Modified nucleic acid aptamer-paclitaxel conjugate, preparation method therefor, and use thereof
By non-covalently modifying the nucleic acid aptamer paclitaxel conjugate with G4 ligand, the problems of insufficient targeting and stability in the existing technology are solved, and better tumor targeting and anti-cancer effects are achieved.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- INCREASEPHARM TIANJIN INST CO LTD
- Filing Date
- 2025-11-21
- Publication Date
- 2026-05-28
AI Technical Summary
Existing nucleic acid aptamer-paclitaxel conjugates have shortcomings in terms of cancer cell targeting and stability, which affects their anti-cancer efficacy.
The nucleic acid aptamer paclitaxel conjugate was non-covalently modified with G4 ligand. The specific method involved inserting the G4 ligand into the G4 plane of the AS1411 aptamer and preparing the modified conjugate through a specific ratio and purification steps.
It improves tumor targeting and the ability to inhibit tumor cell proliferation and migration, enhances the specific recognition and efficacy of cancer cells, and significantly inhibits tumor growth.
Smart Images
Figure CN2025136931_28052026_PF_FP_ABST
Abstract
Description
A modified nucleic acid aptamer-paclitaxel conjugate, its preparation method and uses Technical Field
[0001] This invention relates to the field of pharmaceutical technology, specifically to a modified nucleic acid aptamer paclitaxel conjugate, its preparation method, and its uses. Background Technology
[0002] Cancer is the second leading cause of death from cancers of the female reproductive system, and the second most common gynecological malignancies. First-line treatments for cancer include debulking surgery and paclitaxel-based combination chemotherapy. PTX is widely used as a first-line drug in clinical chemotherapy for various cancers. However, PTX's low water solubility hinders its direct clinical application. Furthermore, PTX lacks specificity in distinguishing between cancer cells and normal cells, often leading to serious adverse reactions.
[0003] Nucleic acid aptamers are short, single-stranded RNA or DNA oligonucleotide fragments screened using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) technique. They can specifically bind to various molecular targets. Compared to peptides and small molecule ligands, nucleic acid aptamers exhibit higher affinity and selectivity for their targets, with dissociation constants in the nanomolar (nmol) range. Furthermore, nucleic acid aptamers offer several advantages over antibodies, including small size, low immunogenicity, and low production cost. These unique characteristics allow nucleic acid aptamers to be used not only as drugs but also as drug delivery vectors. For example, AS1411, a 26-base DNA aptamer with a G-quadruplex structure that selectively binds nucleolinin, has entered Phase II clinical trials. Recent studies have shown that nucleolinin exists on the cell membranes of certain cancers (such as lung cancer, breast cancer, and kidney cancer) at varying expression levels. Antitumor drugs modified with AS1411 can enhance the specific recognition of tumor cells, thereby leading to subsequent endocytosis through the interaction between AS1411 and nucleolin.
[0004] Patent WO2017128173 discloses aptamer conjugates of paclitaxel or its derivatives, their preparation methods, and applications. The conjugate structure comprises three parts: the anticancer drug paclitaxel (PTX), the nucleic acid aptamer AS1411, and a linker. This patent discloses the structures and preparation methods of a series of conjugates, as well as their applications in treating breast cancer, colon cancer, bronchial cancer, ovarian cystic adenocarcinoma, or endometrial cancer. However, these paclitaxel aptamer conjugates exhibit poor stability and low targeting, thus affecting their anticancer efficacy.
[0005] Therefore, there is an urgent need in this field to address the problem of low stability and targeting of nucleic acid aptamer-paclitaxel conjugates for cancer cells, thereby improving their anti-cancer effects. Technical issues
[0006] The purpose of this invention is to overcome the shortcomings of the prior art and provide a modified nucleic acid aptamer paclitaxel conjugate that has better tumor targeting than the unmodified nucleic acid aptamer paclitaxel conjugate, while also enhancing the ability to inhibit tumor cell proliferation and migration. Technical solutions
[0007] To solve the above problems, the technical solution adopted by the present invention is as follows:
[0008] In a first aspect, the present invention provides a modified nucleic acid aptamer-paclitaxel conjugate, the modified nucleic acid aptamer-paclitaxel conjugate comprising paclitaxel, a succinic acid linker, and a G4 ligand-modified AS1411 aptamer, the structure of which is as follows:
[0009]
[0010] Wherein, X is an AS1411 aptamer modified with G4 ligand, wherein the G4 ligand is inserted into the G4 plane of the AS1411 aptamer via a non-covalent bond.
[0011] Figure 1 shows a schematic diagram of the modified nucleic acid aptamer-paclitaxel conjugate provided by this invention. The figure shows that the terminal amino group of AS1411 is coupled to paclitaxel via a succinic acid linker, and the G4 ligand is inserted into the G4 plane of AS1411 via a non-covalent bond. Therefore, the modification described in this invention refers to the non-covalent modification of the G4 ligand.
[0012] This invention achieves better tumor targeting by non-covalently modifying the nucleic acid aptamer paclitaxel conjugate with G4 ligand, while also enhancing its ability to inhibit tumor cell proliferation and migration.
