow and differentiates them into bone marrow-derived DC cells through the stimulation of GM-CSF. Although in the culture process, in addition to DC, it may also be accompanied by the production of macrophages and granulocytes, but because macrophages usually have a strong ability to adhere to the bottom of the dish, granulocytes are usually in a suspended state, and BMDCs usually have the characteristics of semi-attached and semi-suspended. Therefore, by utilizing the characteristic differences between different cells, BMDCs cells can be simply separated. The extraction and culture methods refer to and improve the methods reported in the relevant research.

[0187] 1.4 Investigation of the Cellular Uptake Ability of Nanoemulsion

[0188] DiD, a highly lipophilic fluorescent dye, is used instead of the encapsulated drug. Cells that take up DiD will become fluorescent, and the efficiency of cellular uptake is investigated by measuring the proportion of fluorescent cells. The present invention investigated the uptake of nanoemulsions in mouse lung cancer cells LLC, human normal lung epithelial cells BEAS-2B, 293T cells that overexpress ACE2 receptors (ACE2-293T), and human lung cancer cells A549.

[0189] 1.5 Investigation of the anti-tumor ability of nanoemulsion in vitro

[0190] 1.5.1 Investigation of the ability of nanoemulsion to kill tumor cells

[0191] The present invention adopts CCK-8 method to detect tumor cell activity.

[0192] 1.5.2 Investigation of the Killing Ability of Nanoemulsion on Tumor Spheroids

[0193] The present invention uses 3D Cell Viability Assay to detect changes in tumor sphere viability after drug administration

[0194] 1.6 Investigation of the antibacterial ability of nanoemulsion in vitro

[0195] 1.6.1 Determination of Minimum Inhibitory Concentration of Rice Milk

[0196] The MIC was determined using the resazurin colorimetric method.

[0197] 1.6.2 Investigation of the ability of nanoemulsion to eliminate symbiotic bacteria of tumor cells

[0198] According to the different characteristics of E.coli and L.iners, different experiments were designed for investigation.

[0199] For E. coli, its growth and metabolism rate is relatively fast, and the colonies on Columbia blood agar plates are 2-4 mm gray-white circles, which are easy to identify and count. Therefore, counting the colonies by dropping the plates can more intuitively reflect the antibacterial effect.

[0200] For L. iners, its growth and metabolism rate is slow, the environment required is harsh, and the colonies on Columbia blood agar plates are small, transparent circles that are difficult to identify and count. Therefore, it is difficult to directly count the colonies using the drop plate method. D-alanine is an important component of bacterial cell wall peptidoglycan and is easily taken up by living bacteria and metabolized into cell wall peptidoglycan. Therefore, fluorescently labeled D-alanine can achieve specific and efficient labeling of bacteria in the presence of bacterial cells. Therefore, blue fluorescently labeled D-alanine (HADA) is used to indirectly measure bacterial content.

[0201] 1.6.3 Investigation of the ability of nanoemulsions to eliminate symbiotic bacteria in tumor spheres

[0202] LLC tumor spheres and L. iners were selected as representatives of tumor spheres and bacteria, respectively, and the ability of free SAAP-148 and two nanoemulsions (NP, NA) to eliminate the symbiotic bacteria of tumor spheres was investigated.

[0203] 1.7 In vitro safety study of nanoemulsions

[0204] The present invention examines the cytotoxicity of free drugs and two nanoemulsions to normal cells by using the CCK-8 method.

[0205] 1.8 Proof of concept of nanoemulsion-induced production of tumor antigens and bacterial adjuvants

[0206] The present invention utilizes a Transwell system to investigate the effects of anti-tumor production of tumor antigens and / or antibacterial production of bacterial adjuvants on BMDC maturation in the presence of tumor and bacteria, thereby providing conceptual validation of the present invention. The specific steps are as follows:

[0207] (1) According to the above steps, nanoemulsions co-loaded with CZT and SAAP and modified with ACE2 targeting peptide (CS NA), nanoemulsions single-loaded with CZT and modified with ACE2 targeting peptide (C NA), and nanoemulsions single-loaded with SAAP and modified with ACE2 targeting peptide (S NA) were prepared respectively.

[0208] (2) LLC were seeded at a high density in the upper chamber of a Transwell in advance. After culturing for approximately 4 hours, L. iners were added. BMDCs were collected and seeded in the lower chamber of the Transwell. The previously prepared upper chamber was then placed in the lower chamber, and each preparation was added to the upper chamber.

[0209] (3) After 24 h of co-culture, BMDCs were collected and surface stained with CD11c-superbright600, CD40-PE, CD80-FITC, and CD86-APC, and analyzed by flow cytometry. The supernatant of the upper chamber cell culture was also collected for TNF-α and IL-6 cytokine detection.

[0210] 2. Experimental Results

[0211] 2.1 Cellular uptake ability of nanoemulsion

[0212] Figure 10 The cellular uptake of nanoemulsion is shown, wherein (A) shows the quantitative results of nanoemulsion uptake by flow cytometry; (B) shows the uptake of nanoemulsion observed by confocal fluorescence microscopy, with a scale of 50 nm. The flow uptake results show ( Figure 10-(A)), for 293T cells that contain almost no ACE2 receptors, the percentage of DiD-positive cells in the CS NA group and the CS NP group was low and there was no significant difference; for ACE2-293T cells that highly express ACE2 receptors, the percentage of DiD-positive cells of CSNA increased significantly, indicating that the modification of ACE2 targeting peptide significantly improved the cell's ability to take up nanoemulsion; this improvement effect also existed in LLC and BEAS-2B, among which the improvement ability for LLC was 4 times, while the improvement for BEAS-2B was only 1.5 times, suggesting that the modification of ACE2 targeting peptide may be more significant for the improvement of tumor cell uptake. The significant improvement of tumor cell uptake by CS NA compared to CS NP can also be verified under confocal microscope ( Figure 10 -(B)).

[0213] 2.2 Cellular uptake mechanism of nanoemulsion

[0214] The above results show that the modification of ACE2 targeting peptide significantly improves the uptake ability of nanoemulsion for cells with high expression of ACE2 receptors. In order to investigate whether this enhancement effect is to convert the nanoemulsion entry pathway from other pathways to the ACE2 receptor-mediated entry pathway, the present invention further explores the uptake mechanism of different nanoemulsions. The present invention uses DiD, a strong lipophilic fluorescent dye, instead of the encapsulated drug, and adds competitive inhibitors of different receptors (Table 6) for pretreatment before nanoemulsion administration, and then reveals its uptake pathway by comparing whether the addition of inhibitors affects the uptake efficiency of nanoemulsion. Using 293T cells (ACE2-293T) with high expression of ACE2 receptors as a control, the changes in the uptake pathway of CS NA compared to CS NP are examined to determine the mechanism by which CS NA enhances the uptake of the corresponding cells. Uptake inhibitor entry pathway and concentration.

