Bionic nanoparticle, composition, preparation method and application thereof

Abstract

The invention discloses bionic nanoparticles, a composition, a preparation method and application thereof, and relates to the technical field of red blood cell-cancer cell hybrid membrane bionics. The bionic nanoparticles comprise lipidosome and a hybrid membrane coated on the surface of the lipidosome, drugs are coated in the lipidosome, and the drugs are astragaloside and curcumin; the molar ratio of the medicine to the liposome is 1: 15; the hybrid membrane is a hybrid membrane of an erythrocyte membrane and a cancer cell membrane. The erythrocyte-cancer cell hybrid membrane wraps the outer layer of the liposome phospholipid bilayer to construct the nano targeting drug delivery system, the modification of the hybrid cell membrane integrates the advantages of long circulation, immune escape, homologous targeting and the like, and a new treatment mode is developed for immunotherapy of malignant tumors with low response rate. The hybrid membrane can improve the targeting property of the drug, reduce side effects on patients and improve the tolerance of the patients. The medicine can be coated in an aqueous solution layer of the liposome, so that the water solubility of the medicine is improved, and meanwhile, the anti-tumor effect of the medicine can be enhanced.

Description

Technical Field

[0001] The invention relates to the technical field of red blood cell-cancer cell hybrid membrane bionics, and in particular to a bionic nanoparticle, a composition, a preparation method and an application thereof. Background Art

[0002] Lung cancer, known as "lung accumulation" or "lung congestion" in traditional Chinese medicine, is the second most common cancer in the world and the most lethal, and is a major public health issue worldwide. Current treatments for lung cancer include surgery, radiotherapy, chemotherapy, immunotherapy, and traditional Chinese medicine. Traditional treatments are poorly tolerated by patients and are accompanied by strong side effects, resulting in unsatisfactory treatment results.

[0003] Traditional Chinese medicine believes that the pathogenesis of lung cancer is mainly "deficiency of both qi and yin". Therefore, traditional Chinese medicine often treats lung cancer with tonifying deficiency as the main method, and promoting blood circulation and removing blood stasis, clearing away heat and detoxifying, resolving phlegm and relieving cough, and promoting diuresis and removing dampness as the auxiliary methods. Astragaloside AS, also known as astragaloside IV, is the main active ingredient of "qi-tonifying medicine" astragalus. It has the effects of strengthening the exterior and stopping sweating, astringing sweat and strengthening the body, and enhancing immunity. It is often used to treat lung cancer in traditional Chinese medicine. Curcumin (Cur) is the main active ingredient of the blood circulation and blood stasis medicine turmeric and zedoaria, and has attracted much attention in the field of anti-inflammatory and anti-tumor. Astragalus-turmeric is a common combination of traditional Chinese medicine for the treatment of lung cancer. Free drugs have the disadvantages of poor targeting, low water solubility, wide distribution, and rapid metabolism, which greatly limit the therapeutic effect of drugs. In cell membrane bionics technology, erythrocyte membrane has the advantages of high biocompatibility, long circulation in the body, and escape from immune rejection, while cancer cell membrane has the characteristics of homologous targeting. Therefore, using erythrocyte-cancer cell hybrid membrane to deliver astragaloside I-curcumin is expected to improve the therapeutic effect of lung cancer.

[0004] Although patent CN 115919880 A discloses the use of Chinese herbal ingredients (oxymatrine and astragaloside IV) and their preparations in synergistic anti-tumor effects with PD-1 inhibitors, it only provides a physical combination of oxymatrine, astragaloside IV and PD-1 inhibitors, but does not disclose the synergistic anti-tumor effects of curcumin and astragaloside IV, nor does it provide hybrid membrane bionic technology.

[0005] Although patent CN 116019786 A constructs a red blood cell-cancer cell biomimetic targeted nano drug delivery system for tumor treatment, the patent is for delivering a liposome drug-loaded core composed of a phototherapy agent, a phototherapy adjuvant and a chemotherapy drug.

[0006] In addition, traditional methods of treating lung cancer have disadvantages such as poor prognosis and intolerance in patients. It is expected to treat lung cancer from the perspective of traditional Chinese medicine, but the therapeutic effect of a single drug is often unsatisfactory, and free drugs have poor targeting, large side effects, and poor patient tolerance.

[0007] In view of this, the present invention is proposed. Summary of the invention

[0008] The purpose of the present invention is to provide a bionic nanoparticle, a composition, a preparation method and an application thereof to solve the above technical problems.

[0009] The present invention is achieved in that:

[0010] In a first aspect, the present invention provides a biomimetic nanoparticle, comprising: a liposome and a hybrid membrane coated on the surface of the liposome, wherein the liposome contains a drug, wherein the drug is astragaloside IV and curcumin, wherein the molar ratio of astragaloside IV to curcumin is 1.5:1; and the mass ratio of the drug to the liposome is 1:15;

[0011] The hybrid membrane is a hybrid membrane of red blood cell membrane and cancer cell membrane, the mass ratio of red blood cell membrane to cancer cell membrane is 1:2-3, and the liposome includes phospholipid and cholesterol.

