PH and enzyme double-response type targeted DNA nano-carrier for relieving tumor cell hypoxia as well as preparation method and application of pH and enzyme double-response type targeted DNA nano-carrier

Abstract

The invention discloses a pH and enzyme dual-response type targeted DNA nano-carrier for relieving tumor cell hypoxia as well as a preparation method and application of the pH and enzyme dual-response type targeted DNA nano-carrier. The DNA nano-carrier comprises m tandem repeat units, each tandem repeat unit comprises a long single-stranded DNA repeat unit, three complementary short single-stranded DNAs and siRNA, each of the three complementary short single-stranded DNA sequences comprises a functional region and a base complementary region, and the functional region is a pH-responsive nucleic acid complementary sequence or a nucleic acid aptamer sequence for specifically targeting cancer cells; siRNA is a gene therapeutic agent for relieving tumor hypoxia, an extended positive-sense strand of siRNA comprises a 5 '-terminal extended pH response sequence and a positive-sense strand, and the 5'-terminal extended pH response sequence is complementary with a pH-responsive nucleic acid complementary sequence of the complementary short single-stranded DNA. The nano-drug carrier has excellent drug loading capacity and targeting property, can deliver anti-cancer drugs such as siRNA in a targeted manner, responds to a slightly acidic / enzyme environment of tumor cells and is quickly released, and the anti-tumor treatment effect is improved.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a pH and enzyme dual-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia, its preparation method, and its application. Background Technology

[0002] Due to their inherent biocompatibility and biodegradability, DNA nanostructures are widely used for drug delivery. Among them, pH-responsive DNA nanocarriers, which can respond to changes in the physiological pH environment, can achieve autonomous drug release in the acidic tumor microenvironment, showing great promise in the field of intelligent drug delivery. Furthermore, the sensitivity of DNA nanocarriers to intracellular nucleases also facilitates drug release. However, low drug loading capacity and poor targeting are significant challenges faced by existing pH-responsive DNA nanocarriers as drug delivery systems. Especially in the hypoxic environment of tumors, combined photodynamic and gene therapy strategies place higher demands on the drug loading capacity of drug carriers. It is also noteworthy that siRNAs that alleviate tumor hypoxia by regulating tumor metabolism cannot autonomously reach their sites of action within tumor cells, directly impacting clinical cancer treatment efficacy. Therefore, developing a pH- and enzyme-responsive DNA nanocarrier with high drug loading capacity, strong specific targeting ability, and the ability to effectively alleviate tumor cell hypoxia is a pressing scientific problem to be solved in the biomedical field. Summary of the Invention

[0003] To address the aforementioned technical problems, this invention provides a pH- and enzyme-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia, along with its preparation method and applications. Based on cutting-edge DNA nanotechnology, this invention uses ultra-long single-stranded DNA from rolling circle amplification (RCA) products as a backbone. A large number of pH-responsive sequences, multivalent aptamers, and siRNA gene therapy agents are efficiently self-assembled to construct a pH- and enzyme-responsive DNA nanocarrier with high drug loading capacity, strong specific targeting ability, and simultaneous pH / enzyme controlled release, capable of alleviating tumor cell hypoxia. This carrier can serve as a smart drug delivery platform; by changing different aptamers or anticancer drugs, it can act on various types of cancer cells, significantly improving anticancer efficacy.

[0004] In a first aspect, the present invention provides a pH and enzyme dual-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia, which is achieved through the following technical solution.

[0005] A pH and enzyme dual-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia comprises m tandem repeat units, each tandem repeat unit comprising one long single-stranded DNA repeat unit, three complementary short single-stranded DNAs, and n siRNAs, where m is an integer from 10 to 100; and n is an integer from 1 to 3.

[0006] In the tandem repeat unit, the sequence of the long single-stranded DNA repeat unit is shown in SEQ ID NO.4; the three complementary short single-stranded DNA sequences each include a functional region and a base complement region. The functional region is a pH-responsive nucleic acid complement sequence or a nucleic acid aptamer sequence specifically targeting cancer cells. The base complement region sequence is completely complementary to the long single-stranded DNA repeat unit sequence. The functional sequence and the base complement sequence are linked by 4 T bases. siRNA is a gene therapy agent to alleviate tumor hypoxia. The extended sense strand of siRNA includes a 5' extended pH-responsive sequence and a sense strand. The 5' extended pH-responsive sequence is partially complementary to the pH-responsive nucleic acid complement sequence of the complementary short single-stranded DNA. The sense strand and antisense strand of siRNA are completely complementary.

[0007] Furthermore, the complementary nucleic acid sequence of the pH-responsive short single-stranded DNA is the first 12 bases at the 5' end of the i-motif complementary sequence. The pH-responsive sequence is a DNA sequence that reversibly undergoes conformational transitions between quadruplex and single-stranded structures when the environmental pH is between 5.0 and 7.4. Under acidic environmental stimulation, it transforms from a single-stranded structure to a C quadruplex (i-motif) structure, thereby enabling the rapid release of loaded drugs within tumor cells.

[0008] By adopting the above technical solution, the i-motif sequence is very sensitive to the ambient pH. When the ambient pH drops from neutral (about 7.4) to acidic (<6.0), the i-motif sequence can be transformed from a single-chain structure to a C four-chain structure, so that the loaded drug is released with the conformational change.

[0009] In one specific implementation, one of the three complementary short single-stranded DNA sequences contains an aptamer sequence that specifically targets cancer cells, enabling specific targeting of cancer cells.

[0010] Furthermore, the complementary short single-stranded DNA specific targeting nucleic acid aptamer sequences for cancer cells include the AS1411 aptamer sequence, the MUC1 aptamer sequence, the Cyt-C aptamer sequence, and the TD05 aptamer sequence.

[0011] The aptamers used in this invention are arbitrarily selected, as long as they can specifically target the corresponding cancer cells. An aptamer is a single-stranded DNA or RNA molecule that can bind to its target with high specificity and high affinity. Its binding ability stems from shape complementarity with the homologous target and the synergistic effect of various molecular forces, including hydrogen bonds, electrostatic interactions, and van der Waals forces. The aptamers selected in this invention are cancer cell-targeting aptamers, which can specifically bind to target molecules on cancer cells. For example, the AS1411 aptamer, which targets various cancer cells, specifically binds to nucleolin on the cancer cell membrane surface; or the MUC1 aptamer, which targets human breast MCF-7 cancer cells, specifically binds to mucin 1, which is widely expressed on the MCF-7 cell membrane; or the TD05 aptamer, which targets Burkitt lymphoma cells, specifically binds to 50 immunoglobulin heavy chain molecules of lymphoma cells. A large number of aptamers are attached to one side of the DNA nanocarrier, forming a multivalent aptamer structure.

