Lysosome nitric oxide preparation as well as preparation method and application thereof

Abstract

The invention discloses a lysosome nitric oxide preparation as well as a preparation method and application thereof. The preparation method comprises the following steps: 1) preparing tetrahedral framework nucleic acid with a cell lysosome targeting capability and a hydrophobic microenvironment; 2) providing a hydrophobic molecule capable of controllably releasing nitric oxide as a nitric oxide donor; and (3) enabling the controllable release nitric oxide donor to enter an internal hydrophobic microenvironment of the tetrahedral framework nucleic acid under vortex oscillation, so as to prepare the lysosomal nitric oxide preparation. The lysosome nitric oxide preparation prepared by the method has good lysosome targeting ability and nitric oxide controllable release ability, the lysosome membrane permeability of tumor cells can be influenced by regulating and controlling nitric oxide release, then cytotoxicity is generated, and powerful technical support is provided for developing a novel tumor treatment strategy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more specifically, to a lysosomal nitric oxide controlled release preparation and a preparation method thereof, as well as an application of achieving precise and controlled release of nitric oxide in tumor cell lysosomes. Background Art

[0002] Nitric oxide plays a key role in tumor growth, migration and invasion. Its biological effects are obviously dose-dependent. Low concentrations of nitric oxide can promote tumor proliferation and angiogenesis, while high concentrations of nitric oxide can effectively inhibit tumor cell proliferation and kill tumor cells by inducing cell apoptosis or necrosis. Therefore, in tumor treatment, how to accurately regulate the release concentration and action site of nitric oxide has become one of the research hotspots of tumor targeted therapy.

[0003] Lysosomes of cells are involved in a variety of physiological and pathological processes. Abnormal components in lysosomes may lead to imbalance in cell homeostasis. Therefore, regulating lysosomal function has important therapeutic potential. Studies have shown that lysosomes are important sites of action for nitric oxide, and their activity is affected by changes in nitric oxide concentration. By regulating the spatiotemporal release of nitric oxide in lysosomes, the permeability of lysosomal membranes can be effectively regulated, thereby inhibiting tumor cell growth and inducing their death. However, current nitric oxide gas therapy mainly works through passive release at the tumor site, and it is difficult to achieve precise release and dynamic regulation of nitric oxide in lysosomes, which limits its therapeutic effect.

[0004] Framework nucleic acids show high reproducibility, precise addressability and good biocompatibility, making them a highly promising biomaterial. Among them, tetrahedral framework nucleic acids can efficiently enter cells through endocytosis and accurately locate to lysosomes through microtubule-dependent transport mechanisms. These unique properties provide an ideal carrier for achieving spatiotemporal controllable release of nitric oxide in lysosomes, providing a new solution for improving the killing effect of tumor cells. Summary of the invention

[0005] The purpose of the present invention is to provide a lysosomal nitric oxide preparation and a preparation method thereof, as well as an application for the precise release of lysosomal nitric oxide in tumor cells, thereby solving the problems of insufficient targeting of lysosomes of nitric oxide donor cells in the prior art and the inability to achieve point-specific controlled release of nitric oxide.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] According to a first aspect of the present invention, a method for preparing a lysosomal nitric oxide preparation is provided, comprising the following steps: 1) preparing a tetrahedral framework nucleic acid having a cell lysosome targeting capability and an internal hydrophobic microenvironment; 2) providing a hydrophobic molecule capable of controllably releasing nitric oxide as a nitric oxide donor; and 3) allowing the controllably released nitric oxide donor to enter the internal hydrophobic microenvironment of the tetrahedral framework nucleic acid under the action of vortex oscillation, thereby obtaining a lysosomal nitric oxide preparation.

[0008] In step 1), the tetrahedral framework nucleic acid having a hydrophobic microenvironment and having a cell lysosome targeting capability, the hydrophobic microenvironment is present inside the tetrahedral framework nucleic acid. After the tetrahedral framework nucleic acid having a hydrophobic microenvironment is taken up by the cell, it moves along the microtubule to the lysosome and fuses with it through the caveolin-mediated endocytosis pathway, and is finally located in the lysosome. Therefore, the tetrahedral framework nucleic acid prepared according to the present invention has the ability to target the cell lysosome.

