Preparation and application of hydrogen sulfide response aggregation type gold nanoprobe

Abstract

The invention discloses preparation and application of a hydrogen sulfide response aggregation type gold nanoprobe. The probe has good stability, biocompatibility and functionality, shows good selectivity and sensitivity to hydrogen sulfide, quickly responds to AS1411-Ce6 release and gold nanoparticle aggregation, and can be used for fluorescence imaging and photodynamic photo-thermal combined therapy of tumor cells and tissues overexpressing hydrogen sulfide. In tumor tissue cells, overexpressed hydrogen sulfide promotes the activity of the photosensitizer to be activated, and active oxygen is generated under irradiation of 660 nm light and used for photodynamic therapy; after responding to hydrogen sulfide, the gold nanoparticles aggregate to enhance the tumor retention capacity, meanwhile, absorption of the probe is shifted to a near-infrared region in a red shift mode, and after 808 nm laser irradiation, the temperature is rapidly increased, and the probe is used for tumor cell photothermal therapy. In-vitro and in-vivo studies prove that the probe has good response, detection and imaging capabilities on hydrogen sulfide and an excellent tumor treatment effect.

Description

Technical Field

[0001] The present invention belongs to the field of tumor diagnosis and treatment nanoprobes, and specifically relates to the preparation and application of a hydrogen sulfide responsive aggregated gold nanoprobe. Background Art

[0002] Phototherapy is a promising and highly effective strategy for tumor treatment, with advantages such as low invasiveness, good spatiotemporal controllability, and minimal side effects. There are two main modes of phototherapy, namely photodynamic therapy (PDT) and photothermal therapy (PTT), which kill tumor cells through reactive species (ROS) or high temperature generated by light irradiation, respectively. Although some favorable progress has been made in tumor phototherapy, many solid tumors cannot be effectively inhibited by a single treatment method (using only PDT or PTT) due to the heterogeneity and complexity of tumor tissues. Therefore, the need to develop multifunctional treatment modes with improved therapeutic effects remains very urgent. The development of nanotechnology has enabled us to effectively integrate multifunctional applications into a single nanoplatform, showing enhanced anti-tumor effects through a rationally designed nanoplatform combining PDT and PTT.

[0003] DNA-based nanomachines, especially gold nanoparticle (AuNPs)-DNA nanomachines, show great potential in biomedical applications due to their good optical properties, biocompatibility, and the ability of DNA and AuNPs to be easily designed on demand. By utilizing gold-thiol interactions to modify DNA, AuNPs not only serve as excellent carriers to promote DNA cellular uptake, but also as super fluorescence quenchers for various dyes. In most reports, researchers have developed various AuNPs-DNA nanoprobes for imaging cellular ATP, mRNA or proteins, enzymes, etc. Hydrogen sulfide (H 2 As an important gas molecule, S has shown potential as an important biomarker for responsive therapeutic systems, but has not yet been applied to tumor diagnosis and treatment in AuNPs-DNA-based nanomachine phototherapy. Although the need to develop AuNPs-DNA nanomachines with stimulus responsiveness to the tumor microenvironment (TME) and multifunctional and efficient anti-cancer therapeutic capabilities is very urgent, research in this field is still in its early stages and has not been well met.

[0004] In summary, a hydrogen sulfide-responsive aggregated gold nanoprobe was developed and applied to tumor H 2 The combined photodynamic and photothermal therapy of S response has important development and application prospects. Summary of the invention

[0005] The present invention aims to provide a preparation method and application of a hydrogen sulfide responsive aggregated gold nanoprobe.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows: The first aspect of the present invention provides a hydrogen sulfide responsive aggregated gold nanoprobe, wherein the hydrogen sulfide responsive aggregated gold nanoprobe comprises gold nanoparticles, polyadenylic acid modified nucleic acid aptamer AS1411 and a photosensitizer; the response target of the hydrogen sulfide responsive aggregated gold nanoprobe is hydrogen sulfide; The gold nanoparticles are spherical or quasi-spherical with a particle size of 10 to 100 nm; the sequence of the polyadenylic acid modified nucleic acid aptamer AS1411 is: 5'-AAAAAAAAAATTTGGTGGTGGTGGTTGTGGTGGTGGTGG-3'; and the photosensitizer is any one or more of dihydrochlorin e6, pyropheophorbide-a, protoporphyrin, and zinc phthalocyanine.

