Method for accurately diagnosing early-stage lung cancer by using two-photon near-infrared fluorescent probe targeted lipid droplets

Abstract

The invention discloses a novel method based on a two-photon near-infrared fluorescent probe, which realizes sensitive monitoring of early lung cancer by detecting dynamic changes of lipid droplets in tumor cells and tissues. Comprising the following steps: clinical sample collection, laser microdissection, mass spectrometry detection and proteomics analysis, lipid droplet molecule analysis and detection, cell culture and preliminary verification, chemical structure design of a fluorescent probe, characteristic verification of the fluorescent probe, cellular level verification, animal model experiment and clinical sample verification. The fluorescent probe disclosed by the invention not only can distinguish tumor tissues from normal tissues by specifically detecting the change of the LDs, but also can further identify the type of early-stage lung cancer which is shown as a solid or ground glass-like focus in imaging. Meanwhile, development of the probe brings brand-new tool and method support for clinical diagnosis of early-stage lung cancer, and the diagnosis precision and the success rate of early-stage treatment are expected to be remarkably improved.

Description

Technical Field

[0001] The present invention relates to the technical field of early lung cancer diagnosis, and in particular to a method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe to target fat droplets. Background Art

[0002] Lung cancer is one of the leading causes of cancer-related deaths worldwide. Despite significant advances in treatment in recent years, the five-year survival rate is still less than 20%. Epidemiological studies have shown that the survival rate of patients with early lung cancer has significantly improved, especially for stage 1A lung cancer, with a survival rate of more than 70%. The widespread use of low-dose computed tomography (CT) has greatly improved the detection rate of early lung cancer. In chest CT scans, lung adenocarcinoma (LUAD) can be divided into ground-glass nodular lung adenocarcinoma (GGN-ADC) and solid nodular lung adenocarcinoma (SN-ADC). Clinical studies have shown that SN-ADC is more aggressive and grows faster than GGN-ADC. From a pathological point of view, SN-ADC usually presents as micropapillary or solid structure, with a low degree of differentiation, and at least 20% of the tumor volume is solid. In view of these differences, it is urgent to study more accurate diagnostic methods and technologies from both theoretical and clinical levels to achieve early detection of lung cancer.

[0003] Tumor metabolic reprogramming is an important feature of cancer cells. Lipid droplets (LDs) are key organelles for storing metabolic lipids in cells and play an important role in tumor development. Studies have found that increased activity of sterol regulatory element binding protein 1 (SREBP-1) can promote tumor cell growth and invasion, leading to significant accumulation of lipid droplets in tumor cells. In addition, high expression of cluster of differentiation 36 (CD36) enhances the uptake of exogenous fatty acids, further promoting the accumulation of lipid droplets. As tumor cells proliferate, these lipid droplets provide additional energy for their metabolic needs. Therefore, changes in lipid droplet levels can be used as potential biomarkers for early diagnosis and prognosis of tumors. Fluorescent probe technology has become an important tool for studying organelle function due to its advantages in visualization, sensitivity, and specificity. Two-photon fluorescent probes have multiple advantages over single-photon methods, including reduced photodamage, increased tissue penetration depth, and reduced background fluorescence. These characteristics make them more suitable for detecting changes in lipid droplet polarity in deep tissues. In addition, near-infrared fluorescent probes have shown the potential to detect biomarkers in vivo. Probes that combine two-photon and near-infrared properties can significantly improve the ability to detect changes in lipid droplet polarity, thereby better monitoring physiological and pathological processes. However, current research on the use of highly sensitive fluorescent probes to visualize and monitor lipid droplet changes in early lung cancer is still limited, and new monitoring methods are urgently needed. Summary of the invention

[0004] 1. Technical issues to be resolved

[0005] In view of the shortcomings of the existing technology, the present invention develops a rapid and effective tool for the diagnosis of early lung cancer. The proposed method provides deeper insights into monitoring the behavior of LDs in tumor cells and has potential clinical applications.

[0006] (II) Technical solution

[0007] To achieve the above objectives, the present invention provides the following technical solutions: a method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe targeting lipid droplets, which achieves sensitive monitoring of early lung cancer by detecting the dynamic changes of lipid droplets in tumor cells and tissues, comprising the following steps:

[0008] S1. Clinical sample collection: Clinical samples were collected from lung cancer centers, including normal lung tissue and cancerous lung tissue of patients with early lung cancer. The samples were pathologically confirmed to include two subtypes: solid lung adenocarcinoma and ground-glass lung adenocarcinoma.