[0012] Preferably, the aptamer is the AS1411 aptamer, which can specifically recognize nucleolin protein that is widely expressed on the surface of cancer cells; a large number of aptamers form a multivalent aptamer structure on one side of the DNA nanocarrier, and through multivalent interaction, precise targeting of MCF-7 cells is achieved.

[0013] Furthermore, siRNA is an anti-MCT1 siRNA (siMCT1) that regulates abnormal tumor metabolism to alleviate tumor hypoxia. It inhibits the expression of monocarboxylic acid transporter-1 (MCT1), blocks lactate-stimulated oxidative respiration of tumor cells, and effectively increases local oxygen partial pressure, thereby significantly improving the efficacy of photodynamic therapy and other treatments.

[0014] Furthermore, the 5' end of the extended positive strand of the siRNA is a pH-responsive sequence rich in cytosine, forming an i-motif sequence.

[0015] In one specific embodiment, the present invention provides a pH and enzyme dual-response targeted DNA nanocarrier for alleviating tumor cell hypoxia, comprising 40 tandem repeat units, each tandem repeat unit comprising one long single-stranded DNA repeat unit, three complementary short single-stranded DNAs and two siRNAs; the 40 long single-stranded DNA repeat units are sequentially linked together to form a long single-stranded DNA, the sequence of which is shown in SEQ ID NO:3.

[0016] In the tandem repeat unit, one complementary short single-stranded DNA includes the first 12 bases of the i-motif complementary sequence at the 5' end, as shown in SEQ ID NO:5; another complementary short single-stranded DNA includes the AS1411 aptamer sequence, as shown in SEQ ID NO:6; a third complementary short single-stranded DNA includes the first 12 bases of the i-motif complementary sequence at the 5' end, as shown in SEQ ID NO:7; the siRNA includes an anti-MCT1 siRNA extended sense strand with the i-motif sequence extended at the 5' end, as shown in SEQ ID NO:8; and an anti-MCT1 siRNA antisense strand, as shown in SEQ ID NO:9.

[0017] In the specific embodiment described above, the pH-responsive sequence is directly modified onto the extended positive strand of siMCT1, which alleviates tumor cell hypoxia. The pH-responsive sequence is complementary to 12 bases of the functional sequences of two complementary short single-stranded DNAs. The complementary sequences of the complementary short single-stranded DNAs are 21 bases long, perfectly complementary to one-third of the bases of the repeating unit of the long single-stranded DNA. The self-assembled DNA nanocarrier can alleviate tumor cell hypoxia while exhibiting both pH and enzyme-responsive release and a high number of drug binding sites.

[0018] In this invention, "relieving tumor cell hypoxia" refers to siRNA gene therapy agents that regulate abnormal tumor metabolism to relieve tumor hypoxia. These agents are difficult to effectively enter cancer cells and exert their effects on their own. However, by using a DNA nanocarrier, they can be delivered in a targeted manner and exert gene therapy effects. Compared with other delivery carriers, this DNA nanocarrier has a higher drug loading capacity and more precise targeted release capability.

[0019] In this invention, the "pH response" refers to the use of cytosine-rich i-motif sequences to activate the i-motif sequences through acidic pH activation between the physiological environment (pH 7.4) and the acidic organelles within tumors (pH 5.0), causing a conformational change (folding into a dense quadruplex structure). This enables the rapid release of anticancer drugs from cancer cells. The DNA nanocarrier contains a large number of i-motif sequences, which can rapidly increase the drug accumulation concentration within cancer cells, greatly improving the therapeutic effect and reducing systemic toxicity.

[0020] In this invention, the "enzyme response" refers to the use of the DNA double-stranded structure of the DNA nanocarrier, which is sensitive to intracellular nucleases and is easily cleaved into oligonucleotide chains by nuclease I, leading to double-strand dissociation and thus drug release.

[0021] In this invention, "targeting" refers to multiple aptamer molecules that can specifically target and bind to multiple target molecules on the surface of cancer cells, thereby achieving efficient multivalent binding. Compared with monovalent aptamers, this DNA nanocarrier has a stronger specific targeting ability for cancer cells.

[0022] Secondly, the present invention provides a method for preparing a pH and enzyme dual-responsive targeted DNA nanocarrier to alleviate tumor cell hypoxia, which is achieved through the following technical solution.

[0023] A method for preparing the above-mentioned DNA nanocarrier includes the following steps:

[0024] A long single-stranded DNA with m tandem repeat sequences was synthesized by rolling circle amplification reaction;

[0025] Based on DNA self-assembly technology, the long single-stranded DNA synthesized in step S1 is used as the backbone strand and combined with m*3 complementary short single-stranded DNA strands to obtain a DNA assembly.

[0026] The sense and antisense strands of siRNA are extended to undergo complementary base pairing to obtain a double-stranded siRNA.

[0027] The pH-responsive complementary nucleic acid sequence of the DNA assembly is base-paired with the pH-responsive complementary nucleic acid sequence of the siRNA double-strand extension to construct a DNA nanocarrier.

[0028] The DNA nanocarrier of this invention is composed of m tandem repeat units, and its long single-stranded DNA product exhibits periodicity, high molecular weight, and high programmability. The length of the long single-stranded DNA can be directly controlled by the rolling circle amplification (RCA) reaction time (under the condition that the dNTP concentration is fixed at 7 mM). Based on extensive preliminary experiments, the preferred reaction time range is 5 to 30 minutes, and within this range, a more optimal reaction time is 20 minutes. Under these conditions, long DNA chains with suitable molecular weights that are conducive to subsequent self-assembly can be synthesized.

[0029] Specifically, a method for preparing a pH- and enzyme-responsive targeted DNA nanocarrier to alleviate tumor cell hypoxia includes the following steps:

[0030] S1. In a mixed solution of the circular DNA template shown in SEQ ID NO:1, the DNA primer shown in SEQ ID NO:2, Phi29 DNA polymerase buffer and deoxyribonucleotide triphosphates (dNTPs), Phi29 DNA polymerase was added to initiate the amplification reaction (30℃, 20 min), and then the reaction was immediately terminated (65℃, 10 min) to obtain the long single-stranded DNA shown in SEQ ID NO:3;

[0031] S2. The long single-stranded DNA shown in SEQ ID NO:3 and the complementary short single-stranded DNA shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 are reacted in annealing buffer (containing 10 mM Mg). 2+ Mix thoroughly in TE buffer, centrifuge at low speed, heat at 95°C for 5 min, and then anneal at 25°C to obtain DNA assemblies.

