CN117065041A

CN117065041A - Outer membrane vesicle coated with drug-loaded lipid nanoparticle, and preparation method and application thereof

Info

Publication number: CN117065041A
Application number: CN202310377445.0A
Authority: CN
Inventors: 张娜; 刘金虎; 刘永军; 高彤; 牟伟伟; 梁爽; 黄昕研; 刘暑珺; 刘美辰
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2023-04-04
Filing date: 2023-04-04
Publication date: 2023-11-17
Anticipated expiration: 2043-04-04
Also published as: CN117065041B

Abstract

The invention discloses an outer membrane vesicle coated with drug-loaded lipid nanoparticles, a preparation method and application thereof, wherein the outer membrane vesicle is obtained from a FimH positive strain, is fused with C16-ceramide, and is coated with the drug-loaded lipid nanoparticles. The outer membrane vesicle can enter M1 type macrophages through an endocytic pathway mediated by the small nest protein, so that degradation of nano medicines by strong acid environments such as lysosomes and the like is avoided. The C16-ceramide fused with the outer membrane vesicle can enable the drug-loaded macrophage to release drugs in a non-free form at the tumor site, and the released drugs can be taken up by tumor cells and tumor-related macrophages, so that the effects of regulating the phenotype of the tumor-related macrophages and continuously enhancing the activity of T cells are exerted, and a strong anti-tumor immune response is initiated.

Description

Outer membrane vesicle coated with drug-loaded lipid nanoparticle, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of tumor targeted therapy, and particularly relates to an outer membrane vesicle coated with drug-loaded lipid nanoparticles, a preparation method and application thereof.

Background

Macrophages have the characteristics of flexible self morphology, abundant surface receptors, low immunogenicity and long circulation time, have the cell therapeutic potential of phagocytizing tumor cells, presenting antigens, secreting cytokines and the like, have the drug delivery capability of phagocytizing drug loading, inherent targeting and deep permeation, and are expected to become a dual tool of cell therapy and drug delivery.

In recent years, nano-macrophage drug delivery systems have been developed, which are drug delivery systems formed by loading nano-drugs inside living macrophages. The core technology is that the preparation of the nano particles, the particle size, the surface charge and the shape and the texture of the nano particles can influence the ingestion of macrophages, if the nano particles enter a macrophage lysosome after being ingested, the nano particle structure is degraded, so that the medicine loaded by the nano particles is released in a free form through killing cells or crossing cell membranes, and the tumor treatment effect is seriously influenced. Therefore, what form the nano-drug is loaded inside the living macrophage and then released in what form is of great significance to tumor treatment.

Disclosure of Invention

In one aspect, the invention provides an outer membrane vesicle coated with a drug-loaded lipid nanoparticle.

Further, outer membrane vesicles prior to coating the drug-loaded lipid nanoparticle fuse with C16-ceramide.

Further, outer membrane vesicles prior to coating of drug-loaded lipid nanoparticles were obtained from FimH positive strains. Preferably, the MG1655 strain.

Further, the drug carried by the drug-carrying lipid nanoparticle is R848 and INCB-024360.

R848 is a TLR7/8 pathway agonist, chinese is known as Raximote and English is known as Resiquimod.

INCB-024360 is an IDO1 pathway inhibitor, abbreviated as INCB in this patent.

The important abbreviations in the present invention have the following specific meanings:

OMVs (Outer membrane vesicle, outer membrane vesicles, uncoated lipid nanoparticles)

HCC (Hepatocellular carcinoma )

RIL (Co-carrying R848 and INCB lipid nanoparticle)

RILO (OMV coated with co-supported R848 and INCB lipid nanoparticles)

RILOM1 (M1 type macrophages coated with co-supported R848 and INCB lipid nanoparticle OMVs).

The functional polarization of macrophages is diverse, and is currently considered to be an overlapping continuum of functional states, possessing two extreme states of M1 and M2, and a series of intermediate states that combine the functions of M1 and M2. Wherein, the M1 type macrophage highly expresses CD80 molecules and has the functions of pro-inflammation and anti-tumor; m2 type macrophages highly express CD206 molecules and have anti-inflammatory and antitumor effects. We selected M1 type macrophages for testing.

RILOM1G (M1 type macrophage modified by G12 coating and co-carrying R848 and INCB lipid nanoparticle OMV)

In one aspect, the invention provides a method for preparing outer membrane vesicles coated with drug-loaded lipid nanoparticles, wherein the outer membrane vesicles obtained from FimH positive strains are fused by C16-ceramide, and then the drug-loaded lipid nanoparticles are fused.

Further, RIL was prepared by nano precipitation.

Specifically, 1) R848 and INCB are dissolved in absolute ethanol according to the mass ratio of 1:1, so that an organic phase with a certain drug concentration (drug-to-lipid ratio of 15:100, namely 9mg/mL R848 and 9mg/mL INCB) is obtained.

