Mitochondria-targeted photoresponse nano agonist as well as preparation method and application thereof

Abstract

The invention relates to a mitochondria-targeted photoresponse nano agonist and a preparation method and application thereof.The method comprises the following steps that gold nanoparticle engineered hollow mesoporous silica is obtained in an in-situ growth mode, then the gold nanoparticle engineered hollow mesoporous silica and a mitochondria-targeted type 1 photosensitizer are subjected to ultrasonic dispersion to be uniform, stirring is conducted in a dark place, and the mitochondria-targeted photoresponse nano agonist is obtained; centrifuging and washing to obtain the gold nanoparticle engineered hollow mesoporous silica (HAB) loaded with the mitochondrion-targeting type 1 photosensitizer; in a dark stirring state, dispersing the HAB into the deionized water, and then dropwise adding into the hyaluronic acid solution to obtain the nano agonist. The nano agonist is applied to a light immunotherapy drug, and is used for accurate light-controlled mitochondrial stress and non-agonist-dependent cGAS-STING pathway activation. The nano agonist obtained by the method integrates a plurality of unique attributes, activates an STING signal channel in a cascade amplification manner to enhance immunity, has good safety, and can effectively induce a powerful anti-tumor immune response.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a mitochondrial-targeted light-responsive nano agonist and a preparation method and application thereof. Background Art

[0002] Currently, researchers activate the cGAS-STING pathway by activating cyclic interferon cGAM-AMP synthase, thereby triggering anti-tumor immune activation, which is an important innate immune activation pathway in cancer immunity. Activation of the cGAS-STING pathway can upregulate the secretion of type I interferon (IFN) and proinflammatory cytokines, thereby inducing dendritic cell (DC) maturation, triggering host immune response, and enhancing therapeutic effects. Typically, STING agonists such as natural cyclic dinucleotides (CDNs) and small molecules are designed as nanoplatforms to overcome their shortcomings of metabolic instability and insufficient bioavailability. However, this mainstream strategy still has the risk of dose-dependency, which may lead to unnecessary activation of non-target cells due to overexposure or uncontrolled agonist leakage. The latest studies have shown that the use of drugs to precisely destroy mitochondria can more effectively induce structural and functional disorders of mitochondria, leading to mitochondrial stress. During stress, mitochondria choose to repair damaged DNA, degrade damaged proteins and lipids, or perform mitochondrial unfolded protein responses to maintain their structural and functional integrity. When these rescue measures fail, mitochondria release contents including mitochondrial DNA (mtDNA) into the cytoplasm, which may cause damage to mitochondrial and even nuclear DNA (nDNA), thereby activating the cGAS-STING signaling pathway and triggering an innate immune response.

[0003] The use of photodynamic therapy (PDT) to induce mitochondrial stress and activate the cGAS-STING pathway, thereby avoiding the use of STING agonists, is expected to solve the problem of insufficient immune activation faced by conventional PDT and systemic toxicity caused by small molecule agonists faced by immunotherapy based on the cGAS-STING pathway. It has good clinical application prospects, but this method faces two major challenges. First, the hypoxic conditions in the tumor microenvironment limit the efficacy of PDT, and the photosensitizer's ability to target mitochondria is also limited, making it impossible to achieve precise mitochondrial destruction. In addition, photodynamic therapy alone is not enough to trigger the activation of the cGAS-STING pathway, and mitochondria can self-repair and prevent the leakage of mtDNA. Therefore, the development of a method that can accurately target mitochondria, tandem amplification, and achieve non-agonist-dependent cGAS-STING pathway activation is of great significance in the fields of immunotherapy and cancer photodiagnosis. Summary of the invention

[0004] In view of the current problems of metabolic instability, off-target toxicity and insufficient bioavailability of small molecule agonists, the purpose of the present invention is to provide a mitochondrial-targeted light-responsive nanoagonist and its preparation method and application. The nanoagonist prepared by this method utilizes the nanocascade catalytic reaction of hollow mesoporous silica engineered with gold nanoparticles and a type I photosensitizer BDP loaded with intelligent mitochondrial targeting to achieve powerful PDT treatment and activate the STING pathway to enhance strong immunity. The nanoagonist can not only be precisely controlled, but also has high safety and can effectively induce a strong anti-tumor immune response.

