CN114053437A

CN114053437A - Alkaline phosphatase response bimodal probe P-CyFF-68Ga and preparation method and application thereof

Info

Publication number: CN114053437A
Application number: CN202111345893.XA
Authority: CN
Inventors: 叶德举; 胡宇轩; 缪银杏; 张俊雅
Original assignee: Nanjing University
Current assignee: Nanjing University
Priority date: 2021-11-15
Filing date: 2021-11-15
Publication date: 2022-02-18

Abstract

The invention discloses an alkaline phosphatase response bimodal probe P-CyFF-⁶⁸Ga and a preparation method and application thereof. The probe P-CyFF-⁶⁸Ga is dephosphorylated under the action of ALP to generate an amphiphilic dephosphorylated product and self-assembles to form the fluorescence-enhanced radioactive nanoparticles. The nanoparticles formed by in-situ assembly can stay on the tumor cell membrane and enter the cell through endocytosis, and the increased size is utilized to prolong the stay time of the probe in the tumor cell and enhance the probe in the tumor cellAccumulation at the tumor site. After the inactivated probe is cleared from normal tissues, the probe can carry out high-sensitivity and high-tissue-penetrability imaging detection on tumors by using NIR fluorescence and PET bimodal signals.

Description

An alkaline phosphatase-responsive bisModal probe P-CyFF-68Ga and preparation method and application thereof

Technical Field

The invention relates to molecular imaging, in particular to an alkaline phosphatase response bimodal probe P-CyFF-⁶⁸Ga and a preparation method and application thereof.

Background

In recent years, Positron Emission Tomography (PET) imaging technology has been widely used in clinical imaging diagnosis of tumors. PET imaging uses positron emitted from decaying radionuclide for imaging, has high tissue penetration depth and high sensitivity, and can be used for non-invasive whole body imaging of patients. The combination of PET and fluorescence can provide two high-sensitivity imaging signals at the same time, the accuracy of in-vivo imaging detection is improved by utilizing complementary information, and the diagnosis and treatment of tumors can be realized by utilizing the whole-body imaging of PET and the surgical navigation of fluorescence, so that the dual-mode probe integrating fluorescence/PET has great application potential in the imaging diagnosis of tumors. To date, some fluorescent/PET bimodal imaging probes have been reported to be applied to imaging research of tumors, and these probes are often used for labeling fluorescent and PET imaging groups onto small molecule targeting groups, tumor-specific antibodies or nanoparticles, so as to introduce fluorescent and PET bimodal imaging signals simultaneously. These probes are capable of achieving bimodal imaging detection of tumors to some extent, but still have certain limitations: the small molecular probe has small molecular size, is easy to diffuse and is difficult to stay at a tumor part for a long time, so that the living body imaging time window is short, and the long-time imaging detection of the tumor is not facilitated; the nano probe has large size, can reach a tumor part through an EPR effect and stay in the tumor for a long time, but the large size ensures that the tissue penetration capability of the nano probe is weak, the nano probe is difficult to penetrate into the internal area of the tumor and is easy to be captured by an endothelial reticulum system, the tumor uptake is reduced, and a background signal is enhanced; the antibody has specific tumor targeting capacity, but the blood circulation time is long, potential radiation hazard is brought to patients, and meanwhile, the accumulation at the tumor site is limited by the number of cell antigen proteins. Therefore, the development of a fluorescent/PET bimodal probe with high permeability, low blood circulation time and long tumor residence time has great significance for imaging and diagnosis of tumors.

Disclosure of Invention

The purpose of the invention is as follows: the technical problem to be solved by the invention is to provide an ALP activated dual-mode probe P-CyFF-⁶⁸Ga and ALP activated non-radioactive probe P-CyFF-Ga.

The technical problem to be solved by the invention is to provide the preparation method of the non-radioactive probe P-CyFF-Ga

The technical problem to be solved by the present invention is to provide the above-mentioned dual-mode molecular imaging probe P-CyFF-⁶⁸A method for preparing Ga.

The invention further solves the technical problem of providing the bimodal molecular imaging probe P-CyFF-⁶⁸Application of Ga in preparing a contrast agent for tumor diagnosis.

The technical problem to be solved by the present invention is to provide the above-mentioned bimodal molecular imaging probe P-CyFF-⁶⁸fluorescent/PET bimodal imaging applications of Ga.

The invention idea is as follows: probe P-CyFF-⁶⁸Ga and P-CyFF-Ga have good water solubility and can permeate into the tumor. Under the action of ALP enzyme over-expressed on the surface of tumor cell, P-CyFF-⁶⁸The phosphate radical of Ga and P-CyFF-Ga is hydrolyzed to generate dephosphorylated product CyFF-⁶⁸Ga and CyFF-Ga, resulting in the activation of fluorescence; amphiphilic CyFF-⁶⁸Ga and CyFF-Ga are further assembled under the hydrophobic effect and the pi-pi stacking effect of Cy-Cl and FF to form nano-particles with fluorescent signals and nuclide radioactivity (namely, in-situ co-assembly is carried out to form nano-particles NPs-⁶⁸Ga) and adheres to the cell membrane surface. Nanoparticles formed by in situ assembly in tumor tissue have a larger size than small molecules and can stay at the tumor site for a long time. NPs by means of activated Cy-Cl fluorophores and radionuclides⁶⁸Ga can provide enhanced NIR fluorescence and PET imaging signals at a tumor site, and P-CyFF-Ga and P-CyFF-⁶⁸After Ga is cleared from normal tissues, tumors can be imaged by using bimodal imaging signalsAnd (6) detecting.

In order to solve the first technical problem, the present invention discloses a method such as P-CyFF-⁶⁸Ga, which comprises the following features: (1) ALP recognizes the phosphate group (-PO)₃H₂) (ii) a (2) Hydrophobic NIR fluorophores (anthocyanidin derivatives, Cy-Cl); (3) a hydrophilic NODA ligand of a labeling moiety; (4) a hydrophobic dipeptide (FF) linking fragment capable of facilitating molecular self-assembly.

Wherein, the structure of the dual-mode non-radioactive probe P-CyFF-Ga activated by ALP is as follows:

in order to solve the second technical problem, the invention discloses a preparation method of the probe P-CyFF-Ga, which comprises the following steps:

step a: compound mCy-NH₂Reacting with the compound 1 to obtain a compound 2;

step b: carrying out substitution reaction on the compound 2 to obtain a compound 3;

step c: carrying out substitution reaction on the compound 3 to obtain a compound 4;

step d: carrying out condensation reaction on the compound 4 to obtain a compound P-CyFF-NODA;

step e: chelating the compound P-CyFF-NODA with Ga to obtain the target non-radioactive probe P-CyFF-Ga.

In the step a, the step (c),

wherein the reaction is compound mCy-NH₂Dissolving compound 1, benzotriazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU) and N, N-Diisopropylethylamine (DIPEA) in solvent, and reactingThus, a reaction solution containing compound 2 was obtained.

Wherein said compound 1 was prepared by standard Solid Phase Peptide Synthesis (SPPS) procedure starting from 2-chlorotrityl chloride resin and Fmoc protected phenylalanine. After the solid phase synthesis reaction step was completed, the product was cleaved from the resin using 1% TFA in Dichloromethane (DCM). After removal of the solvent by rotary evaporation in vacuo, cold ether was added to give a precipitate, which was centrifuged to give the crude compound, which was dried and used directly in the next step.

Wherein said compound mCy-NH₂Compound 1, HBTU and DIPEA in a molar ratio of 7: (5-9): (7-11): (15.5-19.5), preferably 7: 7: 9: 17.5.

wherein, the solvent includes but is not limited to tetrahydrofuran, and preferably anhydrous tetrahydrofuran.

Wherein the reaction temperature is 20-30 ℃, and room temperature is preferred.

Wherein the reaction is carried out under stirring.

The reaction time is 1 hour or more, preferably 1 to 5 hours, and more preferably 2 hours.

After the reaction is finished, carrying out vacuum rotary evaporation on the reaction liquid containing the compound 2, purifying and eluting to obtain the compound 2.

