CN116139097A

CN116139097A - Macrophage-loaded nano-composite anti-tumor targeting drug delivery system and preparation method and application thereof

Info

Publication number: CN116139097A
Application number: CN202211463696.2A
Authority: CN
Inventors: 王伟; 傅聪; 唐璐
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2022-11-22
Filing date: 2022-11-22
Publication date: 2023-05-23

Abstract

The invention discloses a macrophage loaded nano-composite anti-tumor targeting drug delivery system, a preparation method and application thereof, wherein the drug delivery system is formed by forming liposome nano-particles by using Toll-like receptor agonists, photothermal response agents, phospholipids and cholesterol, coating bacterial outer membrane vesicles on the outer surfaces of the drug-loaded liposome nano-particles to form drug-loaded nano-composites, and loading the drug-loaded nano-composites into macrophages through co-incubation; the system of the invention combines immunotherapy and combined photothermal therapy to produce immunogenic cell death induced by photothermal effect by taking macrophages and liposome as carriers to improve the immunotherapy effect; the inflammatory tumor microenvironment regulated by the delivery system can further promote the recruitment and accumulation of macrophages, generate positive feedback circulation, and effectively improve tumor immunosuppression microenvironment and inhibit tumor growth and metastasis.

Description

Macrophage-loaded nano-composite anti-tumor targeting drug delivery system and preparation method and application thereof

Technical Field

The invention relates to a targeting drug delivery system, in particular to a macrophage loaded nano-composite anti-tumor targeting drug delivery system, and also relates to a preparation method and application of the targeting drug delivery system.

Background

The tumor microenvironment (Tumor microenvironment, TME) is an internal and external environment for the development of tumorigenesis and has complex composition. The cellular components of TME are composed of tumor-associated macrophages, dendritic cells, lymphocytes, fibroblasts, and the like. Among them, tumor-associated macrophages (Tumor-associated macrophages, TAMs) are one of the most abundant immune cells in TME. Current macrophage-based tumor treatment strategies are inhibiting macrophage recruitment, repolarization and depletion of macrophages. Since M1/M2 imbalance is a key cause of tumor progression and metastatic resistance, activation of innate immunity NF- κb signaling pathway by targeted delivery of Toll-like receptor (TLRs) agonists and repolarization of M2-type TAMs is a promising immunotherapeutic strategy, but targeted delivery of Toll-like receptor agonists has limited drug loading and still presents the problem of low drug loading and safety of abnormal changes to tissues.

Disclosure of Invention

The invention aims to: the invention aims to provide a macrophage loaded nano-composite anti-tumor targeting drug delivery system which has the advantages of large drug loading capacity, strong targeting property, strong safety and strong anti-tumor treatment, and the second aim is to provide a preparation method and application of the drug delivery system.

The technical scheme is as follows: the drug delivery system comprises liposome nanoparticles composed of Toll-like receptor agonists, photothermal response agents, phospholipids and cholesterol, and a drug-carrying nano-composite formed by coating bacterial outer membrane vesicles on the outer surfaces of the liposome nanoparticles, wherein the nano-composite is co-incubated and loaded in macrophages.

Preferably, the Toll-like receptor (TLR 3, TLR7/8 and TLR 9) agonist is one of poly (I: C), imiquimod (R837)/resiquimod (R848) and an oligodeoxynucleotide (CpG ODN) containing cytosine guanine dinucleotides.

Preferably, the photothermal response agent is one of indocyanine green (ICG), carbocyanine Dye (DIR), IR820, IR780, iron phthalocyanine (FePc), chlorin (Ce 6), porphyrin-diketopyrrolopyrrole (Por-DPP), polydopamine (PDA), gold nanoparticles (AuNPs), gold nanorods (AuNRs), carbon Nanotubes (CNTs), carbon Dots (CDs) or Quantum Dots (QDs).

Preferably, the Quantum Dots (QDs) are ultra-micro Black Phosphorus Quantum Dots (BPQDs).

Preferably, the bacterial outer membrane vesicles are from one of E.coli K-12W3110, E.coli BL21 (DE 3), E.coli DH 5. Alpha., E.coli JC8031, A.baumannii, K.pneumoniae, P.aeromonas.

Preferably, the macrophage is one of a mouse mononuclear macrophage leukemia cell (RAW 264.7), a bone marrow-derived macrophage (BMDM), an abdominal cavity-derived macrophage (PM), a peripheral blood-derived macrophage, or an induced pluripotent stem cell-derived macrophage.

Preferably, the phospholipid is one of soybean phospholipid, soybean lecithin, egg yolk lecithin, dimyristoyl lecithin, dipalmitoyl lecithin or distearyl lecithin.

