Preparation method and application of new ferroferric oxide fluorescent nanoprobe

Abstract

The present invention relates to a preparation method and application of a novel ferroferric oxide fluorescent nanoprobe, belonging to the field of biological probes. The present invention uses biocompatible ferroferric oxide nanoparticles (Fe3O4) as a carrier, modifies the surface with PEG, and couples the surface with a polypeptide substrate specifically recognized by matrix metalloproteinase-2 (MMP-2) labeled with fluorescein isothiocyanate (FITC) to prepare a novel MMP-2-specific response nanomolecular probe Fe3O4-PEG-KCPLGVR-FITC. The probe has good biocompatibility, high stability, high specificity, high detection sensitivity, and a strong fluorescent signal. It can be used for the early, accurate, and rapid diagnosis of MMP-2-overexpressing tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of biological probes, and in particular relates to a preparation method and application of a novel ferroferric oxide fluorescent nanoprobe. Background Art

[0002] Malignant tumors are among the most serious threats to human health. Since the five-year relative survival rates for cancer patients treated in the early and late stages vary significantly, early diagnosis of tumors is crucial for cancer treatment. Matrix metalloproteinases (MMPs) are proteinases that hydrolyze the extracellular matrix and play a critical role in tumor growth, invasion, metastasis, and angiogenesis. Therefore, MMPs are considered important biomarkers for early tumor diagnosis. The development of MMP-based probe materials for early diagnosis and treatment of malignant tumors has been a hot topic among scientists.

[0003] Currently, due to their small size, large specific surface area, superparamagnetism, and low toxicity, ferroferric oxide nanoparticles have been used in a variety of research fields, including medicine, biology, and materials science. However, due to the lack of sufficient active groups on the surface of ferroferric oxide nanoparticles, they are prone to coagulation in most solvents and have a certain degree of chemical inertness. Modifying the surface of ferroferric oxide nanoparticles can improve the dispersibility of the particles in solvents and increase their biocompatibility, thereby enabling their use in biomedicine and targeted formulations. At the same time, literature reports that when fluorescent molecules are attached to the surface of metal nanoparticles, they cause a significant fluorescence quenching effect on fluorescent dyes. Therefore, fluorescent "light-up" nanoprobes are gradually being applied in the fields of biological detection, sensing, and bioimaging research.

[0004] New probe materials based on tumor targets are the key to achieving accurate and rapid early diagnosis of tumors. Currently, there is an urgent need for a targeted probe molecule with good biocompatibility, high stability, high specificity, high detection sensitivity and strong fluorescence signal. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a new type of ferroferric oxide fluorescent nanoprobe. The prepared probe has good biocompatibility, high stability, high specificity, high detection sensitivity and strong fluorescent signal. The present invention also provides the application of the probe in the early diagnosis of tumors.

[0006] The preparation method of the novel ferroferric oxide fluorescent nanoprobe of the present invention comprises the following steps:

[0007] (1) reacting azidopropylamine with fluorescein isothiocyanate FITC-NCS to prepare fluorescein isothiocyanate FITC-N3;

[0008] (2) Under the catalysis of cuprous ions, fluorescein azidoisothiocyanate undergoes a click chemistry reaction with the terminal alkyne-modified peptide substrate KCPLGVR, which is specifically recognized by matrix metalloproteinase-2 (MMP-2), to obtain the peptide molecule KCPLGVR-FITC;

[0009] (3) Fe3O4 nanoparticles were prepared by coprecipitation, and dp-PEG-Mal was then added to the surface of the Fe3O4 nanoparticles to obtain Fe3O4-PEG-Mal nanoparticles;

[0010] (4) The thiol group at the end of the polypeptide molecule KCPLGVR-FITC reacts with the maleimide on the surface of the nanoparticle Fe3O4-PEG-Mal to obtain the probe Fe3O4-PEG-KCPLGVR-FITC.

[0011] The novel ferroferric oxide fluorescent nanoprobe of the present invention can be used to prepare tumor diagnostic reagents.

[0012] The probe can be used for early diagnosis of MMP-2 overexpressing tumor cells, and preferably can be used for MMP-2 enzyme activity detection and cell fluorescence imaging of MMP-2 overexpressing tumor cells.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] 1. The probe of the present invention has good biocompatibility and high stability. Surface modification with polyethylene glycol can increase the density of polar functional groups and improve the water solubility and stability of ferrosoferric oxide nanoparticles in aqueous media.

