A mitochondria-targeted nanodevice and its preparation method and application

Abstract

The application belongs to the technical field of biomedicine, and relates to a mitochondrion-targeted nano device and a preparation method and application thereof, the nano device comprising a cascade-activated photodynamic molecular beacon and a mitochondrion-targeted delivery carrier, the cascade-activated photodynamic molecular beacon comprising an APE1 cleavage site and a microRNA recognition region. The cascade-activated photodynamic molecular beacon and the mitochondrion-targeted delivery carrier are self-assembled into a complex through electrostatic adsorption, and cascade activation and signal amplification are realized through the dual response of miR-146a / APE1 which is highly expressed in mitochondria of tumor cells. The application triggers precise and controllable cell pyroptosis through tumor-specific signals, reverses the immune-inhibitory microenvironment, converts the 'cold tumor' into a 'hot tumor', significantly improves the effect of cancer immunotherapy, has good biocompatibility and tumor selectivity, and provides a new strategy and tool for precise treatment of cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to a mitochondrial-targeting nanodevice, its preparation method, and its application. Background Technology

[0002] The immunosuppressive tumor microenvironment leads to low response rates and a high risk of metastasis and recurrence in "cold tumors" to immune checkpoint blockade (ICB) therapy, representing a core bottleneck in clinical treatment. Pyroptosis, a highly immunogenic programmed cell death mechanism, can activate anti-tumor immunity by releasing tumor-associated antigens and pro-inflammatory factors, offering a new direction for overcoming this bottleneck. However, existing pyroptosis inducers lack spatiotemporal controllability and organelle targeting, easily causing off-target damage and limiting their clinical application.

[0003] DNA nanotechnology, with its excellent biocompatibility, programmable structure, and sequence recognition capabilities, has become an ideal platform for building intelligent therapeutic systems. Photodynamic molecular beacons (PMBs), as a combination of DNA nanotechnology and photodynamic therapy (PDT), can improve treatment precision by activating photosensitizers through specific signals. However, traditional PMBs suffer from insufficient signal amplification and a lack of organelle targeting capabilities, making it difficult to achieve efficient induction of tumor cell pyroptosis.

[0004] Mitochondria, as core organelles for cellular energy metabolism and apoptosis regulation, can be targeted with therapies that directly disrupt the energy supply of tumor cells, thereby enhancing therapeutic efficacy. Triphenylphosphine (TPP), a classic mitochondrial-targeting molecule, can achieve specific enrichment through the negative potential of the mitochondrial inner membrane.

[0005] Therefore, constructing mitochondrial-targeted, cascade-activated DNA nanodevices to achieve tumor-specific pyroptosis induction and immune activation has significant clinical value. Summary of the Invention

[0006] In view of the shortcomings of the existing technology, the purpose of this invention is to provide a mitochondrial-targeted nanodevice, its preparation method and application.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides a mitochondrial-targeted nanodevice, the nanodevice comprising a cascade-activated photodynamic molecular beacon and a mitochondrial-targeted delivery carrier, wherein the cascade-activated photodynamic molecular beacon comprises an APE1 cleavage site and a microRNA recognition region.

[0009] To address the issues of low response rates and high off-target toxicity of existing tumor immunotherapy methods, this invention provides a mitochondrial-targeted cascade-activated DNA nanodevice. This device achieves cascade activation through a dual response of tumor-specific microRNA and APE1, triggering precise and controllable pyroptosis via tumor-specific signals. This reverses the immunosuppressive microenvironment, transforming "cold tumors" into "hot tumors," significantly improving the efficacy of cancer immunotherapy. Furthermore, it exhibits good biocompatibility and tumor selectivity, providing a new strategy and tool for precision cancer treatment.

[0010] The cascade activation described in this invention refers to the photodynamic molecular beacon (EMB) relying on the opening and closing of its hairpin structure to achieve activity regulation. In the non-responsive state, the structure remains stable, and methylene blue is closely adjacent to BHQ3, quenching its ability to generate reactive oxygen species (ROS) under light irradiation. After recognizing and binding with miR-146a, APE1 cleaves the AP site to release miR-146a, realizing the cyclic release of methylene blue, which significantly improves the efficiency of photodynamic therapy. This design has been verified by in vitro solution phase fluorescence experiments to demonstrate its response specificity and stability.

