Macrophage-loaded nanocomposite antitumor targeted drug delivery system and preparation method and application thereof

Abstract

The application discloses a macrophage-loaded nanocomposite anti-tumor targeted drug delivery system and a preparation method and application thereof. The drug delivery system is formed by loading a Toll-like receptor agonist, a photothermal response agent, a phospholipid and cholesterol into a liposome nanoparticle, coating a bacterial outer membrane vesicle on the outer surface of the drug-loaded liposome nanoparticle to form a drug-loaded nanocomposite, and loading the drug-loaded nanocomposite into macrophages through co-incubation. The system of the application uses macrophages and liposomes as carriers, combines immunotherapy and combined phototherapy to improve the immunotherapy effect by inducing immunogenic cell death caused by the photothermal effect, and further promotes the recruitment and accumulation of macrophages through the regulation of the inflammatory tumor microenvironment, generates a positive feedback cycle, and effectively improves the tumor immunosuppressive microenvironment and inhibits tumor growth and metastasis.

Description

Technical Field

[0001] This invention relates to a targeted drug delivery system, and more particularly to a macrophage-loaded nanocomposite antitumor targeted drug delivery system, as well as the preparation method and application of the targeted drug delivery system. Background Technology

[0002] The tumor microenvironment (TME) is the internal and external environment for tumor development and progression, and it has complex components. The cellular components of the TME include tumor-associated macrophages (TAMs), dendritic cells, lymphocytes, and fibroblasts. Among them, TAMs are one of the most abundant immune cells in the TME. Current macrophage-based tumor therapy strategies focus on inhibiting macrophage recruitment, repolarizing, and depleting macrophages. Since M1 / M2 dysregulation is a key cause of tumor progression, metastasis, and drug resistance, targeted delivery of Toll-like receptor (TLR) agonists to activate the innate immune NF-κB signaling pathway and repolarize M2-type TAMs is a promising immunotherapy strategy. However, targeted delivery of Toll-like receptor agonists has limited drug loading capacity, leading to safety concerns such as low drug loading and abnormal tissue alterations. Summary of the Invention

[0003] Purpose of the invention: The purpose of this invention is to provide a macrophage-loaded nanocomposite anti-tumor targeted drug delivery system with large drug loading capacity, strong targeting, strong safety and potent anti-tumor therapy. The second purpose is to provide the preparation method and application of the above-mentioned drug delivery system.

[0004] Technical solution: The drug delivery system of the present invention includes liposome nanoparticles composed of Toll-like receptor agonists, photothermal responsive agents, phospholipids and cholesterol, and a drug-loaded nanocomplex formed by coating bacterial outer membrane vesicles on the surface of the liposome nanoparticles. The nanocomplex is co-incubated and loaded into macrophages to form the drug delivery system.

[0005] Preferably, the Toll-like receptor (TLR3, TLR7 / 8 and TLR9) agonist is one of polyinosinic-poly(I:C), imiquimod (R837) / requimod (R848), or an oligodeoxynucleotide containing cytosine guanine dinucleotide (CpG ODN).

[0006] Preferably, the photothermal responsive agent is one of indocyanine green (ICG), carbocyanine dyes (DIR), IR820, IR780, iron phthalocyanine (FePc), dichloroporphyrin (Ce6), porphyrin-diketone pyrrole (Por-DPP), polydopamine (PDA), gold nanoparticles (AuNPs), gold nanorods (AuNRs), carbon nanotubes (CNTs), carbon dots (CDs), or quantum dots (QDs).

[0007] Preferably, the quantum dots (QDs) are ultra-miniature black phosphorus quantum dots (BPQDs).

[0008] Preferably, the bacterial outer membrane vesicles are derived from one of E. coli K-12W3110, E. coli BL21(DE3), E. coli DH5α, E. coli JC8031, A. baumannii, K. pneumoniae, and P. aeruginosa.

[0009] Preferably, the macrophage is one of mouse mononuclear macrophage leukemia cells (RAW264.7), bone marrow-derived macrophages (BMDM), peritoneal macrophages (PM), peripheral blood-derived macrophages, or induced pluripotent stem cell-derived macrophages.

[0010] Preferably, the phospholipid is one of soybean phospholipid, soybean lecithin, egg yolk lecithin, myristoyl lecithin, dipalmitoyl lecithin, or distearate lecithin.

[0011] The method for preparing the macrophage-loaded nanocomposite antitumor targeted drug delivery system of the present invention includes the following steps:

[0012] (1) Preparation of liposome nanoparticles loaded with Toll-like receptor agonists and photothermal responsive agents: phospholipids, cholesterol and Toll-like receptor agonists were dissolved in an organic solvent, rotary evaporated and dried at room temperature, an aqueous solution of photothermal responsive agent was added, rotary hydration was performed and ultrasonic dispersion was performed, and liposome nanoparticles were obtained after water film filtration.

