Near-infrared / glutathione dual-responsive nanocapsules integrating targeting, controllable drug delivery and photoacoustic imaging and preparation method thereof

Abstract

The application discloses a kind of near-infrared / glutathione dual-response nanovesicle integrated targeting, controllable drug delivery and photoacoustic imaging and a preparation method thereof, and the nanovesicle can release doxorubicin controllably after near-infrared / glutathione dual-response and generate reactive oxygen to realize the chemical-photodynamic combined therapy of tumor region, is prepared by doxorubicin amino iridium complex and carboxylated transferrin through amide reaction, wherein the doxorubicin amino iridium complex is prepared by loading doxorubicin in amphiphilic iridium complex containing quaternary ammonium group and disulfide bond;Experimental results show that the nanovesicle can realize controllable release of doxorubicin, and based on light-induced effect, the nanovesicle is excited and generates reactive oxygen, and the nanovesicle can be preferentially targeted and rapidly accumulated in tumor tissue, and plays antitumor therapeutic effect under the tumor microenvironment rich in high concentration of glutathione and local near-infrared stimulation;Meanwhile, the nanovesicle can monitor tumor treatment effect in real time, realize real-time pathological analysis and rapid medical diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a near-infrared / glutathione dual-response nanocapsule integrating targeted, controllable drug delivery and photoacoustic imaging, and its preparation method. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common type of cancer, characterized by a high recurrence rate and difficult-to-control prognosis. Most HCC patients are diagnosed at an advanced stage, primarily due to the carcinoma's insidious nature, often presenting no symptoms in its early stages. To date, treatments for HCC mainly include surgery, liver transplantation, ablation, chemotherapy, and radiotherapy. Considering its high cytotoxicity and phototoxicity, combining chemotherapy with photodynamic therapy can significantly improve the therapeutic efficacy of HCC. For example, Chinese patent CN113512066A discloses a sorafenib-ruthenium complex, its preparation method, and its applications. The sorafenib-ruthenium complex prepared in this invention exhibits low toxicity to Hep-G2 liver tumor cells and normal liver cells under dark conditions; however, its toxicity to liver tumor cells is enhanced under light conditions. It can be used for photoactivated chemotherapy of tumors and has potential applications in the preparation of targeted drugs for liver cancer. Therefore, combining photodynamic / chemotherapy is a promising approach to improve the treatment efficacy of HCC and reduce the mortality rate of liver cancer patients.

[0003] As a crucial component of photodynamic therapy, photosensitizers are activated upon light exposure, generating highly destructive reactive oxygen species (ROS) that induce DNA and mitochondrial damage. Due to their advantages such as minimal invasiveness, high spatiotemporal precision, and controllability, numerous photosensitizers with different compositions and structures have been used to explore treatments for hepatocellular carcinoma. Iridium (Ir) complex-based photosensitizers can generate not only oxygen-independent type I ROS (such as superoxide radicals) through photoinduced electron transfer, but also oxygen-dependent type II ROS (such as singlet oxygen) through photoinduced energy transfer. This dual-mode catalytic generation of ROS by Ir complexes avoids the limitations of photodynamic therapy efficiency under tumor hypoxia and shows promising application prospects for the treatment of highly metastatic, orthotopic hepatocellular carcinoma (Nam JS, et al. J. Am. Chem. Soc. 2016, 138, 10968-10977). Despite these advantages, the application of Ir complex-based photosensitizers in inhibiting tumor growth is still limited by a series of obstacles, including weak hydrophilicity, low targeting ability, and limited intelligent reactivity.

