An inhibitor targeting delivery system for highly malignant triple-negative breast cancer and a preparation method and application thereof

Abstract

The present application relates to the technical field of cancer cell inhibitor targeted delivery system, in particular to an inhibitor targeted delivery system for high-malignant triple-negative breast cancer and a preparation method and application thereof. The delivery system is a nanoparticle structure, which comprises, from inside to outside, a core, a carrier skeleton layer, a crosslinked shell layer and a targeting modification layer. The core is a miR-5134-5p inhibitor, the carrier skeleton layer is polyhistidine, the crosslinked shell layer is methoxy polyethylene glycol-disulfide-carboxyl, and the targeting modification layer is an m6A short nucleic acid sequence. The present application uses the m6A short nucleic acid sequence as a targeting ligand, which can specifically bind to tumor cells with high expression of IGF2BP1, realize active targeting enrichment of the delivery system, trigger carrier disintegration only at tumor cells, realize controllable release of the miR-5134-5p inhibitor, avoid drug leakage in normal tissues, and is suitable for precise treatment of high-malignant triple-negative breast cancer, and has a broad clinical application prospect.

Description

Technical Field

[0001] This invention relates to the field of targeted delivery systems for cancer cell inhibitors, specifically to a targeted delivery system for inhibitors of highly malignant triple-negative breast cancer, its preparation method, and its application. Background Technology

[0002] Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is negative for estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It is characterized by poor differentiation, high invasiveness, high recurrence and metastasis rates, and poor prognosis. Due to the lack of clearly defined molecular target sites, traditional treatment primarily relies on chemotherapy. However, chemotherapy drugs lack tumor specificity, easily leading to severe systemic toxicity and side effects, and patients are prone to developing drug resistance, thus requiring significant improvement in clinical treatment efficacy. In recent years, small nucleic acid drugs (such as microRNA inhibitors) have become a research hotspot in TNBC treatment due to their advantage of precisely regulating tumor-related signaling pathways. Among them, members of the miR-513 family (including miR-5134-5p) have been shown to promote breast cancer progression through mechanisms such as inhibiting progesterone receptor expression and regulating tumor stem cell characteristics, and their inhibitors have significant anti-tumor potential.

[0003] However, the clinical translation of small nucleic acid drugs faces numerous delivery challenges: First, insufficient targeting; traditional nanocarriers often rely on the high permeability and retention effect (EPR) of tumor tissue for passive targeting, resulting in low enrichment efficiency in tumor tissue and easy damage to normal tissues. Second, poor release controllability; the lack of a precise response mechanism to the tumor microenvironment leads to premature leakage of the drug during circulation, reducing treatment efficiency and increasing toxic side effects. Furthermore, existing active targeting ligands (such as folic acid, peptides, and nucleic acid aptamers) suffer from insufficient specificity, susceptibility to shielding, or immunogenicity, limiting the effectiveness of targeted delivery.

[0004] Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is highly expressed in various malignant tumors, including TNBC. By regulating the mRNA stability and translation of target genes such as MYC and CD44, it promotes tumor cell proliferation, invasion, and drug resistance. Its high expression level is closely related to poor patient prognosis, making it an ideal target for precision treatment of TNBC. m6A (N6-methyladenosine) modification is one of the most common epigenetic modifications on eukaryotic mRNA. IGF2BP1, as a typical m6A reading protein, can specifically recognize and bind to m6A modification sites on RNA molecules. Summary of the Invention

[0005] The technical problem to be solved by this invention is to provide a targeted delivery system for inhibitors of highly malignant triple-negative breast cancer, its preparation method and application, using the m6A short nucleic acid sequence as a targeting ligand to specifically bind to TNBC cells with high expression of IGF2BP1, while constructing a reduction-responsive nanocarrier to solve the technical problems of poor targeting and uncontrollable release of small nucleic acid drugs, thus providing a new solution for TNBC treatment.

[0006] To achieve the above objectives, the present invention provides the following technical solution: A targeted delivery system for an inhibitor of highly malignant triple-negative breast cancer, wherein the delivery system is a nanoparticle structure comprising, from the inside out, a core, a carrier framework layer, a cross-linked outer shell layer, and a targeting modification layer; The core is a miR-5134-5p inhibitor, the carrier backbone layer is polyhistidine, the cross-linked outer shell layer is methoxy polyethylene glycol-disulfide bond-carboxyl group, and the targeting modification layer is an m6A short nucleic acid sequence.