2) Dissolving soybean lecithin in Tween-80 buffer solution with a mass fraction of 0.5%, and ultrasonically dissolving to obtain water phase with a certain phospholipid concentration (soybean lecithin concentration 12 mg/mL). According to the basis of the earlier laboratory study, the volume ratio of the aqueous phase to the organic phase was determined to be 10:1.

3) The organic phase was added to the aqueous phase using a microinjection pump at a drop rate of 10mL/h under ice bath magnetic stirring at 600 rpm. Stirring at room temperature under magnetic stirring at 400rpm for 1 hr to volatilize ethanol until no ethanol smell is present, and removing free medicine with 0.22 μm microporous membrane to obtain RIL.

Further, OMVs are extracted and separated by an ultra-high speed centrifugation method.

Specifically, E.coli MG1655 was added to 250mL of LB medium, and incubated with shaking at 37℃and 200 rpm. When the optical density at 600nm is about 1.2, the supernatant is centrifuged at 4000g for 10min at 4 ℃. After suction filtration through a 0.45 μm aqueous microporous membrane, the filtrate was centrifuged at 4000g at 4℃for 10min using an ultrafiltration centrifuge tube having a molecular weight cut-off of 100kDa to obtain 50mL of concentrated filtrate. The concentrate was resuspended in 1 XPBS buffer using a super-high speed centrifuge at 4℃at 150000g for 3h, centrifuged again at 4℃at 150000g for 3h to remove the contaminating protein from the OMVs, and finally the OMVs were resuspended in 1mL of 1 XPBS buffer for further use at-80 ℃.

Further, RILO is prepared by a film extrusion process.

Specifically, C16-ceramide (450. Mu.M) was added to OMVs at a concentration (400. Mu.g/mL) and sonicated at 4℃for 2min to give OMVs enriched in C16-ceramide. Then, OMVs rich in C16-ceramide and freshly prepared RIL are mixed in a volume ratio of 1:1 under vortex conditions, and the RILO is obtained by extruding a film (50 nm) for a plurality of times (12 times).

In one aspect, the invention provides an application of outer membrane vesicles coated with drug-loaded lipid nanoparticles in preparation of a nano-macrophage drug delivery system.

In one aspect, the invention provides an application of outer membrane vesicles coated with drug-loaded lipid nanoparticles in preparing a cell therapy based on living macrophages.

In one aspect, the invention provides the use of outer membrane vesicles coated with drug-loaded lipid nanoparticles to maintain an anti-tumor phenotype in a tumor microenvironment for a live macrophage-based cell therapy.

In one aspect, the invention provides an application of C16-ceramide in regulating and controlling a drug release form of a nano-macrophage drug delivery system.

Compared with the prior art, the invention has the beneficial effects that:

1. the OMV-encapsulated lipid nanoparticle (RIL) fused with C16-ceramide is used for encapsulating and co-carrying R848 and INCB to prepare the OMV-encapsulated RIL (RILO), and the RILO can enter M1 type macrophages through an endocytosis path mediated by the small nest protein, so that the degradation of nano-drugs by strong acid environments such as lysosomes and the like is avoided. In addition, the fused C16-ceramide in OMV can enable the drug-loaded macrophage to release drugs in a non-free form at the tumor site, and the released drugs can be taken in by tumor cells and tumor-related macrophages, so that the effects of regulating the phenotype of the tumor-related macrophages and continuously enhancing the activity of T cells are exerted, and a strong anti-tumor immune response is initiated. The C16-ceramide is a lipid with a conical structure, is helpful for supporting and stabilizing the membrane structure of OMVs, enhances the negative curvature of the OMVs membrane, and plays a role in supporting and stabilizing. Meanwhile, we have found that C16-ceramide can also promote macrophages to release phagocytic drugs again in a non-free form, including forms in which the drugs are encapsulated by exosomes or microvesicles released by macrophages. The patent proves that the nano-drug wrapped by the fused C16-ceramide outer membrane vesicle (Outer membrane vesicle, OMV) can effectively avoid the degradation of the nano-structure, so that the two drugs are released in a non-free form under the condition of optimal proportion, thereby realizing the regulation and control of the drug release form in the M1 type macrophage carrying the nano-drug and exerting the capacity of remodelling TME in all directions.

2. OMV coated nano particles carrying two medicines together exert synergistic effect, and the immune suppression microenvironment is regulated and controlled by regulating the phenotype of tumor-related macrophages and enhancing the activity of T cells, if the nano structure of the nano medicine is degraded in advance and then released, the concentration and the proportion of the medicine at the tumor part can not be controlled, and the expected treatment effect is influenced.

3. Outer membrane vesicles were obtained from FimH positive strains. The invention utilizes the phenomenon that the FimH positive strain in nature can enter cells through the pit protein. RILO prepared by OMVs using this strain was found to be well able to enter M1-type macrophages via the cryptand-mediated endocytic pathway.