The invention relates to a preparation method of a macrophage loaded nano-composite anti-tumor targeting drug delivery system, which comprises the following steps:

(1) Preparing liposome nanoparticles loaded with Toll-like receptor agonists and photothermal response agents: dissolving phospholipid, cholesterol and Toll-like receptor agonist in an organic solvent, rotary evaporating, drying at room temperature, adding a photothermal response agent aqueous solution, rotary hydrating, performing ultrasonic dispersion, and filtering with a water film to obtain liposome nanoparticles.

(2) Preparation of bacterial outer membrane vesicle modified drug-loaded nanocomposites: and (3) preparing a bacterial outer membrane vesicle aqueous solution, uniformly mixing the liposome nanoparticles with the bacterial outer membrane vesicle aqueous solution, and coextruding by a liposome extruder to obtain the drug-loaded nano-composite.

(3) Preparing an anti-tumor targeted drug delivery system: diluting the drug-loaded nano-composite prepared in the step (2) to a proper concentration by using a culture medium, and loading the nano-composite in macrophages after co-incubation to obtain the anti-tumor targeted drug delivery system.

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

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

Preferably, in the step (3), the final concentration of the Toll-like receptor agonist in the drug-loaded nano-composite is 5-15 mug/mL, and the co-incubation time is 2-12 h.

Preferably, the step (1) specifically comprises: dissolving phospholipid, cholesterol and R837 in an organic solvent, removing the organic solvent by rotary evaporation for 15min at 40 ℃ to form a uniform oil film, drying at room temperature, adding a BPQD aqueous solution into the dry oil film, rotary hydrating for 30min at 40 ℃ to wash the oil film and uniformly dispersing the oil film into the aqueous solution, further dispersing the aqueous dispersion under a probe of an integrated ultrasonic cell disruption instrument, and sequentially filtering the nanoparticle solution through a water system microporous filter film with the thickness of 0.45 mu m and a water system microporous filter film with the thickness of 0.22 mu m to finally obtain the liposome RB@Lip loaded with the R837 and the BPQD.

Preferably, the step (2) specifically comprises: and E, centrifuging the culture solution of the coli DH5 alpha bacteria in a refrigerated centrifuge at 4 ℃ and 8000g for 15min, discarding the precipitate, filtering the supernatant through a 0.45 mu m PES vacuum filter, concentrating the supernatant by using a 100kDa Amicon Ultra15 centrifugal ultrafiltration tube, discarding the outer tube liquid, centrifuging the inner tube liquid in an overspeed low-temperature centrifuge at 4 ℃ and 150000g for 2h, discarding the supernatant, and re-suspending the precipitate in 1 XPBS and filtering the precipitate through the 0.22 mu m PES vacuum filter to finally obtain the E.coli DH5 alpha outer membrane vesicle OMVs. And (3) coextruding RB@lip and OMVs in a certain proportion by a liposome extruder for a plurality of times to finally obtain the E.coli DH5 alpha outer membrane vesicle modified drug-loaded liposome RB@OL.

Preferably, the step (3) specifically comprises: 1 to 5 multiplied by 10 ⁶ The RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum, 100U/mL penicillin, 100. Mu.g/mL streptomycin, replaced with serum-free DMEM medium containing RB@OL after 24 hours, and cultured for a period of time to allow the cells to phagocytose the nanocomposites, then the cells were washed several times with pre-warmed PBS buffer to remove free nanocomposites, and the cells were collected with a cell scraper to finally obtain nanocomposite-loaded macrophages RB-OL@M.

The macrophage loaded nano-composite anti-tumor targeting drug delivery system is applied to preparation of drugs for improving tumor immunity inhibition microenvironment and inhibiting tumor growth.

The invention adopts liposome modified by bacterial outer membrane vesicle to load fat-soluble Toll-like receptor agonist and water-soluble photo-thermal response agent to form drug-loaded nano-composite, then the drug-loaded nano-composite enters macrophages through membrane fusion or endocytosis, and is contained in phagolysosomes formed by fused phagolysosomes and lysosomes to form an antitumor targeted drug delivery system for loading therapeutic drugs on the macrophages.

Wherein Gram-negative bacteria (G ^- ) Outer Membrane Vesicles (OMVs) as carriers and immune activators with particle sizes of 20-250nm. OMVs are more readily recognized and phagocytized by macrophages than other exosomes such as mesenchymal stem cell-derived exosomes, because they possess a large number of components from the outer membrane and peripheric parent bacteria and are therefore characterized by high immunogenicity. R837 acts as a TLR-7 agonist, binding to TLR-7 overexpressed in the endosome/lysosome of macrophages, activating innate and adaptive immune responses; r837 can be used for repolarization of TAMs to M1-type macrophages, thereby promoting infiltration of cytotoxic T cells to tumors, thereby inhibiting tumors; when it is not encapsulated, there are problems of poor water solubility and no targeting, and the function of the immunomodulator is limited, which seriously affects the potency of the drug. BP material, especially BPQD has large extinction coefficient, high photo-thermal conversion efficiency and high kidney discharge efficiency, but the material is too fragile and is easy to pass through O in water environment exposed in air ₂ And H ₂ The O reaction degrades, greatly reducing its PTT potency, and the BP is poorly dispersible, which makes it easy to aggregate under physiological conditions, and has the disadvantages of short in vivo circulation time and unstable optical properties, which can be resolved by further coating it.