[0009] S2. Laser microdissection: Laser microdissection is used to separate malignant cells and tumor areas from tissue samples, providing an accurate sample source for subsequent analysis;

[0010] S3, mass spectrometry detection and proteomic analysis: extract tumor tissue proteins, use mass spectrometry technology to analyze the differential proteins and signaling pathways between the two cancer subtypes, and combine bioinformatics methods to analyze the expression of key molecules;

[0011] S4. Lipid droplet molecular analysis and detection: Western blotting and scanning electron microscopy were used to detect the expression levels of lipid droplet-related molecules, and the distribution and aggregation characteristics of lipid droplets in solid lung adenocarcinoma and ground-glass lung adenocarcinoma were compared;

[0012] S5. Cell culture and preliminary validation: Normal epithelial cells and lung cancer cell lines were cultured in a 5% CO2 cell culture incubator at 37°C, and the expression differences of lipid droplet-related molecules in the two were detected;

[0013] S6. Chemical structure design of fluorescent probe: A two-photon fluorescent probe targeting lipid droplet polarity change was designed based on cyanomethylene-benzopyran and N,N-dimethylaniline. The chemical structure of the probe was confirmed by nuclear magnetic resonance and high-resolution mass spectrometry, and its fluorescence properties in different solvents and environmental conditions were further analyzed.

[0014] S7. Verification of the characteristics of fluorescent probes: Detect the fluorescence properties of fluorescent probes: Analyze the weak fluorescence intensity of fluorescent probes in oil and water and under high viscosity conditions; Analyze the photophysical properties of fluorescent probes in various solvents with different polarities, including toluene (TOL), 1,4-dioxane (DIO), ethyl acetate (EA), THF and acetone; Analyze the changes in fluorescence intensity of fluorescent probes at different concentrations in neutral, acidic and alkaline environments;

[0015] S8. Cell-level validation: Add fluorescent probes to normal epithelial cells and lung cancer cell lines, and verify their sensitivity and specificity for detecting early lung cancer through changes in fluorescence intensity;

[0016] S9. Animal model experiments: Establish subcutaneous and tail vein tumor mouse models to evaluate the distribution of fluorescent probes in tumor tissues and the intensity of fluorescent signals, and analyze their safety and background signals in other tissues and organs;

[0017] S10. Clinical sample verification: For clinical samples of different types of early lung cancer, detect the changes in the signal intensity of the fluorescent probe in lung cancer tissue to further confirm its potential in actual clinical applications.

[0018] As a preferred solution, after the sample tissue is collected in S1, the tumor tissue is immediately cooled to 4°C and rinsed with isotonic saline, and then each sample is divided into two parts: one part for histological staining and the other part for mass spectrometry and proteomics analysis.

[0019] As a preferred embodiment, the laser microdissection in S2 includes using a slicer to cut the formalin-fixed paraffin-embedded (FFPE) specimen into 10uM thickness and fix it on a Leica pen film slide (2uM), then the slide is dewaxed with xylene, rehydrated through different gradients of ethanol and water, and stained with hematoxylin; after staining, the slices are dehydrated again through different concentrations of alcohol and xylene, and the malignant and normal areas of the tumor are selected using Leica's LMD 6500 laser microdissection system and collected in a 0.5 ml test tube; the total area collected is 1-5×10 6 mm 2 , equivalent to approximately 5,000 to 25,000 cells.

[0020] As a preferred solution, the specific process of mass spectrometry analysis in S3 is as follows: first, a 15 L volume of peptides is injected and pre-concentrated on a trapping column with 0.1% TFA for 7 minutes (Acclaim ProMap 100, 300 m x 5 mm, C18, 5 m, 100; flow rate 30 L / min), and then they are separated using a 250 min HPLC gradient from 2% to 60% at a flow rate of 200 nL / min in buffer B (80% acetonitrile, 0.5% formic acid); using 70000 m / z Survey scans were performed at a resolution of 200 (60,000 for Q accurate HF) and the scan range was set from 300 to 1650 m / z; the three most abundant MS1 features (charge > 2) were selected for high-energy collisional dissociation fragmentation at a resolution of 17,500 m / z (15,000 for Q accurate HF), dynamic exclusion of sequenced peptides was set to 45 s, ion injection time and ion target value were set to 20 ms and 3 × 106 for survey scans, and ion injection time and ion target value were set to 220 ms and 1 × 10^5 for MS / MS scans, respectively; data acquisition was managed using Xcalibur software (ThermoScientific).

