Photo-activated bispecific nucleic acid aptamer chimera as well as preparation method and use method thereof

Abstract

The invention relates to the technical field of targeted lysosomal protein degradation drugs, and provides a photo-activated bispecific aptamer chimera (PBACs) which comprises a first nucleic acid sequence with a first aptamer sequence and a second nucleic acid sequence with a second aptamer sequence, and the first nucleic acid sequence and the second nucleic acid sequence are paired through a complementary sequence. The first nucleic acid aptamer targets a cell surface transport protein, and the second nucleic acid sequence targets a membrane protein target to be degraded. The second nucleic acid sequence is designed with a drug loading sequence and is accurately modified with a photosensitizer. The photosensitizer can respond to near-infrared light, increase ROS generation in tumor cells and start cell autophagy. The invention also provides a preparation method and a use method of the PBACs. On one hand, the structure of the PBACs can be accurately controlled, and on the other hand, the PBACs open cell autophagy through photoresponse, so that a lysosome targeted protein degradation pathway is strengthened, cloning and migration of tumor cells are greatly limited, and the tumor cell killing effect is remarkably improved.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and in particular, relates to the technical field of drugs targeting lysosomal protein degradation. Background Art

[0002] Targeted Protein Degradation (TPD) is a new protein regulation technology with disruptive significance that has been developed in recent years. It degrades target proteins through ubiquitin proteasomes and lysosomes.

[0003] Among them, the protein degradation targeting chimera (PROTAC) is the most mature and shows broad application prospects in the field of disease treatment. It mainly uses the natural protein degradation system in the cell, the ubiquitin protease system (UPS), to achieve targeted degradation of the target protein (Protein of interest, POI). PROTAC is a bifunctional small molecule that targets POI at one end and recruits E3 ubiquitin ligase at the other end to form a dumbbell-shaped ternary complex. POI is labeled with ubiquitination and then recognized and degraded by the proteasome. In the past 20 years, various degradation systems based on PROTAC technology have emerged in an endless stream, and related research results have exploded in recent years, such as molecular glues, autophagy-targeted chimeras, etc., which have greatly enriched the means of regulating intracellular protein homeostasis. What's more exciting is that as of March 2023, 19 PROTAC drugs have entered clinical research and have initially shown good therapeutic effects.

[0004] Lysosome-Targeting Chimaeras (LYTAC) technology is another new type of targeted protein degradation technology. This technology was developed and designed by the 2022 Nobel Prize winner, Professor Carolyn. R. Bertozzi's team at Stanford University in 2020. LYTAC relies on the lysosomal degradation pathway and for the first time achieves the goal of bringing the target protein on the surface of the tumor cell membrane and the extracellular target protein into the lysosome for degradation, thereby inhibiting tumor development. LYTAC makes up for the technical shortcomings of traditional PROTAC that can only degrade intracellular proteins, and has gradually become a new favorite in the drug research and development community. At present, the use of LYTAC technology has successfully achieved the degradation of membrane proteins such as epidermal growth factor receptor (EGFR), programmed death ligand 1 (PD-L1), transferrin receptor (CD71), and secretory protein apolipoprotein E4 (APOE4). The development of LYTAC provides a new tool for tumor treatment and has become a research hotspot in the development of tumor treatment drugs in recent years.

[0005] Although LYTAC technology has developed rapidly, there are still two key technologies that need to be solved: (1) How to prepare LYTAC molecules with relatively simple structures and precise controllability? The coupling method of avidin and biotin molecules based on traditional technology is not precise enough and there is a great deal of relative uncertainty. Glycosylated LYTAC molecules based on mutual recognition of glycoproteins are prepared by coupling sugar ligands with antibodies. On the one hand, the synthesis of sugar ligands that recognize lysosome targeting receptors (LTRs) is complex, low in yield, and high in cost; on the other hand, sugar ligands targeting LTRs are non-specific for antibody labeling, and the optimal number and optimal coupling point are still unclear. (2) How to improve the tumor treatment efficacy of LYTAC? More and more evidence shows that the degradation efficiency of LYTAC is affected by many factors, such as the affinity between LYTAC molecules and target proteins, intracellular transport and recycling mechanisms, etc. Therefore, the tumor treatment efficacy of LYTAC needs to be improved. Summary of the invention

[0006] In order to solve the above problems mentioned in the prior art, the present invention provides a photoactivated bispecific aptamer chimeras (PBACs), whose molecular structure can be precisely controlled and respond to near-infrared light, thereby enhancing the generation of intracellular ROS and triggering enhanced cellular autophagy, and strengthening the lysosomal pathway through cellular autophagy to target protein degradation, thereby significantly limiting the cloning and migration capabilities of tumor cells, and improving the tumor cell killing effect and the therapeutic effect of LYTAC.