[0011] Mitochondrial-targeted nanodevices (EM / NP) T The device consists of a cascade-activated photodynamic molecular beacon (EMB) and a mitochondrial targeting carrier (PLL-TPP). The EMB employs a hairpin design, with an AP site introduced into the loop region, a photosensitizer methylene blue modified at the 5' end, a quencher BHQ3 modified at the 3' end, and a stem containing a sequence complementary to miR-146a. The mitochondrial targeting carrier (PLL-TPP) is obtained by coupling TPP to polylysine (PLL) via an amidation reaction, and can bind to the EMB via electrostatic adsorption, thus endowing the nanodevice with mitochondrial targeting capability. The response characteristics of the EMB to miR-146a and APE1 were verified by in vitro solution phase fluorescence experiments, and the change in fluorescence intensity directly reflects the activation degree of the EMB. The photosensitizer methylene blue can be activated under 640 nm illumination, generating a large amount of singlet oxygen (…). 1 O2) The generation of singlet oxygen is 2.4 times higher than that of traditional photodynamic molecular beacons, which can efficiently induce tumor cell damage.

[0012] DNA nanodevices (EM / NP) in this invention T miR-146a and apurinyl / depyrimidine endonuclease 1 (APE1) were selected as dual triggers for EMB. miR-146a is a microRNA highly expressed in the mitochondria of tumor cells and is a tumor-specific expression marker; APE1 is a base excision repair enzyme widely present in tumor cells and can specifically cleave AP sites. The synergistic effect of the two achieves cascade activation of EMB, ensuring that the nanodevice initiates subsequent reactions only in tumor cells, thereby improving therapeutic specificity.

[0013] Preferably, the mass ratio of the cascaded activated photodynamic molecular beacon to the mitochondrial targeted delivery vector is 1:(1-5).

[0014] For example, the ratios can be 1:1, 1:2, 1:3, 1:4, 1:5, etc. Other specific point values ​​within the above range can also be selected, which will not be elaborated here.

[0015] Preferably, the mass ratio of the cascaded activated photodynamic molecular beacon to the mitochondrial targeted delivery vector is 1:2.

[0016] Preferably, the cascade-activated photodynamic molecular beacon further includes a quenching group and a photosensitizer.

[0017] Preferably, the photosensitizer includes methylene blue.

[0018] Preferably, the quenching group includes BHQ3.

[0019] Preferably, the quenching group and the photosensitizer are located at both ends of the cascade-activated photodynamic molecular beacon.

[0020] Preferably, the nucleotide sequence of the cascade-activated photodynamic molecular beacon includes any one of methoxy, thio, or L-DNA modifications.

[0021] Preferably, the nanodevice is a hairpin structure, and the ring region includes an APE1 cleavage site.

[0022] Preferably, the microRNA includes miR-146a.

[0023] miR-146a is a specific biomarker highly expressed in mitochondria of tumor cells. Its binding specificity to EMB can ensure the tumor targeting of nanodevices and reduce off-target damage to normal cells.

[0024] Preferably, the nucleotide sequence of miR-146a includes the sequence shown in SEQ ID No:1.

[0025] SEQ ID No:1:

[0026] UGAGAACUGAAUUCCAUGGGUU.

[0027] Preferably, the mitochondrial-targeted delivery carrier comprises any one or a combination of at least two of cationic polymers, liposomes, lipid nanoparticles, or cell membrane biomimetic nanocarriers.

[0028] Preferably, the cationic polymer comprises (4-carboxybutyl)triphenylphosphine bromide-polylysine copolymer.

[0029] Preferably, the (4-carboxybutyl)triphenylphosphine bromide-polylysine copolymer is prepared by a method comprising the following steps:

[0030] (1) A solution of (4-carboxybutyl)triphenylphosphine bromide was mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to obtain a first mixture;

[0031] (2) Mix the first mixture with polylysine and ultrafilter to obtain the final product.

[0032] Preferably, the ratio of (4-carboxybutyl)triphenylphosphine bromide, EDC, NHS and polylysine is (0.1-0.5) mmol:(0.1-0.5) mmol:(0.1-1) mmol:(0.25-2) mg.

[0033] The specific point values ​​in (0.1-0.5) can be 0.1, 0.2, 0.3, 0.4, 0.5, etc.; the specific point values ​​in (0.1-1) can be 0.1, 0.2, 0.5, 0.75, 1, etc.; the specific point values ​​in (0.25-2) can be 0.25, 0.5, 1, 1.5, 2, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0034] Preferably, the ratio of (4-carboxybutyl)triphenylphosphine bromide, EDC, NHS and polylysine is 0.2 mmol:0.2 mmol:0.5 mmol:1 mg.