[0021] As a preferred embodiment, in S5, normal epithelial cells and lung cancer cell lines are cultured in a 5% CO2 cell culture incubator at 37°C. After 24 hours, the culture medium is removed and the cells are fixed with pre-cooled acetone for 20 minutes; the cells are washed three times with PBS, and then incubated with different concentrations of lipid droplet probes (0, 0.5uM, 1uM, 5uM, 10uM, 20uM) for 1 hour; after incubation, the cells are washed with PBS for 5 minutes and stained with DAPI for 10 minutes; finally, 20 μL is applied to a cell slide, the edges are sealed with nail polish, and fluorescence imaging is performed using a multiphoton laser scanning confocal microscope.

[0022] As a preferred solution, the cell level verification specifically includes the following steps:

[0023] Step 1: The cytotoxicity of lipid droplet probes in normal epithelial cells and lung cancer cells was detected at different concentrations and time intervals;

[0024] Step 2: Minimal cytotoxicity was observed, indicating the excellent biocompatibility of the lipid droplet probe;

[0025] Step 2: A549 cells were stained with lipid droplet probe and a commercially available LD-specific dye, LD-Tracker Dark Green. The LD-probe and LD-Tracker Dark Green showed excellent co-localization.

[0026] As a preferred solution, the S9 animal model experiment specifically includes:

[0027] To establish the mouse tumor model, a total of 2×10 6 A549 cells were injected subcutaneously into the right flank of 5-week-old nude mice, and imaging was performed once tumors reached approximately 1 cubic centimeter in size;

[0028] For fluorescence measurements in the mouse model, LD-probes at a concentration of 100 μM were administered intratumorally at the tumor edge or via the tail vein. In vivo imaging was performed 30 minutes after injection using a PerkinElmer IVIS Spectrum with an emission wavelength of 719 nm. After imaging, mice were euthanized, and tumors as well as major organs (lungs, liver, kidneys, spleen, and heart) were quickly collected for ex vivo imaging.

[0029] Fluorescence measurement in lung cancer tissues. First, frozen tissues from SN-ADC, GGN ADC, and adjacent normal tissues were embedded with OCT embedding medium and cut into 10-20 micron slices using a microtome; these slices were fixed on gelatin / potassium dichromate-coated slides and stored at -80°C. The slices were then stained with different concentrations of lipid droplet probes (0.5uM and 1uM) for 30 minutes and with DAPI (1g / ml) for 10 minutes, and fluorescence imaging was performed using a Nikon Ni-E multiphoton laser scanning confocal microscope with an emission wavelength of 719nm.

[0030] As a preferred solution, the S10 for clinical sample verification specifically includes:

[0031] Sample collection, five and six SN–ADC and GGN–ADC samples were collected, respectively, and hematoxylin and eosin (H&E) staining was performed to confirm the diagnosis of LUAD and identify the malignant tumor area;

[0032] In the synthesis of lipid droplet probes, dicyanomethylenebenzopyran and N, N-dimethylaniline were selected as the classic electron-withdrawing group and strong electron-donating group, respectively;

[0033] The spectral response of lipid droplet probe to polarity was used to evaluate the fluorescence performance of lipid droplet probe, which confirmed the typical ICT effect and high sensitivity to polarity.

[0034] Subcellular co-localization experiments were performed to evaluate the specificity of lipid droplet probes in living cells based on their superior performance;

[0035] Visualization of the fluorescence changes of lipid droplet probes in lung cancer cell lines. A549 and HBE cells were treated with lipid droplet probes at concentrations of 0.5uM and 1uM, confirming the specificity and robust targeting ability of lipid droplet probes to LDs.