4. On the basis of a single factor test, preparing RIL by adopting a nano precipitation method, and selecting a surfactant (Tween-80) buffer solution containing soybean lecithin as a water phase; anhydrous ethanol containing R848 and INCB is selected as an organic phase, and the organic phase is added into an aqueous phase, so that the R848 and the INCB are insoluble in water and supersaturated in the aqueous phase rapidly, and an amorphous or crystalline medicament with smaller particle size can be formed. And then preparing RILO by a film passing extrusion method, obtaining OMV rich in C16-ceramide by ultrasonic mode, then mixing OMV rich in C16-ceramide with freshly prepared RIL, passing through a film (50 nm) and extruding for several times, gradually enabling the OMV to completely wrap the RIL in the process of crushing and re-fusing the OMV, and simultaneously limiting the excessive increase of the grain size by the film of 50 nm.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention.

Fig. 1: single factor test results of RIL: effects of soybean lecithin concentration (a), ratio of drug to lipid (b) and tween-80 mass fraction (c) on particle size and DL (n=3).

Fig. 2: single factor test results of RILO: impact of extrusion times (a), OMV concentration (b) on particle size and PDI (n=3).

Fig. 3: microcosmic morphologies (a), zeta potential (b), particle size (c) and protein composition characterization (d) of RIL, OMV and RILO.

Fig. 4: stability of RILO under storage (a) or simulated physiological conditions (b) (n=3).

Fig. 5: schematic of M1 uptake of RILO via the cellular protein-mediated pathway (a) and endocytic pathway of C6-LO were examined (b) (n=3, × P < 0.01).

Fig. 6: TEM images (scale 200nm, a) of RILOM1 at 0h and 48h after preparation and confocal images (scale 10 μm, B) of rhodamine B labeled C6-LOM1 (RAW 264.7) at different time points. Fig. 7: phenotypic analysis of RILOM1G in different culture environments: percentage of M1 or M2 in macrophages (a); M1/M2 ratio (b) (n= 3;ns,no significance).

Fig. 8: phenotypic analysis of RILOM1G (RAW 264.7) in different culture environments: percentage of M1 or M2 in macrophages (a); M1/M2 ratio (b) (n= 3;ns,no significance).

Fig. 9: release profile of R848 (a) or INCB (b) in different formulations (n=3, ×p <0.001, ×p < 0.01).

Fig. 10: r848 was in free or non-free form with a cumulative release ratio (b) at 72h and 72h release profile (a) in different formulations (n=3, P < 0.001).

Fig. 11: INCB accumulates release ratio (b) at 72h of release profile (a) in different formulations (n=3, ×p < 0.001) in free or non-free form.

Fig. 12: m1, RILOM 1G-and RILOM1G released medium TEM images (scale 200 nm) 24h after preparation.

Detailed Description

In order to enable those skilled in the art to more clearly understand the technical scheme of the present invention, the following description describes the technical scheme of the present invention in detail.

1. Experimental materials

1. Reagents and medicines

Soybean lecithin (Shanghai Ai Weita pharmaceutical technologies Co., ltd.); r848 (Shanghai aladine Biochemical technologies Co., ltd.); INCB-024360 (INCB, targetMol, USA); distearoyl phosphatidylethanolamine-polyethylene glycol 5000-maleimide (DSPE-PEG 5k-Mal, sienna-xi biotechnology limited); GPC3 targeting polypeptide (D-Cys-DHLASLWWGTEL, G12), fluorescent FITC modified GPC3 targeting polypeptide (D-Cys-DHLASLWWGTEL-Lys-FITC, G12-FITC) (Nanjing Leon Biotechnology Co., ltd.); mouse monocyte colony stimulating factor (M-CSF, peproTech, U.S.A.); other reagents were analytically pure and purchased from national drug group chemical reagent limited.

2. Instrument and equipment

Microinjection pump (KDS 100 type, KD Scientific Co., U.S.A.); ultra-high speed centrifuges (XPN-100 type, beckmann Kelter life sciences, USA); dynamic light scattering analyzer (Nano-ZS 90 type, malvern Co., UK); transmission electron microscopy (HT 7700 type, hitachi Corp.); cell imaging multifunctional detection system (type 5, bioTek company, usa); flow cytometry (Accuri C6 Plus type, BD Co., U.S.A.).

3. Cells and animals

Mouse bone marrow derived macrophages (Mouse bone marrow-derived macrophage, BMDM) are derived from 6-8 week old female BALB/c mice; mouse mononuclear macrophage leukemia cell line (RAW 264.7) was purchased from Shanghai Fuji Biotech Co., ltd, and cultured in DMEM medium containing 10% FBS. All cells were under standard conditions (37 ℃, 5% co) ₂ ) And (5) culturing. Female BALB/c mice (6-8 weeks old) were purchased from Beijing Bei Fu Biotechnology Co.

4. Reagent preparation

4.1 preparation of Tween-80 buffer solution with certain mass fraction

Precisely weighing 0.250g of Tween-80, adding 1 XPBS buffer solution to 100mL, performing ultrasonic dispersion and dissolution to obtain Tween-80 buffer solution with mass fraction of 0.25%, and preparing Tween-80 buffer solution with mass fraction of 0.5, 1.0 and 1.5% by the same method.

