CN116036042A - A stable exosomal nanoparticle targeting M2 tumor-associated macrophages and its preparation method and application - Google Patents

Abstract

The invention discloses stable exosome nanoparticles targeting M2 type tumor-associated macrophages, a preparation method and application thereof, and belongs to the technical field of exosome preparation and drug gene targeted delivery. The invention aims to provide a stable exosome nanoparticle targeting M2 type tumor-associated macrophages. The invention discloses an exosome nano-particle of a targeted M2 type tumor-associated macrophage, which is formed by fusing exosome secreted by the macrophage and a phospholipid-polyethylene glycol-mannose receptor conjugate. The exosome nano-particles designed by the invention have good stability and can prevent the delivery content from being released in advance in the blood circulation process; the exosome nanoparticle designed by the invention also has excellent tumor targeting effect, especially can target M2 type tumor-associated macrophages which promote tumor growth in tumor tissues, has no damage to normal organs of the whole body, and can deliver contents into cells through membrane fusion and other ways to improve the delivery efficiency.

Description

A stable exosomal nanoparticle targeting M2 tumor-associated macrophages and its preparation method and application

technical field

The invention relates to a stable exosome nanoparticle targeting M2 tumor-associated macrophages, a preparation method and application thereof, and belongs to the technical field of exosome preparation and drug gene targeted delivery.

Background technique

Tumor-associated macrophages (tumor-associated macrophages, TAMs) are one of the most abundant tumor-infiltrating leukocytes in tumor tissues, and are a key factor affecting tumor progression, metastasis, and recurrence, and their presence is usually associated with poor prognosis in solid tumors. TAMs mainly originate from monocyte precursor cells in the circulatory system. In addition, resident macrophages in the tumor microenvironment can also differentiate into TAMs under corresponding stimuli. In solid tumors, TAM represents the main immune cell population, which has plasticity, making it polarized to M1 phenotype or M2 phenotype under the stimulation of corresponding cytokines. The M1 phenotype promotes inflammation and suppresses tumor development, while the M2 phenotype is anti-inflammatory and promotes tumor development. The number of M2-type TAMs in solid tumors is significantly higher than that of M1-type TAMs, which is one of the important reasons why tumors are difficult to cure. Polarization of M2-type TAMs into M1-type TAMs with antitumor effects has become a research hotspot in anticancer therapy. Researchers have discovered a variety of drugs with TAM polarizing effects, but these drugs are small molecular compounds, lack of targeting, and are easily eliminated by metabolism. Efficient delivery of them to tumor tissues still needs to rely on carriers.

Exosomes can not only carry small molecule chemical drugs to optimize the efficiency of old drugs, but also deliver protein or nucleic acid drugs to achieve new therapies. However, exosomes directly secreted from cells often have poor stability and are easily destroyed in the blood circulation after administration, resulting in early release of the drug; in addition, natural exosomes have poor targeting, although they have the ability to homing However, it is still unable to meet the needs of targeted delivery. In order to use it as a delivery carrier to achieve efficient delivery and therapeutic effects, it is necessary to endow exosomes with other functions such as stability and targeting.

Contents of the invention

The purpose of the present invention is to improve the stability of exosomes and tumor tissue targeting. The present invention designs and constructs a stable exosome nanoparticle targeting M2 tumor-associated macrophages, providing a targeted The preparation method of exosome nanoparticles of M2 tumor-associated macrophages improves the stability of exosomes as drug carriers and achieves excellent tumor targeting effects.

The present invention provides an exosome nanoparticle targeting M2 tumor-associated macrophages, the exosome nanoparticle is exosomes secreted by macrophages and phospholipid-polyethylene glycol-mannose receptors formed by fusion.

In one embodiment, the macrophages are mononuclear macrophages.

In one embodiment, the macrophages are Raw 264.7 cells.

In one embodiment, the exosome extraction method is as follows: collect macrophage culture fluid, centrifuge at 900g for 20min to remove dead cells; centrifuge at 3000g for 20min to remove cell debris; centrifuge at 10000g for 30min to further remove cell debris and large particles; Pass the supernatant through a 0.220 μm microporous membrane to remove large particles remaining after centrifugation; centrifuge at 100,000 g for 90 to 150 minutes at a high speed, and there will be white precipitates at the bottom of the centrifuge tube, which are exosomes.

