A nanomaterial of tumor cell membrane coated fullerene and a preparation method and application thereof

Abstract

The application discloses a kind of nanometer material of tumor cell membrane coating fullerene and its preparation method and application.The nanometer material of the application includes cell membrane and drug-loading core;The drug-loading core includes fullerene derivative and lactic acid-glycolic acid copolymer;The cell membrane is wrapped in the surface of the drug-loading core.Amino fullerene, PLGA nanosphere and the advantage of tumor cell membrane are combined in the application, and a kind of nanometer material with stability, electric capacity and target delivery capacity is formed.The nanometer material obtained by the application is more easily passed through EPR effect and the target effect of tumor cell membrane increases the aggregation degree of nanometer material in tumor site, improves the use efficiency of drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological medicine, in particular to a kind of nanomaterial of tumor cell membrane coated fullerene and its preparation method and application. BACKGROUND

[0002] Tumor is a common and serious disease, which has caused a huge burden on global health. At present, the treatment methods for tumor include surgical resection, radiotherapy and chemotherapy, etc. However, these methods have some limitations, such as poor treatment effect, large drug side effects and drug resistance problem, etc. Therefore, it is of great clinical significance to find new tumor treatment strategies.

[0003] In recent years, nanotechnology has received extensive attention in the field of tumor treatment. Nanomaterials can play an important role at the cellular and molecular level due to their unique physical and chemical properties. They have large specific surface area and excellent charge capacity, which can carry and release various therapeutic substances, thereby improving the treatment effect and reducing side effects.

[0004] Fullerene is a special carbon nanomaterial with spherical structure and many excellent properties. Its high stability, strong oxidation resistance and good biocompatibility make it an ideal biological nanomaterial. Fullerene has a large surface area and rich functional groups, which can react with different types of substances, so it has wide application prospects in drug delivery, biological imaging, photothermal therapy, etc. SUMMARY

[0005] To solve the above technical problems, the present application provides the following technical solutions:

[0006] A kind of nanomaterial, the nanomaterial includes cell membrane and drug-loaded core;The drug-loaded core includes fullerene derivative and lactic acid-glycolic acid copolymer (PLGA);The cell membrane is wrapped on the surface of the drug-loaded core.

[0007] According to the embodiments of the present application, the cell membrane is selected from the cell membrane of tumor cells, preferably the cell membrane of colorectal cancer cells, such as MC38 colorectal cancer cells (also known as MC38 tumor cells).

[0008] According to the embodiments of the present application, in the drug-loaded core, the mass ratio of fullerene derivative and lactic acid-glycolic acid copolymer is (1-10):1, for example, 5:1.

[0009] According to the embodiments of the present application, the lactic acid-glycolic acid copolymer can be commercially available, for example, purchased from Macklin Reagent.

[0010] According to an embodiment of the present application, the molecular weight of the lactic acid-glycolic acid copolymer is 1000-20000, for example, 5000, 10000, 15000.

[0011] According to an embodiment of the present application, the lactic acid-glycolic acid copolymer has nanosphere structure.

[0012] According to an embodiment of the present application, the particle size of the nanosphere is 10-200nm, for example, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 150nm, 200nm.

[0013] According to an embodiment of the present application, the fullerene derivative is selected from the group consisting of amino fullerene derivatives having the structure shown in formula (I);

[0014]

[0015] wherein F is fullerene, the fullerene is selected from at least one of the group consisting of empty fullerene, metal fullerene, heterocyclic fullerene and endohedral fullerene;

[0016] wherein R is an amino modification group of the fullerene, one end of R is combined with the fullerene through a nitrogen-containing group, a phenyl group, a mercapto group, and the other end of R is any nitrogen-containing group, the nitrogen-containing group includes any one or several of primary amine, tertiary amine, secondary amine, quaternary amine;

[0017] wherein m is selected from an integer of 1-12, preferably 3, 4, 5, 6, 10.

[0018] According to an embodiment of the present application, R is -NR 1 -R'-NR 2 R 3 , R 1 , R 2 , R 3 may be hydrogen, or optionally substituted C1-C6 alkyl, cycloalkyl, heteroatom-containing alkyl, heterocycloalkyl, aryl, heteroaryl independently or simultaneously, or R 1 and R 2 and / or R 3 together form a cyclic substituent;

[0019] R' is optionally substituted C1-C6 alkyl, cycloalkyl, heteroatom-containing alkyl, heterocycloalkyl, aryl, heteroaryl, or R 1 and R' together form a cyclic substituent; wherein when R 1 and R 2 and / or R 3 together form a cyclic substituent, R' is absent.

