A cisplatin prodrug diagnostic probe P-CyPt and its preparation method and application

Abstract

The present invention provides a cisplatin prodrug diagnostic and therapeutic probe P-CyPt and its preparation method and application, which belong to the technical field of biological probes. The present invention combines the enzyme-mediated in situ self-assembly and the disassembly strategy regulated by the endogenous reducing environment in the cell to design and synthesize the cisplatin prodrug diagnostic and therapeutic probe P-CyPt with the following characteristics: (1) cisplatin tetravalent prodrug nanoparticles (Pt) with enhanced near-infrared fluorescence and photoacoustic signals are formed by self-assembly mediated by extracellular alkaline phosphatase. Ⅳ NPs) are used to enhance tumor uptake and tumor lesion tracking; (2) Glutathione reduction in cells regulates Pt Ⅳ NPs disassemble to release cisplatin prodrug and small molecule near-infrared dye Cy‑COOH for in vivo anti-tumor and drug release monitoring; the cisplatin prodrug probe P‑CyPt has great applications in near-infrared fluorescence and photoacoustic dual-modality imaging of tumors, preparation of anti-tumor prodrugs, and preparation of tumor diagnostic imaging agents.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological probes, and in particular relates to a cisplatin prodrug diagnostic probe P-CyPt and a preparation method and application thereof. Background Art

[0002] Cisplatin is a first-line chemotherapy anticancer drug used clinically to treat various malignant tumors. However, due to nonspecific biodistribution and insufficient tumor uptake, it can cause strong systemic toxicity during treatment, such as common nephrotoxicity, neurotoxicity, and tumor resistance, which limits its therapeutic efficacy. Although a large number of cisplatin prodrugs and nanocarrier-based delivery systems have been developed to overcome the side effects and drug resistance caused by cisplatin itself, the anti-cancer effect of this type of probe in vivo is still unsatisfactory due to the low effective concentration of cisplatin in tumor tissues and the development of multidrug resistance.

[0003] Self-assembly and disassembly are ubiquitous, reversible processes in living organisms, involved in many important physiological and pathological processes. Numerous studies have reported on synthesizing nanostructures and microstructures through self-assembly to perform higher-order complex functions, or on regulating drug release and degradation of complex structures through disassembly. Stimuli-responsive in situ self-assembly processes, in particular, allow for the in situ synthesis of nanostructures in vivo, providing an efficient approach for the in situ synthesis of macromolecules. In this field, replacing pre-assembled nanomaterials in vitro with small molecules with well-defined chemical structures offers certain advantages. Due to their small size, small molecules can rapidly penetrate diseased tissues and, under the influence of biological targets, undergo structural transformations, self-assembling in situ into nanoscale aggregates, thereby slowing the diffusion of probe molecules and prolonging their residence time in the target tissue. This biological target-triggered in situ self-assembly strategy has been widely applied in molecular imaging, drug delivery, and other fields. Combining in situ self-assembly with stimulus-triggered disassembly strategies can not only enhance the delivery of prodrug molecules to deep diseased tissues through in situ self-assembly, but also enable on-demand release of drug molecules in diseased tissues through controlled disassembly, thereby significantly improving the therapeutic efficacy of drug molecules in vivo. However, due to the highly dynamic and extremely complex living environment, designing a small molecule prodrug capable of in situ self-assembly and controllable disassembly for in vivo drug delivery and release remains a challenging problem. Summary of the Invention

[0004] In view of the shortcomings of the prior art, the present invention provides a cisplatin prodrug diagnostic probe P-CyPt and its preparation method and application. The present invention combines the enzyme-mediated in situ self-assembly and the intracellular endogenous reducing environment disassembly strategy to design and synthesize a cisplatin prodrug diagnostic probe P-CyPt with the following characteristics: (1) Enzyme-activated self-assembly outside the cell to form cisplatin prodrug nanoparticles (Pt Ⅳ (1) NPs are used for near-infrared fluorescence and photoacoustic dual-modality imaging of tumor foci; (2) in the intracellular reducing environment, they are stimulated to disassemble and release cisplatin prodrug and small molecule near-infrared dye Cy-COOH for in vivo anti-tumor and drug release process monitoring. The cisplatin prodrug diagnostic probe P-CyPt has great applications in near-infrared fluorescence and photoacoustic dual-modality imaging, preparation of anti-tumor drugs, and preparation of tumor diagnostic imaging agents.

[0005] The present invention first provides a cisplatin prodrug diagnostic and therapeutic probe P-CyPt, wherein the cisplatin prodrug diagnostic and therapeutic probe P-CyPt comprises:

[0006] (1) Phosphate groups (-PO3H2) that can be hydrolyzed by alkaline phosphatase (ALP),

[0007] (2) Hydrophobic near-infrared (NIR) fluorophores (merocyanine derivatives, mCy) for NIR fluorescence and photoacoustic dual-modality imaging;

[0008] (3) a tetravalent cisplatin prodrug fragment that can be reduced by reduced glutathione (GSH) to release the cisplatin original drug;

[0009] (4) Hydrophobic dipeptides (FF) connect the fragments to promote molecular self-assembly.

[0010] Specifically, the structural formula of the cisplatin prodrug diagnostic probe P-CyPt is:

[0011]

[0012] The present invention also provides a method for preparing the above-mentioned cisplatin prodrug diagnostic probe P-CyPt, which specifically comprises the following steps:

[0013] Compound 4, HS-Pt, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), and triethylamine (TEA) are dissolved in a solvent, and then reacted under stirring. After the reaction, the mixture is centrifuged, purified, and freeze-dried to obtain the cisplatin prodrug probe P-CyPt;

[0014] The structural formula of the compound 4 is:

[0015]

[0016] The preparation method of compound 4 is described in Nano Lett., 2021, 21, 10377; the synthesis method of the compound HS-Pt is described in Chem. Sci., 2013, 4, 2605.

[0017] Further, the molar ratio of compound 4, HATU, TEA, and HS-Pt is 1:2.5-5.5:2.5-5.5:1.5-3.5;

[0018] The concentration of the compound 4 solution is 0.004 to 0.024 mmol / mL; the reaction is stirred at -40 to 0° C. for more than 12 hours; and the solvent includes N,N-dimethylformamide (DMF).

[0019] Furthermore, the molar ratio of compound 4, HATU, TEA, and HS-Pt was 1:3:3:2;

[0020] The concentration of the compound 4 solution is 0.014 mmol / mL;

[0021] The reaction is stirred at -20°C for 12 to 26 hours.

[0022] The present invention also provides the use of the above-mentioned cisplatin prodrug diagnostic probe P-CyPt in the preparation of tumor diagnostic imaging agents. Specifically, the application is to prepare alkaline phosphatase (ALP)-positive tumor diagnostic imaging agents, where the alkaline phosphatase is endogenous ALP on the surface of the human cervical cancer HeLa cell or human liver cancer HepG2 cell membrane.

[0023] The present invention also provides the use of the above-mentioned cisplatin prodrug diagnostic and therapeutic probe P-CyPt in constructing a living anti-tumor model; specifically, the living tumor model is a HeLa cell subcutaneous tumor model and a HepG2 liver in situ tumor model.

[0024] The present invention also provides the application of cisplatin prodrug diagnostic probe P-CyPt in near-infrared fluorescence and photoacoustic dual-modality imaging.

[0025] The present invention also provides the use of cisplatin prodrug diagnostic probe P-CyPt in the preparation of anti-tumor drugs.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] In this study, using cisplatin, a first-line chemotherapy anticancer drug, as a model drug, a sequential stimulus-triggered in situ self-assembly and disassembly system was designed, and the cisplatin prodrug therapeutic probe P-CyPt was designed and prepared. In this study, the cisplatin prodrug therapeutic probe P-CyPt can enhance the delivery and release of cisplatin in tumor cells by utilizing in situ self-assembly triggered by extracellular alkaline phosphatase (ALP) and disassembly triggered by intracellular glutathione (GSH).