[0013] (2) Preparation of drug-loaded nanocomposites modified with bacterial outer membrane vesicles: Prepare an aqueous solution of bacterial outer membrane vesicles, mix liposome nanoparticles with the aqueous solution of bacterial outer membrane vesicles evenly, and co-extrude the mixture using a liposome extruder to obtain the drug-loaded nanocomposites.

[0014] (3) Preparation of antitumor targeted drug delivery system: The drug-loaded nanocomposite obtained in step (2) is diluted to an appropriate concentration with culture medium, co-incubated, and then loaded into macrophages to obtain the antitumor targeted drug delivery system.

[0015] Preferably, in step (1), the mass ratio of the phospholipid, cholesterol, Toll-like receptor agonist and photothermal responsive agent is 180:30:20:1 to 180:30:20:5; the Toll-like receptor agonist is imiquimod, and the photothermal responsive agent is ultra-micro black phosphorus quantum dots.

[0016] Preferably, in step (2), the volume ratio of the bacterial outer membrane vesicle aqueous solution to the liposome nanoparticle solution is 1:1 to 1:5.

[0017] Preferably, in step (3), the final concentration of the Toll-like receptor agonist in the drug-loaded nanocomposite is 5–15 μg / mL, and the co-incubation time is 2–12 h.

[0018] Preferably, step (1) specifically involves: dissolving phospholipids, cholesterol, and R837 in an organic solvent, removing the organic solvent by rotary evaporation at 40°C for 15 min to form a uniform oil film, drying at room temperature, adding an aqueous solution of BPQD to the dried oil film, hydrating by rotary evaporation at 40°C for 30 min to wash off the oil film and uniformly dispersing it in the aqueous solution, further dispersing the aqueous dispersion under the probe of an integrated ultrasonic cell disruptor, and then filtering the nanoparticle solution sequentially through 0.45 μm and 0.22 μm aqueous microporous membranes to finally obtain liposomes RB@Lip loaded with R837 and BPQD.

[0019] Preferably, step (2) specifically involves: centrifuging the E. coli DH5α bacterial culture medium at 4°C and 8000g for 15 min in a refrigerated centrifuge, discarding the precipitate, and filtering the supernatant through a 0.45μm PES vacuum filter. Then, the supernatant is concentrated using a 100kDa Amicon Ultra15 centrifugal ultrafiltration tube, discarding the outer tube liquid. The inner tube liquid is then centrifuged at 4°C and 150000g for 2 h in an ultra-low temperature centrifuge, discarding the supernatant. The precipitate is resuspended in 1×PBS and filtered through a 0.22μm PES vacuum filter to finally obtain E. coli DH5α outer membrane vesicles (OMVs). RB@Lip and OMVs are extruded several times using a liposome extruder to finally obtain drug-loaded liposomes modified with E. coli DH5α outer membrane vesicles, RB@OL.

[0020] Preferably, step (3) specifically involves: 1 to 5 × 10 6RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. After 24 h, the medium was replaced with serum-free DMEM medium containing RB@OL. After a period of culture, the cells were allowed to engulf the nanocomplex. The cells were then washed several times with preheated PBS buffer to remove the free nanocomplex. The cells were collected with a cell scraper to obtain macrophages RB-OL@M loaded with the nanocomplex.

[0021] The application of the macrophage-loaded nanocomposite antitumor targeted drug delivery system described in this invention in the preparation of drugs that improve the tumor immunosuppressive microenvironment and inhibit tumor growth.

[0022] This invention utilizes bacterial outer membrane vesicle-modified liposomes to load a lipid-soluble Toll-like receptor agonist and a water-soluble photothermal responsive agent to form a drug-loaded nanocomposite. This nanocomposite is then introduced into macrophages via membrane fusion or endocytosis and contained in phagosomes or phagolysosomes formed by fused phagosomes and lysosomes, forming an anti-tumor targeted drug delivery system for macrophages loaded with therapeutic drugs.

[0023] Among them, Gram-negative bacteria (G) were used. - Outer membrane vesicles (OMVs) serve as carriers and immune activators, with a particle size of 20-250 nm. Compared to other exosomes such as mesenchymal stem cell-derived exosomes, OMVs are more easily recognized and phagocytosed by macrophages because they possess a large number of components derived from the outer membrane and surrounding tissues of their parent bacteria, thus exhibiting high immunogenicity. R837, as a TLR-7 agonist, can bind to TLR-7 overexpressed in macrophage endosomes / lysosomes, activating innate and adaptive immune responses; R837 can be used for the repolarization of TAMs into M1 macrophages, thereby promoting cytotoxic T cell infiltration into tumors and inhibiting tumor growth. However, when not encapsulated, it suffers from poor water solubility and lack of targeting, limiting its immunomodulatory function and severely affecting drug efficacy. BP materials, especially BPQD, have a large extinction coefficient, high photothermal conversion efficiency, and efficient renal excretion. However, the material itself is too fragile and is easily degraded by reacting with O2 and H2O when exposed to water in the air, which greatly reduces its PTT efficiency. In addition, BP has poor dispersibility, which makes it easy to aggregate under physiological conditions. It also has the disadvantages of short in vivo circulation time and unstable optical properties. Further coating can solve these problems.

