Thiol-nucleic acid aptamer and derivative self-assembled nano material thereof for improving stability of nucleic acid aptamer and derivative thereof and application of self-assembled nano material
By combining nucleic acid aptamer or its derivatives with thiols to form self-assembled nanomaterials, the problem of low bioavailability of nucleic acid aptamer in the blood and difficulty in entering cells is solved, and the purpose of improving its stability and therapeutic effect is achieved.
Patent Information
- Application Number
- CN202510094451.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-21
- Publication Date
- 2025-05-09
AI Technical Summary
Nucleic acid aptamers or their derivatives have low bioavailability in the blood, are easily degraded by nucleases, and are difficult to enter cells, limiting their therapeutic potential.
By combining nucleic acid aptamer or its derivatives with thiol, the positive charge and disulfide bond characteristics of thiol are used to form nucleic acid aptamer-thiol self-assembled nanomaterials to improve their stability and intracellular delivery efficiency.
This method effectively protects nucleic acid aptamers from degradation of nucleases, promotes their escape of lysosomes in cells, improves their stability and bioavailability in tumor cells and tissues, and thus enhances their therapeutic effect.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedicine, in particular to the design of a thiol-nucleic acid aptamer and its derivative self-assembly nanomaterial to improve the stability of nucleic acid aptamers and their derivatives and their applications, specifically involving the preparation and application of acid aptamers or aptamer derivatives combined with thiol self-assembly nanomaterials. Technical Background
[0002] Aptamers are single-stranded DNA or RNA sequences that can fold into three-dimensional structures. This enables them to recognize and bind to a wide range of targets, such as small molecules, toxins, ions, or proteins. This versatility makes them valuable tools for bioanalysis, biomedicine, and targeted biological diagnostics. Despite their significant advantages, aptamers' use is limited by serum nucleases and rapid renal excretion. This restricts their bioavailability in the blood and reduces their therapeutic potential. Furthermore, due to their large size and negative surface charge, free aptamers or their derivatives have difficulty entering cells and exerting their biological functions. To address these challenges, various approaches have been developed to improve the stability of aptamers and aptamer-drug conjugates (ApDCs), such as chemical modifications of the phosphate backbone, sugars, and nucleotide bases. Although some progress has been made, these approaches, like traditional modifications, also have limitations.
[0003] Another obstacle to therapeutic efficacy is the problem of cellular uptake, where aptamers or aptamer-drug conjugates (ApDCs) are internalized by cells via endocytosis, which can lead to their sequestration in lysosomes, which contain hydrolases capable of degrading nucleic acids. To overcome this challenge, various strategies have been explored to improve the intracellular delivery efficiency of aptamers and ApDCs. One approach is to conjugate aptamers or ApDCs to cell-penetrating peptides (CPPs) or lipids to promote cellular entry through alternative mechanisms such as direct transmembrane transport. However, this approach is time-consuming and requires batch-by-batch design. This suggests the need for rational design of aptamers or ApDCs to bypass the lysosomal pathway while maintaining binding capacity.
[0004] Thiols are small organic molecules synthesized from lipoic acid and other compounds. Due to the presence of a guanidine group, they carry a positive charge and readily form ionic bonds with negatively charged aptamers. Furthermore, the disulfide bonds on thiols can form dimers and trimers through cross-linking. Furthermore, due to the presence of disulfide bonds, thiols readily exchange sulfur with Cys on the cell membrane surface, allowing them to enter cells and escape lysosomes. Therefore, the combined self-assembly of thiols with aptamers or their derivatives can be explored to enhance their stability and improve their therapeutic efficacy. Summary of the Invention
[0005] In order to overcome the defects of nucleic acid aptamers or their derivatives, the present invention aims to provide a nucleic acid aptamer or an aptamer derivative combined with thiol.
[0006] Another object of the present invention is to provide a method for preparing nucleic acid aptamers or their derivatives in combination with thiol, which has simple preparation, mild reaction and better stability, and has no effect on the conformation or structure of the nucleic acid aptamers and their derivatives.