[0012] In a second aspect, the present invention also provides a method for preparing bionic nanoparticles, comprising mixing astragaloside IV, curcumin, phospholipids, cholesterol, DSPE-PEG and an organic solvent, and obtaining a liposome film after removing the organic solvent; obtaining liposomes by ultrasonic granulation, and then adding red blood cell membranes and cancer cell membranes, and preparing bionic nanoparticles by ultrasonic treatment.

[0013] In a third aspect, the present invention further provides the use of bionic nanoparticles or the bionic nanoparticles prepared by the above preparation method in preparing a composition.

[0014] In a fourth aspect, the present invention further provides a composition, which comprises the above-mentioned bionic nanoparticles or the bionic nanoparticles prepared by the above-mentioned preparation method.

[0015] The present invention has the following beneficial effects:

[0016] In view of the problem of poor targeting of free drugs in the prior art, the present invention wraps the outer layer of the phospholipid bilayer of liposomes with the hybrid membrane of red blood cells and cancer cells to construct a nano-targeted drug delivery system. The modification of the hybrid cell membrane integrates the advantages of long circulation, immune escape, homologous targeting, etc., and opens up a new treatment mode for malignant tumors with low response rate to immunotherapy. The hybrid membrane can improve the targeting of drugs, reduce side effects on patients, and improve patient tolerance. The phospholipid bilayer of liposomes includes a hydrophobic end and a hydrophilic end. Lipophilic drugs can be coated in the hydrophobic layer of liposomes, thereby improving the water solubility of drugs and enhancing the anti-tumor effect of drugs.

[0017] The in vitro results showed that astragaloside IV and curcumin had a certain inhibitory effect on various cancer cells such as lung cancer cells, and the combined use of the two drugs had a more obvious inhibitory effect, indicating that the bionic nanoparticles have the effect of inhibiting lung cancer cell proliferation and apoptosis; the results of the in vivo lung cancer mouse model showed that the bionic nanoparticle experimental group had a significant tumor growth inhibitory effect, and its tumor inhibition rate was higher than that of the single astragaloside IV and curcumin injection group; during the toxicity evaluation experiment, the model mice were able to tolerate the predetermined dose of bionic nanoparticles, and did not show obvious hair, behavior, eating abnormalities and other characteristics. The weight of the mice increased normally during the treatment, confirming that the bionic nanoparticles provided by the present invention reduced the toxicity of the drug. Therefore, the bionic nanoparticles provided by the present invention have good application prospects in anti-tumor.

[0018] In addition, since curcumin has anti-inflammatory effects, biomimetic nanoparticles loaded with curcumin and astragaloside IV also have good application prospects in the preparation of vaccine compositions. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0020] Figure 1 The results of the astragaloside IV-curcumin drug combination ratio screening experiment are shown in Figure 1. (a) The synergy analysis results based on SynergyFinder, and (b) The analysis results of the combination index method.

[0021] Figure 2 The results of the hybrid membrane ratio screening experiment are shown in Figure 1. (a) The statistical results of the fluorescence absorption signal of the nanoparticle uptake by RAW264.7 cells when the ratio of red blood cells to cancer cells is 1:1, 1:2, 1:3, and 1:5;

[0022] Figure 3 The results of characterization of CM@AC lip particle size (a) and potential (b);

[0023] Figure 4 This is the TEM result of CM@AC lip;

[0024] Figure 5 The results of the stability test of CM@AC lip ((a) The statistical results of particle size stability within one month of storage at 4°C; (b) The statistical results of PDI within one month of storage at 4°C;

[0025] Figure 6The results of the toxicity experiments of AS (a) and Cur (b) on LLC cells are shown;

[0026] Figure 7 The statistical results of cell survival rate in the toxicity experiment of biomimetic nanoparticles on LLC cells (Ⅰ: AS, Ⅱ: Cur, Ⅲ: CM@AC lip);

[0027] Figure 8 Figure 1 is the experimental result of the cell invasion experiment. (a) is a microscopic picture of cells in the drug-treated group that passed through the polycarbonate membrane of the Transwell chamber; (b) is a statistical result of the number of cells in the drug-treated group that passed through the polycarbonate membrane of the Transwell chamber;

[0028] Fig. 9 This is the flow cytometric graph of the cell apoptosis experiment;

[0029] Fig.10 The biodistribution diagram of biomimetic nanoparticles loaded with fluorescent molecule ICG in vivo ((a) in vivo fluorescence images of tumor-bearing mice at different time points; (b) curve of fluorescence intensity variation at the tumor site over time (n=3)). DETAILED DESCRIPTION

[0030] References to embodiments of the present invention will now be provided in detail, one or more examples of which are described below. Each example is provided as an explanation rather than a limitation of the present invention. In fact, it will be apparent to those skilled in the art that various modifications and variations may be made to the present invention without departing from the scope or spirit of the present invention. For example, a feature illustrated or described as part of one embodiment may be used in another embodiment to produce a further embodiment.