[0007] Furthermore, the polyhistidine is wrapped around the outside of the miR-5134-5p inhibitor, the methoxy polyethylene glycol-disulfide bond-carboxyl group is wrapped around the outside of the polyhistidine, and multiple m6A short nucleic acid sequences are covalently coupled to the surface of the methoxy polyethylene glycol-disulfide bond-carboxyl group.

[0008] Furthermore, the 5′ or 3′ end of the m6A short nucleic acid sequence is modified with an amino group, which is coupled to the carboxyl group of the methoxy polyethylene glycol-disulfide bond-carboxyl group.

[0009] Furthermore, the m6A short nucleic acid sequence can specifically recognize and bind to the IGF2BP1 protein in highly malignant triple-negative breast cancer cells, achieving targeted delivery.

[0010] A method for preparing an inhibitor targeted delivery system for highly malignant triple-negative breast cancer. (1) The miR-5134-5p inhibitor solution was slowly added dropwise to the polyhistidine solution at a nitrogen-to-phosphorus ratio of (10-15):1. The mixture was stirred while adding the solution and allowed to stand at room temperature to react. The PHH / miR-5134-5p inhibitor nanoparticles were formed by self-assembly using the interaction of positive and negative charges. (2) Add Mal-SS-COOH solution to the PHH / miR-5134-5p inhibitor nanoparticle solution obtained in step (1), mix well, add mPEG-SH solution, and react at room temperature in the dark. Mal-SS-COOH and mPEG-SH undergo a specific Michael addition reaction, forming an mPEG-SS-COOH shell in situ on the surface of the PHH / miR-5134-5p inhibitor nanoparticles, to obtain mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticles; (3) Add EDC•HCl and NHS to the mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticle solution obtained in step (2) and activate the carboxyl groups on the surface of the support at room temperature; add an amino-modified m6A short nucleic acid sequence solution and react at room temperature in the dark to achieve covalent coupling between the m6A short nucleic acid sequence and the mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticles to obtain the m6A / mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticle delivery system.

[0011] Furthermore, In step (1), Polyhistidine solution was prepared at a concentration of 1 mg / mL using 0.01 M acetate-sodium acetate buffer at pH 4.5-5.5; miR-5134-5p inhibitor solution was prepared at a concentration of 1-20 μM using DEPC enzyme-free water. The reaction time at room temperature is 25-35 min.

[0012] Furthermore, In step (2), The Mal-SS-COOH solution concentration was 1 mg / mL, prepared using DEPC enzyme-free water; The concentration of mPEG-SH was 5 mg / mL, prepared using 0.01M acetate-sodium acetate buffer solution at pH 4.5-5.5; The mass ratio of Mal-SS-COOH to PHH added in step (1) is 1:6-1:9; The molar ratio of Mal-SS-COOH to mPEG-SH is 1:1 to 1:1.2. The reaction time at room temperature in the dark is 30-50 minutes. After the reaction was completed, the mixture was centrifuged at 5000-8000 rpm for 30-40 min using a 3k MWCO ultrafiltration tube and then reconstituted with 1×PBS buffer to complete the buffer system replacement.

[0013] Furthermore, In step (3), The concentration of the amino-modified m6A short nucleic acid sequence solution was 0.1 mg / mL, and it was prepared using DEPC enzyme-free water. The final concentrations of EDC•HCl and NHS were both 5 mM. The activation time at room temperature was 30-60 min. After activation, the free activating reagents were removed by ultrafiltration through a 3 kMW CO2 ultrafiltration tube and then reconstituted with 1×PBS.

[0014] Furthermore, In step (3), The mass ratio of the amino-modified m6A short nucleic acid sequence to the Mal-SS-COOH used in step (2) is 1:30-1:50, and the reaction time is 12-14 h at room temperature in the dark. After the reaction was completed, the sample was centrifuged at 5000-8000 rpm for 30-40 min using a 3k MWCO ultrafiltration tube. The flow-through was discarded, and 1×PBS buffer was added to the ultrafiltration tube to reconstitute the retentate. The centrifugation and washing were repeated 3 times.

