Enzyme-responsive peptide-based pna targeting delivery system, and preparation method and application thereof

Abstract

The application provides an enzyme-responsive peptide-based nucleic acid targeting delivery system and a preparation method and application thereof, and relates to the technical field of biotechnology.The delivery system comprises nanomicelles and CD44-targeting nucleic acid aptamers connected to the outer layer of the nanomicelles; the nanomicelles are composed of enzyme-responsive peptides and CD24 siRNA adsorbed to the enzyme-responsive peptides; the enzyme-responsive peptide is a hydrophobic functional group-CGGFLG-HHHKKHHHKKK; and the CD24 siRNA targets and silences a CD24 gene.The delivery system can effectively target triple-negative breast cancer cells, and improves the targeting and drug loading capacity of a targeted drug for triple-negative breast cancer tumors.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an enzyme-responsive peptide-based nucleic acid targeted delivery system, its preparation method, and its application. Background Technology

[0002] Breast cancer is the leading cause of cancer death among women. Triple-negative breast cancer (TNBC) is a type of breast cancer that is negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor HER-2, accounting for 12-17% of all breast cancer cases. Compared to other types of breast cancer, TNBC is highly aggressive, has a high risk of distant metastasis, and a poor prognosis; the 5-year survival rate for stage III-IV patients is less than 15%. Due to the lack of specific surface receptors associated with breast cancer, effective treatment with highly specific drugs is not possible. Current drug treatment for TNBC is limited to early surgical resection supplemented by conventional chemotherapy, primarily anthracyclines, cisplatin, and paclitaxel, with relatively limited efficacy. Clinical data indicate that TNBC is prone to developing resistance to chemotherapy drugs, is prone to recurrence, and easily metastasizes to other major tissues and organs, with a 5-year recurrence and metastasis risk as high as 20%.

[0003] Currently, significant challenges remain in tumor immunotherapy. For example, drug delivery systems often have large molecular weights, making them difficult to deliver to the tumor microenvironment. Furthermore, many commercially available transfection reagents, such as liposomes, generally carry a strong positive charge, resulting in significant cytotoxicity. Other obvious issues include poor targeting of delivery carriers, hindering the accumulation of nanomedicines within tumor cells, and the potential for toxicity to normal cells.

[0004] Given the high immunogenicity of tumor-associated neuroblastoma (TNBC) and the abundance of tumor-infiltrating lymphocytes (TILs) at the tumor site, which enhances the immune response, the emergence of anti-tumor immunotherapy, represented by immune checkpoint inhibitors (ICIs), has provided a novel strategy for TNBC treatment in recent years. However, multiple phase II / III clinical trials utilizing approved antibody-based immune checkpoint inhibitors such as CTLA4 and PD-1 / PD-L1 for TNBC treatment have shown that their use alone or in simple combination with chemotherapy drugs is not effective in improving the survival rate and quality of life of TNBC patients, highlighting the urgent need to explore new immunotherapeutic targets.

[0005] Despite some progress in the research on TNBC treatment, early diagnosis and precise and effective treatment of tumor metastasis remain problems and challenges in the treatment of triple-negative breast cancer. (1) Antibody-based immune checkpoint inhibitors cannot specifically target immune cells in tumor tissues for immune regulation. Widespread expression of immune checkpoints can easily cause non-specific immune responses in normal tissues and organs, resulting in damage to normal tissue cells, and large molecular antibodies are difficult to effectively penetrate into the tumor. (2) Immune checkpoint inhibitors alone are not effective in treating TNBC, and there is an urgent need to develop new therapeutic targets and combine them with other treatment methods. Multiple clinical trial data show that the use of immune checkpoint inhibitors alone (such as antibody blockers such as PD-L1 and CTLA4) is not effective in treating TNBC tumors, and simple combination with chemotherapy drugs cannot effectively improve the treatment effect. (3) Due to the poor in vivo stability of drugs and the tendency to produce off-target effects.

[0006] Therefore, how to improve targeted drugs for TNBC tumors is a problem that needs to be solved.

[0007] In view of this, the present invention is hereby proposed. Summary of the Invention

[0008] The purpose of this invention is to provide an enzyme-responsive peptide-based nucleic acid targeted delivery system that improves the targeting and drug loading capabilities of targeted drugs for triple-negative breast cancer tumors, and can effectively inhibit the proliferation and metastasis of triple-negative breast cancer tumor cells.

[0009] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0010] In a first aspect, an enzyme-responsive peptide-based nucleic acid targeted delivery system is provided, the delivery system comprising nanomicelles and a CD44-targeted nucleic acid aptamer attached to the outer layer of the nanomicelles;

[0011] The nanomicelles consist of enzyme-responsive peptides and CD24 siRNA adsorbed onto the enzyme-responsive peptides.

[0012] The enzyme-responsive peptide has a hydrophobic functional group -CGGFLG-HHHKKHHHKKK;

[0013] The CD24 siRNA targets and silences the CD24 gene.

[0014] In an optional embodiment, the hydrophobic functional group is a C18 aliphatic chain.

[0015] In an optional implementation, the sense strand of the CD24 siRNA is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4.

[0016] In an optional implementation, the CD44-targeting nucleic acid aptamer is DNA.

[0017] In an optional embodiment, the 3' end of the CD44-targeting nucleic acid aptamer is connected to the hydrophilic end of the enzyme-responsive peptide.

[0018] In an optional embodiment, the 3' end of the CD44-targeting nucleic acid aptamer is modified with a cholesterol group.

[0019] In an optional embodiment, the 3' end of the CD44-targeting nucleic acid aptamer contains a single-stranded polynucleotide.

[0020] In an optional embodiment, the 3' end of the CD44-targeting nucleic acid aptamer has a single-stranded polynucleotide of the same length as the CD24 siRNA.

[0021] In an optional embodiment, the nucleotide sequence of the CD44-targeting nucleic acid aptamer is shown in SEQ ID NO.2.

[0022] In an optional embodiment, the enzyme-responsive peptide is C18-CGGFLG-HHHKKHHHKKK; the sense strand of the CD24 siRNA is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4; the nucleotide sequence of the CD44-targeting aptamer is shown in SEQ ID NO.4, and the 3' end of the CD44-targeting aptamer is modified with a cholesterol group.

[0023] In an optional embodiment, the mass ratio of nitrogen to phosphorus in the delivery system is (10-2):1.

[0024] In an optional embodiment, the CD44-targeting nucleic acid aptamer accounts for 1 to 10% of the total molar amount of CD24 siRNA and CD44-targeting nucleic acid aptamer.

[0025] Secondly, a method for preparing the enzyme-responsive peptide-based nucleic acid targeted delivery system of the first aspect is also provided, comprising: firstly mixing CD24 siRNA and CD44 targeted nucleic acid aptamer according to the formula amount to obtain a polynucleotide mixture system; then mixing the polynucleotide mixture system into a solution containing enzyme-responsive peptides, and incubating to obtain the delivery system.