[0024] By loading liposoluble R837 and water-soluble BPQD onto OMVs-modified Lip to form RB@OL nanocomposites, a system for co-incubating macrophages to load therapeutic drugs was formed. This approach simultaneously addresses the issues of poor water solubility, lack of targeting, and low potency of R837, as well as the problems of easy degradation, low PTT efficacy, poor dispersibility, short in vivo circulation time, and unstable optical properties of BPQD. It synergistically achieves targeted delivery of R837 and improves the stability of BPQD, while also enhancing photothermal effects, thereby improving the overall efficacy of the drug delivery system.

[0025] This nanodelivery system targets the RB@OL portion of macrophage phagosomes, releasing cells into the tumor microenvironment and penetrating deep tissues. OMVs and R837 activate TLR-7 receptors within tumor-associated macrophages, polarizing them from the M2 phenotype to the M1 phenotype, while simultaneously secreting cytokines such as TNF-α, IL-1β, IFN-γ, and IL-6. It promotes the recruitment and maturation of antigen-presenting dendritic cells, thereby initiating an immune response; it also promotes the infiltration of cytotoxic T cells, improving the tumor immunosuppressive microenvironment. Under 808nm laser irradiation, BPQD generates local heat, promoting complete drug release, inducing tumor cell apoptosis, and inhibiting tumor growth and metastasis. This nanodelivery system acts as an immunomodulator and photothermal responsive agent by targeting the tumor microenvironment, achieving combined tumor and deep tissue immunotherapy with phototherapy, producing significant anti-tumor effects.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0027] (1) The antitumor targeted drug delivery system of the present invention has an encapsulation structure that improves the stability of liposomes, prevents leakage of Toll-like receptor agonist drugs, and promotes the induction of tumor cell apoptosis and drug release and penetration under laser irradiation. On the other hand, it maintains the antitumor phenotype of macrophage carriers, and the safety of the targeted drug delivery system is greatly improved.

[0028] (2) The encapsulation structure of the drug delivery system of the present invention greatly increases the drug loading capacity of cells, significantly improves the tumor targeting efficiency of liposomes and the accumulation and efficacy of therapeutic drugs at the tumor site, and increases the circulation time of drugs in vivo; the combined effect of laser irradiation on apoptosis induction of mouse breast cancer cells shows that the percentage of early apoptosis cells can reach up to 23.7% and the percentage of late apoptosis cells can reach up to 37.5%, which is 5 times higher than the apoptosis rate of cancer cells with liposomes loaded with immunomodulators acting alone, and has excellent tumor killing effect. Attached Figure Description

[0029] Figure 1 Figure A shows the characterization results of RB@Lip nanoparticles in Example 1, where Figure A is the particle size diagram and transmission electron microscope (TEM) image of RB@Lip nanoparticles, and Figure B is the TEM image of BPQD.

[0030] Figure 2 Figure A shows the characterization results of OMVs in Example 2, where Figure A is a transmission electron microscope image of OMVs and Figure B is an SDS-PAGE electrophoresis image of OMVs.

[0031] Figure 3 The fluorescence colocalization map of the RB@OL nanocomposite in Example 2.

[0032] Figure 4 The particle size distribution and transmission electron microscopy (TEM) image of the RB@OL nanocomposite in Example 2 are shown.

[0033] Figure 5 This is a photothermal imaging image of the RB@OL nanocomposite in Example 2;

[0034] Figure 6 Figure A shows the stability results of the RB@OL nanocomposite in Example 2. Figure B shows the changes in particle size and PDI of the aqueous dispersion of the RB@OL nanocomposite and the changes in particle size and PDI of the 10% fetal bovine serum dispersion.

[0035] Figure 7 This is an inverted fluorescence microscope image of the uptake of C6@OL nanocomposite by RAW264.7 cells in Example 3;

[0036] Figure 8 The cytotoxicity of the RB@OL nanocomposite in Example 4 is shown in Figure A, which shows the cytotoxicity of free R837 to RAW264.7, 4T1 and L929; Figure B shows the cytotoxicity of the OL carrier to RAW264.7, 4T1 and HUVEC; and Figure C shows the cytotoxicity of the RB@OL nanocomposite to RAW264.7.

[0037] Figure 9 Figure A shows the cell migration-scratch healing experiment results of the RB@OL nanocomposite in Example 5. Figure B shows the scratch healing rate of RAW264.7 cells with RB@OL nanocomposite containing different concentrations of R837, and Figure B shows the scratch healing rate of 4T1 cells.