4.2 preparation of HCC conditioned Medium

HCC conditioned medium consisted of an equal volume of DMEM medium containing 10% fbs and culture supernatant after 48H incubation of H22 cells.

2. Experimental method and experimental results

Preparation of RILO and optimization of Process recipe

1.1 preparation of RIL and Single factor test investigation

The RIL is prepared by a nano precipitation method. R848 and INCB are dissolved in absolute ethyl alcohol according to the mass ratio of 1:1, and an organic phase with a certain drug concentration is obtained. Dissolving soybean lecithin in Tween-80 buffer solution with a certain mass fraction, and performing ultrasonic dissolution to obtain a water phase with a certain phospholipid concentration. According to the basis of the earlier laboratory study, the volume ratio of the aqueous phase to the organic phase was determined to be 10:1. The organic phase was added to the aqueous phase using a microinjection pump at a drop rate of 10mL/h under ice bath magnetic stirring at 600 rpm. Stirring for 1h under the condition of magnetic stirring at room temperature and 400rpm to volatilize the organic solvent and remove the free medicine by using a microporous filter membrane of 0.22 mu m to obtain RIL.

Taking particle size and Drug Loading (DL) as indexes, respectively examining the influences of 3 factors such as soybean lecithin concentration (9, 12, 15, 18mg/mL, fixed drug-to-lipid ratio of 12:100 and tween-80 mass fraction of 0.5%), drug-to-lipid ratio (the sum of R848 and INCB mass and the mass ratio of soybean lecithin of 9:100, 12:100, 15:100 and 18:100, fixed soybean lecithin concentration of 12mg/mL and tween-80 mass fraction of 0.5), tween-80 mass fraction (0.25, 0.5, 1.0 and 1.5 percent, fixed soybean lecithin concentration of 12mg/mL and drug-to-lipid ratio of 15:100), and screening to obtain a process recipe with smaller particle size and higher drug loading.

In order to screen the optimal process recipe for preparing RIL, 3 factors such as soybean lecithin concentration (9, 12, 15, 18 mg/mL), ratio of medicine to fat (sum of R848 and INCB mass to soybean lecithin mass, 9:100, 12:100, 15:100, 18:100) and tween-80 mass fraction (0.25, 0.5, 1.0, 1.5%) are examined by single factor with the DL of particle size, R848 and DL of INCB as indexes. As shown in FIG. 1 (a), the particle size of RIL decreased and increased as the concentration of soybean lecithin increased, and the particle size was minimized when the concentration of soybean lecithin was 12 mg/mL. As shown in FIG. 1 (b), as the ratio exceeds 15:100, the DL growth rate of R848 and INCB begins to slow as the ratio continues to increase. As shown in FIG. 1 (c), when Tween-80 was 0.5% by mass, RIL had both smaller particle size and higher DL. Therefore, the optimal process recipe for RIL is selected with a soybean lecithin concentration of 12mg/mL, a drug-to-lipid ratio of 15:100, and a Tween-80 mass fraction of 0.5%.

1.2 extraction separation of OMVs

Extracting and separating OMV by ultra-high speed centrifugation. Coli MG1655 was added to 250mL of LB medium and incubated with shaking at 37℃and 200 rpm. When the optical density at 600nm is about 1.2, the supernatant is centrifuged at 4000g for 10min at 4 ℃. After suction filtration through a 0.45 μm aqueous microporous membrane, the filtrate was centrifuged at 4000g at 4℃for 10min using an ultrafiltration centrifuge tube having a molecular weight cut-off of 100kDa to obtain 50mL of concentrated filtrate. The concentrate was resuspended in 1 XPBS buffer using a super-high speed centrifuge at 4℃at 150000g for 3h, centrifuged again at 4℃at 150000g for 3h to remove the contaminating protein from the OMVs, and finally the OMVs were resuspended in 1mL of 1 XPBS buffer for further use at-80 ℃. OMVs were quantified using the method of biquinine formate (bicinchoninic acid, BCA) according to kit instructions, using total protein content as an indicator.

1.3 preparation of RILO and Single factor test investigation

RIL is prepared by a film extrusion method. The C16-ceramide (450. Mu.M) was added to a concentration of OMVs and sonicated at 4℃for 2min to give OMVs enriched in C16-ceramide. Then, OMVs rich in C16-ceramide and freshly prepared RIL are mixed in a volume ratio of 1:1 under vortex conditions, and the RILO is obtained by extruding through a membrane (50 nm) for a plurality of times.

Taking particle size and PDI as indexes, respectively examining the influence of 2 factors of extrusion times (6, 9, 12, 15 and 18 times, and fixing OMV total protein concentration of 400 mug/mL) and OMV total protein concentration (200, 400, 600, 800 and 1000 mug/mL, and fixing extrusion times of 12 times), and screening to obtain a process prescription capable of wrapping RIL by OMVs.