[0013] Preferably, the CD spectrum of the coupling compound shows a positive peak at 265 nm.
[0014] Preferably, the G4 ligand is 360A iodide.
[0015] In this invention, the 360A iodide is as follows:
[0016] ;
[0017] Preferably, the loading ratio of the G4 ligand to AS1411 is 1:1. This ratio is a molar ratio and can be obtained by quantitative analysis of the loaded G4 ligand and AS1411 using HPLC and Nanodrop, respectively.
[0018] In a second aspect, the present invention provides a method for preparing the modified nucleic acid aptamer-paclitaxel conjugate described in the first aspect, the method comprising the following steps:
[0019] The modified aptamer-paclitaxel conjugate was obtained by mixing an aqueous solution of the nucleic acid aptamer-paclitaxel conjugate (PSaA) with a dimethyl sulfoxide solution of the G4 ligand in an ice bath and purifying the mixture.
[0020] Preferably, the ratio of AS1411 aptamer to G4 ligand in the nucleic acid aptamer-paclitaxel conjugate is 1:10. This ratio is the molar ratio.
[0021] Preferably, the mixing time is 20 min to 60 min, for example 30 min, 40 min, 50 min, etc.
[0022] Preferably, the purification method includes ultrafiltration.
[0023] [Correction 20.01.2026 based on Rule 91] Preferably, the method for preparing the modified nucleic acid aptamer-paclitaxel conjugate is as follows (as shown in step iV of Figure 2):
[0024] [Corrected on January 20, 2026, according to Rule 91]
[0025] In an ice bath, the aqueous solution of the nucleic acid aptamer-paclitaxel conjugate was added to the dimethyl sulfoxide solution of G4 ligand at a ratio of AS1411 aptamer to G4 ligand of 1:10. The mixture was stirred for 20 min to 60 min, and the product was purified by ultrafiltration to obtain the modified nucleic acid aptamer-paclitaxel conjugate.
[0026] [Corrected according to Rule 91, 20.01.2026] Preferably, the preparation method of the nucleic acid aptamer-paclitaxel conjugate includes the following steps (as shown in Figure 2):
[0027] [Corrected on January 20, 2026, according to Rule 91]
[0028] (1) Paclitaxel (PTX) was dissolved in anhydrous dichloromethane (DCM) and reacted with acetic anhydride and anhydrous pyridine (Py) at room temperature. After purification, a white powdery paclitaxel succinic acid conjugate (PTX-Sa) was obtained.
[0029] (2) Paclitaxel succinic acid coupling compound, N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) were dissolved in anhydrous tetrahydrofuran (THF), stirred at room temperature, and recrystallized to obtain a white filter cake solid, which is the paclitaxel succinic acid activated ester derivative (PTX-Sa-NHS).
[0030] (3) Add NaHCO3 aqueous solution and PTX-Sa-NHS solution to amino-derived nucleic acid aptamer NH2-AS1411, react, add triethylamine acetic acid (TEAA) buffer solution to terminate the reaction, purify, and obtain the nucleic acid aptamer paclitaxel conjugate.
[0031] Preferably, in step (1), the reaction time is 1-3 days, for example 2 days.
[0032] Preferably, in step (1), the purification method is silica gel chromatography, and the mobile phase of silica gel chromatography is a dichloromethane / methanol mixed solution with a volume ratio of 20 / 1 to 3 / 1.
[0033] Preferably, in step (2), the stirring time is 1-3 days, for example 2 days.
[0034] Preferably, in step (2), the recrystallization method is as follows: the sample is dissolved in an ether solvent and recrystallized at 2-8°C (e.g., 3°C, 4°C, 5°C, 6°C, 7°C, etc.).
[0035] Preferably, the ether solvent includes diethyl ether and / or methyl tert-butyl ether.
[0036] Preferably, in step (3), the paclitaxel succinate activated ester derivative solution is an N,N-dimethylformamide solution of the paclitaxel succinate activated ester derivative.
[0037] Preferably, in step (3), the reaction temperature is 20-35°C, such as 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, etc.
[0038] Preferably, in step (3), the reaction time is 2-6 h, for example 3 h, 4 h, 5 h, etc.
[0039] Preferably, in step (3), the purification method is high performance liquid chromatography.
[0040] Preferably, the preparation method of the nucleic acid aptamer-paclitaxel conjugate is as follows:
[0041] Paclitaxel (PTX) was dissolved in anhydrous dichloromethane (DCM) and reacted with acetic anhydride and anhydrous pyridine (Py) at room temperature for 3 days. The mixture was purified by silica gel chromatography using a dichloromethane / methanol mixture with a volume ratio of 20 / 1 to 3 / 1 as the mobile phase to obtain a white powdery paclitaxel succinic acid conjugate (PTX-Sa).
[0042] (2) Paclitaxel succinic acid coupling compound, N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) were dissolved in anhydrous tetrahydrofuran (THF) and stirred at room temperature for 3 days. The sample was dissolved in diethyl ether and recrystallized at 4°C to obtain a white filter cake solid PTX-Sa-NHS.