[0009] Preferably, in step 1), the tetrahedral framework nucleic acid is composed of four DNA single strands, and the hydrophobic group can be modified at the 5' end or 3' end of each DNA single strand. By designing different DNA single strand sequences, the opening position of the DNA double strand formed during the synthesis of the tetrahedral framework nucleic acid can be controlled, so that the hydrophobic group is modified at different positions on the edge.

[0010] It should be understood that the four DNA chains with hydrophobic alkyl chains at the ends are thermally annealed to form a tetrahedral framework nucleic acid, and these hydrophobic groups are directed toward the interior of the structure to form an internal hydrophobic microenvironment of the tetrahedral framework nucleic acid. It should be understood that the hydrophobic alkyl chains in the embodiments are not limited thereto, and alkyl alcohols, cholesterol, and polycyclic aromatic hydrocarbon structures are all applicable to the present invention.

[0011] In step 2), the controllable release modes include: light stimulation response, pH response and magnetic field response.

[0012] Preferably, in step 2), the controlled release of nitric oxide is a light-stimulated response release, and the amount of nitric oxide released can be controlled by regulating the illumination time to achieve controlled release of nitric oxide.

[0013] Preferably, in step 2), the nitric oxide donor BDOH-NO capable of releasing has an excitation wavelength of 555 nm and an emission wavelength of 570 nm. It should be understood that the donor is only used as an example of a preferred embodiment and is not intended to be limiting. In fact, any nitric oxide donor having an aromatic nitrosamine structure is suitable for the present invention. The nitric oxide donor includes: BDOH-NO (chemical full name: (E)-N-(4-(2-(5,5-difluoro-10-(4-hydroxyphenyl)-1,7,9-trimethy1-5H-4λ 4 ,5λ 4 -dipyrr olo[1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)vinyl)-2-methoxyphenyl)-N-methoxylnitr ous amide) and IBDOH-NO (the full chemical name is (E)-N-(4-(2-(5,5-difluoro-10-(4-hydroxyphenyl)-2,8-diiodo-1,7,9-trimethyl-5H-4λ) 4 ,5λ 4 -dipyrrolo [1,2-c:2',1'-f][1,3,2]diazaborinin-3-yl)vinyl)-2-methoxyphenyl)-N-methoxylnitrous amide) and other hydrophobic molecules containing aromatic N-nitrosamine structures.

[0014] It should be understood that the nitric oxide donor is not necessarily an aromatic nitrosamine structure, and also includes nitrates, diazonium diol salts and S-nitrosothiols, etc., but the way they release NO is different. Other structures require pH hydrolysis, thermal decomposition, etc., but the aromatic nitrosamine structure has a good photolysis efficiency. The principle is that when the aromatic nitrosamine is irradiated with light, the molecule absorbs light energy, causing the electrons of the nitrosamine group to transition to an excited state. In the excited state, the NN bond of the aromatic nitrosamine is easily broken, thereby releasing nitric oxide. Therefore, the present invention preferably has an aromatic nitrosamine structure. Nitric oxide donors.

[0015] Preferably, in step 3), the tetrahedral framework nucleic acid and the nitric oxide donor are mixed in a molar ratio of 1:10 to 1:500.

[0016] According to a second aspect of the present invention, a lysosomal nitric oxide preparation prepared according to the above-mentioned preparation method is provided, wherein the lysosomal nitric oxide preparation is prepared by encapsulating a nitric oxide donor in the internal hydrophobic microenvironment of a tetrahedral framework nucleic acid under the drive of hydrophilic and hydrophobic interactions, wherein the lysosomal nitric oxide preparation has lysosomal targeting capability and can achieve controllable release of nitric oxide in lysosomes through light regulation.

[0017] According to a third aspect of the present invention, a lysosomal nitric oxide preparation is provided for achieving precise and controllable release of lysosomal nitric oxide in tumor cells, thereby killing tumor cells. After being taken up by cells, the lysosomal nitric oxide preparation is retained in the lysosomes of tumor cells, and the controllably released nitric oxide accumulates in the lysosomes, thereby affecting the permeability of the lysosomal membrane of tumor cells, thereby generating cytotoxicity and exerting a good tumor cell killing effect.

[0018] It should be understood that in the prior art, the release of nitric oxide preparations in tumor cells can achieve a therapeutic effect by triggering the release of nitric oxide through light, heat, acidic tumor environment or enzyme activity, but the existing nitric oxide preparations have low biocompatibility and leakage risks, and it is difficult to achieve the precise release of nitric oxide in lysosomes.