In order to screen the optimal process recipe for preparing RILO, 2 factors of extrusion times (6, 9, 12, 15, 18 times) and OMV total protein concentration (200, 400, 600, 800, 1000. Mu.g/mL) were examined in a single factor by taking particle size and PDI as indexes. As shown in fig. 2 (a), when the number of extrusion times was small, the particle size did not increase after extrusion was completed compared to RIL and OMV, indicating that OMV did not completely encapsulate RIL; when the number of extrusion times reaches 12 or more, the particle size is increased compared with the particle sizes of RIL and OMV, and the PDI value is small. As shown in FIG. 2 (b), as OMV concentration increases, RIL is gradually encapsulated by OMVs, reaching the minimum concentration of OMVs required to encapsulate the RIL when the OMV concentration is 400 μg/mL. Thus, the number of presses was chosen 12 times and OMV concentration 400 μg/mL was used as the optimal process recipe for RILO.

Characterization of RIL, OMV and RILO

The relevant properties of freshly prepared RIL, OMV and RILO were evaluated as follows under the optimal process recipe conditions.

2.1 particle size potential analysis

Zeta potential, particle size and PDI of RIL, OMV and RILO were measured separately using a dynamic light scattering analyzer.

2.2 microscopic morphological investigation

Microscopic morphology was observed by transmission electron microscopy. RIL, OMV and RILO were prepared by a solution dispersion-dropping method, stained with 1.0% phosphotungstic acid, dried, mounted on a sample rod, and observed by means of a transmission electron microscope.

2.3 characterization of protein composition

Protein components of RIL, OMV and RILO were compared using SDS-PAGE protein electrophoresis. Preparing 10% of PAGE gel, heating and denaturing protein components of a sample, loading the sample with a loading amount of 20 mug per sample, sequentially carrying out electrophoresis separation under the conditions of 70V, 30min and 120V for 80min, dyeing and decoloring by using coomassie blue G250 staining solution, and carrying out gel imaging.

2.4 determination of drug loading and encapsulation Rate

After 10 times of methanol vortex demulsification, the content of R848 and INCB in RIL and RILO is measured by adopting a high performance liquid chromatography, and is substituted into a standard curve for calculation, and the drug loading and encapsulation efficiency (encapsulation efficiency, EE) are calculated according to the following formula respectively.

The calculation formula of DL (%) is: dl=w _{The mass of a certain drug in the lipid nanoparticle} /W _{Total mass of lipid nanoparticles} ×100％.

The calculation formula of EE (%) is: ee=w _{The mass of a certain drug in the lipid nanoparticle} /W _{The quality of the medicine is added during preparation} Stability investigation of x 100%2.5rilo

To evaluate the stability of RILO under physiological conditions, RILO was separately incubated with 1×pbs buffer, DMEM medium containing 10% fbs, 10% fbs or physiological saline, and then on a shaker at 37 ℃, and the particle size and Zeta potential of RILO were measured at preset time points.

Further, RILO was stored at 4℃and the particle size and Zeta potential were measured daily for 7 days to evaluate the storage stability of RILO.

Inspired by the ease of phagocytosis of bacteria and other pathogens by macrophages in nature, OMVs obtained from non-pathogenic escherichia coli MG1655 medium by multiple centrifugation and ultrafiltration were selected as important components of RILO. RIL was prepared by extrusion of RIL through a membrane with OMV enriched in C16-ceramide. As shown in FIG. 3, the transmission electron microscope image and the particle size potential analysis show that the particle size of RILO is 50.54+/-0.41 nm, the morphology is round and the obvious core-shell structure is presented, which indicates that RILO is successfully prepared. Three batches of reproducibility of RIL, OMV and RILO particle size, PDI, potential and drug loading were examined as shown in tables 1 and 2. The DL of R848 and INCB in RILO is 6.17+ -0.02% and 5.13+ -0.15%, respectively, which indicates that the drugs are successfully entrapped and meet the requirements of the subsequent experiments. The protein components in RIL, OMV and RILO were detected by SDS-PAGE protein electrophoresis, and the results of fig. 3 (d) confirm that the protein components derived from OMV were fully retained in RILO, indicating that RILO possesses biological properties associated with OMV protein components. Further, the stability of RILO under storage and simulated physiological conditions was examined (fig. 4), and the results indicate that the particle size of RILO and Zeta potential were maintained under storage and physiological conditions for at least 14 days and 48 hours, respectively.

Table 1: reproducibility of RIL, OMV and RILO particle size, PDI and potential (n=3)

Table 2: reproducibility of RIL, OMV and RILO drug loading (n=3)

Preparation and characterization of rilom1

3.1 extraction and Induction method and identification of BMDM

Bone marrow cells were isolated from femur and tibia of 6-8 week old female BALB/c mice and then cultured in BMDM growth medium (dmem+10% fbs+20ng/mL M-CSF) under standard conditions. On day 3, the medium was replaced with fresh BMDM growth medium and non-adherent cells were removed. Day 7, brilliant Violet 421 was used ^TM Immunofluorescent double staining of the F4/80 antibody and the PerCP/cyanine5.5-CD11b antibody was performed to assess BMDM formation.