Wherein the eluent for elution is CH₂Cl₂And CH₃OH solution of said CH₂Cl₂And CH₃The volume ratio of OH is preferably 100: 1-10: 1.

in the step (b), the step (c),

dissolving the compound 2 and DIPEA in a first solvent, stirring, and adding a diethyl chlorophosphate solution to perform a first reaction to obtain an intermediate; and dissolving the intermediate in a second solvent, and carrying out a second reaction with trimethyl bromosilane (TMS-Br) to obtain a reaction solution containing the compound 3.

Wherein, the reaction dissolves compound 2 and DIPEA in a first solvent under the protection of inert gas.

Wherein the first reaction solvent includes but is not limited to dichloromethane, preferably anhydrous dichloromethane.

Wherein the molar ratio of compound 2 to DIPEA is 1: 7-13, preferably 1: 10.

wherein the stirring temperature is 20-30 ℃, and preferably room temperature.

Wherein the stirring time is 2min or more, preferably 2 to 20min, and more preferably 10 min.

Wherein, the solvent of the diethyl chlorophosphate solution includes but is not limited to Dichloromethane (DCM).

Wherein the addition of the solution of diethyl chlorophosphate is dropwise.

Wherein the concentration of the diethyl chlorophosphate solution is 0.02-0.22mmol/mL, preferably 0.12 mmol/mL.

Wherein the molar ratio of the compound 2 to the diethyl chlorophosphate is 1: 1-3, preferably 1: 2.

wherein the time of the first reaction is 0.5h or more, preferably 0.5 to 3.5h, and more preferably 2 h.

After the first reaction is finished, diluting the reaction solution, extracting, drying an organic layer, filtering, and performing rotary evaporation on the obtained filtrate to obtain an intermediate.

Wherein the dilution is with DCM; it is preferably diluted with 20mL of DCM.

Wherein the extraction is with saturated Na₂CO₃Aqueous solution, 1M HCl and saturated aqueous NaCl solution were extracted 3 times.

Wherein the drying is carried out by using anhydrous Na as the organic layer₂SO₄And (5) drying.

Wherein, the intermediate is dissolved in a second solvent under the protection of inert gas; preferably under nitrogen protection.

Wherein the second solvent includes, but is not limited to, DCM.

Wherein the dosage ratio of the compound 2 to the second solvent is 6 mmol: 400-600mL, preferably 6 mmol: 500 mL.

Wherein the molar ratio of the compound 2 to TMS-Br is 1: 15-25, preferably 1: 20.

wherein the temperature of the second reaction is 20-30 ℃, and room temperature is preferred.

The time for the second reaction is 12 hours or longer, preferably 12 to 36 hours, and more preferably 24 hours.

After the second reaction is finished, quenching the reaction, dripping a methanol solution of DIPEA, adjusting the pH value to be neutral, performing rotary evaporation, purifying and freeze-drying to obtain a compound 3.

Wherein the quenching solvent is methanol quenching.

Wherein the concentration of the DIPEA solution in methanol is 0.14-0.34mmol/mL, and preferably 0.24 mmol/mL.

Wherein the purification is by preparative HPLC.

Wherein the freeze-drying is freeze-drying by using a freeze-dryer.

In the step c, the step (c),

wherein the reaction is a mixed solution of a compound 3, piperidine and a solvent, adding an HCl solution to adjust the pH value, and purifying to obtain a compound 4.

Wherein the solvent includes but is not limited to DMF, preferably anhydrous DMF.

The reaction is preferably carried out by dissolving the compound 3 in a solvent, stirring, adding a piperidine solution, and reacting.

Wherein the dosage ratio of the compound 3 to the solvent is 0.0125-0.0325mmol/mL, preferably 0.0225 mmol/mL.

Wherein, in the piperidine solution, the solvent of the solution includes but is not limited to DMF, preferably anhydrous DMF; the volume fraction of the piperidine and the solvent is 3% -7%, preferably 5%; the volume of the piperidine solution is 1-3mL, preferably 2 mL.

Wherein the reaction temperature is low temperature, and is preferably ice bath.

Wherein the reaction is carried out under stirring.

Wherein the reaction time is 5-15min, preferably 10 min.

And after the reaction is finished, adjusting the pH solvent to be an HCl solution, wherein the concentration and the volume are 1mol/L and 2mL respectively.

Wherein the purification is by preparative HPLC.

In the step d, the step (c),

wherein the reaction is that the compound 4, NODA-GA-NHS ester and DIPEA are dissolved in a solvent for reaction and purification to obtain the compound P-CyFF-NODA.

Wherein said compound 4: NODA-GA-NHS ester: the molar ratio of DIPEA is (1-3): (1-3): 1, preferably 2: 2: 1.

Wherein the reaction is carried out under stirring.

Wherein the reaction time is 1 hour or more, preferably 3 hours.

Wherein the purification is by preparative HPLC.

Wherein the lyophilization is performed using a lyophilizer.

In the step e, the step (c),

wherein the reaction is compound P-CyFF-NODA and GaCl₃Dissolving in solvent, reacting, and purifying to obtain compound P-CyFF-Ga.

Wherein, the solvent of the compound P-CyFF-NODA includes but is not limited to DMF, GaCl₃The solvent of (2) is secondary water.

Wherein the compound P-CyFF-NODA is firstly dissolved in DMF, and then diluted HCl with the concentration of 1mol/L is added.

Wherein, the compound P-CyFF-NODA and the dilute HCl need to be uniformly mixed and stirred for 5 minutes.

Wherein, the compounds P-CyFF-NODA and GaCl₃In a molar ratio of 1: (5-15), preferably 1: 10.

Wherein, the reaction environment needs to be protected from light.

Wherein the reaction time is overnight reaction.

Wherein the purification is by preparative HPLC.

Wherein the freeze-drying uses a freeze-dryer.

In order to solve the third technical problem, the present invention discloses the dual-mode molecular imaging probe P-CyFF-⁶⁸Method for preparing Ga, i.e. acidic containing⁶⁸Ga³⁺The solution is incubated with P-CyFF-NODA to obtain the P-CyFF-containing-⁶⁸A solution of Ga.

Wherein said acidic group contains⁶⁸Ga³⁺The pH of the solution of (a) is 3.0 to 5.0, preferably 4.0.

Wherein said acidic group contains⁶⁸Ga³⁺The pH of the solution of (a) is adjusted by adding sodium acetate.

Wherein said acidic group contains⁶⁸Ga³⁺The dosage ratio of the solution to the P-CyFF-NODA is 1.4 mL: (40-80) μ g, preferably 1.4 mL: 60 μ g.

Wherein the incubation temperature is 30-44 ℃, preferably 37 ℃.

Wherein the incubation time is 10-20min, preferably 15 min.

In order to solve the fourth technical problem, the invention discloses the target nonradioactive probe P-CyFF-Ga and/or the bimodal molecular imaging probe P-CyFF-⁶⁸Application of Ga in preparing a contrast agent for tumor diagnosis.

Wherein, the application is preferably the target nonradioactive probe P-CyFF-Ga and the bimodal molecular imaging probe P-CyFF-⁶⁸Application of Ga in preparing tumor diagnosis contrast agent, more preferably P-CyFF-Ga and P-CyFF-⁶⁸The mixed solution of Ga is used for preparing a tumor diagnosis contrast agent; wherein the P-CyFF-Ga and P-CyFF-⁶⁸The Ga mixed solution is preferably P-CyFF-⁶⁸Ga (200. mu. Ci) and P-CyFF-Ga (50. mu.M) were mixed in a volume of 200. mu.L.

Wherein the tumor diagnosis contrast agent is preferably an alkaline phosphatase-positive tumor contrast agent.

Wherein the alkaline phosphatase is preferably endogenous ALP on the surface of HeLa cell membrane.

In order to solve the fifth technical problem, the invention discloses the target nonradioactive probe P-CyFF-Ga and/orThe dual-mode molecular imaging probe P-CyFF-⁶⁸Application of Ga in tumor diagnosis fluorescence imaging.

Wherein, the application is preferably the target nonradioactive probe P-CyFF-Ga and the bimodal molecular imaging probe P-CyFF-⁶⁸The application of Ga in tumor diagnosis fluorescence imaging is further preferably P-CyFF-Ga and P-CyFF-⁶⁸The mixed solution of Ga is used for tumor diagnosis fluorescence imaging; wherein the P-CyFF-Ga and P-CyFF-⁶⁸The Ga mixed solution is preferably P-CyFF-⁶⁸Ga (200. mu. Ci) and P-CyFF-Ga (50. mu.M) were mixed in a volume of 200. mu.L.