[0031] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan. This technique is fully explained in the literature, such as Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989); Oligonucleotide Synthesis (MJ Gait, ed., 1984); Animal Cell Culture (RI Freshney, ed., 1987); Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction (PCR: The Polymerase Chain Reaction) (Academic Press, Inc.); Reaction" (Mullis et al., eds., 1994); and Current Protocols in Immunology (JE Coligan et al., eds., 1991), each of which is expressly incorporated herein by reference.

[0032] In a first aspect, the present invention provides a biomimetic nanoparticle, comprising: a liposome and a hybrid membrane coated on the surface of the liposome, wherein the liposome contains a drug, wherein the drug is astragaloside IV and curcumin, wherein the molar ratio of astragaloside IV to curcumin is 1.5:1 to 2:1, and the mass ratio of the drug to the liposome is 1:15 to 1:20;

[0033] The hybrid membrane is a hybrid membrane of red blood cell membrane and cancer cell membrane, the mass ratio of red blood cell membrane to cancer cell membrane is 1:2 to 1:3, and the liposome includes phospholipids and cholesterol.

[0034] The molar ratio of astragaloside IV to curcumin is, for example, 1.5:1, 1.6:1, 1.7:1, 1.8:1, 1.9:1 or 2:1. The mass ratio of the drug to the liposome is, for example, 1:15, 1:16, 1:17, 1:18, 1:19 or 1:20. The mass ratio of the red blood cell membrane to the cancer cell membrane is, for example, 1:2, 1:2.1, 1:2.5, 1:2.7 or 1:3.

[0035] In view of the problem of poor targeting of free drugs in the prior art, the present invention wraps the outer layer of the liposome phospholipid bilayer with the hybrid membrane of red blood cells and cancer cells to construct a nano-targeted drug delivery system. The modification of the hybrid cell membrane integrates the advantages of long circulation, immune escape, homologous targeting, etc., and opens up a new treatment mode for malignant tumors with low response rate to immunotherapy. The hybrid membrane can improve the targeting of drugs, reduce side effects on patients, and improve patient tolerance.

[0036] In view of the poor anti-tumor effect, the present invention provides a liposome drug delivery solution. The phospholipid bilayer of the liposome includes a hydrophobic end and a hydrophilic end. The fat-soluble drug can be encapsulated in the hydrophobic layer of the liposome, thereby improving the water solubility of the drug and enhancing the anti-tumor effect of the drug.

[0037] In order to improve the synergistic effect of astragaloside IV and curcumin, the present invention performed synergistic analysis and combined index analysis based on SynergyFinder, and found that when the molar ratio of astragaloside IV to curcumin was 1.5:1, the inhibitory effect on tumors was stronger, and within a larger concentration range, the two showed additive (Cur at 0-320 μM, AS at 200-640 μM) or synergistic (Cur at 0-60 μM, AS at 0-160 μM) effects.

[0038] In order to increase and reduce the uptake of bionic nanoparticles by macrophages, the present invention has screened and found that when the ratio of red blood cells to cancer cells is 1:2, it has better macrophage escape and tumor cell targeting functions and can be enriched in the tumor site for a longer time. The cancer cell membrane in the hybrid membrane can improve the distribution of drugs in the body through active targeting, enhance tumor targeting, and increase accumulation.

[0039] In a preferred embodiment of the present invention, the molar ratio of phospholipids to cholesterol in the liposome is 4:1 to 5:1. Adding cholesterol to phospholipids can increase the stability of liposomes in the blood, making them less likely to rupture; reduce the permeability of water-soluble molecules to the liposome membrane; increase the stability of liposomes, and prolong their existence time in the blood circulation.

[0040] In a preferred embodiment of the present invention, the hydrophobic end of the liposome is further cross-linked with DSPE-PEG to improve the stability and long circulation performance of the preparation.

[0041] In a preferred embodiment of the present invention, the mass ratio of DSPE-PEG to the drug is 1:1 to 1:3. Under the above mass ratio conditions, the prepared liposomes have good stability and good encapsulation efficiency.

[0042] In a preferred embodiment of the present invention, the relative molecular mass of PEG in DSPE-PEG is 1000-3000, for example, 1000, 1500, 1800, 2000, 2500 or 3000.

[0043] The liposome provided by the invention has two or more phospholipid bilayers and a particle size of about 100 nm.

[0044] In a preferred embodiment of the present invention, the average particle size of the bionic nanoparticles is 111.86±4.12 nm.