[0035] Preferably, the mixing temperature in step (1) is 40-80 °C and the time is 15-60 min.

[0036] Temperatures can be selected from 40 ℃, 50 ℃, 60 ℃, 70 ℃, 80 ℃, etc., and time can be selected from 15 min, 30 min, 45 min, 60 min, etc. Other specific values ​​within the above range can also be selected, which will not be elaborated here.

[0037] Preferably, the mixing temperature in step (1) is 60 °C and the time is 30 min.

[0038] Preferably, the mixing time in step (2) is 6-48 h, for example, 6 h, 12 h, 24 h, 32 h, 48 h, etc. Other specific point values ​​within the above range can be selected, which will not be elaborated here.

[0039] Preferably, the mixing time in step (2) is 24 h.

[0040] In a second aspect, the present invention provides a method for preparing a mitochondrial-targeted nanodevice according to the first aspect, the method comprising: mixing a mitochondrial-targeted delivery carrier with a cascade-activated photodynamic molecular beacon and incubating it to obtain the nanodevice.

[0041] Preferably, the incubation is carried out in the dark.

[0042] Preferably, the incubation time is 10-120 min, for example, 10 min, 15 min, 30 min, 60 min, 120 min, etc. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0043] Preferably, the incubation time is 30 minutes.

[0044] The assembly effect was characterized by transmission electron microscopy to ensure that the nanodevices had uniform morphology and good dispersion.

[0045] Thirdly, the present invention provides a combination pharmaceutical composition comprising the mitochondrial-targeted nanodevice and the αPD-1 antibody described in the first aspect.

[0046] Fourthly, the present invention relates to the use of a mitochondrial-targeting nanodevice as described in the first aspect or a combined pharmaceutical composition as described in the third aspect in the preparation of a medicament with antitumor efficacy.

[0047] Preferably, the antitumor effects include inducing tumor cell pyroptosis, remodeling the tumor immune microenvironment, or enhancing the therapeutic efficacy of immune checkpoint blockade.

[0048] Preferably, the tumor includes breast cancer or osteosarcoma.

[0049] Targeted delivery: EM / NP T By leveraging the cationic properties of PLL-TPP to form electrostatic adsorption with the negative charge of EMB, and utilizing the mitochondrial targeting capability of TPP, efficient mitochondrial-targeted delivery is achieved, ensuring that nanodevices are precisely enriched in the mitochondria of tumor cells.

[0050] Cascade activation: miR-146a, highly expressed in the mitochondria of tumor cells, binds to EMB, opening the hairpin structure. APE1 cleaves the DNA / RNA hybrid strand, releasing miR-146a, thus achieving the cyclic activation of EMB by miR-146a; under 640 nm light irradiation, activated methylene blue produces a large amount of 1 O2 initiates the photodynamic therapy process.

[0051] 1O2-induced mitochondrial damage triggers pyroptosis in tumor cells. Damage-associated molecular patterns (DAMPs) released during pyroptosis promote dendritic cell (DC) maturation and T cell activation, reshaping the tumor immune microenvironment. Combined with anti-PD-1 antibodies, it can further enhance the anti-tumor immune response and effectively inhibit tumor metastasis and recurrence. It has significant therapeutic effects on various tumor cell types, including MCF-7, 4T1, and 143B, enabling precision treatment of various solid tumors and demonstrating promising clinical application potential.

[0052] The DNA nanodevices (EM / NP) of the present invention T In tumor cells, it can achieve cascade activation through a dual miR-146a / APE1 response, generating a large amount of [unclear - possibly a specific substance or process] under 640 nm light illumination. 1 O2 effectively induces pyroptosis in tumor cells; while in normal cells, due to the low expression level of miR-146a, EMB cannot be effectively activated, thereby effectively reducing off-target toxicity and improving treatment safety.

[0053] Compared with the prior art, the present invention has the following beneficial effects:

[0054] (1) Strong spatiotemporal controllability: This invention adopts a synergistic design of miR-146a / APE1 dual response and photoactivation to achieve mitochondrial-specific pyroptosis induction in tumor cells, which can precisely control the timing and site of action of treatment, significantly reduce off-target toxicity, improve the safety and specificity of treatment, and solve the technical problem of severe off-target damage in traditional photodynamic therapy.