The tumor diagnosis fluorescence imaging is preferably alkaline phosphatase positive tumor diagnosis fluorescence imaging, and is further preferably alkaline phosphatase positive tumor diagnosis fluorescence/PET bimodal imaging.

In the fourth and fifth technical problems, the target nonradioactive probe P-CyFF-Ga and the bimodal molecular imaging probe P-CyFF-⁶⁸Ga utilizes enzyme to mediate to carry out self-assembly and is applied to the targeting bimodal formation of tumors; preferably, the target nonradioactive probe P-CyFF-Ga and the bimodal molecular imaging probe P-CyFF-⁶⁸Ga is applied to targeted bimodal imaging of alkaline phosphatase positive tumors by utilizing a process of response to alkaline phosphatase (ALP) and self-assembly; further preferably, the target nonradioactive probe P-CyFF-Ga and the bimodal molecular imaging probe P-CyFF-⁶⁸Ga is dephosphorylated under the action of ALP and is assembled in situ to form nanoparticles; the obtained nanoparticles rely on the Cy-Cl fluorophore and radionuclide to be activated to provide enhanced NIR fluorescence and PET imaging signal at the tumor site, P-CyFF-Ga and P-CyFF-⁶⁸After Ga is cleared from normal tissue, tumors can be detected using bimodal imaging signals.

The invention applies a controllable in-situ self-assembly strategy to construct an ALP activated multi-modal molecular imaging probe for multi-modal imaging analysis of in-vivo horizontal tumors. An ALP activated NIR fluorescence/PET bimodal probe was constructed combining an enzymatic fluorescence activation reaction with an in situ self-assembly strategy. The probe can be selectively activated by ALP and assembled to form nanoparticles with fluorescent signals and nuclide radioactivity, and stay and accumulate at the tumor site. The probe can perform fluorescence activation reaction under the action of ALP to form fluorescence activated nano particles, the fluorescence of the fluorescence activated nano particles is about 70 times, so that high-sensitivity detection can be performed on the ALP by using a fluorescence signal, and the detection limit of the fluorescence activated nano particles on the ALP is about 0.04U/L. At the cellular level, the probes are able to enhance the fluorescent signal and accumulation of radioisotopes on ALP positive cells using ALP mediated membrane surface in situ self-assembly, thereby discriminating at the cellular level between high and low ALP expressing cell lines for bimodal imaging. With this probe we achieved NIR fluorescence/PET bimodal imaging detection of ALP positive tumors at the in vivo level.

Has the advantages that: compared with the prior art, the invention has the following advantages:

the invention provides a bimodal molecular imaging probe P-CyFF-⁶⁸Ga, which is capable of dephosphorylation under ALP, to generate amphiphilic dephosphorylated products and self-assemble to form fluorescent-enhanced radioactive nanoparticles. The nanoparticles formed by in situ assembly can stay on the tumor cell membrane and enter the interior of the cell through endocytosis, and the increased size is utilized to prolong the stay time of the probe in the tumor cell and enhance the accumulation of the probe at the tumor site. After the inactivated probe is cleared from normal tissues, the probe can carry out high-sensitivity and high-tissue-penetrability imaging detection on tumors by using NIR fluorescence and PET bimodal signals. The successful application of the probe provided by the invention provides a new idea for constructing a fluorescence/PET combined activated molecular probe, and is expected to be applied to imaging detection and fluorescence operation navigation of ALP positive tumors. Meanwhile, the invention only needs a single probe, and can realize simultaneous imaging of fluorescence and PET through single injection.

Drawings

The foregoing and/or other advantages of the invention will become further apparent from the following detailed description of the invention when taken in conjunction with the accompanying drawings.

FIG. 1(a) P-CyFF-⁶⁸Schematic representation of the chemical structure of Ga and ALP mediated self-assembly to form nanoparticles. (b) Schematic representation of P-CyFF-Ga for in vivo fluorescence/PET bimodal imaging of ALP high expressing tumors.

FIG. 2. dephosphorylation of P-CyFF-Ga response to ALP and in vitro characterization of self-assembly. (a) HPLC analysis of P-CyFF-Ga (5. mu.M) before and after 10, 20, 30min incubation with ALP (100U/L). (b) DLS analysis of P-CyFF-Ga (100. mu.M) and ALP (500U/L) at 37 ℃ for different times, respectively. (c) TEM imaging of P-CyFF-Ga (100. mu.M) after incubation with ALP (500U/L) for 30min in Tris buffer (pH 8.0) at 37 ℃. Scale bar: 500 nm. (d-e) UV-vis absorption spectrum and (e) fluorescence spectrum of P-CyFF-Ga (2.5. mu.M) incubated with ALP (100U/L) in Tris buffer (pH 8.0) at 37 ℃ for 0-25 min. The excitation wavelength of the fluorescence spectrum is 680 nm. (f) P-CyFF-Ga in Tris buffer (pH 8.0) with MMP-2, GGT, cathepsin B (CTB), Caspace-3, ALP or ALP and inhibitor Na thereof₃VO₄Or fluorescence spectra after incubation with PBS for 30 minutes.

FIG. 3.P-CyFF-⁶⁸Radiolabelling and dephosphorylation of Ga. (a) P-CyFF-activated after Ga68 labeling⁶⁸Ga and free form⁶⁸HPLC analysis of Ga contrast. (b) P-CyFF-⁶⁸Radioactive HPLC analysis after 0-4 hours of incubation of Ga at 37 ℃. (c) P-CyFF-⁶⁸Radioactivity HPLC analysis before and after reaction of Ga with ALP.

FIG. 4. mu.M P-CyFF-Ga (2.5. mu.M) in buffer with Tis, 100U/L ALP and Na₃VO₄(10mM) mixture, 100U/L mixture of ALP and phosphate (10mM), DMEM, 10% FBS-containing DMEM, or 10% FBS plus Na₃VO₄Fluorescence intensity of P-CyFF-Ga changed within 1 hour of incubation in DMEM (10 mM).

FIG. 5 Probe cytotoxicity assay. Human cervical cancer HeLa cells were incubated with different concentrations of P-CyFF-Ga (0, 2.5, 5, 10, 25, 50, 100 μ M) for 24h and cell viability was determined by MTT assay (n ═ 5).

FIG. 6. analysis of the imaging detectability of probes for endogenous ALP in living cells. (a) HeLa cells incubated with Lyso-tracker (green) and Hoechst 33342 (blue)And then incubated with P-CyFF-Ga (5. mu.M, red) for different time periods for fluorescence imaging of the cells within 60 minutes. Scale bar: 20 μm. (b) ICP-MS analysis showed that HeLa cells took up Ga content after 1h incubation of P-CyFF-Ga (5, 10 and 20. mu.M). (c) Probe generation TEM and STEM image analysis of NPs, and elemental mapping of Cl and Ga. The scale bar is 500 nm. (d) HeLa cells and P-CyFF-⁶⁸Intracellular isotopic abundance analysis after 0-4 hours of Ga (1. mu. Ci) incubation. (e) Adding P-CyFF-activated HeLa cells⁶⁸Isotopic abundance analysis after 60min incubation of Ga (1. mu. Ci) with P-CyFF-Ga (5, 10, 20. mu.M). (f) NIR fluorescence (λ ex/em 670/(750 ± 50) nm) and PET imaging of cell suspensions from different treatment groups. (g) The mean FL intensity of the cell suspension (grey) is compared to the radioactive PET signal intensity (black) in the f plot. (h) ICP-MS analysis of Ga content of treated cells. Treatment group I: HEK293T cell and P-CyFF-⁶⁸Ga (5. mu. Ci) was incubated for 1 hour; II: HeLa cells and P-CyFF-⁶⁸Ga (5. mu. Ci) and P-CyFF-Ga (10. mu.M) were incubated for 1 hour; III: HeLa cells and P-CyFF-⁶⁸Ga (5. mu. Ci) was incubated for 1 hour.

FIG. 7.HPLC analysis of medium and cell lysates after 1 hour incubation of P-CyFF-Ga in the amounts of 5. mu.M (a), 10. mu.M (b) and 20. mu.M (c) with HeLa cells. (d) And quantitatively analyzing the P-CyFF-Ga uptake of HeLa cells in the percentage of the incubated probe after incubation of P-CyFF-Ga at different concentrations by using HPLC.