[0007] Another object of the present invention is the use of nucleic acid aptamers or aptamer derivatives in combination with thiols.
[0008] In order to achieve the above-mentioned object, the present invention provides, on one hand, a nucleic acid aptamer or its derivative combined with a thiol, wherein the nucleic acid aptamer or its derivative combined with a thiol comprises a nucleic acid aptamer or its derivative and a thiol, wherein the derivative of the nucleic acid aptamer comprises a nucleic acid aptamer-drug conjugate; the thiol is a positively charged small molecule, and the nucleic acid aptamer or its derivative and the thiol are bound by ionic bonds and disulfide bonds; wherein, in the nucleic acid aptamer or its derivative combined with the thiol, the N:P ratio of the thiol to the nucleic acid aptamer is (1-30):1.
[0009] The present invention utilizes thiols as a highly efficient delivery system for nucleic acid aptamers and their derivatives. This has not been reported to date. Thiols can effectively protect nucleic acid aptamers and their derivatives from degradation by nucleases and allow them to escape lysosomes in cells, thereby increasing their stability in tumor cells and tissues, thereby improving their bioavailability. Furthermore, the present invention is universally applicable; all nucleic acid aptamers and their derivatives can be stabilized and escape lysosomes by combining them with thiols.
[0010] Furthermore, in the combination of the nucleic acid aptamer or its derivative with thiol, the N:P ratio of thiol to nucleic acid aptamer is preferably 15-20:1, more preferably 15:1.
[0011] The thiol is a positively charged small molecule containing a disulfide bond.
[0012] The nucleic acid aptamer is one or more nucleic acid aptamers including Sgc8, cMET, MJ5C, AS1411, XQ-2D, or a combination thereof.
[0013] The nucleic acid aptamer drug conjugate (ApDCs) is selected from one or more of gemcitabine (Gem)-aptamer drug conjugate, monomethyl auristatin E (MMAE)-aptamer drug conjugate, cisplatin aptamer drug conjugate or a combination thereof.
[0014] The nucleic acid aptamer derivatives include, but are not limited to, conjugates of nucleic acid aptamers with drugs, contrast agents such as gadolinium (Gd), iron (Fe), manganese (Mn), etc.
[0015] The derivatives are chemical drugs, antibody-drug conjugates, and aptamer conjugates for imaging diagnosis; the molecules in the nucleic acid aptamer-aptamer-drug conjugates are one or more of Gem, MMAE, cisplatin, doxorubicin, etc.
[0016] The chemotherapy drug Gem in the nano drug carrier prepared in the present invention can be replaced with MMAE, cisplatin, 5-Fu, doxorubicin, etc.
[0017] The nucleic acid aptamer in the nano drug carrier prepared in the present invention can be replaced with XQ-2d, cMET, MJ5C, AS1411, etc.
[0018] The present invention also provides a method for preparing the above-mentioned nucleic acid aptamer or aptamer derivative combined with thiol, comprising the following steps: The thiol and the nucleic acid aptamer or its derivative are mixed and assembled into a nanomaterial at room temperature (e.g., 22-28°C); The mixing is selected from blowing with a gun, ultrasonic 10s or vortex shaking.
[0019] The mixing is preferably vortex mixing.
[0020] The present invention also provides an application of the nucleic acid aptamer or aptamer derivative combined with thiol in tumor medicine.
[0021] This invention combines thiols with nucleic acid aptamers and their derivatives to form biocompatible nanomaterials through self-assembly for use in pharmaceutical applications, particularly in animal therapy. The assembly conditions for thiols, nucleic acid aptamers, and their derivatives are simple and mild. By replacing the nucleic acid aptamer, the stability of various aptamers and their ability to escape lysosomes can be improved, resulting in a highly universal approach.