[0019] However, the lysosomal nitric oxide preparation provided according to the present invention has good lysosomal targeting, and the nitric oxide donor therein can control the release amount of nitric oxide in a variety of ways (such as regulating the illumination time), so the present invention can achieve accurate and controllable release of nitric oxide in lysosomes of tumor cells, and then produce cytotoxicity by affecting the permeability of lysosomal membranes, and provide technical support for the development of new tumor treatment strategies. Although before this, the team of the present invention has improved the performance of probes in tumor imaging by preparing near-infrared fluorescent tetrahedral probes and tetrahedral-near-infrared quantum dot probes, these technologies are mainly focused on tumor imaging and targeted imaging, and do not involve treatment mechanisms. The present invention realizes for the first time that nitric oxide preparations are encapsulated in tetrahedral framework nucleic acids through hydrophilic and hydrophobic effects, and the lysosomal membrane permeability of tumor cells is affected by regulating the release of nitric oxide, thereby producing cytotoxicity and killing tumor cells, thereby achieving tumor treatment. Therefore, the prior art has never disclosed this scheme or given similar technical inspiration.

[0020] The inventive points of the present invention mainly lie in the following three aspects: 1) Lysosomal targeting: The present invention realizes the precise release of nitric oxide in lysosomes for the first time through the lysosomal targeting ability of tetrahedral framework nucleic acids; 2) Nitric oxide release: The present invention releases nitric oxide through light stimulation response, pH response or magnetic field response, and further realizes the controllable release of nitric oxide; 3) Therapeutic mechanism: The present invention regulates the release of nitric oxide in lysosomes, affects the permeability of lysosomal membranes, thereby inhibiting the growth of tumor cells and inducing their death, providing a new strategy for tumor treatment.

[0021] The effect of the present invention relative to the existing technical progress is that the lysosomal nitric oxide preparation provided by the present invention has good lysosomal targeting ability and precise controllable release of nitric oxide. In practical applications, the lysosomal membrane permeability of tumor cells can be affected by regulating the release of nitric oxide, thereby producing cytotoxicity. Therefore, the present preparation can well achieve precise controllable release of nitric oxide in lysosomes, and has the potential to solve the problem of precise controllable release of nitric oxide in lysosomes of tumor cells.

[0022] In summary, according to a lysosomal nitric oxide preparation and a preparation method thereof, as well as an application of the precise release of lysosomal nitric oxide in tumor cells provided by the present invention, the precise and controllable release of nitric oxide in lysosomes is achieved by encapsulating the nitric oxide donor in a tetrahedral framework nucleic acid, and the preparation method has good lysosomal targeting and biocompatibility. The present invention provides strong technical support for the development of new tumor treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1A The absorption spectra of BDOH-NO and TDF-NO in Example 1 and the successful encapsulation of BDOH-NO verified by agarose are shown;

[0024] Figure 1B The atomic force microscope scanning result of TDF-NO in Example 1 is shown;

[0025] Figure 2 The successful encapsulation of BDOH-NO and IBDOH-NO by TDF was verified by agarose electrophoresis in Example 2;

[0026] Figure 3 The quantitative statistical results of the co-localization efficiency of BDOH-NO and TDF-NO with 4T1 cell lysosomes in Example 3 are shown;

[0027] Figure 4 The stability of TDF-NO in an acidic environment was verified by agarose electrophoresis in Example 4;

[0028] Figure 5The quantitative statistical results of the concentration of nitric oxide in lysosomes of 4T1 cells at different illumination times after TDF-NO treatment in Example 5 are shown;

[0029] Figure 6 The figure shows the changes in the lysosomal membrane permeability of 4T1 cells at different illumination times after TDF-NO treatment in Example 6;

[0030] Figure 7 The cytotoxicity data of Example 7 under different illumination times after TDF-I-NO treatment are shown. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods for which specific conditions are not specified in the following examples are carried out according to conventional methods and conditions, or are selected according to the product specifications.

[0032] In the following specific embodiments, the present invention uses BDOH-NO and IBDOH-NO as the main nitric oxide donors, and uses tetrahedral framework nucleic acids with hydrophobic alkyl chains to encapsulate the nitric oxide donors, thereby giving them lysosomal targeting capabilities and constructing lysosomal nitric oxide preparations. The preparations are applied to tumor treatment to achieve accurate and controllable release of nitric oxide in lysosomes. The following examples specifically illustrate the implementation effects of the present invention, in which 4T1 breast cancer cells are selected as tumor cell models.

