Preparation and application of macrophage membrane biomimetic polymer nanoparticles

Abstract

The application discloses a kind of macrophage membrane biomimetic polymer nanoparticles and its application, the nanoparticle includes macrophage membrane, hyaluronic acid and cationic polymer nanoparticle, the macrophage membrane is extracted from RAW 264.7 cell, the polymer nanoparticle is prepared by cinnamaldehyde grafting polyethyleneimine and Fmoc-arginine grafting polyethyleneimine self-assembly.The macrophage membrane biomimetic polymer nanoparticles prepared in the application has the advantages of simple preparation, good reproducibility, high stability, etc., by increasing the uptake of M2 type macrophage, responsive release cinnamaldehyde and up-regulation intracellular ROS level, reprogramming tumor-associated macrophage to M1 phenotype, thereby effectively improving tumor microenvironment and playing tumor cell killing effect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to preparation and application of a macrophage membrane biomimetic polymer nanoparticle. BACKGROUND

[0002] Cancer is the most important factor threatening human health and life, among which breast cancer is the most common one, and the treatment method is still mainly surgery. Among them, cancer immunotherapy is a new generation of tumor treatment method developed rapidly after traditional treatment methods such as surgery, radiotherapy and chemotherapy, and has great clinical application prospect. However, due to the existence of tumor immunosuppressive microenvironment, only part of the solid tumor patients have response to this kind of treatment, resulting in poor clinical effect and prognosis. Among them, tumor-associated macrophages (TAMs) are an important part of tumor microenvironment, accounting for about 50% of tumor immune cells, and mainly in the form of immunosuppressive M2-TAMs, which promote tumor growth and metastasis. Therefore, in view of the high plasticity of macrophages and the key role in cancer immunity, polarizing immunosuppressive M2-TAMs into immunostimulatory M1-TAMs to reverse TME will be an extremely effective treatment strategy for cancer immunotherapy.

[0003] In recent years, functional nanodrugs based on enhanced permeability and retention (EPR) effect have been developed to enrich tumor areas to reverse TME. The main advantages of nanodrugs and delivery systems based on nanotechnology are passive targeting and reducing systemic toxicity reactions, but at the same time, there are also disadvantages such as easy to be recognized and removed by the immune system, and low passive targeting efficiency. Cell membrane biomimetic technology is a simple top-down method, which uses cell membrane as a carrier. The prepared biomimetic nanomedicine not only has the physicochemical properties of the nanocarrier itself, but also has the biological properties of natural cells, which can improve biocompatibility, and realize long-term circulation and active targeting delivery in vivo, thereby making up for the shortcomings of traditional drug delivery systems.

[0004] The "cold" tumor microenvironment contains a large number of M2-type macrophages that promote tumor growth, anti-inflammatory cytokines and immunosuppressive cells, and intratumoral infiltration of cytotoxic T cells is difficult, resulting in poor anti-tumor immunotherapy effect. Among them, tumor-associated macrophages (TAMs) are the most abundant immune cells in tumor tissue, and play a key role in regulating anti-tumor immunity. TAMs can differentiate into two opposite functional phenotypes, one is M1 phenotype promoting inflammation and killing tumor, and the other is M2 phenotype promoting inflammation and tumor. Based on the plasticity of TAMs, reprogramming M2-type TAMs into M1-type TAMs is an effective strategy to alleviate tumor immunosuppressive microenvironment. However, most of the current preparations do not have good safety, effectiveness and biocompatibility, and cannot precisely target M2-type TAMs to exert drug efficacy. SUMMARY

[0005] In order to solve the technical problem that the existing preparation cannot accurately target M2 type TAMs to exert drug efficacy, the application provides a preparation method and application of a macrophage membrane biomimetic polymer nanoparticle.

[0006] The technical scheme adopted by the application is as follows:

[0007] A macrophage membrane biomimetic polymer nanoparticle comprises a cationic polymer nanoparticle inner core and a macrophage membrane shell, and the surface of the cationic polymer nanoparticle inner core is coated with hyaluronic acid.