[0015] Application of a targeted delivery system for an inhibitor of highly malignant triple-negative breast cancer in the preparation of drugs for treating highly malignant triple-negative breast cancer.

[0016] Compared with the prior art, the beneficial effects of the present invention are: This application uses the m6A short nucleic acid sequence as a targeting ligand, which can specifically bind to tumor cells that highly express IGF2BP1, enabling active targeted enrichment of the delivery system. It triggers vector disintegration only at tumor cells, achieving controlled release of miR-5134-5p inhibitors and avoiding drug leakage in normal tissues. It is suitable for the precision treatment of highly malignant triple-negative breast cancer and has broad clinical application prospects. Attached Figure Description

[0017] Figure 1 This is a WB image showing the results of detecting IGF2BP1 protein expression levels in cancer cells of this invention. Figure 2 This is a flow cytometry diagram of the blank control group of this invention; Figure 3 This is a flow cytometry diagram of the free miR-5134-5p inhibitor group of this invention; Figure 4 Flow cytometry diagram of the delivery system group of the present invention; Figure 5 This is a photograph of a tumor in vivo in mice, as shown in the present invention. Figure 6 This is a comparison chart of tumor weight in mice in vivo experiments according to the present invention; Figure 7 This is the immunohistochemical result of the blank control group of this invention; Figure 8 These are the immunohistochemical results of the free miR-5134-5p inhibitor group of this invention; Figure 9 The results of immunohistochemical experiments on the delivery system group of this invention are shown. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] The reagents used in this embodiment are: Polyhistidine (PHH): molecular weight 15 kDa, purity ≥95%, purchased from Sigma-Aldrich; Mal-SS-COOH: Purchased from Xi'an Qiyue Biotechnology; mPEG-SH: molecular weight 5 kDa, purchased from MedChemExpress (MCE); Amino-modified m6A short nucleic acid sequence: 5'-NH2 modified (C6 linker arm), purity ≥98%, purchased from Sangon Biotech. EDC•HCl: purity ≥99%, purchased from Sigma-Aldrich; NHS: Purity ≥98%, purchased from Sigma-Aldrich; RNA extraction kit, purchased from QIAGEN (Cat no. / ID. 217204); The RNA rapid quantification kit was purchased from Sangon Biotech.

[0020] A targeted delivery system for an inhibitor of highly malignant triple-negative breast cancer, wherein the delivery system is a nanoparticle structure comprising, from the inside out, a core, a carrier framework layer, a cross-linked outer shell layer, and a targeting modification layer.

[0021] The core is a miR-5134-5p inhibitor, the carrier backbone layer is polyhistidine, the cross-linked outer shell layer is methoxy polyethylene glycol-disulfide bond-carboxyl group (mPEG-SS-COOH), and the targeting modification layer is an m6A short nucleic acid sequence. The polyhistidine encapsulates the outside of the miR-5134-5p inhibitor, and the mPEG-SS-COOH encapsulates the outside of the polyhistidine. Multiple m6A short nucleic acid sequences are covalently coupled to the surface of the mPEG-SS-COOH. The 5′ end of each m6A short nucleic acid sequence is modified with an amino group (-NH2), which couples with the carboxyl group of mPEG-SS-COOH via an EDC / NHS condensation reaction, ensuring the stable binding of the m6A short nucleic acid sequences to the mPEG-SS-COOH surface. These m6A short nucleic acid sequences can specifically bind to the IGF2BP1 protein in highly malignant triple-negative breast cancer cells, achieving targeted delivery.

[0022] The miR-5134-5p inhibitor was prepared by chemical synthesis of the nucleic acid sequence disclosed in patent CN 119925612 B, and the nucleotide base sequence is shown in SEQ ID NO. 1 of the sequence listing.

[0023] The m6A short nucleic acid sequence is a synthetic RNA oligonucleotide in which adenine A is methylated at the N6 position (m6A). The m6A short nucleic acid sequence used in this embodiment is 5'-GGACU-3', in which adenine A is methylated at the N6 position (m6A), which can specifically bind to IGF2BP1 protein, and the 5' end is modified with an amino group.

