CN111905105B

CN111905105B - Protein nano-drug for cancer targeted therapy and preparation method thereof

Info

Publication number: CN111905105B
Application number: CN202010628742.4A
Authority: CN
Inventors: 关燕清; 张令坤; 焦宇萱; 唐云志
Original assignee: South China Normal University
Current assignee: South China Normal University
Priority date: 2020-07-02
Filing date: 2020-07-02
Publication date: 2023-05-05
Anticipated expiration: 2040-07-02
Also published as: CN111905105A

Abstract

The invention discloses a protein nano-drug for cancer targeted therapy and a preparation method thereof, wherein the protein nano-drug is an exosome transferred with a human p53 protein of which covalent cross-linking is carried out on Triphenylphosphine (TPP). The protein nano-drug delivery system TPP/p53@Exos developed by the invention can target and identify specific cancer cells, can efficiently inhibit the growth of the cancer cells by activating cell endogenous apoptosis channels, has no toxic or side effect, and provides a new means for cancer treatment. The preparation method of the nano-drug delivery system has higher protein load rate (75%), and can also be used for protecting and targeting delivery of other protein drugs.

Description

Protein nano-drug for cancer targeted therapy and preparation method thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to a protein nano-drug for targeted treatment of cancer and a preparation method thereof.

Background

With the increase of morbidity and mortality, cancer has become a major cause of death in chinese diseases and is also a major public health problem.

Chemotherapy and radiotherapy are currently the most prevalent modes of cancer treatment, and face the problems of cancer cell resistance and toxic side effects to the body. Cancer Stem Cells (CSCs) can effectively evade the clearance of chemotherapy and radiation therapy by expressing ATP-binding cassette (ABC) transporters, anti-apoptotic proteins and DNA repair enzymes, resulting in drug tolerance and tumor recurrence. However, chemotherapy drugs based on small molecular weights (alkylating agents, anthracyclines, microtubule inhibitors, antimetabolites, platinum drugs, topoisomerase inhibitors, tyrosine kinase inhibitors, histone deacetylase inhibitors, etc.) often have side effects on patients, greatly reducing the quality of life of the patients [ Park, s.b. et al chemotherapy-Induced Peripheral Neurotoxicity: a Critical analysis.ca. Cancer j. Clin.63,419-437 (2013) ].

TP53 oncogene is the most common mutant gene in all cancer types, and TP53 gene mutation is present in 50% of tumors. TP53 mutations account for approximately 40% of all breast cancers, with highest frequency in basal-like subtypes (80%) and HER 2-rich subtypes (72%) and lower frequencies in luminal a subtype (12%) and luminal B subtype (29%) of triple negative breast cancers.

The human TP53 gene is located at 17P13.1 (chromosome 17 short arm 1 region 3 band 1 sub-band), consists of 11 exons and 10 introns, is transcribed into 2.5kb mRNA, encodes 393 amino acid proteins, and has a molecular weight of 43.7kDa. Since the protein contains a large amount of proline, the electrophoresis speed is slow, and the protein band appears at 53kDa as shown in Marker, which is designated as p 53. p53 is a highly labile transcription factor that is activated by a variety of different stresses (including oncogene activation, DNA damage, hypoxia and nutritional deprivation, etc.) to form tetramers that are stabilized against ubiquitination and proteasome degradation, and enter the nucleus, bind to p53 responsive elements located in its promoter region, transactivate target genes, and coordinate inhibition of numerous downstream responses of tumors (including regulation of cell cycle, proliferation, senescence, differentiation, apoptosis, iron death, DNA repair, metabolism, angiogenesis and autophagy).

In addition to functioning as transcription factors, p53 also plays an important role in mediating transcription-independent cell death processes. Recently a gene construct fused to p53 and BH3 proteins (both proteins contain mitochondrial targeting signals) has shown more remarkable apoptosis-inducing effects in vitro by enhancing the mitochondrial targeting effect of transcribed p53 proteins, suggesting a great potential for the mitochondrial apoptosis pathway of p53 in cancer treatment.

p53, a single chain protein, has an N-terminal residue, which, due to its physiological charge, is most likely exposed to aqueous solvents, thus revealing a specific site of chemical modification. For the human p53 protein expressed by the prokaryotic escherichia coli, the N-terminal methionine is cut off after synthesis, the N-terminal is changed into glutamic acid, and the amino group provided by the N-terminal methionine can be used as an acylation modification site.

The most commonly used acylation catalysts are catalytic systems consisting of water-soluble 1-ethyl- (3-dimethylaminopropyl) -carbodiimide hydrochloride (edc.hcl) and N-hydroxysuccinimide (NHS) capable of catalyzing the formation of amide bonds. The mechanism is as follows: EDC activates the carboxyl groups of reactant 1 to form O-acylisourea that is more susceptible to acylation with amino groups (which is readily hydrolyzed to carboxylic acid in a few minutes), while NHS converts O-acylisourea to an active ester (with a half-life of up to several hours at around ph 7.0) providing more sufficient reaction time for crosslinking reactant 2 with reactant 1 to provide amino groups. EDC and NHS are usually in excess to ensure optimal catalytic effect. Notably, if reactant 2 providing an amino group has a carboxyl group at the same time, its carboxyl group may also be activated by EDC/NHS, thereby producing a non-target product. To avoid the above problems, after the EDC is activated in the reaction 1, a proper amount of a reducing agent such as β -mercaptoethanol or triethylamine may be added to quench the remaining excess EDC.

In order to avoid the neutral lysine, glutamic acid, aspartic acid, etc. residues exposed on the surface of p53 protein as chemical modification sites as far as possible, the slightly acidic reaction conditions (pH 4.5-7) can be controlled.

Since proteins do not readily penetrate the cell membrane and wild-type p53 protein is readily hydrolyzed in the cell (half-life of 20 min), intracellular delivery of p53 protein also requires the reliance on stable, specific and efficient nanocarriers. Exosomes (Exosomes, english abbreviations Exos) are non-binary options for natural nano-protein drug carriers. Exosomes are the smallest nanoscale unilamellar vesicles found today, with particle sizes between 30 and 150nm, secreted by mammalian cells, and widely found in body fluids such as saliva and urine. In addition to having a finer particle size than microvesicles (100-1000 nm), exosomes have a unique origin. The interior of the exosome is typically loaded with protein and nucleic acid fragments, which are mediators of close and remote communication between cells, and play an important role in inflammation, immune response, angiogenesis, cell death, neural degeneration, and cancer migration. However, the mechanism by which exosomes are internalized by cells and whether specific is not known, and their uptake pathways may be more dependent on the type of recipient cell.