[0215] Table 6 Abbreviations, concentrations and cellular entry pathways of competitive inhibitors

[0216] abbreviation Uptake competitive inhibitors Preparation concentration (μg / mL) Endocytosis pathway Control none / / 4℃ Store at 4℃ / Energy suppression <![CDATA[ACE2]]> <![CDATA[ACE2-targeting peptide AY]]> 300 <![CDATA[Mediated by ACE2 receptor]]> Nystatin Nystatin 250 Caveolin-mediated DS Dextran sulfate 100 scavenger receptor-mediated Amiloride Amiloride 26 Macropinocytosis Chlo Chlorpromazine 70 Clathrin-mediated PLL Polylysine 200 Adsorption-mediated MβCD Methyl β-cyclodextrin 120 Lipid raft-mediated

[0217] The results are as follows Figure 11 As shown, Figure 11The cellular uptake mechanism of the nanoemulsion is shown. The relative percentages and significant differences in uptake positive rates after the addition of different receptor competitive inhibitors were calculated, with no competitive inhibitor added as the reference. For both cell types, the uptake of CS NPs and CS NAs was energy-dependent and could be significantly inhibited at 4°C. CS NP uptake by both cells was primarily dependent on scavenger receptors, macropinocytosis, and clathrin-mediated entry pathways. Competitive inhibition of all three pathways reduced CS NP uptake to approximately 50%-75%. However, CS NA uptake by both cells was primarily dependent on the ACE2 receptor. Competitive inhibition of this receptor significantly reduced CS NA uptake to approximately 30%-50%. The approximately 20% reduction in uptake caused by competitive inhibition of the scavenger receptor and macropinocytosis pathways was negligible. Therefore, it can be concluded that for both ACE2-293T and LLC, CS NA enhances nanoemulsion uptake primarily by diverting CS NP uptake from other pathways to the ACE2 receptor-mediated entry pathway.

[0218] 2.3 Tumorsphere penetration ability of nanoemulsion

[0219] The present invention uses LLC tumor spheres as an example, using DiD, a highly lipophilic fluorescent dye, instead of the encapsulated drug, and the position distribution of DiD fluorescence is used to show the penetration depth of the preparation in the tumor spheres. Figure 12 As shown, Figure 12 The tumor sphere penetration ability of nanoemulsion is shown. The confocal Z-axis scanning interval is 20 μm. CS NA can penetrate deeper into the tumor sphere, while CSNP can only penetrate to the periphery of the tumor sphere.

[0220] 2.4 Antitumor activity of nanoemulsion in vitro

[0221] 2.4.1 Tumor cell killing ability of nanoemulsion

[0222] The present invention investigated the in vitro killing ability of two drug-loaded nanoemulsions (CS NP and CS NA) and their active ingredients (CZT and SAAP) on three lung cancer cells (LLC, TC1, A549) using the CCK-8 method. 50 Value Figure 13 As shown, Figure 13 The dose-effect curve and IC of nanoemulsion and active ingredients in killing lung cancer cells are shown. 50 Values, where (A) represents LLC lung cancer cells; (B) represents TC1 lung cancer cells; (C) represents A549 lung cancer cells. 50 The values ​​show that the antibacterial component SAAP-148 has a negligible killing effect on lung cancer cells. In addition, CZT, CS NP, and CS NA all have good tumor cell killing ability.

[0223] 2.4.2 Tumor spheroid killing ability of nanoemulsion

[0224] Taking LLC tumor spheres as an example, by detecting the changes in tumor sphere activity after drug administration, we investigated whether the modification of ACE2 targeting peptides can promote the killing ability of anti-tumor drugs on tumor spheres. Figure 14 As shown, Figure 14 The figure shows the changes in tumor sphere viability after nanoemulsion treatment; CS NA has the strongest tumor sphere killing ability compared with free CZ T and CS NP, which may be attributed to the improved tumor sphere penetration ability exhibited by nanoemulsion.

[0225] 2.5 In vitro antibacterial ability of nanoemulsion

[0226] 2.5.1 Minimum inhibitory concentration of nanoemulsion

[0227] The present invention selected Gram-positive bacteria S.au and Gram-negative bacteria E.coli as common facultative anaerobic bacteria representatives, and Gram-positive bacteria L.iners as anaerobic bacteria representatives, and investigated the antibacterial ability of free SAAP-148 and two nanoemulsions (CS NP, CS NA). 600 Because the absorbance value is affected by the presence of the nanoemulsion, this experiment did not use the absorbance method. Instead, the MIC was determined using the resazurin colorimetric method. Resazurin is a weakly fluorescent blue dye that, after bacterial uptake, is reduced by metabolic intermediates to the highly fluorescent resorufin, which then appears pink. Fluorescence detection can circumvent the interference of nanoemulsions on absorbance. Figure 15 Visualization results of MFI values ​​after treating different bacteria with different concentrations of antimicrobial drugs ( Figure 15 The in vitro antibacterial ability of the nanoemulsion is visualized based on the fluorescence intensity of the metabolites. The blue color represents the fluorescence of the unmetabolized resazurin itself, and the pink color represents the fluorescence of the resazurin metabolites, which are positively correlated with the metabolic activity of the bacteria. The white check mark represents the MIC value, which is the minimum concentration point that completely inhibits the metabolic growth of the bacteria. The MFI is positively correlated with the metabolic activity of the bacteria and can be used to reflect the bacterial growth. The MIC is usually the minimum concentration that can inhibit bacterial growth, that is, the minimum concentration that keeps the bacteria from metabolizing and producing fluorescent substances. Figure 15 The concentrations corresponding to the white checkmarks are shown in the table. The MIC results show that free SAAP-148, CSNP, and CSNA all have good inhibitory effects on the three bacteria.

[0228] 2.5.2 Ability of Nanoemulsion to Eliminate Tumor Cell Symbiotic Bacteria

[0229] The present invention selected Gram-negative bacteria E. coli as a representative of facultative anaerobic bacteria and Gram-positive bacteria L. iners as a representative of anaerobic bacteria, and investigated the ability of free SAAP-148 and two nanoemulsions (NP, NA) to eliminate the symbiotic bacteria of LLC lung cancer cells. Figure 16 As shown, Figure 16 The ability of nanoemulsion to eliminate tumor cell symbiotic bacteria is shown, including: (A) drop plate photo and (B) colony count of the original solution calculated based on the 10-3 dilution factor after nanoemulsion eliminated tumor cell symbiotic E. coli; (C) microplate reader HADA average fluorescence intensity statistics and (D) flow cytometry quantitative results of HADA positive cells after nanoemulsion eliminated tumor cell symbiotic L. iners. It can be seen that whether it is for lung cancer symbiotic E. coli ( Figure 16 -(A) / (B)) or L.iners( Figure 16 Compared with the SAAP-148 and CSNP groups, the CSNA group showed a stronger ability to eliminate commensal bacteria. This effect may be attributed to the enhanced tumor cell uptake of the nanoemulsion, which enables the effective elimination of some intracellular commensal bacteria.

[0230] 2.5.3 Ability of Nanoemulsion to Eliminate Symbiotic Bacteria in Tumor Spheroids

[0231] The present invention selected LLC tumor spheres and L. iners as representatives of tumor spheres and symbiotic bacteria, respectively, and indirectly measured the ability of free SAAP-148 and two nanoemulsions (NP, NA) to eliminate tumor sphere symbiotic bacteria by HADA. Figure 17 As shown, Figure 17 The data demonstrate the ability of nanoemulsions to eliminate symbiotic bacteria in tumor spheres, specifically demonstrating the statistical results of the mean fluorescence intensity (HADA) of the microplate reader after nanoemulsions eliminated the symbiotic Lactobacillus iners in tumor spheres. CS NA exhibited a stronger ability to eliminate symbiotic bacteria in tumor spheres than both the SAA P-148 and CS NP groups. This effect may be attributed to the enhanced tumor sphere penetration demonstrated by the nanoemulsions, enabling the effective elimination of bacteria colonized within the tumor spheres.

[0232] 2.6 In vitro cellular safety of nanoemulsions

[0233] The present invention investigated the cytotoxicity of free drug and two nanoemulsions in human embryonic lung fibroblasts (HELF) and human normal lung epithelial cells (BEAS-2B) by CCK-8 method, aiming to make a preliminary evaluation of the safety of nanoemulsions. Figure 18 As shown, Figure 18The safety of the nanoemulsion and its active ingredients on in vitro cells is shown, including: (A) cytotoxicity of the nanoemulsion and its active ingredients on HELF; (B) cytotoxicity of the nanoemulsion and its active ingredients on BEAS-2B. The antibacterial ingredient SAAP-148 showed no significant cytotoxicity to either cell type; free CZT showed significant cytotoxicity above 13.9 μM; CS NA, through encapsulation and modification of CZT, was able to reduce its cytotoxicity, showing significant cytotoxicity only at 111 μM; however, CS NP, which also encapsulated and modified CZT, also showed significant cytotoxicity, presumably due to an overly positive potential.