[0004] To address the aforementioned limitations, designing Ir complexes containing functionalized functional groups is a promising strategy for hepatocellular carcinoma therapy. Firstly, quaternary ammonium (QA), a widely used hydrophilic functional group, has been shown to modulate host-ligand-based... and auxiliary ligands The hydrophilic and hydrophobic properties of transition metal complexes. In particular, QA can modulate the photophysical / electrochemical properties of Ir complexes by regulating charge redistribution and maintaining the dynamic balance within the host / auxiliary ligands. In the presence of QA, Ir complexes not only exhibit good biocompatibility but also avoid fluorescence aggregation-induced quenching by reducing the probability of π-π stacking of the complex (An LL, J. Cleaner Prod. 2022, 368, 133219). Secondly, transferrin (TF), a receptor-mediated protein, can target primary proliferating malignant cells containing transferrin receptors (mainly transferrin receptor 2) and is a key ligand for achieving precise localization based on Ir complexes. Furthermore, after accurate localization to the tumor site, how to achieve intelligent controlled release is another important factor affecting antitumor efficacy. On the one hand, near-infrared irradiation (NIR) is an important exogenous stimulus for Ir complexes, which helps trigger intelligent drug release and induce the generation of different types of reactive oxygen species. On the other hand, due to the high concentration of glutathione (GSH) in the tumor microenvironment, using GSH as an important exogenous stimulus can achieve the controlled release of chemotherapeutic drugs at the tumor site (Liu J, J. Am. Chem. Soc. 2022, 144(11): 4799-4809.). In this context, synthesizing Ir complexes with dual NIR / GSH responses is a strategy to fully improve the drug utilization of nanoparticles at the tumor site and achieve high-yield reactive oxygen species to damage tumor cells. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a near-infrared / glutathione dual-response nanocapsule that integrates targeted, controllable drug delivery and photoacoustic imaging, as well as its preparation method.

[0006] The present invention adopts the following technical solution:

[0007] A near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging, wherein the nanovesicles can controllably release doxorubicin and generate type I / II reactive oxygen species to achieve chemophotodynamic therapy of tumor regions, is prepared by an amide reaction of doxorubicin aminoiridium complex and carboxylated transferrin.

[0008] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0009] (1) Dissolve 4-(2-pyridyl)benzaldehyde in ethanol and slowly add sodium borohydride under ice-water bath conditions. Stir overnight at room temperature to obtain compound 1.

[0010] (2) Dissolve compound 1 obtained in step (1) in dichloromethane, and add phosphorus tribromide dropwise under ice-water bath conditions, stirring overnight at room temperature; then add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2;

[0011] (3) Dissolve compound 3 obtained in step (2) in tetrahydrofuran, and slowly add N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained.

[0012] (4) Thionyl chloride was slowly added dropwise to a dichloromethane solution containing 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4;

[0013] (5) Dissolve compound 4 obtained in step (4) in tetrahydrofuran containing triethylamine, and slowly add tetrahydrofuran containing cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0014] (6) Dissolve compound 3 obtained in step (3), compound 5 obtained in step (5) and hydrated iridium trichloride in ethylene glycol ethyl ether, heat and reflux overnight under nitrogen atmosphere to obtain aminoiridium complex Ir-NH2;

[0015] (7) Disperse the ethanol containing doxorubicin in the aqueous solution of Ir-NH2 obtained in step (6), mix and stir thoroughly to obtain the doxorubicin aminoiridium complex DOX@Ir-NH2;

[0016] (8) The aqueous solution containing transferrin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is slowly added to the aqueous solution of DOX@Ir-NH2 obtained in step (7). After thorough mixing, the nanovesicles DOX@Ir-TF are obtained.

[0017] Further, in step (1), the molar ratio of 4-(2-pyridyl)benzaldehyde to sodium borohydride is 3:5; the volume of ethanol is 20-60 mL; the stirring speed is 100-700 rpm; after stirring overnight, the organic phase is extracted with dichloromethane; the solvent is removed by rotary evaporation under reduced pressure to obtain compound 1 as a white powder.

[0018] Furthermore, in step (2), the reaction mass of compound 1 is 0.4-0.7 g, the volume of dichloromethane is 20-60 mL, and the volume of phosphorus tribromide is 0.5-4.0 mL. After stirring overnight, the organic phase is extracted with dichloromethane, and the solvent is removed by rotary evaporation under reduced pressure to obtain compound 2 as a white powder.

[0019] Further, in step (3), the molar ratio of compound 2 to N,N-dimethylhexadecane-1-amine is 2:3, the volume range of tetrahydrofuran is 20-70 mL, and after stirring overnight, impurities are removed to obtain compound 3; the impurity removal process is as follows: the reaction liquid is centrifuged at a rate of 12000-18000 rps and a temperature range of 17-21 °C, and then the precipitate is washed three times with ethanol, and after vacuum drying, a light pink powder compound 3 is obtained.

[0020] Furthermore, in step (4), the amount of 2,2'-bipyridine-4,4'-dicarboxylic acid ranges from 4.0 to 7.0 mmol, the volume of thionyl chloride ranges from 20 to 60 mL, and after stirring overnight, the solvent is removed by rotary evaporation under reduced pressure to obtain a light green powder compound 4.

