CN112156189B - HER2+ breast cancer targeted protein composite nanoparticle and preparation method and application thereof - Google Patents

HER2+ breast cancer targeted protein composite nanoparticle and preparation method and application thereof Download PDF

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CN112156189B
CN112156189B CN202010681821.1A CN202010681821A CN112156189B CN 112156189 B CN112156189 B CN 112156189B CN 202010681821 A CN202010681821 A CN 202010681821A CN 112156189 B CN112156189 B CN 112156189B
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关燕清
张令坤
唐云志
焦宇萱
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South China Normal University
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Abstract

The invention discloses a HER2+ breast cancer targeted protein composite nanoparticle and a preparation method and application thereof, wherein the HER2+ breast cancer targeted protein composite nanoparticle is a micelle loaded with a compound formed by crosslinking an ER antibody and a recombinant p53 protein, and the micelle is formed by activating the HER2 antibody, covalently crosslinking phycocyanin and then crosslinking the activated HER2 antibody and paclitaxel through an ester bond. The P53 composite protein nanoparticles successfully synthesized by the invention can enter HER2 positive breast cancer cells in a targeted manner, the concentration of P53 protein closely related to the apoptosis process in the breast cancer cells is increased, the condition that the expression quantity of related protein is too low due to P53 gene mutation is changed, the problem of low expression quantity of exogenous genes in cancer cells in gene therapy is solved, and the problems of canceration, metastasis and the like of the cells due to P53 gene mutation are solved.

Description

HER2+ breast cancer targeted protein composite nanoparticle and preparation method and application thereof
Technical Field
The invention relates to the technical field of biomedicine, in particular to HER2+ breast cancer targeted protein composite nanoparticles and a preparation method and application thereof.
Background
The worldwide cancer analysis report of 2018-2020 indicates that the incidence of breast cancer is always the first malignant tumor of women worldwide and is the first cause of death of women of 40-55 years old. Currently, breast cancer treatment is mainly focused on surgical treatment, chemotherapy, radiotherapy, endocrine treatment, molecular targeted therapy and the like. Although the treatment method is more perfect, due to the heterogeneity of the breast cancer, 20-30% of the breast cancer highly express HER2 protein, and the problems of tumor drug resistance, relapse, metastasis and the like exist to different degrees after treatment. Therefore, finding a suitable method to solve the problems of tumor drug resistance, metastasis and the like is a hot spot of current research and a difficult point to overcome.
HER2 positive breast cancer patients have poor outcome and poor prognosis for both endocrine and standardized therapies, with survival rates that are only half that of HER2 negative breast cancer patients. At present, clinically, the medicines for HER-2 positive patients mainly comprise trastuzumab, pertuzumab, lapatinib and the like. A plurality of clinical trials comprising NSABP B-31, NCCTG N9831, HERA, BCIRG006, finHer, PACS04 and the like prove that in an anthracycline taxane adjuvant therapy scheme, the combination of trastuzumab can further reduce the relapse risk of patients. The antibody in the targeted therapy can utilize a specific target antigen to identify tumor cells, and because the targeted therapy has good targeting property and better clinical curative effect, the targeted drug becomes a research hotspot in the field of biological pharmacy in recent years. However, the antibody drugs currently used in clinical practice have many disadvantages, such as that intact antibody molecules have poor penetration due to their large molecular weight and are difficult to penetrate through connective tissue to reach solid tumor sites; the clinical dosage is large, the production cost is high, and most of the drugs have strong side effects. Trastuzumab administered to patients with advanced breast cancer, when administered alone, has adverse effects in more than 40% of patients, with approximately 70% of patients developing drug resistance (Nielsen FC, hansen VO,
Figure GDA0002810490730000011
CS.Nat Rev Cancer 2016,16:599-621；MillisSZ,Gatalica Z,Winkler J,et al.Clin Breast Cancer 2015,15(6):473-481；Nixon N,Verma S.A value-based approach to treatment of HER2-positive breast cancer:examining the evidence.Am Soc Clin Oncol Educ Book 2016；36:e56–63；von Minckwitz G,Procter M,de Azambuja E,et al.Adjuvant pertuzumab and tras-tuzumab in early HER2-positive breast cancer.N Engl J Med 2017；377(2):122–31)。
the targeted drug delivery system can deliver drugs to target organs and target cells, so that the drugs at the pathological change part are highly concentrated, the treatment effect is improved to the maximum extent, and the targeted drug delivery system is considered to be an optimum anticancer preparation, thereby being highly regarded by the medical field of various countries. Aiming at the research fields of antibody-mediated targeting, microcarrier-mediated targeting, breast cancer stem cell targeting and the like, the method lays a theoretical foundation for the research of breast cancer treatment. The targeted drug selectively kills tumor cells depending on target molecules, and is safer and more efficient than cytotoxic drugs. It should be noted that tumor survival does not depend on a single target, but only one target is inhibited, and the tumor may still have metastasis and recurrence, so the multi-target effect will be the development trend of targeted drugs.
The TP53 oncogene is the most common mutant gene in all cancer types, accounting for approximately 30% of all breast cancers. Under normal conditions, intracellular p53 protein is maintained at a very low level, and under various stresses (such as DNA damage, oncogene activation, etc.), p53 protein undergoes post-translational modification, resulting in its activation, stabilization and accumulation in the cell. When tumors occur, p53 is activated in a large amount to induce the up-regulation or down-regulation of target genes, and further cell cycle arrest, DNA repair, aging or apoptosis occur, so that the p53 and the compound thereof are taken as targets, and the method becomes a research hotspot for developing new anti-cancer drugs at present. Mutation of the P53 gene in breast cancer mostly occurs in the DNA binding domain, resulting in the inhibition of P53 protein synthesis, further causing the disturbance of cell division cycle and apoptosis, and the cell is transformed into an immortalized state.
Combination therapy based on intelligent cancer nanomedicine. Integration of the nano-drug into a multi-modal chemotherapy regimen can reduce side effects, improve patient compliance and improve quality of life; by apportioning multiple drugs, synergistic drug action is promoted by improved control over pharmacokinetic and pharmacodynamic interactions; co-treatment with approved anti-cancer drugs helps to enhance the delivery efficiency of the vascular and tumor microenvironment, thus enhancing the accumulation, penetration and efficacy of cancer nano-drugs. Local combination: the tumor microenvironment is changed through radiotherapy and ultrasonic treatment to increase the blood vessel perfusion and permeability and improve the accumulation, permeability, retention and efficacy of the cancer nano-drugs; hyperthermia can be used to locally trigger the release of a payload of a temperature sensitive liposome, resulting in an increase in drug. The design of small, highly efficient and low-toxic target drugs has become a new trend in antibody drug development.
In summary, conventional cancer therapies today have limitations in improving breast cancer survival and reducing poor patient prognosis. Targeted therapy, photodynamic therapy and protein drug therapy have become the treatment of choice for breast cancer as emerging therapeutic approaches. The nano micelle as a nano carrier can be developed and used for protecting and targeted delivery of protein drugs.
