Novel nanoparticle drug delivery system for targeted therapy of TNBC

Abstract

The invention discloses a novel nanoparticle drug delivery system for targeted therapy of TNBC, and relates to the technical field of targeted inhibition and treatment of TNBC. According to the novel nanoparticle drug delivery system for targeted therapy of TNBC, the preparation method of the novel nanoparticle drug delivery system comprises the following specific steps: S1, synthesizing; the preparation method comprises the following steps: synthesizing B I-PEG-COOH and B I-PEG-PTX with tumor targeting capability; s2, material characterization; using Fourier transform infrared spectroscopy and 1HNMR to verify the structural characteristics of the B I-PEG-COOH and B I-PEG-PTX samples obtained in the step S1; s3, preparing a nano-micelle; and after the verification is passed, preparing the nano-micelles BBR (at) PTX NPs according to the B I-PEG-COOH and the B I-PEG-PTX obtained in the step S1. According to the invention, the nanocluster has a targeting capability, and chemotherapeutic drugs flowing to normal tissues are reduced, so that the toxic influence on the normal tissues is reduced, the dual drug delivery system is beneficial to increase of the tumor targeting capability, novel high-efficiency and low-toxicity nano treatment is realized, and the application prospect is wide. And a further optimized strategy is provided for clinical chemotherapy sensitization of a nanoparticle drug delivery system.

Description

Technical Field

[0001] The present invention relates to the technical field of TNBC targeted inhibition and treatment, and in particular to a novel nanoparticle drug delivery system for targeted treatment of TNBC. Background Art

[0002] Triple-negative breast cancer (TNBC) is a special subtype of breast cancer in which the expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER-2) are all negative. Its direct cause is still unclear, and it may be related to genetic factors, hormones, environment, age, gender and underlying diseases. Patients often show symptoms such as palpable breast lumps, or swelling, deformation and skin changes of the breasts, nipple discharge, breast pain, etc. Compared with other subtypes, triple-negative breast cancer is prone to visceral metastasis, such as lung metastasis, brain metastasis and bone metastasis.

[0003] For the treatment of TNBC, the nano delivery system for targeting and inhibiting triple-negative breast cancer siRNA combined with drugs disclosed in China Invention Publication No. CN115025045A, and its preparation method and application, the system is composed of 4 DNA single strands with a length of 63 nt to form a nanostructure with high mechanical strength, called TDN. Each single strand contains three sequence blocks, each sequence block is complementary to the sequence of another single strand, in this way, after hybridization, the four triangular DNA helices form a rigid tetrahedral structure, each side is 20 bp, and each vertex has a single oligonucleotide, and the four single-stranded DNAs are mixed in TM buffer (10mM, Tris-HCl, 12.5mM, MgCl2·6H2O, pH8.0) at the same concentration. Rapidly heat to 95°C for 10 minutes, then cool to 4°C for 20 minutes, then lower the temperature to 4°C for 1 hour to obtain Td-siRNA nanoparticles. The nano-delivery system prepared by combining traditional anticancer drugs with nanotechnology and RNA therapy can significantly improve the therapeutic effect of DOX / PTX, providing a new direction and method for the targeted treatment of triple-negative breast cancer.

[0004] Current clinical results show that TNBC targeted inhibition and treatment of R and PTX may lead to drug resistance and toxicity after high-dose single or combined use. The drugs in the above-mentioned nano-delivery system include doxorubicin hydrochloride or paclitaxel, while commonly used chemotherapy drugs such as paclitaxel, doxorubicin hydrochloride and doxorubicin have limited efficacy in TNBC patients. Therefore, the present invention provides a further optimization strategy for clinical chemotherapy sensitization of nanoparticle drug delivery systems based on the ability of the nanoparticle co-delivery system to improve drug solubility and enhance drug targeting to tumors. Summary of the invention

[0005] In view of the deficiencies of the prior art, the present invention provides a novel nanoparticle drug delivery system for targeted treatment of TNBC, which solves the problems raised in the above background technology.

[0006] To achieve the above objectives, the present invention is implemented by the following technical scheme: a novel nanoparticle drug delivery system for targeted treatment of TNBC, the preparation of the novel nanoparticle drug delivery system comprising the following specific steps:

[0007] S1: Synthesis; Synthesis of BI-PEG-COOH and BI-PEG-PTX with tumor targeting ability;

[0008] S2: Material characterization: Fourier transform infrared spectroscopy and 1H NMR were used to verify the structural characteristics of the BI-PEG-COOH and BI-PEG-PTX samples obtained in S1;

[0009] S3: Preparation of nanomicelles; after verification, nanomicelles BBR@PTX NPs were prepared based on BI-PEG-COOH and BI-PEG-PTX obtained in S1.

[0010] A further improvement of the technical solution of the present invention is that the synthesis step of the BI-PEG-COOH obtained in S1 comprises the following specific steps:

[0011] 0.4 g BI, 0.376 g EDC and 0.188 g NHS were added to 3 ml dimethyl sulfoxide, activated at 40°C for 2 h, and then 0.2 g NH2-PEG-COOH was added and reacted for 24 h. The resulting reaction solution was dialyzed in deionized water using a 2000 kDa dialysis bag, and the deionized water was replaced every 30 min. The solution was freeze-dried after 1 day to obtain the product BI-PEG-COOH.

[0012] A further improvement of the technical solution of the present invention is that the synthesis step of the BI-PEG-PTX obtained in S1 comprises the following specific steps:

[0013] 3 mg PTX and 10.2 mg EDC were added together to 6 ml dimethyl sulfoxide, completely dissolved in a flask, and activated at 40° C. for 6 hours to obtain a reaction solution. The condensing agent, catalyst and 24 mg BI-PEG-COOH were added together to 12 ml dimethyl sulfoxide, completely dissolved in a flask and activated at 40° C. for 30 minutes to obtain another reaction solution. The two obtained reaction solutions were mixed and reacted at 40° C. under light-shielding conditions for 48 hours. The reaction solution was dialyzed with a 2000 KDa dialysis bag, deionized water was filtered, and the solution was freeze-dried after 1 day to obtain the product BI-PEG-PTX with a hydrophobic feed ratio of 1:8.

[0014] The further improvement of the technical solution of the present invention is that the condensing agent is selected as 1-ethyl-carbodiimide hydrochloride, and the specific amount is 14.4 mg; the catalyst is selected as 4-dimethylaminopyridine, and the specific amount is 14.4 mg.

