Glutathione response confinement aggregation ratio type photoacoustic imaging probe and preparation method thereof
By designing a glutathione response limited-domain aggregation ratio-type photoacoustic imaging probe, the mechanism of reducing the distance between AuNP@MnO2 under GSH conditions is solved, and the imaging resolution and detection accuracy are improved through ratio imaging technology.
Patent Information
- Application Number
- CN202510059344.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-15
- Publication Date
- 2025-05-27
AI Technical Summary
The structure of existing nanomaterials is uncontrollable under complex physiological conditions, resulting in weakening or disappearing physical and chemical properties, making it difficult to achieve assembly and regulation under disease physiological conditions. At the same time, single-band photoacoustic imaging technology is prone to produce large measurement errors in the quantitative analysis of signal molecules in biological bodies.
A glutathione response limited-domain aggregation ratio-type photoacoustic imaging probe was designed. By forming a mesoporous silica coating between AuNP@MnO2 in gold nanovesicles, the degradation of the MnO2 layer under GSH conditions reduces the distance between AuNP@MnO2, thereby enhancing the plasma coupling effect, thereby realizing the generation of ratio-type photoacoustic signals.
This probe achieves highly controllable aggregation of gold nanovesicles under GSH conditions, enhances imaging resolution, reduces the misjudgment rate, and optimizes probe performance through ratio imaging method to improve detection accuracy.
Smart Images
Figure CN120037410A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a glutathione-responsive confined aggregation ratio-type photoacoustic imaging probe and a preparation method thereof.
Background Art
[0002] Due to its local surface plasmon resonance (LSPR) effect, gold (Au) nanoparticles have tunable and good optical absorption characteristics and have long been widely used as photoacoustic (PA) imaging contrast agents. By changing the particle size, inter-particle distance, and morphological characteristics of AuNPs, their absorption peaks can be changed, thereby changing their PA imaging signals. In 2011, Duan et al. innovatively prepared AuNPs modified with amphiphilic polymers and assembled them into gold nanovesicles. Due to the LSPR effect between adjacent AuNPs within the vesicle shell structure, their absorption was significantly higher than that of AuNPs. Therefore, the precise manipulation of the absorption intensity and wavelength range of plasmonic gold nanostructures through adjustment and self-assembly methods has opened a new door for the preparation of PA imaging contrast agents. However, due to the extremely complex tumor microenvironment, the upper-layer structures and properties of pre-self-assembled nanomaterials usually change under complex physiological conditions, including uncontrollable dissociation, aggregation, or transformation of the structures, accompanied by the weakening or even disappearance of their physicochemical properties. How to achieve the assembly regulation of nanomaterials under pathophysiological conditions and then achieve controllable physicochemical properties is a highly challenging scientific problem in the field of medical materials.
[0003] In addition, photoacoustic (PA) imaging has excellent spatio-temporal resolution and can be used to track the distribution of cells, nanoparticles, and tumor markers in vivo, map related images, and diagnose tumors. However, when using single-band PA imaging technology for quantitative analysis of signal molecules in vivo, since its imaging signal completely depends on the strength of the PA signal, this method is prone to large measurement errors. At the same time, there are many factors in the body that have nothing to do with the measurement results (such as the dosage of the probe, background signal, and uneven distribution of the probe in the lesion, etc.) that will affect the accuracy of imaging measurement. Ratiometric fluorescence imaging is based on two signal inputs, one is sensitive to specific target signals, and the other remains unchanged (usually used as an internal reference signal). Such a ratiometric imaging method has an embedded self-correction function, effectively overcoming the interference of external factors. Using this ratiometric imaging method to optimize the performance of the probe can solve many challenges encountered by traditional probes and provide a more reliable detection method. Therefore, it is crucial to design PA probes with reference signals and response signals for precise tracking and quantitative detection of in vivo biomarkers. More importantly, due to its local surface plasmon resonance (LSPR) effect, tunability, and good light absorption characteristics, AuNPs have been widely used as PA imaging contrast agents. When changing the particle size, particle spacing, and morphological characteristics of AuNPs, the absorption peak can be adjusted to generate ratiometric PA signals.