[0043] (3) Add NaHCO3 solution and PTX-Sa-NHS solution to NH2-AS1411, react at 30°C for 2 h, add triethylamine acetic acid (TEAA) buffer solution to terminate the reaction, and purify by high performance liquid chromatography to obtain the nucleic acid aptamer paclitaxel conjugate.
[0044] Thirdly, the present invention provides the use of the modified nucleic acid aptamer-paclitaxel conjugate described in the first aspect in the preparation of antitumor drugs.
[0045] Preferably, the antitumor drug includes drugs for ovarian cancer or breast cancer. Beneficial effects
[0046] Compared with the prior art, the present invention has the following beneficial effects:
[0047] The modified nucleic acid aptamer-paclitaxel conjugate provided by this invention involves the coupling of a nucleic acid aptamer and paclitaxel via a linker bond. The target conjugate is obtained by modifying the structure of the nucleic acid aptamer using a chemical method with G4 ligand. This conjugate exhibits better tumor targeting, ultimately specifically targeting tumor cell surface proteins to achieve anticancer activity, while significantly enhancing efficacy. Therefore, it can be used to treat cancer, especially ovarian cancer. Attached Figure Description
[0048] Figure 1 is a schematic diagram of the modified nucleic acid aptamer-paclitaxel conjugate provided by the present invention.
[0049] Figure 2 shows the synthetic route of the modified nucleic acid aptamer paclitaxel conjugate in the embodiments of the present invention.
[0050] Figure 3 is the mass spectrum of AS1411 (starting material).
[0051] Figure 4 is the mass spectrum of PTX-Sa (intermediate 1).
[0052] Figure 5 shows the 1H NMR spectrum of PTX-Sa (intermediate 1).
[0053] Figure 6 shows the carbon NMR spectrum of PTX-Sa (intermediate 1).
[0054] Figure 7 is the single crystal diffraction pattern of PTX-Sa (intermediate 1).
[0055] Figure 8 is the mass spectrum of PSaA (intermediate 2).
[0056] Figure 9 shows the CD spectra of PSaA and PSaA360.
[0057] Figure 10a shows the spectrum of the blank sample in the 360A standard curve determination.
[0058] Figure 10b shows the spectrum of 0.625 nmol 360A in the 360A standard curve determination.
[0059] Figure 10c shows the spectrum of 1.25 nmol 360A in the 360A standard curve determination.
[0060] Figure 10d shows the spectrum of 2.5 nmol 360A in the 360A standard curve determination.
[0061] Figure 10e shows the spectrum of 5 nmol 360A in the 360A standard curve determination.
[0062] Figure 10f shows the spectrum of 10 nmol 360A in the 360A standard curve determination.
[0063] Figure 11 shows the 360A standard curve.
[0064] Figure 12a shows the spectrum of PSaA360.
[0065] Figure 12b shows the spectrum of PSaA360.
[0066] Figure 12c shows the spectrum of PSaA360.
[0067] Figure 13 is the mass spectrum of PSaA-cy3.
[0068] Figure 14 shows the flow cytometry analysis of SKOV3 cells after incubation with Cy3-labeled AS1411, A360, PSaA, and PSaA360 for 3.5 hours.
[0069] Figure 15 shows fluorescence images of SKOV3 cells after incubation with Cy3-labeled AS1411, A360, PSaA, and PSaA360 for 3.5 hours.
[0070] Figure 16 shows the fluorescence intensity of SKOV3 cells after incubation with Cy3-labeled AS1411, A360, PSaA, and PSaA360 for 3.5 hours.
[0071] Figure 17 shows the cell viability analysis of 293T cells after treatment with PBS, PTX, PTX360 and PSaA360 for 48 hours.
[0072] Figure 18 shows the cell viability analysis of SKOV3 cells after treatment with PBS, PTX, PTX360 and PSaA360 for 48 hours.
[0073] Figure 19 shows the cell viability analysis of SKOV3 cells after treatment with AS1411, A360, 360A, PTX, PTX360 and PSaA360 for 24 hours.
[0074] Figure 20a is a cell cycle diagram of SKOV3 cells after incubation with PBS at a dose of 200 nM (calculated by free AS1411) for 48 hours.
[0075] Figure 20b is a cell cycle diagram of SKOV3 cells after incubation with PTX at a dose of 200 nM (calculated by free AS1411) for 48 hours.
[0076] Figure 20c is a cell cycle diagram of SKOV3 cells after incubation with PTX360 at a dose of 200 nM (calculated by free AS1411) for 48 hours.
[0077] Figure 20d is a cell cycle diagram of SKOV3 cells after incubation with PSaA360 at a dose of 200 nM (calculated by free AS1411) for 48 hours.
[0078] Figure 21 is a comparison of cell migration rates under the action of PBS, PTX, PTX360 and PSaA360 as determined by the wound healing assay.
[0079] Figure 22 shows images of SKOV3 cell migration in the scratch zone under the action of PBS, PTX, PTX360 and PSaA360.
[0080] Figure 23 is a quantitative analysis of SKOV3 cell migration in the scratch area under the action of PBS, PTX, PTX360 and PSaA360.