[0020] Further, R 2 , R 3 is simultaneously hydrogen.

[0021] According to an embodiment of the present application, in the amino fullerene derivative, the fullerene is at least one of a hollow fullerene, a metallofullerene, a heterofullerene and an endohedral fullerene, preferably the fullerene is selected from one or more of C 2n , M@C 2n , M2@C 2n , MA@C 2n , M3N@C 2n , M2C2@C 2n , M2S@C 2n , M2O@C 2n and M x A 3-x N@C 2n , wherein M and A are both metal elements, and the M and A are both selected from any one of Sc, Y and lanthanide metal elements; preferably, the fullerene is selected from one or more of C 2n , wherein 2n is the number of carbon atoms, 30≤n≤60; preferably, the fullerene is selected from one or more of C60, C70, C76, C78, C80, C84; more preferably, the fullerene is selected from one or more of C60, C70.

[0022] According to an embodiment of the present application, in the amino fullerene derivative, the amino modification group R of the fullerene is selected from one or more of (2-aminoethyl)amino, (3-aminopropyl)amino, (4-aminobutyl)amino, (5-aminopentyl)amino, (2-aminophenyl)amino, (3-aminophenyl)amino, (4-aminophenyl)amino, (4-amino)piperidinyl, piperazinyl, 4-aminophenyl, (4-aminomethyl)phenyl, (4-aminophenyl)mercapto, (4-aminocyclohexyl)mercapto, 4-piperidinylmercapto, (1-aminoethyl)mercapto, (1-aminopropyl)mercapto, (1-aminobutyl)mercapto, (1-aminopentyl)mercapto.

[0023] The inventors found that tumor cell membrane is an important biomembrane structure with different characteristics from normal cell membrane. The specific antigens and surface receptors on tumor cell membrane make it a marker of tumor cells, which provides an opportunity for targeting tumors. By using tumor cell membrane to wrap nanomaterials, specific recognition and targeted delivery of tumors can be achieved. The main component of tumor cell membrane is lipid bilayer, which contains abundant membrane proteins. These proteins can bind to corresponding ligands, thereby achieving specific recognition and binding of tumor cells. By wrapping tumor cell membrane on the surface of nanomaterials, the nanomaterials can have external characteristics similar to tumor cells, allowing them to better interact with tumor cells in vivo. The wrapping layer of tumor cell membrane can provide protection for nanomaterials and enhance the stability of nanomaterials. In addition, through specific recognition and binding of tumor cell membrane, nanomaterials can accurately target tumor tissues, thereby improving treatment efficacy and reducing damage to normal cells.

[0024] The application also provides a preparation method of the above-mentioned nanomaterial, which comprises:

[0025] S1, preparing poly (lactic-co-glycolic acid) nanospheres;

[0026] S2, preparing a drug-loaded core: loading fullerene derivatives on the poly (lactic-co-glycolic acid) nanospheres to obtain the drug-loaded core;

[0027] S3, preparing a cell membrane;

[0028] S4, mixing the drug-loaded core obtained in step S2 with the cell membrane obtained in step S3, and coating the drug-loaded core with the cell membrane to obtain the nanomaterial.

[0029] According to an embodiment of the application, in step S1, the specific steps of preparation are as follows: dissolving PLGA in an organic solvent to obtain a PLGA solution, adding the solution drop by drop into water, stirring, ultrafiltration, and then resuspending in water to prepare poly (lactic-co-glycolic acid) nanospheres.

[0030] Preferably, the organic solvent is selected from acetone, dichloromethane, ethyl acetate, etc.

[0031] Preferably, the volume ratio of water to PLGA solution is 1-5:1, for example, 3:1.

[0032] According to an embodiment of the application, in step S2, the specific steps of loading are as follows: under ultrasonic conditions, the poly (lactic-co-glycolic acid) nanospheres are added to a solution of fullerene derivatives, and then stirred and filtered. In the present application, the stirring and filtering can be performed by methods known in the art, for example, ultrafiltration after stirring for 48 hours.