[0234] 2.7 Proof of Concept of Nanoemulsion-Induced Production of Tumor Antigens and Bacterial Adjuvants

[0235] After confirming that nanoemulsions have good anti-tumor and antibacterial effects, the present invention aims to verify the concept of the basic idea of ​​"nanoemulsions clearing tumors and symbiotic bacteria and then producing tumor antigens and bacterial adjuvants" in the inventive concept. The present invention uses a Transwell system, inoculating LLC and L. iners in the upper chamber and BMDCs in the lower chamber, respectively, to examine the effects of anti-tumor production of tumor antigens and / or bactericidal production of bacterial adjuvants on BMDC maturation, thereby verifying the concept of the invention. The results are as follows Figure 19 As shown, Figure 19 The proof of concept of nanoemulsion-induced production of tumor antigens and bacterial adjuvants is shown, indicating that the nanoemulsion has antibacterial and / or antitumor effects and stimulates BMDCs to mature, express co-stimulatory molecules and secrete cytokines; after CS NA clears tumors and bacteria, the BMDCs maturation ability is stronger than that caused by CNA clearing tumors and S NA clearing bacteria, indicating that the inventive concept of the present invention is established.

[0236] The present invention evaluates the uptake ability of different cells on nanoemulsions, and through positive and negative control experiments, clarifies the key role of modification of ACE2 targeting materials in improving the uptake efficiency of ACE2 high-expressing cells.

[0237] The present invention utilizes a pretreatment method with multiple receptor competitive inhibitors to compare and analyze the dependence of nanoemulsions of modified and unmodified ACE2-targeting materials on different endocytic pathways. The results show that ACE2 receptor-mediated endocytosis dominates in modified nanoemulsions, while other pathways (such as scavenger receptors, macropinocytosis, and clathrin-mediated uptake) are relatively less important. This result not only reveals the molecular basis of the ACE2 targeting strategy, but also provides a theoretical basis for the design of nanoemulsions.

[0238] Furthermore, the present invention evaluated the ability of the nanoemulsion to penetrate tumors by constructing a 3D tumor spheroid model. Fluorescent dye labeling results intuitively demonstrated the advantages of the modified nanoemulsion in penetrating into the tumor spheroids, providing evidence for its enhanced tumor-killing ability. Next, the present invention investigated the anti-tumor and antibacterial properties of the nanoemulsion at both the 2D and 3D levels. The results demonstrated that the nanoemulsion modified with the ACE2-targeting material exhibited excellent efficacy in both areas, with the effects being even more pronounced at the 3D level.

[0239] In the cell safety assessment, the present invention compared the toxicity of free drugs and different preparations in normal human lung cells and human embryonic lung fibroblasts, and verified the improvement effect of ACE2 targeted modification in reducing drug toxicity.

[0240] Finally, the present invention conducted a proof-of-concept study on the immune activation induced by nanoemulsion after tumor and commensal bacteria clearance using the Transwell system, demonstrating the advantages of this strategy in inducing the expression of co-stimulatory molecules and cytokine secretion in BMDCs.

[0241] Example 3 Study on tissue targeting of targeted nanoemulsion

[0242] 1. Experimental Methods

[0243] 1.1 Investigation of the distribution of intravenously injected nanoemulsion in the lung and in situ lung tumors

[0244] In this study, the distribution of DiD-loaded nanoemulsion in the lung and tumor was investigated by frozen section method, and the content of coumarin-6-loaded nanoemulsion in the lung and tumor was quantified by flow cytometry. The two methods together explained the distribution of nanoemulsion in the lung and tumor after intravenous injection.

[0245] 1.2 Co-localization of intravenously injected nanoemulsion and ACE2 receptor

[0246] In this experiment, coumarin-6 was used as a model drug to trace the distribution of nanoemulsion in vivo, and the co-localization of nanoemulsion with ACE2 receptors in the lungs and lung tumors was investigated by section staining.

[0247] 2. Experimental Results

[0248] 2.1. Distribution of intravenously injected nanoemulsion in various tissues

[0249] The present invention utilizes DiD, a highly lipophilic fluorescent dye, to replace the encapsulated drug, and the distribution of the preparation in each organ is reflected by the fluorescence intensity of DiD in each organ. Figure 20The distribution of DiD in various tissues at different time points after intravenous injection of nanoemulsion is shown, where (A) represents the fluorescence intensity of DiD in the heart (Heart, H), liver (Liver, L), spleen (Spleen, S), lung (Lung, Lu), kidney (Kidney, K), and tumor (Tumor, T) observed by IVIS at different time points; (B) represents the semi-quantitative statistical results of fluorescence in lung tissue at different time points. Figure 20 -(A) The IVIS images shown show that the nanoemulsion modified with ACE2 targeting peptide has the highest distribution in the lungs at all three time points compared with the unmodified group. Figure 20 -(B) Semi-quantitative results of lung tissue fluorescence showed that the fluorescence intensity of DiD increased at 10 h compared with that at 4 h, and decreased slightly at 15 h. It is speculated that the peak distribution of DiD in the lung is around 10 h.

[0250] 2.2. Distribution of intravenously injected nanoemulsion in the lung and in situ lung tumors

[0251] After demonstrating that modification of ACE2 targeting peptides can enhance the distribution of nanoemulsions in the lung tissue of healthy mice, we further explored whether modification of ACE2 targeting peptides can enhance the distribution of nanoemulsions in lung tumors. Figure 21 The distribution of intravenously injected nanoemulsion in the lung and in situ lung tumors is shown, including: (A) DiD fluorescence intensity in lung and lung tumor sections, scale bar is 100μm; (B) Coumarin-6 positive rate in lung and lung tumors by flow cytometry. The present invention established an in situ lung cancer mouse model and investigated the distribution of DiD-loaded nanoemulsion in the lung and tumors by frozen section method ( Figure 21 -(A)), and the content of coumarin-6 nanoemulsion in lung and tumor was quantified by flow cytometry ( Figure 21 -(B)). Together, these two methods demonstrate that nanoemulsions modified with ACE2-targeting peptides not only enhance their distribution in the lungs but also significantly enhance their distribution in lung tumors after intravenous injection.

[0252] The present invention used an IVIS imaging system to investigate the organ distribution of DiD-labeled nanoemulsions at different time points. The results demonstrated that nanoemulsions modified with ACE2-targeting materials significantly enhanced DiD fluorescence signals in the lungs. Next, to further verify whether the modified nanoemulsions, while increasing lung accumulation, also increased distribution within lung tumors, an orthotopic lung cancer mouse model was constructed. Using confocal microscopy and flow cytometry quantitative analysis, the present invention visually demonstrated enhanced accumulation of the nanoemulsions modified with ACE2-targeting materials in lung and tumor tissues. Furthermore, the present invention compared ACE2 receptor expression levels in various organs of healthy mice and mice with lung cancer. The results demonstrated that orthotopic lung cancer modeling significantly increased ACE2 receptor levels in lung tissue, while a certain level of ACE2 expression was also detected in lung tumor tissue. This conclusion further supports the finding that nanoemulsions modified with ACE2-targeting materials achieve precise delivery to the lungs and tumor sites, and provides a biological basis for the present invention's use of the nanoemulsions in the treatment of lung cancer.