FIG. 8. mice receiving different treatments were imaged for 0, 30, 60, 90 and 120 minutes for (a) FL, (b) mean fluorescence intensity in the tumor, and (c) tumor to background signal ratio (TBR).

Figure 9. mice receiving different treatments at 0, 30, 60, 90 and 120 minutes (a) PET imaging, (b) mean radioactivity signal in the tumor, and (c) tumor to background signal ratio (TBR).

Detailed Description

The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials are commercially available, unless otherwise specified.

The "%" in the following examples is a molar percentage unless otherwise specified.

Reagents and instrumentation: all ofChemicals are commercially available (e.g., Aldrich, Adamas). High-sugar Dulbecco's Modified Eagle's Medium (DMEM), Fetal Bovine Serum (FBS), penicillin/streptomycin were purchased from Thermo (Shanghai, China). 3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide (MTT) kit was purchased from KeyGen Biotech Co. Ltd.¹H-NMR and¹³C-NMR spectra were obtained using a 400MHz Bruker Avance III 400 nuclear magnetic instrument. High Performance Liquid Chromatography (HPLC) using Thermo Scientific Dionex Ultimate 3000, eluent CH₃CN/H₂O(1‰CF₃COOH). Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS) analysis measurements were performed using AB SCIEX 4800Plus MALDI TOF/TOFTM mass spectrometer UV-Vis spectra using an Ocean Optics Maya 2000Pro spectrometer. The fluorescence spectra were measured using a HORIBA Jobin Yvon Fluoromax-4 fluorometer. Dynamic Light Scattering (DLS) analysis was measured using a 90Plus/BI-MAS device (Bruk Highen, USA). TEM images were obtained using a JEM-1011 transmission electron microscope (JEOL, Ltd., Japan) at an accelerating voltage of 100 kV. Fluorescence images of cells were acquired using a Leica TCS SP8 confocal laser scanning microscope or an Olympus IX73 fluorescence inverted microscope. MTT assays were acquired using a microplate reader (Tcan). In vivo fluorescence images were acquired using the IVIS luminea XR III system and fluorescence intensity was quantified by measuring circle area signals using the Living image software (PerkinElmer). Inductively coupled plasma mass spectrometry (ICP-MS) analysis was obtained using an Optima 5300DV plasma mass spectrometer (PE, America).

Example 1: design, Synthesis and characterization of probes

The scheme S1 shows the structural design of the probe P-CyFF-Ga.

Synthesis scheme S1

Reaction conditions are as follows: (a) HBTU, DIPEA, THF, r.t.,2h, 89%; (b) (i) diethyl chlorophosphate, DIPEA, r.t.,2 h; (ii) TMS-Br, r.t.,24h, 76%; (c) piperidine, 91% at 0 ℃ for 10 min; (d) NODA-GA-NHS ester, DIPEA, DMF, r.t.,3h, 56%; (e) GaCl₃,HCl,pH 2-3,r.t.,12h,87％

Synthesis of Compound 1: compound 1 was synthesized by standard Solid Phase Peptide Synthesis (SPPS) procedure (nat. protocol, 2007,2,3247) starting with 2-chlorotrityl chloride resin and Fmoc-protected phenylalanine. After the solid phase synthesis reaction step was completed, the product was cleaved from the resin using 1% TFA in DCM. After removal of the solvent by rotary evaporation in vacuo, cold ether was added to give a precipitate, which was centrifuged to give the crude compound, which was dried and used directly in the next step. Yield: 95mg (89%).¹H NMR(400MHz,DMSO-d₆)δ8.29(d,J＝7.8Hz,1H),7.87(d,J＝7.6Hz,2H),7.61(dd,J＝19.2,9.2Hz,3H),7.40(td,J＝7.4,2.9Hz,2H),7.35–7.21(m,10H),7.21–7.12(m,2H),4.54–4.44(m,1H),4.28(td,J＝10.9,3.7Hz,1H),4.15–4.09(m,2H),3.38(q,J＝7.0Hz,1H),3.09(dd,J＝13.9,5.2Hz,1H),2.96(dt,J＝14.0,7.5Hz,2H),2.73(dd,J＝13.6,11.1Hz,1H).MS:calcd.For C₃₃H₃₁N₂O₅ ⁺[(M+H)⁺]:534.2155；MALDI-MS found:m/z 534.6171.

Synthesis of Compound 2: first, we synthesized the NIR fluorophore mCy-NH according to the previously reported method (anal. chem.2018,90,2875)₂. Then mCy-NH is added₂(34mg, 0.07mmol), Compound 1(37mg, 0.07mmol), HBTU (34mg, 0.09mmol) and DIPEA (30. mu.L, 0.175mmol) were dissolved in a solution of anhydrous THF (5mL) and stirred at room temperature for 2 hours. After completion of the reaction, THF was removed by rotary evaporation under vacuum. The residue was purified by flash chromatography on silica gel using CH₂Cl₂/CH₃A mixed solution of OH (100: 1100 mL, 50: 150 mL, 10: 1100 mL) was used as eluent to give Compound 2 as a blue solid. Yield: 61mg (89%).¹H NMR(400MHz,DMSO-d₆)δ8.51(d,J＝15.0Hz,1H),8.20(d,J＝7.7Hz,1H),8.15(t,J＝5.6Hz,1H),7.83(d,J＝7.6Hz,2H),7.76(dd,J＝7.5,1.2Hz,1H),7.67(d,J＝3.0Hz,1H),7.64(s,1H),7.58–7.52(m,3H),7.45(t,J＝7.5Hz,1H),7.37(ddd,J＝7.5,4.6,1.5Hz,3H),7.29–7.18(m,11H),7.15–7.09(m,2H),7.06(s,1H),6.52(d,J＝14.9Hz,1H),4.48(td,J＝8.1,5.8Hz,1H),4.37–4.19(m,3H),4.14–4.04(m,3H),3.22(d,J＝6.7Hz,2H),3.00(dd,J＝13.7,5.8Hz,1H),2.91(dt,J＝13.6,4.7Hz,2H),2.75–2.59(m,5H),1.85(dq,J＝14.9,7.3Hz,2H),1.80–1.64(m,8H).MS:calcd.For C₆₁H₅₈ClN₄O₆ ⁺[M⁺]:977.4039；MALDI-MS found:m/z 977.3083.

Synthesis of Compound 3: compound 2(59mg, 0.06mmol) and DIPEA (105. mu.L, 0.6mmol) were dissolved in 5mL of anhydrous DCM under nitrogen and stirred at room temperature for 10min, then diethyl chlorophosphate (17.5. mu.L, 0.12mmol) was dissolved in 1mL of DCM and added dropwise to the reaction solution, and the solution was stirred at room temperature for 2 h. The reaction mixture was then diluted with 20mL of DCM and saturated Na₂CO₃The aqueous solution, HCl (1M) and saturated aqueous NaCl solution were washed 3 times, and the organic layer was washed with anhydrous Na₂SO₄And (5) drying. The precipitate was removed by filtration and the solvent was removed by rotary evaporation and the residue was dissolved in 5mL of DCM under nitrogen. TMS-Br (158. mu.L, 1.2mmol) was added rapidly to the solution and the reaction mixture was stirred at room temperature for 24 hours. After completion of the reaction, the reaction solution was added dropwise to 50mL of methanol at-20 ℃ to quench the reaction, and DIPEA (210. mu.L, 1.2mmol) dissolved in 5mL of cold methanol was added dropwise to the mixture to adjust the pH to neutral. The solvent was then removed by rotary evaporation and the residue was purified by preparative HPLC to give compound 3 as a blue solid by lyophilization. Yield: 48mg (76%).¹H NMR(400MHz,DMSO-d₆)δ8.52(d,J＝15.0Hz,1H),8.19(d,J＝7.7Hz,1H),8.14(t,J＝5.6Hz,1H),7.81(d,J＝7.6Hz,2H),7.76(dd,J＝7.5,1.2Hz,1H),7.67(d,J＝3.0Hz,1H),7.62(s,1H),7.58–7.52(m,3H),7.45(t,J＝7.5Hz,1H),7.37(ddd,J＝7.5,4.6,1.5Hz,3H),7.29–7.18(m,11H),7.15–7.09(m,2H),7.06(s,1H),6.52(d,J＝14.9Hz,1H),4.48(td,J＝8.1,5.8Hz,1H),4.37–4.19(m,3H),4.14–4.04(m,3H),3.22(d,J＝6.7Hz,2H),3.00(dd,J＝13.7,5.8Hz,1H),2.92(dt,J＝13.6,4.7Hz,2H),2.75–2.59(m,5H),1.86(dq,J＝14.9,7.3Hz,2H),1.80–1.64(m,8H).MS:calcd.For C₆₁H₅₉ClN₄O₉P⁺[M⁺]:1057.3703；MALDI-MS found:m/z 1057.3013.