In one embodiment, the phospholipid-polyethylene glycol-mannose receptor conjugate is distearoylphosphatidylethanolamine-polyethylene glycol-fucose.

In one embodiment, the mannose receptor conjugate is a sugar substance that specifically binds mannose receptors on the surface of M2 tumor-associated macrophages with mannose, fucose or N-acetylglucosamine.

In one embodiment, the mass ratio of exosomes to phospholipid-polyethylene glycol-mannose receptor conjugate is 100:(1-10).

In one embodiment, the mass ratio of exosomes to phospholipid-polyethylene glycol-mannose receptor conjugate is 20:1.

The present invention provides a method for preparing the above exosome nanoparticles, the steps of the preparation method are as follows: exosomes and phospholipid-polyethylene glycol-mannose receptor conjugates are prepared at 4°C and pH=7.4 Stir for 1.5 h under the same conditions to obtain an exosome solution targeting M2 tumor-associated macrophages.

The application of the above-mentioned exosome nanoparticles provided by the present invention in the preparation of carriers carrying drugs or genes.

Beneficial effects: the purpose of the present invention is to provide a stable exosome nanoparticle targeting M2 tumor-associated macrophages and its preparation method to solve the above-mentioned background technical problems. The exosome nanoparticles designed in the present invention have good stability and can prevent the delivery contents from being released in advance during blood circulation; the exosome nanoparticles designed in the present invention also have excellent tumor targeting effects, especially targeting tumors M2-type tumor-associated macrophages in tissues that promote tumor growth have no damage to normal organs throughout the body, and can deliver content into cells through membrane fusion and other ways to improve delivery efficiency.

The present invention provides a stable exosomal nanoparticle targeting M2 tumor-associated macrophages, which is composed of DSPE-PEG-Fucose and Raw 264.7 cell-derived exosomes. The mass ratio of Raw 264.7 cell-derived exosomes to DSPE-PEG-Fucose was 20:1. The concentration of Raw 264.7 cell-derived exosomes was 1 mg/mL and that of DSPE-PEG-Fucose was 1 mg/mL. DSPE-PEG-Fucose is distearoylphosphatidylethanolamine-polyethylene glycol-fucose, which is added to fuse with Raw 264.7 cell-derived exosomal membrane lipids, and has the ability to target M2 type tumor-associated macrophages and The function of tumor cells can better target tumor tissue. Raw 264.7 cells are mouse mononuclear macrophages, and the exosomes derived from them are extracellular vesicles with a particle size of about 30-150nm and a phospholipid bimolecular membrane. Biologically active proteins, cytokines, and RNA can play certain biological effects. Raw264.7 cell-derived exosomes take advantage of their homing function and are fused with DSPE-PEG-Fucose to increase the ability to target M2 tumor-associated macrophages.

The invention innovatively proposes to combine fucose, mannose, N-acetylglucosamine and other polysaccharides capable of specifically binding to mannose receptors with PEGylated phospholipids to form conjugates, and fuse them into external In secretory body. The PEGylated phospholipid has the functions of emulsification and solubilization, and can also endow the nanoparticles with the effect of long circulation in vivo. Mannose receptors are highly expressed on macrophages, especially M2 macrophages, and on the surface of most tumor cells. Polysaccharides such as fucose, mannose, and N-acetylglucosamine are specific ligands for mannose receptors. , using receptor-ligand interactions to achieve precise targeting. This design can improve the stability of exosomes and the targeting of M2 tumor-associated macrophages. It is an efficient and stable nano-drug delivery carrier, which can be widely used in the field of drug or gene delivery in the future, especially in tumors. The field of anticancer therapy for tissue-targeted drug delivery.

Description of drawings

Figure 1 is a particle size diagram of exosomal nanoparticles targeting M2 tumor-associated macrophages and unmodified exosomes provided in Example 1;

Figure 2 is a Zeta potential diagram of exosome nanoparticles targeting M2 tumor-associated macrophages and unmodified exosomes provided in Example 1;

Figure 3 is a TEM image of exosome nanoparticles targeting M2 tumor-associated macrophages provided in Example 1;

Fig. 4 is a particle size change diagram of exosome nanoparticles targeting M2 tumor-associated macrophages provided in Example 2 and unmodified exosomes kept in PBS buffer solution of pH = 7.4 for seven days;

Figure 5 is a diagram of the particle size change of exosome nanoparticles targeting M2 tumor-associated macrophages provided in Example 2 and unmodified exosomes in PBS containing 10% fetal bovine serum for 24 hours;

Fig. 6 is a data diagram of the effect of exosome nanoparticles targeting M2 tumor-associated macrophages on the cell viability of macrophages provided in Example 2.