In the case where polarized BMDM is required to be M1 type macrophages (M1), the medium is replaced with stimulation medium on day 7. Namely, M1 was obtained by stimulating with DMEM medium containing FBS (10%), LPS (100 ng/mL) and IFN-. Gamma.s (20 ng/mL) for 24 hours, and immediately used in the subsequent experiments.

Preparation of 3.2RILOM1

Co-incubating M1 with RILO gives RILOM1. Depending on the purpose of the experiment, experiments were performed using 150mm diameter dishes (30 mL of culture system), 12 well plates (1 mL of culture system), 24 well plates (600. Mu.L of culture system) and 96 well plates (100. Mu.L of culture system), respectively. RILO was added at a density of about 1X 10 at 200. Mu.g/mL (quantified on the basis of the concentration of R848) ⁵ Individual cells/cm ² In M1 type macrophage of (C) at 37deg.C, 5% CO ₂ Incubating for 2h, discarding supernatant, and washing with DMEM medium to remove non-ingested RILO to obtain RILOM1. RILOM1 (RAW 264.7) and RILM1 (RAW 264.7) were prepared in the same manner. BMDM is a macrophage obtained by in vitro induction of mononuclear cells extracted from mouse bone marrow; RAW264.7 is a mouse mononuclear macrophage leukemia cell line, which is a macrophage cell line capable of continuous passage.

Characterization of 3.3RILOM1

Freshly prepared RILOM1 was taken according to the conditions described above for "preparation of RILOM 1" and characterized as follows.

3.3.1 endocytic pathway investigation

To assess the endocytic pathway of RILO in M1, experiments were performed using hydrophobic fluorescent-labeled lipid nanoparticles. Specifically, coumarin 6 (C6) was used in place of the drug to prepare C6-L and C6-LO according to the process recipe under items "1.1" and "1.3". Genistein (200. Mu.M), methyl-beta-cyclodextrin (800. Mu.M), chlorpromazine (50. Mu.M) and cytochalasin D (5. Mu.M) were added to a density of 1X 10, respectively ⁵ Individual cells/cm ² (4.5X10 in 12-well plate) ⁵ Individual cells/well) for 30min. Subsequently, with the inhibitor concentration maintained, C6-L or C6-LO (200 ng/mL) was added to each well, and after incubation for 2 hours, cells were collected and detected by flow cytometry after washing 2 times with 1 XPBS buffer.

FIG. 5 (a) is a schematic representation of uptake of RILO by M1 via the cryptand-mediated pathway, by preincubating M1 with different endocytic pathway inhibitors, the extent to which the different endocytic pathways are affected by endocytic inhibitors was assessed based on C6 fluorescence intensity for comparison of the primary endocytic pathway for M1 uptake of C6-L or C6-LO. As shown in FIG. 5 (b), the flow cytometer detection results showed that uptake of C6-L and C6-LO was completely blocked at 4deg.C, indicating that uptake of C6-L and C6-LO by M1 was through an energy-dependent endocytic pathway. It was further found that inhibitors of the cellular protein mediated pathway (genistein and methyl- β -cyclodextrin) both significantly inhibited cellular uptake of C6-LO, whereas inhibitors of the clathrin mediated pathway (chlorpromazine) or inhibitors of the megacell pathway (cytochalasin D) did not affect cellular uptake of C6-LO, indicating that C6-LO is taken up by M1 mainly through the cellular protein mediated pathway, to a significantly different extent (P <0.01 ) than the cellular protein mediated pathway inhibitors have on the entry of C6-L into M1. In conclusion, the OMV is coated to enable the C6-LO to be absorbed by M1 mainly through a small nest protein mediated path, so that the damage of the strong acid environment of the lysosome in cells to the nano-drug structure is avoided, and reliable guarantee is provided for the exertion of the RILOM1 therapeutic effect.

3.3.2 investigation of nanostructure stability

The nanostructure stability of RILO in RILOM1 was evaluated using transmission electron microscopy. Fresh RILOM1 was taken, cultured in DMEM medium supplemented with 10% FBS for 0 and 48 hours, respectively, RILOM1 was collected at a preset time point, fixed with 2.5% glutaraldehyde aqueous solution, and then sent for transmission electron microscopy.

The nanostructure stability of RILO in RILOM1 was further evaluated using a laser confocal microscope. Selecting C6 to replace the medicine, selecting a rhodamine B marked DSPE marked lipid carrier, and preparing rhodamine B marked C6-LO according to the technological prescription under the item of 1.3. RAW264.7 cells were polarized to M1 (RAW 264.7) in polylysine coated 20mm glass bottom confocal dishes. Subsequently, the medium was replaced with DMEM medium containing rhodamine B labeled C6-LO (C6 was quantified as 200 ng/mL) and incubation was continued for 2h. After washing with 1 XPBS buffer, incubation with DMEM medium supplemented with 10% FBS for different times (0, 24 and 48 h), washing with 1 XPBS buffer at the corresponding times, blocking, alexa647-F4/80 antibody stained the cell membrane and visualized using a confocal laser imaging system.

