A glutathione-activated co-assembled nanoprobe and its preparation method and application

Abstract

The present invention provides a glutathione-activated co-assembled nanoprobe and a preparation method and application thereof, belonging to the technical fields of chemical synthesis, biological analysis and detection, tumor imaging and treatment. In the present invention, amphiphilic small molecule probes 1-Zn-PPA and 1-NLG are constructed, and the probes 1-Zn-PPA and 1-NLG can be co-assembled in a solution to form co-assembled nanoprobes 1-NPs. The nanoprobes 1-NPs can accumulate at the tumor site under the tumor EPR effect and the targeting action of cRGD on the probe surface, and disassemble under the action of GSH present in the tumor reducing environment to release Gd-containing hydrophilic small molecule 2-Gd, light / sound-sensitive drug Zn-PPA-SH and immune adjuvant drug NLG919. The nanoprobes 1-NPs can realize combined sonodynamic therapy / photodynamic therapy / immunotherapy of living tumors. The nanoprobes 1-NPs have good applications in fluorescence imaging, magnetic resonance imaging, and the preparation of tumor treatment drugs and tumor-killing devices.

Description

Technical Field

[0001] The present invention belongs to the technical fields of chemical synthesis, biological analysis and detection, tumor imaging and treatment, and particularly relates to a glutathione-activated co-assembled nanoprobe and a preparation method and application thereof. Background Art

[0002] Cancer (also known as tumor) is a malignant disease associated with abnormal cell proliferation, differentiation, migration, and invasion. Currently, there are multiple treatments for cancer, including surgery, chemotherapy, radiotherapy, and photodynamic therapy. However, these existing treatments lack the ability to monitor and regulate specific sites. Therefore, researchers have turned their attention to the tumor environment itself.

[0003] The special environment in which tumor cells are located is called the tumor microenvironment, which is closely related to the occurrence, development and metastasis of tumors. This environment mainly includes many overexpressed enzyme receptors, overexpressed enzymes and some special physical and chemical properties, among which the glutathione (GSH) content in tumors is higher than that in normal tissues. Therefore, researchers have developed some GSH-activated diagnostic and therapeutic probes based on this characteristic to improve the accuracy of tumor diagnosis and enhance the effect of drugs. These probes often achieve imaging and treatment of tumors by covalently binding fluorescent imaging groups and drug fragments. Although existing probes can achieve the diagnosis and treatment of tumors to a certain extent, there are still certain limitations: a single imaging mode is difficult to achieve accurate imaging of tumors, and a single treatment method has limited therapeutic ability for tumors, especially for some tumors that are prone to recurrence and metastasis. Therefore, it is necessary to develop a tumor diagnostic and therapeutic probe that can accurately image tumors and has more effective treatment capabilities. Summary of the Invention

[0004] In response to the shortcomings of the prior art, the present invention provides a glutathione-activated co-assembled nanoprobe, its preparation method, and application. In the present invention, amphiphilic small molecule probes 1-Zn-PPA and 1-NLG are constructed. The probes 1-Zn-PPA and 1-NLG can be co-assembled in solution to form co-assembled nanoprobes 1-NPs. The nanoprobes 1-NPs can accumulate at the tumor site under the tumor EPR (high permeability and retention of solid tumors) effect and the targeting effect of cRGD on the probe surface. Under the action of GSH present in the tumor reducing environment, they disassemble and release the hydrophilic small molecule 2-Gd containing Gd, the photo / sonosensitive drug Zn-PPA-SH, and the immune adjuvant drug NLG919. The nanoprobes 1-NPs can achieve combined sonodynamic therapy / photodynamic therapy / immunotherapy for living tumors. The nanoprobes 1-NPs have excellent applications in fluorescence imaging, magnetic resonance imaging, and the preparation of tumor treatment drugs and tumor-killing devices.

[0005] The present invention first provides an amphiphilic small molecule probe, which comprises the following structure:

[0006] (1) Rigidly linked backbone aminofluorescein fragment (AO-Luc);

[0007] (2) tumor-targeting cRGD fragments;

[0008] (3) DOTA-Gd fragments that can be used for MR imaging;

[0009] (4) disulfide bonds that can be cleaved by GSH;

[0010] (5) Zn-PPA fragments for acousto-photodynamic therapy or NLG919 fragments of the IDO1 inhibitor for immunotherapy.

[0011] Specifically, the amphiphilic small molecule probe containing the Zn-PPA fragment that can be used for acousto-photodynamic therapy is denoted as 1-Zn-PPA, and its structural formula is:

[0012]

[0013] The amphiphilic small molecule probe containing the IDO1 inhibitor NLG919 fragment that can be used for immunotherapy is denoted as 1-NLG, and its structural formula is:

[0014]

[0015] The present invention also provides a method for preparing the above-mentioned amphiphilic small molecule probe, wherein the amphiphilic small molecule probe includes 1-NLG and 1-Zn-PPA, and the specific steps are as follows:

[0016] (1) Preparation of intermediates:

[0017] Preparation of compound 1: 1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethanol (denoted as compound NLG919), 4-dimethylaminopyridine (DMAP) and triphosgene (bis(trichloromethyl)carbonate) were dissolved in solvent 1, heated with stirring under reflux to carry out reaction 1. After completion of the reaction, solvent 1 and residual triphosgene were removed to obtain intermediate A; solvent 2 and 2-[2-(pyridyl)thio]ethanol were added to intermediate A, and reaction 2 was carried out under stirring. After completion of the reaction, vacuum rotary evaporation, elution, and purification were carried out to obtain 1-cyclohexyl-2-(5H-imidazo[5,1-a]isoindol-5-yl)ethyl (2-(pyridin-2-yl disulfide)ethyl) carbonate, denoted as compound 1;

[0018] Preparation of compound 2: Pyropheophorbide a (denoted as compound PPA), 2-(pyridin-2-ylthio)ethane-1-amine, benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) and N,N-diisopropylethylamine (DIPEA) were dissolved in solvent 3 and reacted under stirring. After the reaction, the mixture was vacuum rotary evaporated, purified, and eluted to obtain compound 2;

[0019] Preparation of compound 3: 2-amino-5-cyanobenzothiazole (NH2-CBT), DMAP and triphosgene (bis(trichloromethyl)carbonate) were dissolved in solvent 4, heated with stirring under reflux to carry out reaction 4, and after completion of the reaction, solvent 4 and triphosgene were removed to obtain intermediate B;

[0020] 2-Triphenylmercaptoethanol and solvent 5 are added to intermediate B and reacted under stirring conditions. After the reaction is completed, vacuum rotary evaporation, elution, and purification are performed to obtain intermediate C;

[0021] Solvent 6, tris(2-carboxyethyl)phosphine, cysteine, and N,N-diisopropylethylamine (DIPEA) were added to intermediate C to carry out reaction 6. After the reaction, manganese dioxide was added and reacted under stirring conditions to carry out reaction 7. After the reaction, vacuum rotary evaporation, elution, and purification were carried out to obtain compound 3.

[0022] Preparation of compound 4: Compounds amino PEG alkynyl (NH2-PEG4-Alkyne), N-alpha-fluorenylmethoxycarbonyl-N-epsilon-tert-butyloxycarbonyl-L-lysine (Boc-Lys(Fmoc)-OH), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), and DIPEA were dissolved in solvent 7 and reacted under stirring conditions 8. After completion of the reaction, the mixture was vacuum evaporated, eluted, and purified to obtain intermediate D;

[0023] The intermediate D is dissolved in a solvent 8 containing trifluoroacetic acid, and then reacted 9. After the reaction is completed, the trifluoroacetic acid and the solvent 8 are removed by vacuum rotary evaporation to obtain compound 4;

[0024] Preparation of compound 5: Compound 3, compound 4, HBTU and DIPEA were dissolved in solvent 9 to react 10, and solvent 9 was removed by rotary evaporation to obtain intermediate E;

[0025] The intermediate E is dissolved in a solvent 10 containing piperidine to carry out reaction 11. After the reaction is completed, vacuum rotary evaporation, purification, and elution are performed to obtain compound 5.