7 is a data diagram of the targeting of M2 macrophages and tumor cells by exosomal nanoparticles targeting M2 tumor-associated macrophages and unmodified exosomes provided in Example 2.

Figure 8 shows the distribution of exosome nanoparticles targeting M2 tumor-associated macrophages provided in Example 2 and unmodified exosomes at the tumor site after tail vein injection into tumor-bearing mice.

Detailed ways

In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, the present invention can be implemented in many other ways different from those described here, and those skilled in the art can make similar improvements without departing from the connotation of the present invention, so the present invention is not limited by the specific implementations disclosed below.

A stable exosomal nanoparticle targeting M2 tumor-associated macrophages, its preparation method and application provided in the embodiments of the present invention will be described in detail below.

Distearoylphosphatidylethanolamine-polyethylene glycol-fucose was purchased from Xi'an Ruixi Biotechnology Co., Ltd., product number: R-DP-130.

Example 1.

The preparation method of stable exosome nanoparticles targeting M2 tumor-associated macrophages comprises:

S1: Preparation of exosomes derived from Raw264.7 cells: culture Raw264.7 cells under normoxic conditions, collect the culture supernatant of the above cell culture medium, centrifuge at 900g for 20min to remove dead cells; centrifuge at 3000g for 20min to remove cell debris; Centrifuge at 10,000g for 30min to further remove cell debris and large particles; pass the supernatant through a 0.220μm microporous membrane to remove large particles remaining after centrifugation; centrifuge at 100,000g for 90-150min at ultra-high speed, gently take out the centrifuge tube, and carefully suck off the culture medium At this time, it can be seen that there are white precipitates at the bottom of the centrifuge tube, which are exosomes. Add 1.5mL pre-cooled PBS solution to the precipitate, gently blow and beat to resuspend the exosomes, and transfer them into a special centrifuge tube again. Purify by ultracentrifugation at 100,000 g for 90-150 min; then suck off the PBS solution, add 0.2 mL of pre-cooled PBS solution to the precipitate and resuspend by blowing. The protein concentration was determined by the BCA method and diluted to a 1 mg/mL exosome solution as solution A.

S2: Prepare a pH=7.4 PBS solution containing 1 mg/mL DSPE-PEG-Fucose as solution B. Mix 1mL of solution A with 0.05mL of solution B (the mass ratio of Raw264.7 cell-derived exosomes to DSPE-PEG-Fucose is 20:1), and stir at 4°C for 1.5h to obtain a stable M2-targeted tumor-associated Exosomal nanoparticles solution of macrophages.

S3: Centrifuge the above synthesized exosome nanoparticle solution fused with DSPE-PEG-Fucose at 100,000g for 90-150min at high speed, discard the supernatant, add 0.5mL PBS 7.4 solution to resuspend the precipitate, and centrifuge again at 100,000g for 90-150min After 150 minutes, the supernatant was discarded, and the precipitate was collected and stored at -80°C for later use.

The stable exosomal nanoparticles targeting M2 tumor-associated macrophages prepared in this example were diluted to 0.1 mg/mL and subjected to a hydraulic diameter test (DLS). The particle size results are shown in Figure 1. Exosome nanoparticles with a uniform diameter in the range of 30-150nm; the Zeta potential results are shown in Figure 2, and negatively charged exosome nanoparticles were obtained.

The stable exosomal nanoparticles targeting M2 tumor-associated macrophages prepared in this example were diluted to 0.1 mg/mL, and dozens of drops were dropped onto the carbon-supported copper grid, dried overnight and then subjected to transmission electron microscopy. A microscope (TEM) took pictures of the nanoparticle morphology, as shown in Figure 3.