[0007] The second aspect of the present invention provides a method for preparing the above-mentioned hydrogen sulfide responsive aggregated gold nanoprobe, which comprises synthesizing gold nanoparticles, then modifying the surface of the gold nanoparticles with poly(A)-modified nucleic acid aptamer AS1411, and then combining a photosensitizer with the poly(A)-modified nucleic acid aptamer AS1411 modified on the surface of the gold nanoparticles, thereby preparing the hydrogen sulfide responsive aggregated gold nanoprobe.

[0008] Furthermore, the preparation method of the above-mentioned hydrogen sulfide responsive aggregated gold nanoprobe comprises the following steps: 1) Synthesize gold nanoparticles by reducing tetrachloroauric acid with trisodium citrate; 2) Adding polyadenylic acid modified nucleic acid aptamer AS1411 solution to the gold nanoparticle solution and mixing, adding 500mM sodium citrate-hydrochloric acid buffer solution with a pH of 3 after standing for 5 minutes, shaking and adding 500mM sodium citrate-hydrochloric acid buffer solution with a pH of 3 again, standing for 5 minutes, adding 500mM HEPES solution with a pH of 7.6 to adjust the pH of the system to neutral, standing for 10 minutes, and then centrifuging, discarding the supernatant, washing the precipitate with 10mM PB buffer solution with a pH of 7.4 containing 20mM NaCl, and resuspending it in 10mM PB buffer solution with a pH of 7.4 to obtain AuNPs-AS1411 probe solution; 3) Centrifuging the AuNPs-AS1411 probe solution obtained in step 2), discarding the supernatant, and resuspending the precipitate in 20 mM PB buffer to obtain a resuspended AuNPs-AS1411 probe solution; adding the photosensitizer solution to the obtained resuspended AuNPs-AS1411 probe solution, mixing, standing for 2 hours, and then centrifuging, discarding the supernatant, and resuspending the precipitate in 20 mM PBS buffer to obtain a hydrogen sulfide responsive aggregated gold nanoprobe solution; The molar ratio of the gold nanoparticles to the poly(A)-modified nucleic acid aptamer AS1411 is 1:50-300, and the molar ratio of the poly(A)-modified nucleic acid aptamer AS1411 to the photosensitizer is 1:1-20.

[0009] The third aspect of the present invention provides the use of the above hydrogen sulfide responsive aggregated gold nanoprobe in the preparation of a hydrogen sulfide concentration quantitative reagent.

[0010] The fourth aspect of the present invention provides the use of the above-mentioned hydrogen sulfide responsive aggregated gold nanoprobe in the preparation of a fluorescent imaging agent.

[0011] The fifth aspect of the present invention provides the use of the above hydrogen sulfide responsive aggregated gold nanoprobe in the preparation of tumor treatment agents; Among them, the treatment includes photodynamic therapy, photothermal therapy, and photodynamic and photothermal combined therapy.

[0012] The beneficial effects of the present invention are mainly reflected in the following aspects: (1) The synthesis steps of the hydrogen sulfide responsive aggregated gold nanoprobes of the present invention are simple, easy to operate, highly reproducible, and the raw materials used have good biocompatibility; (2) The hydrogen sulfide-responsive aggregated gold nanoprobes of the present invention have hydrophilic protective groups on the surface and are evenly dispersed in particle size, thus ensuring their physiological safety, stability and effectiveness; (3) The hydrogen sulfide-responsive aggregated gold nanoprobes of the present invention can specifically and sensitively respond to changes in hydrogen sulfide in the tumor microenvironment, release the loaded photosensitizer, induce gold nanoaggregation to produce near-infrared absorption, and achieve hydrogen sulfide in the tumor microenvironment. 2 S-responsive tumor imaging and photodynamic combined with photothermal therapy applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :(A) Schematic diagram of the synthesis of hydrogen sulfide-responsive aggregated gold nanoprobes (AAC). (B) Schematic diagram of the response of hydrogen sulfide-responsive aggregated gold nanoprobes and in vivo photodynamic combined with photothermal therapy.

[0014] Figure 2 :(A) Synthesis and response of hydrogen sulfide responsive aggregated gold nanoprobe (AAC) 2 Schematic diagram of aggregation and fluorescence recovery after S; (B) Transmission electron microscopy image of AAC nanoprobe; (C) Response of AAC nanoprobe to H 2 Transmission electron microscopy images of the aggregated samples after S; (D) Different nanoprobes and their responses to H 2 (DLS) graph of particle size change after S; (E) Zeta potential graph of gold nanoparticles (AuNPs), gold nanoparticles-DNA (AuNPs-AS1411) and AAC nanoprobes; (F) Different nanoprobes and their response H2 Absorption spectra after S; (G) Different nanoprobes and their responses H 2 Fluorescence spectrum after S.