The purification method of exosomes is various, the most common method is ultracentrifugation (not less than 100,000Xg), or a combination of filtration and ultracentrifugation, filtering out cell debris and large-sized Exovesicles (EVs) with a filter membrane, and separating exosomes by ultracentrifugation. Various stimuli such as Ca ²⁺ Ionophore, hypoxia, etc. can induce cells to secrete EVs, and can also be used for improving the output of exosomes. For the loading of hydrophilic proteins after vesicle separation and purification, the main obstacle is how to cross the proteins across the lipid bilayer membrane. In this regard, electroporation is a relatively mature technique, which was used for loading chemotherapy drugs into liposomes in early stages, and the principle is to induce lipid membranes to form pores by controlling voltage, thereby encapsulating the drugs.

Membrane proteins and inclusions of tumor cell exosomes often play a role in suppressing immune cells and promoting tumor proliferation, and thus tumor-derived exosomes are rarely used as nanocarriers. However, with intensive research into the communication roles of exosomes between allogeneic cells, tumor-derived exosomes have also gradually demonstrated a potential in targeting drugs to homologous cancer cells. However, the research in this respect is very lacking.

Disclosure of Invention

The invention aims to provide a protein nano-drug for targeted cancer treatment and a preparation method thereof. Specifically, a construction strategy of a recombinant p53 protein-exosome nano-drug for cancer targeted therapy and a preparation method thereof are disclosed, wherein the recombinant p53 protein-exosome nano-drug can effectively induce apoptosis of p53 mutant or non-mutant breast cancer cells in vitro. The preparation method comprises the following steps: firstly, obtaining human p53 protein from transgenic engineering bacteria, and catalyzing amidation reaction between (3-carboxypropyl) -Triphenylphosphine (TPP) and p53 protein by using a catalyst EDC/NHS to prepare TPP/p53 recombinant protein (figure 1 a); and extracting exosomes secreted by cancer cells subjected to ultraviolet light stimulation or heat shock treatment by adopting an ultracentrifugation technology (figure 1 b), and introducing the TPP/p53 recombinant protein into the exosomes by adopting an electroporation technology to prepare the TPP/p53@exo recombinant p53 protein-exosome nano-drug (figure 1 c). The recombinant p53 protein-exosome nano-drug can be applied to the promotion treatment of breast cancer, and the natural nano-carrier exosome can identify homologous cancer cells and deliver TPP/p53 recombinant protein into the cancer cells; the mitochondrial targeting molecule TPP can deliver exogenous recombinant p53 protein to the outer mitochondrial membrane under the drive of mitochondrial membrane potential; the exogenous recombinant p53 protein can act with the protein related to the apoptosis of the outer mitochondrial membrane to finally induce the apoptosis of breast cancer cells.

The first object of the invention is to provide a protein nano-drug for targeted cancer treatment.

The second object of the invention is to provide a preparation method of protein nano-drugs for targeted cancer treatment.

The third object of the invention is to provide the protein nano-drug for cancer targeted therapy prepared by any one of the preparation methods.

The fourth object of the invention is to provide the application of the protein nano-drug for cancer targeted therapy in preparing cancer targeted therapeutic agent.

The above object of the present invention is achieved by the following means:

the invention is developed by taking a mouse model constructed by a humanized breast cancer cell line MCF-7 and SK-BR-3 and a murine cell line 4T1 as main experimental objects. Firstly, catalyzing covalent cross-linking of (3-propylcarboxyl) -triphenylphosphine bromide (TPP for short) and purchased humanized p53 protein expressed by transgenic engineering bacteria to prepare TPP/p53 recombinant protein; and extracting Exosomes (Exosoms, english is called Exos for short) secreted by cancer cells in the breast cancer cell culture supernatant by adopting an ultracentrifugation technology, and introducing the TPP/p53 recombinant protein into the Exosomes by utilizing an electrotransformation technology to prepare a protein nano-drug delivery system TPP/p53@Exos.

The invention performs a series of characterization on the nano-drug delivery system through a series of detection technologies such as Fourier infrared spectrum, nuclear magnetic resonance hydrogen spectrum, circular two-phase chromatography, transmission electron microscope and the like; the targeting capability and the growth inhibition effect of the nano drug delivery system on cancer cells in vitro are explored through experiments such as immunofluorescence, CCK-8 cell counting analysis, fluo-3 AM calcium ion fluorescence detection, cell scratch and the like; the invention builds a breast cancer model of a 4T1 mouse, records the change of tumor volume after in-situ administration for 28 days, and explores the killing effect of the nano drug delivery system on breast cancer cells in vivo and toxic and side effects on organisms through H & E staining, immunohistochemistry, hematology examination and the like. The result shows that the protein nano-drug delivery system TPP/p53@Exos can target and identify specific cancer cells, can efficiently inhibit the growth of the cancer cells by activating cell endogenous apoptosis channels, has no toxic or side effect, and provides a new means for cancer treatment. The preparation method of the nano-drug delivery system has higher protein load rate (75%), and can also be used for protecting and targeting delivery of other protein drugs.

The invention discloses a protein nano-drug for targeted cancer treatment, which is an exosome of a human p53 protein into which triphenylphosphine is transferred and covalently crosslinked.

Preferably, the cancer is breast cancer and the exosomes are extracted from breast cancer cell culture supernatants.

The invention also claims a preparation method of the protein nano-drug for targeted cancer treatment, which comprises the following steps:

covalently crosslinking triphenylphosphine containing carboxyl functional groups with a humanized p53 protein to obtain a recombinant protein TPP/p53;

extracting exosomes;

transferring the prepared recombinant protein TPP/p53 into the extracted exosomes.

Preferably, the cancer is breast cancer and the exosomes are extracted from the cell culture supernatant of breast cancer stimulated by uv irradiation.

More preferably, the ultraviolet light power is 25W and the irradiation time period is 30min.

Preferably, the method for covalently crosslinking Triphenylphosphine (TPP) containing carboxyl functional groups with human p53 protein is: preparing a saturated solution of triphenylphosphine containing carboxyl functional groups, adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl) and N-hydroxysuccinimide (NHS), and activating carboxyl groups of the triphenylphosphine containing carboxyl functional groups; the EDC is quenched by adding beta-mercaptoethanol, the p53 solution is mixed with the activated TPP solution, reacted and purified.

The triphenylphosphine containing carboxyl functional groups is (3-propylcarboxyl) -triphenylphosphine bromide.

More preferably, the saturated solution of triphenylphosphine containing a carboxyl function is a saturated phosphate buffer salt solution of triphenylphosphine containing a carboxyl function, and the saturated phosphate buffer salt solution has ph=6.2.

More preferably, the carboxyl group reaction time of the activated TPP is 10 to 20 minutes.