[0028] Compared with the small molecule cisplatin or pre-prepared Pt(IV) nanoparticles in the prior art, the cisplatin prodrug diagnostic probe P-CyPt in the present invention is applied to the imaging and treatment of subcutaneous and liver in situ tumor models in mice, and its anti-tumor therapeutic effect is significantly enhanced. In addition, the present invention uses complementary near-infrared fluorescence (FL) and photoacoustic (PA) dual-modal imaging to monitor this sequentially activated in situ self-assembly and disassembly process in cells and living tumors in real time. This strategy overcomes the major challenge of less than 1% of the target area of nano drug molecules after systemic administration, and effectively improves the application of cisplatin prodrugs in anti-tumor.

[0029] The cisplatin prodrug diagnostic probe P-CyPt of the present invention contains a phosphate group that can be specifically hydrolyzed by the ALP enzyme highly expressed on the surface of tumor cells. The hydrolyzed probe molecule CyPt can self-assemble in situ around the ALP enzyme on the cell membrane surface to form nanoparticles Pt IV On the one hand, due to the enhanced fluorescence and photoacoustic signals of the leaving probe molecules of the phosphate group, the fluorescence / photoacoustic dual-modal signal distinction of the tumor lesion site is achieved. On the other hand, due to the in situ self-assembly, the cisplatin prodrug nanoparticles Pt IV NPs can adhere to the tumor cell membrane, overcome the diffusion of small molecule cisplatin drugs in tumor tissue, and thus increase the tumor cell's sensitivity to Pt IV NPs uptake. When cisplatin prodrug nanoparticles Pt IV After NPs enter the tumor cells, under the condition of high concentration of GSH in the tumor cells, tetravalent Pt IV It is reduced to divalent cisplatin and quickly releases the cisplatin original drug molecule. With the release of cisplatin, the Pt IV The NPs disassemble, releasing the free small-molecule dye Cy-COOH, which further enhances intracellular fluorescence and weakens the photoacoustic signal at 750nm. The disassembly process also accelerates the release of cisplatin molecules within the cells, rapidly increasing the concentration of cisplatin within tumor cells and killing them. In normal tissues and organs, ALP expression is low, preventing the probe P-CyPt from being activated. This leads to rapid clearance of the hydrophilic cisplatin prodrug P-CyPt within the body, thereby reducing the systemic toxic side effects of the cisplatin prodrug.

[0030] The cisplatin prodrug diagnostic probe P-CyPt provided in the present invention can realize ALP-activated near-infrared fluorescence and photoacoustic dual-modal imaging signals, thereby enabling high-sensitivity and high-penetration-depth detection and imaging of ALP-positive tumors in vivo, and through the ALP enzyme-mediated in situ self-assembly and intracellular glutathione-triggered disassembly strategy, "visualized" tumor treatment guided by near-infrared fluorescence and photoacoustic dual-modal imaging of the tumor area can be achieved.

[0031] In summary, the cisplatin prodrug diagnostic probe P-CyPt achieves near-infrared fluorescence / photoacoustic dual-modal imaging of tumor sites through in situ self-assembly mediated by ALP enzyme and disassembly triggered by intracellular GSH. This strategy can significantly enhance the uptake of cisplatin by ALP-positive tumors and reduce the GSH concentration in tumor cells, thereby effectively inhibiting tumor growth in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Synthesis route of cisplatin prodrug therapeutic probe P-CyPt.

[0033] Figure 2 Design of the cisplatin prodrug therapeutic probe P-CyPt (a) and its mechanism of action (b).

[0034] Figure 3 HPLC tracking (a) and DLS analysis (b) of the ALP enzyme-mediated in situ self-assembly and GSH-triggered disassembly performance of the cisplatin prodrug diagnostic probe P-CyPt.

[0035] Figure 4 The enzymatic cleavage of tetravalent platinum prodrug nanoparticles (Pt IV NPs) transmission electron microscopy (TEM) analysis, where the scale bar is 200 nm.

[0036] Figure 5 Pt is formed after incubation of P-CyPt with ALP IV TEM images (a) and STEM images (b-d) of NPs, where Figure c is for Pt IV The analysis results of chlorine element in NPs, Figure d is the analysis of Pt IV Analysis results of platinum element in NPs.

[0037] Figure 6 The tetravalent platinum prodrug nanoparticles (Pt IV UV-visible absorption spectrum (a) and fluorescence emission spectrum (b) of NPs), where the lower figure is when ALP enzyme is added, and the upper figure is when GSH is added.

[0038] Figure 7To form tetravalent platinum prodrug nanoparticles (Pt IV Figure 3. Transition of photoacoustic signals during the assembly and disassembly processes of NPs.

[0039] Figure 8 The following images show the cell imaging of the probe P-CyPt in HeLa cells, HeLa cells pretreated with Na3VO4, and HEK-293T cells after 30 minutes of incubation. Cy-Cl is the fluorescence channel of the dye mCy, and merge is the overlay of the Cy-Cl channel and the Hoechst 33342 channel.

[0040] Figure 9 The cell cluster imaging images of the probe P-CyPt under different conditions (a), the fluorescence images under the corresponding conditions (b), the photoacoustic signal images under the corresponding conditions (c), and the HPLC analysis images of the cell lysate (d).

[0041] Figure 10 This is the TEM imaging of the probe P-CyPt after incubation with HeLa cells for 1 hour. The images are N, L, Cl, M, C and Pt respectively.

[0042] Figure 11 For the probe P-CyPt, Pt IV Figure 3 (a) shows the ratio of elemental platinum uptake to the total amount by tumor cells in the NPs, CDDP and Na3VO4 control groups, and the distribution of P-CyPt and CDDP in the subcellular organelles of HeLa cells.

[0043] Figure 12 For the probe P-CyPt, Pt IV The cytotoxicity of NPs and CDDP on different cells. In the figure, a is P-CyPt, CDDP, and Pt IV NPs were co-incubated with different cells for 48 h. Figure b shows the IC values measured after the compounds were co-incubated with cells for 2 h, the culture medium was removed, washed three times with PBS, and then replaced with fresh culture medium for another 48 h. 50 value.

[0044] Figure 13 For the probe P-CyPt and Pt IV The permeability of the 3D tumor cell spheroid model of NPs. 0-200 μm represents the depth of the 3D tumor cell spheroid, and Cy-Cl and PI correspond to the fluorescence imaging under this channel.

[0045] Figure 14 For the probe P-CyPt and Pt IVNear-infrared fluorescence image (a) and near-infrared fluorescence intensity quantification value image (c), photoacoustic imaging image (b) and photoacoustic intensity quantification value image (d) of NPs in HeLa subcutaneous tumor model mice.

[0046] Figure 15 (a) shows the injection probe P-CyPt and Pt IV Analysis of lysate of tumor tissue after NPs injection; (b) is the injection of probes P-CyPt, CDDP and Pt IV The Pt element content in different organs and tumor tissues of mice with HeLa subcutaneous tumor model after NPs addition. The bar graphs from left to right are P-CyPt, CDDP and Pt IV NPs.

[0047] Figure 16 Schematic diagram of tumor treatment of HeLa mice with probe P-CyPt (a), tumor growth during treatment (b), weight of mouse tumor tissue after treatment (c), and changes in mouse weight during treatment (d).

[0048] Figure 17 For the probes P-CyPt, PBS, CDDP and Pt IV Figure 3 Changes in blood urea nitrogen (a) and creatinine (b) after 21 days of NPs treatment in HeLa subcutaneous tumor model mice.

[0049] Figure 18 For the probe P-CyPt and Pt IV Near-infrared fluorescence imaging of NPs in HepG2 orthotopic liver tumor model mice (a) and quantification of near-infrared fluorescence intensity (b).

[0050] Figure 19 Schematic diagram of tumor treatment of HepG liver orthotopic tumor model mice with probe P-CyPt (a), tumor growth during treatment (b), and bioluminescence imaging monitoring of HepG liver orthotopic tumor model mice during treatment (c).

[0051] Figure 20 Figure 3 shows the bioluminescence imaging of liver tumors in mice with HepG liver in situ tumor model treated with probe P-CyPt and its control group 15 days after treatment (a), near-infrared fluorescence imaging (b), and the correlation between the relative intensities of bioluminescence and near-infrared fluorescence in the tumor area (c).

[0052] Figure 21 The clearance of probe P-CyPt in urine (a) and feces (b) of mice within 0-12 hours and the clearance of Pt IV Elimination of NPs in urine (c) and feces (d) of mice within 0-12 h.