[0007] The specific technical scheme is as follows: the light-activated bispecific nucleic acid aptamer chimera comprises a first nucleic acid sequence, a second nucleic acid sequence and a photosensitizer; The first nucleic acid sequence includes: a first nucleic acid aptamer sequence and a first complementary sequence, wherein the first nucleic acid aptamer specifically recognizes and binds to a cell surface transport protein; The second nucleic acid sequence includes: a second nucleic acid aptamer sequence, a second complementary sequence, and a drug-carrying sequence, the second nucleic acid aptamer specifically recognizes and binds to the membrane protein target to be degraded, and the second complementary sequence and the first complementary sequence can achieve precise base complementary pairing; The photosensitizer is precisely modified on the drug-carrying sequence of the second nucleic acid sequence; the photosensitizer responds to near-infrared light, increases ROS generation in tumor cells, and initiates cell autophagy. The first nucleic acid sequence is designed to carry a nucleic acid aptamer sequence targeting IGFIIR and a complementary sequence; the second nucleic acid sequence is designed to carry a nucleic acid aptamer sequence, a complementary sequence and a drug-carrying sequence of Sgc8; the sequence of IGFIIR is TTGCCAGTGATTGATCCA; the sequence of Sgc8 is GGATCAATCACTGGCAA.

[0008] By designing the first and second complementary sequences and precisely modifying the photosensitizer on the drug-carrying sequence of the second nucleic acid aptamer, the product structure of the present invention can be stably and precisely controlled.

[0009] Preferably, the second nucleic acid aptamer is a tumor-targeting DNA nucleic acid molecule. Particularly preferably, the second nucleic acid aptamer is Sgc8, AS1411 or XQ-2d nucleic acid aptamer.

[0010] Preferably, the membrane protein target to be degraded and recognized and bound by the second nucleic acid aptamer is PTK7, nucleolin protein, PDL1 protein or PD1 protein.

[0011] The second nucleic acid aptamer can help the present invention target tumor cells, and the membrane protein it binds to can be a protein closely related to the occurrence and development of tumor cells, so that the lysosomal targeted protein degradation that the present invention intends to open and strengthen can effectively interfere with the occurrence and development of tumors. Among the nucleic acid aptamers and membrane proteins mentioned above, the Sgc8 nucleic acid aptamer recognizes the PTK7 protein on the surface of tumor cells, and PTK7 is related to the growth and infiltration of tumors; the nucleolin protein bound by the AS1411 nucleic acid aptamer is a binding protein for several ligands related to angiogenesis and tumorigenesis; the XQ-2d nucleic acid aptamer is a pancreatic cancer-specific nucleic acid aptamer that specifically binds to CD71, which is a transferrin receptor that mediates the absorption of iron by cells; and the PD-1 protein and PD-L1 protein on the tumor cell membrane can affect the growth of tumor cells through signaling pathways.

[0012] Preferably, the cell surface transporter includes: lysosome-targeting receptor (LTR), proteasome transporter, mannose 6-phosphate receptor (M6PR) or lysosomal integral membrane protein 2 (LIMP-2), etc. These transporters have been verified to be able to activate lysosomal transport, and can recognize and bind to the first nucleic acid aptamer of the product PBACs of the present invention, and carry the PBACs of the present invention and the target membrane protein to be degraded bound thereto into the cell to activate the subsequent protein degradation of the lysosomal pathway.

[0013] Preferably, the photosensitizer is a first-generation, second-generation, third-generation or fourth-generation photosensitizer small molecule drug that can respond to near-infrared light. Photosensitizers are commonly used drugs for photodynamic therapy of tumors. They respond to light in cells to produce reactive oxygen species, produce cytotoxic effects, and achieve photodynamic therapy. Among them, the first-generation photosensitizers are mainly hematoporphyrins; the second-generation photosensitizers are dihydrochlorins, including 5-aminolevulinic acid, hemoporfin, verteporfin, etc.; the third-generation photosensitizers are derivatives of the second-generation photosensitizers coupled with different targeting molecules, and the fourth-generation photosensitizers include further derivatives of the first three with other functions or some new photodynamic nanomaterials. Including hydrophobic Ce6, indocyanine green, 5-ALA, and pyropheophorbide A (PA) commonly used in photodynamic therapy, each generation of photosensitizers contains hydroxyl groups, carboxyl groups or unsaturated bonds that can achieve precise covalent bonding with nucleic acid molecules through chemical means, so they can all be precisely modified on the drug-carrying sequence designed by the present invention. The drug loading sequence may be only a single drug loading site, and may be designed at the 3' end or 5' end of the second nucleic acid sequence or at any position that does not affect the functional folding of the second nucleic acid aptamer or the pairing of complementary sequences.

