A glutathione-resistant dual protease-responsive gold nanoparticle fluorescent probe and a preparation method thereof

Abstract

The application discloses a glutathione-resistant dual-protease-responsive gold nanoparticle fluorescent probe and a preparation method thereof, and belongs to the technical field of nanobiomaterials and tumor molecular diagnosis. Two alkynylated fluorescently labeled peptide chains which are respectively specific and sensitive to HNE and MMP-1 are co-incubated with gold nanoparticles under alkaline conditions to form a dual-protease-responsive fluorescent nanoparticle probe through gold-alkyne bond self-assembly; the structure of the probe is (X) n -gold nanoparticle-(Y) m X is an MMP-1 substrate peptide with an amino acid sequence as shown in SEQ ID NO. 1, Y is an HNE substrate peptide with an amino acid sequence as shown in SEQ ID NO. 2, the N-termini of the MMP-1 substrate peptide and the HNE substrate peptide are respectively labeled with a first fluorophore and a second fluorophore, and the C-termini are modified by alkylation and covalently connected to the surface of the gold nanoparticles through an Au-C=C bond; the probe utilizes dual-channel fluorophores which are quenched due to being close to the gold nanoparticles, and when target enzymes exist, the peptide chains are cut to release the fluorophores to restore fluorescence, so that NSCLC cells and normal cells can be accurately distinguished.

Description

Technical Field

[0001] This invention belongs to the field of nanobiomaterials and tumor molecular diagnostics technology, specifically relating to a dual protease-responsive fluorescent nanoprobe based on a gold-acetylene bond (Au-C≡C) interface, its preparation method, and its application in cell and tissue viability imaging and early stratified diagnosis of non-small cell lung cancer (NSCLC). Background Technology

[0002] Early diagnosis of non-small cell lung cancer (NSCLC) is crucial for improving patient survival. Current imaging and tissue biopsy methods have limited sensitivity and specificity for detecting small, early-stage lesions, with approximately 70% of patients diagnosed at an advanced stage. Tumor development and progression are often accompanied by abnormally high expression and enhanced activity of specific proteases. For example, matrix metalloproteinase-1 (MMP-1) and human neutrophil elastase (HNE) are significantly upregulated in the early stages of NSCLC and are involved in extracellular matrix degradation and inflammatory microenvironment remodeling, respectively. Co-expression of these two proteases is highly correlated with tumor invasiveness.

[0003] However, existing detection strategies based on single protease biomarkers have the following drawbacks: 1) Insufficient specificity: Single biomarkers can be elevated even in benign lesions or under individual differences, easily leading to false positives. 2) Intracellular environment interference: Traditional gold-thiol (Au-S) bond-based nanoprobes are prone to ligand substitution in cytoplasm with high concentrations of biothiols (such as glutathione, GSH), resulting in leakage of fluorescence background and signal distortion. 3) Lack of logical discrimination ability: A single signal cannot reflect the functional state of multi-enzyme synergy, making it difficult to achieve accurate phenotypic stratification.

[0004] Therefore, there is an urgent need to develop a nanoprobe that can resist intracellular thiol interference, enable simultaneous detection of multiple enzyme activities and perform logical interpretation, so as to improve the accuracy and specificity of early diagnosis of NSCLC. Summary of the Invention

[0005] This invention provides a dual-protease-responsive gold nanoparticle fluorescent probe against glutathione and its preparation method. The dual-protease-responsive gold nanoparticle fluorescent probe uses gold nanoparticles (Au NPs) as the core and covalently immobilizes two fluorescently labeled enzyme substrate peptides through a stable Au-C≡C interface, which respectively respond to MMP-1 and HNE activities, and achieves accurate differentiation between NSCLC cells and normal cells based on dual-channel fluorescence signals.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, the present invention provides a dual-protease-responsive fluorescent nanoprobe that resists glutathione, wherein the structure of the dual-protease-responsive fluorescent nanoprobe is (X). n -Gold nanoparticles-(Y) mX is the MMP-1 substrate peptide with the amino acid sequence shown in SEQ ID NO. 1, and Y is the HNE substrate peptide with the amino acid sequence shown in SEQ ID NO. 2. The N-terminus of the MMP-1 substrate peptide and the HNE substrate peptide are labeled with a first fluorophore and a second fluorophore, respectively. The C-terminus of both peptides is modified with alkyne and then covalently linked to the surface of gold nanoparticles through Au-C≡C bonds.