[0038] Figure 10 The graph shows the effect of different formulation groups on RAW264.7 cell polarization induction in Example 6;

[0039] Figure 11 The graph shows the effect of different formulation groups on the maturation of DC2.4 cells in Example 7;

[0040] Figure 12 This is a diagram showing the induction of apoptosis in 4T1 cells by different formulation groups in Example 8;

[0041] Figure 13 This is a distribution map of the fluorescently labeled RB-OL@M in tumor-bearing mice in Example 9;

[0042] Figure 14 This is a distribution map of the fluorescently labeled RB-OL@M in isolated tissues in Example 9;

[0043] Figure 15 The image shows the H&E staining results of mouse heart, liver, spleen, lung, and kidney tissue sections, used in the in vivo safety evaluation of RB-OL@M in Example 10. Detailed Implementation

[0044] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0045] Example 1

[0046] Preparation and characterization of RB@Lip nanoparticles

[0047] (1) Preparation of RB@Lip nanoparticles

[0048] 18 mg of soybean lecithin, 3 mg of cholesterol, and 2 mg of R837 were accurately weighed and dissolved in 5 mL of a 1:4 (v / v) methanol / chloroform mixture. After complete dissolution, the solution was transferred to a brown gaiwan flask and evaporated under reduced pressure in a 40°C water bath for 15 min. Once the solvent had completely evaporated and a uniform lipid film had formed at the bottom of the flask, the flask was dried in a vacuum desiccator for 2 h. Then, 5 mL of 100 μg / mL BPQD aqueous dispersion was added to the flask and hydrated at 40°C for 30 min. After complete hydration of the lipid film, the resulting lipid suspension was sonicated in an ice bath for 15 min at 200 W with an on-time of 2 s and a off-time of 2 s to obtain lipid nanoparticles. Finally, the solution was filtered sequentially through 0.45 μm and 0.22 μm aqueous microporous membranes to obtain an aqueous solution of RB@Lip nanoparticles.

[0049] (2) Characterization of RB@Lip nanoparticles

[0050] The particle size of RB@Lip nanoparticles in aqueous solution was measured using dynamic light scattering (DLS), and the results are shown in the attached figure. Figure 1 As shown, the average particle size of the nanoparticles is 81.3 ± 2.4 nm. The structures of BPQD and RB@Lip nanoparticles in aqueous solution were observed by transmission electron microscopy (TEM), and the results are shown in the attached figure. Figure 1 As shown, the actual nanoparticle size is close to the detected particle size, and the structure of BPQD encapsulated in the Lip is clearly visible, indicating that RB@Lip nanoparticles were successfully prepared.

[0051] Example 2

[0052] Preparation, characterization and property study of RB@OL nanocomposites

[0053] (1) Extraction, concentration and purification of OMVs

[0054] E. coli DH5α cells were cultured overnight in 500 mL LB broth (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0) with shaking at 37°C and 200 rpm. [The text abruptly ends here, likely due to an incomplete sentence or missing information.] 600 Once the concentration reaches 1.2-1.5, the bacterial cells are removed by centrifugation at 4°C and 8000g for 15 min in a refrigerated centrifuge. The resulting supernatant is then filtered through a 0.45μm PES vacuum filter for sterilization, followed by concentration using a 100kDa Amicon Ultra15 centrifuge tube. The concentrate is then centrifuged at 4°C and 150000g for 2 h in a Beckman ultracentrifuge. The precipitate is then resuspended in 1×PBS and passed through a 0.22μm PES vacuum filter to remove any contaminants introduced during the process. The concentrate is then stored at -20°C until use.

[0055] (2) Characterization of OMVs

[0056] The structure of the OMVs aqueous dispersion was observed by transmission electron microscopy (TEM), and the results are shown in the attached figure. Figure 2 As shown in Figure A, the prepared E. coli outer membrane vesicles have a double-membrane vesicle structure; SDS-PAGE electrophoresis was used to analyze the full protein profile of E. coli DH5α and its outer membrane vesicles OMVs, and the results are shown in the attached figure. Figure 2 As shown in B, the OMVs and OMVs / NPs groups have consistent protein bands. The concentration of OmpA, a characteristic protein of OMVs, is not different between the two groups, indicating that OMVs are successfully coated on the Lip surface without affecting its structural properties and biological activity.

[0057] (3) Preparation of RB@OL nanocomposite

[0058] First, the drug-loaded liposomes and the E. coli outer membrane vesicle suspension were mixed in a certain proportion and physically extruded through a 100 nm polycarbonate membrane several times. Then, the mixture was centrifuged at 4 °C and 14000 g for 5 min to remove the free vesicles. The resulting precipitate was the RB@OL nanocomposite.