[0024] A method for preparing an inhibitor targeted delivery system for highly malignant triple-negative breast cancer. (1) Polyhistidine was dissolved in 0.01M acetate-sodium acetate buffer at pH 5.0 to prepare a PHH solution with a concentration of 1 mg / mL. The miR-5134-5p inhibitor was dissolved in DEPC enzyme-free water to prepare a miR-5134-5p inhibitor solution with a concentration of 10 μM. 375 μL of miR-5134-5p inhibitor solution was slowly added dropwise to 3.0 mL of PHH solution while stirring and mixing. The solution was allowed to stand at room temperature for 30 min. The PHH / miR-5134-5p inhibitor nanoparticles were formed by self-assembly using the interaction of positive and negative charges. (2) Prepare a 5 mg / mL mPEG-SH solution using 0.01M, pH 5.0 acetate-sodium acetate buffer. Dissolve Mal-SS-COOH in DEPC enzyme-free water to prepare a 1 mg / mL Mal-SS-COOH solution. Add 400 μL of Mal-SS-COOH solution to the PHH / miR-5134-5p inhibitor nanoparticle solution obtained in step (1), mix well, and then add 147 μL of mPEG-SH solution. Stir magnetically at 200 rpm for 10 min to mix well. React at room temperature in the dark for 45 min. Mal-SS-COOH and mPEG-SH undergo a specific Michael addition reaction, forming an in-situ mPEG-SS-COOH shell on the surface of the PHH / miR-5134-5p inhibitor nanoparticles, thus obtaining mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticles. After the reaction is complete, use a 3kMWCO Centrifuge the ultrafiltration tube at 7000 rpm for 30 min, then reconstitute with 1×PBS buffer to a final volume of 2.0 mL to complete the buffer system replacement. (3) The amino-modified m6A short nucleic acid was dissolved in DEPC enzyme-free water to prepare an m6A short nucleic acid solution with a concentration of 0.1 mg / mL. EDC•HCl and NHS mixture was added to the nanoparticle solution obtained in step (2) until the final concentration was 5 mM. The solution was activated at room temperature for 45 min to activate the carboxyl groups on the surface of the carrier. After the reaction was completed, the solution was added to a 3k MWCO ultrafiltration tube and centrifuged at 7000 rpm for 30 min at 4℃. 1×PBS was added to the ultrafiltration tube to reconstitute to 1.9 mL. 100 μL of amino-modified m6A short nucleic acid solution was added, stirred and mixed, and reacted at room temperature in the dark for 12 h to achieve covalent coupling of the m6A short nucleic acid sequence with the mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticles. After the reaction was completed, the solution was added to a 3k MWCO ultrafiltration tube and centrifuged at 7000 rpm for 30 min at 4℃. The flow-through was discarded and 1×PBS was added to the ultrafiltration tube. The retentate was reconstituted with buffer, and the mixture was centrifuged and washed three times. The final volume was adjusted to 2.0 mL to obtain the m6A / mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticle delivery system.

[0025] The assembly principle of this application is as follows: In an acidic environment, polyhistidine is fully protonated, making the imidazole group of polyhistidine positively charged and the phosphate backbone of miR-5134-5p inhibitor negatively charged. When the two come into contact, the positively charged PHH group inserts into the gap of the negatively charged phosphate backbone of miR-5134-5p inhibitor, compressing the long-chain nucleic acid of miR-5134-5p inhibitor into a nanoscale dense core, forming a dense core structure. The maleimide (Mal) of Mal-SS-COOH undergoes a specific Michael addition reaction with the thiol group (-SH) of mPEG-SH, generating mPEG-SS-COOH in situ on the surface of nanoparticles; The 5′ end of the m6A short nucleic acid sequence is modified with -NH2. EDC / NHS activates the carboxyl group of mPEG-SS-COOH to generate an active ester, which then forms a stable amide bond with the amino group of the m6A short nucleic acid sequence, thus stably fixing the m6A short nucleic acid sequence on the surface of mPEG-SS-COOH.

[0026] 1. DLS detection: The particle size, PDI, and Zeta potential of the PHH / miR-5134-5p inhibitor nanoparticles, mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticles, and m6A / mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticle delivery system obtained in steps (1), (2), and (3) of the above preparation method were detected using dynamic light scattering. The results are shown in Table 1 below.