[0015] A further improvement of the technical solution of the present invention is that the S2 for verifying the obtained BI-PEG-COOH and BI-PEG-PTX samples comprises the following specific steps:

[0016] Take a small amount of the BI-PEG-COOH and BI-PEG-PTX sample powders, dry the water under an infrared lamp, grind and press with KBr to form a 500 cm 2 Up to 4000cm 2 The infrared spectrum of the sample was obtained by scanning, and then 10 mg of BI-PEG-COOH and BI-PEG-PTX samples were dissolved in 1 ml of tritiated dimethyl sulfoxide, loaded into the infrared spectrometer NMR sample tube, and the 1H NMR spectrum was scanned by 0ppm-15ppm absorption to complete the structural feature verification of the BI-PEG-COOH and BI-PEG-PTX samples.

[0017] A further improvement of the technical solution of the present invention is that the preparation step of the nano-micelle BBR@PTX NPs includes the following specific steps:

[0018] 8 mg BI-PEG-PTX nanoconjugate was dissolved in 6 ml dimethyl sulfoxide, loaded into a 2000 kDa dialysis bag, dialyzed in deionized water, changing the water once every 1 hour, and dialyzed for 12 hours to obtain loaded PTX NPs. 1 mg BBR and 8 mg BI-PEG-PTX were dissolved in 6 ml dimethyl sulfoxide and mixed in the obtained PTX NPs, mixed evenly, loaded into a 2000 kDa dialysis bag, dialyzed for 12 hours, changing the water once, and dialyzed in deionized water for 12 hours to obtain the dual-loaded nanoparticles BBR@PTX NPS.

[0019] A further improvement of the technical solution of the present invention is that the preparation of the novel nanoparticle drug delivery system also includes evaluating the effectiveness of the BBR@PTX NPs obtained in S3, including characterization of the BBR@PTX NPs under a microscope, drug loading and release rate evaluation.

[0020] A further improvement of the technical solution of the present invention is that the characterization of the BBR@PTX NPs under a microscope comprises the following steps:

[0021] The prepared PTX NPs and BBR@PTX NPs nanomicelle solutions with a concentration of 1 mg / ml were placed in a dynamic light scattering instrument. Each sample was tested three times for 1 minute each time. Transmission electron microscopy was used to characterize the size and morphology of the synthesized nanoclusters. The samples were observed by dynamic light scattering and dropped onto a copper mesh coated with a carbon support film. The samples were dried naturally after the solvent dried, and then dyed with a dyeing catalyst. Finally, their morphology was observed and scanned in a transmission electron microscope to complete the characterization of the BBR@PTX NPs under the microscope and determine whether the prepared BBR@PTX NPs met the requirements.

[0022] A further improvement of the technical solution of the present invention is that the dyeing catalyst is selected to be 2% phosphotungstic acid.

[0023] A further improvement of the technical solution of the present invention is that the specific operation of evaluating the drug loading and release rate of BBR@PTX NPs is as follows:

[0024] 5 mg of freeze-dried BBR@PTX NPs were dissolved in 5 ml of distilled water to prepare a BBR@PTX NPs solution with a concentration of 1 mg / ml, and the UV absorbance was measured at 230 °C. The concentration was calculated according to its standard curve, and the loading amount was calculated using the wavelength method;

[0025] 10 mg of free drug and drug-loaded nanoparticles were accurately weighed and dissolved in 5 ml of PBS, pH 7.4. After dissolution, the mixture was transferred to a 2000 kDa dialysis bag. The dialysis bag was dialyzed in a small beaker containing 50 mL of PBS, pH 7.4 or 50 mL of PBS, pH 5.5, respectively. The dialysis bag was shaken on a shaker at 37°C and 75 pm. The PBS in the dialysis bag was replaced at different times, and the absorbance of the volume Vt and PBS was measured at 230 nm and 340 nm, respectively, to calculate the contents of the two released drugs.

[0026] Compared with the prior art, the beneficial effects of the present invention are: the targeting ability of nanoclusters reduces the flow of chemotherapy drugs to normal tissues, thereby reducing the toxic effects on normal tissues; the dual drug delivery system helps to increase tumor targeting ability, realizes a new type of high-efficiency and low-toxic nanotherapy, and provides a further optimized strategy for clinical chemotherapy sensitization of nanoparticle drug delivery systems;

[0027] The nano-connector carrier BI-PEG-PTX was formed by the reaction of NH2-PEG-COOH with -COOH of BI and -OH of PTX, followed by chemical synthesis. The successful synthesis of the nano-carrier PTX NP was characterized by Fourier transform infrared spectroscopy and nuclear magnetic resonance hydrogen spectroscopy. BBR@PTX NP had better size distribution and morphology.

[0028] By including PEG structure in the material composition, RAW264.7 was successfully prevented from engulfing and clearing the nanomaterial, achieving immune escape, reducing the decomposition of the material by lysosomes in tumor cells, and improving bioavailability. The BBR in the material also stimulated tumor cells 489 to generate ROS, reducing cell membrane potential;

[0029] BBR@PTX NPs have good biosafety, and the BBR@PTX NPs group showed more apoptotic tumor cells in H&E staining, which proved that the BBR@PTX NPs treatment group caused systemic apoptosis of a large number of cancer cells, showing good tumor targeting, efficacy and biosafety in vivo, providing a new option for the clinical treatment of TNBC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of a novel nanoparticle drug delivery system for targeted treatment of TNBC;

[0031] Figure 2 Schematic diagram of the synergistic inhibition of MDA-MB-231 cell viability by BBR and PTX in Example 4 of the present invention;

[0032] Figure 3 This is a diagram of the preparation and characterization experiment of BBR@PTX NPs in Example 3 of the present invention;

[0033] Figure 4 The figure is the experimental result of the release and encapsulation efficiency of BBR and PTX in the evaluation of two drug loading and release rates in the examples of the present invention;

[0034] Figure 5 This is a graph showing the experimental results of in vitro determination of the in vitro toxicity of BBR@PTX with free drugs or drugs in combination with MDA-MB-231 cells in Example 5 of the present invention;

[0035] Figure 6 This is a schematic diagram of the cell migration and invasion experiment in Example 3 of the present invention;

[0036] Figure 7 This is an experimental diagram of lysosomal escape and immune escape in Example 3 of the present invention;

[0037] Figure 8 Figure 2 is the in vivo anti-tumor effect of BBR@PTX NPs in Example 6 of the present invention.