[0053] Figure 22 For HPLC analysis of P-CyPt, Pt IV The contents of NPs in urine (a) and feces (b), and the contents of Pt elements in urine (c) and feces (d) in different treatment groups analyzed by ICP-OES. DETAILED DESCRIPTION

[0054] 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.

[0055] Unless otherwise specified, the "%" in the following examples refers to molar percentage.

[0056] Reagents and Instruments: All chemical reagents and solvents were purchased from J&K Technology Co., Ltd. (Shanghai, China), TCI Chemical Industry Development Co., Ltd. (Shanghai, China), and Sigma-Aldrich. Analytical solvents and reagents were chromatographic grade, and routine reagents were analytical grade without further purification. Cisplatin was purchased from Sigma-Aldrich. N-Fmoc-D-phenylalanine was purchased from GL Biochemical (Shanghai) Co., Ltd. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) kit, propidium iodide (PI), Hoechst 33342, and Annexin V-FITC / PI apoptosis staining kit were all from KeyGen Biotech Co., Ltd. (Nanjing, China).

[0057] 1 H-、 13 C- and 195Pt-NMR spectra were obtained at 298 K on a Bruker DRX-400 spectrometer using DMSO-d6 or Chloroform-d as solvents. Chemical shifts (δ) are reported in ppm, with singlets, doublets, triplets, quartets, dd (doublet of doublets), multiplets, and broad peaks represented by s, d, t, q, dd, and m, respectively. Coupling constants (J) are reported in Hz. The number of hydrogen atoms is determined by integrating the spectra and is labeled nH. High-performance liquid chromatography (HPLC) was performed on a ThermoScientific Dionex Ultimate 3000 using CH3CN / H2O (1‰ CF3COOH) as eluent. Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis was performed using an AB SCIEX 4800Plus MALDI TOF / TOF™ mass spectrometer. UV-Vis spectra were measured using an Ocean Optics Maya 2000Pro spectrometer. Fluorescence spectra were obtained using a HORIBA Jobin Yvon Fluoromax-4 fluorimeter. Inductively coupled plasma optical emission spectrometer (ICP-OES) analysis was performed using an Avio 500 (PE, USA). Fluorescence images of cells and tissue sections were acquired using an Olympus IX73 inverted fluorescence microscope. In vivo fluorescence images were acquired using an IVIS Lumina XR III system, and fluorescence intensity was quantified by measuring the signal within the enclosed area using Living Image software (PerkinElmer). Photoacoustic images were acquired using a Vevo 2100LAZR (FUJIFILM VisualSonics, Canada). Blood count data were analyzed using a BC2800Vet.

[0058] The human cervical cancer HeLa cells, human umbilical vein endothelial HUVEC cells, and human kidney HEK-293T cells used in the following examples were purchased from the Shanghai Stem Cell Institute, Chinese Academy of Sciences, and cultured in Dulbecco's Modified Eagle Medium (DMEM). The culture medium was supplemented with 10% (v / v) fetal bovine serum (FBS) and 100 units of penicillin and 100 units of streptomycin per ml of culture medium. All cells were cultured at 37°C in a humidified atmosphere (5% CO2).

[0059] The specific methods of the cytotoxicity study (MTT) involved are as follows:

[0060] Cultured tumor cells were seeded in a flat-bottom 96-well plate (5000 cells per well, 100 μL of DMEM per well) and incubated overnight at 37°C. The DMEM was aspirated from each well, and P-CyPt, CyPt, and CDDP were added to DMEM at various concentrations (0, 1, 2, 5, 10, 20, 30, 50, 100, and 200 μM) with 100 μL added to each well. After a 2-hour incubation, fresh medium was replaced and incubated for an additional 48 hours. Alternatively, after 48 hours of incubation, 50 μL of MTT solution (1 mg / mL in PBS buffer) was added to each well. The cells were incubated at 37°C for an additional 4 hours, after which the solution was carefully removed from each well. 150 μL of DMSO solution was added to each well to dissolve the purple crystals that formed. The absorbance (OD) of each well was measured at 490 nm using a microplate reader (Tcan). The absorbance of blank cells (OD control) was used as a control, and the percentage of viable cells in each well was calculated by OD / OD control × 100%, and each experiment was repeated three times.

[0061] The design (a) and mechanism of action (b) of the cisplatin prodrug diagnostic probe P-CyPt designed in the present invention are as follows: Figure 2 shown.

[0062] Example 1:

[0063] The synthetic route of cisplatin prodrug diagnostic probe P-CyPt is as follows Figure 1 As shown in a, the specific synthesis process is as follows:

[0064] (1) Synthesis of cisplatin prodrug diagnostic probe P-CyPt:

[0065] Under N2, compound 4 (25 mg, 0.03 mmol), HATU (34.1 mg, 0.09 mmol) and TEA (12.5 μL, 0.09 mmol) were stirred in 5 mL of dry DMF at -20 ° C for 10 minutes, and HSPt (24.9 mg, 0.06 mmol) in 2 mL of DMF was added dropwise. The reaction solution was then stirred at -20 ° C for 12 hours. The solvent was then evaporated and the residue was purified by semi-separation HPLC. After lyophilization, a blue solid (16.9 mg, yield: 45%) was obtained, which is the cisplatin prodrug diagnostic probe P-CyPt.

[0066] The cisplatin prodrug diagnostic probe P-CyPt 11H NMR: (400 MHz, DMSO-d6) δ 8.33 (d, J = 13.9 Hz, 1H), 8.16 (s, 1H), 8.09 (d, J = 7.1 Hz, 1H), 8.04 (d, J = 7.2 Hz, 1H), 7.67 (d, J = 7.1 Hz, 1H), 7.61 (d, J = 4.7 Hz, 2H), 7.54 (s, 1H), 7.46 (t, J = 7.5 Hz, 1H), 7.37 (t, J = 7.3 Hz, 1H), 7.23 (s, 1H), 7.16 (d, J = 5.5 Hz, 4H), 7.09 (d, J = 7.0 Hz, 3H), 7.04 (d, J = 7.2 Hz, 3H), 6.47 (d, J = 14.6 Hz, 1H), 5.96 (s, br, 6H), 4.40–4.24 (m, 4H), 3.19–3.16 (m, 2H), 2.95 (dd, J = 13.5, 4.8 Hz, 1H), 2.86–2.78 (m, 2H), 2.61 (d, J = 13.6 Hz, 5H), 2.31 (s, 1H), 2.16 (dd, J = 21.2, 8.9 Hz, 4H), 1.83 (s, 2H), 1.71 (s, 2H), 1.64 (s, 6H). 13 13C NMR (101 MHz, DMSO-d6) δ 180.45, 178.48, 172.40, 171.61, 171.42, 159.92, 152.27, 151.82, 145.72, 142.59, 141.68, 138.29, 138.17, 131.50, 129.63, 129.50, 129.39, 128.86, 128.55, 128.38, 127.76, 126.79, 126.56, 123.29, 121.37, 117.26, 114.74, 113.83, 108.08, 105.56, 54.90, 54.77, 51.19, 43.62, 40.57, 39.32, 37.91, 37.55, 36.58, 32.47, 32.17, 29.01, 27.97, 27.70, 24.88, 24.11, 20.23.C 50 H 59 Cl3N6O 11 PPt + [M] +:1249.2667(97.4%),1250.2688(100.0%),1250.2700(52.7%),1251.2637(93.4%),1251.2689(74.6%),1251.2721(54.1%),1252.2658(95.9%),1252.2671(50.5%),1252.2723( 40.3%), 1253.2660 (71.5%), 1253.2608 (29.9%), 1253.2692 (51.8%), 1253.2719 (21.2%), 1254.2693 (38.7%), 1254.2629 (30.6%), 1255.2630 (22.9%), 1255.2689 (20.3%); ESI-MS found: m / z 1249.2645, 1250.2686, 1251.2689, 1252.2689, 1253.2682, 1254.2675, 1255.2673. This shows that the cisplatin prodrug diagnostic probe P-CyPt was successfully prepared.

[0067] In order to subsequently investigate the performance of the cisplatin prodrug diagnostic probe P-CyPt, the enzymatic cleavage product CyPt and the product Cy-COOH after CyPt releases Pt were also prepared in this example.