[0032] S3. The extended sense strand of siMCT1 shown in SEQ ID NO:8 and the antisense strand shown in SEQ ID NO:9 were placed in annealing buffer (containing 10 mM Mg). 2+ vortex centrifuge in TE buffer, heat treat at 95°C for 5 min, and then anneal to 25°C to obtain siMCT1 double strands;

[0033] S4. Mix the DNA assembly with the siMCT1 double strand evenly and place it in a 37℃ metal bath for 4 hours to construct a pH and enzyme dual-responsive targeted DNA nanocarrier that alleviates tumor cell hypoxia.

[0034] More specifically, a method for preparing a pH- and enzyme-responsive targeted DNA nanocarrier to alleviate tumor cell hypoxia includes the following steps:

[0035] S1. Preparation of long single-stranded DNA:

[0036] Using the circular DNA template shown in SEQ ID NO:1, a rolling circle amplification (RCA) reaction was performed. In the reaction system, the DNA primers shown in SEQ ID NO:2 (2 μL, 10 μM), the circular DNA template (2 μL, 10 μM), 10×Phi29 DNA polymerase buffer (2 μL), dNTPs (2 μL, 7 mM), and ddH2O (11.5 μL) were added sequentially. Finally, Phi29 DNA polymerase (0.5 μL, 10 U / μL) was added to start the amplification reaction (30℃, 20 min). After the reaction was completed, the Phi29 DNA polymerase was rapidly inactivated (65℃, 10 min) to obtain the long single-stranded DNA shown in SEQ ID NO:3.

[0037] S2. Preparation of DNA assemblies:

[0038] Add 5 μL of the long single-stranded DNA from step S1, 5 μL each of the complementary short single-stranded DNAs shown in SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:7 at a concentration of 8 μM, and 2×TE annealing buffer (containing 10 mM Mg2+). 2+Add 20 μL to a centrifuge tube, vortex centrifuge, place in a PCR instrument (95℃, 5 min), and then gradually cool (1℃ / min) to 25℃ to obtain DNA assemblies;

[0039] Preparation of S3.siMCT1

[0040] The siMCT1 extended sense strand (12 μM, 10 μL) shown in SEQ ID NO:8 and the antisense strand (12 μM, 10 μL) shown in SEQ ID NO:9 were prepared in 2×TE annealing buffer (containing 10 mM Mg). 2+ The sample was centrifuged in 20 μL and placed in a PCR instrument (95 °C, 5 min), then gradually cooled (1 °C / min) to 25 °C to prepare siMCT1.

[0041] S4. Preparation of DNA Nanocarriers

[0042] Take 20 μL each of the DNA assembly from step S2 and the siMCT1 double strand from step S3 and place them in a centrifuge tube. Incubate them in a metal bath at 37°C for 4 h to construct a pH and enzyme dual-responsive targeted DNA nanocarrier that alleviates tumor cell hypoxia.

[0043] Thirdly, the present invention provides the use of a pH and enzyme dual-responsive targeted DNA nanocarrier to alleviate tumor cell hypoxia, which is achieved through the following technical solution.

[0044] The application of the above-mentioned DNA nanocarrier in the preparation of antitumor drugs.

[0045] Fourthly, the present invention provides a pH and enzyme dual-responsive targeted DNA nanocarrier-photosensitizer complex for alleviating tumor cell hypoxia, which is achieved through the following technical solutions.

[0046] A pH- and enzyme-responsive targeted DNA nanocarrier-photosensitizer complex for alleviating tumor cell hypoxia, comprising the aforementioned DNA nanocarrier and a photosensitizer or photochemotherapy combination drug.

[0047] Furthermore, when the DNA nanocarrier forms a complex with the photosensitizer, the concentration ratio of the DNA nanocarrier to the photosensitizer is (0.2-5):400; the photosensitizer is a phenothiazine photosensitizer, including toluidine blue O, methylene blue and thionine;

[0048] Furthermore, when the DNA nanocarrier forms a complex with the photochemotherapy combination drug, the photochemotherapy combination drug includes a photosensitizer and a chemotherapeutic drug, and the concentration ratio of the DNA nanocarrier, photosensitizer, and chemotherapeutic drug is (0.2-5):400:300; the photosensitizer is a phenothiazine photosensitizer, including toluidine blue O, methylene blue, and thionine; the chemotherapeutic drug is an anthracycline chemotherapeutic drug, including doxorubicin, daunorubicin, and demethoxydaunorubicin.

[0049] In this invention, there are no particular limitations on the method of loading photosensitizers or photochemotherapy combination drugs. For example, in one embodiment, the photosensitizer or photochemotherapy combination drug can be bound to the DNA double helix via non-covalent binding, such as intercalation, trenching, or electrostatic interaction, to form a DNA nanocarrier-photosensitizer complex. In this case, the photosensitizer can be a phenothiazine photosensitizer such as toluidine blue O (TBO), methylene blue (MB), and thionine (THN), whose planar aromatic ring structure can be intercalated between adjacent base pairs of the DNA double helix.

[0050] In this invention, a photochemotherapy combination drug can also be bound to the DNA double helix to form a DNA nanocarrier-photosensitizer complex. In this case, the photosensitizer can be toluidine blue O (TBO), and the chemotherapeutic drug can be anthracycline chemotherapeutic drugs such as doxorubicin (DOX), daunorubicin (DNR), and demethoxydaunorubicin (IDA), whose aromatic groups can be partially intercalated between the base pairs of the DNA double helix, undergoing π-π stacking interactions with the bases to prepare a DNA nanocarrier combined with drug delivery system.

[0051] Fifthly, the present invention provides a method for preparing a pH and enzyme dual-responsive targeted DNA nanocarrier-photosensitizer complex that alleviates tumor cell hypoxia, which is achieved through the following technical solutions.

[0052] A method for preparing the above-mentioned pH and enzyme dual-responsive targeted DNA nanocarrier-photosensitizer complex for alleviating tumor cell hypoxia involves mixing the DNA nanocarrier with a photosensitizer or a photochemotherapy combination drug in a certain proportion and incubating at 37°C for 30-60 min to obtain the DNA nanocarrier-photosensitizer complex.

[0053] In one embodiment, preferably, 2 nM DNA nanocarriers are incubated with 400 nM photosensitizers at 37°C for 30 min to obtain DNA nanocarrier-photosensitizer, with 200 photosensitizers loaded on each DNA nanocarrier.

[0054] This application has the following beneficial effects.