[0026] Preparation of compound 6: Compound 5, DOTA activated ester, and DIPEA were dissolved in solvent 11 to react 12. After the reaction was completed, solvent 11 was removed by rotary evaporation under reduced pressure to obtain intermediate F;

[0027] Intermediate F and GdCl3 were dissolved in solvent 12 to react 13. After the reaction, vacuum rotary evaporation, purification, and freeze-drying were performed to obtain compound 6.

[0028] Preparation of compound 7: Compound 6, azide-substituted RGDfK cyclic peptide (cRGD-N3), and copper powder were dissolved in solvent 13 to carry out reaction 14. After the reaction, vacuum rotary evaporation, purification, and lyophilization were performed to obtain compound 7.

[0029] (2) Preparation of amphiphilic small molecule probes:

[0030] The preparation method of the 1-NLG is:

[0031] Compound 7 is dissolved in a solvent 14 containing trifluoroacetic acid and triisopropylsilane to carry out reaction 15. After the reaction is completed, the solvent 14 is removed by rotary evaporation under reduced pressure, cold ether is added to collect the precipitate, and then the precipitate and compound 1 are dissolved in the solvent 14 to carry out reaction 16. After the reaction is completed, vacuum rotary evaporation is carried out, purification is carried out, and lyophilization is carried out to obtain 1-NLG;

[0032] The preparation method of 1-Zn-PPA comprises: dissolving compound 7 in a solvent 14 containing trifluoroacetic acid and triisopropylsilane to carry out reaction 17; after the reaction, removing solvent 14 by rotary evaporation under reduced pressure; adding cold ether to collect a precipitate; then dissolving the precipitate, compound 2, and zinc chloride in solvent 14 to carry out reaction 18; after the reaction, carrying out rotary evaporation under vacuum, purifying, and freeze-drying to obtain 1-Zn-PPA;

[0033] Specifically, in step (1), during the preparation of compound 1, the molar ratio of compound NLG919, triphosgene, 2-[2-(pyridyl)thio]ethanol, and DMAP is 1:0.33:1:1 to 1:1:3:10;

[0034] The solvent 1 is any one of toluene, nitrobenzene, chlorobenzene or benzene, preferably toluene;

[0035] Solvent 2 is any one of dichloromethane, chloroform, tetrahydrofuran or acetonitrile, preferably dichloromethane;

[0036] The conditions of reaction 1 are: reaction at 80-140°C for 1-8 hours, preferably at 110°C for 3 hours;

[0037] The conditions of the reaction 2 are: reacting at 20-30° C. for 1-8 hours, preferably reacting at room temperature for 2 hours.

[0038] Specifically, in step (1), during the preparation of compound 2, the molar ratio of compounds PPA, 2-(pyridin-2-yldisulfide)ethylamine, HBTU, and DIPEA is 1:1:1:1 to 1:2:2:6;

[0039] The solvent 3 includes any one of dichloromethane, chloroform, tetrahydrofuran or acetonitrile;

[0040] The reaction 3 is carried out at 20-30° C. for more than 1 hour, preferably 1-5 hours, and more preferably 2 hours.

[0041] Specifically, in step (1), during the preparation of compound 3, the molar ratio of NH2-CBT, triphosgene, DMAP, 2-triphenylmercaptoethanol, cysteine, tris(2-carboxyethyl)phosphine, N,N-diisopropylamine and manganese dioxide is 1:0.33:1:1:1:1:1:1:1 to 1:1:3:3:3:3:6:20;

[0042] The solvent 4 is any one of toluene, nitrobenzene, chlorobenzene or benzene, preferably toluene;

[0043] Solvent 5 is any one of dichloromethane, chloroform, tetrahydrofuran or acetonitrile, preferably dichloromethane

[0044] Solvent 6 is a mixed solution of liquid A and liquid B in a volume ratio of 1:4 to 4:1, wherein liquid A is dichloromethane or chloroform, and liquid B is methanol or acetonitrile; preferably, a dichloromethane / methanol mixed solution in a volume ratio of 1:1.

[0045] The conditions of reaction 4 are: reaction at 80-140°C for 1-8 hours, preferably at 110°C for 3 hours;

[0046] The conditions of the reaction 5 are: reacting at 20-30°C for 1-8 hours, preferably at room temperature for 2 hours;

[0047] The conditions of reaction 6 are: reacting at 20-30°C for 1-8 hours, preferably at room temperature for 1 hour;

[0048] The conditions of reaction 7 are: reaction at 20-30° C. for 8-24 hours, preferably at room temperature for 14 hours.

[0049] Specifically, in step (1), during the preparation of compound 4, the molar ratio of NH2-PEG4-Alkyne, Boc-Lys(Fmoc)-OH, HBTU and DIPEA is 1:1:1:1 to 1:2:2:6; the content of trifluoroacetic acid in the solvent is 5%-90% v / v, preferably 5%;

[0050] The solvent 7 includes but is not limited to dichloromethane, chloroform, tetrahydrofuran or acetonitrile, preferably tetrahydrofuran;

[0051] The solvent 8 includes but is not limited to dichloromethane, chloroform, tetrahydrofuran or acetonitrile, preferably dichloromethane;

[0052] The conditions of reaction 8 and reaction 9 are both to react at 20-30° C. for 1-8 hours, and preferably to react at room temperature for 2 hours.

[0053] Specifically, in step (1), during the preparation of compound 5, the molar ratio of compound 3, compound 4, HBTU and DIPEA is 1:1:1:2 to 1:3:3:6; the content of piperidine in the solvent is 5-90% v / v, preferably 20%;

[0054] The solvent 9 includes but is not limited to dichloromethane, chloroform, tetrahydrofuran or acetonitrile, preferably anhydrous tetrahydrofuran;

[0055] The solvent 10 includes but is not limited to acetonitrile, dimethyl sulfoxide, N,N-dimethylformamide, preferably N,N-dimethylformamide;

[0056] The conditions of reaction 10 and reaction 11 are both to react at 20-30° C. for 1-8 h, preferably at room temperature for 2 h.

[0057] Specifically, in step (1), during the preparation of compound 6, the molar ratio of compound 5, DOTA activated ester, DIPEA, and GdCl3 is 1:1:1:1 to 1:2:6:10;

[0058] Wherein, the solvent 11 includes but is not limited to chloroform, tetrahydrofuran, acetonitrile, dichloromethane, preferably anhydrous dichloromethane;

[0059] The solvent 12 includes but is not limited to acetonitrile, dimethyl sulfoxide or a mixed solvent of N, N-dimethylformamide and water, preferably a mixed solvent of N, N-dimethylformamide and water in a volume ratio of 1:1;

[0060] The conditions of reaction 12 are: reacting at 20-30° C. for 4-12 h, preferably at room temperature for 8 h;

[0061] The conditions of reaction 13 are: reaction at 20-30° C. for 6-24 h, preferably at room temperature for 14 h.

[0062] Specifically, in step (1), during the preparation of compound 7, the molar ratio of compound 6, cRGD-N3, and copper powder is 1:1:0.2-1:3:3;

[0063] The solvent 13 includes but is not limited to a mixed solvent of dimethyl sulfoxide, N,N-dimethylformamide, tert-butanol and water, preferably a mixed solvent of tert-butanol and water in a volume ratio of 1:1;

[0064] The reaction 14 is carried out at 20-30° C. for 6-24 h, preferably at room temperature for 14 h.