[0015] Figures 3A to 3C :(A) AAC nanoprobe response to H 2 Schematic diagram of gold nanoparticle aggregation and fluorescence recovery after S; (B) AAC nanoprobe responds to different concentrations of H 2 The absorption spectrum changes after S, the inset is the corresponding color change diagram; (C) The AAC nanoprobe responds to different concentrations of H 2 Fluorescence spectrum changes after S.

[0016] Figures 3D to 3G :(DE) Absorption spectrum changes of AAC nanoprobe in response to different interferents; (FG) Fluorescence spectrum changes of AAC nanoprobe in response to different interferents.

[0017] Figure 4 :(A) AAC nanoprobe responds to Na 2 After S, the reactive oxygen species generation capacity was detected by reactive oxygen species probe under 660nm laser irradiation for different time periods. (B) The response of AAC nanoprobe to Na 2 After S, the temperature changes with time under different powers of 808nm laser irradiation.

[0018] Figure 5 :(A) AAC nanoprobe in tumor cells (SMMC-7721) under light (660nm laser, 0.10W / cm 2 ,5min) and without light, with DCFH-DA as the cell active oxygen imaging probe; (B) Evaluation of the AAC nanoprobe on the phototherapy of tumor cells SMMC-7721, in which the 660nm laser (0.10W / cm 2 ,5min), 808nm laser (1W / cm 2 ,10min).

[0019] Figure 6 :(A) AAC nanoprobes and PBS were injected into mouse tumors at different time points, and 808 nm laser (1 W / cm 2 , 10min) irradiation and photothermal heating imaged by a thermal imager; (B) After the AAC nanoprobe was injected into the mouse tumor, the fluorescence changes at the tumor site (right circle) and subcutaneous site (left circle) were detected. The fluorescence signals were excited at 635nm and collected at 650-700nm.

[0020] Figure 7:(A) Schematic diagram of mouse tumor-bearing model and probe injection and phototherapy timeline;(B) Changes in tumor volume over time after treatment with AAC nanoprobes and PBS combined with different phototherapy conditions;(C) Changes in mouse body weight during treatment;(D) Weights of ex vivo tumors in mice in different treatment groups after treatment;(E) Photos of ex vivo tumors in mice in different treatment groups after treatment;(F) H&E, Ki67 and TUNEL staining of ex vivo tumors in mice in different treatment groups after treatment. DETAILED DESCRIPTION

[0021] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further described below in conjunction with specific implementation methods, but the present invention is not limited thereto.

[0022] The sequence of the polyadenylic acid modified nucleic acid aptamer AS1411 (polyA-AS1411) involved in the embodiments of the present invention is: 5'-AAAAAAAAAATTTGGTGGTGGTGGTTGTGGTGGTGGTGG-3'.

[0023] Example 1: Synthesis and Application Principle of Hydrogen Sulfide Responsive Aggregated Gold Nanoprobe The hydrogen sulfide responsive aggregated gold nanoprobe (AAC probe for short) of the present invention is composed of gold nanoparticles (AuNPs), polyA-AS1411 and photosensitizer (such as Figure 1 In the tumor microenvironment (TME), hydrogen sulfide (H 2 Under in situ activation, polyA-AS1411 and photosensitizer are released from the surface of AAC probe, triggering the aggregation of gold nanoparticles and the red shift of absorption to the near-infrared region, enhancing the retention of photosensitizer and gold nanoparticles in tumors, and then activating the released DNA-Ce6 for photodynamic therapy (660nm laser) and the aggregated AuNPs for photothermal therapy (808nm laser), achieving a combined anticancer phototherapy effect (such as Figure 1 B). Specifically: the AAC probe consists of AuNPs (as nanocarriers, nanoquenchers, and photothermal therapeutic agents after aggregation) and polyA-modified DNA (AS1411 nucleic acid aptamer, as a tumor targeting ligand, Ce6 carrier and stabilizer of AuNPs). PolyA-AS1411 forms a G-quadruplex structure under physiological conditions and can target nucleolar proteins highly expressed in tumors. PolyA-AS1411 is modified to the surface of AuNPs under pH = 3.0 citrate buffer conditions and used as a Ce6 carrier. At the same time, it improves the stability of AuNPs in buffer or physiological conditions. In the absence of hydrogen sulfide, the AAC probe has no obvious phototoxicity and produces very low ROS or photothermal effects under laser irradiation. However, when the AAC probe is internalized into the tumor site, H in the TME 2S can release polyA-AS1411 from the surface of AuNPs, which turns on the photoactivity of Ce6 under 660nm laser irradiation (fluorescence imaging and photodynamic therapy effect) and promotes the aggregation of AuNPs (the absorption peak red-shifts to the near-infrared region), achieving good photothermal ablation under 808nm irradiation. 2 The transition from single particles to aggregated states triggered by S further enhances the retention of nanoprobes at the tumor site for fluorescence imaging and promoting the combined phototherapy effect. The efficacy of tumor phototherapy is improved by combining oxygen-dependent photodynamic therapy (PDT) with oxygen-independent photothermal therapy (PTT).