[0055] (2) Significant therapeutic effect: The cascade activation mechanism increases the ROS generation level by 2.4 times compared with the traditional photodynamic molecular beacon, which can efficiently induce tumor cell pyroptosis and significantly inhibit tumor growth. Experimental verification shows that the average tumor weight is reduced by 5.7 times compared with the PBS group, and the therapeutic effect is better than the existing photodynamic therapy technology.

[0056] (3) Outstanding immune regulation ability: Pyroptosis of tumor cells can effectively promote the maturation of DCs (CD80). + CD86 + (Increases the ratio by 2.5 times), enhances the body's anti-tumor immune response; when used in combination with anti-PD-1, it can effectively block tumor lung metastasis and induce the body to produce long-term immune memory, reduce the risk of tumor recurrence, and achieve synergistic effects of "treatment-immunity";

[0057] (4) Good biocompatibility: In vitro experiments show that EM / NP T It exhibits weak toxicity to normal cells; in vivo experiments showed no damage to vital organs such as the heart, liver, spleen, lungs, and kidneys, and no significant abnormalities in blood biochemical indicators. It has excellent biocompatibility and good potential for in vivo application.

[0058] (5) Wide applicability: The DNA nanodevice (EM / NP) provided by this invention T It is effective against a variety of tumor cells, including MCF-7, 4T1, and 143B, and is not limited by tumor cell type. It provides a universal technology platform for the treatment of various solid tumors, has a wide range of applications, and has good clinical translational value. Attached Figure Description

[0059] Figure 1 The fluorescence values ​​are the test results of the test tube solution phase in Example 1.

[0060] Figure 2 The image shows the morphology of the composite formed by electrostatic assembly of the cationic support (PLL-TPP) prepared in Example 2 and EMB. Figure A is a transmission electron microscope image (scale bar = 40 nm), and Figure B is a particle size distribution result.

[0061] Figure 3 This is a confocal fluorescence result image of the mitochondrial targeting ability test in Example 3.

[0062] Figure 4 This is the calculated result of the confocal fluorescence result of the mitochondrial targeting ability test in Example 3.

[0063] Figure 5 This is the result of cytotoxicity in Example 4.

[0064] Figure 6 This is the confocal fluorescence result of live / dead staining in Example 5.

[0065] Figure 7 This is the result of pyroptosis of MCF-7 cells in Example 6.

[0066] Figure 8 These are the results of pyroptosis of 4T1 and 143B cells in Example 6, where Figure A shows the pyroptosis results of 4T1 cells and Figure B shows the pyroptosis results of 143B cells.

[0067] Figure 9 This is the result of in vivo anti-tumor activity in Example 7.

[0068] Figure 10 This is the result of the changes in the proportion of immune cells in the spleen and tumor tissue in the in vivo anti-tumor results of Example 7, where Figure A represents DC and Figure B represents CD4. + T cells, C represents CD8 + T cells, and D represents Treg cells.

[0069] Figure 11 The results are the mouse blood biochemical results from the in vivo anti-tumor results in Example 7.

[0070] Figure 12 This is the result of in vivo anti-lung metastasis in Example 8.

[0071] Figure 13 This is the result of the in vivo tumor volume growth curve in mice after re-challenge in Example 9.

[0072] Figure 14 This is an image showing the results of changes in the proportion of memory T cells in the spleen of mice in vivo during re-challenge in Example 9. Detailed Implementation

[0073] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0074] The sources of the active ingredients in the products involved in the following examples and comparative examples are as follows (only the active ingredients are shown; other necessary excipients contained in commercially available raw materials are not described):

[0075] Opti-MEM was purchased from Gibco;

[0076] APE1 was purchased from New England Biotechnology Co., Ltd., USA;

[0077] The nuclear dye Hoechest 33342, the mitochondrial green fluorescent probe MitoTracker Green, the live / dead cell dye Calcein-AM / PI, and Annexin V-FITC were purchased from Beyotime Biotechnology Co., Ltd.

[0078] CCK8 and PLL were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0079] (4-Carboxybutyl)triphenylphosphine bromide was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0080] EDC and NHS were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0081] Cell lines (HEK293T, MCF-7, 143B, 4T1) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0082] The CY5 fluorescently labeled EMB nucleic acid sequence was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0083] The methylene blue-labeled EMB and miR-146a nucleic acid sequences were purchased from Hippo Biotechnology Co., Ltd.

[0084] Example 1

[0085] Preparation and characterization of EMB

[0086] This experiment completes the design of EMB and verifies the rationality of its sequence design and its response characteristics to miR-146a and APE1.