[0038] Fig. 9 Representative H&E staining images of the main organs of experimental mice in all embodiments of the present invention;

[0039] Fig.10This is an experimental diagram of the in vivo distribution experimental scheme in Example 7 of the present invention. DETAILED DESCRIPTION

[0040] Various exemplary embodiments, features and aspects of the present application will be described in detail below with reference to the accompanying drawings. The same reference numerals in the accompanying drawings represent elements with the same or similar functions. Although various aspects of the embodiments are shown in the accompanying drawings, the drawings are not necessarily drawn to scale unless otherwise specified.

[0041] The word “exemplary” is used exclusively herein to mean “serving as an example, example, or illustration.” Any embodiment described herein as “exemplary” is not necessarily to be construed as preferred or advantageous over other embodiments.

[0042] In addition, in order to better illustrate the present application, numerous specific details are provided in the specific embodiments below. It should be understood by those skilled in the art that the present application can also be implemented without certain specific details. In some examples, methods, means, and elements well known to those skilled in the art are not described in detail in order to highlight the subject matter of the present application.

[0043] Reference Figure 1 - Fig.10 The present invention provides further optimization for the clinical chemotherapy sensitization of the nanoparticle drug delivery system and provides seven technical solutions: Example 1, a new nanoparticle drug delivery system for targeted treatment of TNBC, the preparation of the new nanoparticle drug delivery system includes the following specific steps:

[0044] S1: Synthesis; Synthesis of BI-PEG-COOH and BI-PEG-PTX with tumor targeting ability;

[0045] S2: Material characterization: Fourier transform infrared spectroscopy and 1H NMR were used to verify the structural characteristics of the BI-PEG-COOH and BI-PEG-PTX samples obtained in S1;

[0046] S3: Preparation of nanomicelles; after verification, nanomicelles BBR@PTX NPs were prepared based on BI-PEG-COOH and BI-PEG-PTX obtained in S1.

[0047] The synthesis steps of BI-PEG-COOH obtained in S1 include the following specific steps:

[0048] 0.4 g BI, 0.376 g EDC and 0.188 g NHS were added to 3 ml dimethyl sulfoxide, activated at 40 ° C for 2 h, and then 0.2 g NH2-PEG-COOH was added and reacted for 24 h. The resulting reaction solution was dialyzed in deionized water using a 2000 kDa dialysis bag, and the deionized water was replaced every 30 min. The solution was freeze-dried after 1 day to obtain the product BI-PEG-COOH.

[0049] The synthesis steps of BI-PEG-PTX obtained in S1 include the following specific steps:

[0050] 3 mg PTX and 10.2 mg EDC were added together to 6 ml dimethyl sulfoxide, completely dissolved in a flask, and activated at 40°C for 6 hours to obtain a reaction solution. The condensing agent, catalyst and 24 mg BI-PEG-COOH were added together to 12 ml dimethyl sulfoxide, completely dissolved in a flask and activated at 40°C for 30 minutes to obtain another reaction solution. The two obtained reaction solutions were mixed and reacted at 40°C under light-shielding conditions for 48 hours. The reaction solution was dialyzed with a 2000 KDa dialysis bag, deionized water was filtered, and the solution was freeze-dried after 1 day to obtain the product BI-PEG-PTX with a hydrophobic feed ratio of 1:8.

[0051] The condensation agent is 1-ethyl-carbodiimide hydrochloride, and the specific amount is 14.4 mg. The catalyst is 4-dimethylaminopyridine, and the specific amount is 14.4 mg.

[0052] S2 The verification of the obtained BI-PEG-COOH and BI-PEG-PTX samples includes the following specific steps:

[0053] Take a small amount of BI-PEG-COOH and BI-PEG-PTX sample powder, dry the water under infrared lamp, grind and press with KBr to form 500cm 2 Up to 4000cm 2 The infrared spectrum of the samples was obtained by scanning, and then 10 mg of BI-PEG-COOH and BI-PEG-PTX samples were dissolved in 1 ml of tritiated dimethyl sulfoxide, loaded into the infrared spectrometer NMR sample tube, and the 1H NMR spectrum was scanned by 0ppm-15ppm absorption to complete the verification of the structural characteristics of the BI-PEG-COOH and BI-PEG-PTX samples.

[0054] The preparation steps of nano-microclusters BBR@PTX NPs include the following specific steps:

[0055] 8 mg BI-PEG-PTX nanoconjugate was dissolved in 6 ml dimethyl sulfoxide, loaded into a 2000 kDa dialysis bag, dialyzed in deionized water, changing the water once every 1 h, and dialyzed for 12 h to obtain loaded PTX NPs. 1 mg BBR and 8 mg BI-PEG-PTX were dissolved in 6 ml dimethyl sulfoxide and mixed in the obtained PTX NPs, mixed evenly, loaded into a 2000 kDa dialysis bag, dialyzed for 12 h, changing the water once, and dialyzed in deionized water for 12 h to obtain dual-loaded nanoparticles BBR@PTX NPS.

[0056] Example 2: Based on the BBR@PTX NPS nanoparticles prepared above, the effectiveness of the nanoparticles still needs to be evaluated, including the characterization of BBR@PTX NPs under a microscope, drug loading and release rate evaluation.

[0057] The characterization of BBR@PTX NPs under microscope includes the following steps:

[0058] The prepared PTX NPs and BBR@PTX NPs nanomicelle solutions with a concentration of 1 mg / ml were placed in a dynamic light scattering instrument. Each sample was tested three times for 1 minute each time. Transmission electron microscopy was used to characterize the size and morphology of the synthesized nanoclusters. The samples were observed by dynamic light scattering and dropped onto a copper mesh coated with a carbon support film. The samples were dried naturally after the solvent dried, and then dyed with a dyeing catalyst. Finally, their morphology was observed and scanned in a transmission electron microscope to complete the characterization of the BBR@PTX NPs under the microscope and determine whether the prepared BBR@PTX NPs met the requirements.

[0059] The dyeing catalyst is 2% phosphotungstic acid.