[0026] In an optional embodiment, the concentration of the enzyme-responsive peptide in the solution is 0.8–4 mg / mL; the concentrations of CD24 siRNA and CD44 targeting nucleic acid aptamer are each independently 20 μM; and the polynucleotide mixture system and the enzyme-responsive peptide solution are mixed in equal volumes.

[0027] In an optional embodiment, the concentration of the enzyme-responsive peptide in the solution is 4 mg / mL.

[0028] The application of the enzyme-responsive peptide-based nucleic acid targeted delivery system of the first aspect, or the preparation method of the second aspect, in the preparation of drugs for treating, preventing or alleviating tumors.

[0029] In an optional implementation, the tumor includes triple-negative breast cancer.

[0030] Fourthly, a drug is also provided, comprising the enzyme-responsive peptide-based nucleic acid targeted delivery system of the first aspect and the active pharmaceutical ingredient encapsulated therein.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The enzyme-responsive peptide-based nucleic acid targeted delivery system provided by this invention includes an enzyme-responsive peptide, a CD44-targeting nucleic acid aptamer, and CD24 siRNA. The enzyme-responsive peptide used in this invention, possessing delivery capability, contains an enzyme cleavage site. In vivo, it is recognized and hydrolyzed by hydrolytic enzymes, releasing bound substances and achieving a therapeutic effect. The CD24 siRNA, as a small interfering RNA designed to target the CD24 gene, a proto-oncogene in tumor cells, can knock out the highly expressed CD24 gene on the surface of triple-negative breast cancer cells at the gene editing level, achieving a gene silencing effect on tumor cells and further promoting the phagocytosis of tumor cells by immune cells, thus achieving a tumor immunotherapy effect. The CD44-targeting nucleic acid aptamer enables the delivery system to target tumor cells, avoiding off-target effects caused by the expression of CD24 protein on the surface of immune T cells and B cells.

[0033] The delivery system provided by this invention blocks the CD24 / Siglec-10 immune checkpoint pathway by silencing CD24 gene expression, thereby inhibiting the proliferation and metastasis of TNBC cells. Based on this, a shell-core structured nanomicelle is developed using its own double-stranded matrix as a backbone, enabling long-term in vivo circulation and effectively protecting siRNA from degradation. Using these nanomicelles as carriers and modifying their surface with CD44-aptamer, nanoprobes are developed for imaging and localization of primary and metastatic TNBC tumor sites. The invention also focuses on developing an immunotherapy-chemotherapy combined delivery nanomedicine system, which promotes TAM immune phagocytosis through CD24 regulation, further enabling synergistic treatment of TNBC in combination with chemotherapy, while significantly reducing immune-related toxicities.

[0034] The delivery system provided by this invention delivers drugs specifically and precisely to the tumor site, achieving synergistic effects of immunotherapy and chemotherapy while significantly reducing toxic side effects, which will provide important clinical significance for the treatment of triple-negative breast cancer. Attached Figure Description

[0035] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0036] Figure 1 This is a schematic diagram illustrating the structure and function of the delivery system provided in an embodiment of the present invention;

[0037] Figure 2 Electrophoresis images of PM@siRNA samples with different N / P ratios in Example 1;

[0038] Figure 3 This is a graph showing the Zeta potential results of PM@siRNA samples with different N / P ratios in Example 1.

[0039] Figure 4 This is a graph showing the particle size results of PM@siRNA samples with different N / P ratios in Example 1.

[0040] Figure 5 Electrophoresis images of Apt-PM@siRNA samples with different CD44 Apt percentages prepared in Example 2;

[0041] Figure 6 This is a graph showing the Zeta potential results of Apt-PM@siRNA for samples with different CD44 Apt percentages in Example 2.

[0042] Figure 7 The particle size of the Apt-PM@siRNA samples representing the percentage of CD44 Apt in Example 2;

[0043] Figure 8 The immunoblotting method used in Example 3 was used to detect the silencing effect of PM-siRNA-10%Apt on the CD24 gene in MDA-MB-231, EMT6 and 4T1 cells, respectively.

[0044] Figure 9 The silencing effect of PM-siRNA-10%Apt on the CD24 gene in EMT6 cells was detected by real-time quantitative PCR in Example 3.

[0045] Figure 10 The real-time quantitative PCR detection of the silencing effect of PM-siRNA-10%Apt on the CD24 gene in 4T1 cells was performed in Example 3.

[0046] Figure 11 The results are from the cell migration experiment in Example 4;

[0047] Figure 12 The results of the cell migration rate in the cell migration experiment in Example 4 are statistical results.

[0048] Figure 13 In Example 5, the targeting effect of Apt-PM@siRNA was examined using laser confocal fluorescence microscopy.

[0049] Figure 14 In Example 5, the targeting effect of Apt-PM@siRNA at different time intervals was examined using laser confocal fluorescence microscopy.

[0050] Figure 15 This is a curve showing the change in tumor volume of the tumor model mice in Example 6 over time.

[0051] Figure 16 The image shows a photograph of the tumor in the tumor model mouse in Example 6 after intervention with Apt-PM@siRNA;

[0052] Figure 17 This is the mass spectrum of the enzyme-responsive peptide PM used in the examples. Detailed Implementation

[0053] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. 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.

[0054] In a first aspect, the present invention provides an enzyme-responsive peptide-based nucleic acid targeted delivery system, comprising an enzyme-responsive peptide, CD24siRNA, and a CD44-targeting nucleic acid aptamer.

[0055] The structural diagram and mechanism of action of the enzyme-responsive peptide-based nucleic acid targeted delivery system are as follows: Figure 1 As shown, CD24 siRNA adsorbs onto an enzyme-responsive peptide with a hydrophobic N-terminus and a hydrophilic C-terminus, allowing the adsorbed CD24 siRNA-containing peptide to assemble into nanomicelles. The outer layer of the nanomicelles is linked to a CD44-targeting nucleic acid aptamer, guiding the delivery system to target cells with CD44 surface molecules. After the cells take up the targeting system, intracellular hydrolases cleave the enzyme cleavage sites in the enzyme-responsive peptide, causing the CD24 siRNA to target and silence the CD24 gene. If the delivery system also contains a drug active ingredient, the drug active ingredient is released.

[0056] The enzyme-responsive peptide with delivery capability is a hydrophobic functional group -CGGFLG-HHHKKHHHKKK (SEQ ID NO.1). This peptide has been shown to have a relatively broad delivery effect on breast cancer and prostate cancer cells, and can be released at specific sites in the tumor microenvironment. Through the CGGFLG restriction enzyme sequence in its molecular structure, it can be recognized and hydrolyzed by hydrolytic enzymes in vivo, thereby releasing the bound CD24 siRNA to achieve a certain therapeutic effect. Its N-terminal hydrophobic functional group is hydrophobic, and exemplary hydrophobic functional groups include, but are not limited to, the C18 fatty acid chain; HHHKKHHHKKK is a hydrophilic end, enabling it to assemble into micelles.