[0087] (1) EMB sequence design

[0088] Based on the sequence (5'-3') of miR-146a, a hairpin structure sequence containing an AP site was designed, with methylene blue modified at the 5' end and BHQ3 modified at the 3' end. The EMB nucleotide sequence (5'-3') is shown in SEQ ID No:2.

[0089] SEQ ID No:2:

[0090] CGTAACAACCCATGGATTCAGTTCTCAGTTACG.

[0091] The first base (C) at the 5' end and the first base (G) at the 3' end are modified with methoxy groups; the idSp between the 15th base (G) and the 16th base (A) at the 5' end is the AP site. The methoxy group modification can enhance the stability of EMB and prevent it from being degraded by intracellular nucleases.

[0092] (2) Annealing treatment

[0093] The 10 μM EMB sequence was dissolved in annealing buffer (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2), heated at 95 °C for 5 min, and then slowly cooled to room temperature to promote the formation of a stable hairpin structure. It was then stored at 4 °C for later use to ensure the stability of the EMB structure in subsequent experiments.

[0094] (3) Response characteristics verification

[0095] EMB, miR-146a, and APE1 were incubated in a reaction buffer (20 mM Tris-HCl (pH 7.4), 150 mM KCl, 1 mM MgCl2, 2 mM ATP, 0.05 mg / mL BSA) at 37 °C for 1 h to simulate the intracellular physiological environment. Changes in fluorescence intensity were detected using a fluorescence spectrometer. The results are as follows: Figure 1 As shown, the fluorescence intensity is significantly enhanced only when miR-146a and APE1 are present together, confirming that EMB can be activated by the dual response of miR-146a / APE1, and its hairpin structure can open and close normally, meeting the design requirements.

[0096] Example 2

[0097] EM / NP T Assembly and characterization

[0098] This experimental example demonstrates the completion of a DNA nanodevice (EM / NP). T The assembly of the assemblies was performed, and the morphology and assembly effect were verified by characterization.

[0099] (1) Amide reaction

[0100] 0.2 mmol (4-carboxybutyl)triphenylphosphine bromide was dissolved in PBS buffer (pH 7.4), and 0.2 mmol EDC and 0.5 mmol NHS were added. The carboxyl group was activated by stirring at 60 °C for 30 min to prepare for subsequent coupling with PLL. Then, 1 mL of 1 mg / mL PLL solution was added and stirred at room temperature for 24 h to ensure that TPP and PLL were fully coupled, thus preparing the mitochondrial targeting vector PLL-TPP.

[0101] (2) Purification

[0102] Unbound TPP was removed by ultrafiltration using 3 kDa ultrafiltration centrifuge tubes to obtain high-purity PLL-TPP carrier, which was stored at 4 °C for later use to avoid unbound TPP affecting subsequent EM / NP processes. T The assembly effect.

[0103] (3) Assembly

[0104] EMB and PLL-TPP were mixed in 10 mM HEPES buffer at a mass ratio of 1:2 and incubated at room temperature in the dark for 30 min. The cationic properties of PLL were utilized to form electrostatic adsorption with the negative charge of EMB, achieving EM / NP... T Efficient assembly.

[0105] (4) Characterization

[0106] Transmission electron microscopy was used to examine EM / NP T Morphological characterization was performed. The results are as follows: Figure 2 As shown in Figures A and B, uniform spherical particles with an average diameter of 32.5 ± 6.3 nm were observed, confirming the EM / NP pattern. T The assembly was successful, and the morphology was uniform and the dispersion was good, which can meet the needs of subsequent cell and in vivo experiments.

[0107] Example 3

[0108] Mitochondrial Targeting Experiment

[0109] This experimental example validates the DNA nanodevice (EM / NP) T ) intracellular mitochondrial targeting capabilities.

[0110] MCF-7 cells with Cy5-labeled EM / NP TAfter co-incubation for 4 h and washing three times with PBS, MCF-7 cells were incubated with MitoTracker Green (mitochondrial green fluorescent probe) working solution at 37 °C for 30 min, washed three times with PBS, and then incubated with Hoechst 33342 for another 30 min. After washing three times with PBS again, confocal imaging was performed using a Leica SP8 STED 3X camera. Pearson correlation coefficients were then analyzed using ImageJ software with the JACOP plugin to assess colocalization. Results are as follows: Figure 3-4 As shown: EM / NP T After entering the cell, it is highly localized in the mitochondria, and the Pearson correlation coefficient is high, confirming that PLL-TPP can deliver EMB to the mitochondria.