[0045] In a second aspect, the present invention also provides a method for preparing bionic nanoparticles, comprising mixing astragaloside IV, curcumin, phospholipids, cholesterol, DSPE-PEG and an organic solvent, and obtaining a liposome film after removing the organic solvent; obtaining liposomes by ultrasonic granulation, and then adding red blood cell membranes and cancer cell membranes, and preparing bionic nanoparticles by ultrasonic treatment.

[0046] In addition to ultrasonic granulation, in other embodiments, repeated freezing and thawing or homogenization methods can also be used to cause the rupture of the liposomes and the reorganization and reclosing of the liposomes, and finally form single-layer liposomes with relatively uniform sizes.

[0047] In a preferred embodiment of the present invention, the organic solvent is removed by rotary evaporation or rotary vacuum drying; the rotary evaporation temperature is 40° C., the rotation speed is 52 rpm / min, and the time is 2 h.

[0048] In a preferred embodiment of the present invention, the organic solvent is a fat-soluble solvent.

[0049] In a preferred embodiment of the present invention, after removing the organic solvent to obtain the liposome film, a water-soluble solvent is added for hydration, so that the lipids automatically form liposomes with double or multiple phospholipid bilayers in the water-soluble solvent. The water-soluble solvent is, for example, selected from PBS solution.

[0050] In a preferred embodiment of the present invention, the hydration temperature is 40°C ± 0.5°C, the hydration time is 30-40 min, and the rotation speed is 50-60 rpm / min.

[0051] In a preferred embodiment of the present invention, the conditions for ultrasonic granulation are: power 20% to 30%, ultrasonic treatment for 3 to 5 seconds, stop for 1 to 2 seconds; ultrasonic granulation for a total of 3 to 5 minutes.

[0052] In a preferred embodiment of the present invention, the concentration of the red blood cell membrane solution is 0.29 mg / mL, the concentration of the cancer cell membrane solution is 0.58 mg / mL, and the mixing volume ratio of the red blood cell membrane solution to the cancer cell membrane solution is 2:1 to 2.5:1.

[0053] In a preferred embodiment of the present invention, the ultrasonic parameters for preparing the hybrid membrane are: ultrasonic power of 8% to 10%, ultrasonic treatment for 2 to 3 seconds, stop for 1 to 2 seconds, and a total of 1 to 2 minutes. Under the above ultrasonic treatment conditions, liposomes of relatively uniform size can be quickly formed.

[0054] In a third aspect, the present invention further provides the use of bionic nanoparticles or the bionic nanoparticles prepared by the above preparation method in preparing a composition.

[0055] In order to further improve the targeting effect of the composition, other substances such as antibodies can be attached to the surface of the liposomes to play a targeting function in the body, so that most of the liposomes are aggregated to the target cells.

[0056] In a preferred embodiment of the present invention, the composition is a drug or vaccine composition.

[0057] When the composition is a drug, the drug may further include pharmaceutically acceptable excipients, including but not limited to fillers, lubricants, disintegrants, binders, glidants, and the like.

[0058] In the preferred technical scheme of the present invention, the pharmaceutically acceptable excipients include, but are not limited to, polyvinyl pyrrolidone and its derivatives, polyvinyl alcohol and its derivatives, methyl cellulose and its derivatives, ethyl cellulose and its derivatives, hydroxypropyl cellulose and its derivatives, starch and its derivatives, polyethylene glycol and its derivatives, lactose, sucrose, mannitol, trehalose, sorbitol, dextrin, microcrystalline cellulose, acrylic resin, calcium hydrogen phosphate, calcium stearate, sodium stearyl fumarate, silicon dioxide, titanium dioxide, talc, indigo or a combination thereof.

[0059] In a preferred embodiment of the present invention, the drug is used for oral administration or injection.

[0060] In a preferred embodiment of the present invention, the drug is used to prevent or treat tumors.

[0061] In a preferred embodiment of the present invention, the vaccine composition is used to fight against segmented RNA viruses.

[0062] The segmented RNA virus is selected from at least one virus in the order Bunyavirales and the family Orthomyxoviridae;

[0063] In a preferred embodiment of the present invention, the segmented RNA virus is a segmented negative-strand RNA virus.

[0064] In a preferred embodiment of the present invention, the segmented RNA virus is selected from at least one virus of the families Phenuiviridae, Nairoviridae and Arenaviridae under the order Bunyavirales;

[0065] In a preferred embodiment of the present invention, the segmented RNA virus is selected from at least one virus of Severe fever with thrombocytopenia syndrome virus (SFTSV), Heartland virus (HRTV) and Guertu virus (GTV) under the family Leukoviridae;

[0066] In a preferred embodiment of the present invention, the segmented RNA virus is selected from at least one of Songling virus (SGLV) and Crimean Congo hemorrhagic fever virus (CCHFV) under the Nairoviridae family;

[0067] In a preferred embodiment of the present invention, the segmented RNA virus is selected from at least one of the lymphocytic choriomeningitis virus LCMV, Machupo virus (MACV) and Lassa virus under the Arenaviridae family;

[0068] In a preferred embodiment of the present invention, the segmented RNA virus is selected from at least one virus of the genus Influenza A virus, genus Influenza B virus, genus Influenza C virus and genus Togotovirus under the family Orthomyxoviridae.