[0007] The fluorophore is quenched due to proximity to the gold nanoparticles, and fluorescence is restored when the target enzyme cleaves the peptide chain to release the fluorophore.

[0008] Preferably, the sequence of SEQ ID NO. 1 is VPMSMRGG; the sequence of SEQ ID NO. 2 is QPMAVVQSVPQK.

[0009] Preferably, the first fluorophore is fluorescein isothiocyanate (FITC), and the second fluorophore is 5-carboxytetramethylrhodamine (5-TAMRA).

[0010] Preferably, the average diameter of the gold nanoparticles is 10-20 nm.

[0011] Preferably, the average diameter of the gold nanoparticles is 15-20 nm.

[0012] Preferably, n and m represent the total number of peptide molecules loaded on the surface of each gold nanoparticle, where 500≤n≤600 and 500≤m≤600.

[0013] Secondly, the present invention provides a method for preparing the above-mentioned dual-protease responsive fluorescent nanoprobe, comprising: preparing gold nanoparticles by reducing chloroauric acid with sodium citrate; modifying the terminal carboxyl groups of the MMP-1 substrate peptide labeled with the first fluorophore and the HNE substrate peptide labeled with the second fluorophore by alkyne modification; and co-incubating the two alkyne-modified substrate peptides with gold nanoparticles under alkaline conditions to form a probe by self-assembly of gold-alkyne bonds.

[0014] Preferably, the alkynylation modification includes dissolving the MMP-1 substrate peptide labeled with a first fluorophore and the HNE substrate peptide labeled with a second fluorophore in ethanol, adding equimolar amounts of EDC and NHS to activate the carboxyl group, and then reacting with propargylamine in the dark for no less than 24 hours, followed by dialysis purification and lyophilization; the molar ratio of the MMP-1 substrate peptide, HNE substrate peptide, EDC, NHS and propargylamine is 1:1:63.3:63.3:0.75. Preferably, the self-assembly includes: taking a gold nanoparticle solution, adding NaOH to adjust the pH, adding two alkydinated peptide chain solutions in a certain proportion, and incubating in the dark for no less than 36 hours. After centrifugation and washing, a dual-peptide modified nanoprobe is obtained; the molar ratio of the gold nanoparticles, alkydinated MMP-1 substrate peptide, and alkydinated HNE substrate peptide is 1:1036:128.

[0015] Thirdly, the present invention provides the application of the above-mentioned dual-protease-responsive fluorescent nanoprobe in the preparation of reagents or kits for detecting non-small cell lung cancer.

[0016] Fourthly, the present invention provides a method for identifying non-small cell lung cancer cells for non-diagnostic purposes, comprising: incubating the cells to be tested with the above-mentioned dual protease-responsive fluorescent nanoprobes; detecting the fluorescence signals of the first fluorophore and the second fluorophore; and determining that the cells have NSCLC malignant characteristics if both channels are higher than a preset threshold.

[0017] Preferably, the cells to be tested are derived from cell suspension, tissue homogenate, or body fluid.