[0057] CD44 is a tumor cell surface marker that is highly expressed in various solid tumors, including TNBC, and is not expressed or is expressed at low levels in normal tissues. CD44-targeting aptamers are nucleic acid aptamers that can target CD44. By screening and utilizing high-affinity, low-molecular-weight CD44-targeting aptamers as delivery systems to target the receptor on the surface of tumor cells, it is possible to effectively increase the uptake of CD24 siRNA in tumor cells, reduce the off-target effects of CD24 siRNA, and further avoid the associated immune side effects through specific release in tumor cells.

[0058] In an optional implementation, the CD44-targeting nucleic acid aptamer is DNA.

[0059] In an optional embodiment, the 3' end of the CD44-targeting nucleic acid aptamer is connected to the hydrophilic end of the enzyme-responsive peptide.

[0060] In an optional embodiment, the 3' end of the CD44-targeting aptamer contains a single-stranded polynucleotide segment. This segment does not form a stem-loop structure with other parts of the CD44-targeting aptamer; it is intended to mimic a single RNA strand and act as a bridge, being adsorbed by the enzyme-responsive peptide along with the CD24 siRNA. Optionally, the length of this single-stranded polynucleotide segment acting as a bridge is the same as the length of the CD24 siRNA.

[0061] In an optional embodiment, the 3' end of the CD44-targeting nucleic acid aptamer is modified with a cholesterol group for linking with an enzyme-responsive peptide.

[0062] In an optional embodiment, the nucleotide sequence of the CD44-targeting nucleic acid aptamer is as follows:

[0063] CCAAGGCCTGCAAGGGAACCAAGGACACAGTTTTTTTTTT-TTGAACA TAAAAGAGAAAG(SEQ IDNO.2)

[0064] CD24 siRNA targets and silences the CD24 gene. CD24 (Cluster of Differentiation 24, a thermostable antigen) is a highly glycosylated glycosylphosphatidylinositol-anchored surface protein, which has been shown to be a proto-oncogene in various tumors, promoting tumor proliferation and invasion during tumor progression. In breast cancer, high expression of CD24 increases patient tolerance to the estrogen receptor blocker tamoxifen; in TNBC, CD24 plays an important regulatory role in tumor progression and metastasis, and as an oncogene regulator, it promotes intratumoral angiogenesis by stimulating the EGFR / Stat3 / Scr signaling pathway. Recent experiments have shown that CD24 protein also plays an important role in tumor immune escape. Compared to normal breast cells and other subtypes of breast cancer cells, CD24 expression was significantly increased in TNBC cells. Furthermore, screening of TNBC patients revealed that, compared to the widespread expression of CD47, which also acts on the TAM "Don't eat me" signaling pathway, CD24 was more concentrated on the surface of tumor cells, and its expression level was much higher than other immune checkpoint inhibitors such as PD-L1. Given the proto-oncogene nature of CD24, compared to achieving single-immunotherapy effects by inhibiting other immune checkpoint pathways such as PD-L1, it is believed that specifically inhibiting the expression of CD24 protein on the tumor surface through gene editing can not only relieve its immunosuppressive effect on TAM and achieve immunotherapy, but also regulate downstream pathways within tumor cells, inhibiting the growth of TNBC tumors.

[0065] Similar to other immune checkpoint pathways, CD24 protein is not only highly expressed on the surface of TNBC cells, but also on the surface of immune T cells and B cells. Therefore, actively targeting the TNBC cell-specific surface receptor CD44 can effectively reduce the off-target effects of CD24 siRNA.

[0066] The CD24 siRNA can be any siRNA known in the art that has been proven to silence CD24, and this invention does not limit this. An exemplary CD24 siRNA's sense strand is shown in SEQ ID NO.3, and its antisense strand is shown in SEQ ID NO.4.

[0067] In an optional embodiment, the enzyme-responsive peptide is C18-CGGFLG-HHHKKHHHKKK; the sense strand of the CD24 siRNA is shown in SEQ ID NO.3, and the antisense strand is shown in SEQ ID NO.4; the nucleotide sequence of the CD44-targeting aptamer is shown in SEQ ID NO.4, and the 3' end of the CD44-targeting aptamer is modified with a cholesterol group.

[0068] In an optional embodiment, the mass ratio of nitrogen to phosphorus in the delivery system is (10 to 2):1, for example, but not limited to 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or 2:1, preferably 10:1.

[0069] In an optional embodiment, the CD44-targeting nucleic acid aptamer accounts for 1 to 10% of the total molar amount of CD24 siRNA and CD44-targeting nucleic acid aptamer, for example, but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10%.

[0070] Secondly, a method for preparing the enzyme-responsive peptide-based nucleic acid targeted delivery system of the first aspect is also provided. The preparation method includes first mixing CD24 siRNA and CD44 targeted nucleic acid aptamers according to the formula to obtain a polynucleotide mixture system; then mixing the polynucleotide mixture system into a solution containing enzyme-responsive peptides, and incubating to obtain the delivery system.

[0071] In an optional embodiment, the concentration of the enzyme-responsive peptide in the solution is 0.8–4 mg / mL, the concentration of CD24 siRNA is 20 μM, the concentration of CD44 targeting nucleic acid aptamer is 20 μM, and the polynucleotide mixture and the enzyme-responsive peptide solution are mixed in equal volumes. This mixing method yields a delivery system with an N / P ratio of 10:1 to 2:1. More preferably, the concentration of the enzyme-responsive peptide in the solution is 4 mg / mL, which yields a delivery system with an N / P ratio of 10:1.

[0072] In an optional implementation, CD24 siRNA and CD44 targeting nucleic acid aptamer are mixed and left to stand for 5 minutes.

[0073] In an optional embodiment, the incubation condition is to let it stand at room temperature for 30 minutes.

[0074] Thirdly, the application of the enzyme-responsive peptide-based nucleic acid targeted delivery system of the first aspect, or the preparation method of the second aspect, in the preparation of drugs for treating, preventing, or alleviating tumors is also provided. The tumor can be a tumor expressing CD44 and CD24, such as triple-negative breast cancer.

[0075] Fourthly, a drug is also provided, comprising the enzyme-responsive peptide-based nucleic acid targeted delivery system of the first aspect and the active pharmaceutical ingredient encapsulated therein.

[0076] The active pharmaceutical ingredient may be any known, conventional drug in the art that has therapeutic or replicative therapeutic effects on tumors, including but not limited to methotrexate, fluorouracil, mercaptopurine, hydroxyurea, cytarabine, nitrogen mustard, cyclophosphamide, thiotepa, cisplatin, mitomycin, bleomycin, camptothecin, podophyllotoxin, actinomycin D, doxorubicin, daunorubicin, vincristine, paclitaxel, cephalotaxine alkaloids, and L-asparaginase.

[0077] The present invention will be further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present invention in any way.