[0022] The present invention utilizes a one-step self-assembly strategy to enhance the stability and therapeutic efficacy of nucleic acid aptamers or aptamer-drugs through a lysosomal-independent pathway, including the synthesis of thiols, the preparation and application of thiols and nucleic acid aptamers, and other methods. The thiol-aptamer self-assembly comprises at least one nucleic acid aptamer or its derivative and a thiol. The thiol-aptamer assembly can enhance the targeting and stability of Sgc8, reduce lysosomal capture of aptamers and drug conjugates, and enhance the therapeutic efficacy of Gem-Sgc8. The same self-assembly strategy has been successfully applied to other aptamers, such as MJ5C and cMET, demonstrating the versatility of our method. The preparation method is simple and controllable, with a mild and rapid reaction regime and good reproducibility. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Hereinafter, the embodiments of the present invention will be described in detail with reference to the accompanying drawings. Figure 1 is the structural formula of the thiol used in the present invention; Figure 2 Provide a technical roadmap for the synthesis of nucleic acid aptamers combined with thiols; Figure 3 This is a TEM image of the nanomaterial of nucleic acid aptamer combined with thiol; Figure 4 This is the DLS image of the nanomaterials of nucleic acid aptamer combined with thiol; Figure 5 The zeta potential of the aptamer-thiol nanomaterial; Figure 6 The stability of nucleic acid aptamer-combined thiol nanomaterials in serum; Figure 7 The figure shows the binding ability of nucleic acid aptamer-combined thiol nanomaterials to colon cancer HCT116 cells and human B lymphocyte Ramos cells; Figure 8 Images of the uptake of nucleic acid aptamer-combined thiol nanomaterials by colon cancer HCT116 cells at different time periods; Figure 9 Aptamer-combined thiol nanomaterials escape lysosomal from colon cancer HCT116 cells; Figure 10 Figure 1 shows the results of a CCK-8 experiment using aptamer-combined thiol nanomaterials to treat colon cancer HCT116; Figure 11 Image of aptamer-combined thiol nanomaterials used to treat mice bearing colon cancer; Figure 12 TEM images of aptamer-combined thiol nanomaterials and other aptamers such as MJ5C and cMET; Figure 13 DLS images of aptamer-combined thiol nanomaterials and other aptamers such as MJ5C and cMET; Figure 14 Zeta potential images of aptamer-combined thiol nanomaterials and other aptamers such as MJ5C and cMET; Figure 15 Images of the binding of aptamer-combined thiol nanomaterials with other aptamers such as MJ5C, cMET, MDA-MB-231 cells, and Hela cells. DETAILED DESCRIPTION
[0024] The following are experimental methods in the implementation examples. Unless otherwise specified, they are all conventional methods. The experimental materials used in the following examples were purchased from conventional biochemical reagent manufacturers unless otherwise specified.
[0025] Example 1: Synthesis of positively charged thiols 3.8 mmol of lipoic acid and 5.0 mmol of CDI were placed in a round-bottom flask; then, Add 25 ml of anhydrous DCM to the round-bottom flask; 5.0 mmol) was placed in the flask; next, Add 25 ml of anhydrous DCM to the flask, and add the resulting solution dropwise to a pre-cooled mixture of 7 ml of DCM and 2 ml of ethylenediamine; After stirring at 0°C for 40 minutes, the reaction was allowed to cool to room temperature. After washing with brine and drying over Na2SO4, the organic solvent was removed by rotary evaporation, and the residue was redissolved in DCM, followed by the addition of 1H-pyrazole-1-carboxamidine hydrochloride. After stirring for 4 hours, the organic solvent was removed by rotary evaporation; the final monomer product was obtained by washing the residue with Et2O. The thiol structural formula is as follows Figure 1 The zeta potential is 17.0±2.8 mV, as shown in Figure 5 As shown, thiols are positively charged small molecules.
[0026] Example 2: Aptamer NPs were prepared by the following steps: Figure 2 As shown: Thiol monomers (disulfide monomers) were dissolved in methanol and mixed with Sgc8 aptamers in aqueous solution at different N:P ratios at room temperature, then vortexed and allowed to stand for 15 minutes; the resulting nucleic acid aptamer-combined thiol nanomaterial NPs were stored at 4°C for future use.