[0060] The specific operation of BBR@PTX NPs drug loading and release rate evaluation is as follows:

[0061] 5 mg of freeze-dried BBR@PTX NPs were dissolved in 5 ml of distilled water to prepare a BBR@PTX NPs solution with a concentration of 1 mg / ml, and the UV absorbance was measured at 230 °C. The concentration was calculated according to its standard curve, and the loading amount was calculated using the wavelength method;

[0062] The specific calculation formula is:

[0063] E = A / CL;

[0064] A230=EPTX 230nm*CPTX*L+EBBR340nm*CBBR*L;

[0065] A340=EPTX 340nm*CPTX*L+EBBR 340nm*CBBR*L;

[0066]

[0067] 10 mg of free drug and drug-loaded nanoparticles were accurately weighed and dissolved in 5 ml PBS, pH 7.4. After dissolution, they were transferred to a 2000 kDa dialysis bag. The dialysis bag was dialyzed in a small beaker containing 50 mL PBS, pH 7.4 or 50 mL PBS, pH 5.5, respectively. The dialysis bag was shaken on a shaker at 37 °C and 75 pm. The PBS in the dialysis bag was replaced at different times, and the absorbance of the volume Vt and PBS was measured at 230 nm and 340 nm, respectively, to calculate the content of the two released drugs.

[0068] The specific calculation formula is:

[0069] Wherein, drug refers to the total weight of any drug in the dialysis bag, t refers to the time point of PBS replacement; t = 1, 2, 4, 8, 16, 24, 48 h, VO, CO equal to zero;

[0070] Good drug loading efficiency can show good anti-tumor effect. According to the formula, the drug interception efficiency of the synthesized nanocapsules was calculated. It was found that the drug loading rates of PTX and BBR in BBR@PTX NPs were 9.5% and 28.06%, and the encapsulation rates were 8.6% and 33.5%, respectively. Figure 4 As shown in Figure 2, an ideal drug carrier not only requires a high drug loading capacity but also has good targeting performance. This study used pH 7.4 and pH 5.5 to simulate the in vivo physiological environment and tumor microenvironment, respectively. Figure 4 As shown in Figures A and B, PTX and BBR were released from BBR@PTX NPs in a pH-dependent manner. Compared with pH 7.4, pH 5.5 released PTX and BBR more easily, indicating that BBR@PTX NPs are particularly suitable for tumor therapeutic drug delivery. In order to further detect the effect of tumor cells on the content of micelles, indole green fluorescent dye (ICG) was used instead of BBR to construct ICG@BIPEG.PTX. The results are shown in Figures A and B. Figure 4As shown in C, the red fluorescence of the cells weakened 2 hours after administration, indicating that the nanomaterial loaded with I CG was slowly absorbed into the cells. After 4 or 8 hours of culture, brighter red fluorescence appeared on the cell membrane surface. At this time, BI in the nanocarrier may bind to the BI receptor on the cell membrane surface, and is internalized into the MDA-MB-231 cells through receptor-mediated endocytosis, taken up into the cells, and then releases the fluorescent substance I CG in the weakly acidic environment of the tumor, so that the red fluorescence of CG appears in the saturated matrix and emits a nuclear signal. Within 8 hours of the experiment, the cell uptake of I CGOPTXNPs gradually increased, indicating that the synthesized chemiluminescent material induced continuous hepatic uptake. Figure A is the cumulative release rate of PTX of Free PTX and BI-PEG-PTX at different pH values ​​(5.0 and 7.4), and B is the free BBR and BBR@PTX at different pH values ​​(5.0 and 5.0). Cumulative release rate of BBR from NPs, C is the cellular uptake of 231 cells treated with nanocarrier B-PEG-PTX loaded with fluorophore indocyanine green (ICG) for 2, 4 and 8 h after treatment by laser confocal microscopy (n3), scale bar: 25 μm, p ≈ 0.05, **.p < 0.01,

[0071] Example 3: After the nano-micelles BBR@PTX NPs required for TNBC targeted treatment are prepared as described above, the effects of targeted inhibition and targeted treatment of TNBC need to be verified, so a series of experiments need to be conducted;

[0072] Specifically, TNBC cancer cell lines were cultured, and upon completion, cytotoxicity tests, cell uptake, immune escape and lysosomal escape experiments were performed, cell migration and invasion experiments were performed, ROS levels and mitochondrial membrane potential were measured, apoptosis was detected by flow cytometry, in vivo studies were performed in xenograft tumor models, and histopathological analysis and in vivo distribution studies were performed;

[0073] In order to test the required TNBC cancer cell lines, MDA-MB-231 cells (TNBC cancer cell line) and RAW264.7 cells need to be cultured in DMEM medium containing 10% FBS (fetal bovine serum) and 1% streptomycin / penicillin, and the environmental conditions of the culture medium are 37°C and 5% CO2;

[0074] After the cell culture was completed, multiple groups of MDA-MB-231 cells for the test were obtained. At this time, a cytotoxicity test was performed. The specific steps were as follows: MDA-MB-231 cells were inoculated in triplicate (3×10 cells) into 96-well plates, cultured overnight, and each group was replaced with fresh culture medium containing different concentrations of PTX, BBR and PTX+BB compound drugs. After 48 hours of culture, 10% CCK8 reagent was added to evaluate cytotoxicity, and SynergyFinder3.0 was used to fit the synergistic index. In the other two 96-well plates, double-loaded and single-loaded nanomicelles were respectively laid, and these nanomicelles contained the same amount of drug concentration. After 24 hours of culture, CCK-8 was added to detect its concentration cytotoxicity to determine whether it met the test requirements.

[0075] In the process of TNBC targeted inhibition and treatment, after the nanoparticle carrier enters the cell, it is encapsulated in the body through the membrane vesicle and delivered into the lysosome, and finally escapes from the lysosome and is successfully released into the cytoplasm. During the escape process, the nanoparticle carrier protects the loaded drug from being degraded by the acid hydrolase in the lysosome. Therefore, the novel nanoparticle drug delivery system proposed in the present invention needs to ensure that it can complete the escape and successfully deliver. In order to verify whether it can be successfully delivered, cell uptake, immune escape and lysosomal escape experiments are designed;

[0076] Specifically, I CG (indocyanine green fluorescent dye) was used instead of BBR to construct I CG@PTX NPs. In brief, MDA-MB-231 cells and macrophage RAW264.7 cell lines were seeded on a cell climbing rack placed in a 6-well plate and then incubated with I CG dye-labeled PTX NPs at 37 °C for 2, 4, and 8 hours, the cell nuclei were stained with DAP| dye, the cells were fixed with paraformaldehyde (4%), and the fluorescence images were observed under a laser confocal microscope;