[0253] Example 4 In vivo pharmacodynamic study of lung-targeted nanoemulsion in the treatment of in situ bacterial symbiotic lung cancer

[0254] 1. Experimental methods:

[0255] 1.1 Establishment of in situ lung cancer model or in situ bacterial symbiotic lung cancer model

[0256] In this experiment, tumor cells were inoculated into the lungs of mice by surgical injection. The specific method is as follows:

[0257] (1) Preliminary preparation of mice: 5-week-old C57BL / 6 mice were given 0.2 mg / mL doxycycline in sterile drinking water for 5 days to eliminate the interference of existing bacteria in the body. They were then given sterile drinking water for 3 days to metabolize the doxycycline in the body. 2 days before tumor implantation, the chest cavity and left ribs of the mice were depilated using a shaver and depilatory cream. Care was taken to avoid irritation to the skin caused by residual depilatory cream.

[0258] (2) Cell line selection and preparation: To facilitate the observation of lung cancer cell growth in vivo, LLC-Luc cells with a luciferase (Luc) label were selected. LLC-Luc cells in the logarithmic growth phase were collected and counted, resuspended in pre-cooled sterile PBS, and then mixed with matrigel at a volume ratio of 1:1 to ensure that the tumor cell concentration after mixing was 5e5 cells / 100 μL. The mixture was placed on ice for use. Note that everything in contact with the matrigel during the operation needs to be pre-cooled and placed on ice to prevent the matrigel from solidifying.

[0259] (3) Preparation before tumor grafting: All instruments were first disinfected by soaking in ethanol and then in 50 μg / mL gentamicin. Mice were anesthetized with tribromoethanol gas. After the mice were anesthetized from the excited state to the deep anesthesia state, they were fixed in the right lateral position and maintained under low-dose tribromoethanol inhalation. The chest surface was disinfected with iodine tincture.

[0260] (4) Tumor grafting in mice: The incision was made about 1 cm above the lower edge of the left rib of the mouse (i.e., between the fourth and fifth ribs). After hair removal, two relatively thick blood vessels extending longitudinally were visible at this site. The left lung (largest lobe) of the mouse was located between these two blood vessels. The skin was lifted with tweezers, and a small incision of about 5 mm was first made in the epidermis with surgical scissors. The underlying subcutaneous tissue and muscle tissue were further cut along this small incision, taking care to avoid the abundant blood vessels in this area. The lungs were pink and white, visible through the pleura, and contracted and expanded at the same frequency as the mouse's breathing. 50 μL of the mixed cell suspension was taken, and after locating the left lung, the needle was inserted about 3 mm and the tumor cells were slowly injected. After the injection, the needle was stopped for about 5 seconds, and then the needle was slowly removed by rotating left and right. This was recorded as day 0.

[0261] (5) Mouse wound closure: Use wound clips to close the skin incision of the mouse, and then drip 50 μg / mL gentamicin and iodine to prevent wound infection. Place the mouse in a right lateral position in a warm environment until the mouse wakes up, then return it to the original cage and continue to raise it. The wound will heal after about a week.

[0262] (6) This modeling method can capture Luc bioluminescence using IVIS on the 5th day after tumor implantation.

[0263] 1.2 Construction of an in situ bacterial symbiotic lung cancer model in mice

[0264] In this experiment, tumor cells and bacteria were co-inoculated into the lungs of mice by surgical injection. The rest of the method was consistent with the above method except for the second step, in which the second step was modified as follows:

[0265] Bacterial and Cell Line Selection and Preparation: To characterize the in vivo growth of lung cancer cells, luciferase-tagged LLC-Luc cells were selected. Lactobacillus iners, a strain of bacteria that has been shown to promote lung cancer progression, was used to establish a bacteria-tumor cell symbiosis model. Bacteria were added at the desired ratio according to the experimental requirements. LLC-Luc cells containing Lactobacillus iners were harvested and counted, resuspended in pre-chilled sterile PBS, and mixed with Matrigel at a 1:1 volume ratio to achieve a tumor cell concentration of 5e5 cells / 100 μL. The mixture was placed on ice until ready for use. All items that came into contact with Matrigel must be pre-chilled and kept on ice to prevent the Matrigel from solidifying.

[0266] 2. Experimental Results

[0267] 2.1 Antitumor effect of nanoemulsion on in situ bacterial symbiotic lung cancer

[0268] 2.1.1 Bioluminescence results of mouse tumors

[0269] The treatment plan of the in situ bacterial symbiotic lung cancer model specifically includes: constructing an in situ lung cancer model or an in situ bacterial symbiotic lung cancer model, and the day of tumor inoculation is recorded as day 0. On the 5th day, the Luc bioluminescence is photographed by IVIS, and the groups are divided accordingly to ensure that the conditions of each group are basically the same. Tail vein administration begins on the 6th day, and is administered once every 3 days, for a total of 4 times. The dosage is 5 mg / kg of CZT equivalent. In the embodiment of the present invention, an LLC-Luc in situ bacterial symbiotic lung cancer model is constructed and administered. During the treatment process, the present invention monitors the bioluminescence signals of mice to track the growth trend of tumors in the lungs of each group of mice. Figure 22 The results of IVIS bioluminescence detection during the treatment of LLC-Luc in situ bacterial symbiotic lung cancer mice are shown, including: (A) representative images and (B) semi-quantitative trend graphs. The initial n = 8 for each group, and statistics were stopped when more than 4 mice died. Significant differences were calculated based on the bioluminescence intensity on day 17. Figure 22 -(A) shows the semi-quantitative statistical results of bioluminescence. Figure 22 -(B). As can be seen, bioluminescence in the untreated control group increased rapidly over time, and the growth rate continued to increase. By day 17, the lung tumors were already quite severe, with the tumor lesions spreading from the left lung to the right lung. Among the treatment groups, the CS NA group showed the best effect, with the daily increase in bioluminescence remaining at the lowest level. By day 23, it only reached the level of the other treatment groups on day 11. The remaining treatment groups showed some effect, but were inferior to the CS NA group.

[0270] 2.1.2 Monitoring of mouse survival

[0271] An LLC-Luc in situ bacterial symbiotic lung cancer model was constructed and drug administration was performed. By monitoring the survival of mice and drawing the survival curves of each group of mice, the median survival time of each treatment group could be calculated. The results are as follows: Figure 23 As shown, Figure 23 The survival curves and median survival of mice bearing LLC-Luc orthotopic bacterial symbionts after treatment are shown. The CS-NA group had the longest survival, with a median survival of 41.5 days, 23 days longer than the untreated group and approximately 1.8 times that of the CS, CS NP, and SNA treatment groups. The C-NA group had the second-best median survival of 33.5 days, but this was still 8 days shorter than the CS-NA group.

[0272] 2.1.3 Photography and H&E sections of mouse lung tissue after treatment

[0273] Constructing LLC-Luc in situ bacterial symbiotic lung cancer model and administering drug, Figure 24 Figure 2 shows the lung tissue of mice treated with LLC-Luc in situ bacterial symbiosis for lung cancer. (A) shows a photograph of lung tissue; (B) shows a representative H&E section of lung tissue. After the treatment cycle, the lung tissue containing the tumor was dissected and photographed ( Figure 24 -(A)), representative samples were selected for paraffin sectioning and H&E staining ( Figure 24 -(B)), it can be seen intuitively from the figure that the CS NA group had the smallest lung tumor area after treatment.

[0274] 2.1.4 The proportion of tumor cells in lung cells in mice after treatment

[0275] An LLC-GFP-Luc in situ bacterial symbiotic lung cancer model was constructed and drug administration was performed. After the administration period, the GFP positive rate in the single cell suspension of lung tissue was detected to reflect the content of tumor cells. Figure 25 As shown, Figure 25 Shows LLC-GFP-Luc orthotopic bacterial symbiosis after treatment of lung cancer mice with GFP + The proportion of GFP-positive tumor cells in the lungs was significantly reduced in all three groups of nanoemulsions modified with ACE2-targeting materials compared to the untreated group and the other two treatment groups. However, although the average value of the CS NA group was the smallest, it was not statistically different from the other two groups.