[0081] Figure 24 shows the acrylamide gel electrophoresis analysis of S1411, A360, PSaA, and PSaA360.
[0082] Figure 25 is a melt chain temperature analysis diagram of AS1411, A360, PSaA, and PSaA360.
[0083] Figure 26a is a comparison of the body weights of BALB / c nude female mice in the blank control group, PTX, PTX360, and PSaA360 groups.
[0084] Figure 26b is a comparison of tumor volume between the blank control group, PTX, PTX360 and PSaA360 groups.
[0085] For example, 26c is a comparison chart of tumor weight and size between the blank control group, PTX, PTX360 and PSaA360 groups.
[0086] Figure 27 shows the results of histological examination.
[0087] Figure 28 shows the results of immunohistochemical analysis.
[0088] Figure 29 shows the results of the fluorescence TUNEL analysis.
[0089] Figure 30a is a comparison of albumin levels in liver and kidney function tests.
[0090] Figure 30b is a comparison of alkaline phosphatase levels in liver and kidney function tests.
[0091] Figure 30c is a comparison of total protein levels in liver and kidney function tests.
[0092] Figure 30d is a comparison chart of urea levels in liver and kidney function tests. The best embodiment of the present invention
[0093] The preparation method of the nucleic acid aptamer-paclitaxel conjugate of the present invention is as follows:
[0094] (1) Paclitaxel (PTX) was dissolved in anhydrous dichloromethane (DCM) and reacted with acetic anhydride and anhydrous pyridine (Py) at room temperature for 3 days. The mixture was purified by silica gel chromatography using a dichloromethane / methanol mixture with a volume ratio of 20 / 1 to 3 / 1 as the mobile phase to obtain a white powder of paclitaxel succinic acid coupling compound (PTX-Sa).
[0095] (2) Paclitaxel succinic acid coupling compound, N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) were dissolved in anhydrous tetrahydrofuran (THF) and stirred at room temperature for 3 days. The sample was dissolved in diethyl ether and recrystallized at 4°C to obtain a white filter cake solid PTX-Sa-NHS.
[0096] (3) Add NaHCO3 solution and PTX-Sa-NHS solution to NH2-AS1411, react at 30°C for 2 h, add triethylamine acetic acid (TEAA) buffer solution to terminate the reaction, and purify by high performance liquid chromatography to obtain the nucleic acid aptamer paclitaxel conjugate. Embodiments of the present invention
[0097] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.
[0098] Example 1
[0099] This embodiment provides a modified nucleic acid aptamer-paclitaxel conjugate, the preparation method of which is as follows:
[0100] (1) Preparation of PSaA
[0101] PTX (7.761 g, 9.088 mmol) was dissolved in anhydrous DCM (400 ml) and reacted with acetic anhydride (1.31 g, 11.1 mmol) and anhydrous pyridine (Py) (2.7 ml, 33.5 mmol) at room temperature for 3 days. After the reaction was completed, the compound was purified by silica gel chromatography (DCM / MeOH = 20 / 1~3 / 1) to give a white powder, compound PTX-Sa (8.7 g).
[0102] PTX-Sa (3.7017, 3.880 mmol), NHS (451.4 mg, 3.925 mmol), and DCC (831.1 mg, 4.028 mmol) were dissolved in 120 mL of anhydrous THF. After stirring at room temperature for 3 days, the solution was dissolved in 500 mL of diethyl ether and recrystallized at 4°C. 4.2 g of a white filter cake solid (PTX-Sa-NHS) was obtained, with a yield of approximately 100%.
[0103] 50 mM NaHCO3 solution (60 μL) and PTX-Sa-NHS solution (120 μL) were added to 100 nmol NH2-AS1411, and the reaction was carried out at 30°C for 2 h. The reaction was then terminated by adding 2 M TEAA solution (100 μL, pH=7.28). The crude mixture was then purified by high performance liquid chromatography (HPLC) to obtain PSaA.
[0104] (2) Preparation of PSaA360
[0105] In an ice bath, PSaA aqueous solution was added to DMSO solution of 360A iodide at a ratio of DNA: 360A iodide = 1:10. After mixing for 30 minutes, the product was purified by ultrafiltration to obtain paclitaxel conjugate modified with 360A iodide (PSaA360).
[0106] The synthesis route of this embodiment is shown in Figure 2.
[0107] Performance testing
[0108] (a) Product characterization
[0109] (1) Product confirmation
[0110] [Corrected from Rule 91, 03.02.2026] The starter, intermediate, and final product of Example 1 were characterized by mass spectrometry, 1H NMR, 1C NMR, single-crystal diffraction, and circular dichroism (CD) spectroscopy. Specific experimental instruments and methods are as follows:
[0111] Mass spectrometry: Waters UPLC Q-TOP System; Agilent 6540
[0112] 1H NMR spectrum: Bruker DRX-400
[0113] Carbon NMR spectrum: Bruker DRX-400
[0114] Single crystal diffraction: Bruker D8 Venture
[0115] [Corrected from Rule 91, 03.02.2026] Circular dichroism (CD) determination:
[0116] The conformation of the final product was analyzed using a J-715 circular dichroism (CD) spectrophotometer. Background signal from the blank buffer was measured and subtracted from the CD spectrum. The measurement chamber was deoxygenated with dry, purified nitrogen (99.99%) prior to the assay and maintained under a nitrogen atmosphere throughout the experiment. CD spectra were collected at 0.1 nm intervals, using two scans at a time constant of 4 s, within the wavelength range of 320–220 nm.