[0059] (4) Characterization of RB@OL nanocomposites

[0060] Fluorescence colocalization investigation: RB@OL nanocomposites (DC@OL) co-labeled with DiI and C6 were prepared using the optimal formulation and process, replacing OMVs with OMVs-DiI and coumarin 6 (C6) instead of R837. Fluorescence was observed using confocal microscopy. Results are attached. Figure 3 As shown, the red fluorescence of DiI and the green fluorescence of C6 co-occur, indicating that OMVs were successfully modified onto the Lip surface. (Scale bar: 10 μm)

[0061] The particle size of the RB@OL nanocomposite aqueous solution was measured using dynamic light scattering (DLS), and the results are shown in the attached figure. Figure 3 As shown, the average particle size of the nanocomposite is 126.4 ± 3.8 nm; the structure of the RB@OL nanocomposite in aqueous solution was observed by transmission electron microscopy (TEM), and the results are shown in the attached figure. Figure 4 As shown, the actual size of the nanocomposite is similar to the detected particle size, and it is larger in size and more rounded in shape than RB@Lip nanoparticles. The outermost layer has an obvious shell structure, indicating that OMVs were successfully modified on the Lip surface and the RB@OL nanocomposite was successfully prepared.

[0062] (5) Properties of RB@OL nanocomposites

[0063] To investigate the photothermal conversion capability of RB@OL nanocomposites, four groups were set up: PBS, BPQD, Lip-BPQD, and OMVs / Lip-BPQD. Sample solutions were measured and placed in EP tubes, and then tested using an 808nm laser emitter (1.0W / cm²). 2 The tube was irradiated for 10 minutes, and the thermal radiation images of the EP tube and the temperature change of the dispersion were recorded using a thermal imager during the irradiation process. The results are attached. Figure 5 As shown, neither liposome encapsulation nor membrane vesicle modification affects the photothermal properties of BPQD, and there is no significant difference in photothermal conversion capacity between BPQD and its aqueous dispersion.

[0064] The particle size stability of the RB@OL nanocomposite aqueous dispersion during the standing process was investigated, and the results are shown in the attached figure. Figure 6 As shown in Figure A; the particle size stability of the RB@OL nanocomposite during incubation was investigated using an in vitro environment containing 10% fetal bovine serum to simulate the in vivo environment. The results are shown in the attached figure. Figure 6 As shown in B, with the extension of standing / incubation time, the particle size and PDI of the nanocomposite fluctuated, with particle size changes all <20% and PDI all <0.3, indicating that the aqueous dispersion of the nanocomposite and its in vitro serum had good stability.

[0065] Example 3

[0066] Construction of the RB-OL@M system

[0067] (1) Cultivation of RAW264.7

[0068] RAW264.7 macrophages (passage 5 or less) were selected and seeded into cell culture flasks. They were cultured in DMEM complete medium containing 100 μg / mL streptomycin, 100 U / mL penicillin, and 10% bovine serum emtansone (BSE) peptide, and incubated at 37°C, 5% CO2, and 95% humidity. When cell confluence reached 70-80%, the cells were passaged and digested with 0.25% trypsin at room temperature for 1 min. Digestion was stopped when the cells became rounded, and the adherent cells were gently detached by pipetting. The cell suspension was collected and centrifuged at 1000 rpm for 5 min to pellet the cells. The supernatant was discarded, and the cells were resuspended in fresh complete medium. Finally, the cells were seeded at an appropriate cell density into sterile cell culture flasks and cultured as described above.

[0069] (2) Construction of RB-OL@M system

[0070] RAW264.7 cells in the logarithmic growth phase and in good growth condition were selected and cultured at a concentration of 3 × 10⁻⁶ cells / cells. 5 Cells were seeded at a concentration of [number] cells / well in 12-well plates and cultured in DMEM complete medium for adhesion. Then, the medium was replaced with serum-free DMEM containing C6, Lip-C6, and OMVs / Lip-C6 and incubated for 2–12 hours. After incubation, cells were washed three times with 1×PBS buffer to completely remove untaken C6. Cells were then fixed with 500 μL of 4% (w / w) paraformaldehyde for 20 min, washed with PBS, incubated with 250 μL of DAPI staining solution for 10 min, washed with PBS, the washings were discarded, and cells were infiltrated with 500 μL of PBS. Finally, cell uptake was detected using an inverted fluorescence microscope. Results are attached. Figure 7 As shown, the modification with OMVs makes the fluorescently labeled nanoparticles more easily taken up by macrophages, and the OMVs / Lip-C6 group has the highest fluorescence value.

[0071] Example 4

[0072] In vitro cytotoxicity study of RB@OL nanocomposite against RAW264.7 macrophages and 4T1 tumor cells.