[0005] To achieve the above-mentioned purpose of the invention, the present invention provides a method for preparing a mitochondrial-targeted light-responsive nanoagonist, comprising the following steps:

[0006] S1. After a certain mass fraction of hollow mesoporous silica and chloroauric acid are homogenized by ultrasonication, hollow mesoporous silica engineered with gold nanoparticles is obtained by in-situ growth;

[0007] S2, dispersing the gold nanoparticle-engineered hollow mesoporous silica prepared in step S1 into water and ultrasonically obtaining a suspension; dissolving the mitochondrial type I photosensitizer BDP in an organic solvent and adding the suspension to the suspension, stirring in the dark, centrifuging to remove the unloaded mitochondrial type I photosensitizer BDP, and washing to obtain the gold nanoparticle-engineered hollow mesoporous silica loaded with the mitochondrial type I photosensitizer BDP, referred to as HAB;

[0008] S3. Disperse the HAB prepared in step S2 into deionized water and then add dropwise into the hyaluronic acid solution while stirring in the dark to obtain a nano agonist, referred to as HABH.

[0009] Furthermore, the specific preparation process of step S1 is as follows:

[0010] S1-1, dispersing the hollow mesoporous silica in ultrapure water, and uniformly dispersing by ultrasonication to obtain a uniformly dispersed hollow mesoporous silica suspension;

[0011] S1-2, adding a certain amount of chloroauric acid trihydrate to the hollow mesoporous silica suspension obtained in step S1-1, and ultrasonically homogenizing; the mass ratio of chloroauric acid trihydrate to hollow mesoporous silica is 1:(1-10);

[0012] S1-3. Under stirring conditions, a certain amount of sodium borohydride aqueous solution is added dropwise to the mixed solution obtained in step S1-2. After the addition is completed, stirring is continued for 30 to 60 minutes at a temperature of 24 to 37° C. to obtain a purple-red suspension, which is washed by centrifugation 3 times and dried to obtain hollow mesoporous silica engineered with gold nanoparticles.

[0013] Preferably, in step S1-3, the molar concentration of the sodium borohydride is 0.1-1M; the volume ratio between the sodium borohydride aqueous solution and the mixed solution obtained in step S1-2 is (0.1-0.2):2.

[0014] Preferably, in step S2, the mass ratio between the mitochondrial targeted type 1 photosensitizer BDP and the gold nanoparticle engineered hollow mesoporous silica is (0.1-0.4):1.

[0015] Furthermore, the specific preparation process of step S3 is as follows:

[0016] S3-1, dissolving hyaluronic acid in deionized water, and stirring to obtain a uniform hyaluronic acid solution;

[0017] S3-2, dispersing the HAB prepared in step S2 in deionized water to obtain a HAB suspension; adding the HAB suspension dropwise into the hyaluronic acid solution under strong stirring, and continuing to stir in the dark to obtain a nanoagonist suspension;

[0018] S3-3, dialyzing the nanoagonist suspension in a dialysis bag with a molecular weight cutoff of 250 kDa for 24 hours to remove free hyaluronic acid, thereby obtaining the nanoagonist.

[0019] Preferably, the mass ratio of the hyaluronic acid in step S3-1 to the HAB in step S3-2 is 1:1; the mass concentration of the hyaluronic acid solution is 2 mg / mL, and the molecular weight of the hyaluronic acid is 110-220 kDA.

[0020] In order to achieve the above-mentioned purpose of the invention, the present invention also provides a mitochondrial-targeted light-responsive nanoagonist prepared by the above-mentioned preparation method.