[0036] Visualization of lipid droplet probe fluorescence changes in mouse tumor models, where lipid droplet probes (100uM) were directly administered into subcutaneous tumors in mice, including direct injection of lipid droplet probes (100uM) into normal mice and mice bearing tumors;

[0037] Visualization of lipid droplet probe fluorescence changes in early lung adenocarcinoma. Tissues of SN-ADC, GGN-ADC, and adjacent normal lung tissue (normal) were further stained. The tissues were cut into 10-micron sections and incubated with lipid droplet probes.

[0038] (III) Beneficial effects

[0039] Compared with the prior art, the present invention provides a method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe targeting lipid droplets, which has the following beneficial effects:

[0040] The present invention has developed a multi-mode two-photon fluorescent probe for monitoring LD changes in early lung cancer, which enables accurate diagnosis of various types of early lung cancer. The probe effectively distinguishes tumor cells from normal cells and accurately distinguishes tumors from normal tissues in tumor-bearing mouse models. In addition, this fluorescent probe can not only distinguish tumors from normal tissues by specifically detecting LDs changes, but also distinguish SN-ADC from GGN-ADC at an early stage, and accurately identify the pathological type of early lung cancer by targeting the changes in LDs in tumor cells and tissues. This breakthrough provides crucial theoretical support and innovative diagnostic tools for the clinical identification of early lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 Schematic diagram of the experimental design of SN-ADC samples of the present invention;

[0042] Figure 2 Schematic diagram of CT images and H&E staining images of SN-ADC and GGN ADC of the present invention;

[0043] Figure 3 Schematic diagram of the fluorescence performance of the lipid droplet probe of the present invention Figure 1 ;

[0044] Figure 4 Schematic diagram of the fluorescence performance of the lipid droplet probe of the present invention Figure 2 ;

[0045] Figure 5 Schematic diagram of the distribution of LDs in epithelial cells and various lung cancer cell lines of the present invention;

[0046] Figure 6 Schematic diagram of the excellent biocompatibility of the lipid droplet probe of the present invention Figure 1 ;

[0047] Figure 7 Schematic diagram of the excellent biocompatibility of the lipid droplet probe of the present invention Figure 2 ;

[0048] Figure 8 This is a schematic diagram of the co-localization image of the LD-probe and LD-Tracker of the present invention;

[0049] Fig. 9 This is a visualization diagram of the fluorescence changes of the lipid droplet probe of the present invention;

[0050] Fig.10 A schematic diagram of LD formed by the lipid droplet probe of the present invention;

[0051] Fig.11 This is a visualization diagram of the fluorescence changes of the lipid droplet probe in the mouse tumor model of the present invention;

[0052] Fig.12 Schematic diagram of fluorescence imaging of tumor-bearing mice of the present invention;

[0053] Fig.13 This is a visualization diagram of the fluorescence changes of the lipid droplet probe in early lung adenocarcinoma of the present invention;

[0054] Fig.14 1HNMR spectrum schematic diagram of the LD-probe of the compound of the present invention;

[0055] Fig.15 13CNMR spectrum schematic diagram of the LD-probe of the compound of the present invention;

[0056] Fig.16 Schematic diagram of the HRMS spectrum of the composite laser probe of the present invention;

[0057] Fig.17 This is a schematic diagram of the process of diagnosing early lung cancer according to the present invention. DETAILED DESCRIPTION

[0058] In order to better understand the purpose, structure and function of the present invention, the method of the present invention for accurately diagnosing early lung cancer by targeting lipid droplets with a two-photon near-infrared fluorescent probe will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0059] Clinically, solid lung adenocarcinoma (SN-ADC) grows faster and is more invasive than ground glass adenocarcinoma (GGN-ADC). Metabolic reprogramming is a key feature of tumor cells and may promote the rapid progression of early lung cancer. Here, the present invention used laser microdissection technology to accurately separate the malignant regions from five SN-ADC and six GGN-ADC samples for proteomic analysis. Bioinformatics analysis, Western blotting, and electron microscopy performed in this study revealed significant upregulation of lipid droplet-associated protein (PLIN2) and a significant increase in lipid droplet (LD) accumulation in SN-ADC tissues. Therefore, a new two-photon near-infrared fluorescent probe was developed to monitor the dynamic changes of LDs in lung cancer cells. In addition, experimental validation confirmed the effectiveness of the lipid droplet probe (LD-probe) in monitoring LD changes in tumor cells and distinguishing LD differences between tumor and normal tissues. In particular, the probe successfully detected significant differences in LD behavior between SN-ADC and GGN-ADC in early lung cancer. Therefore, the present invention has developed a rapid and effective tool for the diagnosis of early lung cancer. The proposed method provides deeper insights into monitoring the behavior of LDs in tumor cells and has potential clinical applications.