Disclosure of Invention
The invention aims to provide HER2+ breast cancer targeted protein composite nanoparticles and a preparation method and application thereof.
The first purpose of the invention is to provide a HER2+ breast cancer targeted protein composite nanoparticle.
The second purpose of the invention is to provide a preparation method of HER2+ breast cancer targeted protein composite nanoparticles.
The third purpose of the invention is to provide the HER2+ breast cancer targeted protein composite nanoparticle prepared by any one of the preparation methods.
The fourth purpose of the invention is to provide the application of the HER2+ breast cancer targeted protein composite nanoparticle in preparing a medicament for treating breast cancer.
The above object of the present invention is achieved by the following scheme:
in the invention, a heterobifunctional cross-linking agent SPDP is utilized to introduce a pyridine disulfide group into a free amino group of a p53 protein molecule, and the pyridine disulfide group is reduced into a sulfhydryl group under the action of DTT so as to form disulfide bond to be cross-linked with an antibody anti-ER of an estrogen receptor; firstly, taking photosensitizer phycocyanin and chemotherapeutic drug paclitaxel as substrates, synthesizing a compound with one hydrophilic end (phycocyanin end) and one hydrophobic end (paclitaxel end) through chemical catalysis, modifying an antibody anti-HER2 of a human epidermal growth factor receptor 2 on phycocyanin, and synthesizing PTC/PC/anti-HER2; then, foreign p53 protein covalently bound by anti-ER is loaded, and the final nano protein complex p53/anti-ER @ PTX/PC/anti-HER2 is synthesized. The protein nano-composite with the effect of inhibiting the proliferation of the breast cancer cells is delivered into the tumor cells through an intravenous injection administration mode and the targeting effect of anti-HER2 protein molecules. The programmed death of the breast cancer cell is induced by improving the expression quantity of apoptosis-related protein in the breast cancer cell, thereby achieving the effect of efficiently inhibiting the growth of the cancer cell. Experiments prove that the drug delivery system has low toxic and side effects and obvious inhibition effect on the growth of cancer cells, and provides a drug synthesis concept of crosslinking drug molecules and different targeting protein molecules to overcome tumor heterogeneity, and the self-synthesized protein nano-composite is a novel multi-targeting drug delivery system.
Therefore, the invention claims a HER2+ breast cancer targeted protein composite nanoparticle, wherein the targeted protein composite nanoparticle is a protein composite nanoparticle loaded with a protein composite p53/anti-ER in a PTX/PC/anti-HER2 micelle; wherein, the protein compound p53/anti-ER is a protein compound obtained by cross-linking an apoptosis-related protein p53 and an estrogen receptor antibody anti-ER by disulfide bonds;
the PTX/PC/anti-HER2 micelle is a protein compound obtained by crosslinking phycocyanin PC and an antibody anti-HER2 of a human epidermal growth factor receptor 2 through amido bonds, wherein the phycocyanin PC in the protein compound PC/anti-HER2 is crosslinked with paclitaxel PTX through ester bonds, and the protein compound is obtained by crosslinking the phycocyanin PC/paclitaxel compound/the antibody PTX/PC/anti-HER2 of the human epidermal growth factor receptor 2 through ester bonds to form the micelle PTX/PC/anti-HER2.
Paclitaxel (PTX) is the most widely used anticancer drug and is used to treat various types of malignant diseases. Paclitaxel (PTX) is a class of taxanes, an antineoplastic drug that affects microtubule stability, and is a widely used chemotherapeutic drug in a wide variety of cancers. The paclitaxel is widely used for treating breast cancer, ovarian cancer, partial head and neck cancer and lung cancer in clinic, and the current obstacle to the treatment of the cancer by using the paclitaxel is not the problem of the effectiveness of the paclitaxel but the toxicity problem of the paclitaxel. PTX is a major anticancer chemotherapeutic drug and its use faces global challenges of reducing side effects and improving drug efficacy. The development of drug resistance in patients is also a major obstacle during clinical treatment, and drug combination is a common method for treating drug resistance. At present, various paclitaxel drugs are on the market, such as taxol, paclitaxel injection, paclitaxel microemulsion injection, paclitaxel polymer micelle for injection and the like.
Phycocyanin is a bioactive compound extracted from algae such as spirulina with the relative molecular mass of 30kDa, is in a hollow tooth margin disc-shaped conformation, has photosensitive characteristics possibly related to the structure of an open-chain tetrapyrrole compound in a molecular structure, and has the advantages of antitumor activity, safety, no toxicity and no immunogenicity. Phycocyanin can also remove free radicals of damaged nerve cells, and avoid DNA oxidative damage, thereby preventing nerve cell apoptosis caused by free radicals. Gantar used 10% of the conventional dose of topotecan and phycocyanin in combination to treat prostate cancer (LNCa P) and the results showed that the effect of the combination was much higher than the effect of either alone. The combined use of phycocyanin and topotecan activates the expression activities of Caspase 9 and Caspase-3, increases the expression level of oxygen free Radicals (ROS), induces the apoptosis of tumor cells, and reduces the side effects of topotecan when used alone.
The invention also provides a preparation method of the HER2+ breast cancer targeted protein composite nanoparticle, which comprises the following steps:
s1, phycocyanin PC and an antibody anti-HER2 of a human epidermal growth factor receptor 2 are crosslinked by an amido bond to obtain a hydrophilic protein compound PC/anti-HER2;
s2, crosslinking paclitaxel PTX and phycocyanin PC through ester bonds to obtain a protein complex phycocyanin/paclitaxel complex/human epidermal growth factor receptor 2 antibody PTX/PC/anti-HER2, and self-assembling to form micelle PTX/PC/anti-HER2;
s3, cross-linking the apoptosis-related protein p53 and an antibody anti-ER of an estrogen receptor by using a disulfide bond to obtain a protein compound p53/anti-ER;
s4, dispersing the micelle PTX/PC/anti-HER2, mixing the dispersed micelle PTX/PC/anti-HER2 with the protein complex p53/anti-ER, and loading the protein complex p53/anti-ER into the PTX/PC/anti-HER2 micelle so as to obtain the final nanoparticles p53/anti-ER @ PTX/PC/anti-HER2.
The invention improves the concentration of the P53 protein in the cancer cell by giving the exogenous human P53 protein, and solves the problems of the reduction of the expression quantity of the pro-apoptotic protein P53 and the like caused by P53 gene mutation. The increase of the concentration of the P53 protein can promote the synthesis of other action proteins of an apoptosis pathway or block the progress of a cell cycle, thereby accelerating the apoptosis process of cancer cells or inhibiting the cell proliferation of the cancer cells.
Preferably, in step S1, catalysts NHS and EDC catalyze phycocyanin PC to crosslink with an antibody anti-HER2 of human epidermal growth factor receptor 2 through amide bonds under the catalysis of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride.