The Lip modified by OMVs is used for loading fat-soluble R837 and water-soluble BPQD to form an RB@OL nano-composite, and a macrophage loading therapeutic drug system is formed by co-incubation, so that the problems of poor water solubility, no targeting property and low potency of R837, and the problems of easiness in degradation of BPQD, low PTT efficiency, poor dispersibility, short in-vivo circulation time and unstable optical property are solved; the targeted delivery of R837 is cooperatively realized, the stability of BPQD is improved, and a better photo-thermal effect is exerted, so that the overall drug effect of the drug delivery system is improved.

The nano delivery system is positioned in the RB@OL part in the phagosome of the macrophage to release cells into the tumor microenvironment and permeate into deep tissues, OMVs and R837 activate TLR-7 receptors in the tumor-associated macrophage to polarize the tumor-associated macrophage from an M2 phenotype to an M1 phenotype, and simultaneously secrete cytokines such as TNF-alpha, IL-1 beta, IFN-gamma, IL-6 and the like; promoting recruitment and maturation of antigen presenting dendritic cells, thereby initiating an immune response; promote infiltration of cytotoxic T cells and improve tumor immunosuppression microenvironment. The BPQD generates local heat under the irradiation condition of 808nm laser to promote the complete release of the medicine, induce the apoptosis of tumor cells and inhibit the growth and metastasis of the tumor. The nano delivery system plays roles of an immunomodulator and a photothermal response agent through the targeted TME, realizes combined treatment of tumor and deep immunotherapy and phototherapy thereof, and generates remarkable anti-tumor effect.

The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages:

(1) According to the anti-tumor targeted drug delivery system, on one hand, the stability of liposome is improved, toll-like receptor agonist drugs are prevented from leaking, the anti-tumor targeted drug delivery system is promoted to induce tumor cell apoptosis under laser irradiation, and drug release and permeation are promoted, on the other hand, the anti-tumor phenotype of macrophage carriers is maintained, and the safety of the targeted drug delivery system is greatly improved;

(2) The coating structure of the drug delivery system greatly improves the drug loading capacity of cells, remarkably improves the tumor targeting efficiency of liposome and the accumulation and drug effect of therapeutic drugs at tumor positions, and increases the circulation time of the drugs in vivo; the apoptosis induction effect of the laser irradiation combination on the breast cancer cells of the mice can reach 23.7 percent at the highest, and the percentage of late apoptosis can reach 37.5 percent at the highest, thereby improving the apoptosis rate of the cancer cells with single action compared with the liposome loaded with the immunomodulator by 5 times and having excellent tumor killing effect.

Drawings

FIG. 1 is a graph of characterization results of RB@lip nanoparticles in example 1, wherein graph A is a particle size graph and a transmission electron microscope graph of the RB@lip nanoparticles, and graph B is a transmission electron microscope graph of the BPQDs;

FIG. 2 is a graph showing the results of characterization of OMVs in example 2, wherein A is a transmission electron microscope image of OMVs and B is an SDS-PAGE electrophoresis image of OMVs;

FIG. 3 is a graph of fluorescence co-localization of RB@OL nanocomposites in example 2

FIG. 4 is a graph of particle size and transmission electron microscopy of RB@OL nanocomposites of example 2;

FIG. 5 is a photo-thermal imaging of RB@OL nanocomposite of example 2;

FIG. 6 is a graph of the stability results of the RB@OL nanocomposite of example 2, wherein graph A is a graph of the particle size and PDI change measured for the stability of an aqueous dispersion of the RB@OL nanocomposite, and graph B is a graph of the particle size and PDI change measured for the stability of a 10% fetal bovine serum dispersion;

FIG. 7 is an inverted fluorescence microscope image of the uptake of C6@OL nanocomposites by RAW264.7 cells in example 3;

FIG. 8 is a cytotoxicity study of RB@OL nanocomposites in example 4, wherein Panel A is the cytotoxicity profile of R837 free drug against RAW264.7, 4T1 and L929, panel B is the cytotoxicity profile of OL vectors against RAW264.7, 4T1 and HUVEC, panel C is the cytotoxicity profile of RB@OL nanocomposites against RAW 264.7;

FIG. 9 is a graph showing the results of cell migration-scratch healing experiments for RB@OL nanocomposites of example 5, wherein A is a graph showing the results of scratch healing rates of RB@OL nanocomposites containing different concentrations of R837 on RAW264.7 cells, and B is a graph showing the results of scratch healing rates on 4T1 cells;

FIG. 10 is a graph showing the polarization induction of RAW264.7 cells by different formulation groups in example 6;

FIG. 11 is a graph showing the induction of DC2.4 cell maturation by various formulation groups of example 7;

FIG. 12 is a graph of apoptosis induction of 4T1 cells by different formulation groups of example 8;

FIG. 13 is a plot of the in vivo profile of fluorescently labeled RB-OL@M of example 9 in tumor bearing mice;

FIG. 14 is a plot of the in vitro tissue distribution of the fluorescence labeled RB-OL@M of example 9;

FIG. 15 is a graph showing the results of H & E staining of heart, liver, spleen, lung, kidney tissue sections of mice, as assessed by in vivo safety of RB-OL@M in example 10.