[0021] Further, in step (5), the mass range of compound 4 is 100-400 mg, the volume range of tetrahydrofuran and triethylamine used to dissolve compound 4 is 30-70 mL and 100-400 μL, respectively; the volume range of tetrahydrofuran used to dissolve cystamine dihydrochloride is 20-60 mL, after stirring overnight, the organic phase is extracted with dichloromethane, and the solvent is removed by rotary evaporation under reduced pressure to obtain compound 5 as a white powder.

[0022] Furthermore, in step (6), the mass ranges of compound 3, compound 5 and hydrated iridium trichloride are 50-300 mg, 30-70 mg and 10-30 mg, respectively. After rotary evaporation under reduced pressure to remove the solvent, a yellow powdery aminoiridium complex Ir-NH2 is obtained.

[0023] Furthermore, in step (7), the mass ratio of doxorubicin to Ir-NH2 is 1:1, the mass range of doxorubicin is 1 to 15 mg, the volume range of ethanol is 5 to 40 mL, and the solvent is removed by rotary evaporation under reduced pressure to obtain a yellow powder of doxorubicin aminoiridium complex DOX@Ir-NH2.

[0024] Furthermore, in step (8), the mass ratio of transferrin and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:2, and the volume of secondary water ranges from 1 to 10 mL; the mass of DOX@Ir-NH2 and N-hydroxysuccinimide is 1 mg, and the volume of secondary water ranges from 1 to 10 mL.

[0025] As can be seen from the above description of the present invention, compared with the prior art, the beneficial effects of the present invention are:

[0026] First, the DOX@Ir-TF nanovesicles prepared in this invention, after intravenous injection, precisely target the tumor region, induce reactive oxygen species (ROS) generation following a dual NIR / GSH response, and controllably release chemotherapeutic drugs. Cellular experiments demonstrate that the DOX@Ir-TF nanovesicles possess specific targeting to the cell membrane and highly efficient cellular uptake capabilities. Simultaneously, in vivo experiments prove their outstanding anticancer activity, exhibiting significant inhibitory effects on hepatocellular carcinoma under synergistic chemo / photodynamic therapy. Furthermore, the prepared DOX@Ir-TF nanovesicles also demonstrate impressive photoacoustic imaging capabilities, which facilitates real-time monitoring of the nanovesicles' therapeutic process in vivo, enabling rapid pathological analysis or diagnosis of tumors. In summary, this single-molecule nanovesicle delivery system possesses targeted delivery, dual-response drug release, ROS generation, and real-time monitoring capabilities, demonstrating significant therapeutic efficacy against hepatocellular carcinoma.

[0027] Second, the presence of QA and TF in the DOX@Ir-NH2 nanovesicles prepared by this invention ensures that the complex self-assembles to form a nanovesicle structure with an internal hydrophobic and external hydrophilic structure; and the active sites in QA are beneficial to improving the photophysical properties of DOX@Ir-NH2 nanovesicles and enhancing their photoinduced oxygen production capacity.

[0028] Third, the DOX@Ir-NH2 nanovesicles prepared in this invention exhibit good biocompatibility and the ability to target tumor cells, and can synergistically regulate the drug release rate through dual responses of exogenous NIR and endogenous GSH, demonstrating excellent chemotherapy efficacy.

[0029] Fourth, through its dual-response properties, the prepared DOX@Ir-NH2 nanovesicles can dynamically regulate reactive oxygen species levels and improve oxidative stress in the tumor microenvironment, thereby reducing chemotherapy drug resistance and achieving chemo / photodynamic therapy for liver cancer. Attached Figure Description

[0030] Figure 1 This is a schematic diagram illustrating the preparation process and application of GSH / NIR dual-response nanovesicles based on the present invention.

[0031] Figure 2 The following are synthetic route diagrams for different types of Ir complexes in Examples 1-10, where i: is the synthetic route diagram for the aminoiridium complex Ir-NH2; ii: is the synthetic route diagram for the dual-responsive DOX@Ir-NH2 nanovesicles.