[0065] Specifically, in step (2), in the preparation of 1-NLG: the molar ratio of compound 7 to compound 1 is 1:1 to 1:2; the volume ratio of trifluoroacetic acid, triisopropylsilane and solvent 14 is 5:94:1 to 90:5:5;

[0066] During the preparation of 1-Zn-PPA, the molar ratio of compound 7, compound 2, and zinc chloride is 1:1:1 to 1:2:10; the volume ratio of trifluoroacetic acid, triisopropylsilane, and solvent 14 is 5:94:1 to 90:5:5.

[0067] Specifically, in step (2), the solvent 14 comprises a mixed solution of liquid A and liquid B, wherein liquid A is dichloromethane or chloroform, and liquid B is methanol or acetonitrile; preferably, a dichloromethane / methanol mixed solution with a volume ratio of 1:1;

[0068] The temperature of the reaction 15-18 is 20-30°C for 1-6 hours, preferably room temperature for 2 hours.

[0069] The present invention also provides a glutathione-activated co-assembled nanoprobe, which is recorded as 1-NPs. It is obtained by co-assembling the amphiphilic small molecule probes 1-Zn-PPA and 1-NLG. The molar ratio of 1-Zn-PPA and 1-NLG is 10:0-0:10 and is not 0. The optimal ratio is 1:1.1.

[0070] The present invention also provides the use of the above-mentioned 1-Zn-PPA, 1-NLG or 1-NPs in fluorescence imaging.

[0071] The present invention also provides the use of the above-mentioned 1-Zn-PPA, 1-NLG or 1-NPs in magnetic resonance imaging.

[0072] The present invention also provides the use of the above 1-NPs in magnetic resonance / fluorescence dual-modality imaging.

[0073] The present invention also provides the use of the above-mentioned 1-NPs in the combined treatment of sonodynamic therapy / photodynamic therapy / immunotherapy of living tumors.

[0074] The present invention also provides the use of the above-mentioned 1-Zn-PPA, 1-NLG or 1-NPs in preparing a drug for treating tumors or preparing a device for killing tumors.

[0075] The tumors include lung cancer, gastric cancer, liver cancer, kidney cancer, breast cancer, pancreatic cancer, colorectal cancer, ovarian cancer, prostate cancer, thyroid cancer, esophageal cancer, head and neck cancer, melanoma, glioma, acute myeloid leukemia, etc. Compared with the prior art, the present invention has the following advantages:

[0076] Magnetic resonance / fluorescence dual-modality imaging can achieve high-resolution, high-sensitivity, and precise imaging of tumors. At the same time, the combination of acousto-photodynamic therapy and immunotherapy can achieve more effective treatment of tumors and inhibit tumor metastasis. The glutathione-activated co-assembled nanoprobe 1-NPs provided in the present invention can be used for magnetic resonance / fluorescence dual-modality imaging of living tumors. In the present invention, a 1-Zn-PPA probe containing a Zn-PPA fragment that can be used for acousto-photodynamic therapy and a 1-NLG probe containing an IDO1 inhibitor NLG919 fragment that can be used for immunotherapy are co-assembled in a solution to form co-assembled nanoprobe 1-NPs, which provides a new idea for constructing magnetic resonance / fluorescence dual-modality imaging and an activated molecular probe for the combination of acousto-photodynamic therapy and immunotherapy, and is expected to be applied to the imaging detection and treatment of malignant tumors.

[0077] The co-assembled nanoprobe 1-NPs provided in the present invention have a high longitudinal relaxation rate, quenched near-infrared fluorescence and acousto-photodynamic activity, and IDO1 inhibitory activity due to intermolecular aggregation. The co-assembled nanoprobe 1-NPs can aggregate at the tumor site under the EPR effect and the targeting effect of cRGD on the probe surface, and disassemble and release 2-Gd, Zn-PPA-SH, and NLG919 under the action of GSH in the tumor reduction environment. In addition, the released 2-Gd can be quickly cleared, which can reduce the toxic side effects caused by long-term accumulation of heavy metals in the body; the released Zn-PPA-SH can bind to albumin in tumor cells, restore the near-infrared fluorescence signal, and prolong the retention time of the drug in the tumor, enhance the acousto-photodynamic effect, and kill tumor cells; the released NLG919 reduces the ratio of kynurenine to tryptophan, reduces the inhibitory effect of regulatory T cells on toxic T cells, and further enhances the tumor killing effect. The co-assembled nanoprobe 1-NPs can effectively accumulate at the tumor site, and after being activated by GSH and under the combined action of light and ultrasound, can effectively inhibit the growth, recurrence and metastasis of the tumor. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is the synthetic route of the amphiphilic small molecule probes 1-Zn-PPA and 1-NLG.

[0079] Figure 2 Chemical structures of the amphiphilic small molecule probes 1-Zn-PPA (a) and 1-NLG (b).

[0080] Figure 3 Diagram of the process of co-assembly of amphiphilic small molecule probes 1-Zn-PPA and 1-NLG in solution to form probe 1-NPs.

[0081] Figure 4 This is a diagram showing the mechanism by which probe 1-NPs kill tumors in mice after being injected into the tail vein.

[0082] Figure 5 The DLS analysis diagram of probe 1-NPs in solution, and the inset is the TEM image.

[0083] Figure 6 (a) The UV absorption spectrum and (b) the fluorescence spectrum of probe 1-NPs in PBS solution or DMSO solution.

[0084] Figure 7 HPLC analysis (a) and fluorescence spectrum (b) of probe 1-NPs before and after incubation at 37°C and GSH for 2 h.

[0085] Figure 8 The DLS analysis graphs of probe 1-NPs before and after incubation at 37°C and GSH for 20, 40, 60, 80, 100, and 120 minutes.

[0086] Figure 9 This is a diagram showing the relaxation rate measurement of probe 1-NPs before and after incubation with GSH at 37°C for 2 h.

[0087] Figure 10 The figure shows the release of Zn-PPA-SH and NLG919 from probe 1-NPs in the presence or absence of GSH.

[0088] Figure 11 This is a diagram of the DCF fluorescence enhancement of probe 1-NPs in the presence or absence of GSH and under different treatment conditions.

[0089] Figure 12 Magnetic resonance imaging images (a) and quantitative data of magnetic resonance signals in the tumor at 0, 2, 4, and 8 h after probe 1-NPs were injected into mice via the tail vein (b).

[0090] Figure 13 Figure 3 shows the near-infrared fluorescence imaging images of the tumor at 0, 4, 8, 12, 24, 48, and 72 h after probe 1-NPs were injected into mice via the tail vein (a) and the quantitative data of the near-infrared fluorescence signal in the tumor (b).

[0091] Figure 14 Fluorescence imaging and quantitative data of isolated mouse organs.

[0092] Figure 15Fluorescence imaging of mouse tumor sections. Scale bar: 200 μm.

[0093] Figure 16 The biodistribution of Zn and Gd at different time points after tail vein administration.

[0094] Figure 17 The treatment flow chart of 4T1 mice (a) and the mouse tumor volume growth curve (b).

[0095] Figure 18 Representative images of tumors at 27 days after treatment with different treatment conditions.

[0096] Figure 19 is the survival rate of mice under different treatment conditions within 100 days.

[0097] Figure 20 H&E and TUNEL staining images of tumor sections from mice in different treatment groups.

[0098] Figure 21 Figure 3 Calreticulin content (a), high mobility group protein 1 content (b), dendritic cell maturation rate (c), necrosis factor-α content (d), interleukin-6 content (e), and interferon-γ content (f) in the tumors of mice in different treatment groups.