[0033] According to an embodiment of the present application, the solution of the fullerene derivative comprises the fullerene derivative and a solvent.

[0034] According to an embodiment of the present application, the concentration of the fullerene derivative in the solution of the fullerene derivative is 0.1-10 mg / mL, for example 1 mg / mL.

[0035] According to an embodiment of the present application, the solvent is selected from at least one of ethanol, water, methanol, ethyl acetate, for example 4% ethanol aqueous solution.

[0036] According to an embodiment of the present application, the ultrasonic can be selected using conditions known in the art, for example under 100 W ultrasonic power.

[0037] According to an embodiment of the present application, in step S2, the mass ratio of the lactic acid-glycolic acid copolymer nanosphere and the fullerene derivative is (1-10):1, for example 5:1.

[0038] According to an embodiment of the present application, in step S3, the cell membrane is selected from the above-mentioned tumor cell membrane, for example MC38 tumor cell.

[0039] According to an embodiment of the present application, in step S3, the step of preparing the cell membrane is as follows: after the tumor cells are collected by digestion and centrifugation, a hypotonic solution is added, and then gradient centrifugation is performed to obtain the cell membrane.

[0040] According to an embodiment of the present application, the gradient centrifugation specifically comprises: centrifuging at a first centrifugal force, at a second centrifugal force and at a third centrifugal force for a period of time, respectively. Preferably, the first centrifugal force is (100-1000) x g, for example 500 x g. Preferably, the second centrifugal force is (5000-20000) x g, for example 10000 x g. Preferably, the third centrifugal force is (50000-200000) x g, for example 100000 x g. Illustratively, the gradient centrifugation comprises: centrifuging at 500 x g for 10 minutes, centrifuging at 10000 x g for 10 minutes, and centrifuging at 100000 x g for 60 minutes, respectively.

[0041] According to an embodiment of the present application, the hypotonic solution comprises: Tris-HCl solution, KCl solution, MgCl2 solution, protease inhibitor tablet.

[0042] According to an embodiment of the present application, the cell membrane can be further resuspended in a buffer solution to obtain a cell membrane buffer. In the present application, the buffer solution can be selected from the buffer solutions known in the art, for example PBS buffer solution.

[0043] According to an embodiment of the present application, in step S4, the mass ratio of the drug-loaded core to the cell membrane is 0.5-2:1, for example, 1:1.

[0044] According to an embodiment of the present application, in step S4, the coating can be performed by using methods known in the art, for example, using a film extruder.

[0045] According to an embodiment of the present application, in step S4, after the coating, filtration can be further performed. The filtration can be performed by using methods known in the art, for example, using a 400 nm filter membrane.

[0046] According to an embodiment of the present application, the nanomaterial is prepared by the preparation method as described above, and has the meanings as described above.

[0047] The present application also provides use of the nanomaterial as described above in preparation of a drug for blocking tumor cell cycle, wherein preferably, the drug for blocking tumor cell cycle is a drug for degrading cyclin, more preferably, the cyclin is selected from Cyclin D1.

[0048] The present application also provides use of the nanomaterial as described above in preparation of a CDK inhibitor drug; preferably, the CDK is selected from one or more of CDK4 and CDK6.

[0049] The present application also provides use of the nanomaterial as described above in preparation of a drug for up-regulating autophagy activation protein of tumor cells; preferably, the autophagy activation protein is selected from one or more of PSAP, CTSL, CTSD, or an intermediate or mature protein thereof.

[0050] In any of the uses of the nanomaterial as described above, the tumor is selected from one or more of liver cancer, lung cancer, colorectal cancer, kidney cancer, pancreatic cancer, bone cancer, breast cancer, ovarian cancer, prostate cancer, esophageal cancer, gastric cancer, oral cancer, nasal cancer, laryngeal cancer, cholangiocarcinoma, cervical cancer, uterine cancer, testicular cancer, meningioma, skin cancer, melanoma, lymphoma, glioma, leukemia, or sarcoma; preferably, the tumor is selected from one or more of non-small cell lung cancer, breast cancer, glioma, liver cancer, and prostate cancer.