[0027] The size and morphology of Sgc8 NPs were observed using TEM. Figure 3 The average diameter is 185±43 nm. Dynamic light scattering (DLS) test proves that the dispersion is good. Figure 4 As shown in Figure 2, the Sgc8 nucleic acid aptamer is negatively charged, the thiol monomer is positively charged, and the zeta potential of the assembled Sgc8 NPs increases, as shown in Figure 2. Figure 5 shown.
[0028] Next, we explored whether the above method could be applied to other aptamers. To achieve this, we mixed the MJ5C aptamer with a thiol monomer following the assembly procedure of Sgc8. DLS and zeta potential showed that the monomer and MJ5C could form nanoparticles at a 15:1 N:P ratio. The MJ5C NPs had a diameter of approximately 100 nm ( Figure 12When treated with MDA-MB-231 cell lines, enhanced fluorescence intensity was observed in cells treated with MJ5C aptamers, random sequence, and random NPs, as determined by flow cytometry ( Figure 15 a). CLSM studies also confirmed that the binding capacity of MJ5C NPs was higher than that of MJ5C aptamers ( Figure 15 a).
[0029] The versatility of the cMET aptamer self-assembly method was further tested. Similar assembly results were observed when the cMET aptamer was mixed with thiol monomers in the same ratio ( Figure 13 b and 14b).
[0030] like Figure 15 As shown in b, cMET NPs exhibited higher binding capacity than cMET aptamers. These findings indicate that the self-assembly approach is universal, making it potentially applicable to all aptamers to improve their recognition ability and stability.
[0031] Example 3: Binding of nucleic acid aptamer nanomaterials (aptamer NPs) to cells: To test the specificity and binding capacity of aptamer NPs, HCT116 and Ramos cells were harvested and washed three times with wash buffer (5 mM MgCl2, 4.5 g / L glucose in Dulbecco's PBS). The cells were then incubated with Sgc8, Sgc8 NPs, Gem-Sgc8, GEM-Sgc8 NPs, Random, and random nanomaterial NPs in binding buffer (5 mM MgCl2, 4.5 g / L glucose, 0.1 mg / mL yeast tRNA, and 1 mg / mL BSA in Dulbecco's PBS) at 4°C. After a 30-minute incubation, the cells were washed with wash buffer and recorded by flow cytometry. The Sgc8 aptamer can recognize and bind to the membrane protein PTK7 on HCT116 cells. Therefore, to evaluate whether the binding ability of Sgc8 NPs to HCT116 cells was affected after assembly, the cells were further treated with Sgc8 aptamers and Sgc8 NPs. Figure 7 a shows that the shift of the maximum fluorescence emission peak of Sgc8 NPs is greater than that of the Sgc8 aptamer, which means that the self-assembly strategy enhances the recognition and binding ability of the Sgc8 aptamer. Similar results were observed in cells treated with Gem-Sgc8 NPs. In contrast, no specific binding was observed in Ramos cells treated with Sgc8 NPs compared with random NPs and Gem-Sgc8 NPs ( Figure 7b). Collectively, these results indicate that the binding selectivity of Sgc8 NPs and Gem-Sgc8 NPs is not affected by self-assembly.
[0032] Example 4: It is clear that aptamer NPs have higher cellular uptake than unassembled aptamers Cells were plated at 5 × 10 4 Cells were seeded at a density of 100 cells / mL in 24-well plates and allowed to grow for 24 hours. The cells were then treated with Sgc8, Sgc8 NPs, Gem-Sgc8, GEM-Sg8 NPs, random, and random NPs (aptamer concentration, 250 nM) for 1 and 6 hours, respectively. The cells were collected, washed with PBS, and analyzed by flow cytometry. Next, after 24 hours of culture, the cells were incubated with Sgc8, Sgc8 NPs, Gem-Sgc8, Gem-Sg8 NPs, random, and random NPs for 1 and 6 hours, respectively.