More preferably, the method comprises the following steps:
s11, carrying out light-shielding reaction on EDC, NHS and PC in a PBS buffer solution;
s12, reacting the reaction solution with an HER2 antibody in a dark state;
s13, freeze drying and storing.
Further preferably, in step S11, the molar ratio of EDC, NHS and PC is 5 to 2:5 to 2:3 to 1.
Still more preferably, in step S11, the molar ratio of EDC, NHS and PC is 2:2:1.
more preferably, in step S11, the reaction is carried out for 30 to 60min in the absence of light.
Still more preferably, in step S11, the reaction is carried out for 30min in the absence of light.
More preferably, in step S12, the molar ratio of phycocyanin to HER2 antibody is 1: 10-1:
more preferably, in step S12, the molar ratio of phycocyanin to HER2 antibody is 100:1.
more preferably, in step S12, the reaction is carried out at 0-37 ℃ in the absence of light and with a magnetic stirrer of 500-3000 rmp for 12-48 h.
Still more preferably, in step S12, the reaction is carried out at 4 ℃ in the absence of light and with a magnetic stirrer 1000rmp for 24h.
Further preferably, in step S13, the mixture is freeze-dried and stored at 4 ℃.
Preferably, in step S2, catalysts dicyclohexylcarbodiimide/4-dimethylaminopyridine DCC and 4-dimethylaminopyridine DMAP are used to catalyze the ester bond crosslinking of paclitaxel PTX and phycocyanin PC.
More preferably, the method comprises the following steps:
s21, mixing a DMSO solution of a protein compound PC/anti-HER2 with dicyclohexylcarbodiimide DCC and 4-dimethylaminopyridine DMAP for light-resistant reaction;
s22, mixing the mixture with paclitaxel for reaction, and reacting in a dark place;
and S23, freeze drying and storing.
Further preferably, in step S21, the molar ratio of PC/anti-HER2, DCC and DMAP is 5 to 1:3 to 1:3 to 1.
Still more preferably, in step S21, the molar ratio PC/anti-HER2, DMAP for DCC is 3:3:1.
more preferably, in step S21, the reaction is carried out for 30 to 60min in the absence of light.
Still more preferably, in step S21, the reaction is carried out for 30min in the absence of light.
Further preferably, PC/anti-HER2: the molar ratio of PTX is 10-1.
Still more preferably, in step S22, PC/anti-HER2: the molar ratio of PTX is 1:3.
more preferably, in step S21, the reaction is carried out for 10 to 60min under protection from light.
Still more preferably, in step S21, the reaction is carried out for 30min in the absence of light.
More preferably, in step S23, the reaction is carried out with a magnetic stirrer at 1000 to 3000rmp and at 0 to 37 ℃ for 12 to 48 hours in the absence of light.
Still more preferably, in step S23, a magnetic stirrer 1800rmp,4 ℃ is protected from light for 24 hours.
Preferably, in step S3, the apoptosis-related protein p53 is chemocatalytically cross-linked with an antibody anti-ER of an estrogen receptor with disulfide bonds using catalysts of N-hydroxysuccinimide 3- (2-pyridinedimercapto) propionate SPDP and dithiothreitol DTT.
The invention realizes the cross-linking of two proteins by forming a disulfide bond between a P53 protein and an ER antibody by a heterogeneous functional group cross-linking agent SPDP. The protein closely related to the apoptosis path is applied to the targeted drug, and the design idea of the targeted drug is widened. The synthesized protein particles can specifically target HER2 positive breast cancer cells to realize accurate drug delivery, and disulfide bonds formed by crosslinking are easy to specifically degrade in the cells, so that the action protein is released, and a good treatment effect is achieved.
More preferably, the method comprises the following steps:
s31, mixing and reacting SPDP ethanol solution with p53 protein, and concentrating;
s32, reacting the product obtained in the previous step with a DTT solution, and concentrating;
s33, mixing the SPDP ethanol solution with ER antibody protein for reaction, and concentrating;
and S34, uniformly mixing the product obtained in the step S32 and the product obtained in the step S33 at room temperature, reacting, and concentrating.
Further preferably, in step S31, the mass ratio of SPDP to p53 protein is 10 to 5.
Still more preferably, in step S31, the mass ratio of SPDP to p53 protein is 8.
Further preferably, in step S31, the reaction is carried out at room temperature for 10 to 60min.
Still more preferably, in step S31, the reaction is carried out at room temperature for 30min.
Further preferably, in step S32, the mass ratio of the DTT to the p53 protein is 1000 to 100:10 to 1.
Still more preferably, in step S32, the mass of the DTT and p53 proteins is 500:1.
further preferably, in step S32, the reaction is carried out at room temperature for 10 to 60min.
Still more preferably, in step S32, the reaction is carried out at room temperature for 30min.
Further preferably, in step S33, the mass ratio of SPDP to ER antibody protein is 10 to 5.
Still more preferably, in step S33, the mass ratio of SPDP to ER antibody protein is 8.
More preferably, in step S33, the reaction is carried out at room temperature for 10 to 60min.
Still more preferably, in step S33, the reaction is carried out at room temperature for 30min.
Further preferably, in step S34, the molar ratio of the p53 protein to the ER antibody protein is 10 to 1
Still more preferably, in step S34, the p53 protein and the ER antibody protein are used in a molar ratio of 1.
More preferably, in step S34, the reaction is carried out at room temperature for 10 to 60min
Still more preferably, in step S34, the reaction is carried out at room temperature for 30min.
More preferably, the concentration is performed by using PEG20000 as dialysate, dialyzing at 0-37 deg.C for 12-48 h, and replacing the dialysate 1-5 times.
Still more preferably, the concentration is performed using PEG20000 as dialysate, dialyzing at 4 ℃ for 24h, and changing the dialysate 3 times.
Preferably, in step S4, the micelles PTX/PC/anti-HER2 are broken up by ultrasound.
More preferably, the method comprises the following steps:
s41, dispersing the PBS buffer solution of micelle PTX/PC/anti-HER2 by using an acoustic disperser;
and S42, mixing the product of the previous step with the product of the step S3.
More preferably, the concentration of the micelle PTX/PC/anti-HER2 in the PBS buffer solution of the micelle PTX/PC/anti-HER2 is 0.1 to 0.5 μ g/mL, the dispersed micelle PTX/PC/anti-HER2 is mixed with the product of the step S3, and the volume ratio of the two needles is 5 to 1:5 to 1.
Still further preferably, the concentration of micelle PTX/PC/anti-HER2 in the PBS buffer solution of micelle PTX/PC/anti-HER2 is 0.125 μ g/mL, the micelle PTX/PC/anti-HER2 after being sprinkled is mixed with the product of step S3, and the volume ratio of the two needles is 1:1.
even more preferably, the ultrasonic disperser breaks up 5 to 30S at 50 to 200W.