Detailed Description

The technical scheme of the invention is further described below with reference to the accompanying drawings.

Example 1

RB@lip nanoparticle preparation and characterization

(1) Preparation of RB@lip nanoparticles

18mg of soybean lecithin, 3mg of cholesterol and 2mg of R837 are precisely weighed and dissolved in 5mL of mixed solvent of methanol/chloroform with the ratio of 1:4 (v/v), the mixture is transferred into a brown eggplant-shaped flask after the medicine is completely dissolved, the mixture is subjected to reduced pressure rotary evaporation for 15min in a water bath with the temperature of 40 ℃, and after the solvent is completely evaporated and a uniform lipid film is formed at the bottom of the flask, the mixture is placed in a vacuum dryer for drying for 2h. Then 5mL of 100 mug/mL BPQD aqueous dispersion is measured and added into a flask, hydration is carried out for 30min at 40 ℃, and after lipid membrane hydration is completed, the obtained lipid suspension is subjected to ice bath ultrasonic treatment for 15min under the conditions of 200W and 2s of on time and 2s of off time, so as to obtain the lipid nanoparticle. Finally, the solution is filtered by a water system microporous filter membrane with the thickness of 0.45 μm and a water system microporous filter membrane with the thickness of 0.22 μm to obtain the RB@Lip nanoparticle aqueous solution.

(2) Characterization of RB@lip nanoparticles

Measuring the particle size of the RB@Lip nanoparticle aqueous solution by adopting a dynamic light scattering method (DLS), wherein the average particle size of the nanoparticle is 81.3+/-2.4 nm as shown in a figure 1; the structure of the BPQD and RB@lip nanoparticle aqueous solution is observed through a Transmission Electron Microscope (TEM), the result is shown in the attached figure 1, the actual nanoparticle size is similar to the detected particle size, the structure of the BPQD encapsulated in the Lip is clearly visible, and the RB@lip nanoparticle is successfully prepared.

Example 2

RB@OL nanocomposite preparation, characterization and property research

(1) OMVs extraction, concentration and purification

E. coli DH 5. Alpha. Cells were cultured overnight in 500mLLB broth (1% peptone, 0.5% yeast extract, 1% NaCl, pH 7.0) and incubated with shaking at 37℃and 200 rpm. At OD ₆₀₀ After reaching 1.2-1.5, bacterial cells were removed by centrifugation in a refrigerated centrifuge at 4℃for 15min at 8000g, the resulting supernatant was sterilized by filtration through a 0.45 μm PES vacuum filter, and then concentrated using a 100kDa Amicon Ultra15 centrifugal ultrafiltration tube, the resulting concentrate was centrifuged in a Beckman ultra-low temperature centrifuge at 4℃for 2h at 150000g, and the pellet was resuspended in 1 XPBS to remove the contaminants introduced during the operation by passing through a 0.22 μm PES vacuum filter and stored at-20℃until use.

(2) Characterization of OMVs

Observing the structure of OMVs aqueous dispersion by a Transmission Electron Microscope (TEM), and preparing the structure of the outer membrane vesicle of the obtained escherichia coli in a double-layer membrane vesicle shape as shown in a figure 2A; the SDS-PAGE electrophoresis of E.coli DH5 alpha and its outer membrane vesicle OMVs were analyzed for whole protein profile, and the results are shown in FIG. 2B, wherein the OMVs and OMVs/NPs have identical protein bands, and the OMVs characteristic protein OmpA concentration is not different in the two groups, which indicates that OMVs are successfully coated on the Lip surface and the structural characteristics and the biological activity are not affected.

(3) Preparation of RB@OL nanocomposites

Mixing the drug-loaded liposome and the outer membrane vesicle suspension of the escherichia coli according to a certain proportion, physically extruding for a plurality of times through a 100nm polycarbonate membrane, and centrifuging for 5min at the temperature of 4 ℃ and the condition of 14000g to remove free vesicles, wherein the obtained precipitate is the RB@OL nano-composite.