[0078] The following materials and reagents are involved in the following implementation:

[0079] Reference material for CD24-targeted monoclonal antibody drug (provided by Hangzhou Huaan Biotechnology Co., Ltd.); Real-time quantitative PCR transcription kit (purchased from Shanghai Yisheng Biotechnology Co., Ltd.); Agarose gel powder (purchased from Shanghai Yisheng Biotechnology Co., Ltd.); Streptomycin / penicillin bispecific antibody (purchased from Shanghai Yisheng Biotechnology Co., Ltd.), trypsin (purchased from Shanghai Yisheng Biotechnology Co., Ltd.), FBS (purchased from Shanghai Yisheng Biotechnology Co., Ltd.), and culture medium FBS (purchased from Shanghai Yisheng Biotechnology Co., Ltd.). Cell lines: MDA-MB-231 cell line, EMT6 cell line (purchased from Pronosei Biotechnology Co., Ltd.), and 4T1 cell line (purchased from ATCC).

[0080] In the following examples, the enzyme-responsive peptide is abbreviated as PM, and its hydrophobic functional group is a C18 fatty chain. The CD44-targeting nucleic acid aptamer is abbreviated as CD44 Aptamer or CD44 Apt, which is DNA with a cholesterol group modified at the 3' end. The delivery system composed of PM and CD24 siRNA is denoted as PM@siRNA, and the delivery system composed of PM, CD24 siRNA, and CD44 Aptamer is denoted as Apt-PM@siRNA.

[0081] The sequence information of CD24 siRNA, PM, and CD44 Aptamer used in the following examples is shown in Table 1:

[0082] Table 1 Sequence Information

[0083]

[0084] The preparation method of PM used in Examples 1 to 6 is described in Example 7.

[0085] Example 1

[0086] (I) Experimental Methods:

[0087] 1. Preparation of PM@siRNA:

[0088] First, we used the PM@siRNA system to investigate the ratio of peptides to nucleic acids in the delivery system. When synthesizing nanoparticles using charged raw materials, the ratio of positive to negative charges affects the stability, potential, and other properties of the nanoparticles. In nucleic acid-peptide nanomicelles, the positive charge is usually a peptide with ionizable ammonium (N) ions, while the negative charge is a nucleic acid molecule with a large number of phosphate (P) ions. The two can bind together via electrostatic adsorption. Therefore, an unreasonable ratio may lead to defects such as excessively large particle size and poor stability. Previous experiments have verified the relationship between the concentration and amount of PM and siRNA and the mass ratio of nitrogen to phosphorus (N / P ratio) in PM@siRNA. We assumed that the optimal binding ratio in this system would be the same and conducted experimental verification.

[0089] W = Mv × N

[0090] Where W is the molar concentration, Mv is the molar mass, and N is the molar concentration.

[0091] Based on the above formula and known conditions, it can be finally calculated that when the N / P Ratio is 10:1, the PM molar concentration is 4 mg / mL, which is used for subsequent solution preparation.

[0092] Specifically:

[0093] The PM structure is Stearyl-CGGFLGHHHKKHHHKKK, the molecular formula is C108H172N34O19S, and the final molecular weight is 2447 g / mol.

[0094] The CD24 siRNA stock solution concentration was 20 uM. Based on the RNA stock solution concentration, the PM stock solution concentration was deduced to prepare for subsequent cell-level experiments.

[0095] Detailed calculation steps:

[0096] (1) The concentration of P in 20 μM siRNA: 21 bp (number of bases) × 2 (siRNA is double-stranded) × 20 μM = 840 μM

[0097] (2) Based on previous literature review (see the literature Co-delivery of IKBKE siRNA and cabazitaxel by hybrid nanocomplex inhibits invasiveness and growth of triple-negative breast cancer, DOI:10.1126 / sciadv.abb0616), the optimal binding ratio under the same binding system is 10:1. Therefore, assuming that the ratio is the same in this system, the concentration of N can be calculated. N: 10 × 840 μM = 8400 μM. The structure of PM is shown in formula (Ⅰ).

[0098]

[0099] (3) According to the above formula, 1 mol of PM contains 5 mol of N, so the concentration of PM is 8400 μM / 5 = 1680 μM.

[0100] (4) Based on the mass spectrometry analysis results ( Figure 17 Given that the molar mass of PM is 2447 g / mol, we can deduce from w = Mw × N that the final molar concentration of PM is 2447 g / mol × 10⁻¹⁰. -6 ×1680mol / L≈4mg / ml (when the N / P ratio is 10:1).

[0101] 1.1 Preparation method of PM@siRNA with N / P ratio = 10:1:

[0102] (1) Weigh 4 mg of the synthesized PM pure product, add 1 mL of ultrapure water to dissolve it, prepare a PM mother liquor with a concentration of 4 mg / mL, and sonicate for 20 s to aid dissolution, so as to form a stable dispersed phase.

[0103] (2) The mouse-derived CD24 siRNA purchased from Suzhou Gemma Gene Co., Ltd. was used to perform a binding reaction with PM peptides. 1 OD siRNA was taken and 125 μL of DEPC water was added to prepare a 20 μM stock solution for subsequent reactions.

[0104] (3) After the two are pipetted and mixed, the reaction is carried out. The reaction time is usually 30 minutes.

[0105] 1.2 Dilute the 4 mg / mL PM stock solution to prepare working solutions with N / P ratios of 5:1, 4:1, 3:1, 2:1, and 1:1 for PM@siRNA, corresponding to PM concentrations of 2 mg / mL, 1.6 mg / mL, 1.2 mg / mL, 0.8 mg / mL, and 0.4 mg / mL, respectively. Mix the PM working solutions of different concentrations with an equal volume of 20 μM CD24 siRNA stock solution, incubate at room temperature for 30 minutes, and then bring the volume to 1 mL.

[0106] 2. Agarose Gel Electrophoresis: Agarose gel electrophoresis uses agarose as a medium to separate and screen macromolecules such as proteins or nucleic acids. Under the influence of an applied electric field, charged particles are separated according to their differences in charge and number, exhibiting a molecular sieving effect. This experiment aims to examine the binding ability of PM to RNA and determine the optimal binding ratio. The specific experimental steps are as follows:

[0107] (1) Preparation of 1% agarose gel: Clean the gel tray and assemble the reaction apparatus, including the comb. Weigh 0.6 g of agarose powder and dissolve it in 60 mL of 1×TAE buffer. Mix the two solutions and pour them into an Erlenmeyer flask, shaking well. Place the Erlenmeyer flask in a microwave oven and heat for 1–2 minutes until the agarose powder is completely dissolved and the solution is clear and transparent. Remove the Erlenmeyer flask and allow it to cool to approximately 60–70 degrees Celsius. Add 6 μL of GelRed dye to the solution in the flask, shake the gel in the Erlenmeyer flask thoroughly, and pour it into a prepared tray. Allow the gel to cool and solidify.

[0108] (2) Sample loading: The reaction system ratio is the same as that for particle size and potential measurement. Incubate at room temperature for 30 min, then add ultrapure water and 6×DNA / RNA Loading buffer, and finally adjust the system volume to 20 μL. Then load the sample and run electrophoresis at 80 mV for 40 min.