[0014] The present invention also provides a method for preparing a photoactivated bispecific nucleic acid aptamer chimera (PBACs), comprising the following steps: Step 1: Design a first nucleic acid sequence and a second nucleic acid sequence; the first nucleic acid sequence includes a first nucleic acid aptamer sequence and a first complementary sequence; the first nucleic acid aptamer specifically recognizes and binds to a cell surface transport protein; the second nucleic acid sequence includes a second nucleic acid aptamer sequence, a second complementary sequence, and a drug-carrying sequence; the second nucleic acid aptamer specifically recognizes and binds to a membrane protein target to be degraded; the second complementary sequence and the first complementary sequence can achieve precise base complementary pairing; Step 2: Precisely modifying the photosensitizer on the drug-carrying sequence of the second nucleic acid sequence; Step 3: The first nucleic acid sequence and the second nucleic acid sequence are complementary paired in an annealing environment to prepare PBACs.

[0015] Preferably, the modification method used in the precise modification of the photosensitizer by the second nucleic acid aptamer is one of the following reactions: click chemistry azidation modification, cycloaddition reaction of alkyne and azide, cycloaddition reaction of cycloalkyne and azide, condensation reaction of non-alcohol carbonyl compounds, addition reaction of thiol and carbon-carbon multiple bonds, or dehydration condensation reaction of amino or carboxyl groups. These reactions are commonly used technical means in the art to achieve precise modification of nucleic acid sequences and photosensitizers.

[0016] The present invention also provides a method for using a photoactivated bispecific nucleic acid aptamer chimera (PBACs): the PBACs are used to activate and enhance targeted protein degradation in the lysosomal pathway. Preferably, the PBACs are used in the preparation of drugs related to tumor treatment.

[0017] The present invention provides a near-infrared light-activated bispecific nucleic acid aptamer chimera, which has the potential to enhance the degradation of targeted proteins in the lysosomal pathway by enhancing cell autophagy. The product of the present invention can accurately target cell membrane proteins, activate the cell autophagy program through near-infrared light response, and significantly enhance the degradation ability of the lysosomal degradation pathway on targeted proteins under the promotion of the cell autophagy program, greatly reduce the migration ability of tumor cells, inhibit cell cloning, and effectively kill tumor cells.

[0018] Specifically: On the one hand, the PBACs of the present invention can transport tumor cell membrane proteins into lysosomes through the lysosomal pathway, triggering protein degradation; on the other hand, under near-infrared light irradiation, the photoresponse reaction increases the ROS content in tumor cells, regulates cellular oxidative stress and triggers cellular autophagy. The initiation of the cellular autophagy program can greatly activate the related reactions of the lysosomal degradation pathway, significantly improve the degradation efficiency of lysosomes on target membrane proteins, limit the migration and cloning of tumor cells, and kill tumor cells through photodynamic therapy, thereby improving the tumor treatment efficacy of the LYTAC platform.

[0019] The present invention verifies the effectiveness of the relevant preparation method for accurately synthesizing PBACs by using the Sgc8-PA and IGFIIR nucleic acid aptamer embodiments. On the other hand, it verifies the two technical effects of using PBACs as drugs for tumor treatment: (1) IGFIIR nucleic acid aptamers can bind to lysosomal shuttle receptors on the cell surface to activate the lysosomal degradation pathway; (2) Sgc8-PA can be precisely modified by click chemistry azide modification to recognize membrane protein targets and respond to near-infrared light, thereby initiating cell autophagy to enhance lysosomal degradation of membrane proteins.

[0020] In summary, the PBACs constructed by the present invention, as a multifunctional light-triggered LYTAC platform, have the characteristics of precise modular design, simple preparation, small off-target side effects, and high degradation efficiency, and will provide a promising transformation platform for cell-specific degradation of membrane proteins. At the same time, the preparation process of the present invention is simple, efficient and economical, and has application prospects for large-scale production. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 :Schematic diagram of the synthesis of photoactive bispecific aptamerchimeras (PBACs).

[0022] Figure 2 : The sequences of the Sgc8 aptamer and IGFIIR aptamer used in the examples.

[0023] Figure 3 : Table of dosages of components used in the synthesis of different PBACs (5 μM / 10 μM / 20 μM) in the examples.

[0024] Figure 4 : Electropherograms of Sgc8-PA, IGFIIR aptamer, and 5 μM, 10 μM, and 20 μM PBACs.

[0025] Figure 5 : Confocal microscopy images of targeted binding of Sgc8-PA and PBACs.

[0026] Figure 6 : Confocal microscopy images of cellular uptake and cytoplasmic distribution of PBACs.

[0027] Figure 7 : Lysosomal co-localization map of PBACs.

[0028] Figure 8 : PTK7 protein degradation under treatment with different concentrations of PBACs (0, 100, 200 and 500 nM).

[0029] Fig. 9 : Expression of autophagy-related proteins p62 and LC3 in PBACs under NIR (near infrared) treatment.

[0030] Fig.10 :The effect diagram of the generation of intracellular reactive oxygen species in PBACs under NIR treatment.

[0031] Fig.11 : PTK7 protein degradation in PBACs treated with NIR and autophagy inhibitors (CQ, 3-MA).

