Highly internalizing aptamers targeting hepatocarcinoma cells and uses thereof

Abstract

The application discloses a high internalization nucleic acid aptamer targeting liver cancer cells, and a nucleotide sequence of the high internalization nucleic acid aptamer is shown as SEQ ID NO. 3. The application also provides a use of the high internalization nucleic acid aptamer in preparation of a medicine for treating liver cancer. The application is a cell index enrichment ligand system evolution technology which takes a complete liver cancer cell line as a target and screens a cell index by integrating an internalization function. The nucleic acid aptamer is screened by the cell index enrichment ligand system evolution technology, can be specifically combined with human liver cancer cells, and has a high internalization efficiency. The nucleic acid aptamer has a good use in preparation of a medicine for targeted treatment of liver cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to an aptamer, specifically a highly internalized nucleic acid aptamer for targeting liver cancer cells and its application. Background Technology

[0002] Currently, the clinical diagnosis and treatment of liver cancer still faces severe challenges. In early diagnosis, widely used serum biomarkers such as alpha-fetoprotein (AFP) have unsatisfactory sensitivity and specificity, leading to missed diagnoses in many early-stage patients. In terms of treatment, besides surgical resection, chemotherapy and targeted therapy are the main methods; however, traditional small-molecule chemotherapy drugs suffer from poor specificity, significant toxic side effects, and a tendency to develop drug resistance.

[0003] In recent years, targeted therapy has become a research hotspot in cancer treatment. While monoclonal antibody drugs have achieved significant results, they suffer from inherent limitations such as large molecular weight, poor tissue penetration, high production costs, and the potential to trigger immune responses. Therefore, developing novel targeted molecules is crucial for advancing precision diagnosis and treatment of liver cancer.

[0004] Aptamers are small single-stranded DNA or RNA oligonucleotide fragments obtained through in vitro screening techniques (SELEX). Like antibodies, they bind to target molecules (such as proteins, cells, and even tissues) with high affinity and specificity, hence the name "chemical antibodies." Compared to traditional monoclonal antibodies, aptamers have the following significant advantages:

[0005] Small molecular weight: makes it easy to penetrate tumor tissue.

[0006] Easy to synthesize in vitro: Produced through chemical synthesis, with small batch-to-batch differences and stable and controllable quality.

[0007] Low immunogenicity: It is not likely to trigger an immune response in the body.

[0008] Easy to modify and label: Various functional groups (such as fluorescent groups, biotin, therapeutic drugs and nanomaterials) can be easily introduced during the synthesis process, thereby constructing a multifunctional integrated diagnostic and therapeutic platform.

[0009] Good stability: Through appropriate chemical modification (such as 2'-fluorination of the RNA backbone or thiophosphate modification of the DNA backbone), its ability to resist nuclease degradation can be significantly enhanced, thus improving its stability in vivo.

[0010] Cell-SELEX technology, used for aptamer screening, is a strategy that uses intact, living cells as screening targets. This technology does not require prior knowledge of specific target molecules on the cell surface; it can directly screen for membrane proteomes in their native state, thereby obtaining aptamers that can recognize disease-related cell surface biomarkers. This opens up possibilities for discovering new tumor biomarkers and developing corresponding targeted tools.

[0011] However, conventional Cell-SELEX technology primarily screens aptamers that bind tightly to the cell surface. For targeted cancer therapy, "internalized aptamers" that can efficiently deliver therapeutic payloads (such as chemotherapy drugs, siRNA, toxins, etc.) into the cell are of unparalleled value. These aptamers can enter the cell through internal mechanisms, thereby directly acting on intracellular targets, greatly improving therapeutic efficacy and reducing off-target toxicity. However, currently, there are still relatively few aptamers that can be efficiently internalized by cells, and there is a lack of mature protocols for efficiently and targetedly screening internalized aptamers. Summary of the Invention

[0012] To address the aforementioned technical problems in the prior art, this invention provides a highly internalized nucleic acid aptamer for targeting liver cancer cells and its application. This highly internalized nucleic acid aptamer for targeting liver cancer cells and its application aim to solve the technical problems of insufficient specificity of liver cancer diagnostic markers and low delivery efficiency of targeted therapeutic drugs in the prior art.