[0109] (3) Exposure: After the gel running is finished, turn off the electrophoresis apparatus, put on rubber and plastic gloves, take out the gel plate, and then place it in the gel imaging instrument for gel imaging exposure to view the gel running results.

[0110] 3. Particle size and potential measurement:

[0111] (1) Rinsing: Rinse the cuvette and potentiometer cell in turn with a wash bottle containing ultrapure water and the sample solution.

[0112] (2) Adding the sample: Use a pipette to mix the sample by blowing and blowing, then take the sample and add it into the test device. The height of the sample solution is about 2 / 3 of the total height of the device.

[0113] (3) Place the sample: Place the quartz cuvette with the frosted side facing you into the clamping groove. Once everything is ready, begin the measurement.

[0114] (4) Measurement: Wipe the surface of the device, place it in the measurement chamber, close the cover, and use a nano laser particle size analyzer to measure the particle size and potential changes of the solution under different N / P ratios. Repeat the operation three times to ensure the accuracy of the data. Then, the measurement results are statistically analyzed.

[0115] (5) After the measurement is completed, take out the sample cell, clean it with ultrapure water, export the experimental data, and finally turn off the nano-laser particle size analyzer and the computer. The experimental operation is completed.

[0116] (II) Experimental Results:

[0117] The binding ratio of PM to CD24 siRNA was preliminarily determined by agarose gel electrophoresis, zeta potential, and particle size measurements. The results showed that at an N / P ratio of 2:1, no free RNA was produced by agarose gel electrophoresis. Figure 2 ), and through the Zeta potential results graph ( Figure 3 This was further verified by the Zeta potential plot, which showed a charge reversal at a ratio of 2:1. Furthermore, as the N / P ratio increased, the proportion of positive charge in the potential also increased, showing a positive correlation. The particle size results were consistent with this, showing a positive correlation. Figure 4 Furthermore, the particle size was smallest at an N / P ratio of 10:1, at 146 nm. These results indicate that an N / P ratio of 10:1 is the optimal delivery system. Under the premise of ensuring complete binding between PM and siRNA, the smallest particle size and slightly positively charged nanoparticle system contribute to achieving the best delivery effect.

[0118] Example 2

[0119] (I) Experimental Methods:

[0120] 1. Preparation of Apt-PM@siRNA:

[0121] A cholesterol group was attached to the 3' end of the CD44 Aptamer to facilitate better binding to the amphiphilic block copolymer PM. Simultaneously, a bonding bridge of the same length as the small interfering RNA sequence was designed and attached to the 3' end of the aptamer. This was intended to mimic a single-stranded RNA, allowing the nucleic acid aptamer carrying the bonding bridge to be adsorbed by PM along with the CD24 siRNA. This also better exposed the functional nucleic acid aptamer target, maximizing its targeting effect. The designed CD44 Aptamer was then used to reconstruct and correct its secondary structure using a DNA foldingform aptamer secondary structure prediction website. Ultimately, this ensured that the secondary structure of the aptamer with the bonding bridge sequence was a single conformation, maintaining its stability.

[0122] The connection effect between the aptor target and the system was tested after the design and structural verification.

[0123] Aptamer targets with gradient proportions (1-10% of the mass of CD44 Aptamer to the total molar ratio of CD44 Aptamer and CD24 siRNA) were ligated to PM@siRNA to successfully construct the Apt-PM@siRNA final delivery system. First, aptamer targets at different proportions were mixed with siRNA and allowed to stand for 5 minutes. Then, PM peptide solution was added to bind with the siRNA, and the N / P ratio was adjusted to 10:1. Finally, the mixture was thoroughly mixed by pipetting and allowed to stand at room temperature for 30 minutes. After incubation, the particle size and potential of the Apt-PM@siRNA delivery system were measured, and the binding effect was verified by agarose gel electrophoresis. The experimental method is described in Example 1. Finally, the results were analyzed and summarized to determine the final binding system.

[0124] (II) Experimental Results:

[0125] The optimal delivery system was the one prepared in Example 1 with an N / P ratio of 10:1. Based on this system, an aptamer target was added to enhance its targeting and specificity. CD44 Aptamer with a -SH thiol conjugation was selected as the specific target.

[0126] The results showed that when CD44 Apt was loaded at 1–10%, agarose gel electrophoresis revealed no free nucleic acid production. Figure 5 ), Statistical results of the final delivery system of the coupled aptamer target (Zeta potiental) Figure 6 As can be seen, the potential value is positively correlated with the continuous increase of CD44 Apt ratio, and the final delivery system, PM-siRNA-10% Apt, has a potential value of 25mV, which is slightly positively charged, and the particle size is 153nm under the same conditions. Figure 7 It can be internalized and enter the cell.

[0127] Example 3

[0128] Gene silencing effect test:

[0129] (I) Experimental Methods:

[0130] 1. Prepare Apt-PM@siRNA, wherein CD44 Apt accounts for 10% of the nucleic acid (the sum of the molar amounts of CD44 Apt and CD24 siRNA). The proportions of each component are as follows: 1 μL CD44 Apt (20 μM) + 9 μL CD24 siRNA (20 μM) and 10 μL PM are mixed. The specific steps are as follows: (1) 1 μL CD44 Apt (20 μM) + 9 μL CD24 siRNA (20 μM) + 120 μL Opt-MEM, let stand for 5 minutes; (2) 10 μL PM (4 mg / mL) + 120 μL Opt-MEM; (3) 130 μL of system (1) and 130 μL of system (2) were mixed and the final system was 260 μL. Let stand at room temperature for 30 minutes, then add MEM medium to the system and dilute it to the concentration of CD24 siRNA of 100 nM, 50 nM and 25 nM respectively. After incubation for 30 minutes, the in vitro system was added to the well.

[0131] 2. Cell culture:

[0132] Subsequent experiments were conducted using mouse triple-negative breast cancer cells EMT6 and 4T1, respectively.

[0133] (1) Cell passage can be performed in a clean bench only when the confluence of adherent cells reaches 80-90% as observed under a microscope.

[0134] (2) Discard the old culture medium in the cell culture flask, remove the remaining culture medium with a pipette, add about 1 mL of trypsin, gently shake to spread the trypsin digestion solution to the bottom of the culture flask, and when the cell culture flask wall appears white and foggy, and most of the cells are observed to be rounded and detached under a microscope, use a pipette to draw up the trypsin digestion solution and blow it evenly on the cell culture flask wall to blow off the remaining adherent cells. Then transfer the trypsin digestion solution containing cell suspension to a 15 mL pipette.

[0135] (3) Add about 3 mL of culture medium to a 15 mL centrifuge tube (the culture medium for EMT6 cells is RPMI1640, and the culture medium for 4T1 cells is DMEM high glucose), and then centrifuge at 1000 rpm for 5 minutes.