[0021] To achieve the above-mentioned purpose of the invention, the present invention also provides the use of the above-mentioned mitochondrial targeted light-responsive nanoagonist in photoimmunotherapy drugs, wherein the nanoagonist is used for precise light-controlled mitochondrial stress and non-agonist-dependent cGAS-STING pathway activation.

[0022] In the present invention, the encapsulation of hyaluronic acid enables the nanoagonist to actively target CD44-overexpressing tumor cells after tail vein injection, increasing the tumor enrichment of the drug. In addition, after targeting tumor cells, efficient cell endocytosis and release of photosensitizers can be achieved, achieving precise mitochondrial targeting. Under light, the photosensitizer achieves type I photodynamic therapy without oxygen at the mitochondrial site, generates a large number of hydroxyl radicals, and induces mitochondrial stress. At the same time, the hollow silica complex engineered by gold nanoparticles acts as a nanozyme, continuously providing hydrogen peroxide through a glucose oxidase-like cyclic reaction, and the generated hydrogen peroxide is further oxidized into highly toxic hydroxyl radicals, enhancing the oxidative damage of cancer cells. This combined effect significantly promotes the release of mitochondrial DNA (mtDNA), and ultimately achieves non-agonist-dependent cGAS-STING pathway activation.

[0023] Compared with the prior art, the present invention has the following advantages:

[0024] The present invention provides a mitochondrial-targeted photoresponsive nanoagonist for precise light-controlled mitochondrial stress and non-agonist-dependent cGAS-STING pathway activation, thereby achieving efficient photoimmunotherapy. The nanoagonist integrates a variety of unique properties to stimulate and activate the STING signaling pathway in a cascade amplification manner to enhance immunity. The nanoagonist prepared by the present invention has good safety and can effectively induce a strong anti-tumor immune response, and is expected to become a new type of clinical photoimmunostimulatory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is a schematic diagram of the structure of the nano agonist prepared by the present invention;

[0026] Figure 2 is a transmission electron microscope image of the gold nanoparticle-engineered hollow mesoporous silica prepared in Example 1 of the present invention;

[0027] Figure 3 is a statistical diagram of the particle size of gold nanoparticles in the gold nanoparticle-engineered hollow mesoporous silica prepared in Example 1 of the present invention;

[0028] Figure 4 is a scanning electron microscope image of the nanoagonist prepared in Example 1 of the present invention;

[0029] Figure 5 This is a schematic diagram of the cytotoxicity of the nano-agonist prepared in Example 1 of the present invention to mouse breast cancer cells;

[0030] Figure 6 This is a schematic diagram of the subcellular distribution of the nanoagonist prepared in Example 1 of the present invention;

[0031] Figure 7 Schematic diagram of the damage of intracellular DNA of the nanoagonist prepared in Example 1 of the present invention; wherein the γ-H2AX channel is used to observe the damage of intracellular DNA, and the DAPI channel is used to observe the cell nucleus;

[0032] Figure 8 This is the in vivo imaging image of BALB / c mice of the nano agonist prepared in Example 1 of the present invention;

[0033] Fig. 9 This is an in vitro imaging picture of the main organs of BALB / c mice of the nano agonist prepared in Example 1 of the present invention;

[0034] Fig.10 The maturation of dendritic cells in lymph nodes of a mouse breast cancer model was demonstrated after tail vein injection of the nanoagonist of the present invention;

[0035] Fig.11 This is a mouse breast cancer model in which the nano-agonist of the present invention is injected into the tail vein, and a diagram showing the maturation of cytotoxic T cells in the mouse lymph nodes;

[0036] Fig.12 This is a statistical diagram of the tumor treatment effect of the nano-agonist prepared in Example 1 of the present invention;

[0037] Fig.13 is the hematoxylin-eosin staining of the main organs of mice after phosphate-buffered solution or nano-agonist administration in the tail vein;

[0038] Fig.14 This is a graph of various blood routine indicators of mice after tail vein injection of phosphate buffer solution or nano-agonist;

[0039] Fig.15 The photosensitizer BDP used in the embodiment of the present invention is 1 H-NMR spectrum. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with specific embodiments.