[0008] The cationic polymer nanoparticle is formed by self-assembly of polymer A and polymer B, and the mass ratio of polymer A to polymer B is 0.5:1 to 2:1.

[0009] The polymer A is obtained by grafting modification of cinnamaldehyde on polyethyleneimine, and the polymer B is obtained by grafting modification of Fmoc-arginine on polyethyleneimine.

[0010] Further, the mass ratio of the hyaluronic acid to the cationic polymer nanoparticle is 1:1 to 5:1, and the mass ratio of the protein of the macrophage membrane to the cationic polymer nanoparticle is 1:1 to 10:1.

[0011] Further, the mass ratio of the polymer A to the polymer B is 2.5:1.

[0012] Further, the mass ratio of the macrophage membrane, the hyaluronic acid and the cationic polymer nanoparticle is 1:2:1.

[0013] The preparation method of the above-mentioned macrophage membrane biomimetic polymer nanoparticle comprises the following steps:

[0014] Step 1, preparing polymer A and polymer B;

[0015] Step 2, dissolving polymer A and polymer B in ethanol respectively, mixing, then adding dropwise into water to obtain cationic polymer nanoparticles, and then adding hyaluronic acid to obtain cationic polymer nanoparticles coated with hyaluronic acid;

[0016] Step 3, mixing the macrophage membrane and the polymer nanoparticles obtained in step 2, and preparing the macrophage membrane biomimetic polymer nanoparticle by a co-extrusion method.

[0017] In one embodiment of the present application, the preparation process of the polymer A is as follows: 100 mg of PEI (MV=1.8 k) and 100 mg of cinnamaldehyde are dissolved in 2 ml of ethanol respectively, then the cinnamaldehyde ethanol solution is slowly added into the PEI, and stirred at room temperature for 12 h. After the reaction is completed, the organic solvent and free cinnamaldehyde are removed by rotary evaporation and vacuum drying to obtain a light yellow viscous solid PC.

[0018] In one embodiment of the present application, the preparation process of the polymer B is as follows: Fmoc-Arg-OH (0.56 mmol, 22.2 mg), EDC (1.12 mmol, 214.7 mg) and HOBT (1.12 mmol, 151.35 mg) are dissolved in 4 mL of DMF, and reacted at room temperature for 1 h to activate the carboxyl group, then 200 mg of PEI (MV=1.8 k) and DIPEA (1.12 mmol, 145 mg) are added and the reaction is continued for 24 h. Then, the mixture is dialyzed in pure water (MWCO: 1.5 kDa) for 48 h, and PA is prepared after freeze-drying.

[0019] In one embodiment of the present application, step 2 is specifically as follows: 5 mg of PC and 5 mg of PA prepared in step 1 are weighed and dissolved in 50 μL of ethanol respectively, mixed in a ratio of PC:PA=2.5:1, then slowly added into 1 mL of pure water respectively to prepare PCA nanoparticles. Then, HA (MV=10000) is added to neutralize the positive charge to prepare H@PCA.

[0020] In one embodiment of the present application, the extraction method of the macrophage membrane is as follows: RAW 264.7 cells are induced to culture for 24 h using LPS (1 μg / ml), the cells are collected using a cell scraper and resuspended in a hypotonic lysis solution, and after lysis at 4℃ for 24 h, the membrane is further lysed using an ultrasonic disrupter (4℃, 20 min, 100w). Then, the cell suspension is centrifuged at 10000 g for 15 min, and the supernatant is taken; the supernatant is centrifuged at 20000 g for 30 min, and the supernatant is discarded, and the precipitate is the macrophage membrane.

[0021] The macrophage membrane biomimetic polymer nanoparticles are used for preparing a tumor treatment drug.