Summary of the Invention
[0004] The technical problem to be solved by the present invention is to provide a glutathione-responsive confined aggregation ratiometric photoacoustic imaging probe and its preparation method. Under GSH conditions, the distance between AuNPs in the gold nanovesicles decreases, enhancing the plasmon coupling effect between nanoparticles, achieving a high degree of controllability of the aggregation degree of gold nanovesicles, enabling the probe to more sensitively "respond" according to the GSH content in the tumor microenvironment, greatly enhancing the imaging resolution while reducing the misjudgment rate. 2
[0005] The present invention is implemented as follows:
[0006] A glutathione-responsive confined aggregation ratiometric photoacoustic imaging probe, the probe comprising gold nanovesicles, the gold nanovesicles being vesicles AuNP@MnO coated with mesoporous silica 2 Ve@mSiO 2 ;
[0007] The preparation method of the gold nanovesicles is as follows: MnO with GSH responsiveness is in-situ formed on the surface of AuNPs 2 , to obtain AuNP@MnO 2 ; then on AuNP@MnO 2The surface was modified with phosphoric acid-polystyrene block copolymer P-PS; then it was self-assembled to obtain plasma gold nanovesicles AuNP@MnO 2 Ve; finally, mesoporous silica was coated on the surface of AuNP@MnO 2 Ve for modification and stabilization, and finally GSH-responsive confined aggregation gold nanovesicles AuNP@MnO 2 Ve@mSiO 2 were obtained.
[0008] Furthermore, under GSH conditions, the distance between AuNP@MnO inside the vesicles of the gold nanovesicles 2 decreases.
[0009] Furthermore, the particle size of the AuNPs is 8-20 nm.
[0010] Furthermore, the thickness of MnO in the middle layer of the gold nanovesicles 2 is between 2-8 nm; the thickness of the surface-coated mSiO 2 layer is 0.5-10 nm.
[0011] Furthermore, a preparation method of a glutathione-responsive confined aggregation ratio-type photoacoustic imaging probe, the preparation steps of the probe are specifically as follows:
[0012] Step 1. The synthesis steps of AuNPs gold nanoparticles are as follows:
[0013] Chloroauric acid solution was added to ultrapure water, and the mixed solution was refluxed at 100±10 °C for 8±2 min; then sodium citrate solution was quickly added to the mixed solution, and the reaction was carried out for 10±2 min; finally, a blood-red AuNPs solution was obtained; after cooling to room temperature, it was stored in a 4 °C refrigerator for later use;
[0014] Step 2. Synthesis of AuNP@MnO 2 nanoparticles:
[0015] The AuNPs solution was mixed with KMnO 4 under stirring, and the L-AA aqueous solution of ascorbic acid was slowly dropped into the above mixed solution; after the reaction was completed, the obtained AuNP@MnO 2 nanoparticles were collected by centrifugation;
[0016] Step 3. Preparation of AuNP@MnO 2 Ve:
[0017] In the presence of AuNP@MnO 2SH-PEG and phosphoric acid-polystyrene were added to dichloromethane of nanoparticles. After the mixture reacted, the product was collected by centrifugation to remove free polymers, and amphiphilic AuNP@MnO 2 @P-PS NPs was obtained;
[0018] AuNP@MnO 2 @P-PS NPs was dispersed in dichloromethane, and then an aqueous solution of 5% polyvinyl alcohol PVA was added for ultrasonic emulsification; at room temperature, dichloromethane completely evaporated to form AuNP@MnO 2 Ve; then the obtained AuNP@MnO 2 Ve was washed with deionized water to remove excess PVA, and AuNP@MnO 2 Ve was obtained;
[0019] Step 4. Synthesis of AuNP@MnO 2 Ve@mSiO 2 :
[0020] Sodium hydroxide solution was added to cetyltrimethylammonium bromide CTAB solution, and then AuNP@MnO 2 Ve was stirred; then 5% methanol tetraethoxysilane TEOS and 5% APTES were added to the solution, and they were added 3 times every 30 min at 33°C; after the reaction was completed, the product was centrifuged first, and then dispersed in ethanol 2-3 times; after ultrasonic treatment for 0.5 h, centrifugation was performed to obtain vesicles AuNP@MnO 2 Ve@mSiO 2 .
[0021] Further, the molar ratio of each reactant in Step 2 is AuNPs: KMnO 4 : L-AA = 5-10: 0.1-1: 0.01-10.
[0022] Further, the mass ratio of each reactant in Step 3 is AuNP@MnO 2 nanoparticles: SH-PEG: phosphoric acid-polystyrene = 2-10: 0.1-1: 0.5-5.
[0023] Further, the amount of each reactant in Step 4 is AuNP@MnO 2 Ve: CTAB: TEOS: APTES = 2-10: 0.3-2: 0.1-4: 0.1-2.