[0069] In an optional embodiment, the tumor is one or more of a digestive system tumor, a respiratory system tumor, a reproductive system tumor, a musculoskeletal system tumor, a nervous system tumor, an endocrine system tumor, a circulatory system tumor, a urinary system tumor, and a reproductive system tumor.

[0070] In an alternative embodiment, the tumor is a digestive system tumor.

[0071] In a further optional embodiment, the digestive system tumor is one or more of an esophageal tumor, a gastric tumor, a cardia tumor, an intestinal tumor, a liver tumor, and a gallbladder tumor.

[0072] In an optional embodiment, the tumor is colon cancer, rectal cancer, colorectal cancer, and the colorectal tumor includes one or more of polypoid type, stricture type and ulcerative type.

[0073] In a preferred embodiment of the present invention, the above-mentioned tumor is an epithelial tumor, including but not limited to papilloma, gastrointestinal cancer, uterine cancer, ovarian cancer, cervical cancer, lung cancer, adenocarcinoma, breast cancer, adenoma or squamous cell carcinoma.

[0074] The gastrointestinal cancer is selected from the group consisting of esophageal cancer, gallbladder cancer, stomach cancer, liver cancer, pancreatic cancer, bile duct cancer, small intestine cancer, colorectal cancer and anal cancer.

[0075] In an alternative embodiment, the adenocarcinoma includes but is not limited to lung adenocarcinoma, thyroid cancer, salivary gland cancer, or pancreatic cancer.

[0076] In an alternative embodiment, the adenoma includes but is not limited to a cystadenoma, a fibroadenoma, a pleomorphic adenoma or a polypoid adenoma.

[0077] In an alternative embodiment, breast cancer includes but is not limited to breast epithelial cancer;

[0078] Preferably, the breast cancer is ductal carcinoma or lobular carcinoma.

[0079] Breast cancer, preferably stage II to IV and / or poorly differentiated invasive ductal carcinoma, comedo carcinoma, and medullary carcinoma (preferably grade 2).

[0080] Ovarian cancer, serous and mucinous carcinomas (preferably stages Ic to IIIb), granulosa cell tumors, surface epithelial-stromal tumors (adenocarcinomas), cystadenocarcinomas, and endometrioid tumors.

[0081] Uterine cancer, preferably including endometrioid adenocarcinoma (preferably stage I to IIIc).

[0082] Bladder cancer, preferably including transitional cell carcinoma (preferably stage II to IV).

[0083] Lung cancer, preferably includes small cell lung cancer (preferably stage I to IIIb), non-small cell lung cancer (preferably poorly to moderately differentiated squamous and adenocarcinoma) and large cell lung cancer.

[0084] The squamous cell carcinoma is one or more of oral squamous cell carcinoma, pharyngeal squamous cell carcinoma, laryngeal cancer, esophageal squamous cell carcinoma (eg esophageal squamous cell carcinoma), lip squamous cell carcinoma, uterine squamous cell carcinoma, vaginal squamous cell carcinoma and skin squamous cell carcinoma.

[0085] Oral squamous cell carcinoma (OSCC) is also called oral squamous cell carcinoma. Examples of oral squamous cell carcinoma include but are not limited to tongue squamous cell carcinoma.

[0086] In other embodiments, the squamous cell carcinoma may also be squamous cell carcinoma formed by squamous metaplasia in the bronchus, bladder, renal pelvis and other parts.

[0087] When the composition is a vaccine composition, it also includes an adjuvant, such as at least one of a Toll-like receptor agonist, a RIG-I-like receptor agonist, a NOD-like receptor agonist, a C-type lectin receptor, a STING agonist, bacterial toxins and their derivatives, saponins, cytokines and other adjuvants; other adjuvants are selected from at least one of heat shock protein, A151, GTP-GDP, sodium fluoride, alkyl polypropylene ester polymer, and dimethyl dioctadecyl quaternary ammonium bromide (DDA).

[0088] In a fourth aspect, the present invention further provides a composition, which comprises the bionic nanoparticles described above or the bionic nanoparticles prepared by the above preparation method;

[0089] In a preferred embodiment of the present invention, the composition is a drug or vaccine composition.

[0090] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0091] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0092] Example 1

[0093] This embodiment provides a method for preparing biomimetic nanoparticles, and the specific steps are as follows:

[0094] (1) Astragaloside IV (AS), curcumin (Cur), soybean lecithin, cholesterol, and DSPE-PEG2000 were weighed in a certain proportion and dissolved in anhydrous ethanol to form an organic phase. Specifically, AS was 7.85 mg, Cur was 1.79 mg, soybean lecithin was 150.14 mg, cholesterol was 9.27 mg, DSPE-PEG2000 was 9.64 mg, and anhydrous ethanol was 12 mL.