[0033] Example 1 Preparation and Characterization of Lysosomal Nitric Oxide Preparations

[0034] Synthesis of tetrahedral framework nucleic acid (TDF): Four single-stranded DNAs accurately quantified by UV spectrophotometer were mixed in an equimolar ratio in Tris-MgCl2 buffer (10mM tris, 5mM MgCl2, pH=8.0) to make the final concentration of each single strand 1μM. The mixed solution was heated to 95℃ for 15 minutes, and then quickly cooled to 4℃ and maintained for 15 minutes to obtain the TDF structure.

[0035] Preparation of lysosomal nitric oxide preparation (TDF-NO): TDF and nitric oxide donor were thoroughly mixed at a molar ratio of 1:100, vortexed for 10 minutes, and then excess nitric oxide donor was removed by Nap-5 chromatography column to obtain TDF-NO.

[0036] The successful encapsulation of the dye was verified by agarose gel electrophoresis (1%, mass fraction). The sample was electrophoresed at 100V for 30 minutes in 1×Tris-acetate-EDTA buffer (4mM tris, 2mM MgAc2, 0.2mM EDTA, pH=8.0), and then the gel was imaged using the Syngene gel imaging system to evaluate the encapsulation effect. The absorption spectrum of TDF-NO was characterized by UV-visible spectrophotometer. The structure was characterized by atomic force microscopy to observe its morphological characteristics.

[0037] The single-stranded DNA sequences (SEQ ID No. 1-4) used in TDF are shown in Table 1 below.

[0038] Table 1 Single-stranded DNA sequences used in TDF

[0039] D is a modified hydrophobic alkyl chain with the following structural formula:

[0040] Results: Figure 1A The results of agarose gel electrophoresis showed that under the fluorescent gel illuminator, BDOH-NO had no obvious bands and was mainly blocked in the gel pores; while TDF-NO also had a small amount of residue in the gel pores, but an obvious band could be observed below the gel, and this band co-localized with the DNA band of TDF, indicating that TDF could successfully encapsulate BDOH-NO, thus proving the successful synthesis of TDF-NO. The results of the UV-visible spectrophotometer showed that the absorption peak of TDF-NO was at 555nm, which was consistent with the absorption peak of BDOH-NO, further verifying the successful encapsulation of BDOH-NO ( Figure 1A ). The results of atomic force microscopy showed that TDF-NO exhibited a tetrahedral structure of about 9 nm in size, and its size and morphology were consistent with TDF. This result showed that the encapsulation of BDOH-NO did not affect the morphology and size of TDF, further supporting the successful synthesis and structural integrity of TDF-NO ( Figure 1B ).

[0041] Example 2 Encapsulation of different nitric oxide donors by tetrahedral framework nucleic acids

[0042] Two nitric oxide donors, BDOH-NO and IBDOH-NO, were selected and encapsulated with TDF with an internal hydrophobic microenvironment to verify the universality of TDF for encapsulating nitric oxide donors. The synthesis of TDF and the encapsulation method of nitric oxide donors were the same as in Example 1, and agarose gel electrophoresis and gel imaging were the same as in Example 1.

[0043] Results: Figure 2Both nitric oxide donors showed obvious bands under the fluorescent illuminator, and the bands in the fluorescent channel co-localized with the DNA bands, indicating that both nitric oxide donors can be encapsulated in the hydrophobic microenvironment of TDF, proving that TDF carrying a hydrophobic environment has the universality of loading nitric oxide donors.

[0044] Example 3 Comparison of Lysosomal Targeting Ability of Lysosomal Nitric Oxide Preparations and Free Nitric Oxide Donors

[0045] Cell culture. 4T1 cells were purchased from the cell bank of the Typical Culture Collection Committee of the Chinese Academy of Sciences. RPMI1640 medium (containing 10% FBS, 100 units / mL penicillin, 100 μg / mL streptomycin, and 2 mM glutamine) was cultured in a constant temperature and humidity cell culture incubator at 37°C and 5% CO2. After the cells adhered to the wall, TDF-NO and free BDOH-NO were incubated with the cells for 2 hours, respectively, and then lysosomal probes were loaded and the cells were imaged using a laser confocal microscope (Leica sp8) to observe the lysosomal co-localization of TDF-NO and free BDOH-NO in 4T1 cells. The excitation wavelength of BDOH-NO was set to 561 nm, the acceptance wavelength range was set to 570 nm-600 nm, the excitation wavelength of the lysosomal probe was set to 488 nm, and the acceptance wavelength range was set to 510 nm-530 nm.