[0032] Fig.12 : Comparison of cell clone numbers of PBACs under NIR treatment.

[0033] Fig.13 : Comparison of tumor cell migration under NIR treatment of PBACs.

[0034] Fig.14 : Comparison of tumor cell death in PBACs treated with NIR. DETAILED DESCRIPTION

[0035] Unless otherwise specified, the experimental methods used in the following implementation methods are all conventional methods. The reagents and consumables used in the examples can be purchased from normal commercial channels unless otherwise specified.

[0036] The first nucleic acid aptamer in the first nucleic acid sequence designed in the specific implementation of this section targets IGFIIR protein (a lysosomal targeting receptor), and the second nucleic acid aptamer in the second nucleic acid sequence is Sgc8 nucleic acid aptamer, which targets the membrane protein target PTK7 protein related to tumor growth and infiltration, and its drug-carrying sequence is designed at the 5' end of the second nucleic acid sequence, and the photosensitizer carried is pyropheophorbide-a (PA), and the two are precisely coupled by a Cu-free click reaction coupling method. This section also studies the synthesis efficiency, yield, membrane targeting ability, cell uptake ability, lysosomal transport and enrichment efficiency of the PBACs, target protein degradation ability, ROS generation, the effect of cell autophagy on lysosomal pathway protein degradation, and the effect on the cloning / migration / death of tumor cells after significantly reducing the target protein, in order to illustrate the technical effects of the present invention.

[0037] It should be understood that although the present embodiment is a method of realizing the technical solution mentioned in the above invention content, on the basis of sufficient description of the prior art and the present invention, the technical principles and technical effects disclosed in the present embodiment are sufficient to support the solution summarized and intended to be protected by the above invention.

[0038] 1. Preparation of photoactivated bispecific aptamer chimeras (PBACs) 1. Preparation of nucleic acid aptamer-PA 1.1 Preparation of Azide Polyethylene Glycol-PA (PA-PEG-N3) In this example, pyropheophorbide-a (PA) was coupled with polyethylene glycol amine using a classic amidation reaction. N-Hydroxy succinimide (NHS) (22 mg, 0.19 mmol), 1-ethyl-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC∙HCl) (36 mg, 0.19 mmol) and triethylamine (TEA) (52 μL, 0.38 mmol) were added to a solution of PA (50 mg) in dichloromethane (DCM, 10 mL). After stirring at room temperature for 2 h, azido-polyethylene glycol amine (20 mg, 0.09 mmol) was added and stirred at room temperature for 20 h. The reaction mixture was concentrated by rotary evaporation and purified by thin-layer chromatography (TLC) of a mixture of dichloromethane / methanol (20:1, v / v). Then, the silica was removed by flash chromatography after being scraped one by one to obtain azido-polyethylene glycol-PA as a black solid (40 mg, 58%).

[0039] 1.2 Preparation of aptamer-DBCO oligomers The amino-labeled aptamer with a linker sequence (Poly T) and a drug-carrying sequence was precisely designed and prepared using DNA solid phase synthesis technology. Then, dibenzocyclooctyl-C6-N-hydroxysuccinimide (DBCO-C6-NHS) linker was used to react with the labeled amine in the aptamer to form a stable amide bond. The final aptamer-DBCO oligomer was obtained by purification by high performance liquid chromatography. This part was supported by Sangon Biotech Co., Ltd. (Shanghai, China).

[0040] 1.3 Synthesis of aptamer-PA conjugates Aptamer-DBCO and PA-PEG-N3 were coupled via a Cu-free click reaction (see Appendix Figure 1 Schematic diagram of the synthesis ("Copper-free click reaction" step).

[0041] PA-PEG-N3 was added to the PBS buffer (pH 7.4) of nucleic acid aptamer-DBCO. Then, it was stirred at 37 °C for 24 h. Then, the solution was ultrafiltered and centrifuged at 7000 rpm for 3 times, centrifuged for 20 min, dissolved with deionized water, and ultrafiltered to remove unreacted PA-PEG-N3 (molecular cutoff of 3 kD, Millipore). The obtained product was dissolved by blowing in 500 μL of deionized water, extracted with ethyl acetate 5 times, and the ethyl acetate in the upper layer of DMSO was removed. The nucleic acid aptamer-PA conjugate dissolved in the aqueous phase was concentrated in a DNA concentrator (the conditions were set to V-AQ, 45 °C, and overnight concentration for 10 h). The final product was stored in DNase / RNase-free water at 4 °C.

[0042] 2. Preparation of Photoactivatable Bispecific Aptamer Chimeras (PBACs) According to the attached Figure 2 The first nucleic acid sequence (marked in the figure: IGFIIR) is designed, which carries the nucleic acid aptamer sequence and complementary sequence targeting IGFIIR; the second nucleic acid sequence (marked in the figure: Sgc8-PA) is designed, which carries the Sgc8 nucleic acid aptamer sequence, complementary sequence and drug loading sequence (site). Among them, the green sequence is the complementary sequence of Sgc8-PA (GGATCAATCACTGGCAA) and IGFIIR (TTGCCAGTGATTGATCCA), and the red T base (the first at the 5' end of Sgc8-PA) is the connection site of PA.