[0068] The synthesis process of CyPt is as follows:

[0069] Synthesis of compound 5: A mixture of piperidine / DMF (1:19, 8 mL) was stirred in an ice bath for 5 min, followed by the addition of compound 3 (58.7 mg, 0.06 mmol). After stirring at 0°C for 0 min, cold aqueous hydrochloric acid (1 M, 4.37 mL) was immediately added to the reaction solution to adjust the pH to neutral. After purification by semi-fractionated HPLC, compound 5 was obtained by lyophilization as a blue solid (41.7 mg) with a yield of 92%.

[0070] Compound 5 1H NMR(400MHz,DMSO-d6)δ8.88(d,J=7.9Hz,1H),8.57(d,J=14.8Hz,1H),8.33(t,J=5.7Hz,1H),8.12(s,2H),7.77(d,J=7.4Hz,1H),7.71–7.62(m,2H),7.56(t,J=7.7Hz,1H),7.48(t,J=7.4Hz,1H),7.42(s,1H),7.27–7.24(m,4H),7.22-7.20(m,4H),7.18–7.14(m,3H),6.55(d,J=14.9Hz,1H),4.54–4.49(m,1H),4.40–4.26(m,2H),4.04(s,1H),3.31–3.25(m,1H),3.23-3.16(m,1H),3.08(dd,J=14.1,5.2Hz,1H),3.01–2.83(m,4H),2.71–2.65(m,4H),1.92–1.79(m,4H),1.76(s,6H). 13 C NMR(101MHz,DMSO-d6)δ177.81,170.75,168.26,162.81,160.77,157.15,152.75,145.53,142.43,141.76,137.73,135.10,132.96,130.00,129.61,129.40,128.84,128.66,128.51,127.58,127.44,126.93,123.23,118.52,118.43,115.49,115.34,114.61,113.58,104.83,103.60,54.97,53.54,50.83,43.14,38.32,37.36,36.43,31.23,28.84,27.89,23.97,22.66,20.34.MS:calcd.ForC 46 H 48 ClN4O4 + [M] + :755.3359;MALDI-MS found:m / z 755.2030.

[0071] Synthesis of compound CyPt: Under N protection, compound 5 (22.6 mg, 0.03 mmol), HATU (34.1 mg, 0.09 mmol) and TEA (12.5 μL, 0.09 mmol) were stirred in 5 mL of ultra-dry DMF solvent for 10 minutes. HSPt (24.9 mg, 0.06 mmol) dissolved in 2 mL of DMF was then added dropwise and the solution was stirred at -20°C for 12 hours. CyPt (18.3 mg) was obtained as a blue solid by lyophilization in a yield of 52%.

[0072] 1 H NMR(400MHz, DMSO-d6)δ8.56(d,J=14.8Hz,1H),8.11–8.06(m,3H),7.76(d,J=7.1Hz,1H),7.71–7.63(m,2H),7.55(t,J=7.4Hz,1H),7.4 7(t,J=7.4Hz,1H),7.42(s,1H),7.32–7.27(m,1H),7.26–7.20(m,4H),7.18(d,J=7.2Hz,3H),7.13(d,J=6.8Hz,3H),7.09–7.04(m,1H), 6.55(d,J=14.9Hz,1H),6.14(s,br,6H),4.46–4.40(m,2H),4.37–4.28(m,2H),3.28–3.17(m,2H),3.01(dd,J=13.7,5.5Hz,1H),2.94–2 .83(m,2H),2.73-2.67(m,5H),2.31(d,J=6.1Hz,2H),2.27–2.20(m,2H),1.91–1.84(m,2H),1.80(dd,J=11.3,6.0Hz,2H),1.75(s,6H). 13C NMR(101MHz,DMSO-d6)δ180.15,177.81,172.13,171.58,171.29,160.67,152.73,145.53,142 .43,141.72,138.28,138.14,132.81,129.60,129.53,129.42,128.57,128.38,127.55,126.81 ,126.57,123.20,118.43,115.37,114.63,113.64,104.91,103.62,54.78,54.52,50.84,43.2 0,40.59,39.33,37.94,37.60,36.48,31.99,28.86,27.90,27.78,23.98,20.35.MS:calcd.For C 50 H 58 Cl3N6O8Pt + [M] + :1169.3003(97.4%),1170.3024(100.0%),1170.3037(52.7%),1171.2974(93.4%),1171.3026(74.6%),1171.3058(54.1%),1172.2995(95.9%),1172.3007,(50.5%),1172.3060( 40.3%), 1173.2944(29.9%), 1173.3028(51.9%), 1173.3055(21.2%), 1174.2965(30.6%), 1174.3030(38.7%), 1173.2997(71.5%), 1175.2967(22.9%), 1175.3026(20.3%)), ESI-MS found: m / z 1169.3027, 1170.3044, 1171.3024, 1172.3016, 1173.3000, 1174.2998, 1175.2982. This shows that the probe CyPt was successfully prepared after enzyme cleavage.

[0073] The synthesis process of Cy-COOH is as follows:

[0074] Compound 5 (22.6 mg, 0.03 mg) and succinic anhydride (3.6 mg, 0.036 mmol) were dissolved in 5 mL of dry DMSO and kept stirring at room temperature (rt) for 12 hours, then the solvent was evaporated and the residue was purified by semi-fractionated HPLC and lyophilized to give Cy-COOH as a blue solid (18.3 mg), yield: 88%.

[0075] Cy-COOH 1 H NMR (400MHz, DMSO-d6) δ8.55(d,J=14.2Hz,1H),8.18(d,J=7.1Hz,1H),8.08(d,J=7.3Hz,1H),7.97(s,1H),7.75(d,J=6.9Hz,1 H),7.67(d,J=8.1Hz,2H),7.54(t,J=7.7Hz,1H),7.47(t,J=7.4Hz,1H),7.39(s,1H),7.24(d,J=6.8Hz,4H),7.20–7.15(m,3H), 7.14–7.08(m,4H),6.55(d,J=14.9Hz,1H),4.40(dd,J=10.9,5.4Hz,2H),4.31(dd,J=15.6,7.6Hz,2H),3.22(m,2H),3.10–2.9 8(m,1H),2.88(dt,J=12.6,6.1Hz,2H),2.72–2.65(m,5H),2.29(m,4H),1.93–1.83(m,2H),1.78(d,J=6.2Hz,2H),1.75(s,6H). 13 C NMR(101MHz,DMSO-d6)δ177.29,173.88,171.51,171.05,170.77,160.15,156.58,152.22, 145.05,141.93,141.23,137.78,137.69,132.30,129.06,128.91,128.08,127.89,127.04, 126.28,126.09,122.70,117.93,114.82,114.14,113.13,104.35,103.08,54.40,50.33,4 2.69,37.19,37.05,36.00,29.87,29.04,28.36,27.39,27.28,23.46,19.84.MS:calcd.For C 50 H 52 ClN4O7 + [M] + :855.3519; ESI-MS found: m / z 855.3521. This indicates that the probe CyPt reacts with GSH after enzyme cleavage and releases the molecule Cy-COOH after cisplatin is successfully prepared.

[0076] Example 2:

[0077] In this example, the self-assembly and GSH disassembly capabilities of the cisplatin prodrug diagnostic probe P-CyPt probe in response to ALP enzyme cleavage were investigated.

[0078] (1) In vitro investigation of the assembly and disassembly process of the cisplatin prodrug diagnostic probe P-CyPt probe:

[0079] In this example, to evaluate the dephosphorylation and in situ self-assembly process triggered by ALP, a solution of P-CyPt (10 μM) in 1 mL of Tris buffer was incubated with ALP (100 U / L) at 37°C, and samples were taken at 0, 5, 10, 15, 20, and 30 minutes for HPLC and DLS analysis.

[0080] In this example, to evaluate the GSH-triggered reduction and disassembly process, P-CyPt (10 μM) in 1 mL of Tris buffer was first incubated with ALP (100 U / L) at 37°C for 30 minutes. The digested sample solution was then treated with 10 mM GSH at 37°C. The reaction solution was analyzed by HPLC at 10, 20, 30, 40, 50, and 60 minutes. To monitor the disassembly process, 50 mM N-Ethylmaleimide was added to the solution at 10, 20, 30, 40, 50, and 60 minutes to remove GSH, and DLS analysis was performed using a 90Plus / BI-MAS instrument (Brookhaven, USA).