[0051] In any of the uses of the nanomaterial as described above, the drug comprises one or more of the nanomaterial, and a pharmaceutically acceptable carrier, preferably, the preparation form of the drug is selected from one or more of a solution, a granule, a lyophilized powder, an emulsion, a suspension, an oil, and a nano-preparation; preferably, the solution is an injection.

[0052] In any of the uses of the nanomaterial as described above, the drug can further comprise at least one or more anti-tumor drugs, which can be selected from anti-tumor drugs known in the art.

[0053] Advantages

[0054] In the present application, a new type of nanomaterial is formed for treating tumors by using tumor cell membranes to wrap PLGA nanospheres loaded with amino-fullerene, wherein the tumor cell membranes provide a highly stable wrapping layer to protect the amino-fullerene and the PLGA nanospheres from the external environment. At the same time, the antigens and receptors on the tumor cell membranes can achieve specific recognition and targeted delivery to tumor cells, improving the targeting and efficacy of the treatment. The research on the nanomaterial of the present application has important clinical application prospects.

[0055] The present application can combine the advantages of amino-fullerene, PLGA nanospheres and tumor cell membranes by reasonable design and preparation to form a nanomaterial with stability, charging capacity and targeted delivery capacity.

[0056] In the preparation of the nanomaterial of the present application, especially when loading drugs into the nanomaterial, polyvinyl alcohol does not need to be added, and the particle size of the synthesized nanomaterial is about 100 nm, which is more likely to increase the aggregation degree of the nanomaterial in the tumor site through the EPR effect and the targeting effect of the tumor cell membrane, and improve the use efficiency of the drug.

[0057] Definitions and explanations of terms

[0058] The term "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0059] The term "treatment" includes inhibiting, relieving, preventing or eliminating one or more symptoms or side effects associated with the disease, disorder or malady being treated.

[0060] The use of the terms "reduce", "inhibit", "alleviate" or "diminish" is relative to a control. The skilled person will readily determine the appropriate control for each experiment. For example, a reduced response in a subject or cell treated with a compound is compared to the response in a subject or cell not treated with the compound.

[0061] As used herein, the term "effective amount" or "therapeutically effective amount" refers to the amount of a drug or pharmaceutical agent that, when administered to a subject for treating, inhibiting or reducing the one or more symptoms of the disease state being treated, or otherwise providing a desired pharmacologic and / or physiologic effect. The precise amount will vary depending on a variety of factors, such as the subject's dependence variables (e.g., age, immune system health, etc.), the disease or illness, and the treatment administered. The effect of the effective amount can be relative to a control. These controls are known in the art and discussed herein, and can be, for example, the subject's condition prior to administration of the drug or pharmaceutical combination or without administration, or, in the case of a pharmaceutical combination, the effect of the combination can be compared to the effect of administering only one of the drugs.

[0062] The term "excipient" is used herein to include any other compound that can be included in or on the microparticle that is not a therapeutically or biologically active compound. Thus, the excipient should be pharmaceutically or biologically acceptable or relevant, e.g., the excipient is generally not toxic to the subject. "Excipient" includes a single such compound, and is also intended to include multiple compounds.

[0063] The term "pharmaceutical composition" means a composition comprising an aminofullerene derivative and at least one pharmaceutically acceptable ingredient selected from the group consisting of carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavorings, flavorants, lubricants, dispersing agents, temperature-sensitive materials, temperature-regulating agents, adhesion agents, stabilizers, suspending agents, and the like, depending on the mode of administration and the nature of the dosage form.

[0064] The term "fullerene" as used herein is a series of spherical-like cluster molecules composed of an even number of carbon atoms, with 12 pentagons and the rest being hexagons. Fullerenes include hollow fullerenes, endohedral fullerenes, which are fullerenes with metals or clusters of metal atoms encapsulated within the carbon cage structure of the fullerene.

[0065] The term "metal fullerene", "endohedral fullerene" refers to the encapsulation of various different metals or clusters of metal atoms within the carbon cage structure of a fullerene, forming a class of compounds with special structure and properties, which are commonly referred to as endohedral fullerenes, generally represented by the formula M@C 2n where M represents a metal element.

[0066] The term "amino fullerene derivative" refers to the amino-modification of a fullerene, the modified fullerene externally including one or more identical or different amino-containing substituent groups, the above-mentioned modification methods can be modified according to the methods disclosed in the prior art.