Synthesis of Compound 4: compound 3(48mg, 0.045mmol) was dissolved in 2mL of DMF solution and cooled with stirring on an ice bath for 10 minutes.2mL of a 5% piperidine-containing DMF solution was cooled in an ice bath and added to the reaction mixture, and the reaction mixture was stirred for another 10 minutes while cooling in an ice bath. After the reaction time had elapsed, 2mL of a pre-prepared ice HCl (1M) solution was added to the reaction solution to adjust the pH, the mixture was analyzed by HPLC and purified by preparative HPLC, and lyophilized to give dark blue solid compound 4. Yield: 34mg (91%).¹H NMR(400MHz,DMSO-d₆)δ8.96(d,J＝8.0Hz,1H),8.50(d,J＝15.0Hz,1H),8.43(s,1H),8.07(d,J＝5.1Hz,2H),7.97–7.91(m,1H),7.81(dd,J＝7.4,1.2Hz,1H),7.77–7.73(m,1H),7.72(s,1H),7.60–7.54(m,2H),7.52–7.48(m,1H),7.48–7.44(m,1H),7.41(td,J＝7.4,1.3Hz,1H),7.32(s,1H),7.24(d,J＝4.9Hz,4H),7.21–7.13(m,5H),6.63(d,J＝15.1Hz,1H),4.50(m,2H),4.43–4.38(m,1H),3.29(p,J＝6.4Hz,1H),3.24–3.16(m,1H),3.02(m,3H),2.92–2.85(m,2H),2.72–2.62(m,4H),1.88(q,J＝9.5,9.1Hz,2H),1.75(d,J＝1.6Hz,8H).MS:calcd.For C₄₆H₄₉ClN₄O₇P⁺[M⁺]:835.3022；MALDI-MS found:m/z 835.2593.

Synthesis of the Compound P-CyFF-NODA: compound 4(34mg, 0.04mmol), NODA-GA-NHS ester (19mg, 0.04mmol) and DIPEA (35. mu.L, 0.2mmol) were dissolved in 2mL of DMF and the reaction was stirred at room temperature for 3 hours, after completion of the reaction, the mixture was purified by semi-preparative HPLC and lyophilized to give P-CyFF-NODA as a dark blue solid. Yield: 26mg (56%).¹H NMR(400MHz,DMSO-d₆)δ8.52(d,J＝15.0Hz,1H),8.26(d,J＝7.8Hz,1H),8.19(s,1H),8.04(d,J＝8.4Hz,1H),7.81(dd,J＝7.4,1.3Hz,1H),7.77–7.68(m,2H),7.61–7.53(m,2H),7.52–7.45(m,1H),7.32(s,1H),7.25–7.20(m,4H),7.18–7.11(m,5H),7.10–7.06(m,1H),6.63(d,J＝15.1Hz,1H),4.55–4.29(m,5H),3.88(s,2H),3.59(d,J＝18.2Hz,5H),3.27–3.12(m,5H),3.06–2.81(m,10H),2.71–2.65(m,5H),2.15(t,J＝7.5Hz,2H),1.93–1.65(m,14H).MS:calcd.For C₆₁H₇₂ClN₇O₁₄P⁺[M⁺]:1192.4558；MALDI-MS found:m/z 1192.4766.

Synthesis of a non-radioactive probe P-CyFF-Ga: dissolving P-CyFF-NODA (12mg, 0.01mmol) in 0.5mL DMF, adding 0.5mL diluted HCl (1M), stirring for 5min, and mixingAnd (4) uniformity. Adding GaCl₃(18mg, 0.1mmol) was dissolved in 0.2mL of secondary water, added to the reaction mixture, and stirred overnight at room temperature in the dark. After the HPLC check reaction was complete, the mixture was purified using semi-preparative HPLC and lyophilized to obtain P-CyFF-Ga as a dark blue solid. Yield: 11mg (87%). MS calcd. For C₆₁H₆₉ClGaN₇O₁₄P⁺[M⁺]:1258.3579；MALDI-MS found:m/z 1258.7816.

Example 2: radiolabelling protocol for P-CyFF-NODA

5mL of HCl (0.05mol/L) was injected using a disposable syringe⁶⁸Ga³⁺A generator. 0.5, 1.4, 0.7 and 1.4mL of the total⁶⁸Ga³⁺The solution of (1). After the isotope dosing test, the solutions obtained for the fourth and fifth times were transferred into a hot chamber using lead tanks. Then 1.4mL of the solution was added⁶⁸Ga³⁺The solution was accurately transferred to a new centrifuge tube and the pH adjusted to 4.0 using the previously prepared sodium acetate solution. Then, 60. mu.g of P-CyFF-NODA was added, and the mixture was incubated at 37 ℃ for 15 minutes with shaking every 5 minutes to obtain a final solution containing P-CyFF-⁶⁸A solution of Ga. A small amount of the final solution was taken for radioactive HPLC analysis and the other solution was used directly without further purification. Performing P-CyFF-NODA by using one-step method⁶⁸Ga-marked as shown in FIG. 1 (a).

Example 3: probe performance testing

1. Study of self-Assembly in solution

(1) For DLS analysis, P-CyFF-Ga (20. mu.M) was dissolved in 1mL Tris buffer and incubated with ALP (100U/L) for 30min at 37 ℃. DLS analysis was performed on the reaction solution using a 90Plus/BI-MAS apparatus (Brukhein, USA).

As shown in FIG. 1(b), dephosphorylation of the probe under the action of ALP can effectively reduce the hydrophilicity of the probe, generate a dephosphorylated product CyFF-Ga with one hydrophobic end and one hydrophilic end, and can further undergo self-assembly under the actions of hydrophilicity-hydrophobicity and pi-pi stacking to form nanoparticles.

P-CyFF-Ga and P-CyFF-⁶⁸Ga is small in water solubilityMolecular probes, which can penetrate into tumor tissues after systemic administration; under the action of ALP over-expressed on tumor cell membrane, P-CyFF-Ga and P-CyFF-⁶⁸Ga dephosphorylation and in-situ co-assembly to form nano-particle NPs-⁶⁸Ga, the nano-particles formed on the surface of the membrane are easy to stay on the surface of the tumor cells and are endocytosed by the tumor cells, so that the stay in the tumor is increased. NPs by means of activated Cy-Cl fluorophores and radionuclides⁶⁸Ga can provide enhanced NIR fluorescence and PET imaging signals at a tumor site, and P-CyFF-Ga and P-CyFF-⁶⁸After Ga is cleared from normal tissue, tumors can be detected using bimodal imaging signals.

(2) For TEM analysis, P-CyFF-Ga (20. mu.M) was dissolved in 1mL Tris buffer and incubated with ALP (100U/L) for 30min at 37 ℃. The solution was then dropped onto a copper plate coated with a carbon layer, immediately drained and vacuum dried. The samples were then photographed using a JEM-2800 transmission electron microscope.

We first studied ALP response dephosphorylation of P-CyFF-Ga in solution, and analyzed the reaction solution by high performance liquid chromatography after incubating P-CyFF-Ga (5 μ M) with ALP (100U/L) in Tris buffer (pH 8.0) for 30 minutes. As shown in FIG. 2(a), P-CyFF-Ga (t) was obtained after 30 minutes of reaction with ALP_R13.4min) completely disappeared in the reaction solution and at t_RA new peak with Cy-Cl absorption wavelength appeared at 16.3min position, demonstrating that P-CyFF-Ga can be rapidly converted to dephosphorylated product under the catalytic action of ALP. DLS showed no detectable presence of nanoparticles in the solution of P-CyFF-Ga (100. mu.M) before reaction with ALP, as shown in FIG. 2(b), and after P-CyFF-Ga reacted with ALP (500U/L) for 30 minutes, a monodisperse nanoparticle signal peak appeared on DLS with a hydrated particle size of about 56 nm. Transmission Electron Microscopy (TEM) showed that P-CyFF-Ga reacted with ALP to form uniform spherical CyFF-Ga nanoparticles in water as shown in FIG. 2 (c).