[0018] Preferably, the first fluorophore is FITC, and the second fluorophore is 5-TAMRA. The fluorescence intensity of the FITC channel is detected at a wavelength of 494 nm excitation and 518 nm emission, and the fluorescence intensity of the 5-TAMRA channel is detected at a wavelength of 554 nm excitation and 585 nm emission. If the fluorescence intensity of both channels is significantly higher than that of the negative control (such as normal cells or buffer background), it is judged as double enzyme positive, indicating that the sample has the malignant characteristics of NSCLC. If either or both channels are negative, it is judged as a non-malignant phenotype. Further, the double enzyme activity index (e.g., normalized fluorescence intensity product or weighted sum) can be calculated for quantitative stratification.

[0019] Preferably, the incubation time is 1-5 hours; the fluorescence signal is detected by a confocal microscope or a fluorescence spectrophotometer.

[0020] Preferably, the incubation conditions are 37±2℃ and pH 6.2~8.0.

[0021] The fluorescence quenching mechanism of this invention is as follows: the fluorophore on the intact peptide chain is quenched by fluorescence resonance energy transfer (FRET) due to its proximity to the surface of the gold nanoparticle, and the fluorescence is in the "off" state; when the corresponding protease specifically cleaves the peptide chain, the fluorophore is released away from the gold surface, and the fluorescence returns to the "on" state.

[0022] Compared with existing technologies, the beneficial effects of this solution are: High sensitivity and specificity: The dual-protease responsive gold nanoprobe prepared in this invention ensures that it responds only to the target enzyme through dual substrate sequence design, with a detection limit as low as 10 ng / mL, which is significantly better than the traditional ELISA method.

[0023] Excellent intracellular stability: The innovative Au-C≡C linkage completely resists ligand substitution by millimoles of glutathione, ensuring the authenticity of intracellular signals and solving the core defects of traditional Au-S probes.

[0024] Intelligent logic diagnostic capability: Through dual-channel AND gate criterion, only cells with high MMP-1 and HNE activity are judged as positive, which greatly reduces the false positives that may be caused by a single marker and realizes functional activity stratification of NSCLC cells.

[0025] Simple and quick to operate: In situ detection in live cells can be achieved with only one incubation step, without the need for cumbersome washing or secondary amplification steps.

[0026] With broad application prospects, this platform technology is highly scalable. By changing the peptide sequence, it can be used to detect protease combinations related to other diseases, providing a new tool for the precise diagnosis of tumors and other diseases. Attached Figure Description