[0117] The experimental results are as follows:
[0118] Figure 3 shows the mass spectrum of AS1411, Figure 4 shows the mass spectrum of PTX-Sa, Figure 5 shows the 1H NMR spectrum of PTX-Sa, Figure 6 shows the 1C NMR spectrum of PTX-Sa, Figure 7 shows the single-crystal diffraction pattern of PTX-Sa, and Figure 8 shows the mass spectrum of PSaA. These results demonstrate the successful preparation of the aptamer-paclitaxel conjugate (PSaA).
[0119] Figure 9 shows the CD spectra of PSaA and PSaA360. With the introduction of 360A, a sharper positive peak appeared in PSaA360 (at 265 nm), which is attributed to the formation of more G-quadruplex conformations. This proves that 360A successfully modified PSaA to obtain the final product PSaA360.
[0120] (2) Characterization of G4 ligand loading
[0121] Standard curve determination:
[0122] Different amounts of 360A and blank samples were determined by high performance liquid chromatography (Agilent 1260), and the spectra shown in Figures 10a, 10b, 10c, 10d, 10e, and 10f were obtained. The characteristic peak at 1.8-2.0 min is the characteristic peak of 360A. By corresponding the peak area with the amount of 360A, the standard curve of 360A, y = 1402.6x + 78.979, was obtained (as shown in Figure 11).
[0123] Sample determination:
[0124] Three samples of PSaA360 product were subjected to high performance liquid chromatography (HPLC) tests, and the spectra shown in Figures 12a, 12b and 12c were obtained. The data shown in the table below were obtained. It was calculated that the loading of 360A in PSaA360 was approximately 1:1.
[0125]
[0126] (ii) Targeted characterization
[0127] (1) Effect of PSaA360 on cellular uptake
[0128] To detect the uptake by target cells, the following experiment was conducted:
[0129] Fluorescently labeled AS1411-Cy3 was synthesized (mass spectrum shown in Figure 13), and SKOV3 cells were selected as model cells. SKOV3 cells were treated with 10 nmol of Cy3-labeled AS1411 modifiers for 3 hours; these AS1411 modifiers were designated AS1411-Cy3, A360-Cy3, PSaA-Cy3, and PSaA360-Cy3, respectively. The resulting SKOV3 cells were then analyzed using flow cytometry (Beckman Coulter CytoFlex) and fluorescence microscopy (Nikon Eclipse Ts2). PBS was used as a control group, and the same procedure was followed.
[0130] The experimental results are shown in Figures 14-16. As shown in Figure 14, after incubation with the Cy3-labeled AS1411 modifier for 3 hours, the fluorescence intensity of all four experimental groups significantly increased, but stronger changes were observed in A360-Cy3 and PSaA360-Cy3. Furthermore, as shown in Figures 15 and 16, compared to AS1411-Cy3 and PSaA-Cy3, fluorescence imaging showed stronger luminescent signals (red) in A360-Cy3 and PSaA360-Cy3 in SKOV3 cells, indicating that modification of PSaA with 360A can promote the uptake of PSaA360 by SKOV3 cells, thus exhibiting stronger tumor targeting.
[0131] (2) Effects of PSaA360 on cytotoxicity
[0132] I. To demonstrate the in vitro cytotoxicity of PSaA360, the cell viability of SKOV3 and 293T cells was determined using CCK8 assay. The specific method is as follows:
[0133] Cells were seeded at a density of 5 × 10³ cells / well in 96-well plates and grown for 24 hours. PTX, PTX360, and PSaA360 were then added to the culture medium to a final concentration of 100 nM. After 48 hours, cell viability was determined using a Cell Counting Kit-8 (CCK-8) (HY-K0301). After incubating cells with CCK-8 solution for 1 hour, optical density was measured at 450 nm using a microplate reader (SpectraMax i3x). Three different sets of experiments were performed, each repeated three times. Error bars represent the standard deviation of repeated measurements on biological samples when n=3.
[0134] The experimental results are shown in Figures 17-19. As shown in Figure 17, the cell viability of PSaA360 in the 293T cell line was 45.3 ± 5.6 nM, while that of PTX and PTX360 (a mixture of PTX and 360A in equal proportions) was 30.5 ± 8.1 nM and 26.2 ± 3.8 nM, respectively, indicating that the cytotoxicity of PSaA360 was lower than that of PTX and PTX360. When applied to the ovarian cancer cell line SKOV3, PSaA360 significantly inhibited cell proliferation compared with PTX and PTX360. As shown in Figure 18, PTX and PSaA360 significantly reduced the cell viability of SKOV3 cells in a dose-dependent manner. As shown in Figure 19, the survival rate of SKOV3 cells treated with PSaA360 was low at a concentration of 200 nM per group of drugs, reaching approximately 58% after 24 hours.