[0041] Unless otherwise specified, the materials and reagents used in the examples can be obtained from commercial sources.

[0042] The preparation process of the mitochondrial targeted type 1 photosensitizer BDP used in this example is as follows:

[0043] The first intermediate is obtained by using di(benzo[d]thiazol-2-yl)methane in a toluene medium with boron trifluoride ether and triethylamine as additives; the first intermediate is reacted in a 1,2-dichloroethane medium with phosphorus oxychloride and N,N-dimethylformamide as additives to generate a second intermediate through a Vilsmeier-Haack reaction; 1,1,2-trimethyl-1H-benz[e]indole is reacted in an acetonitrile medium with iodoethane as an additive to generate a third intermediate; the second intermediate and the third intermediate are reacted in a toluene and n-butanol medium to synthesize a photosensitizer BDP, whose structural formula is The photosensitizer BDP 1 H-NMR spectrum Fig.15 shown.

[0044] 1H-NMR (400MHz, DMSO-D6) δ (ppm) = 8.86 (d, J = 15.9Hz, 1H), 8.46 (d, J = 8.6Hz, 1H) ,8.40(d,J=3.7Hz,1H),8.32(d,J=9.2Hz,1H),8.23(d,J=7.9Hz,1H),8.14(d,J= 8.6Hz,1H),8.04(d,J=7.9Hz,2H),7.96(d,J=7.9Hz,2H),7.85-7.72(m,2H),7.6 3(t,J=7.6Hz,2H),7.46(d,J=7.9Hz,4H),4.82(d,J=7.3Hz,2H),2.01-2.09(9H).

[0045] Example 1

[0046] A method for preparing a mitochondrial-targeted light-responsive nanoagonist comprises the following steps:

[0047] S1. After a certain mass fraction of hollow mesoporous silica and chloroauric acid are homogenized by ultrasonication, hollow mesoporous silica engineered with gold nanoparticles is obtained by in-situ growth. The specific steps are as follows:

[0048] S1-1, dispersing 2.5 mg of hollow silica in 2.5 mL of deionized water, and dispersing evenly by ultrasonication for 30 minutes to obtain a hollow mesoporous silica suspension with a W / W ratio of 1%;

[0049] S1-2, adding 37.5 μL of 1% chloroauric acid trihydrate (375 μg) to a 1% W / W hollow mesoporous silica suspension, and ultrasonicating for 30 minutes to make it evenly dispersed in the solution;

[0050] S1-3, under vigorous stirring, add 187.5 μL of a 0.5 M sodium borohydride solution to the solution of step S1-2, and continue stirring at 24-37° C. for 30-60 min to obtain a purple-red suspension, and centrifuge the suspension to wash and dry to obtain gold nanoparticle-engineered hollow mesoporous silica;

[0051] S2, weighing 1 mg of the gold nanoparticles engineered hollow mesoporous silica prepared in step S1, dispersing it in 800 μL of ultrapure water, and ultrasonicating it for 15 min to obtain a suspension; then dissolving the photosensitizer BDP in a dimethyl sulfoxide solution at a concentration of 1 mg / mL, then taking 200 μL of the dimethyl sulfoxide solution containing BDP and adding it to the suspension, stirring it for 4 h under light-proof conditions, centrifuging to remove the supernatant, and washing it with ultrapure water to obtain the gold nanoparticles engineered hollow mesoporous silica (HAB) loaded with BDP;

[0052] S3, under light-proof stirring, dispersing the HAB prepared in step S2 into deionized water and then adding dropwise into the hyaluronic acid solution to obtain a nano agonist, referred to as HABH; the specific steps are as follows:

[0053] S3-1, 1 mg hyaluronic acid (M W =110-220 kDa) was dissolved in 0.5 mL of deionized water and stirred for 10 minutes to obtain a 2 mg / mL uniform hyaluronic acid solution;

[0054] S3-2, dispersing 1 mg of the HAB prepared in step S2 in 0.5 mL of deionized water to obtain a HAB suspension; under strong stirring, adding the HAB suspension dropwise into the hyaluronic acid solution, and continuing to stir for 2 hours in a dark environment to obtain a nanoagonist suspension;

[0055] S3-3, dialyze the nanoagonist suspension in a dialysis bag with a molecular weight cutoff of 250 kDa for 24 hours to remove free hyaluronic acid, thereby obtaining a nanoagonist (HABH), the structural diagram of which is shown in FIG. Figure 1 shown.