[0055] (1) The DNA nanocarrier in this invention has the advantages of relieving tumor hypoxia, strong targeting ability, pH and enzyme dual response release, high drug loading capacity, strong versatility and good biocompatibility. It can be used for efficient loading and targeted delivery of various anticancer drugs such as photosensitizers, accurately reaching the site of action of tumor cells and significantly relieving tumor cell hypoxia, thereby enhancing the synergistic anticancer effect of combined therapy. It is an excellent drug delivery carrier.

[0056] (2) The rolling circle amplification (RCA) technology in this invention is based on isothermal enzymatic reaction, which can efficiently synthesize ultra-long single-stranded DNA containing a large number of repeating units. It is easy to operate, has high amplification efficiency, large product molecular weight and strong sequence programmability, which significantly simplifies the preparation process of DNA nanocarriers and reduces dependence on expensive equipment.

[0057] (3) In this invention, tumor cell hypoxia is relieved by inhibiting the expression of monocarboxylic acid transporter 1 (MCT1) through RNA interference (RNAi) technology, blocking the uptake of lactic acid by oxidative cells, reducing their oxygen consumption rate, thereby increasing the local oxygen partial pressure of the tumor, significantly improving the anti-tumor effect of photodynamic therapy and chemotherapy, and effectively reversing the clinical treatment resistance caused by tumor hypoxia.

[0058] (4) The pH and enzyme dual-response targeted delivery in this invention is based on the microacid and nuclease environment of tumor cells. Through the efficient recognition of overexpressed receptors on the tumor surface by multivalent aptamers, the precise delivery and dual-response rapid release of drugs are achieved, while reducing off-target effects, increasing the drug enrichment concentration at the tumor site and enhancing the anti-cancer effect. Attached Figure Description

[0059] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0060] Figure 1This invention presents a schematic diagram illustrating the preparation and application principle of a pH- and enzyme-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia. (Where: a: DNA nanocarrier assembly process; b: The process of non-covalently intercalating the photosensitizer TBO (represented by a hexagon) and the chemotherapeutic drug DOX (represented by a circle) loaded on the DNA nanocarrier into the DNA double strand to form a DNA nanocarrier@TBO@DOX complex, and the drug release under acidic and nuclease conditions; c: The process of the multivalent aptamer of the DNA nanocarrier@TBO@DOX complex precisely targeting and binding to receptors on tumor cells, the multivalent effect promoting endocytosis, the process of the DNA nanocarrier@TBO@DOX complex responding to the acidic environment and nucleases within tumor cells to release the photosensitizer TBO, the chemotherapeutic drug DOX, and siMCT1, and the process of laser irradiation of TBO generating a large amount of reactive oxygen species, siMCT1 silencing MCT1 mRNA, and DOX inhibiting DNA replication and transcription).

[0061] Figure 2 These are gel images and transmission electron microscope images of the rolling circle amplification (RCA) products, DNA assemblies, siMCT1, and DNA nanocarriers characterized in Example 1 of this invention.

[0062] Figure 3 This is a fluorescence quenching spectrum of the binding mode of the anticancer drug and the DNA nanocarrier in Example 2.1 of the present invention (wherein, a: quenching analysis of free TBO and DNA nanocarrier@TBO complex by potassium ferrocyanide fluorescence quencher; b: quenching analysis of free DOX and DNA nanocarrier@DOX complex by potassium ferrocyanide fluorescence quencher).

[0063] Figure 4This is a graph showing the results of loading a single drug onto a DNA nanocarrier in Example 2.2 of this invention (where ad: the fluorescence intensity trends of different concentrations of DNA nanocarriers incubated with 400nM TBO at 25℃, 29℃, 33℃, and 37℃, respectively; the graph is divided into am lines from top to bottom, and the DNA nanocarrier concentrations corresponding to the am lines are 0nM, 0.05nM, 0.1nM, 0.15nM, 0.2nM, 0.25nM, 0.3nM, 0.35nM, 0.4nM, 0.45nM, 0.5nM, 0.55nM, and 0.6nM; fi: the fluorescence intensity trends of different concentrations of DNA nanocarriers incubated with 400nM TBO at 25℃, 29℃, 33℃, and 37℃, respectively; the graph is divided into am lines from top to bottom, and the DNA nanocarrier concentrations corresponding to the am lines are 0nM, 0.05nM, 0.1nM, 0.15nM, 0.2nM, 0.25nM, 0.3nM, 0.35nM, 0.4nM, 0.45nM, 0.5nM, 0.55nM, and 0.6nM; the DNA nanocarrier concentrations incubated with 400nM TBO at 400nM TBO are 0nM, 0.05nM, 0.05nM, 0.05nM, 0.05nM, 0.06nM, 0.05 ... The fluorescence intensity trends of DOX after incubation at 25℃, 29℃, 33℃, and 37℃ are shown in the figure. From top to bottom, the lines are divided into am lines, and the DNA nanocarrier concentrations corresponding to the am lines are 0 nM, 0.05 nM, 0.1 nM, 0.15 nM, 0.2 nM, 0.25 nM, 0.3 nM, 0.35 nM, 0.4 nM, 0.45 nM, 0.5 nM, 0.55 nM, and 0.6 nM, respectively. e: Van't Hoff plot of TBO at four temperatures; j: Van't Hoff plot of DOX at four temperatures.

[0064] Figure 5 This is a graph showing the results of exploring the proportion of combined drugs loaded on DNA nanocarriers in Example 2.3 of this invention;

[0065] Figure 6 This is a diagram showing the release behavior of the drug loaded on the DNA nanocarrier in Example 3 of the present invention under different conditions (where, a: release behavior of TBO in the DNA nanocarrier@TBO complex under different conditions; b: release behavior of TBO in the DNA nanocarrier@TBO@DOX complex under different conditions; c: release behavior of DOX in the DNA nanocarrier@DOX complex under different conditions; d: release behavior of DOX in the DNA nanocarrier@TBO@DOX complex under different conditions).

[0066] Figure 7 This is a diagram illustrating the targeting ability of the DNA nanocarrier on LO2 cells and MCF-7 cells in Example 4 of this invention.

[0067] Figure 8 This is a graph showing the level of reactive oxygen species (ROS) generation in cells of the photosensitizer TBO loaded on a DNA nanocarrier in Example 5 of the present invention (where a: ROS generation after laser irradiation of different samples; b: ROS generation of different samples without laser irradiation).