[0111] Example 4

[0112] Cytotoxicity assay

[0113] This experimental example validates the DNA nanodevice (EM / NP) T It has selective killing ability and cytotoxicity against tumor cells.

[0114] MCF-7 or HEK293T cells were used at approximately 1×10⁻⁶ 4 Cells were seeded at a density of cells / well in 96-well plates and cultured overnight. Cells were then incubated with PBS or EM / NP. T Cells were co-incubated with Opti-MEM at concentrations of 0, 100, 200, 300, and 400 nM for 4 h. Cells were then washed with PBS and placed in the dark or irradiated with a 640 nm laser for 10 min (0.2 W / cm²). 2 The cells were cultured in fresh medium for another 24 h, then the medium was replaced with 100 μL of fresh medium containing 10% CCK-8. The absorbance was measured at 450 nm. Results are as follows: Figure 5 As shown, EM / NP T The half-maximal inhibitory concentration (IC50) of MCF-7 cells under 640 nm illumination was significantly lower than that of HEK293T cells, indicating that it can selectively kill tumor cells.

[0115] Example 5

[0116] Live / dead cell assay

[0117] This experimental example visually verifies EM / NP through live / dead cell staining. T Its ability to kill tumor cells under light conditions.

[0118] MCF-7 cells (2×10) 5The cells were seeded in 35 mm confocal culture dishes and cultured overnight until they adhered. The cells were then incubated with PBS containing EnM / NP... T TM / NP T and EM / NP T Cells were incubated with Opti-MEM (DNA concentration: 200 nM) for 4 h. After washing three times with PBS, the cells were irradiated with a 640 nm laser for 10 min (0.2 W / cm²). 2 Then, staining with Calcein-AM and PI for 30 min was performed. Subsequently, confocal imaging was performed using a Leica SP8 STED 3X. Results are as follows... Figure 6 As shown: EM / NP T Under 640 nm illumination, the proportion of red fluorescence (dead cells) in MCF-7 cells was significantly higher than in other groups, indicating that EM / NP T It exhibits strong killing ability against MCF-7 cells under light conditions.

[0119] Among them, EnM / NP T TM / NP T With EM / NP T The only difference is that the nucleotide sequence of EMB is different, EnM / NP T TM / NP T Preparation method and EM / NP T Consistent.

[0120] TM / NP T The nucleotide sequence of EMB in the sample is shown in SEQ ID No:3.

[0121] SEQ ID No:3:

[0122] CGTAACAACCCATGGAATTCAGTTCTCAGTTACG.

[0123] EnM / NP T The nucleotide sequence of EMB in the sample is shown in SEQ ID No:4.

[0124] SEQ ID No:4:

[0125] CGTAACAAGCCATGCATTCAGTTGTGAGTTACG.

[0126] EnM / NP T TM / NP T The modification of EMB is the same as that of EM / NP. TThat is, the 5' end is modified with methylene blue, the 3' end is modified with BHQ3, and the first base (C) at the 5' end and the first base (G) at the 3' end are modified with methoxy groups, TM / NP T Without AP sites, EnM / NP T AP sites are the same as EM / NP T That is, the idSp contained between the 15th base (G) and the 16th base (A) at the 5' end is the AP site.

[0127] Example 6

[0128] Burning Verification

[0129] This experimental example verifies EM / NP. T Under light conditions, it can induce pyroptosis in tumor cells.

[0130] MCF-7, 4T1 and 143B cells (2×10⁻⁶) were used. 5 The cells were seeded separately in 35 mm confocal culture dishes and incubated overnight until they adhered. The cells were then incubated with PBS, EM / NP... T Cells were incubated with Opti-MEM (DNA concentration: 200 nM) for 4 h. After washing three times with PBS, the cells were irradiated with a 640 nm laser for 10 min (0.2 W / cm²). 2 The cells were incubated at 37 °C for 2 h. To assess the morphology of pyroptosis, cells were stained with Hoechst 33342 and Annexin V-FITC, washed with PBS, and then confocally imaged using a Leica SP8 STED 3X microscope. Results are as follows: Figure 7-8 As shown: EM / NP T Under light conditions, MCF-7, 4T1 and 143B tumor cells can be induced to exhibit pyroptosis characteristics such as cell swelling and plasma membrane blistering.

[0131] Example 7

[0132] In vivo verification of anti-tumor effects.

[0133] This experimental example validates the DNA nanodevice (EM / NP) T Its anti-tumor effects and biosafety in vivo.