[0077] When performing the lysosomal escape ability test, MDA-MB-231 was previously placed in a 6-well plate and incubated with ICG@PTX NPs for 1, 3, 6, and 12 hours. The cells were incubated with preheated and diluted Lysotracker kit at 37°C for 20 minutes, and then incubated with Hoechst 33258 dye, stained for 10 minutes, rinsed with PBS, and the cell crawler was removed and inverted to a drop of anti-fluorescence quencher, and the slide was sealed with nail polish, and the fluorescence image was observed under laser confocal microscopy; as shown in the attached Figure 7As shown, A is the co-localization of I CG@BI-PEG-PTX with lysosomes for 3, 6 and 12 hours. Red fluorescence indicates nanomembranes labeled with I CG dye. Lysosomes and cell nuclei are stained with Lyso-Tracker (green) and Hoechst33258 dye (blue), respectively. The right figure is a quantitative analysis of the co-localization of I CG@PTXNPs LysoTracker green labeled lysosomes, if they are highly co-localized with Pearsons correlation coefficient close to 1 (n = 4), B is macrophage RAW264, 7 images of macrophages cultured with I CG@PTX NPs for different times, the right figure shows the average fluorescence intensity and average value of macrophages.

[0078] From the fluorescence images, we can see that the experimental results show that the nanoparticle carriers are successfully released into the cytoplasm through lysosomal escape. Nanoparticle carriers need to precisely migrate to cells in specific tissues as drug carriers to complete precise treatment, so cell migration and invasion experiments are performed.

[0079] The specific operation is to use the Transwell system with or without Matrigel (Biotroni k) to measure the migration and invasion of 231 cells and the Transwell test by adding free drugs or nano-drugs for culture. Serum-free cell culture medium is used to add 5×10*231 cell suspension to the upper chamber of the cell culture chamber, and culture medium containing 10% fetal bovine serum is added to the lower chamber. After incubation at 37°C for 24 hours, the cells in the upper chamber that have not migrated or invaded are gently wiped with a cotton swab, and then the cells in the lower chamber are fixed with 4% paraformaldehyde for 10 minutes. Finally, the cells in the lower chamber are fixed with 4% paraformaldehyde for 10 minutes. Stain with 0.1% crystal violet solution for 20 minutes, and then the migrating or invading cells are observed and photographed under a Zeiss optical microscope. The results are shown in the attached figure. Figure 6 As shown, A and B are representative cell migration and invasion images of 231 cells treated with PBS, BBR, PBS, PTX, BBR+PTX, PTXNPs and BBR@PTX NPs, respectively, and the corresponding quantitative analysis using Transwell assay (n-3). *: p<0.05. **: p<0.01.

[0080] In order to understand the mechanism of drug therapy of BBR@PTX NPs particles, it is necessary to measure the level of ROS and mitochondrial membrane potential, and finally detect the apoptosis of MDA-MB-231 cells by flow cytometry. The specific operation is to use Annexin V-FI TC / P|AssayKit to detect cell apoptosis. The cell density was adjusted to 4×10% / mL and treated with PTX (0.1pg / mL), BBR (3ug / mL), PTX+BBR (0.1+3ug / mL) or an equal amount of single-loaded PTX NPs and double BBR@PTX NPs nanocells for 24 hours, and then the cells were collected and washed with PBS, centrifuged, and resuspended in 100u l 1× binding buffer, and then dispersed into the staining solution containing 10u l PI and 5u lAnnexin V-FI TC. The cells were stained at room temperature for 15 minutes, and 400u l 1× binding buffer was added in the dark, and the cells were detected by flow cytometry. The presence of apoptosis and the number of apoptosis were obtained according to the test results;

[0081] To achieve the desired effect of reducing drug damage to healthy cells and lowering side effects, traditional chemotherapy drugs will damage normal cells while killing cancer cells, leading to a series of side effects, and precise drug delivery can reduce this non-specific killing and improve the safety of treatment.

[0082] Since BBR combined with PTX synergistically inhibited the viability of MDA-MB-231 cells as described above, this example designed target-active nanomicelles BBR@PTX NPs modified with bi ct in (B l) and detected Fourier transform infrared spectroscopy (FT IR) and 1H nuclear magnetic resonance (HNMR), as shown in the attached Figure 3As shown in the figure, the absorption peak of the carbonyl group (C-0) in the carboxylic acid BI is located at 1700 cm-1. This is because the steric resistance of the polymer PEG causes the wavenumber to move to a higher wavenumber, resulting in the carbonyl group (C-0) peak of the synthetic product BI-PEG-coOH being located at 1725 cm-1, and in the final synthetic product BI-PEG-PTX. For ROC=NR, due to the resonance effect, the amide bond R-CO-NHR resonance is converted into a stretching vibration peak at 1693cm-1, and because the grafted PTX has a large steric hindrance, the wave number of the p-substituted benzene ring in the PTX structure moves from 700 high wave numbers to 800 high wave numbers in the fingerprint region, which can prove that our material is successfully synthesized. At the same time, the NMR spectrum shows that at 3.52ppm, the peak (-ch2ch2-) in the PEG polymer structure undergoes a chemical shift, and the chlorine on the benzene ring of free PTX has a stretching vibration peak at 7-8ppm. Due to the electron donation effect of the grafted PEG, the chemical shift moves to the high field, resulting in the appearance of a benzene ring peak at 8-9ppm for BI-PEG-PTX and a characteristic peak of paclitaxel at 5-6.5ppm, which indicates that the PTX grafting is successful;

[0083] Next, the synthesized nanocarrier BI-PEG-PTX was used to decapsulate BBR, and the drug was decapsulated using the feed ratio PTX:BBR1:10 obtained from the two-drug synergy experiment, and nanocellular BBR@PTX NPs were successfully obtained. The particle size, potential, and morphology of the nanocellular BBR@PTX NPs were detected using dynamic light scattering (DLS) and transmission electron microscopy (TEM), as shown in Figure 2. Figure 3 As shown in C, D, E, and F, the particle size of the synthesized BBR@PTX NPs nanocellular micelles is about 130-150 nm, the pd i is between 0.1 and 0.2, and the distribution is very uniform. The transmission electron microscopy test uses 2% phosphotungstic acid to stain the nanoclusters. Figure 3 As shown in G and H, the morphology is regular spherical and evenly dispersed, and the particle size is consistent with the DLS results. In summary, both BI-PEG-PTX nanocarriers and BBR@PTX NPs have smaller PD I and higher surface potential, and therefore have good stability. Compared with the nanocarriers, BBR@PTX NPs have larger size and lower C potential, indicating that ABBR@PTX NPz micelles were successfully prepared.