[0027] Figure 1 Characterization images of gold nanoparticles (A is a transmission electron microscope (TEM) image of gold nanoparticles reduced by citric acid, B is dynamic light scattering (DLS) data of uncoated gold nanoparticles, and C is a zeta potential map of gold nanoparticles). Figure 2 Characterization diagrams of the dual protease-responsive nanoprobe (A is a transmission electron microscope image of the nanoprobe, B is the dynamic light scattering data of the nanoprobe, and C is the zeta potential diagram of the nanoprobe). Figure 3 Fluorescence calibration and reporter group loading analysis of dual protease-responsive gold nanoparticle fluorescent probes (A is the standard fluorescence calibration curve of FITC-labeled peptide, B is the standard fluorescence calibration curve of 5-TAMRA-labeled peptide, C is the FITC fluorescence intensity released after competitive replacement from the nanoprobe, and D is the 5-TAMRA fluorescence intensity released after competitive replacement from the nanoprobe). Figure 4 To demonstrate the resistance of dual-protease-responsive gold nanoparticle fluorescent probes to temperature changes (A represents the relationship between the thermal stability of the FITC channel and temperature, and B represents the relationship between the thermal stability of the 5-TAMRA channel and temperature). Figure 5To demonstrate the resistance of the dual-protease-responsive gold nanoparticle fluorescent probe to glutathione-induced signal leakage (A shows the FITC fluorescence of the nanoparticle over time in the presence of glutathione, B shows the 5-TAMRA fluorescence of the nanoparticle over time in the presence of glutathione, C shows the relationship between the FITC fluorescence response of the nanoparticle and the glutathione concentration, and D shows the relationship between the 5-TAMRA fluorescence response of the nanoparticle and the glutathione concentration). Figure 6 The enzyme-responsive fluorescence kinetics and quantitative sensing performance of the dual-protease-responsive gold nanoparticle fluorescent probe are shown in Figure 1 (A is the time-dependent FITC fluorescence response curve of the nanoparticle probe to MMP-1, B is the time-dependent 5-TAMRA fluorescence response curve of the nanoparticle probe to HNE, C is the calibration curve of FITC fluorescence intensity versus MMP-1 concentration (0-800 ng / mL), and D is the calibration curve of 5-TAMRA fluorescence intensity versus HNE concentration (0-1000 ng / mL). Figure 7 To assess the stability and selectivity of the dual-channel sensing platform (A: FITC fluorescence recovery of MMP-1 by the nanoprobe under different pH conditions; B: 5-TAMRA fluorescence recovery of HNE by the nanoprobe under different pH conditions; C: FITC channel specificity for MMP-1 in the presence of potential interfering substances; D: 5-TAMRA channel specificity for HNE in the presence of potential interfering substances). Figure 8 To assess the biocompatibility and intracellular uptake behavior of the dual-protease-responsive gold nanoparticle fluorescent probe, A shows the cytotoxicity of gold nanoparticles and nanoprobes in BEAS-2B cells as determined by the MTT assay; B shows the cytotoxicity of gold nanoparticles and nanoprobes in A549 cells as determined by the MTT assay; C shows the quantitative analysis of total cellular fluorescence intensity in A549 cells after different incubation times; and D shows the time-dependent confocal fluorescence image of A549 cells after incubation with the nanoprobe (scale bar = 100 μm). Figure 9 To analyze the activity of MMP-1 / HNE in NSCLC-related and non-malignant cell models using dual-channel intracellular detection technology (A: confocal fluorescence images of BEAS-2B cells, A549 cells, and NEM-pretreated A549 cells after co-incubation with nanoprobes; B: quantitative analysis of total cellular fluorescence intensity in different cell groups; scale bar = 100 μm, *p<0.05, **p<0.01, ***p<0.001); Figure 10This is a proof-of-concept study of orthogonal sensing technology in a tumor-related in vivo environment (A is a schematic diagram of a mouse right subcutaneous NSCLC tumor model, B is a representative in vivo fluorescence image at different time points after local application of nanoprobes, C is the quantitative FITC fluorescence intensity, corresponding to in vivo MMP-1 responsive activation, and D is the quantitative 5-TAMRA fluorescence intensity, corresponding to in vivo HNE responsive activation). Detailed Implementation

[0028] To facilitate understanding of the present invention by those skilled in the art, the technical solution of the present invention will be further described in detail below with reference to embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific application examples. It should be understood that the specific application examples described herein are merely illustrative and not intended to limit the invention.

[0032] Example 1: Preparation of gold nanoparticles (Au NPs) 70 mL of ultrapure water was heated to a vigorous boil, and 700 μL of 20 mg / mL tetrachloroauric acid trihydrate (HAuCl4·3H2O) solution was added. After boiling again, 3.5 mL of 1% (w / v) trisodium citrate solution was quickly added, and the solution color turned wine red within minutes. Boiling was continued for 20 minutes, followed by cooling to room temperature (10–30 °C) to obtain a gold nanoparticle sol, which was stored at 4 °C for later use. Transmission electron microscopy (TEM) confirmed uniform particle size, and dynamic light scattering (DLS) determined the hydrated particle size to be approximately 17.90 nm, with a Zeta potential of approximately -34.6 mV (see [link to TEM]). Figure 1 ).