[0013] This invention provides a highly internalized nucleic acid aptamer that targets liver cancer cells, the nucleotide sequence of which is shown in any one of SEQ ID NO. 1 to 3.

[0014] Further methods include chemically modifying the bases of nucleic acid aptamers, or binding biotin, digoxigenin, fluorescent substances, luminescent nanomaterials, or enzyme labeling to the sequence.

[0015] This invention also provides a method for screening the above-mentioned nucleic acid aptamers, comprising the following steps:

[0016] 1) A process of elimination and screening:

[0017] The ssDNA library was incubated with HL-7702 cells at 4°C. The sequence of the ssDNA library was 5'-FAM-ATCCAGAGTGACGCAGCA-N. 40 -GGACATGGACGGTGAGGT-3'; Centrifuge and collect the supernatant, which is the secondary library with the sequence that binds to normal cells removed;

[0018] 2) A positive screening step: Incubate the reduced supernatant with Huh-7 cells;

[0019] 3) Separation and recovery steps: The ssDNA internalized in the cells is recovered through washing, digestion, cell lysis and extraction precipitation;

[0020] 4) A PCR amplification and single-strand preparation process:

[0021] a. Using the recovered ssDNA as a template, perform asymmetric PCR amplification;

[0022] b. Purify all PCR products using a standard PCR purification kit and elute them in 50-100 μL of sterile water;

[0023] c. Use streptavidin-coated magnetic beads to bind to biotin-labeled PCR products, denature them, and then elute and retain the supernatant;

[0024] d. Neutralize the supernatant by adding an equal volume of Tris-HCl buffer;

[0025] e. The neutralized ssDNA solution is desalted and concentrated, and finally eluted in an appropriate amount of sterile water to obtain a secondary ssDNA library for the next round of screening.

[0026] 5) A step for monitoring and increasing screening pressure;

[0027] a. Take a portion of the FAM-labeled enriched library and incubate it with Huh-7 cells, then monitor the enrichment of binding signals by flow cytometry;

[0028] b. Increasing screening pressure: reducing target cell / ssDNA, shortening time, increasing washing, and strengthening subtraction to eliminate weak binding sequences and enrich high-affinity and high-specificity aptamers;

[0029] 6) A cloning and sequencing process;

[0030] a. The final enriched PCR product is cloned into the T vector, transformed, and at least 100 single clones are selected for sequencing;

[0031] b. Use sequence analysis software to perform multiple sequence alignment, and obtain the above-mentioned nucleic acid aptamers based on sequence families and occurrence frequencies.

[0032] The present invention also provides the use of the nucleotide sequences shown in any of SEQ ID NO. 1 to 2 above in the preparation of molecular probes or detection kits for in vitro diagnostics or in vivo imaging.

[0033] The present invention also provides the use of the nucleotide sequence shown in SEQ ID NO.3 above in the preparation of a medicament for treating liver cancer.

[0034] The present invention also provides the use of the nucleotide sequence shown in SEQ ID NO.3 in the preparation of targeted delivery vectors.

[0035] The present invention also provides a medicament for treating liver cancer, wherein the active ingredient of the medicament comprises an aptamer of a nucleotide sequence as shown in SEQ ID NO. 3.

[0036] The present invention also provides a kit comprising the above-described nucleic acid aptamers.

[0037] This invention designs a function-guided Cell-SELEX screening process, using the human hepatocellular carcinoma cell line Huh-7 as the target cell and the human normal hepatocyte cell line HL-7702 as the subtractive cell. This serves as a key step in the screening process to integrate internalization function enrichment, aiming to directly and efficiently screen for nucleic acid aptamers (Apt-01, Apt-02, Apt-03) that specifically recognize hepatocellular carcinoma cells and have high internalization efficiency. These aptamers exhibit high affinity and high specificity for human hepatocellular carcinoma cell lines. Among them, Apt-03 can be efficiently internalized by hepatocellular carcinoma cells.

[0038] The beneficial effects of this invention are as follows:

[0039] High affinity and high specificity: All three aptamers specifically recognize liver cancer cells, while binding weakly to normal liver cells. They exhibit nanomolar-level high affinity (Kd = 15.2-21.7 nM), meeting the basic requirements for molecular probes and targeting vectors. Apt-03 maintains high affinity (Kd = 43.28 nM) while displaying the highest specificity ratio (6.65), indicating better targeting accuracy in complex biological environments.