Example 2. Application of exosomal nanoparticles targeting M2 tumor-associated macrophages

The application of exosomal nanoparticles targeting M2 tumor-associated macrophages, which has excellent stability, low cytotoxicity, and excellent targeting as a drug delivery carrier:

1. The exosome nanoparticles targeting M2 tumor-associated macrophages prepared in Example 1 have excellent stability.

The exosome nanoparticles targeting M2 tumor-associated macrophages prepared in Example 1 were resuspended in PBS buffer with pH = 7.4, the concentration of exosomes after dilution was 0.1 mg/mL, and the nanoparticles were measured for seven consecutive days The particle size change is shown in Figure 4. The results showed that compared with the exosomes without DSPE-PEG-Fucose fusion, the particle size of the exosome nanoparticles targeting M2 tumor-associated macrophages prepared in Example 2 did not change much, indicating that it could be used in normal tissues The environment is relatively stable and does not aggregate.

The exosome nanoparticles targeting M2 tumor-associated macrophages prepared in Example 1 were resuspended in pH=7.4 PBS buffer containing 10% fetal bovine serum and placed in a constant temperature incubator at 37°C for 24 hours of measurement The particle size variation of inner nanoparticles is shown in Fig. 5. The results show that compared with exosomes without DSPE-PEG-Fucose fusion, the exosome nanoparticles targeting M2 tumor-associated macrophages prepared in Example 2 can maintain the same particle size within 24h, indicating that It is more stable than unmodified exosomes under simulated blood circulation conditions.

2. The exosome nanoparticles targeting M2 tumor-associated macrophages prepared in Example 1 have low cytotoxicity.

Raw264.7 cells were plated in a 96 ^- well plate at 8×10 cells per well, added 200 μL of DMEM medium containing 10% fetal bovine serum and 1% penicillin/streptomycin, and placed at 37°C, 5% CO ₂ Normoxic cultivation in the incubator. After 24 hours, the prepared exosomal nanoparticles targeting M2 tumor-associated macrophages were added at a certain concentration (0, 0.2, 0.5, 1, 2, 3 μg/mL), and three parallel groups were set up for each concentration. . Place in a 37°C, 5% _CO2 incubator for incubation. After culturing for 48 hours, the original medium was removed, and 200 μL of MTT solution (diluted and filtered in PBS, final concentration 0.5 mg/mL) was added to each well, and incubated at 37°C for 4 hours under light-shielding conditions. MTT was discarded, 200 μL DMSO was added to each well, shaken at 37°C for 15 min, and then the absorbance of each well at the wavelength of 490/570 nm was measured with a microplate reader, and the cytotoxicity of the samples at each concentration was compared. The results of cytotoxicity are shown in Fig. 6, indicating that the prepared exosomal nanoparticles targeting M2 tumor-associated macrophages have lower cytotoxicity.

3. The exosomal nanoparticles targeting M2-type tumor-associated macrophages prepared in Example 1 are targeted to M2-type macrophages and tumor cells, and can be used in in vitro cell uptake experiments and in vivo imaging experiments in tumor-bearing mice prove.

In order to obtain tumor-associated macrophages with M2 phenotype for targeting verification, Raw264.7 cells were resuspended in DMEM medium and diluted to 200,000 cells per ml of culture medium, and the cells were plated in six-well plates 400,000 cells per well. Then put the cells in a 5% _CO2 cell incubator for 24 hours, then add interleukin-4 to the culture medium to make the culture medium contain 100ng/mL of interleukin-4, and place in a 5% _CO2 cell incubator to induce culture for 48 hours , that is, the M2 type Raw264.7 cells were obtained, and the cells were used to represent the tumor-associated macrophages of the M2 phenotype.

In order to more intuitively observe the targeting of exosomal nanoparticles targeting M2 tumor-associated macrophages to M2 macrophages and tumor cells, the prepared exosomal nanoparticles were labeled with a commercially available fluorescent probe Cy5. Exosomal nanoparticles targeting M2 tumor-associated macrophages (named Fuc-PEG-EXO(Cy5)) and exosomes directly extracted from macrophages without DSPE-PEG-Fucose fusion (named EXO(Cy5)).