Still further preferably, the ultrasonic disperser breaks up 20S at 100W.
More preferably, shaking and mixing for 5-60 min at room temperature
Still more preferably, shaking and mixing at room temperature for 30min
The HER2+ breast cancer targeted protein composite nanoparticle prepared by any one of the preparation methods also belongs to the protection scope of the invention.
The application of the HER2+ breast cancer targeted protein composite nanoparticle in preparing the medicine for treating breast cancer also belongs to the protection scope of the invention.
Preferably, the medicament improves the expression level of apoptosis-related protein in the breast cancer cells to induce programmed death of the breast cancer cells, thereby achieving the effect of efficiently inhibiting the growth of the cancer cells.
Preferably, the medicament is for a HER2 positive breast cancer patient.
Compared with the prior art, the invention has the following beneficial effects:
the invention is connected with an ER antibody through exogenously synthesized p53 protein, and the PTX/PC/anti-HER2 micelle loads the protein composite nanoparticles to target breast cancer cells positive for HER2 under the condition of ensuring the biological activity of the protein composite nanoparticles. After the synthesized p53 composite protein nanoparticles enter HER2 positive breast cancer cells in a targeted manner, the concentration of p53 protein closely related to the apoptosis process in the breast cancer cells is increased, and the problem of low expression level of exogenous genes in the cancer cells in gene therapy is solved. Meanwhile, the protein composite nanoparticles can promote the expression of other proteins in an apoptosis pathway, promote the apoptosis of cancer cells, inhibit the growth and proliferation of heterogeneous breast cancer cells, and solve the problems of canceration, metastasis and the like of the cells caused by P53 gene mutation to a certain extent. On the other hand, paclitaxel has a good effect in killing cancer cells, and for rapidly dividing tumor cells, paclitaxel freezes mitotic spindles, so that the tumor cells stop in the G2 phase and the M phase until death; paclitaxel also acts on Tumor Necrosis Factor (TNF) receptors on macrophages, and promotes the release of Interleukin (IL) -1, TNF-2, etc., which kills or inhibits the migration of tumor cells.
The P53 composite protein nanoparticles successfully synthesized by the invention can enter HER2 positive breast cancer cells in a targeted manner, the concentration of P53 protein closely related to the apoptosis process in the breast cancer cells is increased, the condition that the expression quantity of related protein is too small due to P53 gene mutation is changed, the problem of low expression quantity of exogenous genes in cancer cells in gene therapy is solved, and the problems of canceration, transfer and the like of the cells due to P53 gene mutation are solved.
Drawings
FIG. 1 is a schematic diagram of the synthesis of p53/anti-ER @ PTX/PC/anti-HER2 protein nanocomposite.
FIG. 2 shows the Critical Micelle Concentration (CMC) of PTX/PC/anti-HER2.
FIG. 3 is a transmission scan of PTX/PC/anti-HER2 micelles.
Fig. 4 shows the results of dynamic light scattering.
FIG. 5 shows the result of H-nuclear magnetic resonance analysis by PTX/PC.
FIG. 6 is a graph showing the effect of different concentrations of p53 nanoparticles on breast cancer MCF7 cells.
FIG. 7 shows the cell viability of CCK-8 after 24h of drug action.
FIG. 8 shows the DAPI staining and nuclear targeting results.
FIG. 9 shows that nano-drugs inhibit the migration of SK-BR-3 cells.
FIG. 10 shows the body weight change of mice.
FIG. 11 is the change in tumor size after drug treatment.
FIG. 12 is the analysis of the effect of natural nano-micelle complex in vivo in inhibiting breast cancer in mouse model.
Figure 13 immunohistochemical detection of Ki67 and BAX expression in tumor tissues: a saline control group; p53 group; PTX/PC group; p53/anti-ER @ PTX/PC/anti-HER2 group.
FIG. 14 shows the measurement of the blood standard index 28 days after the mice are treated.
Detailed Description
The present invention is further described in detail below with reference to specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
1. Cell line
The human breast cancer cell (MCF-7 cell line) is provided by Shenzhen advanced technology research institute of Chinese academy of sciences and is subcultured in the laboratory.
The SK-BR-3 cell line and the 4T1 cell line are provided by the cell resource center of Shanghai bioscience research institute and are subcultured in the laboratory.
2. Main reagent and instrument
p53 protein, phycocyanin, paclitaxel, antibody HER2, antibody ER, SPDP [ N-succinimidyl 3- (2-pyridyldithio) propionate ], DTT (dithiothreitol), PBS phosphate buffer, DMSO (dimethyl sulfoxide), DCC (dicyclohexylcarbodiimide), DMAP (4-dimethylaminopyridine), dialysis bag (500K, 1000K)
Pancreatin and low-sugar DMEM culture media are products of GIBCOBRL company; newborn calf serum is purchased from Hangzhou ilex chinensis bioengineering materials, inc.; the 96-well polystyrene tissue culture substrate is a product of Corning corporation, usa.
Nikon microscope, japan Olympus optical inverted microscope, sigma32184 high speed refrigerated centrifuge, thermo CO2 incubator, jiangsu province gold jar city medical instrument factory 78-1 magnetic stirrer, HV-85 high pressure sterilizer, sterile console, guangzhou Keqiao experimental technology equipment Limited constant temperature water bath pot, etc.
Example 1 preparation of PTX/PC/anti-HER2@ p53/anti-ER protein nanocomposite
1. And (3) catalyzing the photosensitizer Phycocyanin (PC) and an antibody (anti-HER 2) of a human epidermal growth factor receptor 2 to be crosslinked by amide bonds by using a catalyst 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide (NHS/EDC), so as to obtain a hydrophilic protein compound PC/anti-HER2.
The specific operation is as follows:
weighing 22.9mg (120. Mu. Mol) of EDC, 22.8 mg (120. Mu. Mol) of NHS13.8mg (120. Mu. Mol) of PC and 35.16mg (60. Mu. Mol) of PC according to the molar ratio of EDC/NHS =1, EDC =1/2, adding into 10ml of PBS buffer solution, reacting for 30min in the dark, slowly dripping 10. Mu.l of HER2 antibody (0.6. Mu. Mol) after phycocyanin activation, and reacting for 24h under the conditions of 4 ℃, dark and magnetic stirring for 1000 rmp; freeze-drying, storing in a refrigerator at 4 deg.C, and preparing for subsequent experiment.
2. Then, a catalyst dicyclohexylcarbodiimide/4-dimethylaminopyridine (DCC/DMAP) is used for catalyzing hydrophobic chemotherapeutic drug Paclitaxel (PTX) and Phycocyanin (PC) to be crosslinked in an ester bond manner, so that a phycocyanin/paclitaxel compound PTX/PC/anti-HER2 with one hydrophilic end and one hydrophobic end is obtained, and the compound can self-assemble to form micelles under critical concentration.