[0043] Sgc8-PA was prepared by the method of Example 1, and Sgc8-PA (100 μM) and IGFIIR (100 μM) were stored in ddH2O.

[0044] 5, 10 and 20 μL of Sgc8-PA and IGFIIR were added to 1× DPBS buffer (DPBS containing 12.5 mM MgCl2) in a total volume of 100 μL. Figure 3 As shown in the figure, the mixture was gradually heated to 95 °C and maintained for 10 min, and then the mixture was slowly cooled to 4 °C for storage (see Appendix Figure 1 Synthesis schematic diagram (“annealing” step) to prepare PBACs of different concentrations (5 μM / 10 μM / 20 μM).

[0045] 2. Detection of properties of photoactivated bispecific aptamer chimeras (PBACs) 1. Synthesis and yield of PBACs To verify the synthesis effect and yield of the above preparation method, we used PAGE electrophoresis to analyze the distribution of PBACs in the bands. The PAGE electrophoresis results are shown in Figure 4 As shown, lane 2 represents Sgc8-PA, lane 3 represents IGFIIR nucleic acid aptamer, lanes 4, 5 and 6 represent PBACs of different concentrations, and the red band is Sgc8-PA.

[0046] The results showed that the molecular weight of the prepared product increased under red light, indicating that the complementary pairing of Sgc8-PA and IGFIIR nucleic acid aptamer was successfully formed. At the same time, there were no mixed bands in the lane, which directly reflected that the preparation method was not only simple, economical and convenient, but also had a high yield (up to 99%), so no further purification was required, which met the requirements of drug production efficiency and yield in industrial production applications.

[0047] 2. Detection of targeting ability to tumor cell membrane This section of the experiment detected the binding affinity of single-targeted Sgc8-PA and dual-targeted PBACs to HCT 116 cells that overexpress PTK7 and IGFIIR proteins on the cell membrane.

[0048] HCT 116 cells were cultured at 1×10 5 Cells were seeded in confocal dishes at a density of 100 cells / well overnight. Cells were treated with Sgc8-PA and PBACs (500 nM) for 0.5 h at 4 °C. Cells were washed with DPBS and stained with Dio (a green fluorescent lipophilic membrane stain) working solution (1×) for 30 min and Hoechst working solution (1×) for 10 min. Cells were washed with DPBS and imaged with a confocal microscope (Leica TCS SP8).

[0049] Confocal results are as follows Figure 5 As shown, compared with Sgc8-PA, the red fluorescence of PBACs (PA red fluorescence area) was significantly enhanced, and the co-localization effect between the display area and the cell membrane (i.e., the green area stained with DIO) was obvious, showing a better targeting binding ability of PBACs to tumor cell membranes, indicating that the dual targeting effect of PBACs is significantly better than the single targeting of Sgc8-PA.

[0050] 3. Detection of the ability to be taken up by tumor cells In this experiment, the distribution of PBACs in cells was examined to study their ability to be taken up by tumor cells. First, HCT 116 cells were cultured at 1×10 5 Cells were seeded in confocal dishes at a density of 1000 cells / well overnight. Cells were treated with PBACs (500 nM) for different time periods (0 and 24 h), washed 3 times with DPBS, and imaged under a confocal microscope (Leica TCS SP8).

[0051] The results are as follows Figure 6 As shown, the red fluorescence of PBACs is widely distributed in the cytoplasm, indicating that after PBACs bind to PTK7 and IGFIIR proteins, they can be transferred to the cytoplasm through the mediation of surface ligands and receptors.

[0052] 4. Detection of PBACs transport to lysosomes and enrichment efficiency In this experiment, fluorescence microscopy after LysoTracker Green staining was used to analyze the co-localization of PBACs and lysosomes.

[0053] First, HCT 116 cells were cultured at 1 × 10 5 The cells were seeded overnight at a density of 100 nM / well in a confocal dish. Then, the cells were treated with PBACs at different concentrations (0, 100, 200, and 500 nM) for 24 h. After washing with DPBS, the cells were stained with LysoTracker Green working solution for 30 min or with Hoechst working solution (1×) for 10 min. After staining, the cells were washed 3 times with DPBS and imaged using a confocal microscope (Leica TCS SP8), and the Pearson correlation coefficient was calculated using Leica software.

[0054] The results are as follows Figure 7 As shown in the figure, the red fluorescence of PBACs colocalizes well with the green fluorescence of Lyso-Tracker, indicating that PBACs can be transferred and enriched in lysosomes by cells after 24 h of treatment with cells. The Pearson correlation coefficients after treatment with different concentrations of PBACs were 94%, 88%, and 89%, respectively, showing a strong correlation between PBACs and lysosomal locations, reflecting that PBACs have excellent lysosomal transport and enrichment efficiency.