[0081] The analysis results are as follows Figure 3 As shown in a and 3b, the probe P-CyPt undergoes dephosphorylation reaction under the action of ALP enzyme to form CyPt, and the product after enzyme cleavage can self-assemble to form Pt particles with a size of about 160nm. IV NPs, Pt after adding GSH IV NPs can be disassembled from Pt within 60 minutes. IV NPs are converted into small molecule dye Cy-COOH.

[0082] In this embodiment, TEM and STEM analysis were performed on the self-assembly process and the disassembly process. The specific analysis process is as follows:

[0083] P-CyPt (10 μM) in 1 mL of Tris buffer was incubated with ALP (100 U / L) at 37°C for 0, 5, 10, 15, 20, and 30 minutes, respectively. The enzymatically digested solution was then treated with 10 mM GSH in a 37°C water bath for 10, 20, 30, 40, 50, and 60 minutes. Aliquots at designated time points were then dropped onto carbon-coated copper grids and immediately freeze-dried. TEM imaging of the samples was then performed on a JEM-2800 transmission electron microscope. The scale bar is 200 nm. Figure 4 and Figure 5 As shown in Figure a, the probe P-CyPt gradually transforms from a small molecule into spherical nanoparticles after being hydrolyzed by ALP. Upon addition of GSH, the spherical nanoparticles undergo disassembly and reconvert from nanoparticles into small molecules within 60 minutes. P-CyPt (10 μM) in 1 mL of Tris buffer was incubated with ALP (100 U / L) at 37°C for 30 minutes. The sample was then dropped onto a carbon-coated copper grid and immediately freeze-dried for elemental analysis using STEM. Figure 5 Figures b to d are STEM analysis results. It can be seen from the figure that the spherical nanoparticles contain Cl and Pt elements, further confirming that CyPt can form platinum prodrug spherical nanoparticles.

[0084] (2) Investigation of the optical properties of cisplatin prodrug P-CyPt during ALP-mediated self-assembly and assembly:

[0085] For optical analysis of the self-assembly process, P-CyPt (10 μM) in 1 mL of Tris buffer was incubated with ALP (100 U / L) at 37°C for the indicated times (0, 5, 10, 15, 20, and 30 min). For optical analysis of the disassembly process, P-CyPt (10 μM) in 1 mL of Tris buffer was first incubated with ALP (100 U / L) at 37°C for 30 min, and then treated with 10 mM GSH at 37°C for 10, 20, 30, 40, 50, and 60 min for analysis of UV, near-infrared fluorescence, and photoacoustic properties.

[0086] from Figure 6 As can be seen in a, in the UV absorption spectrum, the probe P-CyPt self-assembles after being cleaved by ALP, and its shoulder peak at 750nm is significantly enhanced. After adding GSH, Pt IV NPs disassembled and the shoulder peak at 750nm decreased and then disappeared completely. Figure 6 As can be seen in b, the fluorescence spectrum of the solution at each time point was obtained on the fluorescence spectrometer with an excitation wavelength of 680 nm. IV The fluorescence of NPs is enhanced when they are formed, and the fluorescence signal is further enhanced when GSH is added to disassemble them. Figure 7 It can be seen that during the self-assembly process, as Pt IV The formation of NPs significantly enhanced the photoacoustic signals at 700 & 750 nm. With the addition of GSH, the disassembly process occurred, and the shoulder peak at 750 nm in the solution appeared, resulting in a decrease in the photoacoustic signal at 750 nm, while the photoacoustic signal at 700 nm remained basically unchanged.

[0087] Example 3:

[0088] In this example, the ability of the cisplatin prodrug diagnostic probe P-CyPt probe to perform near-infrared FL and PA dual-modality imaging of ALP-overexpressing tumor cells was studied. The specific investigation steps are as follows:

[0089] Cells (~5×10 4 ) were seeded into glass bottom dishes (In Vitro Scientific, D35-20-1-N) and allowed to grow overnight. P-CyPt or Pt was added to DMEM without FBS. IV NPs (10 μM) were incubated at 37°C for 30 minutes. To inhibit ALP activity, cells were pretreated with the ALP inhibitor Na3VO4 (10 mM) for 20 minutes and then incubated with P-CyPt (10 μM) for an additional 30 minutes. The culture medium was removed and the cells were gently washed once with 1 mL of PBS buffer. After adding fresh culture medium, fluorescence images were acquired on a Leica TCS SP8 confocal laser scanning microscope with an excitation wavelength of 670 nm and an emission wavelength of 690 to 750 nm.

[0090] Figure 8 The cell imaging images of P-CyPt in HeLa cells, HeLa cells pretreated with Na3VO4 (an ALP enzyme inhibitor), and HEK-293T cells after incubation for 30 minutes. It can be seen from the figure that in HeLa cells with high expression of ALP enzyme, the probe can be quickly activated and adsorbed on the cell membrane surface, while in cells with inhibited ALP activity and low expression of ALP enzyme, the probe P-CyPt is almost not activated.

[0091] In this example, the ability of FL and PA dual-modality imaging of ALP activity in cells was also investigated. HeLa or HEK-293T cells were cultured at 4×10 6 Cells were seeded in a 10 cm cell culture dish at a density of 10 cells / well and grown overnight. P-CyPt or Pt was added to 4 mL of DMEM without FBS. IV NPs (10 μM) and incubated at 37 ° C for 30 min. To inhibit ALP activity, cells were pretreated with Na3VO4 (10 mM) for 20 min and then incubated with P-CyPt (10 μM) for another 30 min. To evaluate the in situ formed Pt IVTo investigate the effect of NPs on intracellular GSH, P-CyPt (10 μM) was added to 4 mL of FBS-free DMEM and incubated at 37°C for 30 min. The culture medium was then removed and the cells were incubated for another 3 h after adding fresh culture medium. The culture medium was then removed and the cells were gently washed once with 1 mL of PBS. Trypsin (1 mL) was added to each dish and the cells were separated at 37°C for 2 min. The cell pellets were then collected after centrifugation at 1000 rpm for 4 min. Fluorescence imaging and PA imaging of the cell pellets were obtained on the IVIS Lumina XR III system (λex / em=670 / 750±50 nm) and the VisualSonics Vevo 2100LAZR system, respectively. Near-infrared fluorescence and photoacoustic quantification were performed at wavelengths of 700 and 750 nm for specific values of individual samples. The cell pellet was then lysed with 200 μL of DMSO, and the cell lysate was mixed with 300 μL of cold MeOH and 500 μL of deionized water, and centrifuged at 14,000 rpm for 10 min at 4° C. Aliquots of the supernatant (250 μL) containing the cell lysate were injected into the HPLC system for analysis.

[0092] Figure 9 (a) Images of P-CyPt cell clusters under different conditions. As can be seen from Figures a-c, after 30 minutes of incubation with HeLa cells, the P-CyPt probe is effectively activated and self-assembles, with enhanced fluorescence and photoacoustic signals (700 and 750 nm). As the nanoparticles disassemble after endocytosis, the fluorescence signal further increases, accompanied by a decrease in the photoacoustic signal at 750 nm. This demonstrates the sequentially triggered assembly and disassembly of P-CyPt at the cellular level. HPLC analysis of cell lysates treated under different conditions further confirmed this conclusion.

[0093] To further demonstrate that platinum nanoparticles Pt IV The formation of NPs in cultured HeLa cells. In this example, HeLa cells were cultured at a rate of 2×10 5Cells were seeded at a density of 100 cells / well in a 6-well cell culture plate. After overnight growth, the cells were incubated with or without P-CyPt (10 μM) for 1 hour and gently washed three times with cold PBS buffer. Trypsin (500 μL / well) was then added to each well and incubated at 37°C for 2 minutes to detach the cells. The cell pellets were then collected and lysed by repeated freezing and thawing in liquid nitrogen. The cell lysate was separated according to the reported disruption procedure to obtain different cellular components. Pellet sample N (nuclei) was separated at 600g for 10 minutes; pellet sample L (lysosomes and mitochondria) was separated at 15,000g for 5 minutes; and pellet sample M (plasma membrane) was separated at 100,000g for 60 minutes. The collected solution was drop-cast onto a carbon-coated copper grid and freeze-dried, followed by elemental analysis by TEM and STEM.