[0135] II. To further test the effects of various AS1411 modifiers on the cell cycle, SKOV3 cells were analyzed by flow cytometry, as detailed below:
[0136] Cell cycle analysis was performed according to the instructions of the propidium iodide flow cytometry kit (Solarbio). Briefly, SKOV3 cells (5 × 10⁵ cells / well) were seeded in 6-well plates and incubated overnight. After washing with PBS, cells were incubated with 200 nM PTX, PTX360, and PSaA360 at 37°C for 48 h. At the end of culture, cells were treated with trypsin, washed, and fixed with 75% ethanol on ice. Cells were stored overnight at 4°C, washed, resuspended in 200 μL of propidium iodide + RNase staining solution, and incubated in the dark at 37°C for 30 min. Finally, DNA content was measured by flow cytometry (Beckman Coulter CytoFlex), and the percentage of cells at each stage of the cell cycle was calculated using ModFit software.
[0137] The experimental results are shown in Figures 20a-20d and 21 (in Figure 21, the bars from left to right represent G2 / M phase cells, S phase cells, and G0 / G1 phase cells). Compared with the control group, the percentage of G0 / G1 phase cells in the drug group was significantly reduced, while the number of S phase cells increased. PSaA360 and PTX360 can increase the proportion of G2 / M phase cells in the SKOV3 cell cycle, just as PTX did after 48 hours, which is consistent with the cytotoxicity results.
[0138] The above results indicate that 360A modification enhances the uptake of PTX by cells, thereby enhancing the cytotoxicity of PTX on SKOV3 cells.
[0139] (3) Inhibitory effect of PSaA360 on SKOV3 cell migration
[0140] The effects of PTX, PTX360, and PSaA360 on SKOV3 cell migration were analyzed using a wound healing assay. The specific experimental methods are as follows:
[0141] SKOV3 cells were seeded into six-well plates overnight at 37°C. A cell monolayer was removed by scratching with a sterile micropipette tip. The cells were then incubated with PTX, PTX360, and PSaA360 at 37°C for 24 hours. Images of the central scratched area were observed at 0 and 24 hours using a fluorescence microscope (Nikon Eclipse Ts2).
[0142] The experimental results are shown in Figures 22 and 23. As shown in Figure 22, PTX, PTX360, and PSaA360 significantly inhibited the migration ability of SKOV3 cells. As shown in Figure 23, compared with the PTX and PTX360 treatment groups, PSaA360 further inhibited the migration ability of SKOV3 cells. These results indicate that PSaA360 modified with 360A can enhance the anti-migration effect of PTX in SKOV3 cells, thereby improving the inhibitory effect on tumor metastasis.
[0143] (III) Thermal stability and serum stability of PSaA360
[0144] I. Serum stability
[0145] To be effective targeting tools, each AS1411 modification must resist rapid degradation by nucleases. The stability of each AS1411 structural modification was assessed by incubating it for different times in cell culture medium containing 10% fresh fetal bovine serum (FBS) at 37°C. The AS1411 modifications were then analyzed by agarose gel electrophoresis.
[0146] The experimental results are shown in Figure 24. AS1411 and PSaA partially decomposed after 3 hours and completely disappeared after 24 hours. The electrophoretic bands of A360 and PSaA360 were still clearly visible after 12 hours and did not completely decompose until 48 hours later.
[0147] II. Thermal Stability Analysis
[0148] The thermal stability of the aptamers was assessed by determining the melting temperature (Tm) of each AS1411 modifier. UV melting profiles of the aptamers were monitored using an Agilent Cary 100 UV-Vis spectrometer equipped with a temperature controller. The absorbance of each sample (2.5 μM in binding buffer) was monitored at 260 nm at a heating rate of 1 °C / min from 25 to 95 °C. Each Tm value was calculated using the first derivative of the melting profile using Cary-WinUV software.
[0149] The experimental results are shown in Figure 25. Although the original AS1411 nucleic acid aptamer exhibited relatively high thermostability (Tm = 61.6 °C), the introduction of 360A further increased the Tm values of A360 (Tm = 77.3 °C) and PSaA360 (Tm = 76 °C). However, when 360A was not introduced into the AS1411 sequence, the stability of PSaA showed a decreasing trend (Tm = 57.7 °C). The thermostability results after AS1411 modification were consistent with its serum stability, indicating that PSaA360 has high thermostability and serum stability and can be used for further in vitro activity studies.
[0150] (iv) In vivo testing
[0151] I. Animal experiments on in vivo anti-tumor efficacy
[0152] Animal experiments were conducted on the in vivo antitumor efficacy of the blank control, PTX, PTX360, and PSaA360 groups, respectively. The specific methods are as follows:
[0153] Animal facilities were designed to maintain a controlled environment with constant temperature and a 12-hour light-dark cycle. Mice were provided with free access to food and water throughout the study. Mice were given at least one week to acclimatize before the start of all experiments. All in vivo studies were conducted in accordance with ethical guidelines and approved by the Animal Ethics Committee of Hong Kong Baptist University (REC / 24-25 / 0407).