[0095] (2) The organic solvent was removed by rotary evaporation to obtain a uniformly dispersed liposome film at a temperature of 40° C., a rotation speed of 52 rpm / min, and a time of 2 h.

[0096] (3) Add appropriate amount of PBS to hydrate the film so that it is evenly distributed in the PBS. The amount of PBS added is 5 mL, the hydration temperature is 40°C, the hydration time is 30 min, and the rotation speed is 60 rpm / min.

[0097] (4) By the method of ultrasonic sieving, the liposomes are evenly distributed, and then passed through a 0.45 μm oil-based filter membrane and a 0.22 μm oil-based filter membrane to obtain AC lip with uniform size. The ultrasonic parameters are as follows: power 30%, ultrasonic for 5 s and pause for 1 s, for a total of 5 min.

[0098] (5) Add the erythrocyte membrane solution and cancer cell membrane solution. The concentration of the erythrocyte membrane is 0.29 mg / mL, and the addition amount is 220 μL. The concentration of the cancer cell membrane is 0.58 mg / mL, and the addition amount is 110 μL. The ultrasonic parameters are as follows: power 65 w, ultrasonic for 3 s and pause for 2 s, for a total of 1 min. The biomimetic nanoparticles (CM@Dil lip) are prepared.

[0099] Experimental Example 1

[0100] Screening experiment on the combination ratio of astragaloside (AS) and curcumin (Cur).

[0101] The evaluation of the drug combination synergy is of positive significance in the process of creating new drugs and optimizing clinical treatment plans. SynergyFinder is a fast, reliable and reproducible platform for calculating synergy scores, and is commonly used for the analysis of the synergy of multi-drug combinations. Cur and AS are respectively compounded in a certain proportion within 0 - 240 μM and 0 - 640 μM, and act on tumor cells. The number of cells is 5×10 3 / well and plated in a 96-well plate. The cytotoxicity at different compounding concentrations is calculated by the CCK8 method, and then the synergy analysis is carried out based on SynergyFinder. The results are as shown in Figure 1 (a) shown below. Cur and AS show additive or synergistic effects within 0 - 60 μM and 0 - 160 μM respectively. As shown by the combination index method in Figure 1 (b) below, when Cur-AS (molar ratio 1:1.5), the inhibitory effect is stronger and the two show a synergistic effect within a large concentration range (1 < IC antagonism, CI = 1 additive, CI < 1 synergistic).

[0102] Experimental Example 2

[0103] Screening experiment on the proportion of hybrid membranes.

[0104] The optimal ratio of erythrocytes to cancer cells is screened by the method of cell uptake. First, AS and Cur are wrapped with hybrid membranes in different ratios. When the ratio of erythrocytes to cancer cells is 1:1, 1:2, 1:3, 1:5 (Ⅰ: 1:1, Ⅱ: 1:2, Ⅲ: 1:3, Ⅳ: 1:5), as shown in Figure 2(a) shows that when the ratio of red blood cells to cancer cells is 1:2, RAW264.7 cells (macrophages) have the least uptake of nanoparticles. The ratio of red blood cells to cancer cells is temporarily selected as 1:2, and then the long circulation effect and targeting of nanoparticles are verified on RAW264.7 cells and LLC (lung cancer) cells, respectively. Figure 2 (b) and (c) confirmed that when the ratio of red blood cells to cancer cells was 1:2, it had better macrophage escape and LLC targeting functions at different times (h) (Ⅰ: free Dil, Ⅱ: Dillip, Ⅲ: CM@Dil lip).

[0105] Experimental Example 3

[0106] The ratio of Cur-AS (molar ratio 1:1.5) and red blood cells to cancer cells was set to 1:2, and the biomimetic nanoparticles were prepared by the method of Example 1, and then the particle size potential and 4° C. stability of the nanoparticles were detected.

[0107] Particle size Figure 3 In (a), the particle size of the biomimetic nanoparticles is 111.86±4.12nm. Figure 3 The potential in (b) is -20.8±3.63 mV, and the PDI is 24.57±0.25%.

[0108] TEM morphology characterization results refer to Figure 4 As shown, (b) is a partial enlarged view of (a). TEM morphology shows that the CM@AC lip bionic nanoparticles are spherical, and there is a shadow film on the outer layer, indicating that the hybrid membrane has been coated on the outer layer of the nanoparticles.

[0109] The obtained CM@AC lip was stored at 4 °C and its stability was investigated within one month. Figure 5 As shown, the particle size (a) and PDI (b) change little, indicating that the nanoparticles are relatively stable.