[0084] Example 4: The above experiment proves the targeted inhibition and apoptosis effect of the obtained BBR@PTX NPs on MDA-MB-231 cells, and further research is needed on the treatment and toxicity of ectopic triple-negative breast cancer in vivo.

[0085] Furthermore, in this example, 6-week-old mice were subcutaneously injected with triple-negative breast cancer MDA-MB-231 cells. After 15 days, when the tumor volume grew to 100 mm, the tumor in the mouse disappeared. The mice were divided into six groups and administered via tail vein injection:

[0086] Experimental group 1 was injected with 5% glucose aqueous solution (150 μ l),

[0087] Experimental group 2 was injected with free BBR (4 mg / kg);

[0088] Experimental group three was injected with free PTX (1 mg / kg);

[0089] Experimental group 4 was injected with free BBR (4 mg / kg) and PTX (1 mg / kg);

[0090] Experimental group 5 was injected with PTX NPs (PTX 1 mg / kg);

[0091] Experimental group VI was injected with BBR@PTX NPs (BBR.4 mg / kg; PTX: 1 mg / kg);

[0092] The drug was administered once every 2 days. Before each administration, the weight of the mice was recorded and the tumor volume was measured. Tumor tissues were taken, weighed and photographed, and all tissues were subjected to the next histological analysis.

[0093] The tissues obtained from experimental groups 1 to 6 were fixed with 4% paraformaldehyde and paraffin, embedded, sectioned, and then stained with H&E. For immunohistochemical analysis, the pretreatment of tissue sections was as follows: first, the tumor sections were stained with anti-Ki-67, Bc-2, and TUNEL; then, the tumor sections were stained with anti-Ki-67, Bc-2, and TUNEL, and then observed under bright field microscopy;

[0094] Furthermore, six-week-old mice were subcutaneously injected with triple-negative breast cancer MDA-MB-231 cell lines, and after waiting for 15 days until the tumor volume grew to 500 mm , 100 u l I CG / BBR@PTX NPs micelle solution and free I CG solution ( n = 3) were injected into the tail vein of nude BALB / c mice, and the fluorescence images were observed and captured using the IVI S in vivo imaging system at 8, 24, 36, and 48 h, and the in vivo fluorescence images were collected at 48 h. The main fluorescence detection of the collected organs (heart, liver, spleen, lung, kidney, tumor) was performed for in vitro organ fluorescence imaging distribution;

[0095] The data obtained from the above two experiments were expressed as mean values ​​and standard deviations. The in vitro experiments were performed in three or five independent experiments, and each group was repeated at least three times. All data were analyzed by t-test or Pearson correlation coefficient using GraphPadPrism9.5 software.

[0096] Six-week-old mice were subcutaneously injected with triple-negative breast cancer MDA-MB-231 cells and waited for 15 days. When the tumor volume grew to 100 mm, the tumor in the mouse disappeared. The mice were divided into six groups and injected with drugs through the tail vein: experimental group one was injected with 5% glucose aqueous solution (150u l), experimental group two was injected with free BBR (4mg / kg); experimental group three was injected with free PTX (1mg / kg); experimental group four was injected with free BBR (4mg / kg) and PTX (1mg / kg); experimental group five was injected with PTX NPs (PTX1mg / kg); experimental group six was injected with BBR@PTX NPs (BBR.4mg / kg; PTX:1mg / kg) The results showed:

[0097] As attached Figure 2 As shown, the inhibition of cell viability by BR and PTX was concentration-dependent, and the half-maximal inhibitory concentrations of PTX and BBR on MDA-MB-231 cells were (0.71±0.39μg / mL) and (7.57±2.17μg / mL), respectively;

[0098] In addition to evaluating the synergistic effect of PTX and BBR, this example also used PTX alone or in combination with BBR to treat MDA-MB-231 cells, and evaluated the synergistic effect of free berberine and paclitaxel by different combination treatments of the two drugs, as shown in the attached Figure 2 As shown in C, the inhibition rate of 0.1μg / mLPTX combined with 1μg / mLBBR on 231 cell viability was 67.67%, which was higher than the inhibition rate of 0.1μg / mLPTX and 1μg / mLBBR combined (25.93±3.74% and 4.96±2.12%). The inhibition rate of 0.1μg / mLPTX combined with 4ug / mLBBR on MDA-MB-231 cell viability was 73.56%, which was also stronger than the arithmetic superposition effect of 0.1μg / mLPTX and 4ug / mLBBR on MDA-MB-231 cell viability (25.9.3±3.74% and 28.94±3.26%).

[0099] As attached Figure 2As shown in D, the synergistic score of PTX and BBR calculated in this example is 23.874. SynergyFinder3.0 is used to verify that berberine (BBR) can sensitize paclitaxel (PTX) and exert anti-tumor effects. The synergistic effect is analyzed by synergistic effect, and it is found that the synergistic effect of PTX and BBR is very strong when the concentration ratio is 1:10, which is an effective ratio in this example;

[0100] Attached Figure 2 A in the middle shows the cell viability of MDA-MB-231 cells treated with different concentrations of PTX; B shows the cell viability inhibition of MDA-MB-231 cells treated with BBR; C shows the MDA-MB-231 cells treated with PTX alone or in combination with BBR; D shows the synergy score of PTX combined with BBR calculated using Synergyfinder3.0 software. (n=3, *p<0.05, **; p<0.01, ***; p<0.001).