[0073] RAW264.7, 4T1, L929, and HUVEC cells in the logarithmic growth phase and in good growth condition were seeded at 8 × 10⁻⁶ cells per cell line. 3Cells were seeded at a density of 100 cells / well in 96-well plates with five replicates. Each well contained 100 μL of DMEM / 1640 complete medium and was incubated at 37°C in a 5% CO2 incubator. After cell attachment, cells were cultured for 24 h in DMEM / 1640 basal medium containing R837, OMVs / Lip, and RB@OL, respectively. The working concentrations of R837 were 0.1, 0.5, 1, 2, 5, 10, 15, 20, 30, and 50 μg / mL; the working concentrations of OMVs / Lip were 50, 20, 15, 10, 5, 1, and 0.1 μg / mL (final PC concentration determined); and the working concentrations of RB@OL were 1, 10, 20, 50, and 100 μg / mL (final R837 concentration determined). A control group (no drug administration) and a blank control group (PBS only) were established. The culture medium was then discarded, and the cells were washed with PBS buffer. Next, 120 μL of serum-free medium containing 20 μL of 5 mg / mL MTT solution was added to each well, and the cells were incubated at 37°C in a 5% CO2 incubator for 4–6 h. The culture medium was then discarded, and 150 μL of dimethyl sulfoxide was added to each well. After shaking for 60 s, the absorbance of each well was measured at 490 nm using a microplate reader, and the cell viability was calculated using the formula below. Results are attached. Figure 8 As shown, firstly, the safety of the nanocomposite carrier was verified, and secondly, based on the IC50 value of R837 and the survival results of RB@OL, a safe and optimal dosage could be determined.

[0074] Cell viability = (A Sample -A PBS ) / (A control -A PBS )×100%

[0075] A Sample —Absorbance of the drug-treated cell group

[0076] A PBS —Absorbance of PBS solution

[0077] A control —Absorbance of untreated cell groups

[0078] Example 5

[0079] In vitro cell migration study of RB@OL nanocomposite for RAW264.7 macrophages and 4T1 tumor cells.

[0080] Cell scratch assay: RAW264.7 and 4T1 cells in logarithmic growth phase and in good growth condition were scratched at 5 × 10⁻⁶ cells per cell line. 5 and 3×10 5Cells were seeded at a density of cells / well in 6-well plates. The plates were then gently shaken to ensure a uniform monolayer distribution of cells. When cell confluence was >95%, scratching was prepared. Using a 100 μl yellow pipette tip, the cells were scratched vertically along a sterile ruler. After scratching, the cells were washed three times with 1×PBS buffer to completely remove any scratched cells, and the images were taken and recorded under an inverted microscope (S). 0h Add 2% FBS DMEM and 1640 medium containing different concentrations of R837 (10, 20, 40 μg / mL) of RB@OL nanocomposite, and incubate at 37°C and 5% CO2 for 24 h before photographing (S). 24h The scratch area S was obtained using ImageJ software, and the scratch healing rate was calculated using the following formula. The results are attached. Figure 9 As shown, RAW264.7 was used as a carrier for the nanocomposite. The expected experimental results were that the drug-containing nanocomposite had no or little effect on its migration, that is, its activity and motility were not affected, ensuring that it could successfully deliver the nanocomposite to the tumor microenvironment. Based on this experiment and the results of the cytotoxicity experiment, it was determined that the final concentration of R837 in the nanocomposite was 5-15 μg / mL.

[0081] Scratch healing rate = [1-(S)] 24h / S 0h )]×100%

[0082] Example 6

[0083] Study on the polarization induction effect of RB-OL@M system on RAW264.7 macrophages

[0084] The polarization-inducing effect of the RB-OL@M system on RAW264.7 macrophages was investigated using Transwell cell chambers combined with FITC-CD206 and PE-CD86 antibodies. RAW264.7 macrophages were cultured at 3 × 10⁻⁶ cells / cells. 5 In 12-well plates, seeding density of 1 × 10⁶ cells / well was used. 5 Macrophages were seeded at a density of 1 cell / well in 0.4 μm Transwell chambers. After cell adhesion and normal differentiation, the macrophages in the 12-well plates were incubated with IL-4 for 36 h, followed by co-culture pretreatment for 24 h. The presence of 808 nm and 1.5 W / cm² was then assessed. 2 The ability of the RB-OL@M system and RB@OL nanocomposite to induce macrophage polarization in vitro was compared with that of the untreated control group under 5 min laser irradiation. Results are attached. Figure 10 As shown, compared with other groups, the combined treatment and drug delivery system under RB-OL@M+808nm laser irradiation conditions exhibited the strongest polarization induction effect on mouse mononuclear macrophage leukemia cells RAW264.7, CD86+ CD206 - The cell percentage was 35.0%, verifying that the nano-drug delivery system constructed in this invention has excellent immune activation effects in vitro.