Study of the digestion kinetics of P-CyFF-Ga by ALP

P-CyFF-Ga was diluted with Tris buffer to different concentration probe solutions (0.5, 1, 2,3, 4,5 and 10. mu.M) and 100. mu.L of different concentration probes were takenThe solution was added to a 96-well black plate. To each solution was added 100. mu.L of ALP (60U/L) solution, and Spark was then used immediately^TMA10M Multimode microplate reader (. lamda.ex/em. 680/710nm) recorded the fluorescence intensity generated in each well over the first five minutes. Under the same conditions, the amount of CyFF-Ga species at each reaction time point was determined by a standard curve method of fluorescence intensity-concentration. Kinetic values, in which Km and Vmax are included, were calculated from Lineweaver-Burk plots.

The enzyme kinetics of the probe can be researched by utilizing the fluorescence recovery of P-CyFF-Ga under the catalysis of ALP, and the increase of the fluorescence signal of the first 3 minutes is measured after P-CyFF-Ga is diluted into different concentrations to react with ALP. The maximum reaction rate (Vmax) and apparent Michael constant (Km) of P-CyFF-Ga for ALP reaction were measured by a Lineweaver-Burk curve to be 0.90. mu.M min^-1And 23.70. mu.M. Thus, the kcat/Km value was calculated to be 2.0X 10⁵M^-1s^-1It was demonstrated that P-CyFF-Ga has a very fast response capability to ALP.

Study on detection sensitivity of P-CyFF-Ga to ALP

To evaluate the sensitivity of P-CyFF-Ga for ALP detection, P-CyFF-Ga (2.5. mu.M) was incubated with different concentrations of ALP (0, 0.5, 1, 2, 4, 6, 10, 20, 40, 60, 80 and 100U/L) in Tris buffer at 37 ℃ for 25 minutes, and then the fluorescence spectrum of the reaction solution was obtained using HORIBA Jobin Yvon Fluoromax-4 fluorometer with excitation wavelength of 680 nm. The obtained fluorescence intensity (λ em: 710nm) was plotted against the ALP concentration to obtain a probe for ALP detection in a linear range of 0.5 to 20U/L and a slope k. The detection limit was calculated using the formula 3 σ/k, where σ represents the standard deviation of 11 blank measurements.

With the phosphate removed, Intramolecular Charge Transfer (ICT) of the Cy-Cl fluorophore was restored, with a concomitant change in uv absorption and restoration of fluorescence. After P-CyFF-Ga is incubated with ALP, ultraviolet absorption peaks of the probe at 605nm and 652nm gradually red shift to 690nm along with the extension of the incubation time, meanwhile, new absorption peaks appear from 750nm to 800nm due to the ultraviolet absorption red shift caused by molecular self-assembly, the intensity increases along with the extension of the incubation time, and after about 25 minutes of incubation, the ultraviolet absorption peaks stop changing, which suggests the complete hydrolysis of the phosphate radical of the probe (FIG. 2 d). Meanwhile, the fluorescence spectrum of P-CyFF-Ga showed that after incubation with ALP, the NIR fluorescence of the probe at 710nm was gradually activated and reached a maximum after 25 minutes (FIG. 2 e).

Study of Selectivity of P-CyFF-Ga for ALP detection

We evaluated the specificity of P-CyFF-Ga for ALP detection based on fluorescence. P-CyFF-Ga (2.5 mu M) and 10nmol/L MMP-2, 100U/L GGT, 100U/L cathepsin B, 0.2 mu g/mL Caspase-3, 100U/L ALP or 100U/L ALP in Tris buffer solution and inhibitor Na₃VO₄(1mM) or phosphate (10mM) for 30 minutes at 37 ℃. Fluorescence spectra of each set of solutions were then collected under 680nm excitation.

The enzyme selectivity experiment shows that P-CyFF-Ga is not obviously dephosphorylated under the action of other cell-related proteases (such as MMP-2, GGT, Casepase-3, cathepsin B and the like) and the fluorescence is not activated (FIG. 2 f).

5.P-CyFF-⁶⁸Ga stability study

P-CyFF-⁶⁸Ga is diluted into PBS buffer solution, incubated at 37 ℃, 60 ℃, 70 ℃ and 80 ℃ for different times, and after incubation is completed, an equal amount of solution is taken out for analysis by radioactive HPLC. Analyzing the peak position change on the map to determine P-CyFF-⁶⁸Stability of Ga at different temperatures.

As shown in FIG. 3(a), the probe can be well aligned with each other⁶⁸Ga labeling, no radioisotope peak observed at HPLC retention time front, evidence⁶⁸Complete labelling of Ga. As shown in FIG. 3(b), when P-CyFF-⁶⁸After Ga was dissolved in PBS buffer (pH 7.4) and left at 37 ℃ for a maximum of 4 hours, the signal peak on the radioactive HPLC was hardly changed, demonstrating good stability of the probe under physiological conditions. Furthermore, we also investigated P-CyFF-⁶⁸ALP response to dephosphorylation of Ga. P-CyFF-⁶⁸After Ga reacts with ALP for 30 minutes, the reaction liquid and P-CyFF-⁶⁸The Ga raw solution was subjected to radioactive HPLC analysis, and as shown in FIG. 3(c), the retention time of the probe on the signal peak of radioactive HPLCIncreased after reaction with ALP, which is consistent with the HPLC pattern of the cold compound probe P-CyFF-Ga after ALP, indicating that P-CyFF-⁶⁸In Ga⁶⁸The presence of Ga does not affect the activity of ALP, so P-CyFF-⁶⁸Ga is capable of producing dephosphorylated products in response to ALP.

Stability study of P-CyFF-Ga under physiological conditions

The in vitro stability of P-CyFF-Ga was evaluated in a fluorescence-based manner. First, P-CyFF-Ga (2.5. mu.M) was mixed with the inhibitor Na in Tris buffer (pH 8.0), 100U/L ALP₃VO₄(1mM) or phosphate (10mM), DMEM medium containing 10% Fetal Bovine Serum (FBS), or DMEM medium containing 10% Fetal Bovine Serum (FBS) and Na₃VO₄Incubation was performed at 37 ℃ in DMEM medium (10 mM). The fluorescence intensity at 710nm in each well was recorded every 10min on a microplate reader and lasted for 1h (. lamda.ex/em. 680/710 nm). The probe remained stable under the conditions of FIG. 4 except for the few ascending curves, which indicated that Ga was in physiological conditions without ALP (10% FBS + DMEM + Na)₃VO₄) And the better stability is shown.

7. Cell culture method

Human cervical cancer HeLa cells, liver cancer HepG2 cells and human embryonic kidney HEK293T cells were purchased from Shanghai Stem cell institute of Chinese academy of sciences and cultured in DMEM (Dulbecco's modified Eagle Medium). The medium was supplemented with 10% (v/v) Fetal Bovine Serum (FBS), 100 units penicillin and 100 units streptomycin per ml of medium. All cells were in a humidified environment (5% CO) at 37 ℃₂) Culturing in medium.

8. Cytotoxicity Studies (MTT)

HeLa cells were seeded in flat bottom 96-well plates (5000 cells per well) and incubated overnight at 37 ℃. DMEM was aspirated from each well, and P-CyFF-Ga was added to the DMEM medium to make solutions (0, 2.5, 5, 10, 25, 50 and 100. mu.M) at different concentrations, 100. mu.L per well. After 24 hours of incubation, 50. mu.L of MTT solution (1mg/mL in PBS buffer) was added to each well. The cells were incubated at 37 ℃ for an additional 4 hours, and then the solution in each well was carefully removed. The purple crystals formed in the wells were dissolved by adding 150. mu.L of DMSO solution to each well. The absorbance (OD) of each well solution at 490nm was measured with a microplate reader (Tecan). The absorbance of blank cells (OD control) was used as a control, and the percentage of cell activity in each treatment group was calculated by dividing OD by OD control. FIG. 5 shows that the mortality of HeLa cells after 24 hours incubation with P-CyFF-Ga is negligible, indicating that the probe has good biocompatibility and can be used for imaging detection of ALP activity in living cells.