[0015] 2. The probe of the present invention has high specificity, high detection sensitivity, and a strong fluorescence signal. Biocompatible ferrosoferric oxide nanoparticles, surface-modified with PEG, are then coupled to a fluorescein isothiocyanate-labeled peptide substrate specifically for MMP-2. The probe exhibits strong detection sensitivity and specificity for MMP-2, and also demonstrates excellent cell fluorescence imaging with a strong fluorescence signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 , specific detection of MMP-2 enzyme by Fe3O4-PEG-KCPLGVR-FITC;

[0017] Figure 2 , the synthetic route of KCPLGVR-FITC;

[0018] Figure 3 , H NMR spectrum of FITC-N3;

[0019] Figure 4, FITC-N3 NMR spectrum;

[0020] Figure 5 , high-resolution mass spectrum of FITC-N3;

[0021] Figure 6 , HPLC spectrum of KCPLGVR-FITC;

[0022] Figure 7 , mass spectrum of KCPLGVR-FITC;

[0023] Figure 8 , UV absorption spectra of KCPLGVR-FITC, Fe3O4-PEG-Mal and Fe3O4-PEG-KCPLGVR-FITC;

[0024] Figure 9 , hydrated particle size of Fe3O4-PEG-Mal and Fe3O4-PEG-KCPLGVR-FITC;

[0025] Figure 10 , Zeta potential of Fe3O4-PEG-Mal and Fe3O4-PEG-KCPLGVR-FITC;

[0026] Figure 11 , TEM images of Fe3O4-PEG-Mal and Fe3O4-PEG-KCPLGVR-FITC;

[0027] Figure 12 , Fluorescence spectra of the same probe under different concentrations of MMP-2, where [probe] = 100 μg·mL -1 , [MMP-2] = 320 ng·mL -1 , [inhibitor] = 0.1 mg·mL -1 ;

[0028] Figure 13 , fluorescence spectra of the probe itself, and competition experiments between the probe and MMP-2 and inhibitors, where [probe] = 100 μg mL -1 , [MMP-2] = 320 ng·mL -1 , [inhibitor] = 0.1 mg·mL -1 ;

[0029] Figure 14 , cell fluorescence imaging, where 3T3: 3T3 + probe, MCF-7: MCF-7 + probe, MCF-7 + Inhibitor: MCF-7 + inhibitor + probe, [probe] = 50 μg mL -1 , [inhibitor] = 0.1 mg·mL -1, scale bar = 60 μm. DETAILED DESCRIPTION

[0030] The present invention will be further described below with reference to the following examples. Unless otherwise specified, all raw materials used in the examples are commercially available.

[0031] Example

[0032] The preparation of the novel ferroferric oxide fluorescent nanoprobe described in the present invention uses the following reagents and instruments:

[0033] Ferric chloride (FeCl3), ferrous sulfate heptahydrate (FeSO4·7H2O), sodium hydroxide (NaOH), propylamine azide, triethylamine (TEA), KCPLGVR polypeptide, anhydrous copper sulfate (CuSO4), sodium ascorbate, polyethylene glycol (dp-PEG-Mal, MW=2000), fluorescein isothiocyanate (FITC-NCS), ilomastat (GM6001), recombinant matrix metalloproteinase-2 (MMP-2), DMEM medium, fetal bovine serum, penicillin / streptomycin antibiotics, Hoechst 33342, mouse fibroblasts 3T3, and human breast cancer cells MCF-7.

[0034] Agilent 1260 high performance liquid chromatograph, Shimadzu UV-3600 ultraviolet spectrophotometer, Malvern Nano ZS90 particle size potentiometer, Bruker ultrafleXtreme matrix-assisted laser desorption / time-of-flight mass spectrometer, Edinburgh FLS980 fluorescence spectrometer, FEI Tecnai G2 Spirit transmission electron microscope, and Olympus FV1200 laser confocal microscope.

[0035] The preparation method of the novel ferroferric oxide fluorescent nanoprobe of the present invention comprises the following steps:

[0036] (1) Azide propylamine and fluorescein isothiocyanate FITC-NCS are reacted to prepare fluorescein isothiocyanate FITC-N3:

[0037] like Figure 2 Triethylamine (TEA, 9.8 mg, 0.096 mmol) and azidopropylamine (9.6 mg, 0.096 mmol) were added dropwise to a 1 mL DMF solution of fluorescein isothiocyanate (FITC-NCS, 28 mg, 0.072 mmol) and stirred at room temperature in the dark for reaction. The reaction was followed by HPLC until completion, followed by solvent removal, concentration, and column chromatography separation and purification to obtain a yellow powdery product, fluorescein azidoisothiocyanate (FITC-N3). The product was further confirmed by H NMR, C NMR, and high-resolution mass spectrometry. Figure 3 、 Figure 4 and Figure 5.