[0033] Example 2: Alkylation modification of fluorescently labeled peptides Weigh out fluorescein isothiocyanate (FITC)-labeled MMP-1 substrate peptide (sequence: Ac-FITC-VPMSMRGG-COOH) and 5-TAMRA-labeled HNE substrate peptide (sequence: Ac-5-TAMRA-QPMAVVQSVPQK-COOH) with a purity ≥95%. Dissolve each in anhydrous ethanol at a molar ratio of HNE substrate peptide to MMP-1 substrate peptide of 1:1. Add equimolar amounts of EDC and NHS to achieve a molar ratio of HNE substrate peptide to EDC of 1:63.3. Activate the carboxyl groups at room temperature for 30 minutes. Then add 1.89 × 10⁻⁶ ppm of ethanol. −5 % (w / v) of propargylamine was used to make the molar ratio of HNE substrate peptide to propargylamine 1:0.75, and the reaction was carried out in the dark for 24 hours. The reaction solution was dialyzed in pure water in the dark for 48 hours using a dialysis bag with a molecular weight cutoff of 1 kDa. Both MMP-1 substrate peptide and HNE substrate peptide were successfully alkynylated and stored at -20°C.

[0034] Example 3: Assembly of dual protease-responsive nanoprobes 4 mL of the 3 nM gold nanoparticle solution prepared in Example 1 was placed in a glass bottle, and 120 μL of 0.1 M NaOH solution was added and mixed well. Then, 2.84 mL of the dialyzed alkynylated HNE substrate peptide solution from Example 2 and 0.35 mL of the alkynylated MMP-1 substrate peptide solution were added, making the molar ratio of gold nanoparticles, alkynylated MMP-1 substrate peptide, and alkynylated HNE substrate peptide 1:1036:128. The mixture was incubated at room temperature in the dark for 36 hours. After the reaction, the mixture was centrifuged at 13,000 rpm for 20 minutes, the supernatant was discarded, and the precipitate was washed three times with ultrapure water. Finally, it was resuspended in 10 mM HEPES buffer (pH 7.4) and stored at 4 °C in the dark. The gold concentration was determined to be approximately 1 nM (based on gold nanoparticles) by UV-Vis spectroscopy at 520 nm.

[0035] Example 4: Characterization of Nanoprobes In this embodiment, TEM, DLS, Zeta potential, and UV-Vis spectroscopy were used to characterize the morphology and size of the dual protease-responsive nanoprobe prepared in Example 3.

[0036] TEM showed that the peptide-modified nanoprobe particles remained spherical with no obvious aggregation. DLS measurements indicated that the hydrated particle size increased to approximately 21.46 nm. The Zeta potential changed to -38.3 mV, indicating enhanced electronegativity and good colloidal stability (see...). Figure 2 UV-Vis spectroscopy showed that the surface plasmon resonance peak of the nanoprobe redshifted from 518 nm to 520 nm.

[0037] In this embodiment, the peptide loading was further determined using the selenocysteine ​​(10 mM) competitive substitution method.

[0038] The 1 nM probe was incubated with 10 mM selenocysteine ​​for 12 hours, and the supernatant was collected by centrifugation. The fluorescence intensities of FITC (excitation 494 nm / emission 518 nm) and 5-TAMRA (excitation 554 nm / emission 585 nm) were measured and calculated using the results from the standard curve. The results showed that each gold nanoparticle was loaded with an average of approximately 359 ± 2 FITC-peptides and 215 ± 2 5-TAMRA-peptides (see [link to standard curve]). Figure 3 ).

[0039] Example 5: Stability Test This embodiment first tests the thermal stability of the dual-protease responsive nanoprobe prepared in Example 3. A 1 nM probe solution was heated at 30-80 °C for 5 minutes, and after cooling, the fluorescence intensity of FITC and 5-TAMRA was measured. The thermal stability was evaluated by comparing the changes in fluorescence intensity at different temperatures (see [link to example]). Figure 4 ).

[0040] The results showed that the fluorescence intensity at each temperature fluctuated by less than 10% compared to that at 30℃, indicating that the probe has excellent thermal stability.