[0040] Functional differentiation:

[0041] Apt-01 and Apt-02: Excellent surface binding ability, they are typical surface-bound aptamers (internalization efficiency <16%), suitable for in vitro diagnostics and in vivo imaging.

[0042] Apt-03 possesses excellent internalization capabilities and is a highly efficient internalization aptamer (internalization efficiency = 60%). It has the following unique advantages: it can effectively deliver payloads into the cell interior, making it suitable as a targeted delivery carrier for drugs, siRNA, and nanoparticles. In targeted therapy, it can achieve a "synergistic effect with reduced toxicity." It is expected to become a core component in the development of next-generation early diagnostic reagents for liver cancer, molecular imaging probes, and highly efficient, low-toxicity targeted therapies, demonstrating significant clinical application prospects and market value.

[0043] Innovation at the source: The screening process integrates functional pressure and directly enriches internalized aptamers, making it more efficient.

[0044] Compared with existing technologies, the technical effects of this invention are positive and significant. This invention is a cell index enrichment ligand system evolution technique that uses intact liver cancer cell lines as targets and integrates internalization function screening pressure to obtain nucleic acid aptamers that can specifically bind to human liver cancer cells and have high internalization efficiency. The nucleic acid aptamers of this invention have good applications in the preparation of drugs for in vitro diagnostics, in vivo molecular imaging, or targeted therapy for liver cancer. Attached Figure Description

[0045] Figure 1 This is an affinity curve of the aptamer of the present invention with Huh-7 cells.

[0046] Figure 2 The results show the affinity curve fitting between the aptamer of this invention and Huh-7 cells.

[0047] Figure 3a This is a comparison of dose-response curves for Huh-7 cells.

[0048] Figure 3b This is a comparison of dose-response curves for HL-7702 cells. Detailed Implementation

[0049] Example 1: Aptamer Screening (Cell-SELEX)

[0050] 1.1 Reagent Preparation

[0051] Cell lines:

[0052] Target cells: Human hepatocellular carcinoma cell line Huh-7 (high metastatic potential) (BNCC, BNCC337690).

[0053] Control cells: Human normal hepatocyte cell line HL-7702 (BNCC, BNCC338358).

[0054] ssDNA initial library: 5'-FAM-ATCCAGAGTGACGCAGCA-N 40 -GGACATGGACGGTGAGGT-3' (100 nmol, dissolved in 1 mL PBS) (SEQ ID NO.4).

[0055] Binding buffer: DPBS (containing 5 mM MgCl2, 0.1 mg / mL yeast tRNA, 1 mg / mL BSA) (pH 7.4).

[0056] 1.2 SELEX Screening

[0057] Elimination Filtering:

[0058] a. Combine ssDNA library (1000 pmol) with HL-7702 cells (1×10⁻⁶ pmol). 6 Incubate at 4°C for 60 minutes.

[0059] b. Centrifuge and carefully collect the supernatant, which is the secondary library with the sequences that bind to normal cells removed.

[0060] Positive filtering:

[0061] a. Mix the reduced supernatant with Huh-7 cells (1×10⁻⁶). 6 Reduced to 5×10 starting from round 4. 5 Incubate at 37°C and 5% CO2 for 60 minutes. This temperature allows memory to function.

[0062] Separation and recycling:

[0063] a. Wash three times with pre-cooled binding buffer to remove unbound sequences.

[0064] b. Add 0.25% trypsin (containing EDTA) and digest at 37°C for 5 minutes to dissociate and remove aptamers that remain only on the cell membrane surface.

[0065] c. Centrifuge and discard the supernatant (containing surface-binding sequences), resuspend the cell pellet in cell lysis buffer (containing SDS and proteinase K), and incubate at 55°C for 1 hour.

[0066] d. The ssDNA internalized in cells was recovered by phenol-chloroform-isoamyl alcohol extraction and ethanol precipitation.

[0067] PCR amplification and single-strand preparation:

[0068] f. Using the recovered ssDNA as a template, perform asymmetric PCR amplification. Reaction program: 95℃ 5min → [95℃ 30s → 58℃ 30s → 72℃ 30s] ×25 → 72℃ 5min.

[0069] g. Purify all PCR products using a standard PCR purification kit to remove dNTPs, enzymes, salt ions, etc. Elute in 50-100 μL of sterile water.