[0085] Example 7

[0086] Study on the maturation induction effect of RB-OL@M system on dendritic cells DC2.4

[0087] The ripening and induction effects of the RB-OL@M system on dendritic cell DC2.4 were investigated using Transwell cell chambers combined with FITC-CD80 and PE-CD86 antibodies. Dendritic cell DC2.4 cells were incubated at 2 × 10⁻⁶ cells per cell line. 5 Macrophages were seeded at a density of 1 × 10⁶ cells / well in 12-well plates. 5 Cells were seeded at a density of 1 cell / well in 0.4 μm Transwell chambers. After cell adhesion and normal differentiation, a 24-hour co-culture pretreatment was performed, followed by examination for the presence of 808 nm and 1.5 W / cm². 2 The ability of the RB-OL@M system, RB@OL nanocomposite, OMVs / Lip nanoparticle carrier, and 4T1-DC2.4 co-cultured under 5 min laser irradiation to induce dendritic cell maturation in vitro was compared with that of the untreated control group. Results are attached. Figure 11 As shown, compared with other groups, the combined therapeutic drug delivery system under RB-OL@M+808nm laser irradiation conditions exhibited the strongest maturation induction effect on mouse bone marrow-derived dendritic cells (DC2.4), with a mature cell percentage of 38.2%. Simultaneously, the 4T1-DC2.4 co-culture drug delivery + light irradiation group also showed a certain induction effect, with a mature cell percentage of 13.5%. Heat-induced apoptosis of cancer cells may lead to the release of tumor-associated antigens (TAAs) and related molecular patterns (DAMPs) such as heat shock protein 70 (Hsp70) and high-mobility group box 1 (HMGB1); these substances can induce an effective immune response, and the stimulation is further enhanced with the introduction of immune adjuvants. This verifies that the nanodelivery system constructed in this invention has excellent immune activation activity in vitro.

[0088] Example 8

[0089] Study on the apoptosis-inducing effect of the RB-OL@M system on 4T1 tumor cells

[0090] The apoptosis-inducing effect of the RB-OL@M system on tumor cells 4T1 was investigated using Transwell cell chambers combined with an Annexin V-FITC / PI apoptosis detection kit. Tumor cells 4T1 were inoculated at a concentration of 2 × 10⁶ cells / year. 5Macrophages were seeded at a density of 1 × 10⁶ cells / well in 12-well plates. 5 Cells were seeded at a density of 1 cell / well in 0.4 μm Transwell chambers. After cell adhesion and normal differentiation, a 24-hour co-culture pretreatment was performed, followed by examination for the presence of 808 nm and 1.5 W / cm². 2 The ability of the RB-OL@M system and RB@OL nanocomposite to induce tumor cell apoptosis in vitro was compared with that of the untreated control group under 5 min laser irradiation. Results are attached. Figure 12 As shown, compared with other groups, the combined treatment and drug delivery system under RB-OL@M+808nm laser irradiation conditions had the strongest apoptosis-inducing effect on mouse breast cancer 4T1 cells, with an early apoptosis rate of 23.7% and a late apoptosis rate of 37.5%; verifying that the nano-drug delivery system constructed in this invention has excellent tumor-killing effects in vitro.

[0091] Example 9

[0092] The fluorescently labeled RB-OL@M system was distributed in tumor-bearing mice in vivo and in isolated organs and tissues.

[0093] A mouse orthotopic breast cancer model was established. Three groups (DiR@OL, DiR@M, and DiR-OL@M) were administered the drug via tail vein injection. The distribution of DiR fluorescence emission within the mice was observed using a small animal in vivo imaging system at 1, 4, 8, 12, 24, and 48 hours post-administration. Finally, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, and tumor tissues were dissected. The distribution of DiR fluorescence emission within the excised tissues was then observed using a small animal in vivo imaging system. Results are attached. Figure 13 , 14 As shown, the DiR-OL@M group can efficiently target and accumulate in tumor tissue. There is no significant difference in tumor targeting ability compared with the DiR@M group, indicating that loading nanomedicine into macrophages does not affect their normal activity and migration ability; and it is superior to the DiR@OL group, indicating that loading nanoparticles into macrophages can significantly improve their tumor targeting.

[0094] Example 10

[0095] In vivo safety evaluation of the RB-OL@M system

[0096] Healthy female BALB / c mice were randomly divided into two groups and injected via tail vein with PBS and RB-OL@M, respectively. After treatment, the mice were sacrificed, and the heart, liver, spleen, lungs, and kidneys were dissected and fixed with 4% paraformaldehyde. After fixation, the tissues were embedded in paraffin, and finally, sections were stained with H&E and observed. The results are attached. Figure 15As shown in the scale bar (200 μm), compared with the PBS control group, no pathological changes such as lesions, necrosis, or morphological abnormalities were found in any tissue of the RB-OL@M administration group. Their histological morphology was normal, indicating that the RB-OL@M system has good in vivo safety.

[0097] The macrophage-loaded nanocomposite antitumor targeted drug delivery system of the present invention has a coating structure that significantly increases drug loading capacity. At the same time, it greatly improves safety. The OMVs modified on the surface of liposomes improve the stability of liposomes and prevent leakage of Toll-like receptor agonist drugs, while maintaining the antitumor phenotype of macrophage carriers. It also improves the stability of BPQD and can induce tumor cell apoptosis and promote drug release and penetration under laser irradiation.