[0068] Figure 9This is a graph showing the toxicity results of the single drug TBO and the combined drug TBO@DOX on MCF-7 cells in Example 6 of the present invention, both free and loaded with DNA nanocarriers (with and without siMCT1). (where a: the inhibitory effect of the free drug on MCF-7 cells; b: the inhibitory effect of the drug on MCF-7 cells after loading with DNA nanocarriers). Detailed Implementation

[0069] Preferred embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. Before description, it should be understood that the terminology used in the specification and appended claims should not be construed as limited to its general and dictionary meaning, but rather should be interpreted based on the principle of allowing the inventors to appropriately define the terminology for the best interpretation, and based on its meaning and concept corresponding to the technical level of the invention. Therefore, the description herein is merely a preferred example for illustrative purposes and is not intended to limit the scope of the invention; thus, it should be understood that other equivalent implementations and modifications can be made without departing from the spirit and scope of the invention.

[0070] This invention uses long single-stranded DNA prepared by rolling circle amplification (RCA) as the basic backbone for binding multivalent nucleic acid aptamers and linking i-motif sequences. The i-motif sequences then act as a bridge connecting the long single-stranded DNA and siRNA that alleviates tumor cell hypoxia. This self-assembly yields a pH- and enzyme-dual-response (RAS) targeted DNA nanocarrier for alleviating tumor cell hypoxia. While precisely targeting and rapidly releasing anticancer drugs with dual responsiveness, the siRNA can more continuously and effectively regulate abnormal tumor metabolism to alleviate tumor hypoxia, significantly improving the synergy and effectiveness of photodynamic therapy-based combined cancer treatment strategies. Compared to traditional pH-responsive drug carriers, the pH and enzyme-responsive targeted DNA nanocarrier of this invention, which alleviates tumor cell hypoxia, combines the advantages of high programmability, high drug loading capacity, precise targeting capability, rapid responsive release, and relief of tumor hypoxia, while being simple, versatile, and biocompatible. It shows great potential in the targeted co-delivery of combined drugs and the enhancement of synergistic anti-cancer effects. In particular, in the field of multi-strategy combination therapy for cancer, this carrier provides an excellent solution for overcoming cancer and has broad application potential.

[0071] The preparation and application of pH- and enzyme-responsive targeted DNA nanocarriers for alleviating tumor cell hypoxia are described in the following specific methods.

[0072] Example 1: Preparation method of DNA nanocarriers

[0073] 1.1 Preparation of Rolling Circle Amplification (RCA) Products

[0074] The reaction system was prepared by sequentially adding 2 μL of primer (10 μM, sequence as shown in SEQ ID NO.2), 2 μL of circular DNA template (10 M m, sequence as shown in SEQ ID NO.1), 2 μL of 10×Phi29 DNA polymerase buffer, 2 μL of dNTPs (7 mM), and 11.5 μL of ddH2O. Then, 0.5 μL of Phi29 DNA polymerase (10 U / μL) was added. After vortexing and centrifugation, the mixture was incubated in a 30°C metal bath for 20 min, followed by incubation at 65°C for 10 min to inactivate the Phi29 DNA polymerase and terminate the reaction, yielding the RCA product (long single-stranded DNA, sequence as shown in SEQ ID NO.3). The rolling circle amplification (RCA) product was characterized by agarose gel electrophoresis.

[0075] 1.2 Preparation of DNA assemblies

[0076] Add 5 μL of the RCA product from step 1.1, 5 μL each of complementary short single-stranded DNA S1-S3 (concentration 8 μM, sequence as shown in SEQ ID NO. 5-7), and 2×TE (containing 10 mM Mg). 2+ Add 20 μL of annealing buffer to a centrifuge tube, vortex, and incubate at 95°C for 5 min in a PCR instrument. Then, gradually decrease the temperature (1°C / min) to 25°C to obtain the DNA assembly. Characterize the DNA assembly using agarose gel electrophoresis, such as... Figure 2 As shown in Figure a, with the gradual addition of the three short chains, the molecular weight of the product increases, and self-assembly is successful.

[0077] 1.3 Preparation of siMTC1

[0078] 10 μL of the extended sense strand (12 μM, sequence as shown in SEQ ID NO. 8) and 10 μL of the antisense strand (12 μM, sequence as shown in SEQ ID NO. 9) of siMCT1 were added to 20 μL of 2×TE annealing buffer (containing 10 mM Mg). 2+ The siMCT1 assembly was prepared by vortexing and centrifugation in a PCR instrument (95℃, 5 min), followed by a gradient cooling (1℃ / min) to 25℃. The siMCT1 assembly was characterized by polyacrylamide gel electrophoresis. Figure 2 As shown in b, compared with the extended sense and antisense strands of siMCT1, the product of siMCT1 has a larger molecular weight and clearer and brighter electrophoretic bands, indicating successful preparation.

[0079] 1.4 Preparation of DNA Nanocarriers

[0080] 20 μL each of the DNA assembly from step 1.2 and the siMCT1 from step 1.3 were placed in centrifuge tubes and incubated in a 37°C metal bath for 4 hours to construct a pH- and enzyme-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia. The DNA assembly was characterized using agarose gel electrophoresis, such as... Figure 2 As shown in Figure c, the brightness of the siMCT1 band decreased significantly, while the brightness of the DNA nanocarrier band increased, indicating successful loading of siMCT1 and successful assembly. At pH 5.0, the siMCT1 band was clear, and the DNA nanocarrier released siMCT1, achieving an acid response.

[0081] The DNA nanocarriers were tested using transmission electron microscopy (TEM) at an accelerating voltage of 200 kV. The sample preparation method for TEM was as follows: 5 μL of DNA nanoparticle sample was dropped onto a copper grid, and the sample was tested after complete drying. The DNA nanocarriers were characterized using TEM, such as... Figure 2 As shown in d, the product is linear, and due to its large aspect ratio, some parts are flexibly entangled.

[0082] Table 1 shows the oligonucleotide sequences used in Example 1.

[0083]

[0084]

[0085]

[0086] Note: In complementary short single-stranded DNA S1 and S3, the underlined portion represents the i-motif complementary sequence; in short strand S2, the dashed portion represents the AS1411 aptamer sequence; in the siRNA extended sense strand, the bold portion represents the i-motif sequence, and the italic portion represents the siRNA sense strand. Additionally, to meet the requirements for creating the WIPO Sequence listing, the "U" bases in the siMCT1 extended sense strand and siMCT1 antisense strand are replaced with "T" bases during sequence listing.