(4) Characterization of RB@OL nanocomposites

Fluorescence co-localization investigation: OMVs-DiI was used in place of OMVs, coumarin 6 (C6) was used in place of R837, and a DiI and C6 co-labeled RB@OL nanocomposite (DC@OL) was prepared according to the best-prescription procedure and fluorescence observed using a confocal microscope. As shown in FIG. 3, the red fluorescence of DiI and the green fluorescence of C6 are in the same domain, which indicates that OMVs were successfully modified on the Lip surface. (Scale bar:10 μm)

The particle size of the RB@OL nanocomposite aqueous solution is measured by a dynamic light scattering method (DLS), and the result is shown in figure 3, wherein the average particle size of the nanocomposite is 126.4+/-3.8 nm; the structure of the RB@OL nanocomposite aqueous solution is observed through a Transmission Electron Microscope (TEM), the result is shown in a figure 4, the actual size of the nanocomposite is similar to the detected particle size, compared with the RB@lip nanoparticle, the particle size is larger, the morphology is more round, the outermost layer has an obvious shell structure, and the fact that OMVs are successfully modified on the Lip surface is indicated.

(5) Property study of RB@OL nanocomposites

The photo-thermal conversion capability of the RB@OL nanocomposite was examined, four groups of PBS, BPQD, lip-BPQD and OMVs/Lip-BPQD were set, a sample solution was measured and placed in an EP tube, and a 808nm laser emitter (1.0W/cm ² ) It was irradiated for 10 minutes, and a thermal radiation image of the EP tube during irradiation and a temperature change value of the dispersion were recorded with a thermal imager. As shown in fig. 5, the liposome coating and membrane vesicle modification do not affect the photothermal properties of BPQDs, and the photothermal conversion capability of the aqueous dispersion of BPQDs is not significantly different.

The particle size stability of the RB@OL nanocomposite aqueous dispersion liquid in the standing process is examined, and the result is shown in a figure 6A; the particle size stability of the RB@OL nanocomposite in the incubation process is examined, and an environment containing 10% fetal bovine serum is adopted to simulate an in-vivo environment in vitro, and the result is shown in figure 6B; along with the extension of standing/incubation time, the particle size and PDI of the nano-composite fluctuate up and down, the particle size change is less than 20%, and the PDI is less than 0.3, which shows that the nano-composite aqueous dispersion and the in-vitro serum stability are good.

Example 3

Construction of RB-OL@M system

(1) Culture of RAW264.7

RAW264.7 macrophages within 5 generations are selected, inoculated into a cell culture flask, cultured in a DMEM complete medium containing 100 mug/mL streptomycin, 100U/mL penicillin, 10% peptide bovine serum, and placed at 37 ℃ and 5% CO ₂ And culturing in an incubator with 95% humidity. When the cell fusion reached 70-80%, passaging was performed, and digestion was performed with 0.25% trypsin at room temperature for 1min. Stopping digestion after the cells are digested to be round, and gently blowing the adherent cells to fall off. The cell suspension was collected and centrifuged at 1000rpm for 5min to pellet the cells. The supernatant was discarded, the cells were resuspended in fresh complete medium, and finally inoculated into sterile cell culture flasks at the appropriate cell density and the culture was continued as described above.

(2) Construction of RB-OL@M system

RAW264.7 cells in the logarithmic growth phase and in good growth state were collected at 3X 10 ⁵ The concentration of each/well was inoculated into a 12-well plate, and the wall was cultured in DMEM complete medium. Then the DMEM serum-free medium containing C6, lip-C6 and OMVs/Lip-C6 is replaced for incubation for 2 to 12 hours. After the incubation was completed, the cells were fixed with 4% (w/w) paraformaldehyde at 500. Mu.L for 20min, washed with PBS, incubated with DAPI staining solution at 250. Mu.L for 10min, washed with PBS, the washes were discarded and the cells were infiltrated with 500. Mu.LPBS, and finally examined for uptake by an inverted fluorescence microscope. As shown in FIG. 7, the modification of OMVs resulted in the fluorescent-labeled nanoparticles being more readily taken up by macrophages, with the highest fluorescence values for the OMVs/Lip-C6 group.

Example 4

In vitro cytotoxicity study of RB@OL nanocomplex on macrophage RAW264.7 and tumor cell 4T1

RAW264.7, 4T1, L929 and HUVEC cells in the logarithmic growth phase and in good growth state were inoculated and were 8X 10 respectively ³ The density of each well is inoculated into a 96-well plate, five wells are arranged, 100 mu LDMEM/1640 complete medium is added into each well, and the temperature is 37 ℃ and the CO concentration is 5% ₂ Culturing in a cell culture incubator. Waiting cellAfter adherence, the cells were then cultured for 24h with DMEM/1640 basal medium containing R837, OMVs/Lip and RB@OL, respectively. The working concentration of R837 was 0.1, 0.5, 1, 2, 5, 10, 15, 20, 30, 50. Mu.g/mL, the working concentration of OMVs/Lip was 50, 20, 15, 10, 5, 1, 0.1. Mu.g/mL (PC final concentration determination), and the working concentration of RB@OL was 1, 10, 20, 50, 100. Mu.g/mL (R837 final concentration determination). The non-dosed group was set as the control group and the PBS group alone was set as the blank group. The culture was then discarded and washed with PBS buffer, followed by the addition of 120. Mu.L per well of serum-free medium containing 20. Mu.L of 5mg/mL MTT solution and 5% CO at 37 ℃ ₂ Incubating in a cell incubator for 4-6h. The culture medium was discarded, 150. Mu.L of dimethyl sulfoxide was added to each well, and after shaking for 60 seconds, the absorbance value of each well was measured at 490nm using an microplate reader, and the cell viability was calculated as follows. The results are shown in fig. 8, where firstly the nanocomposite carrier safety was verified and secondly the safe and optimal dosing was determined based on the IC50 values of R837 and the survival results of rb@ol.