[0038] (2) Under the catalysis of cuprous ions, fluorescein azidoisothiocyanate undergoes a click chemistry reaction with the terminal alkyne-modified peptide substrate KCPLGVR, which is specifically recognized by matrix metalloproteinase-2 (MMP-2), to obtain the peptide molecule KCPLGVR-FITC:

[0039] KCPLGVR (21.8 mg, 0.024 mmol) and FITC-N3 (14.6 mg, 0.030 mmol) were reacted by "click" chemistry under the catalysis of sodium ascorbate (4.8 mg, 0.024 mmol) and anhydrous copper sulfate (CuSO4, 1.92 mg, 0.012 mmol) to obtain KCPLGVR-FITC, which was purified by HPLC and its structure was confirmed by HPLC spectrum and mass spectrometry, as shown in FIG. Figure 6 and Figure 7 . Pale yellow solid, MALDI-TOF-MS, C 64 H 89 N 18 O 14 S2[M+H]+, experimental value (calculated value) m / z: 1397.366 (1397.625).

[0040] (3) Ferrous oxide nanoparticles were prepared by coprecipitation method, and dp-PEG-Mal was then modified on the surface of the ferrous oxide nanoparticles to obtain Fe3O4-PEG-Mal nanoparticles:

[0041] Under nitrogen protection, ferric chloride solid (3.88 g, 0.0239 mol) and ferrous sulfate heptahydrate (3.34 g, 0.012 mol) were dissolved in 280 mL of ultrapure water, and stirred mechanically for 1 h to allow Fe 2+ and Fe 3+ After fully dissolving, add 68 mL of a 1.59 mol / L aqueous solution containing 4.32 g of NaOH dropwise until the pH of the solution is approximately 9, at which point a large amount of black precipitate will appear in the solution. Continue stirring for 3 hours to allow for complete precipitation. Separate with a magnet and wash with ultrapure water and then anhydrous ethanol until the precipitate is neutral. Finally, place in a vacuum drying oven and dry at 70°C to obtain Fe3O4 particles. The concentration is calibrated using ICP-MS for later use.

[0042] Ferroferric oxide nanoparticles (160 μg·mL -1dp-PEG-Mal (MW = 2K, 5 mg, 0.5 mL) was added to a solution of Fe₃O₄ (1.0 mL) and sonicated for 15 minutes. The mixture was then stirred in the dark at room temperature for 8 hours. After the reaction, the mixture was centrifuged at 15,000 rpm for 15 minutes and then washed and purified twice to obtain Fe₃O₄-PEG-Mal. The properties and morphology of the modified Fe₃O₄ nanoparticles were characterized using UV-vis, DLS, Zeta potential, and TEM. The corresponding Zeta potential changed to 7.9 ± 0.8 mV, indicating that dp-PEG-Mal was successfully modified on the surface of the Fe₃O₄ nanoparticles.

[0043] (4) The thiol group at the end of the polypeptide molecule KCPLGVR-FITC reacts with the maleimide on the surface of the nanoparticle Fe3O4-PEG-Mal to obtain the probe Fe3O4-PEG-KCPLGVR-FITC:

[0044] PEG-modified Fe3O4 nanoparticles (160 μg·mL -1 KCPLGVR-FITC (50 mg mL -1 , 0.02mL) of DMSO solution, stirred at room temperature in the dark for 1 hour. After the reaction, centrifuged at 15000 rpm for 15 minutes and washed with ultrapure water for purification three times, and then redispersed with ultrapure water for later use. The properties and morphology of the reacted ferroferric oxide nanoparticles Fe3O4-PEG-KCPLGVR-FITC were characterized by DLS, Zeta potential, UV-vis and TEM. The Zeta potential rose to 15.8±0.2mV (as Figure 10 ), indicating that KCPLGVR-FITC was successfully connected to the surface of ferroferric oxide nanoparticles. Figure 8 The UV-visible absorption spectrum showed that the PEG-modified ferroferric oxide nanoparticles had no obvious UV absorption at 490 nm, but the probe Fe3O4-PEG-KCPLGVR-FITC connected with KCPLGVR-FITC exhibited certain UV absorption at 490 nm.

[0045] Table 1 and Figure 9 It was shown that the hydrated particle sizes of PEG-modified ferroferric oxide nanoparticles and the probe Fe3O4-PEG-KCPLGVR-FITC were not much different, both of which were about 45 nm, and TEM results showed (e.g. Figure 11 ), the ferroferric oxide nanoparticles are uniform in size, well dispersed, and have little size variation.