[0101] Example 5: Six-week-old mice were subcutaneously injected with triple-negative breast cancer MDA-MB-231 cell lines, and the tumor volume was grown to 500 mm after 15 days. 100 μl ICG / BBR@PTX NPs micelle solution and free ICG solution (n=3) were injected into the tail vein of nude BALB / c mice. Fluorescence images were observed and captured using the IVI S in vivo imaging system at 8, 24, 36 and 48 hours. After collecting in vivo fluorescence images at 48 hours, the mice were subjected to the main fluorescence detection at 48 hours. The collected organs (heart, liver, spleen, lung, kidney, tumor) were subjected to in vitro organ fluorescence imaging distribution. The results are shown in the attached figure. Figure 5 As shown in the figure, when the c50 of BBR is about 7.56675ug / mL and the IC50 of PTX is about 0.7081ug / mL, it is found that the combination of the two drugs has an inhibitory effect on MDA-MB-231 cells. When the concentration of BBR is 0.03125ug / mL and the concentration of PTX is 0.000781ug / mL, the inhibitory effect on MDA-MB-231 cells is very obvious, and the cell survival rate is about 50%. At this time, the dose of free single drug BBR and PTX is greatly reduced. Reducing the dose can effectively reduce the toxic effect on patients and increase patient compliance. Although the toxicity results of the synthesized nanomicelles on MDA-MB-231 cells are shown in the figure, at the equivalent delivery concentration of PTX, the BBR-encapsulated nanoparticles do reduce the cell viability value of stage III breast cancer cells, as shown in the attached figure. Figure 5In C and D, the results of colony formation and A0 / EB cell apoptosis staining also showed that the synthesized nanocapsules could better induce the colony formation of MDA-MB-231 cells and induce strong apoptosis compared with free BBR or PTX drug treatment. Flow cytometry experiments and fluorescence activated cell sorting methods proved the inhibitory effect of dual-loaded nanoparticles on triple-negative breast cancer cells. The apoptosis of 231 cells treated with different dose groups was analyzed and detected, as shown in the attached figure. Figure 5 As shown in Figure E, the apoptosis rates in the early and late stages of triple-negative breast cancer cells were significantly increased in the BBR combined with PTX treatment group (346 + 1.2%) compared with the BBR combined with PTX treatment group (18.77 + 0.89%) or the PTX treatment group (22.45 + 1.03%). The apoptosis rate of the dual drug-loaded nanoparticles BBR@PTX NPs was 42.3 ± 1.78%, so BBR@PTX NPs has a good in vitro anti-tumor effect.

[0102] Example 6: Inspired by the in vitro experimental results, we will continue to explore the anti-tumor activity of the synthesized BBR@PTX NPs in a triple-negative breast cancer xenograft model. Figure 8 BALB / c nude mice were used as transplanted tumor models. When the subcutaneous tumor volume grew to 100 mm 3 Treatment was given at 1:10 pm, and the tumor volume and body weight of the loaded mice were monitored for 14 days after administration. In the volume change curve, Figure 8 As shown in B, we found that the weight of mice in each group remained basically unchanged. In the tumor volume change curve, the tumor volume of mice in the Ctrl group increased by nearly 15 times, and the growth of tumors in the BBR+PTX group was significantly reduced. After BBR@PTX NPs treated mice, the added value of tumor cells was significantly reduced, and the turnover volume only increased by nearly 4-5 times, with the strongest therapeutic effect. After 14 days of treatment, the mice were euthanized, the tumor tissues were removed and the attached Figure 8 D and E in the middle further confirm the effective inhibitory effect of BBR@PTX NPs nanomicelles on xenograft tumor growth; Figure 8 A is a schematic diagram of the treatment process of striated muscle nude mice, B is the weight record of striated muscle nude mice during treatment, C is the tumor volume of striated muscle nude mice normalized to the baseline value during different drug treatments, D is the weight record of 231 tumors removed from striated muscle nude mice during different drug treatments, E is the physical image record of the removed tumors of striated muscle nude mice (n4), cell H&E staining, Ki-67, Bcl-2 immunohistochemical analysis and TUNEL fluorescence staining, scale bar: 100μm, *: p<0.05, ***: p<0.001.

[0103] For Examples 1 to 6, the nanocell delivery system with clinical transformation potential requires not only a strong therapeutic effect but also a high degree of biosafety. During the administration period, no obvious weight changes or mortality were observed in all groups of mice.

[0104] Further histological analysis of the main organs of rats (heart, liver, kidney) and results were analyzed by H&E staining. Fig. 9 As shown, there was no obvious damage to these tissues in the B8R@PTX NPs group of mice compared with the control group. However, there were some lesions in some tissues, such as obvious damage to the tumor in the free BBR group and the free PTX group, and obvious damage in the free BBR combined with PTX paper, while no obvious abnormalities were found in the BBR PTX NPs group compared with the Ctr group, indicating that the nanobeam co-delivery system we synthesized is not easy to cause obvious organ damage to mice. Fig. 9 These data indicate that BBR@PTX NPs have good biosafety and BI-PEG-PTX (PTXNPs) is a safe and effective tumor chemotherapy drug delivery carrier.

[0105] Example 7: The biodistribution of nanomedicine in nude mice bearing tumors was monitored using the IVI S in vivo imaging system to verify the tumor targeting ability of nanocell micelles BI.PEG-PTX (PTXNPs) and BBR@IPTXNPs. Fig.10 As shown in the figure, the in vivo biodistribution of B-PEG-PTX was evaluated using nanomaterials loaded with ICG. BALB / c nude mice were injected via the tail vein and photographed at 8, 24, 36, and 48 hours. The enhancement of PTxNPs in mice at different time periods was monitored by a visual IS imaging system, as shown in the attached figure. Fig.10 As shown in Figure B, compared with the Free I CG group, PTXNPs began to target tumors 8 hours after administration, and the fluorescence hormone of ICG@PTX NPs at the tumor site increased significantly at 24 hours. In contrast, in mice injected with an equal amount of free I CG, the fluorescence of free I CG was detected at the tumor site, indicating that free I CG itself lacks the accumulation ability to target tumors. After 48 hours, the hormone-treated mice were euthanized, and the main organ tissues (heart, liver, spleen, lung, kidney and tumor) were imaged. The results are shown in the attached figure. Fig.10As shown in Figure C, the results showed that the fluorescence intensity of the tumor tissue in the PTXNPs group was the highest, followed by the liver tissue, and no obvious fluorescence signal aggregation was observed in other tissues. However, in mice injected with free IgG, no fluorescence was detected in the isolated tumors, and the fluorescence mainly appeared in the liver and kidneys, indicating that IgG was metabolized by the liver and kidneys. These data indicate that BIPEG-PTX achieved effective aggregation at the diseased site and may trigger clearance by the liver and kidneys. Finally, the final two drugs, nano-BBR@PTX NPs, were loaded with IgG and injected into three nude mice through the tail vein, and photographed on the 1st, 2nd, and 3rd days. The experimental results are shown in the attached figure. Fig.10 As shown in D and E, it is also proved that the ICG@BBR PTX NPs group has the ability to deliver drugs to tumors in a targeted manner. The results of in vitro organ imaging are consistent with the results of in vivo fluorescence imaging. The fluorescence of the ICG@BBRPTX NPs group is largely accumulated in the tumor, and there is little fluorescence in other organs. The fluorescence of the free ICG group is strong, mainly in the liver and kidneys, and the fluorescence intensity gradually weakens over time, indicating that the free ICG is removed. These results suggest that the nanocellular delivery system has effective tumor targeting ability relative to free drugs, which is mainly due to its biotin receptor-mediated active tumor targeting and passive EPR effect on tumor tissue. The superior tumor accumulation ability of BBR@PTX NPs achieves effective in vivo anti-tumor efficacy and minimal side effects.