[0024] Example 2: Synthesis of hydrogen sulfide responsive aggregated gold nanoprobes (1) Synthesis of gold nanoparticles: Spherical gold nanoparticles with a particle size of about 13 nm were synthesized by sodium citrate reduction. Specifically, all glassware was treated with aqua regia (volume ratio of HNO 3 :HCl=1:3) soak for more than 30min, wash with ultrapure water, and dry for use. Heat 50mL of 1mM tetrachloroauric acid aqueous solution at 110℃ in a 100mL flask until boiling, keep reflux at 110℃ for 15min, and keep stirring rapidly; quickly add 5mL of 38.8mM trisodium citrate solution under stirring, continue heating and stirring for 15min, then remove the heat source, cool to room temperature, stop stirring, and obtain gold nanoparticles (AuNPs) solution, store at 4℃ away from light for use. The concentration of gold nanoparticles is calculated by Lambert-Beer law A=ε*b*c, where A is the absorbance of the solution at 524nm, ε is the molar absorption coefficient of gold nanoparticles, b is the optical path (1cm), and c is the concentration of the absorbing substance. The extinction coefficient of gold nanoparticles at 524nm is ε=2.7×10 8 L×mol -1 ×cm -1 (2) Synthesis of AuNPs-AS1411 nanoprobes: PolyA-AS1411 was modified onto the surface of gold nanoparticles using the acidic (pH = 3) nucleic acid rapid modification method. First, 1.5 mL of 10 nM AuNPs solution was placed in a 2 mL centrifuge tube, and 20 μL of 100 μM polyA-AS1411 solution was added and mixed (molar ratio C AuNPs :C DNA≈1:133), after standing at room temperature for 5 minutes, add 30 μL of 500mM sodium citrate-hydrochloric acid buffer with pH=3, shake well and add 30 μL of 500mM sodium citrate-hydrochloric acid buffer with pH=3 again, stand at room temperature for 5 minutes, add 180 μL of 500mM HEPES solution with pH=7.6 to adjust the pH of the reaction system to neutral, and then stand at room temperature for 10 minutes. After the reaction is completed, centrifuge at 14800rpm for 10 minutes, discard the supernatant to remove the unreacted DNA, centrifuge and wash the precipitate once with 750 μL of 10mM PB buffer (pH=7.4) containing 20mM NaCl, and resuspend with 50 μL of 10mM PB buffer (pH=7.4) to obtain AuNPs-AS1411 nanoprobe solution, which is stored at 4°C in the dark for use.

[0025] (3) Synthesis of AuNPs-AS1411-Ce6 nanoprobe: The AuNPs-AS1411 nanoprobe solution obtained in (2) was centrifuged at 14800 rpm for 10 min, the supernatant was discarded, and 500 μL of 20 mM PB buffer (pH = 7.4) was added to the precipitate to obtain a resuspension of AuNPs-AS1411 nanoprobe; 2.25 μL of A 2mM Ce6 solution (DMSO as solvent) was shaken and allowed to react at room temperature for 2 hours, then centrifuged at 14800rpm for 10 minutes, the supernatant was discarded, and the precipitate was resuspended in 500μL 20mM PBS buffer (pH=7.4) to obtain AuNPs-AS1411-Ce6 nanoprobe solution (also known as AAC nanoprobe solution), which is a hydrogen sulfide-responsive aggregated gold nanoprobe solution, and stored at 4°C away from light for later use. The concentration of AAC nanoprobe is measured by the concentration of gold nanoparticles therein. It is still determined by Lambert-Beer's law, where the extinction coefficient of gold nanoparticles at 524nm is ε=2.7×10 8 L×mol -1 ×cm -1 .