[0046] Table 1 Hydrated particle size and zeta potential of Fe3O4-PEG-Mal and Fe3O4-PEG-KCPLGVR-FITC

[0047]

[0048] The prepared new Fe3O4 fluorescent nanoprobe was tested in vitro and the activity of tumor cells was studied:

[0049] 1. In vitro testing of the sensitivity and specificity of the probe for MMP-2

[0050] Probe Fe3O4-PEG-KCPLGVR-FITC (100 μg mL -1 ) at 37°C with different concentrations of MMP-2 (0, 10, 20, 40, 80, 160, 320, 640 ng·mL -1 )TBS solution (pH=7.2) for 2 hours, and the changes in fluorescence signals in different solutions were detected by fluorescence spectrometer. -1 ) in the presence of Fe3O4-PEG-KCPLGVR-FITC (100 μg·mL -1 ) and MMP-2 (320 ng·mL -1 ) were incubated at 37°C for 2 hours, and then the fluorescence intensity of the corresponding solutions was measured using a fluorescence spectrometer.

[0051] 2. Cell fluorescence imaging

[0052] Mouse fibroblast 3T3 and human breast cancer cell MCF-7 were treated with 50 μg·mL -1 The probe was incubated at 5% CO2 and 37°C for 8 hours. At the same time, 0.1 mg mL -1 Competition experiments were conducted with the MMP-2 inhibitor GM6001, and finally, cell fluorescence imaging was observed using a laser confocal microscope.

[0053] 3. Results and Analysis

[0054] (1) In vitro testing of the sensitivity and specificity of the probe for MMP-2

[0055] like Figure 12 It shows that the same concentration of Fe3O4-PEG-KCPLGVR-FITC (100 μg·mL -1 ) will increase with the increase of MMP-2 dosage. When the MMP-2 dosage reaches a certain concentration (320 ng·mL -1 ), the fluorescence intensity no longer increases significantly, indicating that the probe has mostly reacted with MMP-2 and has a strong detection sensitivity for MMP-2. Figure 13 When the probe reacted with MMP-2, the fluorescence intensity of the solution at 518 nm was significantly enhanced compared to the probe's own background, with an increase of up to 21 times. However, the fluorescence intensity of the solution to which the inhibitor had been added in advance was suppressed, with an increase of only 5 times, indicating that the probe has a certain specificity for MMP-2.

[0056] The above experimental results show that the probe has strong detection sensitivity and good specificity for MMP-2 and can be used for the detection of MMP-2 enzyme activity.

[0057] (2) Cell fluorescence imaging

[0058] Subsequently, tumor cell imaging studies were conducted. Mouse fibroblasts 3T3 and human breast cancer cells MCF-7 were incubated with the probe for 8 hours, and then the cells were imaged using a laser confocal microscope. Figure 14 As shown, MCF-7 cells exhibited a strong green fluorescence signal, and the fluorescence signal could be effectively inhibited by the MMP-2 inhibitor, while only a weak fluorescence signal was detected in 3T3 cells, indicating that the probe can be used for sensitive and specific imaging of MMP-2 overexpressing tumor cells.

[0059] 4. Conclusion

[0060] A new MMP-2-specific fluorescent nanoprobe was designed and synthesized based on ferroferric oxide nanoparticles and a peptide substrate specifically designed to recognize matrix metalloproteinase MMP-2. This probe exhibits strong sensitivity and specificity for MMP-2 in vitro and can be successfully applied to image MMP-2-overexpressing tumor cells, providing important insights and insights for the early, rapid, and accurate diagnosis of MMP-2-overexpressing tumors.

Claims

1. A method for preparing a novel ferroferric oxide fluorescent nanoprobe, characterized in that: The following steps are involved: (1) reacting azidopropylamine with fluorescein isothiocyanate FITC-NCS to prepare fluorescein isothiocyanate FITC-N3; (2) Under the catalysis of cuprous ions, fluorescein azidoisothiocyanate undergoes a click chemistry reaction with the terminal alkyne-modified peptide substrate KCPLGVR, which is specifically recognized by matrix metalloproteinase-2, to obtain the peptide molecule KCPLGVR-FITC; (3) Fe3O4 nanoparticles were prepared by coprecipitation, and dp-PEG-Mal was then added to the surface of the Fe3O4 nanoparticles to obtain Fe3O4-PEG-Mal nanoparticles; (4) The thiol group at the end of the polypeptide molecule KCPLGVR-FITC reacts with the maleimide on the surface of the nanoparticle Fe3O4-PEG-Mal to obtain the probe Fe3O4-PEG-KCPLGVR-FITC.

2. An application of the novel ferroferric oxide fluorescent nanoprobe according to claim 1, characterized in that: The probe can be used to prepare tumor diagnostic reagents.

Images