Most preferably, the carboxyl reaction time of the activated TPP is 15 minutes.

More preferably, the ratio of the amounts of EDC. HCl and NHS is 4-8:1.

Most preferably, the ratio of the amounts of EDC. HCl and NHS substances is 4:1.

More preferably, the ratio of the amounts of EDC. HCl and TPP is 10 to 20:1.

Most preferably, the ratio of the amounts of EDC. HCl and TPP is 20:1.

More preferably, the concentration of the p53 solution is 0.2 to 1mg/mL.

Most preferably, the p53 solution concentration is 0.6mg/mL.

More preferably, the ratio of the amount of substance of the p53 solution to the activated TPP solution is 1:100 to 2000.

Most preferably, the ratio of the amount of substance of the p53 solution to the activated TPP solution is 1:1000.

More preferably, the p53 solution is mixed with the activated TPP solution and reacted for 1 to 4 hours.

Most preferably, the p53 solution is mixed with the activated TPP solution and reacted for 2 hours.

More preferably, the ratio of the amounts of the substances of beta-mercaptoethanol and EDC is 1 to 2:1.

Most preferably, the ratio of the amounts of β -mercaptoethanol and EDC is 1:1.

More preferably, the beta-mercaptoethanol solution is mixed with the activated TPP solution and reacted for 3 to 10 minutes.

Most preferably, the beta-mercaptoethanol solution is mixed with the activated TPP solution and reacted for 5 minutes.

More preferably, the reaction solution is transferred to a 1000-5000 Da dialysis bag for dialysis purification and dialyzed in PBS dialysate for 12-48 hours.

Most preferably, the reaction solution is transferred to a 1000Da dialysis bag for dialysis purification and is permeated in PBS dialysate for 24 hours.

Preferably, the exosomes are extracted using ultracentrifugation techniques.

More preferably, the cell culture supernatant is filtered through a 220 μm microporous filter membrane, and the filtrate is collected; transferring the filtrate into an ultracentrifuge tube, and ultracentrifugating at the temperature of 0-4 ℃ and the speed of 100,000 ～ 150,000 Xg for 1.5-12 h; the supernatant was discarded and the exosome pellet was resuspended using PBS or electrode buffer.

Most preferably, the cell culture supernatant is taken, filtered through a 220 μm microporous filter membrane, and the filtrate is collected; transferring the filtrate into an ultracentrifuge tube, and ultracentrifugating at 4 ℃ for 12h at 103,000Xg; the supernatant was discarded and the exosome pellet was resuspended using PBS or electrode buffer.

Preferably, the prepared recombinant protein TPP/p53 is transferred into the extracted exosomes by using electrotransformation technology.

More preferably, the specific operation of transferring the prepared recombinant protein TPP/p53 into the extracted exosomes by using electrotransformation technology is as follows: suspending the exosomes with an electrode buffer, and performing electroporation treatment to empty the substances in the exosomes; adding DMEM or 1640 basic culture medium for dilution and incubation; purifying exosomes by ultracentrifugation to obtain empty exosome suspension; mixing the empty exosome suspension with a recombinant protein TPP/p53 solution, and carrying out electroporation treatment; incubating and purifying by ultracentrifugation.

More preferably, the medium is selected from DMEM or 1640 minimal medium according to the source cells of the exosomes.

Most preferably, the conditions of the electroporation treatment are that a voltage of 0.5 to 1kV is applied for 2 to 5ms.

More preferably, the conditions of the electroporation treatment are that a voltage of 1kV is applied for 2ms.

More preferably, the ultracentrifugation is purified to 0-4℃and 100,000 ～ 150,000 Xg ultracentrifugation is performed for 1.5-12 hours; the supernatant was discarded and the exosome pellet was resuspended using PBS or electrode buffer.

Most preferably, the ultracentrifugation is pured to 4 ℃,128,000Xg ultracentrifugation for 12h; the supernatant was discarded and the exosome pellet was resuspended using PBS or electrode buffer.

More preferably, the incubation is carried out at 30-40 ℃ for 10-30 min

Most preferably, incubation is carried out at 37℃for 30min

The protein nano-drug for cancer targeted therapy prepared by any one of the preparation methods.

The application of the protein nano-drug for cancer targeted therapy in preparing cancer targeted therapeutic reagent.

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

the material selection and construction method of the nano drug delivery system in the invention has the advantages that: (1) The nano carrier exosome has a smaller particle size compared with other vesicles, so that the nano carrier exosome can effectively penetrate through the vascular wall of tumor tissues, and the drug delivery efficiency is improved; (2) The exosomes extracted by the ultracentrifugation technology keep the membrane protein integrity of the exosomes, can realize the homophilic identification of tumor cells and immune escape related to the four-transmembrane molecular protein CD47 in the protein drug delivery process, and fully exert the biological functions of the exosomes in the drug delivery process; (3) Before the exosomes load p53 protein, the cancerogenic substances such as RNA and the like in the exosomes secreted by cancer cells are discharged by adopting a conductivity technology, so that the effect of promoting the growth and migration of tumors is avoided.

The mechanism of action of the nano drug delivery system in the invention has innovativeness: (1) The natural nano-carrier exosomes can identify homologous cancer cells and deliver the TPP/p53 recombinant protein into the cancer cells; the mitochondrial targeting molecule TPP can deliver exogenous recombinant p53 protein to the outer mitochondrial membrane under the drive of mitochondrial membrane potential; the exogenous recombinant p53 protein can act with the protein related to the apoptosis of the outer mitochondrial membrane to finally induce the apoptosis of breast cancer cells. (2) Starting from the apoptosis signal path, exogenous p53 related to cell endogenous apoptosis is selected as a protein medicine, and is subjected to lipophilic modification, so that the protein medicine can target mitochondria to play a role in promoting cancer cell apoptosis, and is different from the traditional chemotherapy medicine for directly inhibiting normal metabolism of cells, so that the protein medicine is easy to be metabolized by the cells, and has lower toxic and side effects.

The nano-drug delivery system has good practicability: (1) Can effectively induce apoptosis of p53 mutant or non-mutant breast cancer cells in vitro and in vivo; (2) Can also be used to target killing other abnormal tissues, cells or intracellular organelles to treat cancer in situ and metastatic cancers of different molecular subtypes; (3) The method is also used for targeted delivery of other chemicals, proteins, genes and the like for treating other diseases, and only needs to replace exosomes derived from cancer cells with exosomes or vesicles secreted by target cells, replace p53 protein with other apoptosis promoting factors, chemicals, proteins or genes and the like, replace TPP with other organelle targeted preparations and the like.