[0087] Example 2: Anticancer drug loading experiment

[0088] 2.1 Binding mechanism of anticancer drugs and DNA nanocarriers

[0089] The concentrations of the photosensitizer toluidine blue O (TBO) and the chemotherapeutic drug doxorubicin (DOX) were both 400 nM, the concentration of the DNA nanocarrier was 1.2 nM, and the concentrations of the anionic quencher potassium ferrocyanide K4 [Fe(CN)6] were 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM, respectively. The excitation wavelengths of the fluorescence spectra of TBO and DOX were set to 592 nm and 485 nm, respectively, and the emission wavelength ranges were set to 620-745 nm and 525-745 nm, respectively. The detection temperature was 25 °C.

[0090] Preparation methods of DNA nanocarrier@TBO complex and DNA nanocarrier@DOX complex bound to K4[Fe(CN)6]: 400 nM free TBO and 400 nM free DOX were respectively placed with 1.2 nM DNA nanocarrier in TE buffer at pH 7.4 and incubated at 25°C in the dark for 30 min to prepare DNA nanocarrier@TBO and DNA nanocarrier@DOX complexes. Then, K4[Fe(CN)6] at concentrations of 0 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, and 100 mM were added to the DNA nanocarrier@TBO complex, DNA nanocarrier@DOX complex, 400 nM free TBO, and 400 nM free DOX, respectively. After incubation at 25°C in the dark for 30 min, the fluorescence changes of TBO and DOX were measured. The K4[Fe(CN)6] of non-covalently intercalated drugs... sv The value should be significantly smaller than the K of the free drug. sv The quenching constant K is calculated using the Stern-Volmer equation (1). sv .like Figure 3 As shown in 3a and 3b, the Ksv values ​​of both TBO and DOX were significantly reduced after loading with the DNA nanocarrier. This indicates that the DNA nanocarrier binds to both TBO and DOX through intercalation.

[0091]

[0092] Where [Q] is the concentration of K4[Fe(CN)6], and F0 and F are the fluorescence intensities of TBO or DOX in the absence or presence of DNA nanocarriers, respectively.

[0093] 2.2 Loading ratio of single drugs

[0094] The concentrations of the photosensitizer toluidine blue O (TBO) and the chemotherapeutic drug doxorubicin (DOX) were both 400 nM. The concentrations of the DNA nanocarriers were 0 nM, 0.05 nM, 0.1 nM, 0.15 nM, 0.2 nM, 0.25 nM, 0.3 nM, 0.35 nM, 0.4 nM, 0.45 nM, 0.5 nM, 0.55 nM, and 0.6 nM, respectively. The excitation wavelengths of the fluorescence spectra of TBO and DOX were set to 592 nm and 485 nm, respectively, and the emission wavelength ranges were set to 620-745 nm and 525-745 nm, respectively. The detection temperatures were 25℃, 29℃, 33℃, and 37℃, respectively.

[0095] Preparation methods of DNA nanocarrier@TBO complex and DNA nanocarrier@DOX complex: 400 nM TBO or DOX was mixed with 0 nM, 0.05 nM, 0.1 nM, 0.15 nM, 0.2 nM, 0.25 nM, 0.3 nM, 0.35 nM, 0.4 nM, 0.45 nM, 0.5 nM, 0.55 nM, and 0.6 nM DNA nanocarriers, respectively, in TE buffer at pH 7.4. The mixtures were then incubated in the dark at different temperatures of 25℃, 29℃, 33℃, and 37℃ for 30 min, respectively. The fluorescence spectra of TBO and DOX were measured using a fluorescence spectrometer. Figure 4 As shown in the diagram, at different temperatures (25-37℃), the fluorescence intensity of TBO gradually decreased with the increase of DNA nanocarrier concentration. This is because the TBO chromophore overlaps with the base pairs of the DNA nanocarrier, causing the chromophore to be masked, indicating the formation of a DNA nanocarrier@TBO complex. Figure 4 The DOX shown in figure fi exhibits the same trend. This is because the DOX is embedded in the double helix structure of the DNA nanocarrier, leading to energy resonance transfer or a reduction in active surface area, indicating the formation of a DNA nanocarrier@DOX complex. Binding constant (K... a The binding constant (K) and the number of binding sites (n) are important thermodynamic parameters for evaluating the binding process. The binding constant refers to the ability of a drug molecule to stably bind to a DNA nanocarrier; a higher value indicates a stronger binding ability. The number of binding sites refers to the number of sites on the DNA nanocarrier that bind to the drug molecule; a higher value indicates a greater amount of drug loaded. The binding constants (KB) of the DNA nanocarrier@TBO complex and the DNA nanocarrier@DOX complex can be obtained using the Scattchard equation (2, 3). a ) and the number of binding sites (n).

[0096]

[0097] C t =C f +C b (3)

[0098] Among them, C f r represents the concentration of free TBO or DOX, and r is the concentration ratio of bound TBO or DOX to the DNA nanocarrier. f Scattchard plot with r ( Figure 4 (The illustration in the image) obtained n and K a The data are shown in Table 1. The number of binding sites is greater than 800, indicating that the DNA nanocarrier has a high drug loading capacity for both TBO and DOX, especially for DOX. Binding constant (K) a The order of magnitude is approximately 10. 6 This indicates that both TBO and DOX have strong binding affinity to DNA nanocarriers. DOX exhibits a stronger binding affinity than TBO, suggesting a more robust base pair binding between DOX and the DNA nanocarrier. (Except for K...) a In addition to n, this application also calculated the enthalpy change (ΔH), entropy change (ΔS), and Gibbs free energy change (ΔG) of the DNA nanocarrier @TBO and the DNA nanocarrier @DOX complex (4):

[0099] ΔG=-RTlnK a =ΔH-TΔS (4)

[0100] By RlnK a Plotting 1 / T ( Figure 4 e: TBO and Figure 4 The corresponding thermodynamic data obtained for (j:DOX) are shown in Table 1. The ΔG and ΔH values ​​for both TBO and DOX are negative, indicating that their reaction with the DNA nanocarrier is a spontaneous, exothermic process. The ΔH value of the DNA nanocarrier@DOX complex is even more negative, which may be due to the stronger binding ability of DOX to the DNA nanocarrier. Based on the above thermodynamic data, the results were calculated using a type of site model (5) and (6).

[0101]

[0102] C t =C f +C b =C f +nΘM t (6)

[0103] In the formula, Θ represents the ratio of the number of occupied sites to the total number of sites, and M represents... t It is the total concentration of DNA nanocarriers, C t C f and C b These represent the total drug concentration, free drug concentration, and bound drug concentration, respectively, while F and F0 represent the fluorescence intensity of the drug in the presence or absence of the DNA nanocarrier, respectively. According to formulas (5) and (6), given M...t and C t In this case, it can be based on the known K a And n determines the percentage of drug binding C b / C t Given that both TBO and DOX concentrations are 400 nM, and the corresponding DNA nanocarrier concentrations are 2 nM and 1.6 nM, respectively, almost all TBO and DOX are loaded onto the DNA nanocarrier. The binding ratios of TBO, DOX, and DNA nanocarrier are 200:1 and 250:1, respectively.