[0033] After incubation for 1 or 6 h, the uptake of Sgc8 NPs was higher than that of Sgc8 NPs, and the internalization efficiency was time-dependent. The same trend was also observed in Gem-Sgc8 NPs ( Figure 8 a, 8b), indicating that the assembly strategy can also improve the internalization efficiency of aptamers and Gem-Sgc8.
[0034] Example 5: Clarifying the lysosome-independent endocytosis pathway of aptamer NPs Endocytosis plays an important role in the uptake of NPs. Many NPs are internalized into cells and co-localized with lysosomes, leading to lysosomal degradation of the drug, thereby reducing the efficacy of anticancer therapy. To determine their intracellular sublocalization, Sgc8, Sgc8 NPs, and Gem-Sgc8 NPs were incubated with HCT116 cells at different times, and the nucleic acid aptamers and lysosomes were stained with Hoechst and LysoTracker™. Figure 9As shown, the Sgc8 aptamer localizes to the lysosomal compartment, as the majority of the green fluorescence of the Sgc8 aptamer colocalizes with the red dots of the lysosomes, with a high Pearson correlation coefficient (PCC, 0.55). Over time, the Sgc8 aptamer remained sequestered in the lysosomes (PCC = 0.60 after 2 hours and PCC = 0.64 after 4 hours). In contrast, Sgc8 NPs bypassed the lysosomes at early stages. After longer incubation periods, Sgc8 NPs were rarely localized in the lysosomes. Similar to the Sgc8 aptamer, the fluorescence of GEM-Sgc8 overlapped well with LysoTracker at 1, 2, and 6 hours. However, Gem-Sgc8 NPs were rarely trapped in the lysosomes at different incubation times. Therefore, it can be concluded that self-assembly with thiol monomers shows superiority as a delivery system for Sgc8 aptamers or Gem-Sgc8 with minimal lysosomal capture, and both aptamers Sgc8 or Gem-Sgc8 can be delivered to cells via a lysosome-independent pathway.
[0035] Example 6: Efficacy of Gem-Sgc8 NPs The CCK-8 method was used to perform an in vitro (anti-)proliferation assay on HCT116 cells. The cells were placed in the wells of a 96-well plate with 5000 cells per well. After 24 hours, the cells were treated with different concentrations of gemcitabine (GEM), GEM-Sgc8, GEM-Scc8-NP and monomer for 72 hours. The culture medium was discarded and replaced with a medium containing 10% CCK-8 solution. After incubation for 1 to 2 hours, the absorbance was read at 450 nm. The enhanced internalization and lysosomal escape of Gem-Sgc8 may increase its anticancer efficiency
[45] . Figure 10 As shown, Gem-Sgc8 NPs exhibited superior anticancer efficacy compared to Gem or Gem-Sgc8 alone. Furthermore, negligible toxicity was observed in cells treated with the monomer. The enhanced efficacy can be attributed to the unique properties of the nanoparticle formulation, including improved stability, enhanced cellular uptake, reduced lysosomal entrapment, and controlled drug release.
[0036] Inspired by the excellent therapeutic effect in HCT 116 cells, we further tested the in vivo antitumor properties of Gem-Sgc8 NPs. Tumor-bearing BALB / c nude mice were randomly divided into 4 groups of 5 mice each. The drug was administered via the tail vein every 3 days at a dose of 5 mg / kg per mouse. Tumors in PBS-treated mice grew particularly rapidly, reaching approximately 6 times their previous size after 12 days. Mild tumor inhibition was observed in the Gem-treated group, while moderate tumor growth inhibition occurred in the Gem-Sgc8 group. In contrast, the most significant effect was observed in the Gem-Sgc8NPs group, with tumor size reduced by 56%.
Claims
1. A nucleic acid aptamer or an aptamer derivative combined with a thiol, characterized in that: The nucleic acid aptamer or its derivative combined with thiol comprises a nucleic acid aptamer or its derivative and a thiol, and the nucleic acid aptamer or its derivative and the thiol are combined by ionic bonds and disulfide bonds, and the derivative of the nucleic acid aptamer comprises a nucleic acid aptamer drug conjugate; the thiol is a positively charged small molecule, and the thiol is combined with the nucleic acid aptamer and its derivative by ionic bonds and disulfide bonds; in the nucleic acid aptamer or aptamer derivative combined with thiol, the N:P ratio of thiol to the nucleic acid aptamer or aptamer derivative is 1-30:
1.