[0094] Figure 10 This is a TEM image of P-CyPt and HeLa cells after incubation for 1 hour. The images are N, L, Cl, M, C and Pt. As can be seen from the image, Pt can be clearly seen in the cell membrane, mitochondria and lysosomes. IV NPs, indicating that the probe P-CyPt can self-assemble on the surface of HeLa cells to form nanoparticles Pt IV NPs can be effectively taken up into the lysosomes of cells.

[0095] In summary, P-CyPt can self-assemble on the surface of tumor cells to form spherical nanoparticles with uniform particle size. IV NPs, and the self-assembly process was accompanied by the enhancement of near-infrared fluorescence and photoacoustic signals (700&750nm). IV After NPs are taken up by cells, a disassembly process occurs, which leads to a further enhancement of their fluorescence signal, a decrease in the photoacoustic signal at 750 nm, and a basic unchanged photoacoustic signal at 700 nm.

[0096] Example 4:

[0097] In this example, the uptake and cytotoxicity of the probe P-CyPt in tumor cells were studied to prove that the probe P-CyPt can self-assemble after being hydrolyzed by ALP on the cell membrane surface, thereby increasing the tumor cell's ability to absorb platinum prodrug nanoparticles Pt IV NPs are taken up, and then after entering the cell, they interact with GSH to trigger the disassembly process to release cisplatin drugs to kill tumor cells. The specific process is as follows:

[0098] In order to investigate the uptake of platinum prodrug P-CyPt by cells and the distribution of Pt in intracellular subcellular organelles, HeLa cells were cultured at 4×10 6The cells were seeded at a density of 10 cells / well in a 10 cm cell culture dish and allowed to grow overnight. The cells were then incubated with P-CyPt (10 μM), Pt IV NPs (10 μM) and CDDP (10 μM) were incubated for 1 hour. In the P-CyPt+Na3VO4 group, HeLa cells were first incubated with Na3VO4 (10 mM) for 20 minutes, then incubated with P-CyPt (10 μM) for another 60 minutes. After incubation, they were washed three times with PBS, then trypsinized, centrifuged, and counted. For Pt distribution analysis, cells were treated with P-CyPt (10 μM) or CDDP (10 μM) for 1 hour, then washed three times with PBS, and then incubated with fresh medium for another 24 hours. After incubation, they were gently washed once with 2 mL of PBS, then trypsinized, centrifuged, and counted. The cell lysate was then separated according to the reported disruption procedure to obtain different cellular components, including N (nucleus), M (plasma membrane), L (lysosomes and mitochondria), and C (cell membrane). Cells and different cell fragments were digested with concentrated HNO3 (65-70%) at 120℃ for 24h, and the solution was diluted to 5mL 2% HNO3. The concentration of Pt element was measured by ICP-OES, which was further used to calculate the Pt content in cells and subcellular organelles.

[0099] Figure 11 P-CyPt, Pt IV Figure 1 shows the ratio of the uptake of elemental platinum by tumor cells in the NPs, CDDP and Na3VO4 control groups to the total amount (a) and the distribution of P-CyPt and CDDP in the subcellular organelles of HeLa cells. As can be seen from the figure, P-CyPt forms Pt through ALP-mediated in situ self-assembly. IV NPs can be adsorbed on the surface of cell membranes, thus IV NPs and small molecule CDDP have higher tumor cell uptake. The distribution shows that P-CyPt has more nuclei (N), mitochondria and lysosomes than small molecule CDDP.

[0100] In this example, the IC 50 The probes P-CyPt and Pt IV The cytotoxicity of NPs and CDDP on different cells was studied in the following steps:

[0101] Cells were seeded on a flat-bottom 96-well plate (5000 cells / well) and incubated overnight at 37°C. IVNPs or CDDP (0, 1, 2, 5, 10, 20, 30, 50, 100 and 200 μM) were added to DMEM medium (100 μL). They were then divided into two groups for incubation. The first group was incubated with cells for 48 hours, and the second group was first incubated with Pt drugs for 2 hours, washed with PBS, and then incubated with fresh medium for another 48 hours. After the incubation, 50 μL of MTT solution (1 mg / mL in PBS) was added to each well. The cells were kept at 37°C for 4 hours, and then the solution in each well was carefully removed. 150 μL of DMSO was added to dissolve the purple formazan crystals in the wells. The absorbance (OD) at 490 nm in each well was obtained on a microplate reader (Tcan). The absorbance of blank cells (OD control) was used as a control, and the percentage of cell viability in each treatment was calculated by dividing the OD by the OD control, and the corresponding IC was calculated using Prism 7 software. 50 value. Figure 12 For the probe P-CyPt, Pt IV The cytotoxicity of NPs and CDDP on different cells shows that the probe P-CyPt has a better effect on tumor cells with high expression of ALP than Pt IV NPs and CDDP have stronger cytotoxicity, but do not produce obvious cytotoxicity to normal cells.

[0102] In this example, P-CyPt and Pt IV The permeability of the 3D tumor cell spheroid model of NPs was investigated as follows:

[0103] Each well at the bottom of a 96-well plate was covered with 50 μL of hot agarose solution (1.5 w / v%) and cooled to form an agarose layer. HeLa cells were cultured at 2×10 3 The MCSTs were seeded at a density of 10 cells / well into agarose-coated wells and cultured to grow into spheroids. The DMEM medium was replaced with fresh medium every 2 days until the diameter of the tumor spheroids approached 400 μm. Then, MCSTs were incubated with 10 μM P-CyPt or Pt IV The NPs were incubated for 1 hour and washed three times with PBS. After washing, blank DMEM medium was added and cultured for another 24 hours. After the culture was completed, PI (4 μM) was added and cultured for 1 hour. The culture medium was removed and the 3D cell spheroids were gently washed with PBS in sequence and then carefully transferred to a glass bottom dish. Fluorescence images of MCTS were taken under PI and Cy5.5 channels on a Leica TCS SP8 confocal laser scanning microscope. The results are shown in Figure 2. Figure 13 As shown, the molecular probe P-CyPt is compared with Pt IV NPs have stronger penetration ability and can kill deep tissue cells in 3D tumor cell spheres.

[0104] In summary, P-CyPt can self-assemble into spherical nanoparticles Pt through the dephosphorylation of ALP on the cell membrane surface. IV NPs, thereby increasing the uptake of cisplatin prodrug nanoparticles by tumor cells, and releasing cisplatin prodrug by interacting with intracellular GSH to kill tumor cells. IV NPs and CDDP have stronger effects in killing tumor cells, and because the prodrug nanoparticle P-CyPt needs to release the cisplatin original drug through a sequentially triggered assembly and disassembly process, it has lower toxicity to normal cells. At the same time, the small molecule probe prodrug P-CyPt can better penetrate into the interior of the 3D tumor cell spheres and kill cells deep in the tumor, which has better prospects for in vivo application.

[0105] Example 5:

[0106] In this example, the tumor efficacy of P-CyPt was investigated, and the specific investigation steps are as follows:

[0107] Six immunodeficient female BALB / C nude mice aged 6–8 weeks (purchased from the Model Animal Research Center (MARC) of Nanjing University, Nanjing, China) were used in accordance with the regulations of the Institutional Animal Care and Use Committee (IACUC). 2 × 10 6 HeLa cells were used to establish HeLa tumor xenografts. When the average tumor volume reached approximately 100 mm 3 The mice were randomly divided into two groups (n=3).

[0108] For in vivo fluorescence imaging of mice bearing HeLa subcutaneous tumors, P-CyPt or CyPt (100 μM, 200 μL) was injected into the mice via the tail vein. Whole-body fluorescence images were acquired before and 1, 2, 4, and 8 h after injection, and the fluorescence intensity was quantified using ROI measurement using Living Image software (4.5.2, PerkinElmer, MA, USA). When the size of the subcutaneous HeLa tumor grew to approximately 100 mm 3 The mice were divided into two groups. The mice in the first group were intravenously injected with P-CyPt (200 μL, 500 μM). The mice in the second group were intravenously injected with Pt IV NPs (200 μL, 500 μM). PA images of mouse tumors were acquired at 700 and 750 nm before injection and 1, 2, and 4 hours after injection. PA images at 700 and 750 nm were acquired on a Vevo 2100 imaging system (FUJI Visualsonics), followed by quantification of photoacoustic intensity using ROI measurements.