[0136] (4) After centrifugation, discard the supernatant. The bottom is the cell pellet. Add about 4 mL of the corresponding culture medium to the cell pellet, mix the cell pellet by pipetting to form a cell suspension, and divide the cell suspension into different equal parts and transfer them to new culture flasks for culture (for example, when passage culture of EMT6 cells cultured in T25 is performed, it is generally divided into two; when passage culture of 4T1 cells cultured in T25 is performed, it is generally divided into three, and the process is adjusted according to the cell growth rate).

[0137] (5) Finally, tighten the cap of the culture bottle and place it in a cell culture incubator at 37°C and 5% CO2 for culture.

[0138] 3. Cell transfection:

[0139] Cell transfection technology is a necessary test to verify the effect of siRNA gene silencing. Therefore, in this experiment, we transfected the Apt-PM@siRNA prepared in step 1 into mouse triple-negative breast cancer cells. After culturing the transfected cells for 24 or 48 hours, we extracted and experimentally verified the mRNA and protein expression products. Finally, we performed qualitative and quantitative analysis of the expression products based on the results. After culturing for 24 or 48 hours, we used real-time quantitative PCR and Western blotting to perform qualitative or quantitative analysis of the expression products.

[0140] 4. Total RNA extraction from cells

[0141] (1) After transfecting the siRNA complex and culturing the cell plate again for 24 hours, the cell plate was removed and observed under a microscope. When the cell confluence reached more than 90%, RNA extraction was prepared. The supernatant was discarded and the cells were washed three times with PBS. 1 mL of Trizol reagent was added to each well, and the cells were placed on ice and lysed for 30 minutes.

[0142] (2) Transfer the cell lysate containing Trizol to an EP tube that has been soaked in DEPC water, add 200 μl of chloroform organic solvent, mix vigorously and let stand on ice for 5 minutes, then centrifuge at 4°C, 10 min, 12000 rpm.

[0143] (3) Aspirate the contents and transfer them to a new DEPC water-treated EP tube. Add an equal volume of isopropanol, invert the tube to mix, and let it stand on ice for 5 minutes. Then centrifuge at 4°C, 10 min, and 12000 rpm.

[0144] (4) Discard the supernatant. At this point, the white precipitate at the bottom of the EP tube is RNA. Then add 1 mL of 75% ethanol to wash, centrifuge at 4°C for 5 min at 7500 rpm. Repeat this operation twice.

[0145] (5) Remove the supernatant, aspirate the liquid with a small tip, let the precipitate air dry at room temperature for 5-10 min, add 20-30 ul DEPC water to dissolve the RNA precipitate, pipette to mix thoroughly, and incubate at 55-60℃ for 5 min to dissolve the total RNA.

[0146] (6) Use a micro spectrophotometer to detect the concentration and quality of the RNA sample. The OD260 / 280 of RNA that meets the experimental requirements should be between 1.8 and 2.0. Detect RNA bands in a gel containing 1% agarose to determine the integrity of the RNA.

[0147] 5. Synthesis of cDNA extracted from total cellular RNA

[0148] For RNA samples that meet quality standards, cDNA is synthesized using the TaKaRa reverse transcription kit. The specific cDNA synthesis steps are as follows:

[0149] (1) Prepare the reaction solution according to the following system:

[0150] Table 2 cDNA synthesis reaction system

[0151]

[0152]

[0153] (2) Reverse transcription reaction

[0154] After mixing, place the mixture into a PCR instrument. The reverse transcription reaction conditions are: 37℃ for 15 min; 85℃ for 5 s; and terminate the reaction at 4℃.

[0155] 6. qRT-PCR

[0156] Different cDNAs prepared by reverse transcription were used as templates for PCR amplification. The specific qRT-PCR experimental procedures are as follows:

[0157] (1) First, the concentration of the cDNA product obtained by reverse transcription was measured using a micro spectrophotometer. The concentrations of different systems were quantified, generally up to 500 ng or 1 μg. After quantification, the reaction solution was prepared using a real-time fluorescence quantitative PCR reaction kit.

[0158] (2) Prepare the reaction solution according to the following system:

[0159] Table 3 PCR reaction system

[0160] reagents Usage TB Green PremixEx TaqⅡ(Tli RNaseH Plus) 12.5μL PCR Forward Primer 1μL PCR Reverse Primer 1μL DNA template 2μL Sterilized water 8.5μL Total 25μL

[0161] (3) Reaction procedure: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing for 30s, for a total of 40 cycles; 95℃ for 5s; melting curve analysis was performed at 65-95℃. Three technical replicates were set for each sample, and the reaction was performed on a CFX96 real-time quantitative PCR detection system. According to Ct(2 -ΔΔCt ) method to analyze data

[0162] 7. Western blot assay for proteins:

[0163] (1) Cell lysis: Cells were digested with trypsin and collected, and washed with PBS. Cell lysis buffer (100:1) containing protease and phosphatase inhibitors was then added, and the cells were lysed on ice for 30 minutes, with shaking every 5 minutes during the process. Finally, the cells were centrifuged at 4°C, 12,000 rpm for 10 minutes, and the supernatant was collected into a new 1.5 mL EP tube.

[0164] (2) Protein Quantitative Analysis: A standard curve was plotted and the concentration of the target protein was determined using the BCA protein quantification kit. First, BCA standard protein was prepared to a concentration of 0.5 mg / mL. BCA reagent solution A and solution B were thoroughly mixed at a ratio of 50:1 (v:v) to prepare the BCA working solution. The BCA protein standard and the target sample were added according to the system ratios in Table 3.4, and incubated at 37°C for 30 minutes. Then, 200 μL of the sample was added to each well of the microplate (3 replicates), and the absorbance was measured at 562 nm. A standard curve was established with protein concentration (mg / mL) as the X-axis and absorbance as the Y-axis. The concentration of the target sample was then calculated based on the standard curve.

[0165] (3) Protein electrophoresis: Prepare the separating gel and stacking gel according to the ratio in Table 3.5 using the SDS-PAGE gel preparation kit. After the separating gel and stacking gel solidify, place them in the electrophoresis apparatus, add running buffer to the electrophoresis apparatus, and then start loading the samples. After loading the samples, start running the electrophoresis.

[0166] (4) Transfer: After electrophoresis, remove the gel plate and cut the target protein band from the gel plate. Cut a PVDF membrane of the same size and immerse it in methanol solution to activate it for one minute. Then cover it on the gel carrying the target protein. Ensure that no air bubbles are generated during this process. Then clamp the transfer device and start the transfer.

[0167] (5) Blocking: After the transfer, the PVDF membrane is blocked (blocking solution: TBST solution containing 5% skim milk powder). To reduce the influence of nonspecific bands on the experimental results and to achieve the best blocking effect, we generally use a shaker and incubator at room temperature for 1-2 hours.