[0055] 5. Effect of PBACs on the degradation of tumor cell membrane protein (PTK7 protein) This section of the experiment verifies the degradation of PTK7 protein (the targeting protein of Sgc8 nucleic acid aptamer) by PBACs.

[0056] HCT 116 cells were cultured in 6-well plates (3 × 10 5Cells / well) were added overnight and treated with different concentrations of PBACs (0, 100, 200 and 500 nM) for 24 h. After treatment, whole-cell extracts were prepared with protein lysis buffer containing a protease / phosphatase inhibitor cocktail (CST), separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, blocked with 5% albumin, incubated with primary and secondary antibodies, and developed. The antibodies used in this section include PTK7 (17799-1-AP, Proteintech) and β-actin (4967s, CST), which were used to detect the expression of PTK7 and the internal reference β-actin.

[0057] The results are as follows Figure 8 As shown in the figure, after 24 h of PBACs treatment of HCT 116 cells, the expression of PTK7 protein gradually decreased with the increase of PBACs concentration. Compared with the control, 58% of PTK7 protein was degraded when treated with 500 nM PBACs, with significant protein degradation efficiency.

[0058] 6. Effects of PBACs and PBACs+NIR treatment on autophagy of tumor cells In this experiment, immuno-western blotting was used to detect the expression of autophagy-related proteins LC3-II and p62 to verify the effects of PBACs and PBACs+NIR on autophagy.

[0059] HCT 116 cells were cultured in 6-well plates (3 × 10 5 Cells were incubated overnight with 4% paraformaldehyde (PBACs) at 4 °C for 24 h and then treated with different concentrations of PBACs (0, 100, 200, and 500 nM) for 24 h. 2 , 10 min). The old culture medium was discarded, the cells were washed with DPBS three times, 200 μL of trypsin was added for digestion for 3 min, the cells were collected in 1.5 mL EP tubes, centrifuged at 1500 rpm for 4 min, washed with DPBS twice, placed in a liquid nitrogen tank for 1 min to promote cell death, and whole cell extracts were prepared with protein lysis buffer containing a mixture of protease / phosphatase inhibitors, separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to nitrocellulose membranes. Subsequent blocking, incubation with primary and secondary antibodies, and development were performed. The antibodies used included LC3 (3868s, CST), β-actin (4967s, CST), and p62 / SQSTM1 (18420-1-AP, Proteintech). The expression of LC3-II and p62 proteins related to autophagy were detected by immunoblotting.

[0060] The results are as follows Fig. 9 As shown. Under PBACs treatment, the expression of p62 protein gradually decreased, and the 500 nM group was 0.7 times that of the 0 nM group; the expression of LC3-II protein increased, and the 500 nM group was 1.27 times that of the 0 nM group. However, under PBACs and NIR treatment, p62 expression was significantly reduced, and the 500 nM+NIR group was 0.45 times that of the 0 nM+NIR group; LC3-II protein expression was significantly enhanced, and the 500 nM+NIR group was 2.4 times that of the 0 nM+NIR group. It can be seen that PBACs can activate and increase the level of cellular autophagy by responding to near-infrared light.

[0061] 7. Effects of PBACs and PBACs+NIR treatment on ROS-producing capacity of tumor cells In this experiment, confocal microscopy analysis using the DCFH-DA fluorescent probe to detect cellular ROS was used to verify the ROS capacity of PBACs and PBACs+NIR.

[0062] HCT 116 cells were plated on a confocal dish (1 × 10 5 The cells were cultured overnight in 4% paraformaldehyde (PBACs) and treated with different concentrations of PBACs (0, 100, 200, and 500 nM) and near-infrared treatment (660 nM, 0.1 W / cm 2 , 10 min). JC-1 staining solution (1×) / 10μM DCFH-DA was used for staining at 37°C for 30 min, and Hoechst working solution (1×) was used for staining for 10 min. The cells were washed three times with DPBS and imaged with a confocal microscope (Leica TCS SP8).

[0063] The results are as follows Fig.10 As shown, under NIR treatment, the intracellular bright green fluorescence increased sharply with the increase of PBACs treatment concentration, indicating that PBACs+NIR treatment can significantly induce the production of intracellular ROS.

[0064] 8. Effects of PBACs and PBACs+NIR on PTK7 protein degradation in the presence of autophagy inhibitors In this experiment, we compared the degradation of PTK7 protein in PBACs and PBACs+NIR under pretreatment with two autophagy inhibitors, chloroquine (CQ) and 3-methyladenine (3-MA), to explore the effect of the enhanced cellular autophagy by PBACs+NIR on the degradation of membrane proteins in the lysosomal pathway.