[0032] Figure 3The following parameters were used to evaluate the GSH / NIR dual response, drug release, and reactive oxygen species (ROS) generation capabilities of the prepared DOX@Ir-NH2 nanovesicles: i: Schematic diagram of the mechanism of ROS generation by DOX@Ir-TF nanovesicles based on GSH / NIR dual response; ii: Representative transmission electron microscopy after treatment with DOX@Ir-TF, DOX@Ir-TF+GSH-, and DOX@Ir-TF+NIR+GSH-; iii: Dynamic light scattering analysis; V: Particle size distribution map of the corresponding nanovesicles; IV: DOX release curves from DOX@Ir-TF nanovesicles with / without GSH / NIR dual stimulation response; VI: UV-Vis absorption spectra of DOX@Ir-TF nanovesicles with / without GSH response; VII: Fluorescence intensity map of DOX@Ir-TF nanovesicles after co-culturing with ROS fluorescent markers after different irradiation times.

[0033] Figure 4 Representative confocal electron microscope images of reactive oxygen species generation in iii:HepG2 cells from various nanovesicle groups (such as PBS, DOX@Ir-NH2, DOX@Ir-TF, DOX@Ir-NH2+NIR, and DOX@Ir-TF+NIR).

[0034] Figure 5 Photoacoustic imaging of DOX@Ir-TF nanovesicles mediated by NIR / GSH dual response. Detailed Implementation

[0035] The present invention will be further described below through specific embodiments.

[0036] A near-infrared / glutathione dual-responsive nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging is proposed. The nanovesicles can controllably release doxorubicin and generate type I / II reactive oxygen species to achieve chemophotodynamic therapy of tumor regions. It is prepared by an amide reaction of doxorubicin aminoiridium complex and carboxylated transferrin. First, an amphiphilic Ir-NH2 complex with QA group and disulfide bond is prepared. Then, the anticancer drug DOX is loaded into the complex framework to form DOX@Ir-NH2 complex. The amino-exposed DOX@Ir-NH2 complex is combined with carboxylated TF through an amide reaction, and finally DOX@Ir-TF nanovesicles with GSH / NIR dual response are prepared.

[0037] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0038] (1) Dissolve 4-(2-pyridyl)benzaldehyde in ethanol and slowly add sodium borohydride under ice-water bath conditions. Stir overnight at room temperature to obtain compound 1.

[0039] (2) Dissolve compound 1 obtained in step (1) in dichloromethane, and add phosphorus tribromide dropwise under ice-water bath conditions, stirring overnight at room temperature; then add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2;

[0040] (3) Dissolve compound 3 obtained in step (2) in tetrahydrofuran, and slowly add N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained.

[0041] (4) Thionyl chloride was slowly added dropwise to a dichloromethane solution containing 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4;

[0042] (5) Dissolve compound 4 obtained in step (4) in tetrahydrofuran containing triethylamine, and slowly add tetrahydrofuran containing cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0043] (6) Dissolve compound 3 obtained in step (3), compound 5 obtained in step (5) and hydrated iridium trichloride in ethylene glycol ethyl ether, heat and reflux overnight under nitrogen atmosphere to obtain aminoiridium complex Ir-NH2;

[0044] (7) Disperse the ethanol containing doxorubicin in the aqueous solution of Ir-NH2 obtained in step (6), mix and stir thoroughly to obtain the doxorubicin aminoiridium complex DOX@Ir-NH2;

[0045] (8) The aqueous solution containing transferrin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide is slowly added to the aqueous solution of DOX@Ir-NH2 obtained in step (7). After thorough mixing, the nanovesicles DOX@Ir-TF are obtained.

[0046] In step (1), the molar ratio of 4-(2-pyridyl)benzaldehyde to sodium borohydride is 3:5; the volume of ethanol is 20-60 mL; the stirring speed is 100-700 rpm; after stirring overnight, the organic phase is extracted with dichloromethane; the solvent is removed by rotary evaporation under reduced pressure to obtain compound 1, which is a white powder.

[0047] In step (2), the reaction mass of compound 1 is 0.4-0.7 g, the volume of dichloromethane is 20-60 mL, and the volume of phosphorus tribromide is 0.5-4.0 mL. After stirring overnight, the organic phase is extracted with dichloromethane, and the solvent is removed by rotary evaporation under reduced pressure to obtain compound 2 as a white powder.

[0048] In step (3), the molar ratio of compound 2 to N,N-dimethylhexadecane-1-amine is 2:3, the volume range of tetrahydrofuran is 20-70 mL, and after stirring overnight, impurities are removed to obtain compound 3. The impurity removal process is as follows: the reaction solution is centrifuged at a rate of 12000-18000 rps and a temperature range of 17-21 °C, and then the precipitate is washed three times with ethanol. After vacuum drying, compound 3 in light pink powder form is obtained.