The specific operation is as follows:
weighing 58.64mg (9 mu mol) of PC/anti-HER2 solid, adding the solid into 3ml DMSO, fully dissolving the solid, adding 1.85mg (9 mu mol) of DCC and 0.37mg (3 mu mol) of DMAP, reacting at the dark room temperature for 30min, adding 25.59mg (30 mu mol) of paclitaxel PTX after activating phycocyanin carboxyl, reacting at the dark temperature of 4 ℃ on a magnetic stirrer (1800 rmp) for 24h, freezing and drying, storing in a refrigerator at the temperature of 4 ℃, and preparing for subsequent experiments.
3. The catalyst 3- (2-pyridinedimercapto) propionic acid N-hydroxysuccinimide ester/dithiothreitol (SPDP/DTT) is used for chemocatalysis of the crosslinking of the apoptosis-related protein p53 and an antibody (anti-ER) of an estrogen receptor by a disulfide bond to obtain a protein complex p53/anti-ER.
The specific operation is as follows:
dissolving SPDP in absolute ethanol to prepare a 1mg/mL SPDP ethanol solution, and mixing the solution according to the weight ratio of SPDP: the protein is 8:1 (m: m), 48 mul SPDP ethanol solution and 10 mul p53 protein (0.6 mg/mL) react for 30min at room temperature, then the mixture is placed in PBS buffer solution for dialysis for 24h at 4 ℃, the dialysate is changed for 3 times, then PEG20000 solid particles are used for placing in a container, a dialysis bag is placed in PEG20000 solid particles, the volume of the liquid is concentrated to about 1mL by utilizing the water absorption of the PEG20000 solid particles, 3mLDTT solution (10 mg/mL) is added, the mixture is shaken for 30min at room temperature, the mixture is dialyzed for 24h at 4 ℃ in the PBS buffer solution, and the mixture is reserved after the dialysis;
dissolving SPDP in absolute ethanol to obtain a 1mg/mL SPDP ethanol solution, according to the weight ratio of SPDP: adding 48 μ l SPDP and 10 μ l ER protein (0.6 mg/mL) into the protein at a concentration ratio of 8:1 (m: m), reacting at room temperature for 30min, dialyzing in PBS buffer at 4 deg.C for 24 hr, changing dialysate for 3 times, and concentrating to about 1mL with PEG 20000;
mixing the SPDP treated ER antibody protein with the SPDP treated p53 protein (the molar ratio of the dosage of the p53 protein to the dosage of the ER antibody protein is 1 to 5), shaking up at room temperature for reaction for 30min, then placing PEG20000 solid particles into a container, placing a dialysis bag into the PEG20000 solid particles, and concentrating the volume of liquid to about 1mL by utilizing the water absorption of the PEG20000 solid particles to prepare for subsequent experiments.
4. PTX/PC/anti-HER2 micelles are broken through ultrasound, a pre-synthesized protein complex p53/anti-ER is mixed, so that the p53/anti-ER is loaded into the PTX/PC/anti-HER2 micelles, and then the final nanoparticles p53/anti-ER @ PTX/PC/anti-HER2 are obtained.
The specific operation is as follows:
preparing 1mL of PTX/PC/anti-HER2 micellar solution with 0.125 mu g/mL of PBS buffer solution, dispersing by an ultrasonic disperser 100W 20S, then dropping 1mL of p53/anti-ER solution concentrated in the step 3, shaking at room temperature for 30min, wrapping the p53/anti-ER by the PTX/PC/anti-HER2 under the hydrophobic action, and then obtaining the final nano particles of p53/anti-ER @ PTX/PC/anti-HER2.
The synthesis scheme of the p53/anti-ER @ PTX/PC/anti-HER2 protein nano-complex is shown in figure 1.
Example 2 characterization of PTX/PC/anti-HER2@ p53/anti-ER protein Nanocomplexes
1. Ultraviolet full band scanning
1. Experimental methods
The maximum absorption wavelength of the sample in this wavelength range can be obtained by scanning the sample in the wavelength range of 190-900nm by means of an ultraviolet-visible spectrophotometer. Taking p53 protein as a sample 1, synthesizing to obtain a protein compound as a sample 2, taking dispersant water as reference liquid, respectively taking a proper amount of samples in a cuvette, and carrying out spectrum scanning detection in the range of 190-900 nm.
Specifically, PTX/PC solutions with concentrations of 1. Mu.g/mL, 0.5. Mu.g/mL, 0.25. Mu.g/mL, 0.125. Mu.g/mL and 0.0625. Mu.g/mL were prepared, shaken to be sufficiently dissolved, set the wavelength of a spectrophotometer (model) at 590nm, measured the absorption values of PTX/PC micelles with different concentrations at 590nm, and repeated three times for each concentration,
2. results of the experiment
As shown in FIG. 2, CMC of PTX/PC/anti-HER2 was calculated to be 0.125. Mu.g/mL.
2. Scanning electron microscope detection
1. Experimental methods
And synthesizing the protein nano-complex, taking out 20 mu l of the sample after the volume of the sample is 1mL, and sending the sample for detection. Weighing 0.01g of PTX/PC/anti-HER2 powder, dissolving in PBS buffer solution, fully dissolving, centrifuging at 1000r, taking 100 mu of supernatant, placing in an EP tube, and detecting by a transmission electron microscope.
2. Results of the experiment
As a result, as shown in FIG. 3, the synthesized PTX/PC/anti-HER2 micelle was observed to be spherical and uniformly dispersed.
3. Dynamic light scattering detection of particle size
1. Experimental methods
A small amount of certain concentrations of PTX/PC and p53/anti-ER @ PTX/PC/anti-HER2 were added to the cell, characterized by a particle size analyzer (Vector-33, bruker, germany), plotted and analyzed for particle size and distribution.
2. Results of the experiment
Analysis of dynamic light scattering results shows (FIG. 4) that most PTC/PC micelles have the particle size of about 100nm, the particle size is slightly increased after p53/anti-ER @ PTX/PC/anti-HER2 is formed by self-assembly after p53/anti-ER is added, and the particle size of the final synthesized particles is about 80-200 nm.
4. Detection of successful crosslinking of PTX/PC by hydrogen nuclear magnetic resonance spectroscopy
1. Experimental methods
0.01g of PTX, PC and PTX/PC powder are weighed and dissolved in dimethyl sulfoxide-d 6 respectively, the mixture is placed on ice after being fully dissolved, and a 600MHz Fourier transform nuclear magnetic resonance spectrometer (Bruker AVANCE NEO, germany Bruker) is used for detecting the chemical shift.
2. Results of the experiment
The result is shown in figure 5, the benzene ring absorption peak peculiar to paclitaxel is very obvious in the synthesized sample, the displacements are respectively 1-9 in the figure, the benzene ring absorption peaks of paclitaxel in PTX/PC nuclear magnetic resonance H spectrum are overlapped to form a shielding effect, and the ester bond formed by PTX/PC and the benzene ring form rho-pi hyperconjugation phenomenon possibly, so that the electron cloud density on the benzene ring is increased; on the other hand, only three kinds of a, b and c appear on the methyl group on the characteristic peak of phycocyanin, and appear on PTX/PC. Indicating that PTX/PC has been successfully synthesized.