[0276] 2.2 Antibacterial effect of nanoemulsion on in situ bacterial symbiotic lung cancer

[0277] Figure 26 The figure shows the antibacterial effect of LLC-Luc after treatment of mice with lung cancer symbiotic with in situ bacteria. (A) shows the schematic diagram of tumor and bacterial fluorescence characterization; (B) shows the proportion of cells containing bacteria in the lungs of mice after treatment; (C) shows the proportion of tumor cells containing bacteria in the tumors of mice after treatment; (D) shows the comparison of the intracellular and extracellular content of bacteria in the lungs of mice after treatment. Figure 26 -(A)), the bacterial content in the tumor tissues of the CS NA group and the S NA group was relatively low, indicating that both preparations had good antibacterial effects and effectively eliminated tumor symbiotic bacteria. From the bacterial levels in the lung tumors of mice after treatment ( Figure 26 -(B)), except for C NA in the treatment group, there was no significant difference in the other groups. In addition, the bacterial content inside and outside the lungs of mice after treatment ( Figure 26 -(C)), the CS NA group had a better bacterial clearance effect than the other groups.

[0278] In terms of anti-tumor efficacy, the present invention utilizes in vivo bioluminescence imaging to dynamically monitor tumor growth, combined with mouse survival analysis, to reveal the advantages of different treatment strategies in prolonging survival. Furthermore, lung tissue imaging and histopathological H&E staining provide intuitive evidence of tumor progression. Quantitative analysis of GFP-labeled tumor cells using flow cytometry provides objective data support for efficacy evaluation. Through comprehensive evaluation of various indicators, the present invention systematically demonstrates the significant efficacy of CSNA in inhibiting tumor growth, reducing bacterial load, and delaying disease progression.

[0279] In terms of antibacterial effect, the present invention tested the bacterial levels in the lungs and tumors after treatment, proving that the CS NA group was able to eliminate bacteria inside and outside the lungs and tumor cells, fully demonstrating the advantages of nanoemulsion in bacterial clearance.

[0280] Example 5 In vivo pharmacodynamic study of lung-targeted nanoemulsion in the treatment of lung-metastatic bacterial symbiotic lung cancer

[0281] 1. Experimental methods:

[0282] 1.1 Construction of a mouse lung metastasis lung cancer model

[0283] In this experiment, a mouse lung cancer metastasis model was established by injecting tumor cells into the tail vein. The specific method is as follows:

[0284] (1) Preliminary preparation of mice: 5-week-old C57BL / 6 mice were given 0.2 mg / mL doxycycline in sterile drinking water for 5 days to eliminate the interference of existing bacteria in the body. They were then given sterile drinking water for 3 days to metabolize the doxycycline in the body.

[0285] (2) Tumor Implantation in Mice: Following the method described in 1.2.2.4, TC1 cells in the logarithmic growth phase were collected and counted. The cells were resuspended in pre-chilled sterile PBS to a tumor cell concentration of 4e5 cells / 100 μL and placed on ice until use. Each mouse was injected with 4e5 TC1 cells via the tail vein, and the day was designated as day 0. With this modeling method, a small number of obvious lung metastases were observed in the lungs 13 days after tumor implantation.

[0286] 1.2 Construction of a mouse lung metastasis bacterial symbiotic lung cancer model

[0287] This experiment used tail vein injection of tumor cells and nasal instillation of bacteria to establish a lung metastasis bacterial symbiotic lung cancer model. This method is closer to the actual situation, that is, bacteria enter the lungs through the upper respiratory tract. In addition to the above steps, the following additional steps are also included:

[0288] Lactobacillus iners was collected, resuspended in sterile PBS, and administered to mice via nasal drops on days -1, 3, and 5, respectively.

[0289] 2. Experimental Results

[0290] 2.1 Antitumor effect of nanoemulsion on lung cancer with symbiotic bacteria metastasizing to the lung

[0291] A lung metastasis bacterial symbiotic lung cancer model was established and drug administration was performed. On the 22nd day, the lung tissues of the mice were removed and metastasis analysis was performed. Figure 26 Figure 2 shows the anti-tumor effect of TC1 lung metastatic bacterial symbiotic lung cancer mice treated with the drug, where (A) shows Bouin's fixation of mouse lung tissue after treatment and photographed; (B) shows representative H&E sections of mouse lung tissue after treatment, with a 2 mm scale; (C) shows the number of lung metastases in H&E sections of mouse lung tissue after treatment; and (D) shows the ratio of lung metastasis area to lung area in H&E sections of mouse lung tissue after treatment.

[0292] The photo of mouse lung tissue fixed with Bouin's fixative is shown in the figure below. Figure 26 -(A) shows that the tumor tissue is whiter than the lung tissue, which shows that the CS NA group has the best therapeutic effect on tumor lung metastasis. After paraffin sectioning and H&E staining of the lung tissues of each group, the representative results are shown in Figure 2. Figure 26 -(B), and the number of lung metastases was counted ( Figure 26 -(C)), although there was no statistically significant difference between the CS NA group and other nanoemulsions in this indicator, considering the differences in the area of ​​individual metastatic lesions, the ratio of the area of ​​lung metastases to the lung area was further statistically analyzed. The statistical results are shown in Figure 26 -(D), it can be seen that the area ratio of lung metastases to lungs was the smallest after CS NA treatment, indicating that CS NA had the best therapeutic effect.

[0293] 2.2 Antibacterial effect of nanoemulsion on lung cancer cells harboring symbiotic bacteria that metastasize to the lungs

[0294] Figure 27 The figure shows the antibacterial effect of TC1 lung metastasis bacterial symbiotic lung cancer mice after treatment, where (A) represents the bacterial content in the lungs of mice after treatment; (B) represents the L-lactic acid content in the lungs of mice after treatment. Figure 27 -(A)), CS NA group and S NA group both had good antibacterial effects. From the L-lactic acid level in the lungs of mice after treatment ( Figure 27 -(B)), the L-lactic acid content in CSNA, CNA, and CNP is lower. Considering that in addition to Lactobacillus iners, the tumor tissue itself also produces a certain amount of lactic acid, tumor removal can also reduce the lactic acid level to a certain extent.

[0295] Experimental results demonstrated that lung-targeted nanoemulsions exhibit unique advantages in the treatment of lung-metastatic bacterial symbiotic lung cancer. Specifically, the CSNA group demonstrated the greatest anti-tumor efficacy, with the smallest number and area of ​​lung metastases. Furthermore, measurements of bacterial abundance and L-lactic acid levels in lung tissue revealed that the CSNA group also exhibited a significant advantage in antibacterial activity.

[0296] Example 6 Study on the in vivo immunological mechanism of lung-targeted nanoemulsion in the treatment of in situ bacterial symbiotic lung cancer

[0297] 1. Experimental methods:

[0298] 1.1 Investigation of the ability of nanoemulsion therapy to induce tumor immunogenic death and improve the tumor immune microenvironment

[0299] 1.1.1 Preparation of tumor tissue lysate

[0300] An in situ bacterial symbiotic lung cancer model was established and drug administration was initiated. Mice were sacrificed on day 17, and lung tumor tissue containing the tumor was dissected. A portion approximately 3 mm in diameter was isolated, rinsed with saline, and blotted dry with filter paper. The tissue was weighed and placed in a tissue grinding tube. For every 10 mg of tissue, 100 μL of RIPA lysis buffer containing 1× protein inhibitor and 0.1 mg / mL DNase was added. Two 3 mm steel beads and one 2 mm tissue grinding bead were added to each tube. The cells were ground using a cryogenic grinder at 4°C. After thorough grinding, the cells were lysed on ice for 10 min, followed by centrifugation at 12,000 × g for 10 min. The supernatant was transferred to a fresh tube and centrifuged again to remove excess precipitate. The supernatant was collected and temporarily stored at -20°C. Before subsequent use, the protein concentration of each sample was determined using a BCA assay kit. To prevent sample concentrations exceeding the calibration range, the sample was diluted approximately 20-fold before testing.