9. Basic procedure for fluorescence imaging of cells

(1) Fluorescence imaging of cellular uptake

Cells (. about.5X 10)⁴) The cells were seeded on a glass-bottom confocal dish (Invitro Scientific, D35-20-1-N) and allowed to grow overnight. The medium was discarded, DMEM medium without FBS but containing P-CyFF-Ga (20. mu.M) was added to the petri dish and incubated at 37 ℃ for 30 minutes. To inhibit ALP activity, cells were treated with ALP inhibitor Na₃VO₄(10mM) or phosphate (10mM) for 20min, followed by incubation with P-CyFF-Ga (20. mu.M) for 1 h. After the cell incubation was complete, the medium was removed and washed gently once with 1mL PBS buffer. After addition of fresh medium, cells were fluorescence imaged using a Leica TCS SP8 confocal laser scanning microscope with an excitation wavelength of 670nm and an emission wavelength of 690nm to 750 nm.

In real-time imaging experiments of ALP activity in HeLa cells, a solution of P-CyFF-Ga (20. mu.M) was first prepared in FBS-free DMEM medium and added to a HeLa cell-plated confocal culture dish. Cells were fluorescence imaged using a Leica TCS SP8 confocal laser scanning microscope at 5, 10, 20, 30, 40 and 60 minutes with an excitation wavelength of 670nm and an emission wavelength of 690nm to 750nm without washing.

(2) Co-location experiments

To investigate the distribution of P-CyFF-Ga in the subcellular organelles, HeLa cells were incubated for 20min with 2. mu.M Hoechst 33342 and 200nM Lyso Tracker Green DND-26. After washing 3 times with PBS, the cells were incubated with 20. mu. M P-CyFF-Ga for another 30 minutes. The medium was discarded and the cells were gently washed once with 1mL of PBS buffer. After addition of fresh DMEM medium, cells were fluorescence imaged using a Leica TCS SP8 confocal laser scanning microscope. The excitation wavelength of the probe is 670nm, and the emission wavelength is 690nm to 750 nm. Hoechst 33342 has an excitation wavelength of 405nm and an emission wavelength of 420nm to 460 nm. The excitation wavelength of the Lyso Tracker Green DND-26 is 488nm, and the emission wavelength is 500nm to 550 nm.

And incubating the HeLa cells and P-CyFF-Ga for different times, and carrying out confocal fluorescence imaging on the HeLa cells. As shown in FIG. 6(a), after 10 minutes of incubation of HeLa cells with P-CyFF-Ga, NIR fluorescence of Cy-Cl appeared on the surface of HeLa cell membrane, and the fluorescence on the surface of cell membrane further increased with the increase of incubation time, and at the same time, after 20 minutes of incubation with P-CyFF-Ga, spotted fluorescence spots appeared inside HeLa cells, indicating that the dephosphorylated product CyFF-Ga assembled on the membrane was taken up into the interior of the cells by HeLa cells. Co-localization studies using Lyso-tracker found that these fluorescent spots of Cy-Cl overlapped well with the green fluorescence of Lyso-tracker, indicating that the intracellular dephosphorylated product CyFF-Ga was predominantly present in lysosomes. We further imaged the activation and internalization of P-CyFF-Ga in HeLa cells in the same region time-dependently. With the prolonged incubation time, the fluorescence on the cell membrane surface gradually increases and reaches saturation, and meanwhile, the red fluorescence in the cell gradually increases. Meanwhile, FIG. 6(b) ICP-MS analysis shows that after P-CyFF-Ga (5, 10 and 20. mu.M) is incubated for 1h, the Ga uptake content of HeLa cells increases with the increase of the concentration gradient, indicating that activated CyFF-Ga is continuously taken into cell lysosomes. The above results show that P-CyFF-Ga is able to interact with and assemble at the cell membrane surface ALP (fig. 6(c)), and further enter the cell lysosome by endocytosis, thereby prolonging the retention in tumor cells, and image the ALP activity of tumor cells by activated NIR fluorescence.

10. HPLC study of intracellular probe dephosphorylation

Will be about 1X 10⁶HeLa cells were seeded onto 6cm cell culture dishes and incubated overnight at 37 ℃. The medium was changed to 2mL fresh DMEM (FBS-free) containing 5, 10 and 20. mu. M P-CyFF-Ga and the cells were incubated for 1 h. After incubation was complete, the medium was collected.Cells were trypsinized, centrifuged and counted. After lysing the cells with 200 μ L DMSO, the cell lysate was mixed with 300 μ L ice MeOH and 500 μ L deionized water and centrifuged at 14000rpm for 10 minutes at 4 ℃. 250 μ L of cell lysate supernatant and 500 μ L of medium were injected separately into the HPLC system for analysis.

As shown in FIG. 7, after incubation with cells for 1h, only the presence of cleaved CyFF-Ga was observed in the cell lysate, whereas the culture medium was mainly non-cleaved P-CyFF-Ga. With the increasing concentration of the probe incubated with the cells, the retention of the cleaved probe on the cell surface increased, reaching about 39.6% at a P-CyFF-Ga concentration of 20. mu.M.

11. Inside the cell⁶⁸Ga uptake experiments

For isotopic dose testing of the cell pellet, HeLa cells were dosed at 2X 10⁵The density of individual cells/well was seeded on 6-well cell culture plates. After overnight incubation at 37 deg.C, the medium was replaced with 2mL of P-CyFF-supplemented medium⁶⁸Fresh DMEM for Ga, incubated with the cells for different times. To study the effect of cold compounds on isotope uptake, HeLa cells were incubated with a mixture containing P-CyFF-⁶⁸Ga and DMEM at different concentrations of P-CyFF-Ga (5, 10, 20. mu.M) were incubated for 1 hour. After incubation was complete, the medium was removed and washed gently once with 1mL of PBS buffer. Gently washed once with 1mL PBS buffer. Cells were lysed with 500 μ L NaOH (1M), collected and transferred to a new centrifuge tube. And contains P-CyFF-⁶⁸The radioactive dose of Ga (1. mu. Ci) was investigated together with the standard solution. The standard solutions were prepared prior to cell incubation and used to calculate the total dose of radioactivity in the cells at different time points, respectively.

As shown in FIG. 6(d), 1. mu. Ci of P-CyFF-⁶⁸After 30 minutes of Ga incubation, there was approximately 2.5% uptake of the radioactive probe in HeLa cells, with increasing uptake of the radioactive probe at 1 hour up to approximately 5% and flattening out the increase with increasing incubation time, and only about 6% uptake of the radioactive probe at 4 hours. Meanwhile, since the assembly of the probe on the surface of the cell membrane needs to be higher than the Critical Micelle Concentration (CMC) of the probe, the enrichment of the probe on the cell has a certain concentration dependenceThe nature of the plant is determined. The concentration of labeled probe is much lower than the cellular imaging concentration of the cold compound probe described above, and thus may have an effect on the cellular uptake of the probe. Considering the above influencing factors, the low concentration of the labeled probe P-CyFF-⁶⁸The combination of Ga and the high-concentration cold compound probe P-CyFF-Ga can greatly improve the assembly and the retention of the probe on cells. We will use different concentrations of P-CyFF-Ga and 1. mu. Ci of P-CyFF-⁶⁸Ga was mixed and incubated with HeLa cells for 1 hour to quantify the intracellular radioactive dose. As shown in FIG. 6(e), when the concentration of the cold compound probe P-CyFF-Ga was 5. mu.M, the percentage of radionuclide uptake by the cells increased greatly from 5% without addition to about 25%, and reached a saturation uptake of 40% after the concentration reached further 10. mu.M, which is consistent with the percentage of cellular uptake of the cold compound probe analyzed by HPLC as described above. Further cellular Ga uptake analysis showed (FIG. 6h) that the percent uptake of P-CyFF-Ga increased with increasing P-CyFF-Ga incubation and reached saturation of the percent uptake after about 10. mu.M. Labeled probe P-CyFF-relative to low radioactive uptake using labeled probe alone⁶⁸The combined use of Ga and cold compound probe P-CyFF-Ga greatly increased the radioactive uptake of the cells, enabling the enhancement of the PET imaging signal of the cells (FIG. 6 f-g). In summary, P-CyFF-⁶⁸Ga can be activated by endogenous ALP, self-assembled in situ on the surface of cell membrane to form nanoparticles, thereby staying and enriching on the cell⁶⁸The PET signal of Ga images tumor cells highly expressing ALP. By using P-CyFF-⁶⁸The combination of Ga/P-CyFF-Ga can further increase the enrichment of the probe on the cell, so that the tumor can be imaged by utilizing the membrane-bound NIR fluorescence and the PET bimodal signal.