[0154] Eight-week-old female BALB / c nude mice were subcutaneously inoculated with 2×10⁻⁶ scalp irradiation in the axilla. 6SKOV3 cells were collected. After tumor formation within three weeks, mice were randomly divided into four groups of six each for subsequent studies. Mice in each group were intravenously injected with a blank control (PBS buffer), PTX, PTX360, and PSaA360 at a dose of 5.85 µmol / kg (equivalent to 5 mg / kg PTX), twice weekly for four weeks, respectively. The control group received an equal volume of PBS buffer. Tumor volume and mouse weight were recorded every 3-4 days. After treatment, the mice were euthanized, and tumor weight was recorded.
[0155] The experimental results are shown in Figures 26a, 26b, and 26c. Figures 26a and 26b record the body weight (Figure 26a) and tumor size (Figure 26b) of BALB / c nude female mice inoculated with SKOV3 in the control group, PTX, PTX360, and PSaA360 groups, respectively. In the figures, ** P < 0.01, **** P < 0.0001, and two-way ANOVA was used. Figure 26c shows the comparison of tumor weight of xenografted epithelial ovarian tumors in each group 28 days after different treatments. In the figure, * P < 0.05, *** P < 0.001, and one-way ANOVA and Tukey's post-hoc test were used.
[0156] No significant differences in body weight were observed in any of the treatment groups (Figure 26a), indicating good tolerability. Furthermore, the tumors in the PSaA360 treatment group were significantly smaller than those in the PTX group (P<0.01) and the PTX360 group (P<0.01; Figure 26b). In addition, the tumor weight in the PSaA360 group was significantly lower than that in the control group (P<0.001; Figure 26c).
[0157] II. Histological Examination
[0158] Histological examinations were performed on the blank control, PTX, PTX360, and PSaA360 groups, respectively, using the following methods:
[0159] Paraffin sections were sequentially treated with environmentally friendly dewaxing and clearing solutions I and II, anhydrous ethanol I and II, and 75% ethanol, followed by rinsing with tap water. Frozen sections were thawed, fixed with tissue fixative, and then rinsed with running water. Hematoxylin staining involved immersion in hematoxylin staining solution, treatment with hematoxylin differentiation solution and hematoxylin bluing solution, with rinsing interspersed between steps. Eosin staining involved sequential immersion in 85% ethanol, 95% ethanol, and eosin staining solution. During dehydration and mounting, different concentrations of ethanol and xylene solutions were used sequentially, and finally, the sections were mounted with neutral resin. After the above procedures were completed, microscopic examination, image acquisition, and analysis were performed. The results showed that the cell nuclei were blue and the cytoplasm was pink.
[0160] III. Immunohistochemical analysis
[0161] Immunohistochemical analyses were performed on the blank control, PTX, PTX360, and PSaA360 groups, respectively, using the following methods:
[0162] Paraffin sections were dewaxed by soaking in environmentally friendly dewaxing and clearing solutions I-III and anhydrous ethanol I-III, followed by rinsing with pure water. Antigen retrieval was performed using a specific buffer, and slides were rinsed with PBS on a shaker. Subsequently, sections were treated with 3% hydrogen peroxide solution to neutralize endogenous peroxidase activity and rinsed with PBS. Sections were then blocked with 3% BSA to reduce nonspecific binding. Primary antibody KI67 (GB111499, 1:1000) and secondary antibody (GB23303, 1:200) were added sequentially, with thorough rinsing between each step. 3,3'-Diaminobenzidine (DAB) was used for staining to visualize the target antigen, and hematoxylin was used for counterstaining to highlight cellular structures.
[0163] The slides were sequentially dehydrated with ethanol and xylene before being mounted. The prepared slides were then observed and interpreted under a bright-field microscope.
[0164] IV. Fluorescent TUNEL Analysis
[0165] Fluorescence TUNEL analysis was performed on the blank control, PTX, PTX360, and PSaA360 groups, respectively, using the following methods:
[0166] The TUNEL assay was used to detect apoptosis in tumor tissues. First, sections were dewaxed using an environmentally friendly dewaxing solution (Servicebio, G1128) and ethanol (Sinopharm Chemical Reagent Co., Ltd., 100092183), followed by proteinase K repair. After equilibration at room temperature, the sections were covered with a reaction solution containing terminal deoxynucleotidyl transferase (TDT). After incubation, the nuclei were stained with 4',6-diamidinyl-2-phenylindole (DAPI). The slides were then mounted and photographed using a microscope. The results showed that DAPI-stained nuclei appeared blue under ultraviolet light, while apoptotic nuclei labeled with tetramethylrhodamine (TMR) fluorescein using the TUNEL kit (Servicebio, G1502) showed red fluorescence.