[0024] The present invention has the following advantages:
[0025] Ratio-based fluorescence imaging is based on two signal inputs, one sensitive to specific target signals and the other remaining unchanged (usually used as an internal reference signal). Therefore, it has a self-calibration function and effectively overcomes the interference of external factors. The present invention provides a GSH-responsive ratio-based PA imaging probe that can be realized at the in vivo level, namely, a plasmonic gold nanoassembly AuNP@MnO 2 Ve@mSiO 2 , which generates ratio-based photoacoustic signals through a "confined aggregation strategy". The performance of the probe can be optimized by using ratio imaging methods, greatly improving the detection accuracy of the probe.
[0026] For the gold nanovesicles of the ratio-based photoacoustic imaging probe of the present invention, under GSH conditions, the distance between AuNP@MnO 2 inside the vesicles decreases, enhancing the plasmon coupling effect between nanoparticles, resulting in a red shift in absorption. It can be used to monitor / quantify the dynamic process of self-assembly in real time, can be used as a photoacoustic probe to locate tumors, and is used for in vivo tumor PA imaging and tumor volume measurement. At the same time, by introducing a glutathione response mechanism, a high degree of controllability of the aggregation degree of gold nanovesicles is achieved, enabling the ratio-based photoacoustic imaging probe to more sensitively "respond" according to the GSH content in the tumor microenvironment, precisely controlling the physicochemical properties of the nanoprobe as needed, greatly enhancing the imaging resolution while reducing the misjudgment rate.
Description of the Drawings
[0027] The present invention will be further described below with reference to the drawings in conjunction with the embodiments.
[0028] Figure 1 It is a transmission electron microscope (TEM) image of AuNPs prepared in the embodiment of the present invention.
[0029] Figure 2 It is a TEM image of AuNP@MnO 2 prepared in the embodiment of the present invention.
[0030] Figure 3 It is an energy-dispersive X-ray spectroscopy (EDS) elemental distribution map of AuNP@MnO 2 NPs prepared in the embodiment of the present invention.
[0031] Figure 4 It is a TEM image of AuNP@MnO 2 Ve prepared in the embodiment of the present invention.
[0032] Figure 5 It is a TEM image of AuNP@MnO 2 Ve@mSiO 2 prepared in the embodiment of the present invention.
[0033] Figure 6 TEM image of AuNP@MnO 2 Ve@mSiO 2 after being stimulated by 1 mM GSH in the embodiment of the present invention.
[0034] Figure 7 UV absorption spectrum of AuNP@MnO 2 Ve@mSiO 2 after being stimulated by 1 mM GSH in the embodiment of the present invention.
[0035] Figure 8 Photoacoustic imaging map of mouse tumor of the ratio-type photoacoustic imaging probe AuNP@MnO 2 Ve@mS iO 2 prepared in the embodiment of the present invention.
Specific Embodiments
[0036] The technical solutions of the present invention will be clearly and completely described below in conjunction with Figure 1-8 the accompanying drawings and specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. For reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.
[0037] Embodiment
[0038] 1 Preparation steps
[0039] 1.1 Synthesis of phosphoric acid-polystyrene, the specific steps are as follows:
[0040] Step A: Synthesis of initiator. 770 mg (1 eq) of dimethyl 2-hydroxyethylphosphonate is dissolved in 20 mL of dichloromethane, and triethylamine (3 eq) is added. The mixed solution is stirred in an ice-water bath for 30 min to cool down the reaction system. 2-bromo-2-methylpropionyl bromide (1.2 eq) is dissolved in 5 mL of dichloromethane and added dropwise to the above mixed solution at a rate of one to two drops per second; then, after reacting in an ice-water bath for 1 h, the temperature is raised to room temperature and the reaction is carried out for 8 h. After the reaction is completed, the obtained mixed solution is concentrated to 5 mL, then 15 mL of ethyl acetate is added, and then the mixed solution is washed three times with saturated sodium bicarbonate solution and deionized water in sequence. The organic phase of the mixed solution is collected, then anhydrous sodium sulfate is added to remove water, and the excess solvent is removed by rotary evaporation to obtain a viscous yellow-brown liquid.