[0099] Figure 22 Figure 3 shows the T cell infiltration in tumor tissues of mice in different treatment groups.

[0100] Figure 23 The content of helper T cells (a), the content of cytotoxic T cells (b), the ratio of kynurenine to tryptophan (c), the content of regulatory T cells (d), and the ratio of cytotoxic T cells to regulatory T cells (e) in the tumor tissues of mice in different treatment groups, where Ⅰ: normal saline group, Ⅱ: 1-NPs group, Ⅲ: 1-NPs + light group, Ⅳ: 1-NPs + ultrasound group, Ⅴ: 1-Zn-PPA + ultrasound + light group, Ⅵ: 1-NPs + ultrasound + light group. DETAILED DESCRIPTION

[0101] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the scope of protection of the present invention is not limited thereto. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0102] Example 1: Preparation of amphiphilic small molecule probes 1-NLG and 1-Zn-PPA

[0103] The synthetic route of amphiphilic small molecule probes 1-NLG and 1-Zn-PPA is shown in the figure. Figure 1As shown, the specific preparation method is:

[0104] 1. Synthesis of intermediates:

[0105] (1) Synthesis of Compound 1:

[0106] Triphosgene (21 mg, 0.07 mmol), N,N-dimethylpyridine (75.8 mg, 0.62 mmol) and NLG919 (50 mg, 0.18 mmol) were dissolved in 10 mL of anhydrous dichloromethane solution and stirred at room temperature for 1 h under argon protection. After the reaction was completed as monitored by TLC, 2-[2-(pyridyl)thio]ethanol (35 mg, 0.18 mmol) was added to the reaction solution under argon protection. After stirring at room temperature for 3 h, the solvent was evaporated and purified by semi-preparative HPLC to obtain 35 mg of a white solid, recorded as compound 1, with a total yield of 40% for the two steps.

[0107] Compound 1 1 H NMR spectrum: (500 MHz, Chloroform-d) δ9.09 (s, 1H), 8.53 (d, J = 5.0 Hz, 1H), 7.83–7.76 (m, 2H), 7.73–7.68 (m, 1H), 7.67–7.63 (m, 1H), 7.58–7.49 (m, 3H), 7.24 (ddd, J = 6.8, 5.0, 1 .9Hz,1H),5.56(t,J=5.5Hz,1H),4.80(ddd,J=11.3,5.6,2.3Hz,1H),4.32(t,J=6.3Hz ,2H),3.04(t,J=6.4Hz,2H),2.63–2.33(m,2H),1.81–1.56(m,7H),1.31–0.93(m,8H). 13 C NMR (126 MHz, Chloroform-d) δ 154.4, 148.5, 143.2, 138.5, 138.2, 131.1, 129.8, 129.6, 126.5, 124.2, 121.7, 121.5, 120.9, 109.8, 78.3, 65.7, 61.7, 53.4, 42.0, 36.8, 28.0, 27.7, 26.0, 25.7, 25.6., which indicated the successful preparation of compound 1 (NLG-SS-Pyr).

[0108] (2) Synthesis of Compound 2:

[0109] To a solution of PPA (60 mg, 0.10 mmol), 2-(pyridin-2-ylthio)ethane-1-amine (22 mg, 0.12 mmol) and HBTU (49 mg, 0.13 mmol) in 10 mL of anhydrous dichloromethane was added N,N-diisopropylamine (119 μL, 0.65 mmol). The reaction solution was stirred at room temperature for 3 h. After completion of the reaction as monitored by TLC, the solvent was evaporated and purified by column chromatography to obtain 70 mg of a dark green solid, designated as compound 2, with a yield of 92%.

[0110] Compound 2 1 The H NMR spectrum was as follows: (500 MHz, Chloroform-d) δ9.31 (d, J = 9.5 Hz, 2H), 8.52 (s, 1H), 7.92 (dd, J = 17.8, 11.5 Hz, 1H), 7.34 (d, J = 4.4 Hz, 1H), 7.11–7.01 (m, 2H), 6.97 (s, 1H), 6.22 (dd, J = 17.8, 1.3 Hz, 1H), 6.14 (dd, J = 11.6, 1.3 Hz, 1H), 6.06 (t, J = 4.9 Hz, 1H), 5.34–4.9 7(m,2H),4.51–4.47(m,1H),4.43–4.29(m,1H),3.61(q,J=7.4Hz,3H),3.51(s,3H),3.38(s,3H),3.39–3.31(m,2H),3.19(s, 3H),2.66(t,J=5.6Hz,2H),2.57–2.41(m,1H),2.32–2.20(m,1H),1.80(d,J=7.3Hz,3H),1.65(t,J=7.6Hz,4H),1.25(s,2H). 13 C NMR (126MHz, CDCl3) δ196.1,172.3,171.9,160.4,158.5,154.9,150.5,149.5,148.9 ,148.8,144.9,141.5,137.7,137.4,136.4,136.1,136.0,135.8,131.6,130.4,129.1 ,128.2,122.6,121.1,120.9,120.6,119.6,106.0,103.9,97.1,93.1,51.7,49.8,48 .0,38.7,37.0,32.6,30.0,29.7,23.1,19.4,17.4,12.1,11.9,11.2.HRMS:calcd.for C 40 H 42 N6O2S2 + [(M+H)+ ]:703.2889; found 702.9395., which indicates the successful preparation of compound 2.

[0111] (3) Synthesis of compound 3:

[0112] 2-Amino-5-cyano-benzothiazole (220 mg, 1.25 mmol) and DMAP (152 mg, 1.25 mmol) were dissolved in 50 mL of dry toluene. Triphosgene (370 mg, 1.25 mmol) was then dissolved in 5 mL of dry toluene. Under nitrogen, the reaction mixture was added dropwise at 0°C, followed by stirring at 120°C for 3 hours. After the reaction, the toluene was removed by rotary evaporation under reduced pressure, and the residue was dissolved in 15 mL of dry DCM. 2-Triphenylmercaptoethanol (357 mg, 1.0 mmol) was dissolved in 5 mL of dry DCM and added dropwise at 0°C. The reaction was continued at room temperature overnight. After the reaction, the solvent was removed by rotary evaporation under reduced pressure, and the product was purified by column chromatography to obtain a white compound.

[0113] A white compound (250 mg, 0.48 mmol), tris(2-carboxyethyl)phosphine hydrochloride (145 mg, 0.40 mmol), L-cysteine (59 mg, 0.48 mmol), and N,N-diisopropylamine (0.5 mL, 2.88 mmol) were added to a mixed solvent containing 10 mL of dichloromethane and 10 mL of methanol. The reaction solution was stirred at room temperature for 1 hour. After the reaction was completed by HPLC, activated manganese dioxide powder (417 mg, 4.8 mmol) was added to the mixed solution and stirred at room temperature overnight. After the reaction was completed by HPLC, the manganese dioxide powder was removed by filtration using diatomaceous earth. The filtrate was collected, concentrated, and purified by column chromatography to obtain 390 mg of a dark yellow solid, designated as compound 3, with a yield of 79%.

[0114] HRMS of compound 3: calculation for C 33 H 26 N3O4S3 + [(M+H) + ]:624.1085; found 623.6874., which indicates the successful preparation of compound 3.