[0070] h. Use streptavidin-coated magnetic beads to bind to biotin-labeled PCR products, denature them with 0.1M NaOH, and then elute and retain the supernatant.

[0071] i. Neutralize the supernatant (containing the target ssDNA) by adding an equal volume of 1 M Tris-HCl buffer (pH 7.5).

[0072] j. The neutralized ssDNA solution was desalted and concentrated using ethanol precipitation or a dedicated nucleic acid purification column, and finally eluted in an appropriate amount of sterile water to obtain a secondary ssDNA library for the next round of screening.

[0073] Table 1 PCR reaction system

[0074] .

[0075] SEQ ID NO.5:ATCCAGAGTGACGCAGCA (upstream primer);

[0076] SEQ ID NO.6:ACCTCACCGTCCATGTCC (downstream primer).

[0077] 1.3 Monitoring and Incremental Screening Pressure

[0078] c. Starting from round 5, a portion of the FAM-labeled enriched library was incubated with Huh-7 cells at 4°C, and the enrichment of binding signals was monitored by flow cytometry.

[0079] d. Increasing screening pressure: (reducing target cell / ssDNA, shortening time, increasing washing, and strengthening subtraction to eliminate weak binding sequences and enrich high-affinity, high-specificity aptamers;)

[0080] a) Rounds 1-4: The number of cells for both reduction and positive selection is 1×10^6, and the amount of ssDNA is 1000 pmol.

[0081] b) Rounds 5-7: Positive screening cell count reduced to 5×10 5 The amount of ssDNA decreased to 500 pmol.

[0082] c) Rounds 8-10: Positive screening cell count reduced to 2×10 5 The amount of ssDNA was reduced to 200 pmol, and the number of washes was increased to 5.

[0083] 1.4 Cloning and Sequencing

[0084] a. After the 10th round of screening, the final enriched PCR products are cloned into the T vector, transformed, and at least 100 single clones are selected for sequencing.

[0085] b. Multiple sequence alignment was performed using sequence analysis software. Based on sequence family and frequency of occurrence, three representative sequences, Apt-01, Apt-02, and Apt-03, were selected for subsequent characterization. The sequences are shown in the table below.

[0086] Table 2 Aptamer Sequences

[0087] .

[0088] The above-mentioned method for preparing aptamers is as follows:

[0089] The desired sequence was imported into an Oligo 48 synthesizer, and synthesized sequentially from the 3'-5' direction using inverted dT CPG as a carrier. The phosphoramide monomer was dissolved in anhydrous acetonitrile (100 mM) and soaked in molecular sieves for at least 24 hours. Each base linkage or modification involved four steps: deprotection, coupling, capping, and oxidation (or thiolation), yielding a nucleotide sequence with a solid support and protecting groups. Finally, ammonia deprotection and purification were performed to obtain the final product (the specific synthesis of this invention was outsourced to Shanghai Sangon Biotech Co., Ltd.).

[0090] Example 2: Affinity and Specificity Verification

[0091] 2.1 Preparation of Experimental Materials

[0092] Cell lines: Human hepatocellular carcinoma cell line Huh-7, and normal human hepatocytes HL-7702.

[0093] Aptamers: Apt-01, Apt-02, Apt-03, 5' ends were labeled with FAM fluorescence and purified by HPLC.

[0094] 2.2 Experimental Procedure

[0095] Cell preparation:

[0096] a. The day before the experiment, Huh-7 and HL-7702 cells were digested and resuspended, respectively, at a concentration of 5 × 10⁶ cells per tube. 5 Cells were seeded at a density of 1.5 mL in 1 centrifuge tubes. Each concentration point for each aptamer and the control required replicates. A total of 36 tubes of cells were prepared (3 aptamers × 6 concentration points × 2 cell types).

[0097] b. The cells were incubated overnight in a 37°C, 5% CO2 incubator to allow them to adhere to the culture vessel and recover.

[0098] Preparation of aptamer working solution:

[0099] a. Take an appropriate amount of FAM-labeled aptamer stock solution and serially dilute it with pre-cooled binding buffer to prepare working solutions with concentrations of 0, 10, 25, 50, 100, and 200 nM. All operations should be performed on ice in the dark.