[0027] Results analysis: The PHH / miR-5134-5p inhibitor nanoparticles have an average particle size of 88±12 nm, a PDI of 0.18±0.03, and a Zeta potential of +25±3 mV. In a buffer system with pH 4.5-5.5, the imidazole matrix of the polyhistidine side chain is protonated and carries a strong positive charge. It then self-assembles with the negatively charged miR-5134-5p inhibitor through electrostatic attraction, simultaneously nucleating to form a dense and uniform core structure. Excess unbound PHH is distributed on the particle surface, enriching the particle surface with positive charge, thus exhibiting a strong positive potential. The low PDI indicates a narrow particle size distribution, good dispersibility, and no obvious aggregation.

[0028] The average particle size of the mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticles increased to 126±15 nm, the PDI slightly increased to 0.20±0.04, and the Zeta potential flipped from +25±3 mV to -15±3 mV. The slight increase in particle size is mainly due to the assembly of mPEG-SS-COOH on the surface of PHH / miR nanoparticles, forming a flexible hydrophilic shell. The slight increase in PDI is related to the slight difference in the grafting density of the shell, but it still maintains a unimodal distribution without significant particle size broadening or aggregation. The Zeta potential changed from positive to negative, partly because the steric hindrance of the PEG chain shielded the positive charge of the core, and partly because the carboxyl groups at the end of the shell ionized to form negatively charged groups. This potential reversal indirectly proves the successful coating of the PEG shell.

[0029] The average particle size of the m6A / mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticle delivery system further increased slightly to 136±18 nm, the PDI rose to 0.22±0.05, and the Zeta potential changed to -10±2 mV. The slight increase in particle size is attributed to the short m6A nucleic acid being coupled to the amino group on the PHH surface via EDC / NHS activation of the carboxyl group, grafting onto the PEG shell surface. The PDI remained below 0.3, indicating that the particles still possessed good dispersibility. The decrease in the negative charge of the Zeta potential is mainly because the coupling of the EDC / NHS-activated carboxyl group with the aminated m6A consumed some of the negatively charged carboxyl groups on the shell surface, leading to a decrease in the negative charge density on the particle surface. This change can serve as a direct characterization of successful m6A coupling.

[0030] The above results confirm the successful preparation of a uniform and potential-controllable m6A / mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticle delivery system.

[0031] 2. Western blot (WB) detection: Triple-negative breast cancer cells were seeded into culture dishes and cultured until the logarithmic growth phase, then divided into 3 groups: Blank control group (no treatment); free miR-5134-5p inhibitor group (miR-5134-5p inhibitor added to a final concentration of 50 nM); delivery system group (delivery system added to bring the final concentration of miR-5134-5p inhibitor in the system to 50 nM). After culturing for another 48 h, cells from each group were collected, washed twice with pre-cooled PBS buffer, and lysed with RIPA lysis buffer on ice for 30 min, during which time the cells were repeatedly pipetted. The cells were then centrifuged at 4°C and 12,000 rpm for 15 min, and the supernatant was collected to obtain total cell protein. Western blot was performed to detect the expression level of IGF2BP1 protein in the cells.

[0032] Western blot (WB) results of IGF2BP1 protein expression levels in cancer cells of each group are as follows: Figure 1 As shown, Results analysis: Compared with the blank control group, the relative expression level of IGF2BP1 protein in the free miR-5134-5p inhibitor group was slightly decreased, but the difference was not significant. This is because the free miR-5134-5p inhibitor is easily degraded by nucleases secreted by cells in the culture supernatant, making it difficult to exert a regulatory effect. The relative expression level of IGF2BP1 protein in the delivery system group was significantly decreased, indicating that the delivery system can effectively deliver the miR-5134-5p inhibitor into triple-negative breast cancer cells and exert a downregulation effect on IGF2BP1 protein expression, thus verifying the drug loading and release function of the delivery system.