[0049] In step (4), the amount of 2,2'-bipyridine-4,4'-dicarboxylic acid ranges from 4.0 to 7.0 mmol, and the volume of thionyl chloride ranges from 20 to 60 mL. After stirring overnight, the solvent is removed by rotary evaporation under reduced pressure to obtain a light green powder compound 4.

[0050] In step (5), the mass range of compound 4 is 100-400 mg, the volume range of tetrahydrofuran and triethylamine used to dissolve compound 4 is 30-70 mL and 100-400 μL, respectively; the volume range of tetrahydrofuran used to dissolve cystamine dihydrochloride is 20-60 mL. After stirring overnight, the organic phase is extracted with dichloromethane, and the solvent is removed by rotary evaporation under reduced pressure to obtain compound 5 as a white powder.

[0051] In step (6), the mass ranges of compound 3, compound 5 and hydrated iridium trichloride are 50-300 mg, 30-70 mg and 10-30 mg, respectively. After rotary evaporation under reduced pressure to remove the solvent, a yellow powdery aminoiridium complex Ir-NH2 is obtained.

[0052] In step (7), the mass ratio of doxorubicin to Ir-NH2 is 1:1, the mass range of doxorubicin is 1 to 15 mg, the volume range of ethanol is 5 to 40 mL, and the solvent is removed by rotary evaporation under reduced pressure to obtain a yellow powder of doxorubicin aminoiridium complex DOX@Ir-NH2.

[0053] In step (8), the mass ratio of transferrin to 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide is 1:2, and the volume of secondary water ranges from 1 to 10 mL; the mass of DOX@Ir-NH2 and N-hydroxysuccinimide is 1 mg, and the volume of secondary water ranges from 1 to 10 mL.

[0054] Example 1

[0055] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0056] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0057] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0058] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0059] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0060] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0061] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0062] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0063] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0064] Example 2

[0065] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0066] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0067] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.5mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0068] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0069] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0070] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0071] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0072] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0073] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0074] Example 3

[0075] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0076] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0077] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0078] (3) Dissolve 2.0 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0079] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0080] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0081] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0082] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0083] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0084] Example 4

[0085] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0086] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0087] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0088] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0089] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.2 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0090] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0091] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0092] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0093] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0094] Example 5

[0095] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0096] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0097] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0098] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0099] (4) 40 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0100] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0101] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0102] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0103] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0104] Example 6

[0105] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0106] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0107] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0108] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0109] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0110] (5) Weigh 200 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0111] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0112] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0113] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0114] Example 7

[0115] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0116] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0117] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0118] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0119] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0120] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 350 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0121] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0122] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0123] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0124] Example 8

[0125] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0126] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0127] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0128] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0129] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0130] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0131] (6) Dissolve 100 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0132] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0133] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0134] Example 9

[0135] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0136] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0137] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0138] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0139] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0140] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0141] (6) Dissolve 80 mg of compound 3, 70 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0142] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0143] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0144] Example 10

[0145] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0146] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0147] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0148] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0149] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0150] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0151] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 36 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ethyl ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0152] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0153] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0154] Comparative Example 1

[0155] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0156] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0157] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0158] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0159] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0160] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0161] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0162] (7) Disperse 10-15 mL of ethanol containing 10-15 mg of doxorubicin in 20-30 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0163] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 2 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0164] Comparative Example 2

[0165] A method for preparing near-infrared / glutathione dual-responsive nanovesicles integrating targeted, controllable drug delivery and photoacoustic imaging includes the following steps:

[0166] (1) Dissolve 81.9 mM 4-(2-pyridyl)benzaldehyde in 40 ml of ethanol, and slowly add 327.6 mM sodium borohydride under ice-water bath conditions. Stir overnight at room temperature and control the speed at 300 rpm to obtain compound 1 in white powder form.

[0167] (2) Weigh 0.5g of compound 1 and dissolve it in 30mL of dichloromethane. Gradually add 1.0mL of phosphorus tribromide under ice-water bath conditions and stir overnight at room temperature. Then, add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2 as a white powder.

[0168] (3) Dissolve 1.5 mM compound 3 in 40 mL of tetrahydrofuran, and slowly add 4.5 mM N,N-dimethylhexadecane-1-amine at room temperature. After stirring overnight, compound 3 is obtained as a light pink powder.