[0056] from Figure 2 As can be seen from the figure, the gold nanoparticles are evenly distributed on the hollow mesoporous silica, indicating the successful preparation of the hollow mesoporous silica engineered with gold nanoparticles. Figure 3 As shown, Nano measure software was used to count the particle size of gold nanoparticles in the hollow mesoporous silica engineered with gold nanoparticles, and the size of the gold nanoparticles was approximately 2.6-4.3 nm.

[0057] from Figure 4It can be seen that there is a circle of shadow at the edge of the hollow mesoporous silica engineered with gold nanoparticles, indicating that the hyaluronic acid coating is successful and evenly dispersed; after the hyaluronic acid is coated on the outermost layer of HAB, the physiological environment stability and tumor targeting of the nanoagonist are improved.

[0058] Example 2

[0059] The toxicity evaluation of the nano-agonist prepared in Example 1 on mouse breast cancer cells was carried out as follows:

[0060] (1) 5000 mouse breast cancer cells were inoculated into each well of a 96-well plate (100 μL per well) and then cultured at 37°C and 5% CO2 for 24 hours;

[0061] (2) The gold nanoparticle-engineered hollow mesoporous silica (HMSN / AuNPs) prepared in Example 1, the photosensitizer BDP used in Example 1, and the nanoagonist HABH prepared in Example 1 were prepared to corresponding concentrations; wherein the photosensitizer BDP was dissolved in dimethyl sulfoxide to a final concentration of 10 mg / mL; the gold nanoparticle-engineered hollow mesoporous silica was dispersed in ultrapure water to a concentration of 1 mg / mL; and the nanoagonist HABH was dispersed in ultrapure water to a final concentration of 1 mg / mL;

[0062] (3) Take out the 96-well plate from the incubator and discard the culture medium, add 100 μL of RPMI-1640 complete medium to the control group, and add 100 μL of RPMI-1640 complete medium containing free BDP, gold nanoparticle-engineered hollow mesoporous silica, or nanoagonist to the other groups (wherein the concentration of BDP is 0.5, 1, 2, 5, 10, and 20 μg / mL, and the concentration of gold nanoparticle-engineered hollow mesoporous silica is 3.5, 7, 14, 35, 70, and 140 μg / mL), and then incubate at 37°C, 5% CO2 for 24 hours;

[0063] (4) The 96-well plate was removed from the incubator and 10 μL of thiazolyl blue (5 mg / mL) was added to each well. The plate was incubated for 4 hours at 37°C and 5% CO2. The medium was discarded and 150 μL of dimethyl sulfoxide was added to each well. The cytotoxicity was evaluated using an ELISA instrument. The results are shown in Table 2. Figure 5 .

[0064] Figure 5 The experimental results show that compared with other control groups, the nanoagonist of the present invention can cause more efficient killing of cancer cells under the induction of light, which proves the synergistic anti-cancer effect of hollow mesoporous silica nanozymes engineered by gold nanoparticles and the mitochondrial-targeted photosensitizer BDP.