Nanostructure stability of RILO at 0h and 48h in RILOM1 was observed using transmission electron microscopy. As shown in fig. 6 (a), intracellular spherical nanostructures, indicated by red arrows, were observed, indicating that RILO can still maintain nanostructure stability in RILOM1 without degradation over 48h. Representative confocal images also indicate the same test results. As shown in fig. 6 (B), the lipid carrier (red) and the alternative drug (green) were labeled with rhodamine B-labeled DSPE and C6, respectively, and confocal images showed that strong co-localization of the lipid carrier (red) and drug (green) was observed (yellow) within 0, 24, and 48h. The above results mutually confirm that RILO is able to maintain stable nanostructures in M1.

Preparation and characterization of rilom1g

Preparation of 4.1RILOM1G

RILOM1 and DG12 (DSPE)PEG5k-G12, distearoyl phosphatidylethanolamine-polyethylene glycol 5000-G12) to obtain RILOM1G, wherein the hydrophobic long chain of DSPE part in DG12 can be inserted into cell membrane after incubation, and the PEG5k part has stronger hydrophilicity and steric hindrance, thereby anchoring G12 on the cell surface. Specifically, 100 μg/mL DG12 is added to a density of about 1X 10 ⁵ Individual cells/cm ² Freshly prepared RILOM1 was placed at 37deg.C in 5% CO ₂ Incubating for 20min with shaking, discarding supernatant, and washing with 1×PBS buffer to remove residual DG12 to obtain RILOM1G. RILOM1G (RAW 264.7) was prepared in the same manner.

Phenotypic analysis of 4.2RILOM1G

The following characterization was performed under the preparation conditions of RILOM1G under item "4.1" above.

The phenotype of RILOM1G was assessed in different culture environments, including DMEM medium with 10% fbs and HCC conditioned medium. Freshly prepared M1 and RILOM1G were incubated with DMEM medium or HCC conditioned medium containing 10% FBS for 0 or 48h. BMDM was incubated with DMEM medium containing 10% fbs as a control group. Then using Brilliant Violet 421 ^TM -F4/80 antibody, PE-CD80 antibody and APC-CD206 (MMR) antibody label cells, flow cytometric analysis was performed. Similarly, M1 (RAW 264.7) and RILOM1G (RAW 264.7) were prepared using RAW264.7 cells and the same experiment was performed.

The phenotype of RILOM1G in different culture environments (DMEM medium with 10% fbs and HCC conditioned medium) was analyzed by flow cytometry. Using Brilliant Violet 421 ^TM -F4/80 antibody-labeled macrophages, on the basis of which is representative M1 capable of being labeled with PE-CD80 antibody, representative M2 capable of being labeled with APC-CD206 (MMR) antibody. When the ratio of M1 to M2 (M1/M2) is greater than 1, it is indicated that the tested cells are prone to the M1 phenotype; when the ratio of M1 to M2 (M1/M2) is less than 1, it is indicated that the tested cells are prone to the M2 phenotype. As shown in FIG. 7 (a), RILOM1G maintained higher CD80 expression and lower CD206 expression even in HCC conditioned medium as compared to the M1 group. Further, as shown in FIG. 7 (b), the M1/M2 ratio of RILOM1G in DMEM medium and HCC conditioned medium containing 10% FBS was 2.18.+ -. 0.19 and 1.98.+ -. 0.17, respectively. Statistical analysis shows that the two are not presentThe significant difference indicates that RILOM1G can resist immunosuppressive TME, maintaining the original anti-tumor phenotype. Similarly, the same experiment was performed using RAW264.7 cells to prepare M1 (RAW 264.7) and RILOM1G (RAW 264.7), as shown in fig. 8, and the results were consistent.

In vitro release profile investigation of rilom1g

Evaluation of the in vitro Release Curve of 5.1RILOM1G

To obtain different formulation release profiles based on macrophage preparation, 12-well plates were used at 4.5X10 ⁵ Density of individual cells/well RM1G, IM1G, RILOM1G- (RILOM 1G without C16-ceramide) or RILOM1G were prepared and then incubated with DMEM medium for different times (0.5, 1, 2, 4, 8, 12, 24, 48 and 72 h). At preset time points, supernatant samples were collected and rapidly supplemented with an equal volume of DMEM medium until 72h. The content of R848 or INCB in the supernatant samples was measured using high performance liquid chromatography and calculated by substituting the standard curve. The drug release profile of the different formulations was evaluated as a cumulative percent release.

Q _n1 Indicating the cumulative drug release; w is the total drug amount of the release system; c (C) _i1 Is the drug concentration in the supernatant sample corresponding to the sampling point; v (V) ₁ Is the corresponding sample volume of the supernatant at the sampling point.