[0098] Photothermal agents (PTA) convert light energy into heat energy under external light sources such as NIR (500–900 nm). The converted heat is used to raise the temperature of the tumor microenvironment (TME), thereby killing tumor cells. PTA can be broadly classified into inorganic and organic PTA. Inorganic PTA has advantages such as good stability and high photothermal conversion efficiency, and mainly includes noble metals, carbon-based materials, and transition metals. Organic PTA has advantages such as biodegradability and good biosafety, and mainly includes cyanides, porphyrins, and polymer nanoparticles. When PTA is irradiated with light of a specific wavelength, it absorbs energy from photons and transforms from a ground state to an excited state. Then, the electronic excitation energy undergoes vibrational relaxation, a non-radiative decay process. During this process, collisions between the excited PTA and surrounding molecules mediate the return to the ground state. Therefore, the increase in kinetic energy leads to the heating of the surrounding microenvironment.

[0099] The constructed RB-OL@M first utilizes the natural chemotaxis of macrophages to target the tumor microenvironment, which significantly improves the tumor targeting efficiency of liposomes on the one hand, and compensates for their poor deep tumor penetration on the other hand, greatly improving the accumulation and efficacy of therapeutic drugs at the tumor site. Secondly, based on the natural phagocytic capacity of macrophages, bacterial-derived outer membrane vesicles and their internal loads can be effectively accumulated in the cell. At the same time, macrophages loaded with OMVs can not only highly retain their immunogenicity, but also ensure their safety of application and prolong their in vivo circulation time, avoiding the cytokine storm and antibody-specific clearance caused by direct injection of OMVs.

[0100] This invention utilizes macrophages and liposomes as carriers, combining OMVs, R837 immunotherapy with BPQD combined phototherapy. Furthermore, photothermal-induced immunogenic cell death (ICD) can enhance the efficacy of immunotherapy. Moreover, drug delivery via macrophages avoids recognition by the reticuloendothelial system, achieving immune escape and increasing drug circulation time in the body. In addition, the inflammatory tumor microenvironment regulated by this delivery system can further promote macrophage recruitment and accumulation, generating a positive feedback loop and effectively improving the tumor immunosuppressive microenvironment, inhibiting tumor growth and metastasis.

Claims

1. A macrophage-loaded nanocomposite antitumor targeted drug delivery system, characterized in that, The drug delivery system comprises liposomes composed of a Toll-like receptor agonist, a photothermal responsiveness agent, phospholipids, and cholesterol, and a drug-loaded nanocomplex formed by coating bacterial outer membrane vesicles on the outer surface of the liposomes. The nanocomplex is co-incubated and loaded into macrophages. The Toll-like receptor agonist is imiquimod, the photothermal responsiveness agent is ultra-micro black phosphorus quantum dots, the bacterial outer membrane vesicles are derived from Escherichia coli, the macrophages are mouse mononuclear macrophage leukemia cells, and the phospholipids are one of soybean lecithin, soybean lecithin, egg yolk lecithin, myristoyl lecithin, dipalmitoyl lecithin, and distearate lecithin.

2. The preparation method of the macrophage-loaded nanocomposite antitumor targeted drug delivery system according to claim 1, characterized in that, Includes the following steps: (1) Dissolve phospholipids, cholesterol and imiquimod in an organic solvent, rotary evaporate and dry at room temperature, add an aqueous solution of ultra-micro black phosphorus quantum dots, rotary hydrate and ultrasonically disperse, and filter through a water film to obtain liposome nanoparticles; (2) The liposome nanoparticles obtained in step (1) are mixed evenly with an aqueous solution of bacterial outer membrane vesicles from Escherichia coli, and then co-extruded using a liposome extruder to obtain a drug-loaded nanocomposite. (3) After diluting the complex obtained in step (2) with culture medium, the nanocomplex was loaded into mouse mononuclear macrophage leukemia cells after co-incubation to obtain the anti-tumor targeted drug delivery system.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass ratio of phospholipids, cholesterol, imiquimod and ultra-micro black phosphorus quantum dots is 180:30:20:1~5.

4. The preparation method according to claim 2, characterized in that, In step (2), the mass ratio of the aqueous solution of bacterial outer membrane vesicles from Escherichia coli to liposome nanoparticles is 1:2 to 2:

1.

5. The preparation method according to claim 2, characterized in that, In step (3), the final concentration of imiquimod in the complex is 5-15 μg / mL, and the density of mouse mononuclear macrophage leukemia cells is 1.25-6.25 × 10⁻⁶. 5 The number of cells / mL was increased, and the incubation time was 2-12 hours.

6. The use of the macrophage-loaded nanocomposite antitumor targeted drug delivery system according to claim 1 in the preparation of a drug for treating breast cancer.

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