[0065] HCT 116 cells were cultured in 6-well plates (3 × 10 5Cells were pretreated with 3-MA (5 μM) or CQ (5 μM) for 2 h, PBACs (0 and 500 nM) for 24 h, and near-infrared treatment (660 nM, 0.1 W / cm 2 , 10 min). Whole cell extracts were prepared with protein lysis buffer containing a protease / phosphatase inhibitor cocktail, separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes, blocked with 5% albumin, incubated with primary and secondary antibodies, and developed. Western Blot experiments were performed using antibodies PTK7 (17799-1-AP, Proteintech) and β-actin (4967s, CST).

[0066] The results are as follows Fig.11 As shown, under the treatment of PBACs (500 nM), the degradation of PTK7 protein exceeded 40%, and under the treatment of PBACs (500 nM) + NIR, the degradation of PTK7 protein further increased to 80%, indicating that under the treatment of near-infrared light, PBACs can significantly enhance protein degradation by initiating PDT.

[0067] Interestingly, when the autophagy inhibitors 3-methyladenine (3-MA) ​​and chloroquine (CQ) were used to inhibit the cellular autophagy pathway, the PTK7 protein expression in the 3-MA / CQ+PBACs (500 nM)+NIR group was similar to that in the PBACs (500 nM) group without NIR treatment, which was about twice the PTK7 protein expression in the PBACs (500 nM)+NIR group. This indicates that when the autophagy pathway is inhibited, the protein degradation effect mediated by photodynamic therapy of PBACs+NIR is also significantly inhibited. In other words, the PBACs+NIR group responds to near-infrared light to initiate photodynamic therapy, and the ability to degrade proteins in the lysosomal pathway is greatly enhanced beyond expectations by triggering and enhancing cellular autophagy.

[0068] 9. Effects of PBACs and PBACs+NIR treatment on the cloning ability of tumor cells This section experiments to study and compare the effects of PBACs and PBACs+NIR on the plate cloning ability of tumor cells.

[0069] HCT 116 cells were cultured in 6-well plates (200 cells / well) for 48 h, treated with 0 and 500 nM concentrations of PBACs for 24 h, and illuminated with near-infrared light (660 nM, 0.1 W / cm 2) for 10 min. After incubation for 24 h, the cells were washed 3 times with DPBS and cultured for 5 more days with fresh medium. The cells were washed 3 times with DPBS, fixed with 4% paraformaldehyde (1 mL) for 15 min, washed 3 times with DPBS, stained with 0.1% crystal violet for 10 min, dried at room temperature, and the number of cell plate clones was counted.

[0070] The results are as follows Fig.12 As shown in the figure, compared with the 0 nM and 0 nM+NIR treatment groups, the cloning efficiency of the PBACs (500 nM) treatment group was 60% of that of the control group, indicating that 500 nM PBACs treatment can inhibit the cell cloning and proliferation ability by degrading PTK7 protein. On this basis, under PBACs (500 nM) + NIR treatment, the cloning efficiency of tumor cells was reduced to 20% of the control group, reflecting that PBACs photodynamic therapy can significantly inhibit the cloning ability of tumor cells and significantly reduce cell activity.

[0071] 10. Effects of PBACs and PBACs+NIR treatment on tumor cell migration ability In this section, the Transwell experiment was used to study the effects of PBACs and PBACs+NIR on the migration ability of tumor cells.

[0072] HCT 116 cells were cultured in serum-free medium at a density of 2 × 10 5 Cells were seeded in the transwell chamber. 500 µL of complete growth medium was added to the cell dish. Cells were treated with 0 and 500 nM concentrations of PBACs for 24 h and illuminated with near-infrared light (660 nM, 0.1 W / cm 2 ) for 10 min. After 24 h of continuous culture, the upper layer of cells was cleaned with a cotton swab, washed twice with DPBS, and fixed with 4% paraformaldehyde for 10 min. The cells were washed three times with DPBS and stained with 0.1% crystal violet dissolved in DPBS for 30 min. The chamber membrane was obtained, fixed on a slide with neutral gel overnight, and photographed under an upright microscope.

[0073] The results are as follows Fig.13As shown, the 0 nM and 0 nM+NIR treatment groups did not affect the cell migration ability. In the PBACs (500 nM) group, the number of migrating cells was significantly reduced, and the Transwell efficiency dropped to 38%, indicating that the PBACs (500 nM) treatment can inhibit the cell migration ability by degrading the PTK7 protein (PTK7 protein can guide cancer cell movement and metastasis). On this basis, the Transwell efficiency of tumor cells in the PBACs (500 nM) + NIR group was further reduced to 12% of the control group, indicating that the PBACs-mediated photodynamic therapy used in the present invention can further significantly degrade the PTK7 protein on the basis of the lysosomal degradation pathway, significantly interfere with the activity of tumor cells, and significantly inhibit the migration ability of tumor cells.