[0100] Combination reaction:

[0101] a. Remove the cell tube from the incubator and carefully aspirate the supernatant.

[0102] b. Gently wash the cells twice with pre-cooled washing buffer (add 1 mL each time, centrifuge at 800 rpm for 5 minutes and discard the supernatant) to remove the culture medium.

[0103] c. Add 100 μL of the corresponding concentration of aptamer working solution to each tube and gently pipette to mix.

[0104] d. Place all cell tubes in an ice bath or freezer at 4°C and incubate in the dark for 60 minutes. Gently vortex every 15 minutes during this period to ensure full contact between the cells and aptamers. The 4°C condition is intended to inhibit cell memory activity; only surface binding is measured.

[0105] Washing and resuspension:

[0106] a. After incubation, add 1 mL of pre-cooled washing buffer to each tube and gently disperse the cells.

[0107] b. Centrifuge at 800 rpm for 5 minutes at 4°C, and carefully discard the supernatant to remove unbound aptamers.

[0108] c. Repeat this washing step a total of 3 times.

[0109] d. Finally, resuspend the cells in 500 μL of pre-chilled DPBS in each tube and immediately transfer them to flow cytometer-specific sample tubes. Store on ice in the dark until analysis.

[0110] Flow cytometry detection

[0111] a. Using a flow cytometer, FAM fluorescence is excited by a 488 nm laser, and the fluorescence signal is detected in the FITC channel (typically a 530 / 30 nm filter).

[0112] b. Load samples sequentially for testing, collecting 10,000 valid cellular events per tube.

[0113] c. Record the mean fluorescence intensity (MFI) of the FITC channel for each sample.

[0114] 2.3 Data Analysis

[0115] a. Plot fluorescence intensity against aptamer concentration, and calculate the dissociation constant (Kd value) using nonlinear regression fitting (single-point binding model). The smaller the Kd value, the higher the affinity.

[0116] b. At the same concentration, the binding fluorescence intensity of the aptamer to target cells and various control cells was measured separately. Specificity ratio = (target cell fluorescence intensity - background) / (control cell fluorescence intensity - background). A ratio much greater than 1 indicates good specificity.

[0117] c. Affinity test results as follows Figure 1 and Figure 2 As shown, all three aptamers exhibited typical saturation binding curves, indicating their specific binding to Huh-7 cells. The Kd values ​​of Apt-01, Apt-02, and Apt-03 were 32.58, 41.40, and 43.28, respectively, all below 50 nM, demonstrating extremely high affinity.

[0118] d. Specificity test results are shown in Table 3. The signal intensity of all aptamers on Huh-7 cells was significantly higher than that of HL-7702, and the low binding to normal hepatocytes indicated a low risk of off-target effects. Apt-03 showed the highest specificity ratio and the strongest discrimination ability.

[0119] Table 3 Aptamer specificity verification (100 nM)

[0120] .

[0121] Example 3: Verification of Internalization Efficiency

[0122] 3.1 Preparation of Experimental Materials

[0123] Cell lines and aptamers: Same as in Example 1.

[0124] Key reagents:

[0125] Trypsin-EDTA digestion solution (0.25%), preheated at 37°C.

[0126] Acidic washing solution: 0.2 M glycine, 0.15 M NaCl, pH adjusted to 2.5 with HCl. This low pH condition effectively dissociates aptamers bound to the membrane surface.

[0127] 3.2 Experimental Procedure

[0128] Cell preparation:

[0129] a. Huh-7 cells were seeded in 24-well cell culture plates at a density of 2 × 10^5 / well.

[0130] b. Set up 3 experimental groups (total bonding group, internalization group, and surface bonding group), with at least 3 duplicate wells in each group.

[0131] c. The cells were cultured at 37°C in a 5% CO2 incubator for 24 hours to achieve a confluence of 60%-70%.

[0132] Aptamer treatment:

[0133] a. Total binding group: Remove cells from the incubator and gently wash once with binding buffer preheated to 37°C. Then add 500 μL of 200 nM FAM-aptamer (prepared with binding buffer preheated to 37°C). Incubate at 37°C in a 5% CO2 incubator for 120 minutes in the dark.

[0134] b. Internalization group: The treatment is exactly the same as the "overall combination group".