In conclusion, the protein nano-drug delivery system TPP/p53@Exos developed by the invention can target and identify specific cancer cells, can inhibit the growth of the cancer cells efficiently by activating the endogenous apoptosis pathway of the cells, has no toxic or side effect, and provides a new means for cancer treatment. The preparation method of the nano-drug delivery system has higher protein load rate (75%), and can also be used for protecting and targeting delivery of other protein drugs.

Drawings

FIG. 1 shows the preparation process of nanometer TPP/p53@Exos medicine.

FIG. 2 is an infrared spectrum for detecting TPP/p53 chemical bond changes.

FIG. 3 shows the detection of TPP/p53 hydrogen bond changes by nuclear magnetic resonance hydrogen spectroscopy.

FIG. 4 shows the secondary structure of TPP/p53 protein by circular dichroism.

FIG. 5 shows the UV spectrum for determining TPP/p53 crosslinking rate.

FIG. 6 shows immunoblotting for detecting exosome marker membrane proteins.

FIG. 7 shows the detection of exosome forms by immunofluorescence.

FIG. 8 shows the particle size of the exosomes for dynamic light scattering detection.

FIG. 9 shows the detection of TPP/p53 loading into exosomes (scale: 100 nm) by transmission electron microscopy.

FIG. 10 shows the immunofluorescence detection of TPP/p53 loading into exosomes (scale: 200 nm).

FIG. 11 shows the exosome loading of TPP/p 53.

FIG. 12 shows immunofluorescence detection of cancer cell uptake into exosomes (scale: 20 μm).

FIG. 13 shows the mitochondrial targeting effect of immunofluorescence detection of TPP/p53@Exos (scale: 20 μm).

FIG. 14 shows the cell viability of CCK-8 kit for detecting cancer cells 24h after nano-drug action.

FIG. 15 shows Ca of cancer cells after 24h of nano-drug action by Fluo-3 AM kit ²⁺ The content is as follows.

FIG. 16 shows the detection of cancer cell migration after nano-drug action by scratch assay.

Fig. 17 shows tumor volume change during 28 days of drug treatment in breast cancer model in mice.

Figure 18 shows the weight change of mice over the course of 28 days of treatment.

Figure 19 is tumor size 28 days after drug treatment in the breast cancer model of mice.

Fig. 20 is a morphological observation of H & E stained tumor tissue: saline control group; group p 53; a tpexos group; TPP/p53 group; TPP/p53@tExos group, scale: 200 μm;100 μm.

FIG. 21 shows immunohistochemical detection of Bax, bcl-2, caspase-3 and Ki67 expression in tumor tissue: saline control group; group p 53; exo group; TPP/p53 group; TPP/p53@tExos group.

Fig. 22 is a morphological observation of H & E stained tumor tissue: normal group; saline control group and TPP/p53@tpexos treatment group, scale: 100 μm

FIG. 23 shows the blood conventional index test 28 days after the treatment of mice.

Detailed Description

The invention is further illustrated in detail below in connection with specific examples which are provided solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

1. Cell strain

The human breast cancer cell line MCF-7 is provided by a cell resource center of Shanghai bioscience research institute, the human breast cancer SK-BR-3 cell line and the mouse breast cancer 4T1 cell line are provided by advanced technology institute of Shenzhen of China university, and are subjected to subculture in the laboratory.

2. Main reagent and instrument

Recombinant human p53 protein (brand: CUSABIO, cat# CSB-EP024077 HU) (0.6 mg/mL, purity greater than 90%), (3-propylcarboxy) -triphenylphosphine bromide (TPP), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC. HCl), N-hydroxysuccinimide (NHS), phosphate Buffer (PBS), dialysis bag (1 kDa)

Pancreatin, penicillin, streptomycin, high sugar DMEM medium, 1640 medium are all manufactured by gibco company; the new calf serum is purchased from Hangzhou holly bioengineering materials limited company; CD63 antibodies, CD81 antibodies, p53 antibodies; FITC fluorescent secondary antibody, cy5 fluorescent secondary antibody; DAPI; mito-tracker Green mitochondrial active dye liquor; CCK-8 cell counting kit; fluo-3 AM calcium ion probe; other reagents were analytical grade and used without further purification.

Nikon microscope, optical inverted microscope of Olympus corporation, japan, sigma32184 high-speed cryocentrifuge, thermo CO ₂ Incubator, electrotransport (Gene Pulser Xcell Total System), CP80NX ultracentrifuge, jiangsu Jiujin jar medical instrument factory 78-1 magnetic stirrer, HV-85 autoclave, sterile console, guangzhou Kokai bridge laboratory equipment Limited thermostat water bath, etc.

3. Construction of in vitro mouse model and administration mode

4T1 cell culture conditions: 1640 medium containing 10% new calf serum, 37 ℃,5.0% CO ₂ . Preparing 48 healthy female BALB/c mice, and raising until the weight reaches about 15 g; in vitro subculturing 4T1 cells, digesting with 0.25% trypsin solution for 15-30 min, blowing with a pipetting gun, centrifuging at 1000rpm for 5min, discarding supernatant, and re-suspending cells with PBS to adjust the concentration to 1×10 ⁷ individual/mL; preparing 4T1 single cell suspension for later use.

Of these 40 mice were randomized into 5 groups of 8: (1) a control group; (2) p53 treatment group; (3) exosome treatment groups; (4) TPP/p53 treatment group; (5) TPP/p53@Exos treatment group. Sucking 4T1 cell suspension with sterile syringe, inoculating onto mammary gland under lower abdomen of mice, and injecting into each mouse0.1mL; the other 8 were normal groups without any treatment. All mice were in the same feeding environment, given sufficient food and water. Starting from the next day after inoculation, the tumor length and width of the mice were measured daily and the formula v=a×b was used ² 2, calculating tumor volume; when the tumor volume reaches 100mm ³ After that, further experiments were carried out.

Purifying 4T1 cell-derived exosomes, tExos, as protein drug carriers by ultracentrifugation; preparing TPP/p53@tExos, respectively injecting equal-volume p53 protein, exosome, TPP/p53 and TPP/p53@tExos drugs into tumors of mice in an experimental group in situ, injecting equal-volume physiological saline into a control group, and injecting the drugs once every two days, wherein the dosage of each drug is 12 mug/kg (p 53 protein content) for 28 days; tumor volume and body weight of mice were measured every two days, mice were sacrificed after treatment was completed, tumors were dissected and weighed, and data were analyzed and plotted using Graphpad Prism software.

4. Data analysis

Experiments were performed in at least three replicates. All values are expressed as mean ± standard error. Statistical analysis was performed using GraphPad prism6.0 software. The comparison between the two groups uses independent sample T test. Two or more sets of comparisons used one-way analysis of variance (P <0.05 being significantly different and P <0.01 being extremely significantly different).