[0134] (1) Animal model: 6-8 week old female BALB / c mice were subcutaneously inoculated with 4T1 breast cancer cells to establish a tumor-bearing mouse model.

[0135] (2) Treatment plan: When the tumor volume reaches 100 mm 3 Mice were randomly divided into 6 groups (G1: PBS, G2: PBS + light, G3: EM / NP). T G4:EnM / NPT +Lighting, G5:TM / NP T +Light intensity, G6:EM / NP T (+light), intratumoral injection, DNA dose 50 nmol / kg, 4 h after injection, irradiate with 640 nm light for 10 min, once every 3 days, for a total of 3 times.

[0136] (3) Treatment effect: The results are as follows Figure 9 , Figure 10 As shown: EM / NP T + The light-irradiated group showed significant inhibition of tumor growth, with the average tumor weight decreasing by 5.7 times compared to the PBS group; immunological analysis showed that tumor tissue CD4 + CD8 + The proportion of T-cell infiltration doubled, while the proportion of Treg cells decreased from 15.7% to 7.7%.

[0137] (4) Biosafety: Results are as follows Figure 11 As shown, DNA nanodevices (EM / NP) T There was no statistically significant difference in blood biochemical indicators between the device and PBS, indicating that the device has good biosafety.

[0138] Example 8

[0139] Anti-lung metastasis research

[0140] This experimental example validates the DNA nanodevice (EM / NP) T The anti-tumor lung metastasis effect of αPD-1 in combination.

[0141] When the tumor volume reaches approximately 100 mm 3 Mice were randomly divided into 4 groups (PBS, EM / NP) T + Illumination, αPD-1 and EM / NP T +Light +αPD-1). Mice inoculated with 4T1 tumors were injected intratumorally with PBS or EM / NP every 3 days. T (DNA dose: 50 nmol / kg) Three times in total. Four hours after injection, a 640 nm laser at 0.2 W / cm² was used. 2 The tumor was irradiated with a specific intensity for 10 minutes. αPD-1 (5 mg / kg) was administered intraperitoneally for a total of 3 times, every 3 days. On day 9 after the first injection, 1×10⁻⁶ mg / kg was administered intravenously. 6 A lung metastasis model was established using 4T1-Luc cells. Mice were euthanized on day 20, their lungs were removed, and the lung tissue was fixed with Bouin's fixative to observe metastatic nodules. Results are as follows: Figure 12 As shown: EM / NP TThe number of metastatic nodules in the +αPD-1 group was significantly less than in any single treatment group.

[0142] Example 9

[0143] Further Challenge Research

[0144] This experimental example validates the DNA nanodevice (EM / NP) T When used in combination with αPD-1, it can induce long-term immune memory and prevent tumor recurrence.

[0145] A 4T1 tumor model was established on the left side of the mouse back. The tumor volume reached approximately 100 mm. 3 Mice were randomly divided into 4 groups (PBS, EM / NP) T + Illumination, αPD-1 and EM / NP T +Light +αPD-1). Mice inoculated with 4T1 tumors were intraperitoneally injected with PBS or EM / NP every 3 days. T (DNA dose: 50 nmol / kg), injected three times in total. Four hours after injection, a 640 nm wavelength laser at 0.2 W / cm² was used. 2 The tumor was irradiated with an intensity of 10 min. The following day, mice were intraperitoneally injected with αPD-1 (5 mg / kg) three times, every three days. On day 39 after the first injection, 3 × 10⁻⁶ mg / kg of αPD-1 was injected into the tumor. 5 Four T1 cells were subcutaneously seeded into the right back of mice. Tumor volume was then recorded every 4 days. Results are as follows: Figure 13-14 As shown: EM / NP T By powerfully inducing tumor pyroptosis and synergizing with immune checkpoint blockade (ICB), it prevents tumor recurrence by establishing durable systemic anti-tumor immunity.