Example 3 in vitro inhibition of breast cancer MCF7 cell growth experiments
1. Cell culture
Human breast cancer cells (MCF-7 cell line) were provided by Shenzhen advanced technology research institute, university of Chinese academy of sciences; the SK-BR-3 cell line is provided by the cell resource center of Shanghai biological science institute. After the cells were cultured to 80% in a culture flask, the cells were cultured at 1X 10 4 Density per well was plated on 96-well plates and incubated for 12, 24, 36h for subsequent experiments. MCF-7 cell culture conditions were: high-glucose DMEM medium containing 10% newborn calf serum, 37 ℃,5.0% CO 2 . The culture conditions of the SK-BR-3 cells are as follows: 1640 medium containing 10% newborn calf serum, 37 ℃,5.0% CO 2 。
2. Study on in vitro inhibition of MCF-7 growth by protein nanocomposite
(I) detection of effect of p53 nano-particles with different concentrations on breast cancer MCF7 cells
1. Experimental methods
Preparation of cell suspension 10 6 Perml, 100. Mu.l were inoculated into 96-well plates in an amount of 10000/well. Culturing and incubating for 4h at 37 ℃ until the cells adhere to the wall; changing serum-free culture medium, adding p53 protein with different concentrations, and acting for 4h; add 10. Mu.l of CCK-8 (medium was changed before adding CCK-8), enzyme-linked immunosorbent assay, absorbance at 450nm was measured using a microplate reader (Thermo Fisher MK 3). 2. Results of the experiment
As a result, as shown in fig. 6, the semilethality of the cells was p53 concentration =400ng/μ l, and it was found that the cell viability decreased very slowly with the increase of the p53 protein concentration, and there was little difference between the cell viability at 400ng/μ l and 800ng/μ l, so i finally selected the p53 concentration of 400ng/μ l.
(II) CCK-8 cell proliferation assay
1. Experimental method
Preparation of cell suspension 10 6 Perml, 100. Mu.l were inoculated into 96-well plates in the amount of 10000/well. Culturing and incubating for 4h at 37 ℃ until the cells adhere to the wall; changing a serum-free culture medium, adding synthetic composite protein nanoparticles with a certain concentration, and acting for 4 hours; adding 10 μ l CCK-8 (medium exchange before adding CCK-8), performing enzyme-linked immunosorbent assay, and measuring 450nm absorbance with enzyme-linked immunosorbent assay (Thermo Fisher MK 3)And (4) luminosity.
Sample detection: and (5) detecting by using an enzyme-labeling instrument and processing data under the condition of keeping out of the sun.
And (3) data analysis: cell viability = [ (As-Ab)/(Ac-Ab) ] × 100%; cytostatic rate = [ (Ac-As)/(Ac-Ab) ] × 100% (As = number of experimental wells; ab = number of blank wells; ac = number of control wells). The action time is used as the abscissa, and the cell survival rate and the cell inhibition rate are used as the ordinate, and a standard curve is drawn.
2. Results of the experiment
The results are shown in FIG. 7, the cancer cell viability of the nanoparticles p53/anti-ER @ PTX/PC/anti-HER2 after acting on the MCF-7 cell line for 24h is 40% of that of the control group, while the viability of the treatment group of the SK-ER-3 group is only 25% of that of the control group, which indicates that the nanoparticles have stronger killing effect on the SK-BR-3 cell line positive to HER2.
(V) DAPI staining for observing nuclear morphology and nuclear targeting subcellular localization
1. Experimental methods
The cultured cells were removed from the incubator, trypsinized for 2min, and 4mL of culture medium was added to blow the cells. 3mL of cell suspension is added into a centrifuge tube, 11mL of culture solution is added, and cells are blown to be uniformly suspended. And (3) sucking 100 mu L of cell suspension by using a pipette gun, adding the cell suspension into a 24-well plate, and carrying out adherent culture on the cells for 6h. The cultured cells were taken out, the cell waste liquid was aspirated, washed twice with PBS, and fresh medium was added. Adding quantitative protein composite particles, incubating for 4h, absorbing waste liquid, and washing twice with PBS. Adding prepared DAPI staining solution for staining for 10min, sucking waste liquid, and washing twice with PBS. Adding 1mL TBS for membrane permeation treatment for 5min, washing with PBS for 3 times, adding 1mL primary anti-p53 diluent (1; the cells were washed with pre-cooled PBS 3 times, and then diluted with FITC-fluorescently labeled secondary antibody (1. The pre-cooled PBS was washed 3 times. Add 100. Mu.l DAPI dilutions (1, 1000), stain for 10min at room temperature, wash three times with pre-chilled PBS, take pictures using fluorescence microscopy, and PS process pictures. The pictures were photographed using a fluorescence microscope and processed by PS.
2. Results of the experiment
The result is shown in FIG. 8, the DAPI staining result shows that the activity of the SK-BR-3 cells is reduced after p53/anti-ER @ PTX/PC/anti-HER2 acts for 24h compared with the cell nucleus of the SK-BR-3 cells in the Control group, the morphology is obviously changed, and the cell is changed from a long fusiform to a spherical shape, which indicates that the cells are undergoing apoptosis; the fluorescence of the nanoparticle p53/anti-ER @ PTX/PC/anti-HER2 synthesized by the p53 protein marked by green fluorescence FITC coincides with the blue fluorescence of the MCF-7 cell nucleus, which indicates that the nanoparticle has the effect of targeting the MCF-7 cell nucleus.
(VII) Nano-drug for inhibiting cancer cell migration
1. Experimental methods
SK-BR-3 cells were trypsinized, plated into 6-well plates and 5X 10 cells were added per well 5 Culturing the individual cells in an incubator overnight; after the cells adhere to the wall, 4-5 parallel lines are scribed in the hole by using a sterilized toothpick, and the toothpick is vertical to the wall of the hole; injecting PBS along the hole wall, rinsing for 3 times, removing the scratched cells, and paying attention to gentle action; two groups are set: (1) p53/anti-ER @ PTX/PC/anti-HER2; (2) a control group; respectively adding a serum-free culture medium containing p53/anti-ER @ PTX/PC/anti-HER2 and a serum-free culture medium, and respectively treating for 24h and 48h; the cells were observed under a microscope, photographed, and analyzed for cell spacing using Image J software.
2. Results of the experiment
As shown in FIG. 9, the cell migration experiment shows that the cell gap of the SK-BR-3 breast cancer cell treated by p53/anti-ER @ PTX/PC/anti-HER2 is 82% of the original cell gap after 24h and is 80% of the original cell gap after 48h, indicating that the cell is hardly migrated under the drug treatment, while the cell gap of the control group is obviously narrowed after 24h, only 50% of the original cell gap is 50% of the original cell gap and only 30% of the original cell gap after 48h, indicating that the synthesized drug has an inhibitory effect on the migration of the cancer cell.