EXAMPLE 1 preparation of TPP/p53@Exos

1. Preparation and characterization of TPP/p53

1. Experimental method

Dissolving (3-propylcarboxyl) -triphenylphosphine bromide in hot phosphate buffered saline (PBS, ph=6.2) to give a saturated TPP solution at a concentration of about 0.46 mg/mL; EDC and NHS (20:1, n/n) are added to activate the carboxyl of TPP, and the reaction is carried out for about 15min; adding p53 solution (0.6 mg/mL) into activated TPP solution (1:1000, n/n), reacting for 2h at room temperature, synthesizing TPP/p53; quenching EDC (1:1, n/n) by adding beta-mercaptoethanol for 5min; the reaction solution was transferred to a dialysis bag (1000 Da) and dialyzed against PBS dialysate for 24 hours.

By FT-IR Fourier infrared spectroscopy and ¹ H-NMR hydrogen spectrum detects the chemical bond change of TPP/p 53.The secondary structure of recombinant p53 was detected by circular dichroism. And scanning the maximum absorption peak of the TPP solution by using an ultraviolet spectrophotometer (UV-1800), drawing a standard curve of TPP by taking the TPP solutions with different concentrations as an abscissa and the maximum absorbance as an ordinate, and calculating the residual TPP content by analyzing the absorbance of the TPP/p53 dialysate so as to further calculate the crosslinking rate.

2. Experimental results

(1) FIG. 2 shows that the center of the-OH stretching vibration frequency in the infrared spectrum of p53 is at 3394cm ^-1 The center of the stretching vibration frequency of the-NH 2 is 2915cm ^-1 The stretching vibration frequency of the amide band is 2847cm ^-1 And 1647cm ^-1 Between them. Since TPP is slightly soluble in PBS, the characteristic peaks of low concentration TPP samples are weaker. In the infrared spectrum of TPP, the C=C stretching vibration frequency of the benzene ring is 1474cm ^-1 And 1561cm ^-1 The C=O stretching vibration center of the carboxyl is positioned at 1636cm ^-1 The telescopic vibration frequency of the benzene ring hydrogen-H is at 3326cm ^-1 And the frequency of stretching vibration of hydroxyl-OH (3374 cm) ^-1 ) And (5) overlapping. The infrared characteristic peak of TPP/p53 is also weaker due to low solute concentration. By careful comparison, benzenoid hydrogen-H, C =o and benzenoid c=c are red shifted compared to TPP, meaning enhanced instability of TPP/p53, and also meaning successful cross-linking of TPP with p53 protein.

(2) As shown in FIG. 3, TPP has 22 kinds of substituted hydrogens in total, 15 kinds of aromatic ring hydrogens (chemical shift between 7.5 and 8.5 ppm) in equal proportion, 3 kinds of alkane hydrogens (chemical shift between 2.5 and 4.5ppm, peak area is twice that of aromatic ring hydrogens, two kinds of hydrogens are very similar, and are seemingly overlapped) and one kind of carboxyl hydrogen. The remaining peaks may be due to sample purity problems. The reaction product TPP/p53 recombinant protein sample is dissolved in polar solution, and has hydrogen spectrum similar to TPP aromatic ring hydrogen (chemical shift between 7.0 and 8.0 ppm) and complex hydrogen spectrum of p53 protein (chemical shift between 0 and 4.5 ppm), and both peaks move to low shift region than TPP and p 53. In addition, a new peak appears at chemical shift 4.7ppm, presumably the newly formed amide hydrogen, indicating that the TPP and p53 proteins are not simply mixed but chemically reacted. FIG. 4 shows a circular dichroism spectrum of human p53 protein and TPP/p53 recombinant protein, both of which are negative peaks at 222nm and 208nm and positive peaks at 191nm, which are characteristic of the conformation in which an alpha-helix exists. The difference between the round two chromatograms of the TPP/p53 sample and the p53 sample is that the peaks of TPP/p53 become more sharp. In addition, the overall peak amplitude of TPP/p53 is lower than that of p53, which may be caused by the lower concentration of TPP/p53 recombinant protein. In conclusion, the secondary structure of TPP/p53 is somewhat altered from that of p53, but the alpha helix structure of p53 protein is generally retained.

(3) The experiment first measured the ultraviolet spectra of different concentrations of TPP solution, and the result shows that the maximum absorption peak of TPP is 267nm as shown in FIG. 5.

The absorbance of TPP solutions at 267nm, 0.008mg/mL, 0.010mg/mL, 0.012mg/mL, 0.014mg/mL, 0.016mg/mL, 0.018mg/mL, 0.020mg/mL, 0.022mg/mL, 0.024mg/mL, 0.026mg/mL, respectively, was then measured, and the results were shown in fig. 5, with the standard curve equation y=35.5x+0.198 for TPP at 267nm, r2= 0.99759. The absorbance of the TPP/p53 dialysate after the reaction was 0.704, and the residual TPP concentration in the solution was calculated to be 21.84. Mu.g/mL by substituting the above standard curve equation. The content of TPP in the pre-reaction solution was 1 mL. Times.0.46 mg/mL=460. Mu.g, the concentration of TPP in the post-reaction TPP/p53 dialysate was 21.84. Mu.g/mL, the content of TPP remaining in the reaction was 21.84. Mu.g/mL. Times.21 mL= 458.64. Mu.g, the content of TPP in the reaction was 460. Mu.g to 458.64. Mu.g=1.36. Mu.g (i.e., 3.17.times.10) ^-9 mol). The crosslinking rate of TPP and p53 was 1.36. Mu.g.460. Mu.g=0.30%. Since the content of p53 is 10. Mu.L.times.0.6 mg/mL=0.006 mg (i.e., 1.37.times.10) ^- ¹⁰ mol), whereby the reaction mass TPP is obtained: p53≡23:1, i.e. on average about 23 TPP molecules per p53 protein molecule are linked.