[0060] This study used laser microdissection (LMD) to precisely select malignant tumor tissues from primary lung cancer samples, including five SN-ADCs and six GGN-ADCs (Scheme 1). Proteomic sequencing and analysis showed a significant increase in the expression of perilipin-2 (PLIN2) in SN-ADCs, accompanied by a large number of LDs. To facilitate the detection of LD levels, a fluorescent probe was developed, featuring the electron-withdrawing properties of a dicyanomethylenebenzopyran structure and N,N-dimethylaniline as an electron donor group. The results showed that the lipid droplet probe effectively monitored the changes in LDs using lung cancer cells and mouse tumor models. In addition, LD-probe was able to distinguish SN-ADCs from GGN-ADCs in early lung cancer. By targeting the polarity changes of LDs, this study introduces a new tool for identifying different types of lung cancer at an early stage, thereby providing important clinical utility.

[0061] Example 1

[0062] Specifically, Figure 1-17 As shown, the present invention provides a method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe to target lipid droplets, which realizes sensitive monitoring of early lung cancer by detecting the dynamic changes of lipid droplets in tumor cells and tissues, and includes the following steps:

[0063] S1. Clinical sample collection: Clinical samples were collected from lung cancer centers, including normal lung tissue and cancerous lung tissue of patients with early lung cancer. The samples were pathologically confirmed to include two subtypes: solid lung adenocarcinoma and ground-glass lung adenocarcinoma.

[0064] S2. Laser microdissection: Laser microdissection is used to separate malignant cells and tumor areas from tissue samples, providing an accurate sample source for subsequent analysis;

[0065] S3, mass spectrometry detection and proteomic analysis: extract tumor tissue proteins, use mass spectrometry technology to analyze the differential proteins and signaling pathways between the two cancer subtypes, and combine bioinformatics methods to analyze the expression of key molecules;

[0066] S4. Lipid droplet molecular analysis and detection: Western blotting and scanning electron microscopy were used to detect the expression levels of lipid droplet-related molecules, and the distribution and aggregation characteristics of lipid droplets in solid lung adenocarcinoma and ground-glass lung adenocarcinoma were compared;

[0067] S5. Cell culture and preliminary validation: Normal epithelial cells and lung cancer cell lines were cultured in a 5% CO2 cell culture incubator at 37°C, and the expression differences of lipid droplet-related molecules in the two were detected;

[0068] S6. Chemical structure design of fluorescent probe: A two-photon fluorescent probe targeting lipid droplet polarity change was designed based on cyanomethylene-benzopyran and N,N-dimethylaniline. The chemical structure of the probe was confirmed by nuclear magnetic resonance and high-resolution mass spectrometry, and its fluorescence properties in different solvents and environmental conditions were further analyzed.

[0069] S7. Verification of the characteristics of fluorescent probes: Detect the fluorescence properties of fluorescent probes: Analyze the weak fluorescence intensity of fluorescent probes in oil and water and under high viscosity conditions; Analyze the photophysical properties of fluorescent probes in various solvents with different polarities, including toluene (TOL), 1,4-dioxane (DIO), ethyl acetate (EA), THF and acetone; Analyze the changes in fluorescence intensity of fluorescent probes at different concentrations in neutral, acidic and alkaline environments;

[0070] S8. Cell-level validation: Add fluorescent probes to normal epithelial cells and lung cancer cell lines, and verify their sensitivity and specificity for detecting early lung cancer through changes in fluorescence intensity;

[0071] S9. Animal model experiments: Establish subcutaneous and tail vein tumor mouse models to evaluate the distribution of fluorescent probes in tumor tissues and the intensity of fluorescent signals, and analyze their safety and background signals in other tissues and organs;

[0072] S10. Clinical sample verification: For clinical samples of different types of early lung cancer, detect the changes in the signal intensity of the fluorescent probe in lung cancer tissue to further confirm its potential in actual clinical applications.