[0067] As used herein, the term "tumor" refers to or describes the physiological condition in mammals, especially humans, that is typically characterized by unregulated cell growth. Examples of tumors include, but are not limited to, solid tumors, carcinomas, lymphomas, blastomas, sarcomas, and leukemias.

[0068] As used herein, the term "cell cycle" refers to the entire process a cell undergoes from the completion of one division to the end of the next, and is divided into two phases, interphase and mitotic phase. The interphase is further divided into three phases, i.e., the pre-DNA synthesis phase (G1 phase), the DNA synthesis phase (S phase), and the post-DNA synthesis phase (G2 phase). The regulation of cell cycle is mainly achieved through the arrest at G1 phase, and G0 phase refers to the state of cell arrest, and G0 phase is a phase in which the cell stops dividing temporarily out of the cell cycle, but under certain appropriate stimulation, it can also enter the cycle.

[0069] As used herein, the term "Cyclin D1" refers to G1 / S-specific cyclin-D1, which belongs to a kind of cell cycle proteins. "Cell cycle proteins" refer to proteins that show synchronous periodic concentration changes with the cell cycle of eukaryotic cells, including cyclins A, B, D, E, G, and H. They bind to key protein kinases (cyclin-dependent kinases, CDKs) and regulate their enzymatic activity, thereby helping to drive and coordinate the progress of the cell cycle.

[0070] As used herein, the term "autophagy" refers to or describes the process of transporting intracellular damaged, denatured or aged proteins and organelles to lysosomes for digestion and degradation. Under normal physiological conditions, cellular autophagy helps the cell to maintain homeostasis; when stress occurs, cellular autophagy prevents the accumulation of toxic or carcinogenic damaged proteins and organelles, and inhibits cell carcinogenesis. During the autophagy process, lysosomes are involved in autophagy regulation. CTSL (Cathepsin L) and CTSD (Cathepsin D) are lysosomal proteases responsible for degrading proteins and activating enzymatic precursors. PSAP (Prosaposin) isolates lipid substrates from the membrane environment, making soluble degrading enzymes more accessible. Both CTSL and CTSD can stimulate the activation of autophagy, and the lack of PSAP can cause dysfunction of autophagy. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 The molecular structure of amino fullerene TAPC is shown;

[0072] Figure 2 The transmission electron microscope (TEM) image of the nanomaterial is shown;

[0073] Figure 3 The Zeta potential of the nanomaterial is shown;

[0074] Figure 4 Gel electrophoresis staining image of cell membrane surface antigen is shown;

[0075] Figure 5 Photos of tumors in each group after treatment are shown;

[0076] Figure 6 Mass of tumors in each group after treatment is shown; DETAILED DESCRIPTION

[0077] The technical solutions of the present application will be further described in detail below in combination with specific examples. It should be understood that the following examples are only illustratively described and explained the present application, and should not be interpreted as limiting the scope of protection of the present application. Any technology realized based on the above description of the present application is covered within the scope intended to be protected by the present application.

[0078] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0079] Example 1: Synthesis of tumor cell membrane-wrapped amino fullerene nanomaterials

[0080] The tumor cell membrane-wrapped amino fullerene-loaded PLGA nanomaterials were prepared according to the following specific steps:

[0081] S1, Synthesis of PLGA nanospheres, prepare 40 mL of PLGA acetone solution with a concentration of 1 mg / mL, fully dissolve and mix, drop into 120 mL of water, stir overnight, use 10k ultrafiltration tube 6000 rpm ultrafiltration for 10 minutes, collect the product as PLGA nanospheres.

[0082] S2, load amino fullerene on PLGA nanospheres, dissolve amino fullerene TAPC (amino fullerene has the structure as shown in Figure 1 in 4% ethanol aqueous solution, prepare TAPC solution with a concentration of 1 mg / mL, in order to increase the stability of amino fullerene, add PEG-PO polymer excipient (the molar ratio of PEG-PO to TAPC is 10:1), and mix uniformly with the TAPC solution. Then, drop 2 mg / mL of PLGA solution under 100 W ultrasonic power, stir for 48 hours, and ultrafiltrate to obtain amino fullerene-loaded PLGA inner core, marked as TAPC-PLGA.