2. Purification and characterization of exosomes

1. Experimental method

Taking new-born calf serum, ultracentrifugating (at 4 ℃ C., 100,000Xg) for 90min, and collecting supernatant to obtain new-born calf serum without exosomes; DMEM high sugar medium containing 10% serum and 1640 medium containing 10% serum were prepared using exosome-free newborn Calf Serum (CS) at 37℃and 5% CO ₂ Culturing MCF-7 and SKBR-3 breast cancer cells in a constant temperature incubator; when the cell density reaches 80%,discarding the culture solution, and washing 3 times by using PBS; digesting with 0.25% trypsin for 2min to obtain single cell suspension, inoculating into 10 culture dishes with diameter of 10cm, and culturing to obtain 1×10 ⁷ A cell; when the cell densities of MCF-7 and SKBR-3 reach 80%, irradiating with 25W ultraviolet light for 30min (or subjecting the cells to heat shock treatment at 43 ℃ for 1 h), and stimulating the cells to secrete exosomes; 37 ℃,5% CO ₂ Incubating for 6 hours in a constant temperature incubator; collecting the cell culture supernatant, filtering with 220 μm microporous membrane, and collecting filtrate; the filtrate was transferred to an ultracentrifuge tube and ultracentrifuged (4 ℃,103,000Xg) for 12 hours; the supernatant was discarded, and the exosome pellet was resuspended using 200. Mu.L of PBS or electrode buffer to give MCF-7 and SK-BR-3 derived exosome suspensions, respectively, which were transferred to 250. Mu.L EP tubes and frozen at-80 ℃. (electrode buffer: 120mmol/L KCl;0.15mmol/L CaCl) ₂ ；10mmol/L K ₂ HPO ₄ ；25mmol/L H&EPES；2mmol/L EGTA；5mmol/L MgCl ₂ The method comprises the steps of carrying out a first treatment on the surface of the 2mmol/L ATP;5mmol/L RGD glutathione);

detecting a fluid diameter of the exosome using a dynamic light scatterometer; detecting the four membrane-spanning proteins CD63 and CD81 marked on the membrane of the exosomes by immunoblotting; exosomes were incubated with CD63 primary antibody (diluted to 1:1000), FITC secondary antibody (1:1000) fluorescently labeled, and the morphology of exosomes was observed under a fluorescence microscope.

2. Experimental results

(1) As shown in FIG. 6, immunoblotting experiments revealed that the purified exosome samples contained exosome marker proteins CD63 and CD81, with a molecular weight of 26kDa.

(2) As shown in FIG. 7, the exosomes were observed under a fluorescence microscope, in approximately spherical form, with a diameter of about 150nm.

3. Preparation and characterization of TPP/p53@Exos

1. Experimental method

Suspending the exosome by using electrode buffer solution, transferring to an electrode cup, applying 1kV voltage for 2ms, and evacuating the substances in the exosome; adding a small amount of DMEM (MCF-7 cell source) or 1640 (SK-BR-3 cell source) minimal medium into the electrode cup according to the cell source of the exosome for dilution, and incubating at 37 ℃ for 30min; purifying exosomes by ultracentrifugation again to obtain empty exosome suspension; according to 1: adding exosome suspension and p53 protein solution into the electrode cup according to the volume ratio of 1, and carrying out electroporation treatment according to the same parameters to load p53 protein into exosome; after re-incubation, the mixture was ultracentrifuged to obtain a TPP/p53@Exos suspension.

In order to detect the load rate of the recombinant protein, ultraviolet spectrum of TPP/p53 is detected by using ultraviolet-visible spectrophotometry, and the maximum absorption peak is determined to be 213nm; taking 3 mug/mL of TPP/p53 solution as mother solution; preparing solutions with mother solution contents of 100%, 75%, 50%, 25% and 0%, respectively, and detecting luminosity at maximum absorption wavelength; drawing a standard curve by taking the concentration of TPP/p53 as an abscissa and the absorbance as an ordinate; TPP/p53 (6. Mu.g/mL) and exosome 1 were then detected separately: 1 absorbance of the mixed solution at the maximum absorption wavelength before and after electroporation; and calculating the concentration of free TPP/p53 in the solution after the exosome electroporation is loaded with TPP/p53 according to a standard curve, and further calculating the loading amount of the protein.

Dynamic light scattering detection of TPP/p53@Exos fluid diameter; detecting the form of TPP/p53@Exos by a transmission electron microscope; the Cy5 fluorescent secondary antibody is used for marking the TPP/p53 recombinant protein, the FITC fluorescent secondary antibody is used for marking the CD63 protein on the surface of the exosome, and the exosome is observed under a fluorescence microscope.

2. Experimental results

(1) The particle sizes of the exosome sample, the empty exosome sample and the TPP/p53@Exos sample were measured using a dynamic light scattering particle sizer, respectively.

As shown in FIG. 8, the exosomes and empty exosomes have heterogeneity in particle size, mostly around 30nm and around 150nm, while the particle size uniformity of TPP/p53@Exos is strong, between 67nm, which is caused by fusion of part of exosomes by two electroporation.

(2) The morphology of the exosomes and the TPP/p53@exos samples were examined separately using transmission electron microscopy, as shown in fig. 9, the exosomes were spherical, approximately 100nm in diameter, and contained a small amount of microparticles inside. TPP/p53@Exos is spherical, has a diameter close to 100nm, and contains 6-7 spherical particles with uniform size in each exosome, which is the TPP/p53 recombinant protein.

(3) The p53 protein antibody was used as primary antibody, cy5 red fluorescent labeling was performed on TPP/p53, while FITC green fluorescent labeling was performed on CD63 protein on exosomes. As shown in fig. 10, in one field of view of the fluorescence microscope, using the green filter, the TPP/p53 recombinant protein labeled with red fluorescence Cy5 was observed, and switching the blue filter, the green fluorescence FITC-labeled exosomes in this region were observed, and from the covered image, it was confirmed that TPP/p53 was successfully loaded into exosomes.

(4) Absorbance at 213nm was measured before and after electroporation of the exosomes with the TPP/p53 cocktail using an ultraviolet spectrophotometer. As a result, as shown in FIG. 11, the ratio of the residual TPP/p53 which was not loaded was 26.65% of the initial content, and the ratio of the TPP/p53 loaded into the exosomes was 73.35% of the initial content, as compared with the standard curve of TPP/p53, as measured by an ultraviolet spectrophotometer.

Example 2 cellular uptake and mitochondrial targeting characterization of TPP/p53@Exos

1. Experimental method

Cellular uptake and mitochondrial targeted delivery of the nanopharmaceutical delivery system TPP/p53@exos was observed by immunofluorescence. Purification of the secreted exosomes of MCF-7 and SK-BR-3, TPP/p53@Exos were prepared separately. TPP/p53@Exos from two cell sources was applied to MCF-7 and SK-BR-3 cells, respectively, at 37℃with 5% CO ₂ Incubating for 2 hours in a constant temperature incubator; cells were fixed with pre-chilled 4% paraformaldehyde for 15min at room temperature; membrane permeation treatment is carried out for 5min by using TBS triethanolamine buffer saline solution; incubation with CD63 primary antibody (dilution 1:100), labeling exosomes, overnight at 4 ℃; incubation for 1h at room temperature using FITC fluorescent secondary antibody (dilution 1:1000); cells were incubated with DAPI dye (dilution 1:1000) at room temperature for 10min, and nuclei were stained; observation was performed using a fluorescence microscope.