2. The nucleic acid aptamer or aptamer derivative combined with thiol according to claim 1, characterized in that: In the nucleic acid aptamer or aptamer derivative combined with thiol, the N:P ratio of thiol to nucleic acid aptamer or aptamer derivative is 15-20:
1.
3. The nucleic acid aptamer or aptamer derivative combined with thiol according to claim 1 or 2, characterized in that: The nucleic acid aptamer is one or more of Sgc8, cMET, MJ5C, AS1411, XQ-2D or a combination thereof.
4. The nucleic acid aptamer or aptamer derivative combined with thiol according to claim 1, characterized in that: The nucleic acid aptamer drug conjugate (ApDCs) is selected from one or more of gemcitabine (Gem)-aptamer drug conjugate, monomethyl auristatin E (MMAE)-aptamer drug conjugate, cisplatin aptamer drug conjugate or a combination thereof.
5. The nucleic acid aptamer or aptamer derivative combined with thiol according to claim 4, characterized in that: The nucleic acid aptamer includes one or more of Sgc8, MJ5C, cMET, XQ-2D, and As1411 nucleic acid aptamers.
6. The nucleic acid aptamer or aptamer derivative combined with thiol according to claim 1, characterized in that: The derivatives are chemical drugs, antibody-drug conjugates, and aptamer conjugates used for imaging diagnosis; the molecules in the aptamer-drug conjugates are one or more of Gem, MMAE, cisplatin, doxorubicin, etc.
7. The method for preparing a nucleic acid aptamer or an aptamer derivative combined with a thiol according to any one of claims 1 to 6, characterized in that: The steps include: The thiol and the nucleic acid aptamer are mixed and assembled into a nanomaterial at room temperature; The mixing is selected from blowing with a gun, ultrasound for 10 seconds or vortex shaking.
8. The method for preparing a nucleic acid aptamer or an aptamer derivative combined with thiol according to claim 7, characterized in that: The synthesis of positively charged thiols follows the following steps: 3.8 mmol of lipoic acid and 5.0 mmol of CDI were placed in a round-bottom flask; then, Add 25 ml of anhydrous DCM to a round-bottom flask; 5.0 mmol) was placed in the flask; next, Add 25 ml of anhydrous DCM to the flask and add the resulting solution dropwise to a pre-cooled mixture of 7 ml of DCM and 2 ml of ethylenediamine; After stirring at 0°C for 40 minutes, the reaction was allowed to cool to room temperature. After washing with brine and drying over Na2SO4, the organic solvent was removed by rotary evaporation, and the residue was redissolved in DCM, followed by the addition of 1H-pyrazole-1-carboxamidine hydrochloride; After stirring for 4 hours, the organic solvent was removed by rotary evaporation; the final monomer product was obtained by washing the residue with Et2O, and the thiol structure is shown in Figure 1. The Zeta potential is 17.0±2.8 mV, as shown in Figure 5, and the thiol is a positively charged small molecule.
9. The method for preparing a nucleic acid aptamer or an aptamer derivative combined with thiol according to claim 7, characterized in that: Prepared by the following steps: The thiol was dissolved in methanol and the Sgc8, MJ5C or cMET aptamers were mixed in aqueous solution at different N:P ratios at room temperature, and then vortexed and allowed to stand for 15 minutes to form a nucleic acid aptamer-thiol-combined nanomaterial, which was stored at 4°C for future use.
10. A nucleic acid aptamer or an aptamer derivative combined with thiol as claimed in claims 1 to 6, comprising at least one nucleic acid aptamer and a gold nanocluster, wherein the nucleic acid aptamer or an aptamer derivative combined with thiol has targeting, stability and biocompatibility.