[0022] The application constructs a macrophage membrane coating modified polymer nanoparticle, which specifically targets M2 type tumor-associated macrophages through macrophage membrane, and improves the biocompatibility and in vivo uptake of the polymer nanoparticle. The biomimetic polymer nanoparticle prepared by the application has the advantages of simple preparation, good reproducibility, high stability and the like, and by increasing the uptake of M2 type macrophages, responsively releasing cinnamaldehyde and up-regulating the intracellular ROS level, reprogramming the tumor-associated macrophages to M1 phenotype, the tumor microenvironment is effectively improved and the tumor cell killing effect is exerted. Advantages

[0023] (1) The macrophage membrane biomimetic polymer nanoparticle prepared by the application has the advantages of simple preparation process, suitable and uniform particle size, high stability, low toxicity and side effects, tumor accumulation and macrophage targeting.

[0024] (2) The macrophage membrane biomimetic polymer nanoparticle prepared by the application improves the M2 type macrophage uptake capacity of the polymer nanoparticle, responsively releases cinnamaldehyde in the intracellular acid, up-regulates the intracellular ROS level of M2-TAMs, reprograms M2-TAMs, enhances the tumor killing capacity thereof, and has high clinical use value. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Fig. 1 is a nuclear magnetic resonance hydrogen spectrum of PC, wherein A is a cinnamaldehyde grafted polyethyleneimine (PEI-Cin, PC) and B is a Fmoc-arginine grafted polyethyleneimine (PEI-Fmoc-Arg, PA).

[0026] Figure 2 Fig. 4 is the particle size and PDI results of the polymer nanoparticles PCA composed of different mass ratios of PC and PA.

[0027] Figure 3 Fig. 5 is the cell activity results of the polymer nanoparticles PCA.

[0028] Figure 4 Fig. 6 is the SDS-PAGE results of the biomimetic polymer nanoparticles.

[0029] Figure 5 Fig. 7 is the particle size distribution and Zeta potential results of the biomimetic polymer nanoparticles.

[0030] Figure 6 Fig. 8 is the cell uptake results of the biomimetic polymer nanoparticles by flow cytometry detection of M2-TAMs. DETAILED DESCRIPTION

[0031] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0032] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0033] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Example 1

[0034] 1. Synthesis of cinnamaldehyde-grafted polyethyleneimine (PC) and Fmoc-arginine-grafted polyethyleneimine (PA)

[0035] (1) Polymer PC was synthesized via Schiff base reaction. Specifically: 100 mg of PEI (MV=1.8k) and 100 mg of cinnamaldehyde were dissolved in 2 mL of ethanol, respectively. Then, the cinnamaldehyde ethanol solution was slowly added dropwise to PEI and stirred at room temperature for 12 h. After the reaction was completed, the organic solvent and free cinnamaldehyde were removed by rotary evaporation and vacuum drying to obtain a pale yellow viscous solid PC.

[0036] Polymer PA was synthesized via an amide reaction. Specifically, Fmoc-Arg-OH (0.56 mmol, 22.2 mg), EDC (1.12 mmol, 214.7 mg), and HOBT (1.12 mmol, 151.35 mg) were weighed and dissolved in 4 mL of DMF. The reaction was carried out at room temperature for 1 h to activate the carboxyl groups. Subsequently, 200 mg of PEI (MV = 1.8 kDa) and DIPEA (1.12 mmol, 145 mg) were added, and the reaction was continued for another 24 h. The prepared mixture was dialyzed in pure water (MWCO: 1.5 kDa) for 48 h and then lyophilized to obtain PA.

[0037] like Figure 1 As shown in Figure A, the characteristic proton peak (C=N) of cinnamaldehyde grafted with polyethyleneimine appears at 8.31 ppm. Calculations using peak area integration show that the PEI-Cin grafting rate is 35%, confirming the successful synthesis of PC. Figure 1 As shown in Figure B, the characteristic peak of PEI appears at 2.5-3 ppm, while the characteristic peak of Fmoc-Arg is at 7-8 ppm. Based on peak area integration, the grafting rate of PEI-Fmoc-Arg is 23.5%.