[0041] Step B: Synthesis of ATRP polymer. 1 g of styrene monomer and 28 μL of N,N,N',N'',N''-pentamethyldiethylenetriamine (PMDETA) were dissolved in 3 mL of anisole, and then 30.3 mg of the above initiator was added. After the mixed solution was degassed for 30 min, 17 mg of copper(I) bromide was weighed and added to the mixed solution, and degassing was continued for 10 min (at this time, the color of the mixed solution was green). The mixed solution was placed in an oil bath at 105 °C and reacted for 12 h (the color of the solution changed to yellow after 30 min, indicating that the reaction occurred). After the reaction, 5 mL of dichloromethane was added for dilution, and copper ions were removed using a silica gel column. The silica gel column was rinsed with dichloromethane, and the product was collected and concentrated by rotary evaporation. The concentrated solution was dropped into diethyl ether, and the precipitated white solid was collected and dried.
[0042] Step C: Demethylation synthesis of phosphate ester. Weigh ATRP polymer (1 eq) and dissolve it in 10 mL of dichloromethane. Trimethylsilyl bromide (5 - 10 eq) was added with stirring, and after reacting for 12 h, 5 mL of methanol was added and the reaction continued for 12 h. The mixed solution was concentrated by rotary evaporation (the final volume was about 8 mL). Then, the mixed solution was dropped into diethyl ether, and the precipitated white solid was collected and dried.
[0043] 1.2 Synthesis of AuNPs gold nanoparticles
[0044] Chloroauric acid solution (1 mL, 20 mg mL -1 ) was added to 200 mL of ultrapure water, and the mixed solution was refluxed at about 100 °C for 8 min; then, 5 mL of a sodium citrate solution containing 12 mg mL -1 was quickly added to the mixed solution, and the reaction was carried out for 10 min; finally, a blood-red 13 nm AuNPs solution was obtained. After cooling to room temperature, it was stored in a refrigerator at 4 °C for standby.
[0045] 1.3 Synthesis of AuNP@MnO 2 nanoparticles
[0046] The AuNPs solution (0.5 mg mL -1 , 100 mL) was mixed with 42 mg of KMnO 4 under stirring. After 5 min, 1 mL of an aqueous solution of ascorbic acid (L-AA) at 50 mg mL -1 was slowly dropped into the above mixed solution. During the reaction, the color of the solution changed from purple-red to yellow-brown and finally to black. The AuNP@MnO 2 nanoparticles obtained were collected by centrifugation.
[0047] 1.4 Preparation of AuNP@MnO 2 Ve
[0048] In 10 mL of dichloromethane containing AuNP@MnO 2 NPs (2 mg mL -1 ), 4 mg of SH-PEG and 8 mg of phosphoric acid-polystyrene were added. After the mixture reacted for 8 h, the product was collected by centrifugation (15,000×g, 20 min) to remove free polymers, and amphiphilic AuNP@MnO 2 @P-PS NPs were obtained.
[0049] The water-in-oil emulsion method was used to prepare AuNP@MnO 2 Ve: 5 mg of AuNP@MnO 2 @P-PS NPs were dispersed in 200 μL of dichloromethane, and then 5 mL of 5% polyvinyl alcohol PVA (Mw: 9000 - 10000) aqueous solution was added for ultrasonic emulsification. At room temperature, dichloromethane completely evaporated to form AuNP@MnO 2 Ve. Finally, the obtained AuNP@MnO 2 Ve was washed three times with deionized water to remove excess PVA.
[0050] 1.5 Synthesis of AuNP@MnO 2 Ve@mSiO 2
[0051] 200 μL of 0.1 M sodium hydroxide (NaOH) solution was added to cetyltrimethylammonium bromide (CTAB) solution, and AuNP@MnO 2 Ve was added and stirred. 120 μL of 5% methanol tetraethoxysilane (TEOS) and 48 μL of 5% APTES were added to the solution three times at 33 °C every 30 min. After reacting for 48 h, the product was centrifuged at 7000 rpm for 10 min, and then dispersed twice in ethanol (2 mg / mL). After ultrasonic treatment for 0.5 h, AuNP@MnO 2 Ve@mSiO 2 with mesoporous silica coating was obtained by centrifugation.
[0052] 2 Experimental Results
[0053] First, 10 nm AuNP was synthesized by the sodium citrate reduction method (as Figure 1 shown). Subsequently, by the in-situ reduction method of KMnO 4 , MnO 2 was coated on the outer surface of AuNPs to obtain AuNP@MnO 2 . As shown in the TEM image of AuNP@MnO Figure 2 , it has a core-shell structure with a particle size of about 13 nm, in which MnO 2 ...2 The layer thickness is about 3 nm (MnO 2 The thickness of the layer can be controlled by adjusting the initial dosages of the reducing agent L-AA and KMnO 4 ). As Figure 3 shown, the STEM-Mapping elemental distribution analysis results of AuN P@MnO 2 nanoparticles further confirmed the MnO 2 layer coated on its surface.