[0169] (4) 35 mL of thionyl chloride was slowly added dropwise to a dichloromethane solution containing 6.0 mM 2,2'-bipyridine-4,4'-dicarboxylic acid, and the mixture was refluxed and stirred overnight under a nitrogen atmosphere to obtain compound 4, which was a light green powder.

[0170] (5) Weigh 150 mg of compound 4 and dissolve it in 35 mL of tetrahydrofuran containing 100 μL of triethylamine, and slowly add 25 mL of tetrahydrofuran containing 300 mg of cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5.

[0171] (6) Dissolve 80 mg of compound 3, 65 mg of compound 5 and 32 mg of hydrated iridium trichloride in 30 mL of ethylene glycol ether, heat and reflux under nitrogen atmosphere and stir overnight to obtain aminoiridium complex Ir-NH2;

[0172] (7) Disperse 10-15 mL of ethanol containing 10-15 mg doxorubicin in 10-15 mg mL of ethanol. -1 After thorough mixing and stirring in an aqueous solution of Ir-NH2, the doxorubicin aminoiridium complex DOX@Ir-NH2 was obtained.

[0173] (8) Slowly add 1 mL of aqueous solution containing 1 mg TF, 1 mg EDC and 1 mg NHS to 6 mL of aqueous solution containing 1 mg DOX-Ir-NH2, and mix thoroughly to obtain nanovesicles DOX@Ir-TF.

[0174] The dual response of DOX-Ir-NH2 nanovesicles prepared in Example 10, Comparative Examples 1 and 2 was evaluated. The results showed that, with other component concentrations remaining constant, the group with higher iridium trichloride hydrate content in the nanovesicles exhibited a more sensitive response to NIR / GSH, a greater DOX release, and a better chemotherapy effect on liver cancer. However, it should be noted that excessive iridium trichloride hydrate can be toxic to normal cells, causing precious metal poisoning and hindering the therapeutic effect of nanovesicles on liver cancer.

[0175] The reactive oxygen species (ROS) and photoacoustic imaging capabilities of the DOX-Ir-NH2 nanovesicles prepared in Example 10, Comparative Example 2, and Comparative Example 1 were evaluated. The results showed that, with other component concentrations remaining constant, the group with a higher concentration of Ir-NH2 aqueous solution exhibited stronger ROS generation and higher photoacoustic imaging intensity, enabling more sensitive monitoring of the liver cancer treatment process. However, it should be noted that excessively high Ir-NH2 aqueous solution concentrations can prevent the formation of nanovesicles.

[0176] In summary, the DOX@Ir-TF nanovesicles prepared in this invention, after intravenous injection, first precisely target the tumor region, induce reactive oxygen species (ROS) generation and controllably release chemotherapeutic drugs after NIR / GSH dual response; and cell experiments show that DOX@Ir-TF nanovesicles have specific targeting to the cell membrane and efficient cellular uptake capacity. Simultaneously, in vivo experiments demonstrate outstanding anticancer activity, showing significant inhibitory effects on hepatocellular carcinoma under synergistic chemo / photodynamic therapy. Furthermore, the prepared DOX@Ir-TF nanovesicles exhibit impressive photoacoustic imaging capabilities, which facilitates real-time monitoring of the nanovesicles' therapeutic process in vivo, enabling rapid pathological analysis or diagnosis of tumors. Overall, this single-molecule nanovesicle delivery system possesses targeted delivery, dual-response drug release, ROS generation, and real-time monitoring capabilities, demonstrating significant therapeutic efficacy against hepatocellular carcinoma.

[0177] The above description is merely a preferred embodiment of the present invention and should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent application and the contents of the specification of the present invention should still fall within the scope of the patent of the present invention.