[0065] Example 3

[0066] The distribution of the nano-agonist prepared in Example 1 in the subcellular space of mouse breast cancer cells is as follows:

[0067] (1) 10,000 mouse breast cancer cells were inoculated into each well of an 8-well plate (200 μL per well) and cultured at 37°C for 24 h;

[0068] (2) The nanoagonist was dispersed in RPMI-1640 complete medium at a BDP concentration of 5 μg / mL and added to an 8-well plate (200 μL per well) and incubated at 37°C and 5% CO2 for 4 h;

[0069] (3) The original culture medium was discarded, and different subcellular organelle dyes were dispersed in phosphate buffer and added to an 8-well plate (200 μL per well) and incubated for different time periods (mitochondrial dye Rhod 123, 2 μg / mL, incubation for 10 minutes; Golgi dye Golgi-Tracker, 5 μM, incubation for 30 minutes; lysosomal dye LysoTracker, 0.5 μM, incubation for 30 minutes; endoplasmic reticulum dye ER-Tracker, 1 μM, incubation for 30 minutes; nuclear dye Hoechst 33342, 1 μg / mL, incubation for 10 minutes). The plates were then washed three times with phosphate buffer and observed using a confocal fluorescence microscope. Confocal fluorescence microscopy imaging observation, using different channels to observe the co-localization of doxorubicin and cell nuclei: BDP in the nanoagonist emits red fluorescence under 552nm excitation light, the cell nucleus dye Hoechst 33342 emits blue fluorescence under 405nm excitation light, and the mitochondrial dye Rhod 123, Golgi dye Golgi-Tracker, lysosome dye LysoTracker, and endoplasmic reticulum dye ER-Tracker emit green fluorescence under 488nm excitation light. Figure 6 .

[0070] Figure 6 In the experiment, the Rhod 123 channel is used to observe mitochondria, the Golgi-Tracker channel is used to observe Golgi bodies, the Lyso-Tracker channel is used to observe lysosomes, the ER-Tracker channel is used to observe endoplasmic reticulum, the Hoechst channel is used to observe cell nuclei, and the nanoagonist channel is used to observe the photosensitizer in the nanoagonist. The superposition channels are the superposition of the nanoagonist channel with the Rhod123 channel, the Golgi-Tracker channel, the Lyso-Tracker channel, the ER-Tracker channel, and the Hoechst channel, respectively. The superposition channels are used to observe the co-localization of the photosensitizer in the nanoagonist and the cell nucleus. Figure 6It can be seen that after the nanoagonist is co-incubated with mouse breast cancer cells, BDP can enter the mitochondria quickly and in large quantities, indicating that the nanoagonist can effectively deliver BDP into the mitochondria of cancer cells.

[0071] Example 4

[0072] DNA damage detection in mouse breast cancer cells. The specific process is as follows:

[0073] DNA damage was detected by γ-H2AX immunofluorescence staining. First, mouse breast cancer cells were inoculated into 8-well cell culture dishes at a density of 5,000 cells per well (200 μL per well) and incubated overnight; then the phosphate buffer solution (control group) and the nanoagonist obtained in Example 1 were incubated for 4 hours, and the treated cells were washed twice with PBS, and fresh culture medium was added; the nanoagonist group was irradiated with white light (20 mW / cm2) on ice for 10 minutes. The 8-well plate was returned to the incubator and incubated for another 2 hours. Then, the culture medium was discarded and the cells were washed 3 times with PBS, fixed with 4% paraformaldehyde for 20 minutes, the paraformaldehyde was discarded, and the cells were treated with 0.5% Triton X-100 for 15 minutes, and finally sealed with 5% BSA at room temperature for 45 minutes. Subsequently, the γ-H2AX primary antibody and the Fluor488-coupled secondary antibody were incubated with the treated mouse breast cancer cells for 1 hour. Fluor488 was aspirated, and the cells were washed twice. Then, nuclear staining solution (DAPI) was added and stained for 5 minutes at room temperature. The cells were washed three times with washing solution. Finally, the DNA damage level of the cells was observed using a confocal microscope. The results are shown in Figure 7 .Depend on Figure 7 It can be seen that after mouse breast cancer cells are treated with nano-agonists, nuclear DNA damage can be effectively induced, indicating that the nano-agonist can activate the STING pathway by damaging DNA.