[0026] Example 3: Characterization of hydrogen sulfide responsive aggregated gold nanoprobes The physical and chemical properties of the AAC nanoprobe synthesized in Example 2 were characterized by transmission electron microscopy, nanoparticle size and Zeta potential analyzer, UV-visible spectrophotometer, and fluorescence spectrometer. Figure 2 A is the synthesis of AAC nanoprobe and its response to Na 2 Schematic diagram of aggregation and fluorescence recovery after S. Figure 2 B is the transmission electron microscopy (TEM) image of the AAC nanoprobe, showing that its particle size is about 13 nm. Figure 2C shows the response of the AAC nanoprobe to Na 2 TEM image of the aggregate after S. Figure 2 The particle size distribution of D shows that the AAC nanoprobe has 2 S is distributed in the range of 13 to 30 nm, and responds to Na 2 After S, the aggregation particle size of AAC nanoprobes became larger. Figure 2 E shows that the Zeta potential of the nanoprobe is negative. Figure 2 F shows that the AAC nanoprobe responds to Na 2 The absorption after S obviously red-shifts to the near-infrared region. Figure 2 G shows response to Na 2 The fluorescence recovery of polyA-AS411-Ce6 was obvious after S, which proved that the present invention successfully constructed the hydrogen sulfide responsive aggregated gold nanoprobe.

[0027] Example 4: Spectral changes of Ce6 released by AAC nanoprobe in response to hydrogen sulfide in vitro, ROS generation under illumination and photothermal heating As shown in Figure 3A, 15 μL of AAC nanoprobe with a concentration of 100 nM (concentration in terms of gold nanoparticles) was added to 285 μL of 20 mM PB buffer (pH = 7.4), and sodium sulfide solution with final concentrations of 0 μM, 5 μM, 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, and 100 μM was added (due to H 2 S is a gas and is not easy to use directly. In the in vitro experiment of the present invention, Na 2 S was used to simulate the reaction), the system was mixed and reacted for 5 minutes, then centrifuged at 13000rpm for 10 minutes, 200μL of the supernatant was taken to measure the fluorescence at 550-750nm, and the Ce6 fluorescence spectrum was measured (Ex=404nm). Or the absorption spectrum of the solution after the reaction was directly tested. At the same time, the absorption and fluorescence spectra of the AAC nanoprobe reacting with different interferents were also measured. The results are shown in Figure 3B. The AAC nanoprobe responded to Na 2 S aggregated, and the color of the solution changed significantly, gradually changing from wine red to blue purple. When the concentration of sodium sulfide reached 40 μM, the color of the solution changed significantly, and the absorption spectrum also red-shifted. As shown in Figure 3C, with the increase of Na 2 As the S concentration increases, the amount of released polyA-AS411-Ce6 increases and the fluorescence gradually increases. Figures 3D to 3G , AAC nanoprobe specifically responds to Na 2 S undergoes absorption and fluorescence changes.

[0028] DPBF (4,6-diamino-2-phenylindole) was used as a reactive oxygen species (ROS) detection probe to measure the ROS production capacity of AAC nanoprobes under 660nm laser irradiation. 40μL of 100nM AAC nanoprobe was added to 160μL of 20mM PB buffer (pH=7.4), and sodium sulfide was added to a final concentration of 50μM. After 10 minutes of reaction, centrifugation was performed, and 100μL of the supernatant was taken for the solution phase illumination ROS production capacity determination experiment. The volume of the system for the ROS production capacity determination was 1mL, and the system contained 100μL of the supernatant after centrifugation of the above nanoprobe and 900μL of 120μM DPBF. A 660nm laser was used for irradiation at a power of 0.1W / cm 2 The total irradiation time was 5 min, and the changes in the absorption spectrum in the range of 300-600 nm were measured once every minute. 415nm The ROS production capacity was determined by the absorbance change at Figure 4 As shown in A, the AAC nanoprobe responds to Na 2 The ROS generation capacity of S gradually increased, proving that the AAC nanoprobe can respond to H 2 S generates reactive oxygen species under light for photodynamic therapy.