[0033] 3. Flow cytometry detection of CD44 / CD24 tumor stem cell phenotype: CD44 is a characteristic surface marker of triple-negative breast cancer stem cells; high expression indicates strong cell stemness and vigorous proliferation and invasion. CD24 is a differentiation-related marker; high expression indicates reduced cell stemness and decreased malignancy. The CD44 / CD24 ratio is a classic tumor stem cell phenotype of triple-negative breast cancer cells, and its ratio directly reflects the strength of tumor stemness. IGF2BP1 is a key regulatory protein for maintaining tumor stem cell stemness. This experiment evaluated the role of delivery systems in inhibiting tumor stemness and reducing malignant phenotypes by detecting changes in the CD44 / CD24 ratio.

[0034] Experimental grouping: Triple-negative breast cancer cells were divided into 3 groups: blank control group (no treatment); free miR-5134-5p inhibitor group (miR-5134-5p inhibitor added to a final concentration of 50 nM); and delivery system group (delivery system added to make the final concentration of miR-5134-5p inhibitor in the system 50 nM), and cultured for 48 h respectively.

[0035] CD44-PE / CD24-FITC double staining flow cytometry was used to detect the expression levels of the tumor stem cell phenotypes CD44 and CD24 in triple-negative breast cancer cells in each group. Quadrant analysis was performed with CD44 as the x-axis and CD24 as the y-axis.

[0036] Results analysis: Figure 2 This is a flow cytometry image of the blank control group. The cells are predominantly of the high-malignancy tumor stem cell phenotype, with CD44 cells in the lower right quadrant. + / CD24 - The cell proportion was 86.25%, while CD44 in the upper left quadrant... - / CD24 + Low-grade malignant differentiated cells accounted for only 10%, indicating that the tumor cells had strong stemness and vigorous proliferation and invasion capabilities.

[0037] Figure 3 This is a flow cytometry image of the free miR-5134-5p inhibitor group, with CD44 in the lower right quadrant. + / CD24 - Cell percentage was 85.38%, with CD44 in the upper left quadrant. - / CD24 + Low-grade malignant cells accounted for only 13.58%, which was not significantly improved compared with the blank control group.

[0038] Figure 4 Flow cytometry plot of the delivery system group, lower right quadrant CD44+ / CD24 - The cell percentage decreased to 63.5%, while CD44 in the upper left quadrant... - / CD24 + The proportion of low-grade malignant cells increased significantly to 32.67%, which was significantly different from the control group.

[0039] In this experiment, treatment with the targeted delivery system significantly inhibited intracellular IGF2BP1 expression, directly leading to a decrease in the proportion of tumor stem cells and a reduction in malignant phenotype. The delivery system group showed a reduction in the high proportion of cells in the lower right quadrant and an increase in low-malignant cells in the upper left quadrant. This was achieved by downregulating IGF2BP1, effectively weakening tumor stemness and eliminating or inhibiting highly malignant and invasive cancer stem cell populations. The overall malignancy and proliferative capacity of the cells were reduced, indicating that the delivery system can achieve anti-cancer and killing effects on triple-negative breast cancer cells.

[0040] 4. In vivo experiments in mice: Establishment of a nude mouse xenograft tumor model: Nine SPF-grade female Balb / C nude mice (4-5 weeks old, weighing 16-18 g) were used as experimental animals. Triple-negative breast cancer cells in good growth condition were digested and centrifuged, and the cell density was adjusted to 2.5 × 10⁷ / mL with PBS. Under aseptic conditions, 0.2 mL of the prepared triple-negative breast cancer cell suspension was subcutaneously injected into the right axilla of each mouse. Visible nodules appeared subcutaneously after 10 days. The nine mice were randomly divided into three groups: a blank control group, a free miR-5134-5p inhibitor group, and a delivery system group, with three mice in each group. Each experimental group received a 100 μL injection via tail vein. The blank control group received 0.9% saline; the free miR-5134-5p inhibitor group received miR-5134-5p inhibitor at a concentration adjusted to 300 nmol / mL with sterile PBS; and the delivery system group received the same final dose as the free miR-5134-5p inhibitor group. The drug was administered once every 3 days for a total of 8 times. After 3 weeks of administration, the mice were euthanized by cervical dislocation under anesthesia, the transplanted tumor was removed, and the tumor weight was measured.