[0074] Example 5

[0075] Evaluation of the tumor enrichment effect of the nano-agonist prepared in Example 1, the specific process is as follows:

[0076] (1) Select 6-week-old BALB / c female mice in good health and inoculate breast cancer cells in situ into their mammary tissue. Wait until the tumor grows to about 50 mm. 3 At 14:00, the weight of the mice was recorded, and the required BDP dose (0.5 mg / kg) was calculated based on the actual weight of the mice;

[0077] (2) The nanoagonist prepared in Example 1 was labeled with DiD and injected into mice via tail vein injection, and its fluorescence distribution in the body and various organs was observed within 24 hours. The results are shown in Figure 8 and Fig. 9 . Figure 8 and Fig. 9 The experimental results showed that the nanoagonist had a good tumor enrichment effect and a relatively ideal tumor retention time.

[0078] Example 6

[0079] The immunostimulatory effect of the nano-agonist prepared in Example 1 was evaluated, and the specific process was as follows:

[0080] Select 6-week-old BALB / c female mice in good health and inoculate breast cancer cells orthotopically into their mammary tissue. When the tumor grows to about 50 mm 3 At the same time, the weight of the mice was recorded, and the required BDP dose (0.5 mg / kg) was calculated based on the actual weight of the mice, and the suspension of the nanoagonist in Example 1 (100 μL, the concentration of BDP was 100 μg / mL) was injected through the tail vein. Mice injected with phosphate buffer by the tail vein were used as the control group. After 7 days of treatment, the lymph nodes were removed and ground into single cells. Staining with PE-labeled CD11c antibody, FITC-labeled CD80 antibody and APC-labeled CD86 antibody was used to evaluate the maturation of dendritic cells. Staining with FITC-labeled CD3 antibody and APC-labeled CD8 antibody was used to evaluate the maturation of cytotoxic T cells, and the results are shown in Figure 2. Fig.10 and Fig.11 .

[0081] Fig.10 and Fig.11 The experimental results showed that the nano-agonist can well stimulate the maturation of dendritic cells (CD11c + CD80 + CD86 + ) and increase cytotoxic T cells (CD3 + CD8 + ) ratio, indicating that the nanoagonist can activate the immune system of mice to kill tumors.

[0082] Example 7

[0083] The in vivo anticancer effect of the nanoreagent prepared in Example 1 was evaluated, and the specific process was as follows:

[0084] Select 6-week-old BALB / c female mice in good health and inoculate breast cancer cells orthotopically into their mammary tissue. When the tumor grows to about 50 mm 3 The weight of the mice was recorded, and the required BDP dose (0.5 mg / kg) was calculated based on the actual weight of the mice. Phosphate buffer (control group) and the nanoagonist obtained in Example 1 were injected into the mice via tail vein injection, and the tumor size was observed and counted. The results are shown in Fig.12. Fig.12 The experimental results showed that after the mice were injected with the nano-agonist through the tail vein, the volume of the tumor was significantly inhibited, which indicates that the nano-agonist has a good anti-tumor effect.

[0085] Example 8

[0086] The in vivo safety of the nano-agonist prepared in Example 1 was evaluated, and the specific process was as follows:

[0087] 6-week-old BALB / c female mice in good health were selected, and the weight of the mice was measured. The required BDP dose (0.5 mg / kg) was calculated based on the actual weight of the mice. The nanoreagent obtained in Example 1 containing the above BPD dose was injected into the mice via tail vein injection. The control group was injected with phosphate buffer. After 14 days, the main organs of the mice were sliced ​​and stained with hematoxylin-eosin, and the health of the tissues was observed under a confocal microscope. The results are shown in Table 1. Fig.13 At the same time, blood was taken from mice for routine blood tests, and the results are shown in Fig.14 .

[0088] Fig.13 and Fig.14 The experimental results showed that after the mice were injected with the nano-agonist via the tail vein, there was no obvious damage to the major organs and all blood routine indicators were normal, which indicated that the nano-preparation had good in vivo safety.