Preparation of TPP/p53 recombinant protein labeled with Cy5 fluorescent secondary antibody

TPP/p53@Exos; the nano drug delivery system is acted on breast cancer cells at 37 ℃ and 5% CO ₂ Incubating for 2 hours in a constant temperature incubator; using Mito-the tracker Green mitochondrial activity fluorescent dye solution (20-200 nM) is used for incubating cells for 30min at 37 ℃ and marking mitochondria; and observing under a fluorescence microscope.

Particle size detection of TPP/p53@Exos samples using dynamic light scattering particle sizer

2. Experimental results

(1) The fluorescence microscope observation results are shown in FIG. 12, and it is known that the exosomes mExos and sExos derived from the MCF-7 and SK-BR-3 cell nuclei have stronger affinity to the breast cancer cells derived from them, as shown in FIG. 12, by labeling the marker protein CD81 on the exosome membrane with FITC fluorescent dye after each of the two breast cancer cell lines is acted on the two breast cancer cell lines for 2 hours.

(2) TPP/p53 recombinant protein labeled with Cy5 red fluorescence

TPP/p53, preparation->

After incubating two breast cancer cells for 2h, mitochondria are marked by using Mito-tracker Green mitochondrial active fluorescent dye, and the observation result of a fluorescent microscope is shown in fig. 13, and the red fluorescent marked TPP/p53 is positioned at the mitochondria of the cancer cells under one view of the fluorescent microscope, so that the TPP/p53@exos has good targeting effect on the mitochondria of the cells.

EXAMPLE 3 characterization of toxicity of TPP/p53@Exos to homologous cancer cells

1. Experimental method

(1) The CCK-8 assay was performed to examine the growth inhibition of TPP/p53@Exos on cancer cells. According to 1X 10 ⁴ Cell density MCF-7 (er+, pr+, HER 2-) cells were seeded on 96-well plates and incubated overnight; the free p53 protein, the empty exosome mExos derived from MCF-7, the TPP/p53 recombinant protein and the TPP/p53@mExos nano drug delivery system (equal p53 molar amount, 600 ng/hole) are respectively acted on MCF-7 cells for 24 hours; to examine the affinity effect of the nano-drug delivery system on homologous breast cancer cell lines, the p53 mutation was used simultaneouslyBreast cancer cell line SK-BR-3 (ER-, PR-, her2+) as control, the same treatment was applied using TPP/p53@preexos; the CCK-8 cell viability detection kit working solution is added for incubation, and an enzyme-labeled instrument (ThermoFisher MK 3) is used for measuring the absorbance of the cells at the wavelength of 450 nm.

(2) Detection of Ca in cancer cells Using Fluo-3 AM calcium ion Probe ²⁺ The content is as follows. According to 5X 10 ⁵ Cell density MCF-7 and SK-BR-3 cells were seeded into 96-well plates, respectively, and incubated overnight for 24h; the free p53 protein, the empty exosome homologous to cancer cells, the TPP/p53 recombinant protein and the TPP/p53@Exos nano-drug homologous to cancer cells (equal p53 molar amount, 600 ng) are respectively acted on the two cells, and incubated for 24 hours; fluo-3 AM (dilution 1:1000) working fluid was used at 37℃with 5% CO ₂ Incubating the cells in a constant temperature incubator for 45min; fluorescence intensity at excitation wavelength 488nm and emission wavelength 525nm was measured for each well using a multifunctional enzyme-labeled instrument (EnSpire).

(3) Cell scratch experiments were performed to examine the migration inhibition effect of TPP/p53@Exos on cancer cells. According to 5X 10 ⁵ SK-BR-3 cells were seeded into 6-well plates and incubated overnight; after the cell density reaches 80%, drawing 4-5 parallel lines in each hole by using the sterilized toothpick; washing away suspended cells, adding TPP/p53@Exos (the p53 protein content is 600 ng), and respectively incubating for 24h and 48h; scratch healing was observed using an optical microscope and the ImageJ software analyzed for scratch width.

2. Experimental results

(1) As shown in FIG. 14, the result of CCK-8 cell viability detection shows that p53 wild strain MCF-7 is obviously inhibited by TPP/p53@mExos compared with p53 mutant strain SK-BR-3, the inhibition rate of TPP/p53@mExos on homologous MCF-7 cells is as high as 79%, and the inhibition rate on non-homologous SK-BR-3 is 39%, so that the source of exosomes in the TPP/p53@Exos system has a key effect on killing cancer cells.

(2) Fluo-3 AM kit for detecting Ca ²⁺ The concentration of TPP/p53@Exos was shown in FIG. 15 to promote the same source of intracellular Ca in MCF-7 and SK-BR-3 breast cancer cells ²⁺ The content is obviously increased, and the pro-apoptosis effect is better than that of obvious free p53 protein, TPP/p53 recombinant protein and TPP/p53@Exos.

(3) The cell scratch experiment detects the effect of the nano particles TPP/p53@Exos on SK-BR-3 cell migration inhibition, and the result is shown in figure 16, wherein the migration inhibition rate of the TPP/p53@Exos on SK-BR-3 cells in 24 hours reaches 50%, and the inhibition rate in 48 hours reaches 59%.

Example 4 in vivo killing effect of TPP/p53@Exos on breast cancer tumor detection

1. Experimental method

The body weight and tumor volume of each group of mice was recorded every 2 days during the 28 days of in situ dosing. After the treatment, mice were sacrificed by spinal dislocation, tumors were dissected, and H & E stained and immunohistochemical analysis was performed as follows.

H & E staining: tumor tissue was fixed for 12h using Bouin fixative (saturated picric acid: formaldehyde: glacial acetic acid=12:5:1); soaking in 70%, 90%, 95% and 100% alcohol for 3-5 min to remove water in the tissue sample; placing the tissue sample into an alcohol xylene mixed solution and a xylene solution for transparent treatment for 10min respectively; the xylene on the surface of the tissue sample is sucked by filter paper, and the sample is sequentially placed in liquid pure waxes I, II and III in an oven for wax permeation treatment for 1h respectively; pouring molten wax into a thin paper box with proper size, embedding a tissue sample in the molten wax, placing the paper box on water, and reducing the temperature to solidify the molten wax; trimming the wax block into cubes with proper size by using a blade, melting and fixing the bottom of the cube on a metal wax holder, and slicing by using a rotary slicing machine; taking a clean glass slide, smearing protein glycerol, dripping distilled water, placing a sample slice on the clean glass slide, baking at a high temperature to melt the wax slice, transferring the wax slice into an oven, and drying at 37 ℃; soaking in xylene for 15-20 min and in mixed solution of xylene and alcohol for 5min; soaking in 100%, 95%, 80% and 70% alcohol for 2-3 min, and soaking in distilled water for 2min; hematoxylin dye staining for 15-20 min; washing with tap water slowly for 15min until the slice turns blue, dripping acidic solution until the slice turns light red, and washing with tap water to recover blue; soaking and dehydrating with 70% alcohol and 80% alcohol successively; dyeing for 3-5 min by using 95% eosin dye; soaking the mixture in xylene for 3 to 5 minutes; the filter paper absorbs the xylene on the slice, adds neutral gum dropwise, covers the cover glass for sealing, and dries in a drying oven at 37 ℃; the samples were observed using a microscope.