[0074] 11. Detection of cell killing effects of PBACs and PBACs+NIR treatment This section uses the Live / Dead experiment to observe the live / dead cell staining results under a confocal microscope to study the cell killing effect of PBACs and PBACs+NIR treatment.

[0075] HCT 116 cells were grown in confocal dishes (1 × 10 5 The cells were cultured overnight in 4% paraformaldehyde (PBACs) and treated with different concentrations of PBACs (0 and 500 nM) and near-infrared treatment (660 nM, 0.1 W / cm 2 Live / Dead staining solution was added and incubated at 37°C for 30 min. The cells were washed three times with DPBS and the stained cells were detected with a confocal microscope (Leica TCS SP8).

[0076] The results are as follows Fig.14 As shown. Compared with the control group, there was no significant difference between the PBACs 500 nM treatment group and the NIR treatment group, reflecting that although the use of PBACs alone can accelerate the degradation of PTK7 protein, it is difficult to directly kill tumor cells, so the cell killing effect is not much different from the control group. However, PBACs (500 nM) + NIR treatment can quickly promote cell death and show excellent tumor cell killing ability.

[0077] The data from the above embodiments show that the method of the present invention can accurately synthesize PBACs drugs, which can initiate the degradation of lysosomal pathway proteins to achieve the degradation of membrane proteins, thereby inhibiting cell proliferation and migration ability; and the photodynamic therapy mediated by the PBACs drugs of the present invention (PBACs+NIR treatment group) can simultaneously activate cell autophagy on the basis of the former, and cell autophagy can greatly improve the efficiency of lysosomal pathway protein degradation, with a significant effect beyond expectations, further inhibiting cell proliferation and migration ability, and greatly killing tumor cells.

[0078] Although the technical solutions of the present invention have been described and listed in detail, it should be understood that it is obvious to those skilled in the art to make modifications to the above-mentioned embodiments or adopt equivalent alternatives. These modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. A light-activated bispecific nucleic acid aptamer chimera, characterized in that: comprising a first nucleic acid sequence, a second nucleic acid sequence and a photosensitizer; The first nucleic acid sequence includes: a first nucleic acid aptamer sequence and a first complementary sequence, and the first nucleic acid aptamer specifically recognizes and binds to the IGFIIR protein; The second nucleic acid sequence includes: a second nucleic acid aptamer sequence, a second complementary sequence, and a drug-carrying sequence, the second nucleic acid aptamer specifically recognizes and binds to the membrane protein target to be degraded, and the second complementary sequence is complementary to the first complementary sequence in base pairing; The photosensitizer is precisely modified on the drug-carrying sequence of the second nucleic acid sequence; the photosensitizer can respond to near-infrared light, increase ROS generation in tumor cells, and initiate cell autophagy; The first nucleic acid sequence is designed to carry a nucleic acid aptamer sequence targeting IGFIIR and a complementary sequence; the second nucleic acid sequence is designed to carry a nucleic acid aptamer sequence, a complementary sequence and a drug-carrying sequence of Sgc8; the sequence of IGFIIR is shown in SEQ2; the sequence of Sgc8 is shown in SEQ1.

2. A light-activated bispecific nucleic acid aptamer chimera according to claim 1, characterized in that: The photosensitizer is a first-generation, second-generation, third-generation or fourth-generation photosensitizing small molecule drug that can respond to near-infrared light.

3. The method for preparing a light-activated bispecific nucleic acid aptamer chimera according to claim 1 or 2, characterized in that: The following steps are involved: Step 1: Designing the first nucleic acid sequence and the second nucleic acid sequence; The first nucleic acid sequence includes a first nucleic acid aptamer sequence and a first complementary sequence; the first nucleic acid aptamer specifically recognizes and binds to a cell surface transport protein; The second nucleic acid sequence includes a second nucleic acid aptamer sequence, a second complementary sequence, and a drug-carrying sequence; the second nucleic acid aptamer specifically recognizes and binds to the membrane protein target to be degraded; the second complementary sequence and the first complementary sequence can achieve precise base complementary pairing; Step 2: Precisely modifying the photosensitizer on the drug-carrying sequence of the second nucleic acid sequence; Step 3: The first nucleic acid sequence and the second nucleic acid sequence are complementary paired in an annealing environment to prepare a light-activated bispecific nucleic acid aptamer chimera.

4. The preparation method according to claim 3, characterized in that: The modification methods used for precise modification of photosensitizers on drug loading sequences are: click chemistry azidation modification, cycloaddition reaction of alkyne and azide, cycloaddition reaction of cycloalkyne and azide, condensation reaction of non-alcohol aldehyde carbonyl compounds, addition reaction of thiol and carbon-carbon multiple bonds, or dehydration condensation reaction of amino or carboxyl groups.

5. Use of the light-activated bispecific nucleic acid aptamer chimera as claimed in claim 1 in the preparation of relevant drugs for the treatment of colorectal cancer.

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