[0038] 2. Preparation of PCA nanoparticles composed of PC and PA in different mass ratios

[0039] PCA nanoparticles were prepared by emulsification evaporation method. Specifically: 5 mg of PC and 5 mg of PA were weighed and dissolved in 50 μl of ethanol, respectively, and mixed at a mass ratio of PC:PA=0:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 1:0, and then slowly added to 1 mL of pure water to prepare PCA nanoparticles with different proportions. The appropriate particle size and PDI were screened by Malvern particle size analyzer.

[0040] As shown in Figure 2 , when the ratio of PC to PA was 1:1-3:1, the particle size of PCA was around 200 nm, and the PDI was in the range of 0.2-0.3. Among them, when PC:PA=2.5:1, the particle size was most appropriate and uniform.

[0041] 3. MTT detection of the biological safety of polymer nanoparticles PCA on RAW 264.7 cells

[0042] The biological safety of polymer nanoparticles PCA on RAW 264.7 cells was evaluated by MTT method. RAW 264.7 cells (6×10 4 cells per well) were inoculated into a 96-well plate and incubated in an incubator for 24 h to adhere. Then the culture medium was discarded, and different mass ratio prepared PCA (including PC:PA=0:1, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 1:0) and different concentrations of PCA (including PCA=0, 2.5, 5, 7.5, 10, 20 μg / mL) were added and incubated for 24 h. Then 20 μL of MTT solution (5 mg / mL) was added to each well and incubated for 4 h. Finally, the culture medium was removed, DMSO was added to dissolve the formazan, and the absorbance of the sample at 492 nm was measured by a microplate reader, and the cell survival rate was calculated.

[0043] As shown in Figure 3 , when PC:PA=2.5:1 and the concentration was less than 7.5 μg / mL, the polymer nanoparticles PCA had no significant toxicity to RAW264.7 cells, and the cell survival rate was more than 80%. Therefore, PC:PA=2.5:1 was selected for subsequent experiments.

[0044] 4. Preparation of biomimetic polymer nanoparticles (MH@PCA)

[0045] The macrophage membrane was extracted by ultracentrifugation. Specifically, after RAW264.7 cells were induced by LPS (1 μg / ml) for 24 h, the cells were collected by using a cell scraper and resuspended in a hypotonic lysis solution (20 mM Tris, 2 mM MgCl2, 10 mM KCl, pH=7.4), and then lysed in a refrigerator at 4°C for 24 h. The membrane was further lysed by using an ultrasonic disrupter (4°C, 20 min, 100 w). Subsequently, the cell suspension was centrifuged at 10000 g for 15 min, and the supernatant was collected. The supernatant was centrifuged at 20000 g for 30 min, and the supernatant was discarded. The obtained precipitate was the macrophage membrane (CM). The membrane protein content was detected by BCA quantitative method.

[0046] Hyaluronic acid (HA) was added in a mass ratio of HA: PCA (mass ratio of PC: PA=2.5:1)=2:1 to incubate for 30 min to neutralize the positive charge of PCA, and H@PCA was prepared. The macrophage membrane and the polymer nanoparticle H@PCA were mixed in a mass ratio of 1:1, and repeated co-extrusion was performed 20 times through a 200 nm polycarbonate porous membrane of an AVANTI micro-extruder to obtain the biomimetic polymer nanoparticle MH@PCA.

[0047] The biomimetic polymer nanoparticles prepared in the above examples were tested as follows.

[0048] 1. SDS-PAGE of the biomimetic polymer nanoparticles

[0049] The concentrated gel and the separation gel required for SDS-PAGE were prepared according to the gel kit, the gel plate was transferred to the electrophoresis device, and an appropriate amount of electrophoresis buffer was added. Then, the sample was loaded: including Marker, H@PCA, CM and MH@PCA (the total amount of protein loaded was 20 μg). Then, the electrophoresis separation was started, the concentrated gel was vertically electrophoresed at 80 V for about 60 min, and the separation gel was vertically electrophoresed at 120 V for about 30 min, until the dye front reached the bottom of the gel. Finally, the gel was stained with Coomassie brilliant blue for 30 min, and then washed and photographed.