[0046] Results: Figure 3 The statistical results of laser confocal imaging showed that TDF-NO and free BDOH-NO co-localized with cellular lysosomes after incubation with 4T1 cells for 2 hours, but the co-localization efficiency of TDF-NO with lysosomes was significantly higher than that of free BDOH-NO.

[0047] Example 4 Evaluation of the stability of lysosomal nitric oxide preparations in an acidic environment

[0048] Since the lysosome presents an acidic environment, in order to evaluate the stability of TDF-NO under acidic conditions, it was incubated with PBS buffer at pH = 5 for 0, 1, 2, 4, 6, 12 and 24 hours, and then analyzed by agarose gel electrophoresis. The agarose gel electrophoresis and gel imaging methods are the same as those in Example 1. The structural stability of TDF-NO in an acidic environment was determined by observing the changes in the bands.

[0049] Results: Figure 4 As shown, the DNA band of TDF-NO remained almost unchanged from 0 to 24 hours, indicating that it still had good structural stability under acidic conditions.

[0050] Example 5 Detection of Controllable Release of Nitric Oxide by Lysosomal Nitric Oxide Preparation in Tumor Cells

[0051] The 4T1 cell culture method was the same as in Example 3. After the cells adhered, TDF-NO was added and incubated with the cells for 2 hours, and then replaced with fresh culture medium. Subsequently, nitric oxide detection probe was added and incubated with the cells for 30 minutes. -2 Under white light irradiation conditions, the cells were exposed for 5, 10, and 15 minutes, respectively. After irradiation, the cells were imaged by laser confocal microscopy, and the fluorescence signal intensity of individual cells was counted using image J to evaluate the ability of TDF-NO to achieve controlled release of nitric oxide in tumor cells under white light exposure. In the experiment, the excitation wavelength of the nitric oxide detection probe was set to 488nm, and the receiving wavelength range was set to 510nm-530nm.

[0052] Results: After light treatment, the nitric oxide detection probe produced obvious fluorescence signals in the cells. The fluorescence intensity analysis results showed that after 5, 10 and 15 minutes of white light exposure, the fluorescence intensity in the cells was 2.1, 8.5 and 22.0, respectively, showing a time-dependent ( Figure 5 ). The results showed that as the white light irradiation time increased, the concentration of nitric oxide in the cell lysosome increased significantly. This result proves that TDF-NO can achieve controllable release of nitric oxide in cells by regulating the duration of light irradiation.

[0053] Example 6 Effect of controlled release of lysosomal nitric oxide preparation on lysosomal membrane permeability

[0054] Acridine orange staining is used to assess lysosomal membrane permeability. When the lysosomal membrane is intact, acridine orange exhibits red fluorescence in the acidic environment of the lysosome. When the lysosomal membrane permeability increases, acridine orange dye leaks into the cytoplasm and escapes from the acidic environment, exhibiting green fluorescence.

[0055] The specific operation is as follows: 4T1 cells were plated at 3.0×10 5 The cells were seeded in a 12-well plate at a density of 100 cells / cm. After the cells attached to the wall, TDF-NO was added and incubated with the cells for 2 h. The culture medium was then replaced with fresh medium and incubated at 10 mW cm -2 White light was irradiated for 5, 10 and 15 minutes. 1.0×10 6 After washing with PBS, acridine orange staining solution was added and incubated in an incubator in the dark for 15 minutes. The cells were then washed with PBS and resuspended, and detected by a fluorescence spectrophotometer with an excitation wavelength of 490 nm and an emission wavelength of 528 nm to evaluate the changes in the green fluorescence signal of the cells.

[0056] Results: After 5, 10, and 15 minutes of light treatment, the green fluorescence intensity was 1.18×10 6 , 1.27×10 6 and 1.36×106 , indicating that acridine orange was redistributed from lysosomes to the cytoplasm, indicating that the permeability of the lysosomal membrane increased, and the lysosomal permeability in 4T1 cells increased with the extension of illumination time ( Figure 6 ), these results indicate that TDF-NO can effectively promote the permeability changes of lysosomal membrane under light conditions.