12. Establishment of mouse tumor model

5-6 week old female Nude mice BALB/c Nude were purchased from the Model Animal Research Center (MARC) of Nanjing university (Nanjing, China) and used according to the regulations of the animal Care and national Committee of the institute of Nuclear medicine, Jiangsu province.

To establish a subcutaneous HeLa tumor, 2X 10 cells were used⁶HeLa cells were suspended in 150. mu.L of DMEM mixture containing 33% matrigelSubcutaneously injected into selected sites on the back of nude mice. Tumor growth to about 100mm after about 2 weeks³For fluorescence and PET imaging.

13. Fluorescence imaging of mouse tumor ALP Activity

In order to carry out in vivo fluorescence imaging on the subcutaneous HeLa tumor of a tumor-bearing mouse, different substances are injected into the mouse body through the tail vein; specifically, I, tail vein injection P-CyFF-⁶⁸Ga (200. mu. Ci) 200. mu.L; II tail vein injection of P-CyFF-⁶⁸Ga (200 mu Ci) and P-CyFF-Ga (50 mu M) mixed solution is 200 mu L; III intratumoral injection of Na₃VO₄After inhibiting the tumor ALP activity for 15 minutes, P-CyFF-one injection is injected into the tail vein⁶⁸Ga (200. mu. Ci) and P-CyFF-Ga (50. mu.M) were mixed in a volume of 200. mu.L. The whole body fluorescence image is collected before injection and 30min, 60min, 90min and 120min after injection.

Whole-body fluorescence images at the indicated time points were acquired using the IVIS luminea XR III imaging system using a 660nm excitation filter and a 710nm emission filter. Each experiment was performed in three mice. Quantitative analysis of fluorescence intensity was performed on a region of interest (ROI) at the tumor site using Living Image Software (4.5.2, PerkinElmer, MA, u.s.a).

As shown in FIG. 8, P-CyFF-Ga can lighten the fluorescence of Cy-Cl at the tumor site and distinguish it from normal tissue, and the tumor has stronger signal relative to the background as the probe in the normal tissue is cleared, so that the outline of the tumor can be divided. And P-CyFF-activated single injection⁶⁸Mouse (I) tumor fluorescence and background of Ga (200. mu. Ci) were nearly identical due to P-CyFF-⁶⁸Ga (200. mu. Ci) concentrations are too low to effectively fluoroimage tumors. When Na is injected into tumor₃VO₄After inhibiting tumor activity, 200. mu.L of injection containing P-CyFF-Ga (50. mu.M) and P-CyFF-⁶⁸The fluorescent signal of the tumor site of the mixed solution of Ga (200 mu Ci) is obviously inhibited, which proves that the fluorescent signal of the cold compound probe at the tumor site is activated by the ALP of the tumor site, and the probe can perform fluorescence imaging detection on the ALP activity of the tumor at the living body level.

14. PET imaging of mouse tumor ALP activity

Two mice were scanned in pairs by an Inveon Dedicated micro-PET scanner. When the scanner starts scanning, injecting different substances through the tail vein, specifically I, injecting P-CyFF-⁶⁸Ga (200. mu. Ci) 200. mu.L; II tail vein injection of P-CyFF-⁶⁸Ga (200 mu Ci) and P-CyFF-Ga (50 mu M) mixed solution is 200 mu L; III intratumoral injection of Na₃VO₄After inhibiting the tumor ALP activity for 15 minutes, P-CyFF-one injection is injected into the tail vein⁶⁸Ga (200. mu. Ci) and P-CyFF-Ga (50. mu.M) were mixed in a volume of 200. mu.L. Throughout the experiment, mice were anesthetized with oxygen containing 2% isoflurane at a flow rate of 1.5L/min. Real-time and dynamic images were processed and acquired using specialized image processing software (ASIPro, siemens), and the first hour of dynamic images was reconstructed and divided into twelve frames. Then, static images were acquired for 90 minutes and 120 minutes, respectively. The ROI was further analyzed and normalized according to the injected dose. The uptake value of the tissue is expressed as a percentage of injected dose per cubic centimeter of tissue (% ID/mL).

Three groups of mice were also PET imaged simultaneously with tumor over time, as shown in FIG. 9, containing P-CyFF-Ga (50. mu.M) and P-CyFF-⁶⁸After the mixed solution of Ga (100 mu Ci) is injected into a mouse body, the probe can generate obvious PET signals at a tumor part and stay in the tumor for a long time, so that the subcutaneous HeLa tumor of the mouse is distinguished from the surrounding normal tissues. Injection of P-CyFF-⁶⁸The PET signal of the mouse tumor of Ga (200 mu Ci) is obviously reduced due to the injected labeled probe P-CyFF-⁶⁸The concentration of Ga is about 1 μ M, which is lower than the CMC of the probe itself, so that in situ self-assembly at the tumor site cannot occur, thereby reducing accumulation at the tumor site. When Na is injected into tumor₃VO₄After tumor activity was inhibited, the PET signal at the tumor site was also significantly decreased, indicating that accumulation of radioactive signal at the tumor site is caused by ALP. The above experimental results show that P-CyFF-⁶⁸Ga can be dephosphorylated under the action of tumor endogenous ALP, and is self-assembled to form nanoparticles at the tumor site under the help of high-concentration P-CyFF-Ga, so that the stay and accumulation of the probe in the tumor are enhanced, and finally the probe can be usedALP positive tumors were detected by bimodal imaging with fluorescence and PET.

The invention provides an alkaline phosphatase response bimodal probe P-CyFF-⁶⁸While Ga, its preparation and application, and many methods and ways for implementing the technical solution have been described above, it should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered as the protection scope of the present invention. All the components not specified in the present embodiment can be realized by the prior art.

Claims

1. A structure shown as a formula P-CyFF-Ga;

2. such as P-CyFF-⁶⁸A structure represented by Ga;

3. the process for producing P-CyFF-Ga according to claim 1, which comprises the steps of:

step a: compound mCy-NH₂Reacting with the compound 1 to obtain a compound 2;

step e: chelating a compound P-CyFF-NODA with Ga to obtain a compound P-CyFF-Ga;

4. the bimodal molecular imaging probe P-CyFF-⁶⁸A method for producing Ga, characterized in that an acidic Ga-containing solution⁶⁸Ga³⁺The solution is incubated with P-CyFF-NODA to obtain the P-CyFF-containing-⁶⁸A solution of Ga.

5. The P-CyFF-Ga of claim 1 and/or the bimodal molecular imaging probe P-CyFF-Ga of claim 2⁶⁸Application of Ga in preparing a contrast agent for tumor diagnosis.

6. The use according to claim 5, wherein said tumor diagnostic contrast agent is an alkaline phosphatase positive tumor contrast agent.

7. The P-CyFF-Ga of claim 1 and/or the bimodal molecular imaging probe P-CyFF-Ga of claim 2⁶⁸Application of Ga in tumor diagnosis fluorescence imaging.

8. The use according to claim 7, wherein said tumor diagnostic fluorescence imaging is alkaline phosphatase positive tumor diagnostic fluorescence imaging.

9. The use of claim 7, wherein said tumor diagnostic fluorescence imaging is alkaline phosphatase positive tumor diagnostic fluorescence/PET bimodal imaging.

10. The use as claimed in any one of claims 5 to 9, wherein the P-CyFF-Ga and the bimodal molecular imaging probe P-CyFF-⁶⁸Ga utilizes enzyme-mediated self-assembly and is applied to targeted bimodal imaging of tumors.