As shown in fig. 9, the release profile of R848 or INCB in the different formulations was examined over 72h, respectively. As shown in FIG. 9, RILOM1G can release R848 cumulatively for 61.79+ -1.45% over 72h, RILOM 1G-can release R848 cumulatively for 50.36+ -1.09% over 72h, and the cumulative percentage of release of R848 in RILOM1G is significantly higher than the cumulative percentage of release of R848 in RILOM1G- (P < 0.001). Similarly, as shown in FIG. 9 (b), the cumulative percentage release of INCB in RILOM1G is significantly higher than the cumulative percentage release of INCB in RILOM1G- (P < 0.01). These results demonstrate that the addition of C16-ceramide promotes the release of drug in RILOM1G. As shown in FIG. 9, the cumulative percent release of RILOM1G group was greater than that of RILOM1G group at 72h, with significant differences, indicating that C16-ceramide was present, promoting drug release in RILOM1G.

Investigation of the in vitro Release form of 5.2RILOM1G

To further obtain a profile of drug release in free and non-free drug form in different formulations prepared based on macrophages, supernatants were collected at different time points according to the method under item "5.1", and centrifuged in ultrafiltration centrifuge tubes with a molecular weight cut-off of 100kDa (5000 g,15 min). By measuring the drug content in the ultrafiltrate, the amount of R848 or INCB released in free form was obtained. The curves of the different formulations for drug release in free form were evaluated as cumulative percent release.

Q _n2 Indicating the cumulative release of drug in free form; w is the total drug amount of the release system; c (C) _i2 The drug concentration in the ultrafiltrate sample at the corresponding sampling point; v (V) ₂ Corresponds to the sample volume of the ultrafiltrate at the sampling point.

The cumulative release of drug in non-free form is achieved by (Q _n1 –Q _n2 ) And (5) performing calculation. Further, the release medium of M1, RILOM 1G-and RILOM1G after 24 hours of preparation was taken and subjected to transmission electron microscopy.

As shown in fig. 10 and 11, the 72h release profile of the drug in the different formulations in free or non-free form was further examined. The free or non-free form of the drug is separated by ultrafiltration centrifuge tubes with a molecular weight cut-off of 100kDa and quantified by HPLC. As shown in fig. 10 (b), the cumulative release of R848 in the RILOM1G in the non-free form was 75.38±1.16% of the sum of the release in the free form and the release in the non-free form over 72 hours, which is significantly higher than the cumulative release ratio of R848 in the RILOM1G in the free form (24.62±1.16%, P < 0.001) and the cumulative release ratio of R848 in the RILOM1G in the non-free form (57.03 ±0.48%, P < 0.001). As shown in FIG. 11 (b), the same conclusion can be reached based on quantitative data analysis of INCB. As shown in FIG. 12, RILOM1G contained a large number of spherical nanoparticles in the release medium 24h after preparation, which was not observed in either M1 group or RILOM 1G-group. These results indicate that RILOM1G releases the drug in a non-free form at a higher rate under the action of C16-ceramide.

Claims

1. An outer membrane vesicle coated with a drug-loaded lipid nanoparticle, wherein the outer membrane vesicle prior to coating the drug-loaded lipid nanoparticle is fused with C16-ceramide.

2. The outer membrane vesicle coated with a drug-loaded lipid nanoparticle of claim 1, wherein the outer membrane vesicle prior to coating the drug-loaded lipid nanoparticle is obtained from a FimH positive strain.

3. The outer membrane vesicle coated with a drug-loaded lipid nanoparticle of claim 1, wherein the drug loaded lipid nanoparticle is R848 and INCB-024360.

4. A method for preparing outer membrane vesicles coated with drug-loaded lipid nanoparticles according to claims 1-3, wherein outer membrane vesicles obtained from FimH positive strains are fused with C16-ceramide and then fused with drug-loaded lipid nanoparticles.

5. The method for preparing outer membrane vesicles coated with drug-loaded lipid nanoparticles as claimed in claim 4, wherein the lipid nanoparticles of R848 and INCB-024360 are co-loaded by a nano precipitation method, the outer membrane vesicles not coated with the lipid nanoparticles are extracted and separated by an ultra-high speed centrifugation method, and the outer membrane vesicles coated with the lipid nanoparticles of R848 and INCB are prepared by a membrane extrusion method.

6. The method for preparing the outer membrane vesicles coated with the drug-loaded lipid nanoparticles as claimed in claim 5, wherein the method for extruding the outer membrane comprises the following specific steps: adding C16-ceramide into outer membrane vesicles of which the concentration is not coated with lipid nanoparticles, performing ultrasonic treatment, then mixing with freshly prepared co-supported R848 and INCB-024360 lipid nanoparticles in a volume ratio of 1:1 under vortex conditions, and performing membrane extrusion for a plurality of times.

7. Use of outer membrane vesicles coated with drug-loaded lipid nanoparticles according to claims 1-3 for the preparation of a nano-macrophage drug delivery system.

8. Use of outer membrane vesicles coated with drug-loaded lipid nanoparticles according to claims 1-3 for the preparation of a live macrophage based cell therapy.

9. Use of outer membrane vesicles coated with drug-loaded lipid nanoparticles according to claims 1-3 to maintain an anti-tumor phenotype in a tumor microenvironment for live macrophage-based cell therapy.

Use of a c 16-ceramide in modulating the drug release form of a nano-macrophage drug delivery system.