[0146] In summary, this study constructed a mitochondrial-targeted, cascade-activated DNA nanodevice (EM / NP). TThe nanodevice comprises a photodynamic molecular beacon (EMB) module and a mitochondrial targeting vector (PLL-TPP) module. The EMB module contains an AP site and uses methylene blue as a photosensitizer and BHQ3 as a quencher, achieving cascade amplification through a dual response of miR-146a / APE1, which is highly expressed in tumor cell mitochondria. The PLL-TPP vector is synthesized via an amidation reaction and achieves specific targeting through the electrostatic attraction between the TPP group and the mitochondrial membrane potential. Upon entering tumor cells, the EMB specifically binds to miR-146a to form a heteroduplex. APE1 cleaves the AP site to release miR-146a, achieving cyclic activation. Under light, the activated photosensitizer efficiently generates ROS, inducing mitochondrial damage and triggering pyroptosis, promoting DC maturation and T lymphocyte activation, and reversing the tumor immunosuppressive microenvironment. In vitro and in vivo experiments have validated that this nanodevice can specifically kill various tumor cells. Combined with anti-PD-1 antibodies, it significantly enhances anti-tumor immune memory, effectively inhibiting tumor growth, metastasis, and recurrence, while exhibiting minimal toxicity to normal tissues. This study constructed a synergistic therapeutic system relying on tumor-specific molecular triggering and mitochondrial-targeted photodynamic therapy. This system possesses advantages such as precise targeting, spatiotemporal controllability, significant immune activation effects, and good biocompatibility, providing a new strategy and tool for precision immunotherapy in cancer. It is applicable to the treatment of malignant tumors and the improvement of immunotherapy efficacy.

[0147] The above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A mitochondrial-targeting nanodevice, characterized in that, The nanodevice comprises a cascade-activated photodynamic molecular beacon and a mitochondrial-targeted delivery vector, wherein the cascade-activated photodynamic molecular beacon includes an APE1 cleavage site and a microRNA recognition region.

2. The mitochondrial-targeted nanodevice according to claim 1, characterized in that, The mass ratio of the cascaded activated photodynamic molecular beacon to the mitochondrial targeted delivery vector is 1:(1-5).

3. The mitochondrial-targeted nanodevice according to claim 1 or 2, characterized in that, The cascade-activated photodynamic molecular beacon also includes a quenching group and a photosensitizer; Preferably, the photosensitizer includes methylene blue; Preferably, the quenching group includes BHQ3; Preferably, the quenching group and the photosensitizer are located at both ends of the cascade-activated photodynamic molecular beacon.

4. The mitochondrial-targeted nanodevice according to any one of claims 1-3, characterized in that, The nucleotide sequence of the cascade-activated photodynamic molecular beacon includes any one of methoxy, thio, or L-DNA modifications; Preferably, the nanodevice is a hairpin structure, and the ring region includes an APE1 cleavage site; Preferably, the microRNA includes miR-146a; Preferably, the nucleotide sequence of miR-146a includes the sequence shown in SEQ ID No:

1.

5. The mitochondrial-targeted nanodevice according to any one of claims 1-4, characterized in that, The mitochondrial targeted delivery carrier includes any one or a combination of at least two of cationic polymers, liposomes, lipid nanoparticles, or cell membrane biomimetic nanocarriers. Preferably, the cationic polymer comprises (4-carboxybutyl)triphenylphosphine bromide-polylysine copolymer; Preferably, the (4-carboxybutyl)triphenylphosphine bromide-polylysine copolymer is prepared by a method comprising the following steps: (1) A solution of (4-carboxybutyl)triphenylphosphine bromide was mixed with EDC and NHS to obtain a first mixture; (2) Mix the first mixture with polylysine and ultrafilter to obtain the final product; Preferably, the ratio of (4-carboxybutyl)triphenylphosphine bromide, EDC, NHS and polylysine is (0.1-0.5) mmol:(0.1-0.5) mmol:(0.1-1) mmol:(0.25-2) mg; Preferably, the mixing temperature in step (1) is 40-80℃ and the time is 15-60 min; Preferably, the mixing time in step (2) is 6-48 h.

6. The method for fabricating mitochondrial-targeted nanodevices according to any one of claims 1-5, characterized in that, The preparation method includes: mixing a mitochondrial targeted delivery vector with a cascade-activated photodynamic molecular beacon and incubating it to obtain the final product.

7. The preparation method according to claim 6, characterized in that, The incubation was carried out in the dark. Preferably, the incubation time is 10-120 min.

8. A combination pharmaceutical composition, characterized in that, The combined pharmaceutical composition comprises the mitochondrial-targeted nanodevice and αPD-1 antibody as described in any one of claims 1-5.

9. The use of the mitochondrial-targeting nanodevice according to any one of claims 1-5 or the combined pharmaceutical composition according to claim 8 in the preparation of a drug with antitumor efficacy.

10. The application according to claim 9, characterized in that, The anti-tumor effects include inducing tumor cell pyroptosis, remodeling the tumor immune microenvironment, or enhancing the efficacy of immune checkpoint blockade therapy. Preferably, the tumor includes breast cancer or osteosarcoma.

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