Cell viability= (a _Sample -A _PBS )/(A _control -A _PBS )×100％

A _Sample Absorbance of drug-treated cell group

A _PBS Absorbance of PBS solution

A _control Absorbance of the non-drug treated cell group

Example 5

In vitro cell migration study of RB@OL nanocomplex on macrophage RAW264.7 and tumor cell 4T1

Cell scratch assay: RAW264.7 and 4T1 cells in the logarithmic growth phase and in good growth state were taken at 5X 10, respectively ⁵ And 3X 10 ⁵ The density of each hole is inoculated into a 6-hole plate, then the cell culture plate is shaken lightly to uniformly distribute the cell monolayer, and the cell fusion degree is achieved>Scratch was prepared at 95%. Scratch along a sterile ruler with a 100 μl yellow gun head kept vertical. After the scratch was completed, the scratched cells were completely removed by washing 3 times with 1 XPBS buffer and photographed under an inverted microscope for recording (S _0h ). Adding a mixture containing R837 (10, 20)40 μg/mL) of RB@OL nanocomposite in 2% FBS DMEM and 1640 medium at 37deg.C, 5% CO ₂ After culturing in a cell incubator for 24 hours, photographing (S) _24h ). Scratch area S was obtained using ImageJ software and scratch healing rate was calculated as follows. As shown in fig. 9, RAW264.7 is used as a carrier of the nano-composite, and the expected experimental result is that the drug-containing nano-composite has no effect or little effect on the mobility thereof, namely, the activity and mobility thereof are not affected, so that the nano-composite can be successfully delivered to the tumor microenvironment; as can be seen from the experimental results of the experiment combined with cytotoxicity, the concentration of the final nanocomposite containing R837 was determined to be 5-15. Mu.g/mL.

Scratch healing rate= [1- (S) _24h /S _0h )]×100％

Example 6

Polarization induction effect research of RB-OL@M system on macrophage RAW264.7

The polarization induction effect of the RB-OL@M system on macrophage RAW264.7 was studied using Transwell cell-chamber binding FITC-CD206 antibody and PE-CD86 antibody. Macrophage RAW264.7 at 3×10 ⁵ Density of individual/well inoculated in 12 well plate, 1×10 ⁵ Inoculating density of each hole into a Transwell chamber with the aperture of 0.4 mu m, incubating macrophages in a 12-hole plate for 36h after cells are attached and differentiated normally, performing co-culture pretreatment for 24h, and then respectively examining whether 808nm and 1.5W/cm exist or not ² The ability of the RB-ol@m system and rb@ol nanocomposite to induce macrophage polarization in vitro compared to untreated control group under laser irradiation conditions for 5 min. As shown in FIG. 10, the combination therapy administration system under the laser irradiation condition of RB-OL@M+808nm has the strongest polarization induction effect on mouse mononuclear macrophage leukemia cells RAW264.7, CD86, compared with other groups ⁺ CD206 ^- The percentage of cells was 35.0%; the nano drug delivery system constructed by the invention is proved to have excellent immune activation effect in vitro.

Example 7

Research on maturation induction effect of RB-OL@M system on dendritic cell DC2.4

Using Transwell cellsCell binding FITC-CD80 antibody and PE-CD86 antibody the maturation-inducing effect of the RB-OL@M system on dendritic cells DC2.4 was studied. Dendritic cell DC2.4 was used at 2X 10 ⁵ Density of cells/well in 12-well plate, macrophage RAW264.7 was seeded at 1×10 ⁵ Inoculating the density of each hole into a Transwell chamber with the aperture of 0.4 mu m, carrying out co-culture pretreatment for 24 hours after the cells are attached and differentiated normally, and then respectively checking whether 808nm and 1.5W/cm exist or not ² The ability of the RB-OL@M system, the RB@OL nanocomposite, the OMVs/Lip nanoparticle carrier and the 4T1-DC2.4 co-culture administration + illumination group to induce dendritic cell maturation in vitro compared with the untreated control group under the laser irradiation condition for 5 min. As shown in fig. 11, the combination therapy administration system under the laser irradiation condition of RB-ol@m+808nm has the strongest maturation induction effect on mouse bone marrow-derived dendritic cells DC2.4, the percentage of mature cells is 38.2%, while the co-culture administration of 4T1-DC 2.4+illumination group also has a certain induction effect, the percentage of mature cells is 13.5%, and the heat-induced apoptosis of cancer cells may lead to the release of Tumor Associated Antigens (TAAs) and associated molecular patterns (DAMPs) such as heat shock protein 70 (Hsp 70) and high mobility group protein B1 (HMGB 1); these substances can induce an effective immune response and the degree of stimulation is further enhanced with the introduction of immune adjuvants. The nano drug delivery system constructed by the invention is proved to have excellent immune activation effect in vitro.