[0135] c. Surface binding assay: Wash cells with binding buffer pre-chilled at 4°C. Then add 500 μL of 200 nM FAM-aptamer (prepared with binding buffer pre-chilled at 4°C). Incubate at 4°C on ice in the dark for 120 minutes.

[0136] Post-treatment and washing:

[0137] a. General combination group

[0138] a) After incubation, discard the supernatant.

[0139] b) Wash the cells twice quickly with washing buffer preheated to 37°C.

[0140] c) Add 300 μL of trypsin digestion solution, digest at 37°C for 1-2 minutes, add complete culture medium to stop digestion, gently pipette to prepare a single-cell suspension, and transfer to a 1.5 mL centrifuge tube.

[0141] d) Centrifuge at 4℃ and 800 rpm for 5 minutes, then discard the supernatant.

[0142] e) Resuspend the cells in 500 μL of pre-cooled DPBS and immediately analyze them using a flow cytometer.

[0143] f) This set of signals represents the total fluorescence of "surface binding + internalization".

[0144] b. Internalization group

[0145] a) After incubation, discard the supernatant.

[0146] b) Step 1: Remove surface binding. Add 300 μL of preheated trypsin digestion solution and digest at 37°C for 5 minutes to fully dissociate the aptamers bound to the membrane surface. Then add an equal volume of complete culture medium to terminate the digestion.

[0147] c) Step 2: Acid washing. Transfer the cell suspension to a 1.5 mL centrifuge tube, centrifuge at 800 rpm for 5 minutes at 4°C, and discard the supernatant. Resuspend the cells in 500 μL of pre-chilled acidic washing buffer and incubate on ice for 5 minutes to thoroughly remove any residual membrane surface binding.

[0148] d) Centrifuge at 4℃ and 800 rpm for 5 minutes, then discard the supernatant.

[0149] e) Resuspend the cells in 500 μL of pre-cooled DPBS and immediately analyze them using a flow cytometer.

[0150] f) This set of signals represents only the fluorescence of the "internalized" aptamers.

[0151] c. Surface bonding group

[0152] a) After incubation, discard the supernatant.

[0153] b) Wash the cells three times with washing buffer pre-cooled to 4°C.

[0154] c) The digestion, centrifugation, and resuspension steps are the same as those in the "total conjugation group".

[0155] d) This set of signals represents only the fluorescence of "surface-bound" fluorescence.

[0156] Flow cytometry detection

[0157] a. Using a flow cytometer, FAM fluorescence is excited by a 488 nm laser, and the fluorescence signal is detected in the FITC channel (typically a 530 / 30 nm filter).

[0158] b. Load samples sequentially for testing, collecting 10,000 valid cellular events per tube.

[0159] c. Record the mean fluorescence intensity (MFI) of the FITC channel for each sample.

[0160] 3.3 Data Analysis

[0161] a. Internalization efficiency (%) = (Fluorescence intensity of internalized group / Fluorescence intensity of total bound group) × 100%.

[0162] b. The results of aptamer in vivo incorporation efficiency are shown in Table 4. The incorporation efficiencies of Apt-01 and Apt-02 are both less than 16%, mainly remaining on the cell surface. The incorporation efficiency of Apt-03 is as high as 60%, which is 4-5 times that of other aptamers, and the incorporation / surface ratio of Apt-03 is >1, indicating that it mainly enters the cell through the internalization pathway.

[0163] Table 4 Comparative Analysis of Adaptor In vivo Efficiency

[0164] .

[0165] Example 4: Targeted drug conjugation and in vitro killing experiment

[0166] 4.1 Experimental Objective

[0167] The function of the highly internalized aptamer Apt-03 as a targeted delivery carrier was verified, and highly selective and efficient killing of liver cancer cells was achieved by conjugating it with the chemotherapeutic drug doxorubicin (Dox).

[0168] 4.2 Experimental Materials and Methods

[0169] 4.2.1 Synthesis and Characterization of Aptamer-Drug Conjugate (Apt-03-Dox)

[0170] Synthetic route: A pH-sensitive hydrazone bond was used for coupling. First, the 3' end of Apt-03 was modified with an amino group (introducing C6-NH2). Simultaneously, the ketone group of Dox was reacted with excess adipic acid dihydrazide (ADH) to generate a Dox-ADH derivative with a reactive hydrazide group at the end. Finally, under EDC / NHS catalysis, the hydrazide group of Dox-ADH was linked to the terminal amino group of Apt-03 to form a hydrazone bond. This bond is stable at neutral blood pH (7.4) and can be rapidly cleaved in the acidic environment of tumor cell endosomes / lysosomes (pH 4.5-5.5), releasing active Dox.