[0115] (4) Synthesis of compound 4:

[0116] To a 50 mL anhydrous tetrahydrofuran solution of amino-PEG-alkynyl (300 mg, 1.30 mmol), Boc-Lys(Fmoc)-OH (666 mg, 1.43 mmol) and HBTU (540 mg, 1.43 mmol) was added N,N-diisopropylamine (770 μL, 4.32 mmol) and stirred at room temperature for 3 h. After the reaction was completed as monitored by TLC, the solvent was evaporated to dryness. Then, 10 mL of a dichloromethane solution containing 10% trifluoroacetic acid was added and stirred at room temperature for 2 h. After the reaction was completed as monitored by HPLC, the solvent was evaporated to dryness and dissolved in a small amount of ethyl acetate. The organic phase was washed three times with a saturated sodium chloride solution, separated, dried, filtered, concentrated, and purified by column chromatography to obtain 760 mg of a colorless oil, recorded as compound 4, with a yield of 85%.

[0117] HRMS of compound 4: calculation for C 37 H 51 N3NaO9 + [(M+Na) + ]:704.3513.; found704.2507. This indicates the successful preparation of compound 4.

[0118] (5) Synthesis of Compound 5:

[0119] To a solution of compound 3 (132 mg, 0.17 mmol), compound 4 (150 mg, 0.22 mmol), and HBTU (72 mg, 0.19 mmol) dissolved in 10 mL of anhydrous tetrahydrofuran was added N,N-diisopropylamine (91 μL, 0.49 mmol), and the reaction mixture was stirred at room temperature for 3 h. After HPLC analysis, the solvent was evaporated, and 5 mL of anhydrous N,N-dimethylformamide containing 20% piperidine was added to the solid residue. The reaction mixture was stirred at room temperature for 2 h. After HPLC analysis, the solvent was evaporated, and the residue was purified by semi-preparative HPLC to obtain 110 mg of a yellow solid, designated as compound 5, with a total yield of 66% over the two steps.

[0120] HRMS of compound 5:calcd.for C 50 H 57 N6O8S3 + [(M+H) + ]:965.3400; found 964.7095. This indicates the successful preparation of compound 5.

[0121] (6) Synthesis of Compound 6:

[0122] To a solution of compound 5 (90 mg, 0.09 mmol) and DOTA activated ester (78 mg, 0.10 mmol) dissolved in anhydrous dichloromethane was added N,N-diisopropylethylamine (DIPEA, 95 μL, 0.54 mmol). The reaction solution was stirred at room temperature overnight under argon protection. After HPLC analysis, the solvent was dried and the solid residue was added with 10 mL of a 1 / 1, v / v % N,N-dimethylformamide / water mixture. Then, gadolinium chloride hexahydrate (568 mg, 1.53 mmol) was added and stirred at room temperature for 10 minutes. Saturated sodium bicarbonate solution was added to adjust the pH of the reaction system to neutral and the mixture was stirred at room temperature overnight. After HPLC analysis, the reaction solution was purified by semi-preparative HPLC to obtain 98 mg of a yellow solid, designated as compound 6, with an overall yield of 70%.

[0123] HRMS of compound 6:calcd.for C 66 H 79 GDJ 10 NaO 15 S3 + [(M+Na) + ]:1528.4027; found1527.4828. This indicates the successful preparation of compound 6.

[0124] (7) Synthesis of Compound 7:

[0125] To a 10 mL mixture of tert-butanol / water (1:1, v / v%) containing compound 6 (95 mg, 0.06 mmol) and cRGD-N3 (51 mg, 0.07 mmol) was added copper powder (16 mg, 0.24 mmol). The mixture was stirred at room temperature for 6 h. After completion of the reaction, as determined by HPLC, the solvent was evaporated and the residue purified by semi-preparative HPLC to afford 115 mg of a yellow solid (85% yield).

[0126] HRMS of compound 7:calcd.For C 93 H 118 GDJ 21 O 22 S3 + [(M+H) + ]:2135.7242; found:2135.6316. This indicates the successful preparation of compound 7.

[0127] 2. Synthesis of amphiphilic small molecule probes 1-NLG and 1-Zn-PPA:

[0128] Synthesis of 1-NLG:

[0129] Compound 7 (48 mg, 0.02 mmol) was dissolved in 5 mL of a mixture of dichloromethane, trifluoroacetic acid, and triisopropylsilane (v:v:v = 50:46:4). The reaction was stirred at room temperature for 1 h. After HPLC detection, the solvent was evaporated to dryness, glacial ether was added, and the pale yellow precipitate was collected by centrifugation and dissolved in 10 mL of methanol. Compound 1 (20 mg, 0.02 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction, the solvent was evaporated to dryness. After HPLC detection, the residue was evaporated to dryness and purified by semi-preparative HPLC to obtain 59 mg of 1-NLG as a white solid with a yield of 67%.

[0130] 1-HRMS of NLG:calcd.for C 95 H 129 GDJ 23 O 25 S4 + [(M+H) + ]:2276.76,found MALDI-MS:m / z2277.70,found HRMS[(M+2H) 2+ ]: m / z 1139.3331. This indicates the successful preparation of compound 1-NLG, whose chemical structure is as follows Figure 2 As shown in b.

[0131] Synthesis of 1-Zn-PPA:

[0132] Compound 7 (38 mg, 0.016 mmol) was dissolved in 5 mL of a mixture of dichloromethane, trifluoroacetic acid, and triisopropylsilane (v:v:v = 50:46:4). The reaction was stirred at room temperature for 1 h. After the reaction was complete as determined by HPLC, the solvent was evaporated, glacial ether was added, and the pale yellow precipitate was collected by centrifugation and dissolved in 10 mL of methanol. Compound 2 (9 mg, 0.016 mmol) was added, and the reaction was stirred at room temperature overnight. After the reaction was complete as determined by HPLC, 20 mg of zinc chloride was added to the reaction solution and stirring was continued at room temperature for 6 h. After the reaction was complete as determined by HPLC, the solvent was evaporated, and the residue was purified by semi-preparative HPLC to obtain 23 mg of a dark green solid, 1-Zn-PPA, in a yield of 61%.

[0133] HRMS of 1-Zn-PPA:calcd.for C 109 H 139 GDJ 26 O 24 S4Z 2+ [M 2+]:2546.79, found MALDI-MS: m / z 2548.60, found HRMS[(M+H+Na) 2+ ]: m / z 1284.5454. This indicates the successful preparation of compound 1-Zn-PPA, whose chemical structure is as follows Figure 2 As shown in a.

[0134] Example 2: Performance evaluation of 1-NPs

[0135] In this embodiment, Figure 3 The probes 1-Zn-PPA and 1-NLG were self-assembled into 1-NPs in solution based on the principle of GSH, and the assembly performance, spectral performance and GSH-activated disassembly performance of 1-NPs in solution were investigated.

[0136] (1) Study on the assembly performance of 1-NPs in solution:

[0137] The probes 1-Zn-PPA and 1-NLG were prepared in a DMSO solution at a molar ratio of 1:1.1. The mixed probe (20 μM) was dissolved in 1 mL of PBS buffer and the reaction solution was mixed and then subjected to DLS analysis.

[0138] The mixed probe (20 μM) was dissolved in 1 mL of PBS buffer, and the solution was added dropwise onto the carbon grid, followed by drainage and vacuum drying. The sample was then photographed using a transmission electron microscope.

[0139] Figure 5 These are the DLS analysis and TEM analysis diagrams of probe 1-NPs in solution. It can be seen from the figure that the probe 1-Zn-PPA and 1-NLG can be co-assembled into nanoparticles with an average particle size of about 100 nm, namely 1-NPs.

[0140] (2) Study on the spectral properties of 1-NPs in solution

[0141] The probes 1-Zn-PPA and 1-NLG were prepared in a DMSO solution at a molar ratio of 1:1.1. The mixed probes (20 μM) were dissolved in 1 mL of PBS buffer or DMSO solution. After mixing, the solution was subjected to UV absorption spectrum test. The test results are shown in Figure 2. Figure 6 As shown in a.