Claims

1. A method for preparing a mitochondrial-targeted light-responsive nanoagonist, characterized in that: The following steps are involved: S1. After a certain mass fraction of hollow mesoporous silica and chloroauric acid are homogenized by ultrasonication, hollow mesoporous silica engineered with gold nanoparticles is obtained by in-situ growth; S2, dispersing the gold nanoparticle-engineered hollow mesoporous silica prepared in step S1 into water and ultrasonically obtaining a suspension; dissolving the mitochondrial type I photosensitizer BDP in an organic solvent and adding the suspension to the suspension, stirring in the dark, centrifuging to remove the unloaded mitochondrial type I photosensitizer BDP, and washing to obtain the gold nanoparticle-engineered hollow mesoporous silica loaded with the mitochondrial type I photosensitizer BDP, referred to as HAB; S3. Disperse the HAB prepared in step S2 into deionized water and then add dropwise into the hyaluronic acid solution while stirring in the dark to obtain a nano agonist, referred to as HABH.

2. The method for preparing a mitochondrial-targeted light-responsive nanoagonist according to claim 1, characterized in that: The specific preparation process of step S1 is: S1-1, dispersing the hollow mesoporous silica in ultrapure water, and uniformly dispersing by ultrasonication to obtain a uniformly dispersed hollow mesoporous silica suspension; S1-2, adding a certain amount of chloroauric acid trihydrate to the hollow mesoporous silica suspension obtained in step S1-1, and ultrasonically homogenizing; the mass ratio of chloroauric acid trihydrate to hollow mesoporous silica is 1:(1-10); S1-3. Under stirring conditions, a certain amount of sodium borohydride aqueous solution is added dropwise to the mixed solution obtained in step S1-2. After the addition is completed, stirring is continued for 30 to 60 minutes at a temperature of 24 to 37° C. to obtain a purple-red suspension, which is washed by centrifugation 3 times and dried to obtain hollow mesoporous silica engineered with gold nanoparticles.

3. The method for preparing a mitochondrial-targeted light-responsive nanoagonist according to claim 2, characterized in that: In step S1-3, the molar concentration of the sodium borohydride is 0.1-1M; the volume ratio between the sodium borohydride aqueous solution and the mixed solution obtained in step S1-2 is (0.1-0.2):

2.

4. The method for preparing a mitochondrial-targeted light-responsive nanoagonist according to claim 1 or 2, characterized in that: In step S2, the mass ratio between the mitochondrial targeted type 1 photosensitizer BDP and the gold nanoparticle engineered hollow mesoporous silica is (0.1-0.4):

1.

5. The method for preparing a mitochondrial-targeted light-responsive nanoagonist according to claim 1 or 2, characterized in that: The specific preparation process of step S3 is: S3-1, dissolving hyaluronic acid in deionized water, and stirring to obtain a uniform hyaluronic acid solution; S3-2, dispersing the HAB prepared in step S2 in deionized water to obtain a HAB suspension; adding the HAB suspension dropwise into the hyaluronic acid solution under strong stirring, and continuing to stir in the dark to obtain a nanoagonist suspension; S3-3, dialyzing the nanoagonist suspension in a dialysis bag with a molecular weight cutoff of 250 kDa for 24 hours to remove free hyaluronic acid, thereby obtaining the nanoagonist.

6. The method for preparing a mitochondrial-targeted light-responsive nanoagonist according to claim 5, characterized in that: The mass ratio between the hyaluronic acid in step S3-1 and the HAB in step S3-2 is 1:1; the mass concentration of the hyaluronic acid solution is 2 mg / mL, and the molecular weight of the hyaluronic acid is 110-220 kDA.

7. A mitochondrial-targeted light-responsive nanoagonist as described in any one of claims 1 to 6.

8. Use of the mitochondrial-targeted photoresponsive nanoagonist according to claim 7 in photoimmunotherapy drugs, wherein the nanoagonist is used for precise light-controlled mitochondrial stress and agonist-independent cGAS-STING pathway activation.

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