M2 type Raw264.7 cells and mouse melanoma B16 cells were spread in 35mm confocal dishes at 1× ¹⁰⁵ cells per well, and cultured in 2 mL of DMEM containing 10% fetal bovine serum and 1% penicillin/streptomycin cultured in a 37°C, 5% CO ₂ incubator. After 24 hours, 2 μg/mL Fuc-PEG-EXO (Cy5) and EXO (Cy5) were added respectively, and then the well plate was placed in a 5% CO ₂ incubator and incubated at 37° C. for 8 hours. After the culture was completed, aspirate the culture medium, add 1mL PBS to wash three times, add 1mL 4% paraformaldehyde, and incubate at 4°C in the dark for 20min. Aspirate the paraformaldehyde, add 300 μL DAPI solution (1 μg/mL), and incubate in the cell culture incubator for 5 minutes. Aspirate the above DAPI solution and wash once with 1 mL of PBS. Then add 1mL PBS, and store in the dark at 4°C. Observe its fluorescence intensity under a confocal microscope, as shown in Figure 7, it can be seen that the prepared exosomal nanoparticles (Fuc-PEG-EXO(Cy5)) targeting M2 tumor-associated macrophages have a strong effect on M2 Type Raw264.7 cells and B16 cells have better targeting and more cellular uptake.

In order to verify the targeted delivery effect of the prepared exosomal nanoparticles targeting M2 tumor-associated macrophages in vivo, B16 tumor cells were injected subcutaneously into the back of C57BL/6 mice. When the tumor volume of the mice reached about 100 mm ³ , the fluorescent probes Cy5, EXO (Cy5) and Fuc-PEG-EXO (Cy5) were injected into the tail vein of the tumor-bearing mice, and the amount of Cy5 in each group was fixed at 60 μg. The mice were sacrificed 6h and 24h after administration, and the tumors were peeled off. Near-infrared fluorescence images of tumors were obtained by Caliper IVIS Lumina II in vivo imaging system (PerkinElmer, Waltham, MA, USA), as shown in FIG. 8 . It was found that the prepared exosomal nanoparticles targeting M2 tumor-associated macrophages (Fuc-PEG-EXO(Cy5)) had more distribution and accumulation in the tumor site, indicating that it had better targeting ability to tumor tissue. good.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (10)

1. An exosome nanoparticle targeting M2 tumor-associated macrophages, wherein the exosome nanoparticle is fused with a phospholipid-polyethylene glycol-mannose receptor conjugate secreted by macrophages.

2. The exosome nanoparticle of claim 1, wherein the macrophage is a mononuclear macrophage.

3. The exosome nanoparticle of claim 1, wherein the macrophage is a Raw264.7 cell.

4. Exosome nanoparticles according to claim 1, characterized in that the extraction method of exosomes is as follows: collecting macrophage culture liquid, centrifuging for 20min at 900g, and removing dead cells; centrifuging at 3000g for 20min to remove cell debris; centrifuging at 10000g for 30min to further remove cell debris and large particles; passing the supernatant through a 0.220 μm microporous filter membrane to remove large particles remained after centrifugation; and (3) performing ultra-high speed centrifugation at 100000g for 90-150 min, wherein white precipitate is formed at the bottom of the centrifuge tube, namely the exosome.

5. The exosome nanoparticle of claim 1, wherein the phospholipid-polyethylene glycol-mannose receptor conjugate is distearoyl phosphatidylethanolamine-polyethylene glycol-fucose.

6. The exosome nanoparticle of claim 1, wherein the mannose receptor conjugate is a carbohydrate that specifically binds mannose, fucose or N-acetamido glucose to mannose receptors on the surface of M2-type tumor-associated macrophages.

7. The exosome nanoparticle according to claim 1, wherein the mass ratio of exosome to phospholipid-polyethylene glycol-mannose receptor conjugate is 100 (1-10).

8. The exosome nanoparticle of claim 1, wherein the mass ratio of exosome to phospholipid-polyethylene glycol-mannose receptor conjugate is 20:1.

9. A method of preparing exosome nanoparticles according to any one of claims 1 to 8, characterized in that the steps of the preparation method are as follows: the exosomes and phospholipid-polyethylene glycol-mannose receptor conjugate are stirred for 1.5h at 4 ℃ and at ph=7.4, to obtain exosome solution targeting M2-type tumor-associated macrophages.

10. Use of exosome nanoparticles according to any one of claims 1-8 for the preparation of a vector for carrying a drug or gene.

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