[0142] Figure 6 a is the UV absorption spectrum of probe 1-NPs in PBS solution or DMSO solution. It can be seen from the figure that the Q band of Zn-PPA is red-shifted, indicating that it forms the assembly 1-NPs.

[0143] For fluorescence spectroscopy analysis, the probes 1-Zn-PPA and 1-NLG were prepared in a DMSO solution at a molar ratio of 1:1.1. The mixed probe (20 μM) was dissolved in 1 mL of PBS buffer or DMSO solution. After mixing, the solution was subjected to fluorescence spectroscopy test.

[0144] Figure 6 b is the fluorescence spectrum of probe 1-NPs in PBS solution or DMSO solution. It can be seen from the figure that in PBS solution, the fluorescence quenching of Zn-PPA at 672 nm also reflects the formation of assembly 1-NPs.

[0145] (3) Study on the GSH-activated disassembly performance of 1-NPs in solution

[0146] For HPLC analysis, the probes 1-Zn-PPA and 1-NLG were prepared in a DMSO solution at a molar ratio of 1:1.1. The mixed probe (20 μM) was dissolved in 1 mL of PBS buffer and incubated with 1 mM GSH at 37°C for 2 h. The solutions were then injected into HPLC for analysis.

[0147] Figure 7 a is the HPLC analysis of probe 1-NPs before and after incubation with GSH at 37℃ for 2h. It can be seen from the figure that after activation by GSH, 1-Zn-PPA (t R =15.5min) and 1-NLG(t R =18.3min) can be effectively reduced to the cleavage product 2-Gd(t R =11.5min)、Zn-PPA-SH(t R =24.8min) and NLG919 (t R =18.5min).

[0148] For fluorescence spectroscopy analysis, the probes 1-Zn-PPA and 1-NLG were prepared in a DMSO solution at a molar ratio of 1:1.1. The mixed probe (20 μM) was dissolved in 1 mL of PBS buffer and incubated with 1 mM GSH at 37°C for 2 h. The fluorescence spectrum of the solution was recorded on a fluorimeter.

[0149] Figure 7 b is the fluorescence spectrum of probe 1-NPs before and after incubation with GSH at 37°C for 2 h. It can be seen from the figure that in the presence of GSH and albumin, the dual-channel fluorescence of 1-NPs at AO-Luc (547 nm) and Zn-PPA-SH (672 nm) was enhanced, which was increased by about 500 and 85.9 times respectively compared with before the addition of GSH.

[0150] For DLS analysis, the probes 1-Zn-PPA and 1-NLG were prepared in a DMSO solution at a molar ratio of 1:1.1. The mixed probe (20 μM) was dissolved in 1 mL of PBS buffer containing 5% albumin. The reaction solution was subjected to DLS analysis before and after incubation with 1 mM GSH at 37°C and at 20, 40, 60, 80, 100, and 120 min.

[0151] Figure 8 This is the DLS analysis of probe 1-NPs before and after incubation with GSH at 37°C for 20, 40, 60, 80, 100, and 120 minutes. It can be seen from the figure that after 1-NPs were incubated with GSH for 2 hours, its average particle size decreased from about 100 nm to about 6 nm, which is similar to the size of albumin, indicating that the disassembly of 1-NPs was effectively mediated by GSH.

[0152] For relaxivity determination, the probes 1-Zn-PPA and 1-NLG were prepared in PBS buffer at a molar ratio of 1:1.1 and then diluted into solutions of five different concentrations (0, 0.025, 0.05, 0.075, and 0.1 mM). The solutions were incubated with 10 mM GSH at 37°C for 2 h. The T1 values in the solutions were inverted and measured on a 0.5T MR scanner. The r1 longitudinal relaxivity was expressed as 1 / T1 and the slope of the fitting curve of the [Gd] concentration.

[0153] Figure 9 The relaxation rate of probe 1-NPs was measured before and after incubation with GSH at 37℃ for 2h. As can be seen from the figure, when 1-NPs decomposed into small molecule product 2-Gd, its r1 value increased from 18.7±0.3mM to -1 s -1 decreased to 7.3±0.2mM -1 s -1 , indicating that after disassembly, the r1 relaxation rate of 1-NPs decreased and T1 increased.

[0154] For drug release assay, the probes 1-Zn-PPA and 1-NLG were prepared in a DMSO solution at a molar ratio of 1:1.1. The mixed probe (20 μM) was dissolved in 1 mL of PBS buffer and incubated with 1 mM GSH at 37°C for 2 h. The solutions were then injected into HPLC to analyze the release of Zn-PPA-SH and NLG919, respectively.

[0155] Figure 10 Zn-PPA-SH and NLG919 released from probe 1-NPs in the presence or absence of GSH. As can be seen from the figure, 1-NPs can quickly release Zn-PPA-SH and NLG919 after incubation with GSH, and achieve complete release within 2 hours.

[0156] For the determination of the probe's ability to generate ROS, the probes 1-Zn-PPA and 1-NLG were prepared in a DMSO solution at a molar ratio of 1:1.1. The mixed probe (20 μM) was dissolved in 1 mL of PBS buffer and incubated with 2 μM DCFH-DA and 1 mM GSH at 37°C for 2 h. The solution was then left untreated, ultrasonically treated (1.0 W / cm2, 50% duty, 1.0 MHz), irradiated with light (130 mW / cm2, 671 nm), and treated with both ultrasound and light. The fluorescence spectra of the solutions were recorded using a fluorometer.

[0157] Figure 11 The DCF fluorescence enhancement of probe 1-NPs under different treatment conditions with or without GSH can be seen from the figure. After co-incubation of 1-NPs with GSH and ultrasound and light irradiation, the ROS generated increased by about 2.5 times and 3.6 times compared with the blank group, respectively, and can be further increased to ~5.8 times under combined ultrasound and light irradiation.

[0158] In summary, the co-assembled nanoprobe 1-NPs can be effectively reduced and disassembled by GSH, releasing the photo / sound-sensitive drug Zn-PPa-SH and the immune adjuvant drug NLG919, and achieving enhanced activation of ROS production under the cascade activation of HSA and the combined action of light / sound.

[0159] Example 3: Application of probe for in situ tumor treatment using magnetic resonance / fluorescence dual-modality imaging

[0160] 1. Establishment of Mouse Tumor Model:

[0161] Experimental animals: 6- to 8-week-old BALB / b female mice (purchased from the Model Animal Research Center (MARC) of Nanjing University (Nanjing, China)) were housed and used in accordance with the regulations of the Institutional Animal Care and Use Committee.

[0162] To establish orthotopic 4T1 tumors, approximately 3 × 10 6 4T1 cells (mouse breast cancer cells, purchased from Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China)) were suspended in a mixture of RPMI1640 cell culture medium (10% FBS) / matrigel (50 / 50, v / v%) and then injected into the lower right mammary pad of mice. Tumors grew to approximately 100 mm 7–10 days after inoculation. 3 The size of the microscope is used for magnetic resonance and fluorescence imaging and tumor therapy.

[0163] 2.1-Magnetic resonance / fluorescence dual-modality imaging applications of NPs probes:

[0164] (1) In vivo magnetic resonance imaging of mice:

[0165] In this example, a PBS solution containing 1-NPs was injected into the tail vein of 4T1 tumor-bearing mice (the injection dosage was 0.03 mmol / kg, based on the dosage of Gd metal ions). Magnetic resonance images of the mice were acquired using a mouse body coil and a 1T small animal magnetic resonance scanner 0, 2, 4, and 8 hours after the injection. The acquired images were processed, and the magnetic resonance signals of the tumor area were circled for quantitative analysis. The analysis results are shown in FIG. Figure 12 shown.