EXAMPLE 4 in vivo growth inhibition of breast cancer MCF7 cells
1. Construction of mouse HER2+ and ER + breast cancer model
40 healthy female BALB/c mice of 5 weeks old were prepared, purchased from the center of laboratory animals of the university of traditional Chinese medicine (tribasic school district) in Guangzhou, and license numbers were produced: SCXK (Guangdong) 2018-0034, and depilating the abdominal mammary gland part of the depilatory cream when the weight of the fed animal reaches about 15 g; in vitro passageSK-BR-3 and MCF-7 cells were cultured, and a mixed suspension of SK-BR-3 and MCF-71 cells was prepared using PBS, and the number of cells was adjusted to 1X 10 7 Per mL; of these, 32 were randomly divided into 4 groups (each: a: blank control group (physiological saline); b: PTX/PC/anti-HER2 group; c: free p53 group; d: p53/anti-ER @ PTX/PC/anti-HER2 group), 8 cells per group, the mixed cell suspension was aspirated by a sterile syringe, inoculated into the lower abdominal mammary fat pad of mice, and 0.1mL was injected per mouse; in addition, 8 were not treated for Normal group. All mice were in the same breeding environment and given sufficient food and water. The mouse tumor length (a) and width (b) were measured daily starting on the second day after inoculation and using the formula V = a × b 2 Calculating the tumor volume; further study was carried out when the tumor volume reached about 80mm 3.
2. Study on in vivo inhibition of MCF-7 growth of protein nanocomposite
(one) Change in body weight of mice during administration
1. Experimental methods
Starting on the second day after dosing, the body weight of the mice was measured every two days, three times per mouse, using a weighing scale.
2. Results of the experiment
The weight change of Balb/c mice successfully modeled as SK-BR-3 breast cancer cells during the administration period, measured every 2 days during the 27 days administration, in FIG. 10, showed that the weight change of each group was small and the weight of p53/anti-ER @ PTX/PC/anti-HER2 treated mice did not decrease, indicating that the drug had no effect on the growth of the mice.
(II) Change in tumor volume in mice after drug treatment
1. Experimental methods
The mouse tumor length (a) and width (b) were measured every two days using a vernier caliper from the second day after the administration, and using the formula V = a × b 2 Tumor volume was calculated three times per mouse.
2. Results of the experiment
The results are shown in fig. 11, the tumor size is measured every 2 days, wherein the tumor volume V = (long diameter x short diameter 2)/2, the tumor long diameter and short diameter are measured by vernier caliper, the data shown in fig. 11 are obtained by calculation, the graph in fig. 11 shows that the tumor volume of the control group mouse is increased linearly, the results of the PTX/PC group and the p53 group are similar, the inhibition effect of the PTX/PC group and the p53 group on the tumor growth is similar, and the tumor volume of the p53/anti-er @ PTX/PC/anti-HER2 treated mouse is obviously inhibited, which indicates that the combined use of PTX/PC and p53 is more effective on the treatment of the breast cancer.
On day 27, the mice were dissected and tumors were weighed, and after the end of the treatment, the tumor volume was minimal in the p53/anti-ER @ PTX/PC/anti-HER 2-treated group, which was about 0.5cm in diameter, while the tumor diameter in the control group was about 1.2cm. The mean tumor mass of the p53/anti-ER @ PTX/PC/anti-HER2 treated group was minimal to less than 0.1g, while the mean tumor mass of the control group mice was close to 0.7g, with significant differences. According to various groups of data, p53 protein has an inhibition effect on tumor growth, the inhibition effect is continuously enhanced along with the encapsulation of anti-ER and PTX/PC micelles, and p53/anti-ER @ PTX/PC/anti-HER2 has an obvious inhibition effect on tumors.
(III) analysis of results of protein Complex inhibition of Breast cancer growth
1. Experimental methods
Respectively injecting p53 protein, free p53, p53/anti-ER @ PTX/PC/anti-HER2 with equal volume and equal protein content into the tumor of a mouse in an experimental group in situ, injecting physiological saline with equal volume into a control group, injecting the medicament once every two days, wherein the administration amount is 12 mu g/kg (p 53 protein content) every time, and keeping for 28 days; tumor volume and body weight were measured every two days, mice were sacrificed after treatment, tumors were dissected and measured and weighed, and plotted using Graphpad Prism 8.0 software analysis.
2. Results of the experiment
The body weight of the mice changed within 28 days of the in situ treatment, and the body weight of the mice of each group was close and increased. No obvious change exists in the treatment process, but under the condition that the tumor weight is not excluded, the analysis shows that the weight of the mice in the Control group has more obvious weight reduction compared with the weight of the mice in the normal group and the drug treatment group. Therefore, the increase of tumor tissues of breast cancer can influence the physical health condition of mice and bring certain side effects. The composition has effect in improving health condition of mouse after drug treatment.
During 28 days of treatment, the tumor volumes of mice in the p53/anti-ER @ PTX/PC/anti-HER2 treated group gradually decreased, while the tumor volumes in the other groups all showed an increasing trend, with the increasing trend being most obvious in the control group, followed by the PTX/PC group. The tumor growth of the p53 protein and PTX/PC protein treatment group is respectively inhibited to a certain extent, and the effect of killing cancer cells by nano-drugs is enhanced after the modification by anti-ER and anti-HER2 antibodies, mainly due to the effect of effectively protecting p53 by PTX/PC micelle coating and the targeting effect of cell membranes and cell nucleuses after the modification by antibodies.
As shown in FIG. 12, the p53/anti-ER @ PTX/PC/anti-HER 2-treated group prolonged the survival rate of the breast cancer mouse model compared to the Control group.