[0041] Subsequently, the resistance of the nanoprobe to glutathione-induced (GSH) signal leakage was investigated to prepare for a biothiol resistance test. First, 1 nM of the probe was co-incubated with 5 mM GSH at 37°C for 0, 1, 2, 4, 8, and 12 hours, and the fluorescence intensity was measured. The time dependence was assessed by comparing the changes in fluorescence intensity at different incubation times. Then, 1 nM of the probe was co-incubated with 0–5000 μM GSH for 12 hours, and the dual-channel fluorescence intensity was measured. The concentration dependence was assessed by comparing the changes in fluorescence intensity at different GSH concentrations.

[0042] The results showed that the fluorescence intensity of the dual channels at each time point was comparable to the initial background value, with no increasing trend over time. Even with a GSH concentration as high as 5 mM, the fluorescence intensity was only 1.6 times the initial background, far lower than the leakage typically observed with traditional Au-S probes, which is several to tens of times lower (see...). Figure 5 ).

[0043] The above results demonstrate that the Au-C≡C bond in the probe has excellent resistance to biothiol substitution.

[0044] Example 6: In vitro enzyme response performance test This embodiment tests the in vitro enzyme response performance of the dual protease-responsive nanoprobe prepared in Example 3. All the following tests were performed at 37°C in 10mM HEPES buffer (pH 7.4).

[0045] Reaction kinetics The 1 nM probe was incubated with 800 ng / mL activated MMP-1 or 1000 ng / mL HNE, and the fluorescence of the corresponding channel was recorded every 10 minutes.

[0046] The results showed that the FITC signal plateaued at approximately 70 minutes, and the 5-TAMRA signal plateaued at approximately 60 minutes. Therefore, the incubation time for subsequent experiments was uniformly set at 70 minutes (see...). Figure 6 (A and B in the text).

[0047] Sensitivity and detection limit The 1 nM probe was incubated with enzyme solutions of different concentrations for 70 minutes, and a standard curve of fluorescence intensity versus enzyme concentration was plotted.

[0048] The results showed that MMP-1 exhibited good linearity in the range of 0-800 ng / mL, with a linear equation F = 0.32 × [MMP-1] + 19.12 and R² = 0.993. The limit of detection (LOD) calculated using a signal-to-noise ratio of 3 was 8.03 ng / mL. HNE also showed good linearity in the range of 0-1000 ng / mL, with a linear equation F = 0.18 × [HNE] + 43.54 and R² = 0.993. The LOD was 9.44 ng / mL (see [link to relevant documentation]). Figure 6 (C and D in the text).

[0049] pH adaptability The 1 nM probe was incubated with the target enzyme solution (MMP-1 nanoparticles or HNE nanoparticles) under different pH conditions for 70 minutes. The fluorescence intensity was recorded with the control of no target enzyme solution (no MMP-1 nanoparticles or no HNE nanoparticles).

[0050] The results showed that the background fluorescence was extremely low in the absence of the enzyme at pH 6.2, 6.6, 7.0, 7.4, 7.8, and 8.0; the fluorescence enhancement amplitude was basically consistent after the addition of the target enzyme, indicating that the probe works stably within the physiological pH range (see...). Figure 7 (A and B in the text).

[0051] Specificity test The 1 nM probe was incubated with PSA, Caspase-3, MMP-2, MMP-12, 10% FBS (Serum), Ca²⁺, Zn²⁺, Na⁺, 5 mM GSH, 100 μM H₂O₂, HNE, and MMP1, respectively, and a blank control was set up.

[0052] The results showed that fluorescence in the corresponding channel was significantly enhanced only when the target enzyme (MMP-1 or HNE) was present; fluorescence changes caused by other interfering substances were not significantly different from those in the blank control. Figure 7 (B and D in the text).

[0053] Example 7: Functional Validation at the Cell Level This embodiment verifies the function of the dual protease-responsive nanoprobe prepared in Example 3 at the cellular level.

[0054] Cell culture Human non-small cell lung cancer cell line A549 and normal human lung epithelial cell line BEAS-2B were cultured in DMEM medium containing 10% FBS at 37°C and 5% CO2.