[0104] Table 1. Thermodynamic parameters of TBO or DOX binding to DNA nanocarriers obtained from fluorescence spectroscopy experiments.

[0105]

[0106]

[0107] 2.3 Loading ratio of combined drugs

[0108] Based on previous experimental experience, to obtain a higher combined drug loading capacity, TBO, which has a weaker binding affinity, was loaded first, followed by DOX. The concentration of TBO was fixed at 400 nM, the concentration of the DNA nanocarrier was 2 nM, and the concentrations of DOX were 0 nM, 100 nM, 300 nM, 600 nM, 800 nM, 1000 nM, and 1500 nM. The excitation wavelength of the DOX fluorescence spectrum was set to 485 nm, the emission wavelength range was set to 525-745 nm, and the detection temperature was 37 °C.

[0109] Preparation method of DNA nanocarrier@TBO@DOX complex: 400 nM TBO and 2 nM DNA nanocarrier were mixed thoroughly in TE buffer at pH 7.4 and incubated at 37°C in the dark for 30 min. Then, 0 nM, 100 nM, 300 nM, 600 nM, 800 nM, 1000 nM, and 1500 nM DOX were added respectively, and incubation was continued in the dark for another 30 min. The fluorescence spectrum of DOX was then measured using a fluorescence spectrometer. Figure 5 As shown, the fluorescence intensity of DOX gradually increases with increasing DOX concentration. This is because the DNA nanocarrier cannot load more DOX at the same time, causing DOX to be released. Therefore, the optimal loading ratio of TBO and DOX on the DNA nanocarrier is 2nM:400nM:300nM = 1:200:150.

[0110] Example 3: Anticancer drug release experiment

[0111] The microplate reader was set to kinetic detection: detection time was 90 min, detection interval was 30 s, detection method was fluorescence intensity detection, detection type was endpoint scan mode, excitation wavelengths of TBO and DOX fluorescence spectra were set to 592 nm and 485 nm, respectively, emission wavelength ranges were set to 620-745 nm and 525-745 nm, respectively, and detection temperature was 37℃.

[0112] DNA nanocarriers and TBO and / or DOX complexes simulate drug release in an acidic and enzymatic environment within cells: 400 nM TBO or 300 nM DOX were incubated with 2 nM DNA nanocarriers in a pH 7.4 buffer solution at 37°C in the dark for 30 min to prepare DNA nanocarrier@TBO complexes and DNA nanocarrier@DOX complexes. Adding 300 nM DOX to the DNA nanocarrier@TBO complex and incubating at 37°C in the dark for another 30 min yielded the DNA nanocarrier@TBO@DOX complex. Adding 8 μL of 0.5 U / μL DNase I nuclease to the DNA nanocarrier@TBO, DNA nanocarrier@DOX, or DNA nanocarrier@TBO@DOX complex, or placing the mixture in a pH 5.0 phosphate buffer solution, caused the nuclease to disrupt the DNA double-strand structure, and the acidic environment of pH 5.0 led to the formation of i-motif structures. Figure 6 As shown, the drug was measured using an enzyme-linked immunosorbent assay (ELISA) reader. Figure 6 a, 6b: TBO release; Figure 6 c, 6d: Fluorescence intensity changes (DOX release). TBO and DOX loaded onto the DNA nanocarrier were stably loaded at pH 7.4 without leakage; in an acidic environment, partial drug release occurred due to i-motif structure formation. Furthermore, nucleases directly accelerated the release of the loaded drug. It was observed that the drug release was fastest under the combined action of acidic conditions and nucleases.

[0113] Example 4: Cancer Cell Targeting Experiment

[0114] The DNA nanocarrier was fixed at 2 nM, with the AS1411 aptamer in the DNA nanocarrier at 80 nM. The DNA nanocarrier was then labeled with the NA-Red nucleic acid probe. The images were observed and photographed using an inverted fluorescence microscope with a phase contrast of Ph2 and a magnification of 40x.

[0115] Targeting ability of DNA nanocarriers on different cell types: LO2 cells and MCF-7 cells were seeded in 96-well plates and incubated for 24 hours. Then, 1 μL of NA-Red-labeled DNA nanocarriers containing the AS1411 aptamer was added, and the cells were incubated at 37°C for 1 hour. Observation using an inverted fluorescence microscope showed that the NA-Red-labeled DNA nanocarriers entered the cells via endocytosis. Figure 7 As shown, the DNA nanocarrier has almost no targeting ability against normal human LO2 cells, but has a highly specific targeting ability against human breast cancer MCF-7 cells.

[0116] Example 5: Intracellular Reactive Oxygen Species Generation Experiment

[0117] The concentration ratio of the immobilized DNA nanocarrier to TBO was 1:200, i.e., the TBO concentration was 400 nM and the DNA nanocarrier concentration was 2 nM. Observation and imaging were performed using an inverted fluorescence microscope with a phase contrast of pH 2 and a magnification of 40x.

[0118] Intracellular reactive oxygen species (ROS) generation capacity: TBO (400 nM) and DNA nanocarrier (2 nM) were vortexed and centrifuged, then incubated at 37°C in the dark for 30 min to obtain the DNA nanocarrier@TBO complex. The DNA nanocarrier@TBO complex (100 μL / well) was added to a 96-well MCF-7 cell culture plate and incubated for another 6 h in a cell culture incubator before being exposed to a 637 nm laser (30 mW / cm²). 2 After irradiation for 5 minutes, continue culturing for 18 hours, and observe using an inverted fluorescence microscope. Figure 8 As shown, in MCF-7 cells, TBO loaded on the DNA nanocarrier still exhibits bright green DCF fluorescence after laser irradiation, indicating that the DNA nanocarrier@TBO complex still maintains a high level of reactive oxygen species generation after entering the cell.

[0119] Example 6: CCK-8 Cytotoxicity Assay

[0120] The concentration ratio of the immobilized DNA nanocarrier to TBO and DOX was 1:200:150. The TBO concentrations were 0.4 μM, 0.8 μM, 1.2 μM, 1.6 μM, and 2 μM; the corresponding DNA nanocarrier concentrations were 0.002 μM, 0.004 μM, 0.006 μM, 0.008 μM, and 0.01 μM; and the corresponding DOX concentrations were 0.3 μM, 0.6 μM, 0.9 μM, 1.2 μM, and 1.5 μM. The microplate reader was used for absorbance detection in endpoint scan mode, with an excitation wavelength of 450 nm and a detection temperature of 37 °C.