[0041] Results analysis: Figure 5 This is a photograph of a tumor in a mouse in vivo experiment. Figure 6This is a comparison of tumor weights in mice during in vivo experiments. The tumor weight in the blank control group was 1.62±0.28g, the tumor weight in the free miR-5134-5p inhibitor group was 0.97±0.16g, and the calculated tumor inhibition rate was 13.01%. The tumor weight in the delivery system group was 0.40±0.18g, and the calculated tumor inhibition rate was 75.31%. Compared with the free miR-5134-5p inhibitor group, the tumor weight showed a significant trend of inhibiting the growth of triple-negative breast cancer cells.

[0042] 5. Immunohistochemical experiment: Immunohistochemical experiments were used to verify the differential expression of IGF2BP1 protein in tumor tissues and normal tissues, as well as the enrichment of the delivery system in mouse tumor tissues. Tumors obtained in Experiment 4 (in vivo mouse experiment) were fixed with 4% paraformaldehyde and prepared into immunohistochemical paraffin sections.

[0043] Figure 7 The results of the immunohistochemical experiment are for the blank control group. Figure 8 The results of immunohistochemical experiments on the free miR-5134-5p inhibitor group are as follows. Figure 9 The results of immunohistochemical experiments for the delivery system group.

[0044] Results analysis: In the blank control group, IGF2BP1 protein showed strong positive expression and dense staining signal in tumor tissue. In the free miR-5134-5p inhibitor group, IGF2BP1 protein also showed strong positive expression, but the staining signal was slightly reduced by visual observation. In the tumor group treated with the delivery system, the positive staining intensity of IGF2BP1 protein was significantly reduced, the brown signal became lighter and the distribution was sparse. This proves that the delivery system can specifically identify and enrich tumor tissue with high IGF2BP1 expression, and effectively downregulate IGF2BP1 protein expression to achieve targeted therapy.

[0045] Application of a targeted delivery system for an inhibitor of highly malignant triple-negative breast cancer in the preparation of drugs against highly malignant triple-negative breast cancer: The drug is administered via intravenous injection. After entering the body, the delivery system achieves long circulation through the mPEG-SS-COOH shell, avoiding clearance by the body's immune system. The short m6A nucleic acid sequence on the surface specifically recognizes and binds to the IGF2BP1 protein on the surface and inside tumor cells that are highly expressed by IGF2BP1, thus enabling the delivery system to actively target and enrich the tumor tissue. After the delivery system is endocytosed by tumor cells into the endosomes, the low pH environment of the endosomes triggers protonation of polyhistidine, producing a proton sponge effect. This causes the carrier backbone layer to swell and disintegrate. Simultaneously, under the influence of a high concentration of reduced glutathione in the tumor cell, the disulfide bonds in mPEG-SS-COOH break, and the cross-linked outer shell disintegrates. Under the synergistic effect of these two responses, the miR-5134-5p inhibitor is released from the carrier, exerting its effect of inhibiting tumor cell proliferation, invasion, and metastasis, while reducing the toxic side effects of the drug on normal tissues.

[0046] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent structural or procedural transformations made based on the content of the present invention specification and drawings, or direct or indirect applications in other related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A targeted delivery system for an inhibitor of highly malignant triple-negative breast cancer, characterized in that: The delivery system has a nanoparticle structure, which, from the inside out, includes a core, a carrier framework layer, a cross-linked outer shell layer, and a targeted modification layer. The core is a miR-5134-5p inhibitor, the carrier backbone layer is polyhistidine, the cross-linked outer shell layer is methoxy polyethylene glycol-disulfide bond-carboxyl group, and the targeting modification layer is an m6A short nucleic acid sequence. The m6A short nucleic acid sequence can specifically recognize and bind to the IGF2BP1 protein in triple-negative breast cancer cells, achieving targeted delivery; The nucleotide sequence of the miR-5134-5p inhibitor is shown in SEQ ID NO. 1; The m6A short nucleic acid sequence is 5'-GGACU-3', and adenine A is methylated at the N6 position; The m6A short nucleic acid sequence is modified with an amino group at the 5′ or 3′ end, which is coupled to the carboxyl group of methoxy polyethylene glycol-disulfide bond-carboxyl group.

2. The inhibitor targeted delivery system for highly malignant triple-negative breast cancer according to claim 1, characterized in that: The polyhistidine is wrapped around the outside of the miR-5134-5p inhibitor, the methoxy polyethylene glycol-disulfide bond-carboxyl group is wrapped around the outside of the polyhistidine, and multiple m6A short nucleic acid sequences are covalently coupled to the surface of the methoxy polyethylene glycol-disulfide bond-carboxyl group.