[0301] 1.1.2 Investigation of the ability of nanoemulsion therapy to induce tumor immunogenic death

[0302] Prepare tumor tissue lysate as described above. Refer to the corresponding kit instructions to measure HMGB-1 concentration in tumor tissue using an HMGB-1 quantitative ELISA kit and ATP concentration in tumor tissue using an enhanced ATP detection kit. Calculate the ratio of the concentration to the total protein amount measured by BCAA to reflect the secretion levels of the two substances in tumor tissue.

[0303] 1.1.3 Investigation of the Ability of Nanoemulsion Therapy to Affect the Tumor Immune Microenvironment

[0304] Prepare a single cell suspension of tumor tissue, divide the cell pellet into 4 parts, stain with flow cytometry antibodies, and then detect by flow cytometry. The corresponding test items and staining scheme for each part are as follows:

[0305] (1) DC recruitment and maturation: L / D-BV510, CD11c-SuperBright600, CD80-FITC, CD40-PE, CD86-APC.

[0306] (2) Macrophage polarization: L / D-BV510, CD45-PECY7, CD11b-FITC, F4 / 80-PE, CD86-APC, CD206-APC-eFluor780.

[0307] (3) NK cell infiltration: L / D-BV510, CD45-PECY7, NK1.1-FITC.

[0308] (4) T lymphocyte activation and Treg: L / D-BV510, CD45-PECY7, CD4-APCeflour780, CD8a-FITC, CD69-PE; CD25-APC, Foxp3-BV421.

[0309] Among them, Foxp3 is a transcriptional regulatory factor expressed in the cell nucleus. Therefore, the cells need to be fixed and the nuclear membrane needs to be punctured before staining. The specific method is described in the instructions.

[0310] 1.1.4 Determination of cytokines in tumor tissue after nanoemulsion treatment

[0311] Prepare tumor tissue lysates, and then use ELISA kits to detect the concentrations of IFNγ, IL-4, IL-6, IL-1β, TNF-α, and Granzyme B cytokines in the lysates. Calculate the ratio of cytokine concentration to total protein measured by BCA to reflect the secretion level of each cytokine in the tumor tissue.

[0312] 1.2 Investigation of the ability of nanoemulsion therapy to activate systemic immune responses and tumor antigen-specific immune responses

[0313] 1.2.1 Detection of immune response intensity in tumor-draining lymph nodes after nanoemulsion treatment

[0314] Single-cell suspensions of mediastinal lymph nodes were prepared and stained with flow cytometry antibodies, followed by flow cytometry. The specific staining scheme was: CD11c-SuperBright600, CD80-FITC, CD40-PE, and CD86-APC.

[0315] 1.2.2 Investigation of splenic T cell activation after nanoemulsion treatment

[0316] Single-cell suspensions were prepared from spleens, stained with flow cytometry antibodies, and then analyzed by flow cytometry. T lymphocyte activation staining protocol was: CD4-APCeflour780, CD8a-FITC, CD69-PE.

[0317] 1.2.3 Intrinsic factor staining of spleen after tumor antigen stimulation after nanoemulsion treatment

[0318] LLC tumor cell lysate was prepared in advance by repeated freeze-thaw cycles in liquid nitrogen and stored at -80°C until needed. A single-cell suspension of spleen cells was prepared and seeded into a 96-well plate. The cells were then mixed and incubated in a 37°C, 5% CO2 incubator for 1 hour. Brefedin A was then added for 5 hours to inhibit cytokine efflux. Surface staining was then performed using the following staining protocol: L / D-BV510, CD4-APC efluo r780, and CD8a-FITC. After staining, the cells were washed with PBS, and the cell pellet was resuspended in 100 μL of fixative solution and fixed at room temperature for 20 minutes. The cells were then washed twice with perforation solution, and the cell pellet was resuspended with 50 μL of perforation solution. 50 μL of flow cytometry antibody buffer was added to prepare flow cytometry antibodies. The staining scheme was: INF-α-eFluor450, IL-2-PEcy5.5, IFN-γ-APC, GraB-PEcy7. The cells were incubated in the dark at 4°C for 40 minutes for staining. After staining, the cells were washed twice with perforation solution, and resuspended in PBS for flow cytometry detection.

[0319] 1.2.4 Investigation of the abundance of spleen memory T cells after nanoemulsion treatment

[0320] LLC tumor cell lysate was prepared in advance by repeated freeze-thaw cycles in liquid nitrogen and stored at -80°C until use. A single-cell suspension of spleen cells was prepared and seeded into a 96-well plate. The cells were then mixed and incubated in a 37°C, 5% CO2 incubator for 72 hours. Surface molecules were then stained using the following staining protocol: anti-CD4-APC-eflour780, anti-CD8a-FITC, anti-CD44-APC, and anti-CD62L-PE.

[0321] 2. Experimental results:

[0322] 2.1 Nanoemulsion therapy induces tumor immunogenic death

[0323] In vitro cell experiments have demonstrated that CZT has the ability to induce ICD in LLC cells. To further confirm whether the preparation of the present invention can also induce ICD of tumors in vivo, the present invention tested the ICD indicators in vivo after treatment. The present invention uses an HMGB-1 quantitative detection ELISA kit to detect the HMGB-1 concentration in the lysate of tumor tissue, and uses an enhanced ATP detection kit to detect the ATP concentration in the tumor tissue lysate, and then calculates the ratio of the concentration to the total protein amount measured by BCA to reflect the secretion level of the two substances in the tumor tissue. The results are as follows: Figure 28 shown. Figure 28 The ICD indicators in the tumor tissue lysate after treatment are shown, where (A) represents the relative content of HMGB-1 and (B) represents the relative content of ATP. HMGB-1 is a protein in the cell nucleus. When ICD occurs in tumor cells, it can be released from the cell to the extracellular space, exerting a pro-inflammatory effect and attracting immune cells (such as dendritic cells) to participate in the anti-tumor immune response. In ICD, HMGB-1 acts as a "danger signal" molecule that helps to initiate and enhance the anti-tumor immune response. The HMGB-1 concentration in the tumor tissue lysate of the CS NA treatment group was significantly higher than that of the other groups ( Figure 28 -(A)), which benefits from the ICD-inducing ability of CZT on the one hand and the ability of this agent to efficiently target the lungs on the other.

[0324] When tumor cells undergo ICD, they also release ATP. ATP, as a signaling molecule, interacts with immune cells (such as dendritic cells and macrophages) through P2 receptors, enhancing anti-tumor immune responses and further prompting the immune system to recognize and eliminate tumor cells. In this experiment, the ATP concentration of tumor cell lysates in the CS NA, C NA, and CS NP groups were significantly higher than those in the no-drug group, the free drug group, and the non-CZT-encapsulated group, but there was no statistically significant difference among the three groups ( Figure 28 -(B)). Considering that ATP molecules are easily degraded and the ATP concentrations in each group were low, this result is for reference only.