[0171] Purification and characterization: The conjugate was purified using high-performance liquid chromatography (HPLC). The absorbance was measured by UV-Vis spectrophotometry at 480 nm (characteristic absorption peak of Dox) and 260 nm (characteristic absorption peak of nucleic acids), and the molecular ratio (DAR) of Dox to Apt-03 in the conjugate was calculated. In this experiment, DAR ≈ 2:1.

[0172] 4.2.2 Cytotoxicity assay (CCK-8 assay)

[0173] Cell culture: Human hepatocellular carcinoma cells Huh-7 and normal human hepatocytes HL-7702 were cultured at 37°C and 5% CO2.

[0174] Experimental grouping and drug administration:

[0175] Cells were seeded at a density of 5 × 10³ cells per well in a 96-well plate and cultured for 24 hours to allow them to adhere to the plate.

[0176] Set up the following 7 treatment groups, with 6 replicates for each concentration:

[0177] Blank control group: only complete culture medium was added.

[0178] Free Dox group: concentration gradients of 0.1, 0.2, 0.5, 1.0, 2.0, and 5.0 μM.

[0179] Random-Dox group (irrelevant sequence coupled control): concentration gradient same as free Dox group.

[0180] Apt-03-Dox group: Concentration gradient same as free Dox group (concentration in Dox).

[0181] Free Apt-03 group (100 nM): used to verify that the aptamer itself is non-toxic.

[0182] Random Seq. group (100 nM): irrelevant sequence control.

[0183] Maximum lethality control group: Add 1% Triton X-100.

[0184] Incubation and testing:

[0185] a. After adding the drug, return the cells to the incubator and continue culturing for 48 hours.

[0186] b. Add 10 μL of CCK-8 solution to each well and continue incubation for 2 hours.

[0187] c. Use an ELISA reader to measure the absorbance (OD value) of each well at a wavelength of 450 nm.

[0188] 4.3 Data Analysis

[0189] Cell viability = [(OD-treated group - OD-maximally-killing group) / (OD-free group - OD-maximally-killing group)] × 100%

[0190] like Figure 3a As shown, for Huh-7 cells, the Apt-03-Dox curve exhibited a steep "S" shape and a significant leftward shift, indicating that the concentration required to achieve the same killing effect was much lower than that of other groups, and its IC50 value was significantly lower. 50The value (~0.35 μM) was significantly lower than that of free Dox and Random-Dox (~1.1 μM), demonstrating that targeted delivery resulted in approximately a 3-fold synergistic effect. The free Dox curve almost overlapped with the Random-Dox curve, located on the right side. As shown in Figure 3b, for HL-7702 cells, the three curves highly overlapped, all located on the right side, indicating that Apt-03-Dox had no additional toxicity to normal cells. The effect of Random-Dox was no different from that of free Dox, and the aptamer itself was non-toxic (Table 5), demonstrating that the improved efficacy depended entirely on the specific targeting and internalization function of Apt-03.

[0191] Table 5. Aptamer autotoxicity control group (treated for 48 hours)

[0192] .

Claims

1. A highly internalized nucleic acid aptamer targeting liver cancer cells, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

3.

2. The highly internalized nucleic acid aptamer targeting liver cancer cells according to claim 1, characterized in that, This includes chemically modifying the bases of nucleic acid aptamers, or binding biotin, digoxigenin, fluorescent substances, nanoluminescent materials, or enzyme labeling to the sequence.

3. The use of the highly internalized nucleic acid aptamer targeting liver cancer cells as described in claim 1 in the preparation of a medicament for treating liver cancer.

4. The use of the highly internalized nucleic acid aptamer targeting liver cancer cells as described in claim 1 in the preparation of a targeted delivery vector.

5. A drug for treating liver cancer, characterized in that, The active ingredient of the drug comprises a highly internalized nucleic acid aptamer targeting liver cancer cells as described in claim 1.

6. A reagent kit, characterized in that, It includes a highly internalized nucleic acid aptamer targeting liver cancer cells as described in claim 1.

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