[0054] Next, AuNP@MnO 2 was surface-modified with P-PS to obtain amphiphilic AuNP@MnO 2 @P-PS, and then AuNP@MnO 2 Ve (as Figure 4 shown) was prepared by the ultrasonic emulsification method. Then, mesoporous silica was coated on the surface of AuNP@MnO 2 Ve to obtain AuNP@MnO 2 Ve@mS iO 2 . mSiO 2 rendered AuNP@MnO 2 Ve@mSiO 2 with good stability. The size and internal microstructure of AuNP@MnO 2 Ve@mSiO 2 vesicles were observed by TEM, and the results showed that AuNP@MnO 2 Ve@mSiO 2 had a typical nanovesicle structure with an average diameter of 140 nm (as Figure 5 shown). In addition, AuNP@MnO 2 NPs were uniformly distributed in the vesicles, and the gap between them was about 8 - 10 nm, which was caused by the blocking effect of the MnO 2 layer. In addition, an outer layer structure with a lower electron density was observed in the AuNP@MnO 2 Ve@mSiO 2 vesicles, indicating that the mSiO 2 coating was uniformly deposited on the outermost layer of the vesicles.
[0055] As Figure 6 shown, when AuNP@MnO 2 Ve@mSiO 2 was incubated with 1 mM GSH, the AuNPs inside gradually confined and aggregated over time. This was because the mSiO 2 coated on the surface of AuNP@MnO 2Permits the free entry and exit of GSH into and out of the vesicles, so the MnO modified on the surface of AuNP 2 layer is etched into Mn 2+ by GSH, and the distance between AuNP@MnO 2 NPs in the vesicles gradually decreases from the original 8 nm until they are closely attached, ultimately resulting in the aggregation of AuNP@MnO 2 NPs in the vesicles into a more compact entity.
[0056] As Figure 7 shown, with the increase in the concentration of GSH, the absorption intensity of AuNP@MnO 2 Ve@mSiO 2 at 880 nm increases with time, indicating the degradation of MnO induced by GSH 2 and the aggregation of Au NPs in the vesicles; while at this time, the absorption of AuNP@MnO 2 Ve@mSiO 2 at 680 nm continuously blue-shifts. This is because the confined aggregation of Au NPs in the AuNP@MnO 2 Ve@mSiO 2 vesicles significantly enhances the plasmon coupling effect between Au NPs, enhancing its absorption intensity at 880 nm. In summary, after GSH response, the confined aggregation of Au NPs in the AuNP@MnO 2 Ve@mSiO 2 vesicles enables them to be used as photoacoustic probes to locate tumors (as Figure 8 ) shown.
[0057] In summary, the present invention has the following advantages:
[0058] Ratio-based fluorescence imaging is based on two signal inputs, one sensitive to a specific target signal and the other remaining unchanged (usually used as an internal reference signal), so it has a self-calibration function and effectively overcomes the interference of external factors. The present invention provides a GSH-responsive ratio-based PA imaging probe that can be realized at the in vivo level, namely the plasmonic gold nanoassembly AuNP@MnO 2 Ve@mSiO 2 , which generates ratio-based photoacoustic signals through the "confined aggregation strategy" and can use the ratio imaging method to optimize the performance of the probe, greatly improving the detection accuracy of the probe.
[0059] For the gold nanovesicles of the ratio-based photoacoustic imaging probe of the present invention under GSH conditions, AuNP@MnO in the vesicles 2The distance between them decreases, enhancing the plasmon coupling effect between nanoparticles, resulting in a red shift in absorption. It can be used as a photoacoustic probe to locate tumors and for in vivo tumor PA imaging and tumor volume measurement. At the same time, by introducing a glutathione-responsive mechanism, a high degree of controllability over the aggregation degree of gold nanovesicles is achieved, enabling this ratio-type photoacoustic imaging probe to more sensitively "respond" according to the GSH content in the tumor microenvironment, precisely controlling the physicochemical properties of the nanoprobe as needed, greatly enhancing the imaging resolution while reducing the misjudgment rate.
[0060] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we described are illustrative rather than used to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be covered by the scope protected by the claims of the present invention.