(IV) mouse tumor cell death-related protein expression
1. Experimental method
Each group of mice was dissected to obtain tumor, heart, liver, spleen, lung and kidney. Embedding in paraffin, wherein the slice thickness is 3-4 μm; baking slices: placing the slices to be sliced on a slicing frame, and baking in a constant temperature oven at 65 ℃ for 20min; dewaxing: soaking the slices in xylene for 5min for 2 times; hydration: putting the slices into 100%, 90%, 85% and 75% gradient alcohol for 2min each; endogenous block: placing the slide in a washing box containing PBS, and washing for 5min for 3 times; wiping the liquid on the glass slide by using filter paper, and circling out a sample by using a grouping pen; dropwise adding H2O2 on the sample to carry out endogenous blockage for 10-15min; the slides were washed 3 times in PBS for 5min each time; membrane breaking: dropping Triton in the sample by 0.1% for 10min; the slides were washed 3 times in PBS for 5min each; antigen retrieval: preparing sodium citrate restoration solution (11.5mL of solution A, 88.5mL of solution B, and mixing uniformly); immersing the glass slide in a washing box containing repairing liquid, putting the glass slide in a pressure cooker containing a proper amount of water, and performing high-pressure repairing for 7-10min; and (3) sealing: cooling the repair liquid to room temperature, dropwise adding goat serum working solution, and sealing in a 37 ℃ wet box for 1h; primary anti-incubation: respectively dropwise adding prepared Bax, caspase 3, bcl-2 and Ki-67 monoclonal antibodies (1; wash 3 times with PBS for 5min each; and (3) secondary antibody incubation: dripping secondary antibody of biotin labeled mountain, and incubating for 1-2h in a room temperature wet box; PBS wash 3 times, each time for 5min; dripping horse radish peroxidase streptavidin HRP-SA (1; washing with PBS for 5min for 3 times; dripping DBA color development solution, and reacting for 2-10min; PBS wash 3 times, each time for 5min; dropping hematoxylin for dyeing for 5-10s, rapidly washing off the dye by using PBS, placing the mixture into hydrochloric acid ethanol for differentiation for 1-2s, soaking the sample in the PBS, and returning blue for 1-2h; after the color of the sample is changed into blue, putting the glass slide into 75%, 80%, 95% and 100% gradient alcohol for dehydration for 1min respectively; placing the sample in two bottles of containers containing xylene solution in sequence, and performing transparent treatment for 2min each; sealing: after the tissue specimen is fully dried, dropwise adding neutral resin on the sample, and covering a cover glass; and (3) observation imaging: after the slide was fixed, the image was observed using a fluorescence inverted microscope.
2. Results of the experiment
FIG. 13 shows the expression of Ki67, bax, bcl2, caspase-3 in the tumor tissues of mice after the administration of the nano-drug by immunohistochemical detection. The results show that the expression of Bax and Caspase-3 in the tumor tissue treated by the laser and natural protein nano micelle is up-regulated, and the expression levels of Ki67 and Bcl2 are obviously down-regulated, which indicates that the nano medicament has better inhibition effect on the growth of tumor cells, promotes apoptosis and inhibits the infiltration capacity of breast cancer cells.
(V) detection of mouse index after blood routine detection and treatment
1. Experimental methods
After the nano-drug is administrated in situ for 28 days, venous blood of each group of mice is extracted from tail veins to prepare anticoagulant (EDTA disodium is added); 20 mu.L of the anticoagulated diluent is taken, and the total number of white blood cells in a blood sample and the contents of various white blood cells (neutrophils (GR%), lymphocytes (LY%), monocytes (MO%)) and the like are detected by a blood cytometer in a conventional way.
2. Results of the experiment
As shown in FIG. 14, in order to quantify the toxicity of the nanoparticles, 28 days after treatment, normal, control and natural complex micellar groups were further subjected to routine blood tests. No significant changes were observed between normal mice, mice given saline and p53/anti-ER @ PTX/PC/anti-HER2. The evaluation of the quantity of the white blood cells, the platelets and the red blood cells of the nanoparticle treatment group shows that the number of the white blood cells, the platelets and the like which cause inflammatory reaction is also in a normal range, and the safety of p53/anti-ER @ PTX/PC/anti-HER2 and the effect of the natural protein bionic nano micelle on targeting tumor cells are better.
It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Claims (10)

1. The HER2+ breast cancer targeted protein composite nanoparticle is characterized in that the targeted protein composite nanoparticle is a protein composite nanoparticle loaded with a protein composite p53/anti-ER in a PTX/PC/anti-HER2 micelle; wherein, the protein compound p53/anti-ER is a protein compound obtained by cross-linking an apoptosis-related protein p53 and an antibody anti-ER of an estrogen receptor by disulfide bonds;
the PTX/PC/anti-HER2 micelle is a protein complex obtained by self-assembling phycocyanin PC in a protein complex PC/anti-HER2 obtained by cross-linking phycocyanin PC and an antibody anti-HER2 of a human epidermal growth factor receptor 2 through amide bonds and then cross-linking phycocyanin PC in the protein complex PC/anti-HER2 with paclitaxel PTX through ester bonds, and an antibody PTX/PC/anti-HER2 of the protein complex phycocyanin/paclitaxel complex/human epidermal growth factor receptor 2 through ester bonds.
2. A preparation method of HER2+ breast cancer targeted protein composite nanoparticles is characterized by comprising the following steps:
s1, crosslinking phycocyanin PC and an antibody anti-HER2 of a human epidermal growth factor receptor 2 by using an amido bond to obtain a hydrophilic protein compound PC/anti-HER2;
s2, crosslinking paclitaxel PTX and phycocyanin PC through ester bonds to obtain a protein complex phycocyanin/paclitaxel complex/human epidermal growth factor receptor 2 antibody PTX/PC/anti-HER2, and self-assembling to form micelle PTX/PC/anti-HER2;
s3, cross-linking the apoptosis-related protein p53 and an antibody anti-ER of an estrogen receptor by using a disulfide bond to obtain a protein compound p53/anti-ER;
s4, dispersing the micelle PTX/PC/anti-HER2, mixing the dispersed micelle PTX/PC/anti-HER2 with the protein complex p53/anti-ER, and loading the protein complex p53/anti-ER into the PTX/PC/anti-HER2 micelle so as to obtain the final nanoparticles p53/anti-ER @ PTX/PC/anti-HER2.
3. The method of claim 2, wherein in step S1, catalysts NHS and EDC, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, catalyze the cross-linking of phycocyanin PC with the antibody anti-HER2 of human epidermal growth factor receptor 2 by amide bond.
4. The method of claim 2, wherein the paclitaxel PTX is crosslinked with the phycocyanin PC via an ester bond using dicyclohexylcarbodiimide/4-dimethylaminopyridine DCC/DMAP as a catalyst in the step S2.
5. The method of claim 2, wherein in step S3, the apoptosis-related protein p53 is chemically catalyzed by disulfide bond cross-linking with an antibody anti-ER of estrogen receptor using catalysts of N-hydroxysuccinimide ester of 3- (2-pyridinedimercapto) propionate SPDP and dithiothreitol DTT.
6. The method of claim 2, wherein in step S4, the micelle PTX/PC/anti-HER2 is dispersed by sonication.
7. The HER2+ breast cancer targeted protein composite nanoparticle prepared by the preparation method of any one of claims 2 to 6.
8. Use of the HER2+ breast cancer-targeted protein composite nanoparticle of claim 1 or 7 for the preparation of a medicament for the treatment of breast cancer.
9. The use of claim 8, wherein the medicament is effective in increasing the expression level of apoptosis-related proteins in breast cancer cells to induce apoptosis in the breast cancer cells, thereby achieving the effect of inhibiting the growth of the cancer cells at a high level.
10. The use of claim 8, wherein the medicament is for a HER2 positive breast cancer patient.
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