Example 8

Apoptosis induction effect research of RB-OL@M system on tumor cell 4T1

And (3) researching the apoptosis induction effect of the RB-OL@M system on the tumor cell 4T1 by combining a Transwell cell chamber with an Annexin V-FITC/PI apoptosis detection kit. 4T1 tumor cells were treated at 2X 10 ⁵ Density of cells/well in 12-well plate, macrophage RAW264.7 was seeded at 1×10 ⁵ Inoculating the density of each hole into a Transwell chamber with the aperture of 0.4 mu m, carrying out co-culture pretreatment for 24 hours after the cells are attached and differentiated normally, and then respectively checking whether 808nm and 1.5W/cm exist or not ² The RB-OL@M system and the RB@OL nanocomposite under laser irradiation conditions for 5min were induced in vitro as compared to untreated control groupsAbility of tumor cells to apoptosis. As shown in fig. 12, compared with other groups, the combined treatment administration system under the laser irradiation condition of RB-ol@m+808nm has the strongest apoptosis induction effect on the breast cancer 4T1 cells of the mice, and the early apoptosis percentage is 23.7% and the late apoptosis percentage is 37.5%; the nano drug delivery system constructed by the invention is proved to have excellent tumor killing effect in vitro.

Example 9

Fluorescence labelled RB-OL@M system distribution in vivo and ex vivo organ tissue of tumor bearing mice

The method comprises the steps of constructing a mouse in-situ breast cancer model, carrying out intravenous injection administration on three groups of tails of DiR@OL, diR@M and DiR-OL@M, and observing the distribution of DiR emission fluorescence in a mouse body in a

living animal imager

1, 4, 8, 12, 24 and 48 hours after administration. Finally, mice were sacrificed and heart, liver, spleen, lung, kidney and tumor tissues were dissected out, and the distribution of DiR-emitted fluorescence in the ex vivo tissues was observed in a small animal biopsy imager. As shown in fig. 13 and 14, the DiR-ol@m group can efficiently target and accumulate in tumor tissues, and compared with the dir@m group, the tumor targeting ability is not significantly different, which indicates that the normal activity and migration ability of the nano-drug loaded in macrophages are not affected; and is superior to the DiR@OL group, which shows that the macrophage loaded nano particle can obviously improve the tumor targeting.

Example 10

In vivo safety assessment of RB-OL@M system

Normal healthy BALB/c female mice were selected and randomly divided into 2 groups, which were injected with PBS and RB-ol@m via tail vein, respectively. Mice were sacrificed after treatment and heart, liver, spleen, lung, kidney were dissected out, fixed with 4% paraformaldehyde, paraffin tissue embedded after fixation was completed, and finally sections were H & E stained and observed. As a result, as shown in FIG. 15 (Scale bar:200 μm), each tissue of the RB-OL@M administration group did not find pathological changes such as lesions, necrosis, morphological abnormality, etc., and its histological morphology was normal, indicating that the RB-OL@M system had good in vivo safety, as compared with the PBS control group.

According to the macrophage-loaded nano-composite anti-tumor targeting drug delivery system, the drug loading capacity is greatly improved through the coating structure; meanwhile, the safety is greatly improved, on one hand, the liposome surface modified OMVs improve the stability of the liposome and prevent Toll-like receptor agonist drugs from leaking, and on the other hand, the antitumor phenotype of the macrophage carrier is maintained; meanwhile, the stability of the BPQD is improved, and under the irradiation of laser, the apoptosis of tumor cells can be induced, and the drug release and permeation can be promoted;

the photothermal conversion agent (photothermal agent, PTA) converts light energy into heat energy under the irradiation of external light sources such as NIR (500-900 nm), and the converted heat is used for increasing the temperature of TME so as to kill tumor cells. The PTA can be roughly divided into an inorganic material PTA and an organic material PTA, and the inorganic PTA has the advantages of good stability, high photo-thermal conversion efficiency and the like and mainly comprises noble metal, carbon base, transition metal and the like; the organic PTA has the advantages of biodegradability, good biological safety and the like, and mainly comprises cyanines, porphyrin, polymer nano-particles and the like. When PTA is irradiated with light of a specific wavelength, it absorbs energy from photons and changes from a ground state to an excited state; the electron excitation energy then undergoes decay in a non-radiative form of vibrational relaxation, in which collisions between the excited PTA and surrounding molecules mediate a return to the ground state. Thus, the increase in kinetic energy results in heating of the surrounding microenvironment.