[0029] Study on the photothermal heating performance of nanoprobes: 1 mL of AAC nanoprobe containing 6 nM AuNPs and 50 μM Na 2 S is placed at 808nm (0.5W / cm 2 , 0.7W / cm 2 , 1W / cm 2 ) laser irradiation for 10 min. By immersing a thermocouple probe (φ=0.5 mm) in the solution, the temperature change of the sample at different times after laser irradiation was monitored in real time. Figure 4 As shown in B, the AAC nanoprobe responds to Na 2 After S, the temperature gradually increased under 808nm laser irradiation, and the temperature increased with the increase of power, proving that the AAC nanoprobe can respond to H 2 S produces photothermal effect under light and is used for photothermal therapy.

[0030] Example 5: Photoactivity and phototherapy effects of AAC nanoprobes at the cellular level like Figure 5 As shown in A, after co-incubation with liver cancer cells (SMMC-7721), AAC nanoprobes were illuminated at 660 nm (0.1 W / cm 2 , 5min), using DCFH-DA as a ROS probe, it showed clear green fluorescence, proving that it can effectively generate reactive oxygen species. Figure 5As shown in B, after incubation of AAC nanoprobe with tumor cells SMMC-7721, the AAC nanoprobe was exposed to 660nm (0.1W / cm 2 , 5min) plus 808nm (1W / cm 2 , 10min) irradiation, has the highest tumor cell killing ability. Therefore, AAC nanoprobes can be used in response to tumor H 2 When illuminated under S conditions, tumor cells are effectively killed through photodynamic and photothermal means.

[0031] Example 6: Photothermal heating of AAC nanoprobes in living tumors and their fluorescence imaging applications Figure 6 As shown in A, after constructing a subcutaneous SMMC-7721 tumor-bearing model in mice, the AAC nanoprobe was injected into the tumor site. As the time after injection increased, the 808nm (1W / cm 2 , 10min) irradiated the tumor site, and its temperature gradually increased. 2h after injection, the tumor temperature rose to about 50℃ (above 45℃ can kill tumor cells). Figure 6 B, After the AAC nanoprobe was injected into the tumor, the fluorescence of the tumor site responded to the tumor H compared with the normal subcutaneous tissue. 2 The fluorescence recovery of S was obvious, which proved that the AAC nanoprobe can be used in response to tumor H 2 S is used for in vivo tumor photothermal heating and fluorescence imaging applications.

[0032] Example 7: Application of AAC nanoprobes in photothermal therapy of living tumors The human hepatoma cell line SMMC-7721 was used to establish a nude mouse xenograft tumor model using BALB / c nude mice. 3 The mice were divided into groups at 14 days (recorded as day 0). On day 0, the newly prepared AAC nanoprobes were injected into the tumor and laser irradiation (660 nm, 0.2 W / cm 2 , 5min; 808nm, 1W / cm 2 , 10min), such as Figure 7 A. The results show that Figure 7 B, The tumor volume of mice in the treatment groups without drug administration and only laser irradiation or drug administration without any laser irradiation increased rapidly, indicating the low tumor inhibition effect of light alone and the low in vivo dark toxicity of AAC nanoprobes. In the treatment groups that were irradiated with a single laser (660nm or 808nm) after drug administration, tumor growth was inhibited to a certain extent, proving that AAC nanoprobes can respond to tumor H 2S can achieve tumor treatment under the condition of single laser irradiation, but the treatment effect is not good enough. When photodynamic / photothermal combined treatment is performed after drug administration, the treatment effect is better than the single treatment group of single laser irradiation after drug administration, and tumor growth is strongly inhibited, which shows the feasibility and effectiveness of optical combined treatment. It shows that AAC nanoprobe has the best tumor inhibition effect under laser combined irradiation, and can perform photodynamic / photothermal combined treatment on tumors. At the same time, Figure 7 As shown in C, during the tumor treatment, there was no significant difference in the weight of mice between different groups, indicating that the AAC nanoprobe has low in vivo toxicity and good safety. The mice were killed after the treatment on the 14th day, and the tumor tissue was removed and weighed ( Figure 7 D) and take photos ( Figure 7 E). The results showed that among the optical therapy groups, the combined therapy group (AAC+660+808nmLaser group) had the smallest tumor mass and size. This proved that AAC nanoprobe combined with laser therapy had a significant inhibitory effect on tumor growth, and the combined therapy group had the smallest tumor. H&E, Ki67 and TUNEL staining were used to observe and compare the histological damage, cell proliferation and apoptosis levels of tumor sections in different treatment groups. The results are shown in Figure 7 F. The results of H&E staining showed that the morphology of tumor cells changed seriously after combined optical therapy; the results of Ki67 staining showed that the combined treatment group also had the strongest inhibitory effect on tumor cell proliferation; the TUNEL staining of the treatment group also showed the most extensive fluorescence, indicating that the apoptosis level of tumor cells in the combined treatment group was the highest. The above results prove that the hydrogen sulfide-responsive aggregated gold nanoprobe of the present invention has the ability to treat tumors in vivo with combined photodynamic / photothermal therapy.