[0109] Figure 14 P-CyPt and Pt IV Near-infrared fluorescence image (a) and near-infrared fluorescence intensity quantification value image (c), photoacoustic imaging image (b) and photoacoustic intensity quantification value image (d) of NPs in HeLa subcutaneous tumor model mice. It can be seen from the figure that the probe P-CyPt can be rapidly activated and self-assembled in the tumor area of the mouse, while generating strong near-infrared fluorescence / photoacoustic signals (700&750nm). As time extends to 4h, the near-infrared fluorescence of the tumor area and the photoacoustic signal at 750nm are significantly reduced after disassembly occurs, indicating that the probe P-CyPt can also undergo assembly and disassembly processes in the tumor area, and can be visualized by near-infrared fluorescence and photoacoustic signals.

[0110] For HPLC analysis of compounds in tumor tissues, P-CyPt (100 μM) or Pt IV NPs (100 μM) were intravenously injected into mice with HeLa subcutaneous tumors in 200 μL of normal saline, and the tumor tissue was removed and cut into small pieces after 4 hours. NEM (100 mM, 200 μL), PBS buffer (200 μL) and RIPA buffer (200 μL) were added to the small pieces to obtain a mixture, and the mixture was sonicated in an ice bath for 5 minutes to prepare a tumor homogenate. Then, 200 μL of DMSO was added to the homogenate, and sonicated for another 5 minutes to dissolve the compound. Finally, 200 uL of methanol was added to the homogenate, and centrifuged at 4,000 rpm for 10 minutes to remove the protein. The supernatant was injected into HPLC to analyze the compound, and the results were examined as shown in FIG. Figure 15 shown.

[0111] Figure 15 (a) is the injection of P-CyPt and Pt IV Analysis of lysate of tumor tissue after Nps; (b) injection of probes P-CyPt, Pt IV The Pt element content in different organs and tumor tissues of mice with HeLa subcutaneous tumor model after NPs and CDDP. As can be seen from the figure, the probe P-CyPt can be effectively activated at the tumor site and release the dye molecule Cy-COOH, and the probe P-CyPt is significantly higher than Pt IV NPs and CDDP can better accumulate in tumor tissues.

[0112] In order to investigate the distribution of Pt in different organs in vivo, P-CyPt (100 μM), Pt IVNPs (100 μM) or CDDP (100 μM) were intravenously injected into mice bearing HeLa subcutaneous tumors in 200 μL of normal saline. Tumors and major organs, including heart, liver, spleen, lungs, and kidneys, were collected and weighed 4 hours after injection. The tissues were cut into small pieces and digested with concentrated HNO3 at 120°C overnight. The residue in each organ was then diluted with 5 ml of 2% HNO3 solution, and the platinum concentration was determined by ICP-OES. The tissue uptake value was calculated and expressed as the percentage of the injected dose per gram of tissue %ID / g for comparison. The results are shown in Figure 2. Figure 15 As shown in b. The accumulation of probe P-CyPt in tumor tissue was the highest, significantly higher than that of control group Pt IV NPs and CDDP, indicating that the sequentially triggered assembly and disassembly strategy can effectively enhance the activation and accumulation of platinum prodrug probe P-CyPt in the tumor area.

[0113] Example 6:

[0114] In this example, near-infrared FL and PA dual-modality imaging-guided treatment of subcutaneous tumors was investigated. The specific steps are as follows:

[0115] When the tumor volume is 70-80 mm 3 The HeLa tumor-bearing mice were randomly divided into four groups: 1 (PBS only), 2 (CDDP), 3 (Pt IV NPs), 4 (P-CyPt). Mice were intravenously injected with PBS (200 μL) or Pt drug (2.25 mg kg -1 Pt), a total of 5 doses. The tumor volume and body weight of the mice were measured every three days. The relative tumor volume of each mouse was calculated as V / V0 (V0 is the tumor volume at the beginning of treatment), and the relative body weight was based on the body weight at the time of initial treatment. All mice were euthanized on day 21, and the tumors of the mice were removed and weighed and recorded. The body weight of the mice was weighed every three days during the treatment to evaluate the biosafety during the treatment process, and independent experiments were performed in four mice in each experimental group.

[0116] Figure 16 Schematic diagram of tumor treatment of HeLa mice with probe P-CyPt (a), tumor growth during treatment (b), weight of mouse tumor tissue after treatment (c), and changes in mouse weight during treatment (d). The figures show that probe P-CyPt can effectively inhibit the growth of HeLa subcutaneous tumors, and the inhibitory effect is significantly better than that of other treatment groups. In addition, the treatment process does not cause a significant decrease in mouse weight, and has good biosafety.

[0117] In this example, blood urea nitrogen (BUN) and creatinine (CRE) were analyzed to investigate the toxicity of the probe P-CyPt to the kidneys. The specific investigation steps are as follows:

[0118] Mice were intravenously injected with P-CyPt, Pt IV NPs, CDDP (2.25 mg kg -1 Pt) or PBS. Mice were intravenously injected with PBS (200 μL) or Pt drug (2.25 mg kg -1 Pt), for a total of 5 doses. After 21 days of treatment, blood (~1.0 mL) was collected from the venous sinus and stored in a 1.5 mL EDTA-coated Eppendorf tube and refrigerated on ice. The collected blood samples were centrifuged at 3500 rpm for 20 min. Blood urea nitrogen (BUN) and creatinine (CRE), indicators of kidney injury, were measured in serum using ELISA kits according to the manufacturer's protocol. The experimental results are shown in Figure 2. Figure 17 (a) and 17 (b). As can be seen from the figure, CDDP can cause significant upregulation of BUN and CRE, leading to certain kidney damage, while cisplatin prodrugs P-CyPt and Pt IV NPs did not produce significant renal toxicity, indicating that the treatment group using cisplatin prodrug could reduce the renal toxicity of CDDP itself.

[0119] In summary, the cisplatin prodrug probe P-CyPt, which can sequentially trigger self-assembly and disassembly, has a significant inhibitory effect on the growth of HeLa subcutaneous tumors (compared to PBS, CDDP and Pt IV NPs reduced tumor volume by ~21.2-, ~15.8-, and 13.0-fold, and did not cause a significant decrease in mouse body weight or obvious kidney damage during the 21-day treatment process. Therefore, the probe P-CyPt has good biocompatibility and low renal toxicity.

[0120] Example 7:

[0121] In this example, a carcinoma in situ model was constructed to perform near-infrared fluorescence imaging and anti-tumor effect evaluation. The specific investigation steps are as follows:

[0122] Construction of carcinoma in situ model: A midline incision was made in the anterior abdominal wall of Balb / c nude mice, and 2×10 6 HepG2 luciferase-transfected (Luc / HepG2) cells were suspended in 100 μL of a 33 v / v% Matrigel and DMEM mixture and injected directly into the left liver lobe. Bioluminescence imaging (BLI) was used to monitor tumor growth in mice. After 10 days, the in situ carcinoma model was successfully established, and the mice had HepG2 in situ liver tumors.

[0123] Bioluminescence Monitoring: D-luciferin (150 mg / kg) was intraperitoneally injected into mice bearing orthotopic HepG2 liver tumors. Ten minutes later, the mice were imaged using the IVIS Lumina XR III system in bioluminescence imaging mode (Open). Regions of interest (ROIs) in the liver were measured using Living Image software, and bioluminescence intensity was quantified.

[0124] To perform in vivo fluorescence imaging of mice bearing HepG2 orthotopic liver tumors, P-CyPt or Pt IV NPs (100 μM, 200 μL) in normal saline were injected into mice. Whole-body fluorescence images were acquired before and 1, 2, 4, and 8 h after injection on an IVISLumina XR III imaging system using a 660 nm excitation filter and a 750 ± 50 nm emission filter, and quantified by ROI measurement using Living Image software.