[0106] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0107] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A novel nanoparticle drug delivery system for targeted treatment of TNBC, characterized in that: The preparation of the novel nanoparticle drug delivery system comprises the following specific steps: S1: Synthesis; Synthesis of BI-PEG-COOH and BI-PEG-PTX with tumor targeting ability; S2: Material characterization: Fourier transform infrared spectroscopy and 1H NMR were used to verify the structural characteristics of BI-PEG-COOH and BI-PEG-PTX samples obtained in S1; S3: Preparation of nanomicelles; after verification, nanomicelles BBR@PTX NPs were prepared based on BI-PEG-COOH and BI-PEG-PTX obtained in S1.

2. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 1, characterized in that: The synthesis steps of the BI-PEG-COOH obtained in S1 include the following specific steps: 0.4 g BI, 0.376 g EDC and 0.188 g NHS were added to 3 ml dimethyl sulfoxide, activated at 40°C for 2 h, and then 0.2 g NH2-PEG-COOH was added and reacted for 24 h. The resulting reaction solution was dialyzed in deionized water using a 2000 kDa dialysis bag, and the deionized water was replaced every 30 min. The solution was lyophilized after 1 day to obtain the product BI-PEG-COOH.

3. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 1, characterized in that: The synthesis steps of BI-PEG-PTX obtained in S1 include the following specific steps: 3 mg PTX and 10.2 mg EDC were added together to 6 ml dimethyl sulfoxide, completely dissolved in a flask, and activated at 40° C. for 6 hours to obtain a reaction solution. The condensing agent, catalyst and 24 mg BI-PEG-COOH were added together to 12 ml dimethyl sulfoxide, completely dissolved in a flask and activated at 40° C. for 30 minutes to obtain another reaction solution. The two obtained reaction solutions were mixed and reacted at 40° C. under light-shielding conditions for 48 hours. The reaction solution was dialyzed with a 2000 KDa dialysis bag, deionized water was filtered, and the solution was freeze-dried after 1 day to obtain the product BI-PEG-PTX with a hydrophobic feed ratio of 1:

8.

4. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 1, characterized in that: The condensation agent is 1-ethyl-carbodiimide hydrochloride, and the specific amount is 14.4 mg. The catalyst is 4-dimethylaminopyridine, and the specific amount is 14.4 mg.

5. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 1, characterized in that: The S2 verification of the obtained BI-PEG-COOH and BI-PEG-PTX samples includes the following specific steps: Take a small amount of the BI-PEG-COOH and BI-PEG-PTX sample powder, dry the water under an infrared lamp, grind and press with KBr to form a 500 cm 2 Up to 4000cm 2 The infrared spectrum of the sample was obtained by scanning the fragments within the range, and then 10 mg BI-PEG-COOH and BI-PEG-PTX samples were dissolved in 1 ml of tritiated dimethyl sulfoxide, loaded into the infrared spectrometer NMR sample tube, and the absorption 1HNMR spectrum was scanned from 0ppm to 15ppm to complete the verification of the structural characteristics of the BI-PEG-COOH and BI-PEG-PTX samples.

6. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 1, characterized in that: The preparation steps of the nano-micelle BBR@PTX NPs include the following specific steps: 8 mg BI-PEG-PTX nanoconjugate was dissolved in 6 ml dimethyl sulfoxide, loaded into a 2000 kDa dialysis bag, dialyzed in deionized water, changing the water once every 1 hour, and dialyzed for 12 hours to obtain loaded PTX NPs. 1 mg BBR and 8 mg BI-PEG-PTX were dissolved in 6 ml dimethyl sulfoxide and mixed in the obtained PTX NPs, mixed evenly, loaded into a 2000 kDa dialysis bag, dialyzed for 12 hours, changing the water once, and dialyzed in deionized water for 12 hours to obtain the dual-loaded nanoparticles BBR@PTX NPS.

7. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 6, characterized in that: The preparation of the novel nanoparticle drug delivery system also includes evaluating the effectiveness of the BBR@PTX NPs obtained according to S3, including characterization of the BBR@PTX NPs under a microscope, and evaluation of drug loading and release rate.

8. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 7, characterized in that: The characterization of the BBR@PTX NPs under a microscope includes the following steps: The prepared PTX NPs and BBR@PTX NPs nanomicelle solutions with a concentration of 1 mg / ml were placed in a dynamic light scattering instrument. Each sample was tested three times for 1 minute each time. Transmission electron microscopy was used to characterize the size and morphology of the synthesized nanoclusters. The samples were observed by dynamic light scattering and dropped onto a copper mesh coated with a carbon support film. The samples were dried naturally after the solvent dried, and then dyed with a dyeing catalyst. Finally, their morphology was observed and scanned in a transmission electron microscope to complete the characterization of the BBR@PTX NPs under the microscope and determine whether the prepared BBR@PTX NPs met the requirements.

9. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 8, characterized in that: The dyeing catalyst is 2% phosphotungstic acid.

10. A novel nanoparticle drug delivery system for targeted treatment of TNBC according to claim 7, characterized in that: The specific operation of the BBR@PTX NPs drug loading and release rate evaluation is as follows: 5 mg of freeze-dried BBR@PTX NPs were dissolved in 5 ml of distilled water to prepare a BBR@PTX NPs solution with a concentration of 1 mg / ml, and the UV absorbance was measured at 230 °C. The concentration was calculated according to its standard curve, and the loading amount was calculated using the wavelength method; 10 mg of free drug and drug-loaded nanoparticles were accurately weighed and dissolved in 5 ml of PBS, pH 7.

4. After dissolution, the mixture was transferred to a 2000 kDa dialysis bag. The dialysis bag was dialyzed in a small beaker containing 50 mL of PBS, pH 7.4 or 50 mL of PBS, pH 5.5, respectively. The dialysis bag was shaken on a shaker at 37°C and 75 pm. The PBS in the dialysis bag was replaced at different times, and the absorbance of the volume Vt and PBS was measured at 230 nm and 340 nm, respectively, to calculate the contents of the two released drugs.

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