[0166] Figure 12 Figures (a) and (b) show MRI images of the tumor at 0, 2, 4, and 8 hours after tail vein injection of probe 1-NPs into mice. The images show a significant increase in the MR signal within the tumor 1 hour after nanoprobe injection, reaching a maximum signal intensity 4 hours after injection. Quantitative analysis of the MR signal at the tumor site revealed an average signal enhancement (%SE) exceeding 80%, demonstrating that NP-cRGDs are effectively delivered to the tumor.

[0167] (2) In vivo fluorescence imaging of mice:

[0168] In this example, 200 μL of PBS solution containing 1-NPs was injected into the tail vein of 4T1 tumor-bearing mice, wherein the concentration of 1-NPs was 200 μM. Fluorescent images of the mice were collected using a small animal fluorescence imager at 0, 4, 8, 12, 24, 48, and 72 hours after injection. The collected images were processed, and the fluorescent signals of the tumor area were circled for quantitative analysis. The analysis results are shown in FIG. Figure 13 shown.

[0169] Figure 13 Figure 1 shows the near-infrared fluorescence imaging of the tumor at 0, 4, 8, 12, 24, 48, and 72 hours after probe 1-NPs were injected into the mouse via the tail vein (a) and the quantitative data of the near-infrared fluorescence signal in the tumor (b). It can be seen from the figure that after the nanoprobe was injected into the body via the tail vein, the near-infrared fluorescence of the tumor site gradually increased, and the fluorescence intensity of the tumor site reached the maximum at 8 hours after injection, indicating that the nanoprobe can be injected into the mouse via α v β3 integrin-mediated active targeting accumulates at the tumor site and can be activated for disassembly by reduced GSH that is highly expressed inside tumor cells.

[0170] (3) In vitro organ fluorescence imaging:

[0171] In this example, 200 μL of PBS solution containing 1-NPs was injected into the tail vein of 4T1 tumor-bearing mice, wherein the concentration of 1-NPs was 200 μM. The mice were killed 24 hours after the injection, and the tumors and major organs including the brain, heart, liver, spleen, kidney, and lungs were isolated and collected, and their fluorescence images were collected. The collected images were processed, and the fluorescence signals of the tumor area were circled for quantitative analysis. The analysis results are shown in FIG. Figure 14 shown.

[0172] from Figure 14 It can be seen that compared with organ tissues, tumors have the strongest fluorescence, among which tumor tissues have the strongest fluorescence, indicating that the nanoprobes have v Active targeting mediated by β3 integrin enabled effective accumulation and activation at the tumor site. Furthermore, a certain intensity of fluorescence was observed in the kidneys and liver, likely due to partial activation of the probe in the liver and the clearance of a portion of the released small molecule photosensitizer product through the kidneys.

[0173] (4) Fluorescence imaging of tumor tissue sections:

[0174] In this example, 200 μL of PBS solution containing 1-NPs was injected into the tail vein of 4T1 tumor-bearing mice, wherein the concentration of 1-NPs was 200 μM. The mice were killed 12 hours after the injection, and the tumors were isolated and collected. The tumor tissues were cut into 10 μm thick slices using a microtome, and fluorescence images of the slices were collected under an inverted fluorescence microscope. The results are shown in FIG. Figure 15 shown.

[0175] from Figure 15 As can be seen in the figure, obvious dual-channel fluorescence of ZnPPA-SH and Oxy-Luciferin was observed in tumor cells, confirming that NP-cRGDs can be effectively activated in tumors.

[0176] (5) Biodistribution test of Zn and Gd:

[0177] In this example, 200 μL of PBS solution containing 1-NPs was injected into the tail vein of 4T1 tumor-bearing mice, wherein the concentration of 1-NPs was 200 μM. The mice were sacrificed 4, 24, or 48 hours after injection, and the tumors and major organs were isolated and collected. The tissues were digested with dilute nitric acid and diluted. After analysis, the Zn and Gd content in each tissue was determined by ICP-MS. The analysis results are shown in Figure 2. Figure 16 shown.

[0178] from Figure 16It can be seen that 4 hours after probe injection, the uptake of Gd(III) and Zn(II) in the tumor reached 25% ID / g and 24% ID / g, respectively. At 24h and 48h, the amount of Zn(II) in the tumor was ~21% ID / g~16% ID / g, and the amount of Gd(III) in the tumor was ~8% ID / g and ~7% ID / g, respectively. This shows that after the nanoprobe is disassembled in a reducing environment, 2-Gd can be quickly cleared from the tumor, and Zn-PPA-SH prolongs its residence time in the tumor due to its binding to albumin inside the tumor, which helps to improve the effect of acousto-photodynamic therapy on the tumor.

[0179] (6) Study on the anti-tumor effect of 1-NPs in vivo:

[0180] When the tumor volume reaches 100 mm 3 The 4T1 tumor-bearing mice were randomly divided into 6 groups and treated as follows:

[0181] I) normal saline; II) 1-NPs; III) 1-NPs + ultrasound; IV) 1-NPs + laser; V) 1-Zn-PPA + ultrasound + laser; VI) 1-NPs + ultrasound + laser;

[0182] In groups II to VI, the dosage of 1-NPs and 1-Zn-PPA was based on 6.35 mg / kg of pyropheophorbide a (Zn-PPA) that chelates Zn ions. Groups III to VI were treated with laser (671 nm, 130 mW / cm 2 ) or ultrasound (2.0W / cm 2 Mice were treated with a 5-minute RT-PCR (50% duty, 1.0 MHz) at the tumor site. Body weight and tumor size were recorded every two days. Seven days after treatment, mice were sacrificed, tumors were collected, and fixed in 4% formalin. After dehydration, H&E and TUNEL staining were performed.

[0183] from Figures 16-19 As can be seen from the figure, the tumor growth curve of group II is slightly slower than that of group I, but the tumor volume continues to increase over time, indicating that 1-NLG in NP-cRGDs has a certain inhibitory effect on tumor growth, but its inhibitory effect is limited. Group V mice showed an effective inhibitory effect on the early growth of tumors under the action of SPDT, but relapsed 15 days after treatment, and the tumor volume increased rapidly. After treatment with 1-NPs + ultrasound + laser (group VI), the tumor growth of mice was greatly slowed down, and the tumors in this group were completely eliminated after 30 days ( Figure 18Compared with other control groups, the survival rate of mice treated with 1-NPs+US+laser (Group VI) was 100% at 100 days, indicating that 1-NPs+US+laser can effectively prolong the survival rate of 4T1 tumor-bearing mice ( Figure 19 ). Hematoxylin and eosin (H&E) and TUNEL staining of tumor tissue sections after different treatments also confirmed that 1-NPs+ultrasound+laser could induce more 4T1 tumor cell death ( Figure 20 ).

[0184] Figure 4 The in vivo tumor-killing mechanism of 1-NPs was demonstrated. 1-NPs were injected into mice via the tail vein. Under the action of surface cRGD, 1-NPs accumulated at the tumor site. Under the action of GSH within the tumor cells, 2-Gd fragments were disassembled and released, restoring fluorescence at 547 nm. The released Zn-PPA-SH then bound to intracellular albumin, restoring near-infrared fluorescence at 677 nm. Under the combined action of ultrasound and light, 1-NPs released ROS, which killed tumor cells and induced immunogenic cell death (ICD). This ROS catalyzed dendritic cell maturation, enhanced antigen presentation, and recruited cytotoxic T cells to kill tumor cells. The released NLG919 inhibited the action of IDO1, downregulated the kynurenine to tryptophan ratio, weakened the inhibitory effect of regulatory T cells on cytotoxic T cells, and further enhanced the anti-tumor activity of cytotoxic T cells.