[0121] Cytotoxicity assay of DNA nanocarriers with TBO and / or DOX complexes: First, DNA nanocarriers were prepared by binding DNA assemblies with siMCT1 in a 37°C constant-temperature metal bath for 4 h. DNA nanocarriers (without siMCT1) were prepared by binding DNA assemblies with only siMCT1 extended positive strands in a 37°C constant-temperature metal bath for 4 h. Then, different concentrations of TBO and DOX were mixed with the DNA nanocarriers, centrifuged at low speed, and incubated at 37°C in the dark for 30 min to obtain DNA nanocarriers@TBO and DNA nanocarriers@DOX. DOX was added to the DNA nanocarrier@TBO complex, and incubated at 37°C in the dark for 30 min to obtain the DNA nanocarrier@TBO@DOX complex. Different complexes (100 μL / well) were added to 96-well MCF-7 cell culture plates and incubated for 6 h in a cell culture incubator before being subjected to a 637 nm laser (30 mW / cm²). 2 After irradiation for 5 minutes, the cells were cultured for another 18 hours. The absorbance at a wavelength of 450 nm was measured, and the cell inhibition rate was calculated. Figure 9 As shown, DNA nanocarriers enhance cell inhibition rate by improving the targeted delivery efficiency of combined drugs, and siMCT1, which alleviates tumor hypoxia, has a synergistic effect on photodynamic therapy. The combined therapy has a particularly significant anti-cancer effect.

[0122] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A pH- and enzyme-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia, characterized in that: It includes m tandem repeat units, each of which consists of one long single-stranded DNA repeat unit, three complementary short single-stranded DNAs, and n siRNAs, where m is an integer from 10 to 100 and n is an integer from 1 to 3. In the tandem repeat unit, the sequence of the long single-stranded DNA repeat unit is shown in SEQ ID NO.4; the three complementary short single-stranded DNA sequences each include a functional region and a base complement region. The functional region is a pH-responsive nucleic acid complement sequence or a nucleic acid aptamer sequence specifically targeting cancer cells. The base complement region sequence is completely complementary to the long single-stranded DNA repeat unit sequence; siRNA is a gene therapy agent to alleviate tumor hypoxia. The extended sense strand of siRNA includes a 5' extended pH-responsive sequence and a sense strand. The 5' extended pH-responsive sequence is partially complementary to the pH-responsive nucleic acid complement sequence of the complementary short single-stranded DNA; the sense strand and antisense strand of siRNA are completely complementary.

2. The pH and enzyme dual-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia according to claim 1, characterized in that: The pH-responsive nucleic acid complementary sequence of complementary short single-stranded DNA is the first 12 bases from the 5' end of the i-motif complementary sequence.

3. The pH and enzyme dual-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia according to claim 1, characterized in that: Nucleic acid aptamer sequences that specifically target cancer cells with complementary short single-stranded DNA include the AS1411 aptamer sequence, the MUC1 aptamer sequence, the Cyt-C aptamer sequence, and the TD05 aptamer sequence.

4. The pH and enzyme dual-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia according to claim 1, characterized in that: siRNA is an anti-MCT1 siRNA that regulates abnormal tumor metabolism to alleviate tumor hypoxia.

5. The pH and enzyme dual-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia according to claim 1, characterized in that: The 5' end extension of the siRNA positive strand is a pH-responsive sequence rich in cytosine, i-motif.

6. The pH and enzyme dual-responsive targeted DNA nanocarrier for alleviating tumor cell hypoxia according to any one of claims 1-5, characterized in that: The DNA nanocarrier comprises 40 tandem repeat units, each of which includes one long single-stranded DNA repeat unit, three complementary short single-stranded DNA units, and two siRNAs. In the tandem repeat unit, one complementary short single-stranded DNA includes the first 12 bases of the i-motif complementary sequence at the 5' end, as shown in SEQ ID NO:5; another complementary short single-stranded DNA includes the AS1411 aptamer sequence, as shown in SEQ ID NO:6; a third complementary short single-stranded DNA includes the first 12 bases of the i-motif complementary sequence at the 5' end, as shown in SEQ ID NO:7; the siRNA includes an anti-MCT1 siRNA extended sense strand with the i-motif sequence extended at the 5' end, as shown in SEQ ID NO:8; and an anti-MCT1 siRNA antisense strand, as shown in SEQ ID NO:

9.

7. A method for preparing a DNA nanocarrier according to any one of claims 1-6, characterized in that: Includes the following steps: A long single-stranded DNA with m tandem repeat sequences was synthesized by rolling circle amplification reaction; Based on DNA self-assembly technology, the long single-stranded DNA synthesized in step S1 is used as the backbone strand and combined with m*3 complementary short single-stranded DNA strands to obtain a DNA assembly. The sense and antisense strands of siRNA are extended to undergo complementary base pairing to obtain a double-stranded siRNA. The pH-responsive complementary nucleic acid sequence of the DNA assembly is base-paired with the pH-responsive complementary nucleic acid sequence of the siRNA double-strand extension to construct a DNA nanocarrier.

8. The use of the DNA nanocarrier according to any one of claims 1-6 in the preparation of antitumor drugs.

9. A pH- and enzyme-responsive targeted DNA nanocarrier-photosensitizer complex for alleviating tumor cell hypoxia, characterized in that: Includes the DNA nanocarrier as described in any one of claims 1-6, and photosensitizers or photochemotherapy combination drugs.

10. The pH- and enzyme-responsive targeted DNA nanocarrier-photosensitizer complex for alleviating tumor cell hypoxia according to claim 9, characterized in that: When the DNA nanocarrier forms a complex with the photosensitizer, the concentration ratio of the DNA nanocarrier to the photosensitizer is (0.2-5):400; the photosensitizer selected is a phenothiazine photosensitizer, including toluidine blue O, methylene blue and thionine; When the DNA nanocarrier forms a complex with the photochemotherapy combination drug, the photochemotherapy combination drug includes a photosensitizer and a chemotherapeutic drug, and the concentration ratio of the DNA nanocarrier, photosensitizer, and chemotherapeutic drug is (0.2-5):400:300; the photosensitizer is a phenothiazine photosensitizer, including toluidine blue O, methylene blue, and thionine; the chemotherapeutic drug is an anthracycline chemotherapeutic drug, including doxorubicin, daunorubicin, and demethoxydaunorubicin.

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