[0033] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A hydrogen sulfide responsive aggregated gold nanoprobe, characterized in that: The hydrogen sulfide responsive aggregated gold nanoprobe comprises gold nanoparticles, polyadenylic acid modified nucleic acid aptamer AS1411 and a photosensitizer; and the response target of the hydrogen sulfide responsive aggregated gold nanoprobe is hydrogen sulfide.

2. The hydrogen sulfide responsive aggregated gold nanoprobe according to claim 1, characterized in that: The gold nanoparticles are spherical or quasi-spherical with a particle size of 10-100 nm; the sequence of the polyadenylic acid modified nucleic acid aptamer AS1411 is: 5'-AAAAAAAAAATTTGGTGGTGGTGGTTGTGGTGGTGGTGG-3'; the photosensitizer is any one or more of dihydrochlorin e6, pyropheophorbide-a, protoporphyrin, and zinc phthalocyanine.

3. A method for preparing a hydrogen sulfide responsive aggregated gold nanoprobe according to any one of claims 1 to 2, characterized in that: Gold nanoparticles were synthesized, and then poly(A)-modified nucleic acid aptamer AS1411 was modified on the surface of the gold nanoparticles. Then, a photosensitizer was combined with the poly(A)-modified nucleic acid aptamer AS1411 modified on the surface of the gold nanoparticles to prepare a hydrogen sulfide-responsive aggregated gold nanoprobe.

4. The preparation method according to claim 3, characterized in that: The following steps are involved: 1) Synthesize gold nanoparticles by reducing tetrachloroauric acid with trisodium citrate; 2) Add polyadenylic acid modified nucleic acid aptamer AS1411 solution to the gold nanoparticle solution and mix well. After standing for 5 minutes, add 500 mM sodium citrate-hydrochloric acid buffer with a pH of 3. After shaking, add 500 mM sodium citrate-hydrochloric acid buffer with a pH of 3 again. After standing for 5 minutes, add 500 mM HEPES solution with a pH of 7.6 to adjust the pH of the system to neutral. After standing for 10 minutes, centrifuge, discard the supernatant, wash the precipitate with 10 mM PB buffer with a pH of 7.4 containing 20 mM NaCl, and resuspend it in 10 mM PB buffer with a pH of 7.4 to obtain AuNPs-AS1411 probe solution; 3) The AuNPs-AS1411 probe solution obtained in step 2) was centrifuged, the supernatant was discarded, and the precipitate was resuspended in 20 mM PB buffer to obtain an AuNPs-AS1411 probe solution resuspension; the photosensitizer solution was added to the obtained AuNPs-AS1411 probe solution resuspension and mixed, and the mixture was allowed to react for 2 h, and then centrifuged, the supernatant was discarded, and the precipitate was resuspended in 20 mM PBS buffer to obtain a hydrogen sulfide-responsive aggregated gold nanoprobe solution.

5. The preparation method according to claim 3, characterized in that: The molar ratio of gold nanoparticles to poly(A)-modified nucleic acid aptamer AS1411 is 1: 50-300, and the molar ratio of poly(A)-modified nucleic acid aptamer AS1411 to photosensitizer is 1: 1-20.

6. Use of the hydrogen sulfide responsive aggregated gold nanoprobe according to any one of claims 1 to 2 in the preparation of a hydrogen sulfide concentration quantitative reagent.

7. Use of the hydrogen sulfide responsive aggregated gold nanoprobe according to any one of claims 1 to 2 in the preparation of a fluorescent imaging agent.

8. Use of the hydrogen sulfide responsive aggregated gold nanoprobe according to any one of claims 1 to 2 in the preparation of tumor therapeutic agents.

9. The use according to claim 8, characterized in that: The treatment includes photodynamic therapy, photothermal therapy, and photodynamic and photothermal combined therapy.

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