[0168] (6) Antibody Incubation: Remove the blocked transfer membrane, discard the blocking solution in the incubation chamber, then put the transfer membrane back in and add primary antibody, anti-CD24 antibody (1:1000), and anti-GAPDH antibody (1:5000) to the incubation chamber. Incubate overnight at 4°C. The next day, remove the membrane to prepare for secondary antibody incubation. The following day, recover the antibody from the protein incubation chamber after primary antibody incubation, and wash the membrane three times with TBST washing buffer for 10 minutes each time, for a total of 30 minutes. Then incubate with secondary antibody (rabbit anti, 1:10000; mouse anti, 1:10000) at room temperature, and wash the membrane three times with TBST washing buffer for 10 minutes each time, for a total of 30 minutes. Finally, expose and develop the membrane.

[0169] Table 4 BCA Protein Quantitative Addition System

[0170] serial number 1 2 3 4 5 6 7 8 sample BCA / μl 0 3 6 12 24 36 48 60 0 <![CDATA[H2O / μl]]> 60 57 54 48 36 24 12 0 57 working fluid 600 600 600 600 600 600 600 600 600 Concentration (mg / mL) 0 0.025 0.05 0.1 0.2 0.3 0.4 0.5 X

[0171] (II) Experimental Results:

[0172] The gene silencing effect of CD24 siRNA conjugated in the Apt-PM@siRNA delivery system was examined, and the results are as follows: Figure 8 As shown in the image, band comparison revealed that the drug-treated group exhibited a certain gene silencing effect compared to the control group. Subsequent quantitative analysis, performed using real-time quantitative PCR, also validated this effect, as shown in the results. Figure 9 and Figure 10 As shown above, the quantitative results indicate that the CD24 siRNA silencing effect was 72% in EMT6 cells and 58% in 4T1 cells, consistent with the WB protein results.

[0173] Example 4: Cell migration inhibition test:

[0174] (I) Experimental Methods:

[0175] 1. Before the scratch assay, cells were first seeded into 6-well plates and cultured in a 37°C, 5% CO2 incubator. When the cell confluence reached 60% or higher, Apt-PM@siRNA was transfected. The preparation and transfection methods for Apt-PM@siRNA were the same as in Example 3. The cells were then cultured in a cell culture incubator for 48 hours. Afterward, the transfected cells were resuspended and re-seeded at 100,000 cells per well. When the cell density reached 90% or higher, cell scratching was initiated. Three concentration gradients of 25, 50, and 100 nM were set. The specific steps of the scratch assay were as follows:

[0176] (1) Scratches: Use a 200 microliter nozzle to make horizontal scratches along the back of the 6-hole plate cover or a ruler, parallel or perpendicular to the horizontal line.

[0177] (2) Washing: Rinse the cells three times with PBS to remove the cells that have been cut off, and add serum-free culture medium.

[0178] (3) Take photos: Take photos under a 4x microscope to ensure that the scratch is centered and vertical. Samples can be taken and photographed at 0, 6, 12 and 24 hours.

[0179] (4) Results analysis: Finally, ImageJ software was used to calculate the mean intercellular distance or the mean scratch area.

[0180] (5) Data processing:

[0181] Cell migration rate = (initial scratch area - scratch area at time t) / initial scratch area.

[0182] (II) Experimental Results:

[0183] The inhibitory effect of CD24 siRNA on the migration ability of triple-negative breast cancer cells was examined, and the results are shown above. Figure 11 and Figure 12 As shown in the figure, the analysis results indicate that three concentration gradients of 25, 50, and 100 nM were established, and the cell migration rate changed to varying degrees with increasing time intervals after administration. The cell migration rate gradually slowed down with increasing RNA concentration, showing a certain degree of negative correlation. Quantitative analysis showed that, compared to the control group, the siRNA-administered groups exhibited a certain inhibitory ability on cell migration at 24 and 48 hours after administration, with 100 nM siRNA showing the strongest inhibitory ability, inhibiting 48% of cell migration at 24 hours and 67% at 48 hours.

[0184] Example 5 Cell uptake test

[0185] (I) Experimental Methods:

[0186] The targeting effect of the delivery system was examined using laser confocal fluorescence microscopy. The specific experimental steps are as follows:

[0187] First, cells were seeded in 12×12mm confocal culture dishes and cultured at 37°C in a 5% CO2 incubator until cell confluence reached 60% or higher, ready for transfection with a nanodelivery system carrying a fluorescent probe. The preparation and transfection methods for Apt-PM@siRNA were the same as in Example 3, except that CD24 siRNA was replaced with Cy5-siRNA. The experiment was divided into four groups: control group, liposome group, PM@siRNA group, and Apt-PM@siRNA group. After the samples were thoroughly mixed and allowed to stand, the samples were aspirated and added dropwise to the confocal culture dish to complete the transfection. After transfection, at the same time intervals, for example, 6 hours after transfection, the fluorescence uptake of transfected cells in different experimental groups was photographed using a laser confocal fluorescence microscope. Three images were taken for each group under different fields of view. Finally, the fluorescence intensity of the images was quantitatively analyzed using ImageJ software. The statistical fluorescence results were then quantitatively analyzed to determine the fluorescence intensity differences between different experimental groups and to identify the delivery system with the optimal cell uptake efficiency. It's important to note that before imaging after transfection, lysosomal probes need to be added to the transfected cells. Lystocker Green (diluted 1:1000) is used here to differentiate the probes from the Cy5-linked siRNA, thus achieving co-localization and determining lysosomal positions. Staining time is generally 30 minutes to 1 hour, followed by DAPI mounting. After determining the optimal delivery system, the cell uptake and final lysosomal release at different time points are examined. Samples are collected at 2, 4, 6, and 24 hours. Fluorescence imaging is then performed to determine cell uptake at different time points, and lysosomal release is imaged after 24 hours. The results are then analyzed.

[0188] (II) Experimental Results:

[0189] First, the targeting effect of Apt-PM@siRNA was validated in tumor cells, and the results showed that ( Figure 13 After the aptamer target was connected, the fluorescence intensity of the siRNA connected to the Cy5 fluorescent probe was significantly enhanced compared with the control group at the same time, which indicates that the specific aptamer target has a certain degree of targeting.

[0190] Subsequently, images were taken at different time intervals, such as 2, 4, 6, and 24-hour intervals. The results showed that ( Figure 14 Analysis revealed that the optimal group of coupled targets exhibited higher fluorescence intensity within the same time interval, indicating better delivery of CD24siRNA and further validating the vector's delivery performance.