[0055] Cytotoxicity assay (MTT method) The two cell types were treated with either 0-3 nM probes or bare gold nanoparticles for 12 hours. The results showed that the cell viability in each concentration group was >90%, indicating that the probes prepared in this invention have good biocompatibility (see...). Figure 8 (A and B in the text).

[0056] Cell uptake and optimal imaging time A549 cells were co-incubated with a 1 nM probe for 1, 2, 3, 4, and 5 hours, washed with PBS, and then subjected to confocal imaging.

[0057] The results showed that fluorescence intensity increased over time, reaching a peak at 4 hours, with no significant enhancement thereafter. Four hours was determined as the standard incubation time (see...). Figure 8 (C and D in the text).

[0058] Differentiating between cancer cells and normal cells using imaging A549 and BEAS-2B cells were incubated with a 1 nM probe for 4 hours before imaging.

[0059] The results showed that A549 cells exhibited bright intracellular fluorescence in both the FITC (green) and 5-TAMRA (red) channels; while BEAS-2B cells showed extremely weak fluorescence in both channels. Quantitative analysis showed that the fluorescence intensity of A549 cells in both channels was significantly higher than that of BEAS-2B (p<0.001) (see...). Figure 9 ).

[0060] Thiol Interference Control Experiment A549 cells were pretreated with 200 μM N-ethylmaleimide (NEM, a thiol blocking agent) for 30 minutes before being incubated with the probe, with the untreated group serving as a control.

[0061] The results showed no significant difference in fluorescence intensity between the NEM-treated and untreated groups, confirming that the intracellular activation of the probe was entirely due to protease cleavage rather than thiol replacement (see [link to study]). Figure 9 ).

[0062] Example 8: Feasibility Verification of In Vivo Imaging This embodiment verifies the feasibility of in vivo imaging using the dual protease-responsive nanoprobe prepared in Example 3 through animal experiments.

[0063] Building animal models BALB / c female mice were subcutaneously injected with 1×10⁻⁶ spores under the right axilla. 6 Each Lewis lung cancer cell (LLC) forms a 5-7 mm tumor in about one week.

[0064] Drug delivery and imaging Mice were randomly divided into two groups of five each. The experimental group received 100 μL of PBS solution containing a probe with a fluorescence equivalent of 200 nM directly injected into the tumor of the model mice; the control group received 100 μL of PBS solution containing a probe with a fluorescence equivalent of 200 nM directly injected into the corresponding site in normal mice. Dual-channel fluorescence images were acquired using a small animal in vivo imaging system before injection and at 2, 4, 8, 12, 24, and 48 hours after injection.

[0065] The results showed that in the experimental group, both FITC and 5-TAMRA dual-channel fluorescence rapidly increased to peak levels 2 hours after injection, then gradually weakened over time, returning to near background levels by 48 hours. In the control group, only weak fluorescence was observed at the injection site, with no specific enhancement. These results indicate that the probe can specifically respond to MMP-1 and HNE activity and generate detectable fluorescence signals in in vivo tumor tissue, and can be metabolically cleared by the body (see...). Figure 10 ).

[0066] In summary, the dual-protease-responsive gold nanoprobe prepared in this invention exhibits high peptide loading efficiency, excellent colloidal stability, and strong resistance to glutathione interference under physiological conditions. Within the clinically relevant concentration range, the probe shows sensitive and linear fluorescence responses to both MMP-1 and HNE, with low detection limits. Live-cell imaging revealed strong dual-channel fluorescence activation in A549 non-small cell lung cancer cells, while fluorescence was negligible in BEAS-2B normal epithelial cells. The dual-enzyme activity index clearly distinguishes between malignant and normal phenotypes. In a mouse subcutaneous tumor model, rapid fluorescence activation appeared in the tumor area after local administration, followed by gradual clearance. This indicates that the probe provides a robust platform for functional activity imaging and activity-based stratification of non-small cell lung cancer. The combination of a stable gold-acetylene nanobiointerface and multiple protease sensing offers a promising strategy for improving the specificity of early lung cancer diagnosis and holds promise for application in other activity-based cancer imaging.