[0110] Experimental Example 4

[0111] The toxicity of astragaloside IV, curcumin and biomimetic nanoparticles on LLC cells was investigated by CCK8 method. 5 / mL and 4×10 5 100 μL of the concentration of cells / mL was spread in a 96-well plate. After the cells attached to the wall, different concentrations of drugs were given for 24h and 48h. After the time was reached, the drug solution was aspirated, CCK8 solution (the ratio of culture medium: CCK8 was 10:1) was added, and the absorbance at 425nm was measured with an enzyme marker after incubation for 2h.

[0112] The results of AS toxicity to LLC cells are as follows Figure 6 (a) shows the IC of 24h 50The IC value was 142.5 μM at 48 h. 50 The value is 108.3 μM; the toxicity results of Cur on LLC cells are as follows Figure 6 (b) shows the IC of 24h 50 The IC value was 29.75 μM at 48 h. 50 The value is 13.49 μM.

[0113] Cytotoxicity of biomimetic nanoformulations and free drugs on LLC cells at 24 h Figure 7 As shown, the IC of AS 50 The IC value of Cur is 169.6 μM. 50 The IC value of CM@AC lip is 43.75 μM. 50 The concentration of 31.41 μM indicates that the toxicity to cells after combined use is stronger than that of single drug. (Ⅰ: AS, Ⅱ: Cur, Ⅲ: CM@AC lip).

[0114] Experimental Example 5

[0115] In this experiment, a cell invasion experiment was conducted to investigate the ability of drugs to inhibit LLC cell invasion using a Transwell chamber. First, the matrix gel and double-free DMEM medium were mixed at a ratio of 1:8 to 1:10 and then spread on the upper chamber. The mixture was solidified at 37±0.5℃ for 3 to 5 hours. After the matrix gel solidified, the cells and drug suspension were added to the upper chamber (the LLC cell concentration was 3×10 6 / mL).

[0116] like Figure 8 As shown in the figure, compared with the control group, the number of cells that passed through the polycarbonate membrane of the Transwell chamber in the drug-treated group was reduced, indicating that both Cur and AS can inhibit tumor cell invasion to a certain extent. Among them, the number of cells in the CM@AC Lip-treated group was the least, indicating that the combination of Cur and AS can play a synergistic role in inhibiting tumor cell invasion. (Ⅰ: Control; Ⅱ: AS; Ⅲ: Cur; Ⅳ: CM@AC lip).

[0117] Experimental Example 6

[0118] This experiment was conducted to test apoptosis. 5 / mL were plated in a 24-well plate. After the cells adhered to the wall, they were treated with drug-containing culture medium. After 24 hours, the drug-containing culture medium was removed, and the cells were washed twice with PBS. After trypsin digestion, the digestion was terminated with complete culture medium, and the cells were centrifuged at 1200rpm for 3 minutes. The cells were collected and three tubes of cells were separated from the PBS. Annexin V-FITC single-stained tubes, PI single-stained tubes and unstained tubes were set up respectively. The remaining experimental groups used Annexin V-FITC / PI double staining method to distinguish the early and late apoptotic cells and dead cells. Single-stained tubes and unstained tubes were required for adjustment and compensation. The apoptosis induction of LLC cells was detected by flow cytometry.

[0119] Fig. 9 The results showed that the AS group had a weaker pro-apoptotic effect, only 6.79±0.15%, which may be related to the drug concentration. Cur had a more obvious effect on promoting apoptosis, 19.37±0.8% at 24h. After 24h of treatment with CM@AC lip, the apoptotic rate increased to 22.72±0.3%, which was higher than the other three groups, indicating that the combination of AS and Cur can produce a synergistic effect of promoting apoptosis.

[0120] Experimental Example 7

[0121] In this experimental example, an in vivo biodistribution experiment was performed.

[0122] The fluorescent molecule ICG was used to replace the drugs encapsulated in liposomes, and the biodistribution of CM@AC lip in vivo was investigated through in vivo imaging of small animals.

[0123] The experimental method is as follows:

[0124] Accurately weigh ICG 2mg, phospholipids: 45mg, cholesterol 9mg, DSPE-PEG 2000 1.44 mg was dissolved in 4 mL of chloroform and 2 mL of methanol suspension, and rotary evaporated at 40 ° C for 30 min, then 5 mL of ultrapure water was added for hydration for 30 min, and the liposome solution was collected and granulated by ultrasound at a power of 30% (195w), with ultrasound for 5 s and 1 s rest for 5 min, and filtered once with a 0.4 μm oil filter membrane and then with a 0.22 μm oil filter membrane to obtain a uniform ICG liposome solution.

[0125] The preparation method of CM@ICG lip is based on ICG lip and is coated with a hybrid membrane.

[0126] 2 mg of ICG was dissolved in 5 mL of water to form the free ICG group.