[0125] Figure 18 P-CyPt and Pt IV Near-infrared fluorescence imaging of NPs in HepG2 orthotopic liver tumor model mice (a) and quantification of near-infrared fluorescence intensity (b). As can be seen from the figure, the probe P-CyPt can be activated by HepG2 orthotopic liver tumors, thereby imaging the orthotopic liver tumors, and the imaging results are consistent with bioluminescence imaging.

[0126] In order to investigate the chemotherapy effect of P-CyPt on HepG2 liver orthotopic tumors, mice with HepG2 liver orthotopic tumors were divided into four groups (n=4 in each group): 1 (PBS only), 2 (CDDP), 3 (Pt IV NPs), 4(P-CyPt). PBS (200 μL) or Pt drugs (CDDP, Pt IV NPs, P-CyPt, 2.25 mg kg -1Pt), for a total of 5 doses. To monitor the therapeutic effect, the treated mice were intravenously injected with D-luciferin (150 mg / kg), and whole-body bioluminescence images were recorded 10 minutes later, repeated every 3 days for 15 days. The bioluminescence intensity of the liver area was then quantified by ROI measurement using Living Image software. On the 15th day, all mice were euthanized and the entire liver of the mice was removed. Then, P-CyPt (10 μM) and D-luciferin (5 mM) were sprayed on the liver surface respectively. Bioluminescence images were obtained within 10 minutes using the bioluminescence imaging mode (Open), and near-infrared fluorescence images were obtained within 30 minutes using a 660 nm excitation filter and a 750 ± 50 nm emission filter ( Figure 20 (b) The ROI of the liver tumor region was measured using Living Image software, and the average intensity of bioluminescence and near-infrared fluorescence was quantified. Four mice were independently tested in each experimental group.

[0127] Figure 19 Schematic diagram of tumor treatment of HepG liver orthotopic tumor model mice by P-CyPt (a), tumor growth during treatment (b), and bioluminescence imaging monitoring of HepG liver orthotopic tumor model mice during treatment (c). It can be seen from the figure that the probe P-CyPt is superior to PBS, CDDP and Pt IV NPs have better anti-HepG liver orthotopic tumor growth effect.

[0128] Figure 20 Figures (a) and (b) show the bioluminescence imaging of the liver region of mice treated with P-CyPt and a control group for HepG orthotopic liver tumors 15 days after treatment. The correlation between the relative intensities of bioluminescence and near-infrared fluorescence in the tumor region (c) is shown. As can be seen from the figures, the sprayed P-CyPt probe can also well indicate the contours of the tumor in the liver region, with high consistency with the bioluminescence.

[0129] In summary, the cisplatin prodrug probe P-CyPt has a significant inhibitory effect on the growth of HepG2 liver orthotopic tumors, and the tumor growth is significantly higher than that of PBS, CDDP and Pt IV Tumor volumes were ∼13.0-, ∼10.7-, and 5.7-fold lower with the NPs, and no metastatic tumor foci were observed in the livers of P-CyPt-treated mice. Furthermore, strong correlation and accuracy were observed between tumor near-infrared fluorescence intensity and bioluminescence intensity in the four groups of livers imaged with P-CyPt, demonstrating the potential use of P-CyPt for fluorescence-guided surgery of residual liver tumors after chemotherapy.

[0130] Example 8:

[0131] In this example, the metabolism and clearance of the probe P-CyPt in vivo were investigated:

[0132] Healthy nude mice were intravenously injected with P-CyPt, Pt IV NPs or CDDP (2.25 mg kg -1 Pt, dissolved in saline, was placed in metabolic cages for 12 hours. Urine and feces were collected at 2 (0-2 hours), 4 (2-4 hours), 8 (4-8 hours), and 12 hours (8-14 hours), diluted with methanol, and centrifuged at 4500 rpm for 10 minutes before filtering through a 0.22 μm syringe filter. The filtered solution was analyzed by HPLC (detection at 660 nm) to determine the characteristics of all mCy-containing compounds. Using established standard curves for P-CyPt, CyPt, and Cy-COOH, the content of each compound in urine and feces was calculated, and these curves were further applied to determine their percentages in urine and feces.

[0133] Figure 21 The clearance of P-CyPt in urine (a) and feces (b) in mice within 0-12 hours and the clearance of Pt IV The clearance of NPs in urine (c) and feces (d) of mice within 0-12 hours. As can be seen from the figure, at 0-2 hours, P-CyPt mainly appeared in urine (~11.3%) rather than in feces, while at 2-4 and 4-8 hours, P-CyPt and Cy-COOH were both clearly present in urine. In feces, P-CyPt (~4.5%) appeared at 2-4 hours, but CyPt (~9.7%) and Cy-COOH (~11.1%) mainly appeared at 4-8 hours. In the following 8-12 hours, Cy-COOH dominated both urine and feces. In contrast, injection of Pt IV Mice treated with NPs showed that CyPt and Cy-COOH were mainly present in feces at 2-4 and 4-8 h.

[0134] Figure 22 For HPLC analysis of P-CyPt, Pt IV The content of NPs in urine (a) and feces (b) and the content of Pt elements in urine (c) and feces (d) in different treatment groups analyzed by ICP-OES. As can be seen from the figure, at 12 h, ~44.8% and ~37.0% of the injected P-CyPt were excreted through the kidneys and hepatobiliary system, respectively; however, ~65.9% of Pt IV NPs are excreted through the hepatobiliary system, which is much greater than the excretion through the renal system (~22.4%). This shows that the small molecule probe P-CyPt can be eliminated through two metabolic pathways: urine and feces, unlike CDDP which is mainly excreted through the kidneys and PtIV NPs are mainly eliminated through fecal metabolism, thus producing less kidney and liver toxicity and having better biosafety.

[0135] In summary, the small molecule cisplatin prodrug probe P-CyPt can be metabolized through both the kidney and the liver and gallbladder, which is different from CDDP which is only cleared by the kidney and Pt IV The clearance of NPs was significantly different only through the liver, and P-CyPt was significantly higher than CDDP and Pt IV NPs can be cleared from healthy mice more quickly, indicating that the probe P-CyPt has lower renal and systemic toxicity in vivo and better biosafety.

[0136] 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. A cisplatin prodrug diagnostic probe P-CyPt, characterized in that The cisplatin prodrug diagnostic probe P-CyPt comprises: (1) Phosphate group (-PO3H2); (2) Quadrivalent platinum prodrugs; (3) hydrophobic dipeptide (FF) linker fragment; (4) Near-infrared fluorescent dyes; The structural formula of the cisplatin prodrug diagnostic probe P-CyPt is: 。 2. A method for preparing a cisplatin prodrug diagnostic probe P-CyPt, characterized in that: include: Compound 4, HS-Pt, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) and triethylamine (TEA) were dissolved in N,N -dimethylformamide, and then react under stirring. After the reaction is completed, centrifuge, purify, and freeze-dry to obtain the cisplatin prodrug diagnostic probe P-CyPt; The structural formula of the compound 4 is: 。 3. The method for preparing the cisplatin prodrug diagnostic probe P-CyPt according to claim 2, characterized in that: The molar ratio of compound 4, HATU, TEA, and HS-Pt is 1:2.5-5.5:2.5-5.5:1.5-3.5; The concentration of the compound 4 solution is 0.004~0.024 mmol / mL.

4. The method for preparing the cisplatin prodrug diagnostic probe P-CyPt according to claim 3, wherein The molar ratio of compound 4, HATU, TEA and HS-Pt is 1:3:3:2; and the concentration of the compound 4 solution is 0.014 mmol / mL.

5. The method for preparing the cisplatin prodrug diagnostic probe P-CyPt according to claim 2, characterized in that: The reaction is stirred at -40~0°C for more than 12 hours.

6. The cisplatin prodrug diagnostic and therapeutic probe P-CyPt according to claim 1 or the cisplatin prodrug diagnostic and therapeutic probe P-CyPt prepared by any one of claims 2 to 5 has the following applications: (1) Use in the preparation of tumor diagnostic imaging agents; and / or (2) Application in the preparation of anti-tumor drugs.

7. The use according to claim 6, characterized in that The tumor diagnostic imaging agent is an alkaline phosphatase-positive tumor diagnostic imaging agent.

8. The use according to claim 7, characterized in that The alkaline phosphatase is endogenous ALP on the surface of human cervical cancer HeLa cells and human liver cancer HepG2 cell membranes.

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