[0167] The results of the above histological examination, immunohistochemical analysis, and TUNEL fluorescence analysis are shown in Figures 27, 28, and 29, respectively. H&E staining, Ki-67 immunohistochemical staining, and TUNEL immunofluorescence analysis were performed on xenograft epithelial ovarian tumor sections from the specified group. Extracellular matrix and cytoplasm stained pink, Ki-67 positive staining was brown, cell nuclei stained blue, and in situ apoptosis stained red. Scale bar: 100 μm. Results are expressed as mean ± standard deviation. n = 6. Histological examination (Figure 27) showed that tumors in the PSaA360-treated group mice exhibited significant nuclear pyknosis and cytoplasmic shrinkage. Ki-67 staining (Figure 28) showed a decreased proliferation index, while terminal deoxynucleotidyl transferase (dUTP) nick-end labeling (TUNEL) fluorescence indicated enhanced apoptotic activity (Figure 29).
[0168] V. Liver and kidney function tests
[0169] Liver and kidney function tests were performed on the blank control, PTX, PTX360, and PSaA360 groups, including albumin (ALB), alkaline phosphatase (ALP), total protein (TP), and urea.
[0170] The test results are shown in Figures 30a-30d, which illustrate the renal and hepatic function of Balb / c nude mice treated with different methods. Data are expressed as mean ± standard deviation. n = 5, and one-way ANOVA and Tukey's post-hoc test were used. A p-value > 0.05 was observed when comparing with the blank control group. The results showed no significant differences in albumin (ALB), alkaline phosphatase (ALP), total protein (TP), and urea levels among the groups (P > 0.05; Figures 30a-30d), confirming the systemic biocompatibility.
[0171] Based on the above in vivo experimental results, the modified nucleic acid aptamer paclitaxel conjugate provided in this application has significant tumor inhibition, reduces the Ki-67 proliferation index, and enhances apoptosis. It can effectively inhibit the growth of epithelial ovarian tumors while maintaining excellent safety in vivo. Industrial applicability
[0172] In summary, the modified nucleic acid aptamer-paclitaxel conjugate provided by this invention, compared to the unmodified conjugate, not only improves the thermostability and serum stability of the conjugate but also effectively enhances the uptake capacity of SKOV3 cells, exhibits higher cytotoxicity to SKOV3 cells, and improves their anti-migration ability within SKOV3 cells. In other words, the modified nucleic acid aptamer-paclitaxel conjugate provided by this invention significantly improves stability, tumor targeting, and antitumor efficacy, while toxicological experiments show that the modified conjugate has high safety, balancing therapeutic efficacy and safety.
[0173] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.
Claims
1. A modified nucleic acid aptamer-paclitaxel conjugate, characterized in that, The modified nucleic acid aptamer-paclitaxel conjugate comprises paclitaxel, a succinic acid linker, and a G4 ligand-modified AS1411 aptamer, the structure of which is as follows: ; Wherein, X is an AS1411 aptamer modified with G4 ligand, wherein the G4 ligand is inserted into the G4 plane of the AS1411 aptamer via a non-covalent bond.
2. The modified nucleic acid aptamer-paclitaxel conjugate according to claim 1, characterized in that, The CD spectrum of the coupling compound shows a positive peak at 265 nm.
3. The modified nucleic acid aptamer-paclitaxel conjugate according to claim 1 or 2, characterized in that, The G4 ligand is 360A iodide.
4. The modified nucleic acid aptamer-paclitaxel conjugate according to claim 1 or 2, characterized in that, The loading ratio of the G4 ligand to AS1411 is 1:
1.
5. A method for preparing a modified nucleic acid aptamer-paclitaxel conjugate according to any one of claims 1-4, characterized in that, The preparation method includes the following steps: The modified nucleic acid aptamer-paclitaxel conjugate was obtained by mixing the aqueous solution of the nucleic acid aptamer-paclitaxel conjugate with the dimethyl sulfoxide solution of the G4 ligand in an ice bath and purifying the mixture.
6. The preparation method according to claim 5, characterized in that, The ratio of AS1411 aptamer to G4 ligand in the nucleic acid aptamer-paclitaxel conjugate is 1:
10.
7. The preparation method according to claim 5, characterized in that, The preparation method of the nucleic acid aptamer-paclitaxel conjugate includes the following steps: (1) Paclitaxel was dissolved in anhydrous dichloromethane and reacted with acetic anhydride and anhydrous pyridine at room temperature. After purification, a white powdery paclitaxel succinic acid conjugate was obtained. (2) Dissolve the paclitaxel succinic acid coupling compound, N-hydroxysuccinimide and dicyclohexylcarbodiimide in anhydrous tetrahydrofuran, stir at room temperature, recrystallize to obtain a white filter cake solid, which is the activated paclitaxel succinic acid ester derivative. (3) Add NaHCO3 aqueous solution and paclitaxel succinate activated ester derivative solution to amino-derived nucleic acid aptamer NH2-AS1411, react, add triethylamine acetic acid buffer solution to terminate the reaction, purify, and obtain the nucleic acid aptamer paclitaxel conjugate.
8. The preparation method according to claim 7, characterized in that, In step (1), the purification method is silica gel chromatography; Preferably, in step (3), the purification method is high performance liquid chromatography.
9. Use of a modified nucleic acid aptamer-paclitaxel conjugate according to any one of claims 1-4 in the preparation of an antitumor drug.
10. The use according to claim 9, characterized in that, The anti-tumor drugs include drugs for ovarian cancer or breast cancer.