[0191] Example 6 Animal Level Experiment

[0192] (I) Experimental Methods:

[0193] 1. Construction of a mouse model of xenograft tumor:

[0194] To investigate the in vivo antitumor effect of the Apt-PM@siRNA nanodelivery system, a mouse xenograft tumor model was established. Female Balb / c mice (6-8 weeks old, ~20g) were purchased from Beijing Huafukang Biotechnology Co., Ltd. (Beijing, China) and maintained under standard feeding conditions. The specific procedures were as follows: hair was removed from the backs of the mice using depilatory cream, and each mouse was subcutaneously injected with EMT6 cells (2×10⁻⁶) suspended in 100 μL PBS. 6 During inoculation, the needle should be inserted horizontally and injected subcutaneously into the mouse. After injection, a small suspension bag should form on the back. Then (1) the mouse should be fed normally. After a period of time, when the tumor volume on the mouse's back reaches 100 mm, 3 Mice were divided into groups for drug administration. The Apt-PM@siRNA group received a dose of 10 mg / kg. Mice were administered the drug via peritumoral injection every two days, and tumor volume was measured. The longest diameter (L) and shortest diameter (W) of the tumor were measured using the formula V = LW. 2 / 2 Calculate tumor volume. Observe for several days until the euthanasia criterion (1000 mm) is reached. 3 The mice were euthanized by cervical dislocation (left and right), the tumors were removed, weighed, photographed, and a tumor growth curve was plotted.

[0195] (II) Experimental Results:

[0196] Animal-level experiments were conducted for validation. First, we used 50W EMT6 cells per female 4-6 week old BALB / c mice for tumor modeling. One week after tumor formation, the tumor volume of all mice in the same batch reached 50 mm. 3 We then administered the drug to mice to verify its therapeutic effect. One week after tumor formation, we treated the mice with the drug. The Apt-PM@siRNA group received a dose of 10 mg / kg via peritumoral injection every two days. The final results are as follows: Figure 15 and Figure 16 As shown in the figure, statistical analysis revealed a significant difference between the siRNA-treated group and the control group.

[0197] Example 7: Preparation method of PM

[0198] The enzyme-responsive peptide used in the above embodiments is named Peptide molecules (PM). It was synthesized using a solid-phase synthesis method. The synthetic route can be summarized as follows: a stable amide bond is generated through an amide reaction between the amino and carboxyl groups of the amino acids in the peptide, thus synthesizing the N'-CGGFLGHHHKKHHHKKK-C' sequence structure. Then, the carboxyl group of stearic acid C18 and the amino group of cysteine ​​C are linked together through an amide reaction, ultimately forming a stable amphiphilic block copolymer PM. The specific synthetic steps are as follows:

[0199] Weigh 0.5 g of Wang resin with a degree of substitution of 1.15 mmol / g and swell it in DMF using a peptide synthesizer for at least 1 hour. Step 1: Connect the first amino acid. Weigh 2.3 mmol Fmoc-Lys(Boc)-OH, 2.3 mmol HoBt, 0.23 mmol DMAP, and 2.3 mmol DIC from the first amino acid precursor, dissolve them in approximately 20 mL of DMF, and add them to the reaction vessel containing the resin. Mix for 2–3 hours. Then add 1.15 mmol acetic anhydride and pyridine, and stir for 30 minutes to end-cap the resin. Step 2: Connect subsequent amino acids. Remove the Fmoc protecting group from the already connected amino acids using 20% ​​piperidine / DMF (v:v). Sequentially weigh 2.3 mmol of each subsequent amino acid precursor, using HCTU and DIEA as catalysts. Dissolve each amino acid separately with 2.3 mmol HCTU and 2.3 mmol DIEA in approximately 30 mL of DMF, and then sequentially add the mixed solutions to the reactor to react with the resin. The linkage status of each amino acid was detected by the ninhydrin reaction. After the N-terminal Cys linkage was complete, the Fmoc protecting group of Cys was removed with 20% piperidine / DMF, and the resin was washed three times each with DMF and DCM. Finally, stearic acid C18 was linked. 2.3 mmol of stearic acid C18, 2.3 mmol of EDCI, and 2.3 mmol of DMAP were weighed and dissolved in 30 mL of DCM. The mixture was added to the reactor and mixed with the resin. The reaction status was also detected by the ninhydrin reaction. After the reaction was complete, the resin was condensed with methanol and dried. The cleavage reagent was prepared (0.5 g of resin was weighed to prepare approximately 10-15 mL of solution): TFA:TIPS:ulpure water = 95:2.5:2.5 (V:V:V). After the cleavage reagent and resin were mixed and reacted for approximately 4 hours, the mixture was filtered through a sintered glass funnel, and the filtrate was collected. Pre-cooled diethyl ether (approximately 20 times the volume of the cleavage reagent) was added to the filtrate for extraction. After thoroughly mixing the ether and filtrate, centrifuge at 14000g and 4℃ for 20 minutes, discard the supernatant, add ether to the precipitate, and repeat the extraction three times. Dry the remaining ether in the precipitate in a vacuum drying oven to obtain the crude PM powder, which is then purified by LC-MS analysis to obtain the final pure product.

[0200] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An enzyme-responsive peptide-based nucleic acid targeting delivery system, characterized in that, The nanomicelle and the CD44-targeting aptamer connected to the outer layer of the nanomicelle; The nanomicelle is composed of an enzyme-responsive peptide and CD24 siRNA adsorbed to the enzyme-responsive peptide; The enzyme-responsive peptide is C18-CGGFLG-HHHKKHHHKKK; The CD24 siRNA targets and silences the CD24 gene; The sense strand of the CD24 siRNA is shown in SEQ ID NO. 3, and the antisense strand is shown in SEQ ID NO. 4; The nucleotide sequence of the CD44-targeting aptamer is shown in SEQ ID NO. 2, and the 3' end of the CD44-targeting aptamer is modified with a cholesterol group for connection with the enzyme-responsive peptide.

2. The delivery system of claim 1, wherein, The mass ratio of nitrogen element and phosphorus element in the delivery system is (2-10):

1.

3. The delivery system of claim 2, wherein, The mass ratio of nitrogen element and phosphorus element in the delivery system is 10:

1.

4. The delivery system of claim 1, wherein, The CD44-targeting aptamer accounts for 1-10% of the total moles of CD24 siRNA and CD44-targeting aptamer.

5. The method for preparing the enzyme-responsive peptide nucleic acid targeting delivery system according to any one of claims 1 to 4, characterized in that, The preparation method comprises the following steps: First, mix CD24 siRNA and CD44-targeting aptamer according to the formula amount to obtain a polynucleotide mixture system; then mix the polynucleotide mixture system into a solution containing an enzyme-responsive peptide, and incubate to obtain the delivery system.

6. The production method according to claim 5, characterized by The concentration of the enzyme-responsive peptide in the solution of the enzyme-responsive peptide is 0.8-4 mg / mL; the concentration of CD24 siRNA and CD44-targeting aptamer is independently 20 μM; and the polynucleotide mixture system and the solution of the enzyme-responsive peptide are mixed in equal volumes.

7. The preparation method according to claim 6, characterized in that, The concentration of the enzyme-responsive peptide in the solution of the enzyme-responsive peptide is 4 mg / mL.

8. Use of the enzyme-responsive peptide-based nucleic acid targeting delivery system of any one of claims 1-4 or the preparation method of any one of claims 5-7 in the preparation of a medicament for treating triple-negative breast cancer.

9. A medicament, characterized by, The enzyme-responsive peptide-based nucleic acid targeting delivery system of any one of claims 1-4 and a pharmaceutically active ingredient wrapped therein.

Images