[0083] S3, large-scale collection of MC38 tumor cell membranes, after digestion and centrifugation of MC38 tumor cells (MC38 tumor cells were purchased from the National Biomedical Experimental Cell Resource Bank), low osmotic solution (low osmotic solution includes: 20 mM Tris-HCl solution, 10 mM KCl solution, 2 mM MgCl2 solution, one piece of protease inhibitor tablet) was added, gradient centrifugation (500xg, centrifugation for 10 minutes to take supernatant; 10000xg, centrifugation for 10 minutes to take supernatant; 100000xg, centrifugation for 60 minutes to take the precipitate), to obtain MC38 cell membranes, and the obtained MC38 cell membranes were resuspended in PBS buffer solution to obtain MC38 cell membrane buffer solution, and the mass volume ratio of MC38 cell membranes to PBS buffer solution was 1:50 (g:mL).

[0084] S4, mixing TAPC-PLGA obtained in step S2 with MC38 cell membrane buffer solution obtained in step S3, wherein the mass ratio of TAPC-PLGA to MC38 cell membrane is 1:1, and coating TAPC-PLGA with MC38 cell membrane with a membrane extruder, passing through a 400 nm filter to obtain tumor cell membrane-coated PLGA nanomaterial loaded with amino fullerene, denoted as TPM.

[0085] Comparative Example 1

[0086] This comparative example uses PEG-PO as an excipient for the comparison group, and the concentration of the aqueous solution is 10 mM, denoted as PEG-PO.

[0087] Comparative Example 2

[0088] This comparative example prepares cell membrane-coated PLGA nanomaterial as a cell membrane comparison group, and the preparation method is the same as that of Example 1, except that in step S4, PLGA obtained in step S1 is mixed with MC38 cell membrane buffer solution obtained in step S3, wherein the mass ratio of PLGA to MC38 cell membrane is 1:1, and PLGA is coated with MC38 cell membrane with a membrane extruder, and passed through a 400 nm filter to obtain cell membrane-coated PLGA nanomaterial, denoted as PM.

[0089] Comparative Example 3

[0090] This comparative example uses TAPC as a fullerene derivative comparison group, and the preparation method is to prepare TAPC into an aqueous solution with a concentration of 1 mM, and add 10 times molar ratio of PEG-PO excipient, mix uniformly, and denote as TAPC.

[0091] Example 2: Morphology test of nanomaterial TPM

[0092] Transmission electron microscopy (TEM, model HT7700) was used to observe and analyze the morphology and structure of nanomaterial TPM, as follows:

[0093] (1) Dissolve the nanomaterial TPM in water to prepare a sample solution.

[0094] (2) Place the carbon film on the sample holder and drop the sample solution on the carbon film to uniformly cover the sample solution.

[0095] (3) After the solvent evaporates, the carbon film covering the sample is tested by transmission electron microscopy to obtain a clear particle image, as shown in Figure 2 .

[0096] Conclusion: The nanomaterial TPM synthesized in Example 1 is spherical with a particle size of about 100 nm; and the tumor cell membrane is completely wrapped around the outer surface of TAPC-PLGA.

[0097] Example 3: Zeta potential test

[0098] 1. Preparation of sample solution:

[0099] Control group: PLGA, TAPC-PLGA, and MC38 cell membrane prepared in Example 1 are dispersed in an aqueous solution, respectively, and are denoted as PLGA group, TAPC-PLGA group, and cell membrane group.

[0100] Experimental group: The nanomaterial TPM of Example 1 is dispersed in an aqueous solution, denoted as TPM group.

[0101] The sample solution to be tested of each of the above groups is left to allow the particles to fully disperse and stabilize.

[0102] 2. The potential test procedure is as follows:

[0103] A Zeta potential measuring instrument (Model NanoZS Zen3600) is used, and the above sample solutions are injected into the measuring pool of the Zeta potential measuring instrument, ensuring that the injected sample liquid fills the measuring pool and that air bubbles are excluded as much as possible to obtain accurate measurement results. The measurement process is started through the control panel or software interface of the instrument. According to the instrument's operation manual, the measurement process is completed, and the test results are shown in Figure 3 .