[0325] 2.2 Nanoemulsion therapy improves the tumor immune microenvironment

[0326] The tumor immune microenvironment is a chronic, uncontrolled inflammatory environment constructed by tumor cells, immune cells, blood vessels, cytokines, etc. This immunosuppressive environment greatly weakens the body's own immune system, inhibiting the normal functions of dendritic cells (DCs), tumor-infiltrating lymphocytes (T cells, B cells) and natural killer (NK) cells. At the same time, it also causes a variety of innate and adaptive immune cells to differentiate from a "tumor-suppressing" state to a "tumorigenic" state, such as regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), further promoting immune escape and tumor progression. Therefore, reversing the immunosuppressive tumor immune microenvironment is a key point in the treatment of tumors. To this end, the present invention detects and analyzes the abundance and subtypes of immune cells and cytokine content at the tumor site.

[0327] 2.2.1 Nanoemulsion therapy improves antitumor immune cell infiltration at the tumor site

[0328] In the process of tumor clearance, immune cells in the tumor site play a vital role. DCs have the function of efficiently capturing and presenting tumor antigens, and their existence is essential for successfully activating immune responses and clearing tumor cells; NK cells can directly locate and destroy tumor cells without the need for pre-activation of rapid killing mechanisms; at the same time, activated CD4 + T cells regulate the immune microenvironment by secreting a variety of cytokines, + T cell cytotoxicity provides synergistic support; activated CD8 + T cells can directly mediate cell apoptosis and thus precisely attack tumors. Figure 29 As shown, Figure 29 The infiltration of immune cells with anti-tumor effects in the tumor site after treatment is shown, where (A) represents the percentage of DCs cells; (B) represents the percentage of NKs cells; (C) represents the percentage of CD4 + The degree of activation of T cells; (D) indicates CD8 + The activation level of T cells. The CS NA treatment group can significantly increase the number of DCs, NK cells, CD4 + T cells and CD8 + The degree of T cell activation therefore helps to break through the tumor's immunosuppressive barrier, stimulate the body's inherent anti-tumor potential, and thus inhibit tumor growth.

[0329] 2.2.2 Regulation of Tregs in tumor sites by nanoemulsion therapy

[0330] In the tumor microenvironment, Tregs are derived from both natural Tregs generated by the thymus and iTregs induced under peripheral conditions. Under the stimulation of hypoxia, acidity, and high concentrations of TGF-β and IL-10 at the tumor site, ordinary T cells are easily converted into Tregs by activating the Foxp3 gene. These Tregs cells can effectively inhibit antigen presentation by DCs and weaken CD8 Tregs by secreting inhibitory factors such as IL-10 and TGF-β and by using direct cell-to-cell contact. + The cytotoxic function of T cells and NK cells forms a solid immune barrier, thereby promoting tumor growth and immune escape. Figure 30 As shown, it shows the proportion of Tregs in the tumor site after treatment. The CS NA treatment group can significantly reduce the proportion of Tregs in the tumor site, thereby helping to reactivate effector immune cells and thus enhance the body's ability to clear tumors.

[0331] 2.2.3 Regulation of macrophage differentiation in tumor sites by nanoemulsion therapy

[0332] In the tumor microenvironment, macrophages have a certain plasticity, and their classification mainly includes M1 macrophages with pro-inflammatory and anti-tumor effects and M2 macrophages with anti-inflammatory and pro-tumor effects. M1 macrophages can not only enhance the presentation of tumor antigens by secreting cytokines such as TNF-α and IL-12, but also directly activate effector T cells and NK cells to help form an immune barrier; while M2 macrophages promote angiogenesis, cell proliferation and tumor immune escape with the help of inhibitory factors such as IL-10 and TGF-β. Figure 31 As shown, Figure 31 The following images show the classification of macrophages in the tumor site after treatment, including: (A) percentage of M1 macrophages; (B) percentage of M2 macrophages; and (C) M1 / M2 ratio. Although the CSNA treatment group did not significantly increase the proportion of M1 macrophages, it significantly reduced the proportion of M2 macrophages, leading to a significant increase in the M1 / M2 ratio. This suggests that CSNA treatment can help reverse the immunosuppressive state, restore the immune system's anti-tumor activity, and thus limit tumor progression and metastasis.

[0333] 2.2.4 Nanoemulsion therapy improves cytokine levels in tumor sites

[0334] In tumor tissues, IFN-γ is mainly produced by activated CD8 +Secreted by T cells and NK cells, it not only directly induces tumor cell apoptosis, but also promotes antigen presentation and immune cell recruitment; GranzymeB is released by cytotoxic lymphocytes, penetrates the tumor cell membrane and activates the endogenous apoptosis pathway, thereby killing tumor cells; TNF-α is produced by macrophages, T cells and other immune cells, and further weakens tumor growth vitality by regulating inflammatory response and inhibiting tumor angiogenesis. Figure 32 As shown, Figure 32 Figure 3 shows the levels of cytokines in the tumor site after treatment: (A) IFN-γ; (B) Granzyme B; (C) TNF-α; and (D) normalized to Z-scores and displayed as a heat map. CSNA treatment significantly increased the levels of these three cytokines, thereby reversing the immunosuppressive state, restoring the immune system's anti-tumor activity, and ultimately limiting tumor growth.

[0335] 2.3 Activation of systemic immune responses and tumor antigen-specific immune responses by nanoemulsion therapy

[0336] While local immune cell regulation is crucial in tumor treatment, the present invention also plays a crucial role in stimulating a systemic immune response by inducing the ICD effect, which causes tumor cells to release antigens and, after sterilization, release bacterial adjuvants. Therefore, the present invention assesses the strength of the systemic immune response by measuring the abundance of immune effector cells in tumor-draining lymph nodes and spleen, as well as the response of these immune effector cells to tumor antigen stimulation.

[0337] 2.3.1 Maturation of DCs in tumor-draining lymph nodes after nanoemulsion treatment

[0338] In tumor-draining lymph nodes, the maturation of DCs is key to their effective induction of anti-tumor immune responses. When DCs encounter tumor antigens, immature DCs are activated and undergo a maturation process, which is manifested by the upregulation of co-stimulatory molecules (such as CD80, CD86) and the enhancement of antigen presentation ability. This maturation process is crucial for activating initial T cells and differentiating them into cytotoxic T lymphocytes (CTLs) that can recognize and kill tumor cells. Mediastinal lymph nodes are generally considered to be the main tumor-draining lymph nodes for lung cancer. This is the key location where tumor antigens are first presented and the immune response is initiated. Therefore, by examining the maturation of DCs in the mediastinal lymph nodes, it can be used to assess the strength of the systemic immune response. For example Figure 33 As shown, Figure 33 The figure shows the maturation of DCs in the mediastinal lymph nodes after treatment. The left figure is a representative flow cytometry plot, and the right figure shows the flow cytometry positive rate statistics. The CSNA treatment group was able to significantly increase the maturation rate of DCs in the mediastinal lymph nodes, indicating that CSNA can promote T cell responses by activating DCs.

[0339] 2.3.2 Activation of spleen T cells after nanoemulsion treatment

[0340] The spleen is an important organ of the immune system and plays a key role in anti-tumor immune response. The spleen is not only a reservoir of T cells, but also the main place where they contact tumor antigens, activate and perform immune surveillance. After tumor-associated antigens are presented to the spleen by DCs, they can trigger T cells to recognize and activate, become effector T cells, and thus enhance the ability of tumor immune surveillance and immune clearance. Figure 34 As shown, Figure 34 Shows the activation of T cells in the spleen after treatment, including: (A) CD4 + T cell activation; (B) CD8 + Activation of T cells. CS NA treatment group can significantly increase the number of CD4 + T cells and CD8 + The proportion of T cells has a significant advantage in inducing spleen immune response.