Claims

1. A near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging, characterized in that: The nanovesicles are capable of controllably releasing doxorubicin and generating type I / II reactive oxygen species to achieve combined chemo-photodynamic therapy of the tumor region. They are prepared by amidation reaction of doxorubicin aminoiridium complex and transferrin. The method for preparing the nanovesicles includes the following steps: (1) 4 -( 2 (-pyridyl)benzaldehyde was dissolved in ethanol, and sodium borohydride was slowly added under ice-water bath conditions. The mixture was stirred overnight at room temperature to obtain compound 1. (2) Dissolve compound 1 obtained in step (1) in dichloromethane, and add phosphorus tribromide dropwise under ice-water bath conditions, stirring overnight at room temperature; then add saturated sodium carbonate solution under ice bath conditions and stir to obtain compound 2; (3) Dissolve compound 2 obtained in step (2) in tetrahydrofuran and slowly add it at room temperature. N , N -Dimethylhexadecane- 1 -amine, after stirring overnight, yield compound 3; (4) Slowly add thionyl chloride dropwise to the solution containing 2 , 2 '-Bipyridine- 4 , 4 Compound 4 was obtained by refluxing and stirring in a dichloromethane solution of diformic acid under a nitrogen atmosphere overnight. (5) Dissolve compound 4 obtained in step (4) in tetrahydrofuran containing triethylamine, and slowly add tetrahydrofuran containing cystamine dihydrochloride. Stir overnight at room temperature to obtain compound 5. (6) Dissolve compound 3 obtained in step (3), compound 5 obtained in step (5), and hydrated iridium trichloride in ethylene glycol ethyl ether, heat and reflux overnight under a nitrogen atmosphere to obtain aminoiridium complex Ir-NH2; (7) Disperse the ethanol containing doxorubicin in the aqueous solution of Ir-NH2 obtained in step (6), mix and stir thoroughly to obtain the doxorubicin aminoiridium complex DOX@Ir-NH2; (8) Containing transferrin, 1 -( 3 -dimethylaminopropyl)- 3 -Ethylcarbodiimide and N An aqueous solution of hydroxysuccinimide was slowly added to the aqueous solution of DOX@Ir-NH2 obtained in step (7), and after thorough mixing, the nanovesicles DOX@Ir-TF were obtained.

2. The near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging according to claim 1, characterized in that: In step (1), 4 -( 2 The molar ratio of pyridyl)benzaldehyde to sodium borohydride was 3:5; the volume of ethanol was 20-60 mL; the stirring speed was 100-700 rpm; after stirring overnight, the organic phase was extracted with dichloromethane; the solvent was removed by rotary evaporation under reduced pressure to obtain compound 1 as a white powder.

3. The near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging according to claim 1, characterized in that: In step (2), the reaction mass of compound 1 ranges from 0.4 to 0.7 g, dichloromethane from 20 to 60 mL, and phosphorus tribromide from 0.5 to 4.0 mL.

4. The near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging according to claim 1, characterized in that: In step (3), compound 2 and N , N -Dimethylhexadecane- 1 The molar ratio of the amines is 2:3, and the volume range of tetrahydrofuran is 20~70 mL.

5. The near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging according to claim 1, characterized in that: In step (4), 2 , 2 '-Bipyridine- 4 , 4 The amount of dicarboxylic acid ranged from 4.0 to 7.0 mmol, and the volume of thionyl chloride ranged from 20 to 60 mL. After stirring overnight, the solvent was removed by rotary evaporation under reduced pressure to obtain a pale green powder, compound 4.

6. The near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging according to claim 1, characterized in that: In step (5), the mass range of compound 4 is 100~400 mg, the volume range of tetrahydrofuran and triethylamine used to dissolve compound 4 is 30~70 mL and 100~400 μL, respectively; the volume range of tetrahydrofuran used to dissolve cystamine dihydrochloride is 20~60 mL, after stirring overnight, the organic phase is extracted with dichloromethane, the solvent is removed by rotary evaporation under reduced pressure, and compound 5 is obtained as a white powder.

7. The near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging according to claim 1, characterized in that: In step (6), the mass ranges of compound 3, compound 5 and hydrated iridium trichloride are 50~300 mg, 30~70 mg and 10~30 mg, respectively. After rotary evaporation under reduced pressure, the solvent is removed to obtain a yellow powdery aminoiridium complex Ir-NH2.

8. The near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging according to claim 1, characterized in that: In step (7), the mass ratio of doxorubicin to Ir-NH2 is 1:1, the mass range of doxorubicin is 1~15 mg, the volume range of ethanol is 5~40 mL, and the solvent is removed by rotary evaporation under reduced pressure to obtain a yellow powder of doxorubicin aminoiridium complex DOX@Ir-NH2.

9. The near-infrared / glutathione dual-response nanovesicle integrating targeted, controllable drug delivery and photoacoustic imaging according to claim 1, characterized in that: In step (8), the transferrin, 1 -( 3 -dimethylaminopropyl)- 3 The mass ratio of -ethylcarbodiimide was 1:2, and DOX@Ir-NH2 and N The mass of each hydroxysuccinimide is 1 mg.

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