[0104] Conclusion: The Zeta potentials of PLGA, TAPC-PLGA, cell membrane, and cell membrane-coated nanomaterial (TPM) are -20.0 mV, 5.6 mV, -8.1 mV, and -15.5 mV, respectively. It can be seen that after being coated with the membrane, the potential of the fullerene derivative changes from positive to negative, which can reduce the non-specific binding of proteins in the blood.

[0105] Example 4: Surface protein expression test

[0106] 1. Tumor cell sample: MC38 colorectal cancer cells (purchased from National Biomedical Experimental Cell Resource Bank).

[0107] 2. Preparation of sample: The MC38 cell membrane prepared in Example 1 was dispersed in PBS buffer solution as a blank control group; the TPM nanomaterial prepared in Example 1 was dispersed in PBS buffer solution as an experimental group.

[0108] 3. Surface protein expression test:

[0109] Coomassie brilliant blue staining to detect membrane protein level:

[0110] 1) Collect the tumor cell sample, use RIPA cell lysis buffer to lyse the tumor cell membrane, release the protein in the tumor cell. Centrifuge the tumor cell lysate, collect the supernatant which contains the extracted tumor cell whole protein. Measure the concentration of the extracted tumor cell whole protein using the protein quantification method. Mix the extracted tumor cell whole protein with the protein loading buffer, the ratio of tumor cell whole protein to protein loading buffer (commercial) is 1:4, to obtain the tumor cell protein sample.

[0111] 2) Heat the tumor cell protein sample, add 30 μg of tumor cell protein sample of the control group and the experimental group to the pre-prepared polyacrylamide gel electrophoresis channel respectively. Add electrophoresis buffer (specifically SDS-PAGE Running Buffer) for electrophoresis, the electrophoresis condition is 130V for 75 minutes.

[0112] 3) After electrophoresis, transfer the above gel to a staining box, and cover the gel with Coomassie brilliant blue dye solution. Staining time is 10 minutes. Take out the gel and wash the gel with washing buffer (specifically TBST solution). Observe the stained gel, record and analyze the position and intensity of the protein bands, the results are shown in Figure 4 .

[0113] Conclusion: By comparison, the protein bands of the experimental group are basically consistent with those of the control group, indicating that the membrane protein expression of the nanomaterial TPM is complete, which is conducive to the presentation of tumor cell membrane antigens and the advantage of homotypic targeting.

[0114] Example 5: Tumor inhibition rate of animal experiment is more than 60%

[0115] Animal strain: Balb / c female mice, 5 weeks old, body weight between 16-20g.

[0116] Tumor model: MC38 colorectal cancer subcutaneous tumor model (use the tumor model obtained by cell inoculation in the above examples).

[0117] Experimental grouping:

[0118] The control group is a physiological saline group (denoted as Control), auxiliary materials in the Preparation Example 1 are taken as a PEG-PO group (the concentration is 10 mM, denoted as PEG-PO), PM nanomaterials prepared in the Preparation Example 2 are taken as a blank tumor cell membrane coated PLGA group (denoted as PM), and TAPC prepared in the Preparation Example 3 is taken as a TAPC group (the concentration of TAPC is 1 mM, denoted as TAPC).

[0119] The experimental group: the nanomaterial TPM prepared in the Preparation Example 1 is taken as a cell membrane coated nanomaterial group (denoted as a TPM group, containing 1 mM TAPC).

[0120] The administration mode: intravenous injection of 0.1 mL.

[0121] The experimental method: 100 μL of MC38 colorectal cancer cells with the concentration of 2 x 10 6 / ml are subcutaneously inoculated, two days after inoculation, 100 μL of TPM material is intravenously injected, the control group is intravenously injected with the same dose of physiological saline, PEG-PO, PM and TAPC, and continuous administration is performed for several days, the tumor size is measured after the start of administration, the inhibition of the tumor by the treatment group is observed, and the inhibition results are shown in Figure 5 and 6 .

[0122] The experimental results: it can be obviously seen from the comparison between the experimental group and the control group that the tumor size Figure 5 and the weight Figure 6 of the experimental group are obviously smaller than those of the three control groups, which indicates that the tumor inhibition effect of the TPM group is remarkable, and the tumor inhibition rate is more than 60%. Therefore, it is indicated that the tumor inhibition effect of the nanomaterial TPM is better, and obviously exceeds the treatment effect of a single component.