[0057] Example 7 Evaluation of the Killing Effect of Lysosomal Nitric Oxide Preparation Controlled Release on Tumor Cells

[0058] 4T1 cells were plated at 1.0 × 10 5 The cells were seeded in a 24-well plate at a density of 100 cells / cm. After the cells attached to the wall, TDF-I-NO was incubated with 4T1 cells for 2 h. The cells were then repeatedly washed with PBS to remove the unbound agent, and fresh cell culture medium was added to each well. -2 The cells were irradiated with white light for 5, 10 and 15 minutes, respectively. After the illumination, they were cultured in the incubator in the dark for 24 hours. After the treatment, the cytotoxicity of the preparation was evaluated using the 3-(4,5-dimethylthiazol-2)-2,5-diphenyltetrazolium bromide (MTT) colorimetric method. First, after washing with PBS, 500 μL of culture medium containing 0.5% MTT was added to each well and cultured for 4 hours. Then, 500 μL of triple dissolution solution was added to each well and allowed to stand at 37°C for 4 hours. Finally, the absorbance was measured at 570 nm using a microplate reader, and the cell viability was calculated to evaluate the cytotoxicity of TDF-I-NO to 4T1 cells under different light treatment conditions.

[0059] Results: Figure 7 After 5, 10 and 15 minutes of white light irradiation, the cell survival rates were 21.2%, 6.2% and 3.9%, respectively, indicating that the killing effect gradually increased with the extension of illumination time. The results proved that TDF-I-NO has a good tumor cell killing effect.

[0060] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiments of the present invention can also be modified in various ways. All simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A method for preparing a lysosomal nitric oxide preparation, characterized in that: The following steps are involved: 1) Modifying the hydrophobic groups in the tetrahedral framework nucleic acid by covalent bonds to prepare a tetrahedral framework nucleic acid with lysosomal targeting capability and a hydrophobic microenvironment; 2) providing a hydrophobic molecule capable of controllably releasing nitric oxide as a nitric oxide donor; as well as 3) The tetrahedral framework nucleic acid and the nitric oxide donor are fully mixed, so that the controllably released nitric oxide donor enters the internal hydrophobic microenvironment of the tetrahedral framework nucleic acid under vortex oscillation, thereby preparing a lysosomal nitric oxide preparation.

2. The preparation method according to claim 1, characterized in that: In step 1), the tetrahedral framework nucleic acid containing a specific hydrophobic microenvironment is formed by thermal annealing of four DNA chains whose ends are modified with hydrophobic groups, and when the tetrahedral framework nucleic acid is formed, these hydrophobic groups are collectively oriented toward the interior of the structure to form a hydrophobic microenvironment.

3. The preparation method according to claim 2, characterized in that: The hydrophobic groups include: alkyl alcohols {HO-(CH2) x -} y -, cholesterol, polycyclic aromatic hydrocarbon structures, where x is the length of the alkyl chain, 1 < x < 100, and y is the number of alkyl chains, 1 < y < 100.

4. The preparation method according to claim 1, characterized in that: In step 2), the nitric oxide donor is a nitric oxide donor having an aromatic nitrosamine structure, including BDOH-NO and IBDOH-NO.

5. The preparation method according to claim 1, characterized in that: In step 2), the controllable release modes include: light stimulation response, pH response and magnetic field response.

6. The preparation method according to claim 1, characterized in that: In step 3), the tetrahedral framework nucleic acid and the nitric oxide donor are mixed at a molar ratio of 1:10 to 1:

500.

7. The preparation method according to claim 1, characterized in that: In step 3), the lysosomal nitric oxide preparation has the ability to target cell lysosomes.

8. A lysosomal nitric oxide preparation prepared according to the preparation method according to any one of claims 1 to 7, characterized in that: The lysosomal nitric oxide preparation is prepared by using a tetrahedral framework nucleic acid with an internal hydrophobic microenvironment, and driving the effective aggregation of nitric oxide donors through hydrophilic-hydrophobic interactions, wherein the nitric oxide donors have both hydrophobicity and the ability to controllably release nitric oxide.

9. Use of the lysosomal nitric oxide preparation according to claim 8 to achieve precise and controllable release of nitric oxide in lysosomes in tumor cells.

10. The use according to claim 9, characterized in that: After being taken up by cells, lysosomal nitric oxide preparations are retained in the lysosomes of tumor cells. The controllably released nitric oxide accumulates in the lysosomes, thereby affecting the permeability of the lysosomal membrane of tumor cells, thereby generating cytotoxicity and exerting a good tumor cell killing effect.

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