The constructed RB-OL@M firstly utilizes the natural chemotactic effect of macrophages to target the tumor microenvironment, so that on one hand, the tumor targeting efficiency of the liposome is remarkably improved, on the other hand, the defect of poor deep permeability of the tumor is overcome, and the accumulation and the drug effect of the therapeutic drug at the tumor position are greatly improved; secondly, based on the natural phagocytic capacity of macrophages, the outer membrane vesicles from bacteria and the internal load thereof can be effectively accumulated in cells, and meanwhile, the macrophage loaded OMVs can not only highly retain the immunogenicity, but also ensure the application safety and prolong the in vivo circulation time, thereby avoiding cytokine storm and antibody specific clearance caused by OMVs direct injection.

The invention uses macrophages and liposome as vectors, combines the immunotherapy of OMVs and R837 and the combined phototherapy of BPQD, and can improve the immunotherapy effect by the immunogenic cell death (Immunogenic cell death, ICD) induced by the photothermal effect. The medicament can be delivered by macrophages, so that the medicament can be prevented from being identified by reticuloendothelial systems, the immune escape is realized, and the circulation time of the medicament in vivo is prolonged; in addition, the inflammatory tumor microenvironment regulated by the delivery system can further promote recruitment and accumulation of macrophages, generate positive feedback circulation, and effectively improve tumor immunosuppressive microenvironment and inhibit tumor growth and metastasis.

Claims

1. The macrophage loaded nano-composite anti-tumor targeting drug delivery system is characterized by comprising liposome nanoparticles composed of Toll-like receptor agonists, photothermal response agents, phospholipids and cholesterol, and a drug-loaded nano-composite formed by coating bacterial outer membrane vesicles on the outer surfaces of the liposome nanoparticles, wherein the nano-composite is co-incubated and loaded in macrophages.

2. The anti-tumor targeted delivery system of claim 1, wherein the Toll-like receptor agonist is one of polyinosinic acid, imiquimod, resiquimod, or an oligodeoxynucleotide comprising a cytosine-guanine dinucleotide.

3. The anti-tumor targeted drug delivery system according to claim 1, wherein the photothermal response agent is one of indocyanine green, a carbocyanine dye, near infrared dye IR820, near infrared dye IR780, iron phthalocyanine, chlorin, porphyrin-diketopyrrole, polydopamine, gold nanoparticles, gold nanorods, carbon nanotubes, carbon dots, or quantum dots.

4. The anti-tumor targeted drug delivery system of claim 3, wherein the quantum dots are black phosphorus quantum dots.

5. The anti-tumor targeted drug delivery system of claim 1, wherein the bacterial outer membrane vesicles are from one of escherichia coli, acinetobacter baumannii, klebsiella pneumoniae, or pseudomonas aeruginosa; the macrophage is one of mouse mononuclear macrophage leukemia cells, bone marrow-derived macrophages, abdominal cavity-derived macrophages, peripheral blood-derived macrophages or induced pluripotent stem cell-derived macrophages; the phospholipid is one of soybean lecithin, egg yolk lecithin, dimyristoyl lecithin, dipalmitoyl lecithin and distearyl lecithin.

6. The method for preparing the macrophage loaded nano-composite anti-tumor targeting drug delivery system as claimed in claim 1, which is characterized by comprising the following steps:

(1) Dissolving phospholipid, cholesterol and Toll-like receptor agonist in an organic solvent, rotary evaporating, drying at room temperature, adding a photothermal response agent aqueous solution, rotary hydrating, performing ultrasonic dispersion, and filtering with a water film to obtain liposome nanoparticles;

(2) Uniformly mixing the liposome nano particles prepared in the step (1) with a bacterial outer membrane vesicle aqueous solution, and coextruding by a liposome extruder to obtain a drug-loaded nano compound;

(3) Diluting the compound prepared in the step (2) with a culture medium, and loading the nano-compound in macrophages after co-incubation to obtain the antitumor targeted drug delivery system.

7. The method according to claim 6, wherein in the step (1), the mass ratio of the phospholipid, cholesterol, toll-like receptor agonist and photothermal response agent is 180:30:20:1-5; the Toll-like receptor agonist is imiquimod, and the photothermal response agent is ultra-miniature black phosphorus quantum dots.

8. The method according to claim 6, wherein in the step (2), the mass ratio of the bacterial outer membrane vesicle aqueous solution to the liposome nanoparticle solution is 1:2 to 2:1.

9. The method according to claim 6, wherein in step (3), the Toll-like receptor agonist in the drug-loaded nanocomposite has a final concentration of 5 to 15. Mu.g/mL and the macrophage has a density of1.25～6.25×10 ⁵ The total incubation time is 2-12 h per mL.

10. The use of the macrophage loaded nanocomposite anti-tumor targeted drug delivery system of claim 1 in the preparation of a medicament for improving tumor immunosuppressive microenvironment and inhibiting tumor growth.