[0123] The above describes the exemplary embodiments of the present application. However, the protection scope of the present application is not limited to the above embodiments. Any modification, equivalent replacement, improvement, etc. made by those skilled in the art within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A nanomaterial, characterized in that, The nanomaterial comprises a cell membrane and a drug-loaded core; the drug-loaded core comprises a fullerene derivative and lactic acid. A glycolic acid copolymer; the cell membrane encapsulates the surface of the drug-loaded core; the cell membrane is selected from the cell membranes of colorectal cancer cells; The fullerene derivative is selected from aminofullerene derivatives having the structure shown in formula (I). Formula (I) Where F is a fullerene, and the fullerene is selected from one of C60, C70, C76, C78, ​​and C84; m is selected from an integer from 1 to 12; Wherein R is the amino-modifying group of the fullerene, and R is selected from one or more of (4-aminobutyl)amino, (5-aminopentyl)amino, (2-aminophenyl)amino, (3-aminophenyl)amino, (4-aminophenyl)amino, (4-amino)piperidinyl, piperazine, 4-aminophenyl, (4-aminomethyl)phenyl, (4-aminophenyl)thiol, (4-aminocyclohexyl)thiol, 4-piperidinylthiol, (1-aminobutyl)thiol, and (1-aminopentyl)thiol.

2. The nanomaterial according to claim 1, characterized in that, The drug-loaded core contains fullerene derivatives and lactic acid. The mass ratio of the glycolic acid copolymer is 1~10:1; The lactic acid The molecular weight of glycolic acid copolymers is 1000-20000.

3. The nanomaterial according to claim 1, characterized in that, The lactic acid The glycolic acid copolymer has a nanosphere structure; The nanospheres have a particle size of 10-200 nm.

4. The method for preparing the nanomaterial according to any one of claims 1-3, characterized in that, The preparation method includes: S1. Preparation of lactic acid Glycolic acid copolymer nanospheres; S2. Preparation of the drug-loaded core: Loading a fullerene derivative onto the lactic acid The drug-loaded core was obtained on glycolic acid copolymer nanospheres; the specific steps of the loading are as follows: under ultrasonic conditions, the lactic acid... The glycolic acid copolymer nanospheres were added to a solution of fullerene derivatives, stirred, and filtered to obtain the product. The fullerene derivative solution comprises a fullerene derivative and a solvent; the concentration of the fullerene derivative in the fullerene derivative solution is 0.1-10 mg / mL; the solvent is selected from at least one of ethanol, water, methanol, and ethyl acetate. lactic acid The mass ratio of glycolic acid copolymer nanospheres to fullerene derivatives is 1~10:1; S3. Prepare a cell membrane; the cell membrane is selected from tumor cell membranes; S4. Take the drug-loaded core obtained in step S2 and mix it with the cell membrane obtained in step S3. After the cell membrane coats the drug-loaded core, the nanomaterial is obtained.

5. The preparation method according to claim 4, characterized in that, In step S1, the specific preparation steps are as follows: PLGA is dissolved in an organic solvent to obtain a PLGA solution. After dissolution, the solution is added dropwise to water, stirred, ultrafiltered, and then resuspended in water to prepare lactic acid. Glycolic acid copolymer nanospheres.

6. The preparation method according to claim 5, characterized in that, The organic solvent is selected from at least one of acetone, dichloromethane, and ethyl acetate; The volume ratio of water to PLGA solution added is 1-5:

1.

7. The preparation method according to claim 4, characterized in that, In step S3, the cell membrane preparation steps are as follows: after the tumor cells are digested and centrifuged, a hypotonic solution is added, and then the cells are centrifuged in a gradient to obtain the cell membrane; The gradient centrifugation specifically includes: centrifuging for a period of time under a first centrifugal force, a second centrifugal force, and a third centrifugal force, respectively; the first centrifugal force is (100-1000)×g; the second centrifugal force is (5000-20000)×g; and the third centrifugal force is (50000-200000)×g.

8. The preparation method according to claim 4, characterized in that, In step S4, the mass ratio of the drug-loaded core to the cell membrane is 0.5-2:

1.

9. The use of the nanomaterial according to any one of claims 1-3 in the preparation of a drug for blocking colorectal tumor cells.

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