[0050] As shown in Figure 4 , the protein band of MH@PCA prepared by the extrusion method had no obvious difference compared with CM, indicating that MH@PCA could maintain the integrity of the protein structure, which was beneficial to its targeted action in vivo.

[0051] 2. Particle size and zeta potential determination of the biomimetic polymer nanoparticles

[0052] The MH@PCA was dissolved in deionized water, and the particle size distribution and zeta potential of the MH@PCA were evaluated by a Malvern particle size analyzer. As shown in Figure 5As shown, the particle size of MH@PCA was 210 ± 5.29 nm, and the zeta potential was -29.36 ± 1.24 mv. Compared with the potential of H@PCA, the potential decreased due to the potential change caused by the modification of macrophage cell membrane on the surface of nanoparticles.

[0053] 3. Investigation of the cellular uptake ability of MH@PCA

[0054] The fat-soluble drug chlorin Ce6 was used to replace cinnamaldehyde and Fmoc-Arg to prepare the polymer PEI-Ce6 by amide reaction. PEI-Ce6 nanoparticles were prepared by the emulsion evaporation method, and then coated with hyaluronic acid (HA) and macrophage membrane (CM) to obtain MH@PEI-Ce6.

[0055] The cellular uptake ability of the preparation was evaluated on RAW 264.7 cells. RAW 264.7 cells were seeded in a 24-well plate at a density of 5 × 10 4 cells / well, and after adhering, IL-4 (10 ng / mL) was added for 24 h to induce M2-TAMs. Then the culture medium was discarded, and Ce6, PEI-Ce6 and MH@PEI-Ce6 were added, respectively. After 4 h of incubation, the cells were washed three times with PBS, and the cells were collected using a cell scraper. Finally, the fluorescence intensity of intracellular Ce6 was determined by a BD flow cytometer.

[0056] As shown in Figure 6 compared with free Ce6, the fluorescence signal of PEI-C6 was enhanced, and after coating with macrophage membrane, the fluorescence signal of MH@PEI-Ce6 was further enhanced, indicating that the modification of macrophage membrane improved the uptake of nanoparticles by cells.

Claims

1. A macrophage cell membrane mimicking polymer nanoparticle, characterized in that, The cationic polymer nanoparticle comprises a cationic polymer nanoparticle core and a macrophage membrane shell, and the surface of the cationic polymer nanoparticle core is coated with hyaluronic acid; The cationic polymer nanoparticle is self-assembled by polymer A and polymer B, and the mass ratio of polymer A to polymer B is 1:1-3:1; The polymer A is obtained by grafting modification of cinnamaldehyde on polyethyleneimine, and the polymer B is obtained by grafting modification of Fmoc-arginine on polyethyleneimine.

2. The macrophage membrane mimicking polymer nanoparticle of claim 1, wherein, The mass ratio of the hyaluronic acid to the cationic polymer nanoparticle is 1:1-5:1, and the mass ratio of the protein of the macrophage membrane to the cationic polymer nanoparticle is 1:1-10:

1.

3. The macrophage membrane mimicking polymer nanoparticle of claim 2, wherein, The mass ratio of the macrophage membrane, the hyaluronic acid and the cationic polymer nanoparticle is 1:2:

1.

4. The macrophage cell membrane mimicking polymer nanoparticle of claim 3, wherein, The mass ratio of the polymer A to the polymer B is 2.5:

1.

5. The method of producing macrophage membrane biomimetic polymer nanoparticles of claim 1, wherein, The method comprises the following steps: Step 1, preparing polymer A and polymer B; Step 2, dissolving polymer A and polymer B in ethanol respectively, mixing, and then adding dropwise into water to obtain cationic polymer nanoparticles, and then adding hyaluronic acid to obtain cationic polymer nanoparticles coated with hyaluronic acid; Step 3, mixing the macrophage membrane and the polymer nanoparticles obtained in step 2, and preparing the macrophage membrane biomimetic polymer nanoparticles by co-extrusion method.

6. The macrophage membrane biomimetic polymer nanoparticles of claim 1 are used for preparing tumor treatment drugs.

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