Claims
1. A glutathione-responsive confined aggregation ratio photoacoustic imaging probe, characterized in that: The probe comprises a gold nanocapsule, wherein the gold nanocapsule is a vesicle AuNP@MnO2 Ve@mSiO2 formed by coating with mesoporous silica; The preparation method of the gold nanovesicles is as follows: in situ forming MnO2 with GSH responsiveness on the surface of AuNPs to obtain AuNP@MnO2; then modifying the surface of AuNP@MnO2 with phosphoric acid-polystyrene block copolymer P-PS; then self-assembling it to obtain plasma gold nanovesicles AuNP@MnO2 Ve; finally, mesoporous silica is coated on the surface of AuNP@MnO2 Ve to modify and stabilize it, and finally GSH responsive confined aggregated gold nanovesicles AuNP@MnO2Ve@mSiO2 are obtained.
2. A glutathione-responsive confined aggregation ratio photoacoustic imaging probe according to claim 1, characterized in that: Under GSH conditions, the distance between AuNP@MnO2 in the gold nanovesicles decreases.
3. A glutathione-responsive confined aggregation ratio photoacoustic imaging probe according to claim 1, characterized in that: The particle size of the AuNPs is 8 to 20 nm.
4. A glutathione-responsive confined aggregation ratio photoacoustic imaging probe according to claim 1, characterized in that: The thickness of the MnO2 in the middle layer of the gold nanocapsule is between 2 and 8 nm; the thickness of the mSiO2 layer wrapped on the surface is between 0.5 and 10 nm.
5. A method for preparing a glutathione-responsive confined aggregation ratio photoacoustic imaging probe, characterized in that: The preparation steps of the probe are as follows: Step 1: The synthesis steps of AuNPs gold nanoparticles are as follows: Add chloroauric acid solution to ultrapure water, and reflux the mixed solution at 100±10℃ for 8±2min; then quickly add sodium citrate solution to the mixed solution and react for 10±2min; finally obtain a blood-red AuNPs solution; after cooling to room temperature, store in a 4℃ refrigerator for later use; Step 2: Synthesis of AuNP@MnO2 nanoparticles: The AuNPs solution and KMnO4 were mixed under stirring, and the ascorbic acid L-AA aqueous solution was slowly dripped into the mixed solution; after the reaction was completed, the obtained AuNP@MnO2 nanoparticles were collected by centrifugation; Step 3, preparation of AuNP@MnO2 Ve: SH-PEG and phosphoric acid-polystyrene were added to dichloromethane containing AuNP@MnO2 nanoparticles. After the mixture reacted, the product was collected by centrifugation to remove free polymers, thereby obtaining amphiphilic AuNP@MnO2@P-PS NPs. AuNP@MnO2@P-PS NPs were dispersed in dichloromethane, and then 5% polyvinyl alcohol (PVA) aqueous solution was added for ultrasonic emulsification; at room temperature, dichloromethane was completely evaporated to form AuNP@MnO2Ve; the obtained AuNP@MnO2 Ve was then washed with deionized water to remove excess PVA, thus obtaining AuNP@MnO2 Ve; Step 4: Synthesis of AuNP@MnO2Ve@mSiO2: Sodium hydroxide solution was added to hexadecyltrimethylammonium bromide (CTAB) solution, and then AuNP@MnO2 Ve was added and stirred; then 5% methanol tetraethoxysilane (TEOS) and 5% APTES were added to the solution at 33°C, three times every 30 minutes; after the reaction was completed, the product was first centrifuged and then dispersed in ethanol for 2 to 3 times; after ultrasonication for 0.5 hours, the product was centrifuged to obtain mesoporous silica-coated vesicles AuNP@MnO2 Ve@mSiO2.
6. The method for preparing a glutathione-responsive confined aggregation ratio photoacoustic imaging probe according to claim 5, characterized in that: The molar ratio of the reactants in step 2 is AuNPs:KMnO4:L-AA=5-10:0.1-1:0.01-10.
7. The method for preparing a glutathione-responsive confined aggregation ratio photoacoustic imaging probe according to claim 5, characterized in that: The mass ratio of the reactants in step 3 is AuNP@MnO2 nanoparticles: SH-PEG: phosphoric acid-polystyrene = 2-10: 0.1-1: 0.5-5.
8. The method for preparing a glutathione-responsive confined aggregation ratio photoacoustic imaging probe according to claim 5, characterized in that: The amount of each reactant in step 4 is AuNP@MnO2 Ve: CTAB: TEOS: APTES = 2-10: 0.3-2: 0.1-4: 0.1-2.