[0067] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A dual-protease-responsive fluorescent nanoprobe that inhibits glutathione, characterized in that, The structure of the dual-protease responsive fluorescent nanoprobe is (X). n -Gold nanoparticles-(Y) m X is an MMP-1 substrate peptide with an amino acid sequence as shown in SEQ ID NO. 1, and Y is an HNE substrate peptide with an amino acid sequence as shown in SEQ ID NO.

2. The N-terminus of the MMP-1 substrate peptide and the HNE substrate peptide are labeled with a first fluorophore and a second fluorophore, respectively. The C-terminus of both peptides is modified with alkyne and then covalently linked to the surface of gold nanoparticles through Au-C≡C bonds.

2. The dual-protease-responsive fluorescent nanoprobe for inhibiting glutathione according to claim 1, characterized in that, The first fluorophore is FITC, and the second fluorophore is 5-TAMRA.

3. The dual-protease-responsive fluorescent nanoprobe for inhibiting glutathione according to claim 1, characterized in that, The gold nanoparticles have an average diameter of 10-20 nm.

4. The dual-protease-responsive fluorescent nanoprobe for inhibiting glutathione according to claim 1, characterized in that, The n and m represent the total number of peptide molecules loaded on the surface of each gold nanoparticle, where 500≤n≤600 and 500≤m≤600.

5. The method for preparing the dual-protease-responsive fluorescent nanoprobe according to any one of claims 1-4, characterized in that, include: Gold nanoparticles were prepared by reducing chloroauric acid with sodium citrate. The terminal carboxyl groups of the MMP-1 substrate peptide labeled with the first fluorophore and the HNE substrate peptide labeled with the second fluorophore were modified with alkynylation. The two alkynylated substrate peptides were co-incubated with gold nanoparticles under alkaline conditions, and probes were formed by self-assembly through gold-alkynyl bonds.

6. The method according to claim 5, characterized in that, The alkyne modification involves dissolving the MMP-1 substrate peptide labeled with a first fluorophore and the HNE substrate peptide labeled with a second fluorophore in ethanol, adding equimolar amounts of EDC and NHS to activate the carboxyl group, and then reacting with propargylamine in the dark for at least 24 hours, followed by dialysis purification and lyophilization; the molar ratio of the MMP-1 substrate peptide, HNE substrate peptide, EDC, NHS and propargylamine is 1:1:63.3:63.3:0.

75.

7. The method according to claim 5, characterized in that, The self-assembly includes: taking a gold nanoparticle solution, adding NaOH to adjust the pH, adding two alkyd peptide chain solutions in proportion, and incubating in the dark for no less than 36 hours; obtaining a double peptide modified nanoprobe after centrifugation and washing; the molar ratio of the gold nanoparticles, alkyd MMP-1 substrate peptide and alkyd HNE substrate peptide is 1:1036:

128.

8. The use of the dual protease-responsive fluorescent nanoprobe according to any one of claims 1-4 in the preparation of reagents or kits for detecting non-small cell lung cancer.

9. A method for identifying non-small cell lung cancer cells for non-diagnostic purposes, characterized in that, include: The cells to be tested are incubated with the dual protease-responsive fluorescent nanoprobe according to any one of claims 1-4; the fluorescence signals of the first fluorophore and the second fluorophore are detected; If both channels have signals higher than the preset threshold, the cell is determined to have malignant characteristics of NSCLC.

10. The method according to claim 9, characterized in that, The incubation was carried out at 37±2℃ and pH 6.2~8.0 for 1-5 hours; the fluorescence signal was detected by confocal microscopy or fluorescence spectrophotometer.

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