Immunohistochemistry: tumor tissues were successively subjected to paraffin embedding, sectioning, dewaxing, hydration, endogenous blocking, antigen recovery, followed by incubation with Bax, bcl-2, caspase-3 and Ki-67 polyclonal antibodies for 2h at room temperature, followed by incubation with biotin-labeled secondary antibodies for 2h. Horseradish peroxidase-streptavidin (HRP-SA) working solution was added for Diaminobenzidine (DAB) development. Subsequently, nuclei were stained using hematoxylin. And (5) microscopic imaging.

2. Experimental results

(1) The tumor volumes of the control and exosome treated groups increased significantly over the 28 day period of in situ dosing treatment with normal and no significant difference in weight (fig. 18), with smaller increases in tumor volumes of the p53 and TPP/p53 treated groups and decreased tumor volumes of the TPP/p53@tpexos treated groups (fig. 17, 19). The final tumor volume of TPP/p53@tExos was 50mm on average ³ The inhibition rate in vitro is 94% compared with 17 times of the control group.

(2) After 28 days of in situ administration, the tumors of each group of mice were dissected and H & E stained, and the apoptosis phenomenon of tumor cells treated by TPP/p53@tExos nano-drug was more remarkable, and the phenomena of chromosome aggregation, deepening of chromatin staining and the like occurred in cell nuclei (FIG. 20).

(3) As shown in FIG. 21, the tumor tissue of the TPP/p53@tExos treated group was severely damaged, and the expression of the pro-apoptotic proteins Bax and Caspase-3 was significantly higher than that of the control group, while the levels of the anti-apoptotic proteins Bcl-2 and Ki-67 were significantly lower than those of the control group, indicating that TPP/53@tExos had strong apoptosis-inducing ability and tumor cell metastasis-inhibiting ability.

Example 5 detection of toxic side effects of TPP/p53@Exo

1. Experimental method

After 28 days of in situ administration, mice were sacrificed by spinal dislocation, groups of mice were dissected for heart, liver, spleen, lung and kidney, and H & E staining was performed. In addition, the blood index of the mice is detected, and the specific method is as follows: blood of each group of mice is extracted from tail vein, EDTA-2Na is added to prepare anticoagulation, and the contents and proportion of leucocytes, lymphocytes, monocytes, neutrophils, erythrocytes and the like are measured to obtain a plurality of blood indexes. Various experimental results

(1) As shown in FIG. 22, the H & E staining results showed no obvious difference in morphology of heart, liver, spleen, lung and kidney between mice in the normal group, the control group and the TPP/p53@tExos treatment group, indicating that the nanoparticles did not cause damage to heart, liver, spleen, lung and kidney.

(2) Blood routine tests were further performed on the normal, control and TPP/p53@tExos treatment groups 28 days after treatment, and the results are shown in FIG. 23. From the reference values for biochemical detection of the blood of mice, no significant changes were found between normal mice, mice given normal saline and TPP/p53@tExos. Evaluation of the numbers of leukocytes, platelets, erythrocytes in the nanoparticle treated group showed that the numbers of leukocytes, platelets, etc. that caused the inflammatory response were also within the normal range.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.

Claims

1. The preparation method of the protein nano-drug for targeted cancer treatment is characterized by comprising the following steps:

covalently crosslinking triphenylphosphine containing carboxyl functional groups with human p53 protein to obtain recombinant protein TPP/p53,

extracting exosomes by adopting an ultracentrifugation technology, wherein the ultracentrifugation technology is 0-4 ℃,100,000-150,000Xg ultracentrifugation for 1.5-12 h;

transferring the prepared recombinant protein TPP/p53 into an extracted exosome;

the cancer is breast cancer, and the exosomes are extracted from a breast cancer cell culture supernatant stimulated by ultraviolet irradiation;

transferring the prepared recombinant protein TPP/p53 into an extracted exosome by using an electrotransformation technology;

Wherein, the prepared recombinant protein TPP/p53 is transferred into the extracted exosome by electrotransformation technology to specifically operate as follows:

suspending the exosome by using electrode buffer solution, carrying out electroporation treatment to empty the substances in the exosome,

adding culture medium for dilution, incubating,

purifying exosomes by ultracentrifugation to obtain empty exosome suspension,

mixing the empty exosome suspension with a recombinant protein TPP/p53 solution, and carrying out electroporation treatment; incubating, and purifying by ultracentrifugation,

the condition of the electroporation treatment is that 0.5-1 kV voltage is applied for 2-5 ms,

the incubation is carried out for 10-30 min at 30-40 ℃,

the ultracentrifugation purification is 0-4 ℃, 100-150, 000 Xg ultracentrifugation for 1.5-12 h;

the method for covalently crosslinking the triphenylphosphine containing carboxyl functional groups and the humanized p53 protein comprises the following steps:

adding 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and N-hydroxysuccinimide into saturated phosphate solution of triphenylphosphine containing carboxyl functional groups, activating carboxyl groups of triphenylphosphine containing carboxyl functional groups,

adding beta-mercaptoethanol to quench EDC, mixing p53 solution with activated TPP solution, reacting for 1-4 h, purifying,

the carboxyl reaction time of the activated triphenylphosphine is 10-20 min,

The ratio of the amounts of the substances of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the N-hydroxysuccinimide is 4-8:1,

the ratio of the amounts of the substances of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride and the triphenylphosphine is 10-20:1,

the concentration of the p53 solution is 0.2-1 mg/mL,

the ratio of the amount of p53 solution to the amount of substance of activated TPP is 1:100-2000,

the ratio of the amount of beta-mercaptoethanol to the amount of the substance of the 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride is 1-2:1,

and mixing the beta-mercaptoethanol solution with the activated triphenylphosphine solution, and reacting for 3-10 min.

2. The protein nano-drug for cancer targeted therapy prepared by the preparation method of claim 1.

3. The use of the protein nano-drug for cancer targeted therapy according to claim 2 for preparing a cancer targeted therapeutic agent.