[0341] 2.3.3 Tumor antigen-specific immune response induced by nanoemulsion therapy

[0342] In the present invention, after co-delivering anti-tumor drugs and antibacterial drugs, the ICD effect can be induced to cause tumor cells to release antigens, release bacterial adjuvants after sterilization, and then stimulate systemic immune responses and even tumor antigen-specific immune responses. Based on this, the present invention isolated the spleens of treated mice in vitro and restimulated them with tumor antigens. The cytokine levels secreted by spleen lymphocytes were detected by staining the intrinsic factor in the spleen lymphocytes to reflect the level of tumor antigen-specific immune response. The results are shown in Figure 2. Figure 35 As shown, Figure 35 The diagram shows the secretion of cytokines by T cells in the spleen after treatment after stimulation with tumor antigens, including: (A) a schematic diagram of two types of T cells secreting cytokines; CD4 + (B) IFN-γ and (C) IL-2 levels secreted by T cells; CD8 + T cell secretion of (D) IFN-γ, (E) TNF-α and (F) granzyme B levels. It can be seen that the treatment of CS NA group can induce a higher level of tumor antigen-specific immune response, specifically: spleen CD4 + After restimulation with tumor antigens, T cells secrete higher levels of IFN-γ and IL-2 ( Figure 35 -(B) / (C)), these two cytokines are important for the subsequent activation of CD8 + T cells and NK cells play an important role; and spleen CD8 + After restimulation with tumor antigens, T cells secrete higher levels of IFN-γ, TNF-α, and granzyme B ( Figure 35-(D) / (E) / (F)), for direct killing of target cells.

[0343] 2.3.4 Generation of splenic memory T cells after nanoemulsion treatment

[0344] In addition to differentiating into effector T cells, T cells can also differentiate into longer-lived memory T cells, thereby achieving a rapid immune response in secondary infections. Memory T cells are mainly divided into central memory T cells (Central Memory Cells, Tcm) and effector memory T cells (Efector Memory T cells, TEM). Tcm exists in peripheral blood and spleen for a long time, and has strong proliferation ability and multipotent differentiation characteristics. When they are exposed to tumor-associated antigens for the second time, they can migrate to stimulate lymphoid tissues and initiate a rapid immune response; TEM exhibits stronger effector functions and can directly kill tumor cells through individual and cytokine secretion. Figure 36 As shown, Figure 36 The abundance of effector memory T cells in the spleen after treatment is shown, including (A) effector memory CD4 + T cells; (B) effector memory CD8 + T cells. CSNA can increase the levels of effector memory T cells in the spleen, which is beneficial for long-term protection of anti-tumor immune responses.

[0345] It can be seen that lung-targeted nanoemulsion therapy can not only induce ICD effect in tumor tissue and release DAMPs molecules, but also play a key role in improving the local immune microenvironment and activating systemic immune response.

[0346] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they become aware of the basic creative concepts. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present invention.

[0347] The above is a detailed introduction to a lung-targeted nanoemulsion preparation, preparation method and application for co-delivering anti-tumor drugs and antibacterial drugs provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only intended to help understand the method and core concept of the present invention. At the same time, for those skilled in the art, according to the concept of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A lung-targeted nanoemulsion formulation for co-delivering anti-tumor drugs and antibacterial drugs, characterized in that: The anti-tumor drug includes at least one anti-tumor drug with immunogenic cell death effect; the antibacterial drug includes at least one antibiotic or antibacterial peptide.

2. The lung-targeted nanoemulsion preparation for co-delivering anti-tumor drugs and antibacterial drugs according to claim 1, characterized in that: The anti-tumor drugs with immunogenic cell death effects include: cyclophosphamide, docetaxel, adriamycin, mitoxantrone, paclitaxel and crizotinib; the antibiotics or antimicrobial peptides include: gentamicin, polymyxin E, moxifloxacin and synthetic antimicrobial anti-biofilm peptide SAAP-148.

3. The lung-targeted nanoemulsion preparation for co-delivering anti-tumor drugs and antibacterial drugs according to claim 1, characterized in that: The nanoemulsion preparation is externally modified with DSPE-PEG 2000 -ACE2 targeting peptides AYQ, GQ, or AY.

4. The lung-targeted nanoemulsion preparation for co-delivering anti-tumor drugs and antibacterial drugs according to claim 1, characterized in that: When the antitumor drug is crizotinib and the antibacterial drug is SAAP-148, the nanoemulsion preparation is an oil-in-water nanoemulsion system, which uses the oil phase to encapsulate hydrophobic crizotinib and adds an anionic surfactant to adsorb positively charged SAAP-148 at the oil-water interface.

5. The lung-targeted nanoemulsion preparation for co-delivering anti-tumor drugs and antibacterial drugs according to claim 3, characterized in that: The aqueous phase components of the nanoemulsion preparation include: SAAP-148, water, phospholipids and sodium deoxycholate; the ratio of SAAP-148, water, phospholipids and sodium deoxycholate is: 1-4 mg: 1-4 mL: 10-15 mg: 1.5-2.5 mg; the oil phase components of the nanoemulsion preparation include: crizotinib, soybean oil and MCT, and the ratio of crizotinib, soybean oil and MCT is: 1-4 mg: 10-15 mg: 10-15 mg.

6. The method for preparing the preparation according to claim 1, wherein The method comprises: Preparation of the aqueous phase: Sterile water for injection or a hydrophilic drug dissolved in sterile water for injection is added to the phospholipids, and the mixture is emulsified by vortexing and heating in a 37°C water bath. A surfactant is then added and further vortexed. The hydrophilic drug is positively charged SAAP-148. Preparation of the oil phase: Soybean oil and MCT are added to the hydrophobic drug, and then a small amount of dichloromethane is used to dissolve the oil phase containing the hydrophobic drug, and the oil phase is fully dissolved by water bath sonication. The hydrophobic drug is crizotinib; Mixed ultrasound: the oil phase was dropped into the water phase while vortexing, and then ultrasonicated with an ice bath probe at 180W for 5s, 5s for 10min, and finally the dichloromethane was removed by rotary evaporation to obtain a nanoemulsion formulation for co-delivering antitumor drugs and antibacterial drugs; DSPE-PEG was modified on the outside of the nanoemulsion using the post-insertion method. 2000 -ACE2 targeting peptide to obtain a lung-targeted nanoemulsion formulation for co-delivery of anti-tumor drugs and antibacterial drugs.

7. The preparation method according to claim 6, characterized in that DSPE-PEG was modified on the outside of the nanoemulsion using the post-insertion method. 2000 -ACE2 targeting peptides include: Take 1 mL of the prepared nanoemulsion preparation, add 1 mg of the prepared lyophilized powder of the targeting material, gently shake and incubate in a 37°C water bath for 2 minutes. The pH should be neutral. During this period, the hydrophobic end of the amphiphilic material can be inserted into the oil phase, and the hydrophilic end faces the water phase.

8. Use of the preparation according to claim 1 in the preparation of a bacterial symbiotic tumor treatment preparation, characterized in that: The anti-tumor drug induces immunogenic cell death to kill the tumor, releases endogenous tumor antigens, and converts dying tumor cells into in situ antigens; and is combined with the delivery of antibacterial drugs to eliminate tumor symbiotic bacteria and release endogenous bacterial adjuvants.

9. The use according to claim 8, characterized in that Modified with DSPE-PEG 2000 -The nanoemulsion preparations of ACE2 targeting peptides AYQ, GQ or AY have lung targeting and tumor targeting capabilities.

10. The use according to claim 8, characterized in that The bacterial symbiotic tumor is an in situ bacterial symbiotic lung cancer, and the preparation is used to inhibit tumor growth, reduce bacterial load, delay the course of the disease, and clear bacteria inside and outside the lungs and tumor cells. Alternatively, the bacterial symbiotic tumor is a lung metastatic bacterial symbiotic lung cancer, and the preparation is used to reduce the number and area of ​​lung metastases. The preparation is also used to reduce bacterial content.

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