3. A method for preparing an inhibitor targeted delivery system for highly malignant triple-negative breast cancer according to any one of claims 1-2, characterized in that: (1) The miR-5134-5p inhibitor solution was slowly added dropwise to the polyhistidine solution at a nitrogen-to-phosphorus ratio of (10-15):

1. The mixture was stirred while adding the solution and allowed to stand at room temperature to react. The PHH / miR-5134-5p inhibitor nanoparticles were formed by self-assembly using the interaction of positive and negative charges. (2) Add Mal-SS-COOH solution to the PHH / miR-5134-5p inhibitor nanoparticle solution obtained in step (1), mix well, add mPEG-SH solution, and react at room temperature in the dark. Mal-SS-COOH and mPEG-SH undergo a specific Michael addition reaction, forming an mPEG-SS-COOH shell in situ on the surface of the PHH / miR-5134-5p inhibitor nanoparticles, to obtain mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticles; (3) Add EDC・HCl and NHS to the mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticle solution obtained in step (2) and activate the carboxyl groups on the surface of the support at room temperature; A solution of amino-modified m6A short nucleic acid sequence was added, and the reaction was carried out at room temperature in the dark to achieve covalent coupling between the m6A short nucleic acid sequence and mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticles, thus obtaining the m6A / mPEG-SS-COOH / PHH / miR-5134-5p inhibitor nanoparticle delivery system.

4. The method for preparing an inhibitor targeted delivery system for highly malignant triple-negative breast cancer according to claim 3, characterized in that: In step (1), Polyhistidine solution was prepared at a concentration of 1 mg / mL using 0.01 M acetate-sodium acetate buffer at pH 4.5-5.5; miR-5134-5p inhibitor solution was prepared at a concentration of 1-20 μM using DEPC enzyme-free water. The reaction time at room temperature is 25-35 min.

5. The method for preparing an inhibitor targeted delivery system for highly malignant triple-negative breast cancer according to claim 3, characterized in that: In step (2), The Mal-SS-COOH solution concentration was 1 mg / mL, prepared using DEPC enzyme-free water; The concentration of mPEG-SH was 5 mg / mL, prepared using 0.01M acetate-sodium acetate buffer solution at pH 4.5-5.5; The mass ratio of Mal-SS-COOH to the PHH added in step (1) is 1:6-1:9; The molar ratio of Mal-SS-COOH to mPEG-SH is 1:1 to 1:1.

2. The reaction time at room temperature in the dark is 30-50 minutes. After the reaction was completed, the mixture was centrifuged at 5000-8000 rpm for 30-40 min using a 3k MWCO ultrafiltration tube and then reconstituted with 1×PBS buffer to complete the buffer system replacement.

6. The method for preparing an inhibitor targeted delivery system for highly malignant triple-negative breast cancer according to claim 3, characterized in that: In step (3), The concentration of the amino-modified m6A short nucleic acid sequence solution was 0.1 mg / mL, and it was prepared using DEPC enzyme-free water. The final concentrations of EDC・HCl and NHS were both 5 mM. The activation time at room temperature was 30-60 min. After activation, the free activating reagents were removed by ultrafiltration through a 3 kMW CO2 ultrafiltration tube and then reconstituted with 1×PBS.

7. The method for preparing an inhibitor targeted delivery system for highly malignant triple-negative breast cancer according to claim 3, characterized in that: In step (3), The mass ratio of the amino-modified m6A short nucleic acid sequence to the Mal-SS-COOH used in step (2) is 1:30-1:50, and the reaction time is 12-14 h at room temperature in the dark. After the reaction was completed, the sample was centrifuged at 5000-8000 rpm for 30-40 min using a 3k MWCO ultrafiltration tube. The flow-through was discarded, and 1×PBS buffer was added to the ultrafiltration tube to reconstitute the retentate. The centrifugation and washing were repeated 3 times.

8. The use of the inhibitor targeted delivery system for highly malignant triple-negative breast cancer according to any one of claims 1-2 in the preparation of drugs for treating highly malignant triple-negative breast cancer.

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