[0127] 100 μL of free ICG group, ICG lip group, and CM@ICG lip group were taken respectively, and the concentration and fluorescence were quantified by microplate reader to ensure the consistency of the initial fluorescence intensity of the three groups. 100 μg / mL ICG concentration was injected into mice through tail vein injection, and then the fluorescence intensity of the tumor site was observed by small animal in vivo imaging at different times.

[0128] The fluorescence intensity of the tumor site is shown in Fig.10 In (a), as the time increases, the ICG fluorescence intensity at the tumor site in the free ICG group gradually weakens and decreases rapidly, and the fluorescence almost completely disappears at 24 hours. The fluorescence intensity of the ICG Lip group decreases relatively slowly, and is always significantly higher than that of the free ICG group, and even retains some fluorescence at 48 hours. The fluorescence of the CM@ICG lip group is always stronger than the other two groups, and still retains some fluorescence at 72 hours. The curve of the change of fluorescence intensity at the tumor site over time is shown in Figure 2. Fig.10 As shown in (b) in .

[0129] The above results show that compared with free ICG and ICG lip, CM@ICG lip has stronger tumor targeting and can be enriched in the tumor site for a longer time, indicating that the cancer cell membrane in the hybrid membrane can improve the distribution of drugs in the body through active targeting, enhance tumor targeting, and increase accumulation.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A biomimetic nanoparticle, characterized in that: It includes: Liposome and a hybrid membrane coated on the surface of the liposome, wherein the liposome contains a drug, wherein the drug is astragaloside IV and curcumin, and the molar ratio of the astragaloside IV to the curcumin is 1.5:1 to 2:1; the molar ratio of the drug to the liposome is 1:15 ～ 1:20; The hybrid membrane is a hybrid membrane of red blood cell membrane and cancer cell membrane, the mass ratio of the red blood cell membrane to the cancer cell membrane is 1:2-1:3, and the liposome includes phospholipid and cholesterol.

2. The biomimetic nanoparticle according to claim 1, characterized in that: The molar ratio of phospholipid to cholesterol in the liposome is 4:1 to 5:

1.

3. The biomimetic nanoparticle according to claim 1, characterized in that: The hydrophobic end of the liposome is relatively outwardly cross-linked with DSPE-PEG; Preferably, the mass ratio of the DSPE-PEG to the drug is 1:1 to 1:3; Preferably, the relative molecular mass of PEG in the DSPE-PEG is 1000-3000.

4. The biomimetic nanoparticle according to claim 1, characterized in that: The average particle size of the bionic nanoparticles is 111.86±4.12 nm.

5. The method for preparing biomimetic nanoparticles according to any one of claims 1 to 4, characterized in that: Astragaloside IV, curcumin, phospholipids, cholesterol, DSPE-PEG and an organic solvent are mixed, and after removing the organic solvent, a liposome film is obtained; the liposome is obtained by an ultrasonic granulation method, and then red blood cell membranes and cancer cell membranes are added, and ultrasonic treatment is performed to prepare bionic nanoparticles.

6. The preparation method according to claim 5, characterized in that: The organic solvent is removed by rotary evaporation or rotary vacuum drying; Preferably, the organic solvent is a fat-soluble solvent.

7. The preparation method according to claim 6, characterized in that: After removing the organic solvent, adding a water-soluble solvent for hydration; Preferably, the hydration temperature is 40°C±0.5°C, the hydration time is 30-40min, and the rotation speed is 50-60rpm / min.

8. The preparation method according to claim 5, characterized in that: The conditions of the ultrasonic granulation are: power 20% to 30% (130w to 195w), each ultrasonic treatment for 3 to 5s, stop for 1 to 2s; the total ultrasonic granulation time is 3 to 5min; Preferably, the ultrasonic granulation further comprises filtering with an oil filter membrane to collect the filtered matter; Preferably, the concentration of the red blood cell membrane solution is 0.29 mg / mL, the concentration of the cancer cell membrane solution is 0.58 mg / mL, and the mixing volume ratio of the red blood cell membrane solution to the cancer cell membrane solution is 2:1 to 2.5:1; Preferably, the ultrasonic parameters during the preparation of the hybrid membrane are as follows: ultrasonic power is 8% to 10%, each ultrasonic treatment is 2 to 3 seconds, and then stopped for 1 to 2 seconds, for a total of 1 to 2 minutes.

9. Use of the bionic nanoparticles according to any one of claims 1 to 4 or the bionic nanoparticles prepared by the preparation method according to any one of claims 5 to 8 in preparing a composition; Preferably, the composition is a pharmaceutical or vaccine composition; Preferably, the drug is used to prevent or treat tumors; Preferably, the vaccine composition is used against segmented RNA viruses.

10. A composition, characterized in that It includes the bionic nanoparticles according to any one of claims 1 to 4 or the bionic nanoparticles prepared by the preparation method according to any one of claims 5 to 8; Preferably, the composition is a pharmaceutical or vaccine composition.

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