[0185] (7) Study on the immune effect of 1-NPs in vivo:

[0186] When the tumor volume reaches 100 mm 3 The 4T1 tumor-bearing mice were randomly divided into 6 groups and treated as follows:

[0187] I) normal saline; II) 1-NPs; III) 1-NPs + ultrasound; IV) 1-NPs + laser; V) 1-Zn-PPA + ultrasound + laser; VI) 1-NPs + ultrasound + laser;

[0188] In groups II to VI, the dosage of 1-NPs and 1-Zn-PPA was based on Zn-PPA 6.35 mg / kg. Groups III to VI were treated with laser (671 nm, 130 mW / cm 2 ) or ultrasound (2.0W / cm 2, 50% duty, 1.0MHz) was used to treat the tumor site of mice for 5 minutes. On the 7th day after administration, the mice were killed to collect blood and tumors and lymph nodes. The tumors or lymph nodes were cut into small pieces and ground through a 40μm cell sieve, and the cell suspension was collected for flow cytometric analysis. The mouse blood was placed in a 4°C refrigerator overnight and then centrifuged to obtain the supernatant. The ELISA kit was used to measure the levels of TNF-α, IL-6 and IFN-γ in the serum. The tumor tissue was lysed, the lysate was collected, and the ratio of kynurenine to tryptophan was analyzed by HPLC. The analysis results are shown in the figure. Figures 21-23 shown.

[0189] from Figure 21 As can be seen from Figures a and 21b, the degree of CRT protein externalization and HMGB1 release rate in the tumors of the 1-NPs+ultrasound+laser (Group VI) treatment group were significantly higher than those of the 1-NPs+ultrasound (Group III) or 1-NPs+NIR (Group IV) treatment groups. Figure 21 As can be seen in c, the proportion of mature DCs in the lymph nodes of group V (50.0±4.1%) and group VI (55.5±2.3%) was significantly higher than that of saline-treated mice (group I, 13.7±1.8%), indicating that ICD induction by 1-NPs+ultrasound+laser helps promote DC maturation in lymph nodes, thereby enhancing antigen presentation and activating downstream immune effects. Figure 21 As can be seen from Figures 2d and 21f, the levels of TNF-α, IL-6, and IFN-γ in mice treated with 1-NPs alone (Group II) were similar to those in the control group of mice treated with normal saline (Group I), while the levels of each cytokine in mice treated with 1-NPs + ultrasound + laser (Group VI) were at higher levels, ~3.0 and ~3.7, respectively.

[0190] from Figure 22 and 23 As can be seen from a to b, the numbers of helper T cells and cytotoxic T cells in groups V and VI increased significantly, indicating that photodynamic therapy can recruit more effector T cells to infiltrate tumor tissue than photodynamic therapy or photodynamic therapy alone.

[0191] from Figure 23 As can be seen in c, compared with the control group I, the ratio of kynurenine to tryptophan in the tumor tissue of group V lacking 1-NLG increased, while the ratio of kynurenine to tryptophan in the tumor tissue of groups III, IV and VI was significantly decreased, indicating that the probe 1-NLG can inhibit the metabolism of tryptophan by inhibiting the activity of IDO1.

[0192] from Figure 23 d and Figure 23As can be seen in Figure e, the content of regulatory T cells marked by Foxp3 and CD25 in tumor cells in group VI was significantly reduced compared with group I, indicating that 1-NPs effectively inhibited the immunosuppressive effect in tumors. By quantifying the cytotoxic T cells and regulatory T cells in tumor cells, it was found that the ratio of cytotoxic T cells to regulatory T cells was the highest in group VI.

[0193] In summary, nanoprobe 1-NPs can activate and produce a stronger ICD effect under the synergistic effect of acousto-photodynamic therapy, catalyze DC cell maturation, recruit effector T cell infiltration and reduce the immunosuppressive effect caused by regulatory T cells, thereby exerting a better anti-tumor effect.

[0194] The embodiments described are preferred implementations of the present invention, but the present invention is not limited to the above implementations. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention are within the scope of protection of the present invention.

Claims

1. An amphiphilic small molecule probe, characterized in that: The probe comprises the following structure: (1) Rigidly linked backbone aminofluorescein fragment AO-Luc; (2) tumor-targeting cRGD fragments; (3) DOTA-Gd fragments that can be used for MR imaging; (4) Disulfide bonds that can be cleaved by GSH; (5) Zn-PPA fragment or NLG919 fragment; The amphiphilic small molecule probe containing the Zn-PPA fragment is denoted as 1-Zn-PPA, and its structural formula is: ； The amphiphilic small molecule probe containing the NLG919 fragment is denoted as 1-NLG, and its structural formula is: 。 2. The method for preparing the amphiphilic small molecule probe according to claim 1, wherein the amphiphilic small molecule probe comprises 1-NLG and 1-Zn-PPA; The preparation method of the 1-NLG is: Compound 7 was dissolved in a solvent containing trifluoroacetic acid and triisopropylsilane to react. After the reaction, the solvent was removed by rotary evaporation under reduced pressure. Cold ether was added to collect the precipitate. The precipitate and compound 1 were then dissolved in the solvent to react. After the reaction, vacuum rotary evaporation was performed, purification was performed, and freeze-drying was performed to obtain 1-NLG. The preparation method of the 1-Zn-PPA is: Compound 7 was dissolved in a solvent containing trifluoroacetic acid and triisopropylsilane to react. After the reaction, the solvent was removed by rotary evaporation under reduced pressure. Cold ether was added to collect the precipitate. The precipitate, compound 2, and zinc chloride were then dissolved in the solvent to react. After the reaction, vacuum rotary evaporation was performed, purification was performed, and freeze-drying was performed to obtain 1-Zn-PPA. The structural formula of the compound 1 is: ； The structural formula of the compound 2 is: ； The structural formula of the compound 7 is: 。 3. The method for preparing an amphiphilic small molecule probe according to claim 2, wherein: During the preparation of 1-NLG: The molar ratio of compound 7 to compound 1 is 1:1 to 1:2; The volume ratio of the trifluoroacetic acid, triisopropylsilane and solvent is 46:4:

50.

4. The method for preparing an amphiphilic small molecule probe according to claim 2, wherein: During the preparation of 1-Zn-PPA: The molar ratio of compound 7, compound 2, and zinc chloride is 1:1:1 to 1:2:10; The volume ratio of the trifluoroacetic acid, triisopropylsilane and solvent is 46:4:

50.

5. The method for preparing an amphiphilic small molecule probe according to claim 2, wherein: During the preparation of 1-NLG, the solvent containing trifluoroacetic acid and triisopropylsilane is dichloromethane, and the solvent in which the precipitate and compound 1 are dissolved is methanol; During the preparation of 1-Zn-PPA, the solvent containing trifluoroacetic acid and triisopropylsilane is dichloromethane, and the solvent for the reaction of the precipitate, compound 2, and zinc chloride in the solvent is methanol; All the reactions involved were carried out at 20-30°C for 1-6 hours.

6. A glutathione-activated co-assembled nanoprobe based on the amphiphilic small molecule probe according to claim 1, characterized in that: The glutathione-activated co-assembled nanoprobe is obtained by co-assembling the amphiphilic small molecule probe 1-Zn-PPA and 1-NLG, and is denoted as 1-NPs.

7. The glutathione-activated co-assembled nanoprobe according to claim 6, characterized in that The molar ratio of 1-Zn-PPA to 1-NLG is 1:1.

1.

8. Use of the glutathione-activated co-assembled nanoprobe according to claim 6 or 7 in the preparation of drugs for treating tumors.

Images