[0073] Specifically, the process is as follows: after the sample tissue is collected in S1 above, the tumor tissue is immediately cooled to 4°C and rinsed with isotonic saline, and then each sample is divided into two parts: one part is used for histological staining, and the other part is used for mass spectrometry and proteomics analysis;

[0074] Laser microdissection in S2 involves using a microtome to cut formalin-fixed paraffin-embedded (FFPE) specimens into 10 μM thickness and fix them on Leica pen film slides (2 μM). The slides are then dewaxed with xylene, rehydrated through different gradients of ethanol and water, and stained with hematoxylin. After staining, the sections are dehydrated again through different concentrations of alcohol and xylene, and the malignant and normal areas of the tumor are selected using Leica's LMD 6500 laser microdissection system and collected into 0.5 ml tubes. The total area collected is 1–5 × 10 6 mm 2 , equivalent to approximately 5000 to 25000 cells;

[0075] The specific process of mass spectrometry detection analysis in S3 was as follows: first, a 15-L volume of peptides was injected and pre-concentrated on a trapping column with 0.1% TFA for 7 min (Acclaim ProMap 100, 300 m x5 mm, C18, 5 m, 100; flow rate 30 L / min), and then they were separated using a 250-min HPLC gradient from 2% to 60% at a flow rate of 200 nL / min in buffer B (80% acetonitrile, 0.5% formic acid); using 70000 m / z . Survey scans were performed at a resolution of 200 (60,000 for Q accurate HF) and the scan range was set from 300 to 1650 m / z; the three most abundant MS1 features (charge > 2) were selected for high-energy collisional dissociation fragmentation at a resolution of 17,500 m / z (15,000 for Q accurate HF), dynamic exclusion of sequenced peptides was set to 45 s, ion injection time and ion target value were set to 20 ms and 3 × 106 for survey scans, and ion injection time and ion target value were set to 220 ms and 1 × 10^5 for MS / MS scans, respectively; data acquisition was managed using Xcalibur software (Thermo Scientific).

[0076] In S5, normal epithelial cells and lung cancer cell lines were cultured in a 5% CO2 cell culture incubator at 37°C. After 24 hours, the culture medium was removed and the cells were fixed with pre-cooled acetone for 20 minutes; the cells were washed three times with PBS and then incubated with different concentrations of lipid droplet probes (0, 0.5uM, 1uM, 5uM, 10uM, 20uM) for 1 hour; after incubation, the cells were washed with PBS for 5 minutes and stained with DAPI for 10 minutes; finally, 20μL was applied on a cell slide and the edges were sealed with nail polish, and fluorescence imaging was performed using a multiphoton laser scanning confocal microscope;

[0077] Furthermore, the cell-level verification specifically includes the following steps:

[0078] Step 1: The cytotoxicity of lipid droplet probes in normal epithelial cells and lung cancer cells was detected at different concentrations and time intervals;

[0079] Step 2: Minimal cytotoxicity was observed, indicating the excellent biocompatibility of the lipid droplet probe;

[0080] Step 2: A549 cells were stained with lipid droplet probe and a commercially available LD-specific dye, LD-Tracker Dark Green. The LD-probe and LD-Tracker Dark Green showed excellent co-localization.

[0081] Furthermore, its S9 animal model experiments specifically include:

[0082] To establish the mouse tumor model, a total of 2×10 6 A549 cells were injected subcutaneously into the right flank of 5-week-old nude mice, and imaging was performed once tumors reached approximately 1 cubic centimeter in size;

[0083] For fluorescence measurements in the mouse model, LD-probes at a concentration of 100 μM were administered intratumorally at the tumor edge or via the tail vein. In vivo imaging was performed 30 minutes after injection using a PerkinElmer IVIS Spectrum with an emission wavelength of 719 nm. After imaging, mice were euthanized, and tumors as well as major organs (lungs, liver, kidneys, spleen, and heart) were quickly collected for ex vivo imaging.

[0084] Fluorescence measurement in lung cancer tissues. First, frozen tissues from SN-ADC, GGN ADC, and adjacent normal tissues were embedded with OCT embedding medium and cut into 10-20 micron slices using a microtome; these slices were fixed on gelatin / potassium dichromate-coated slides and stored at -80°C. The slices were then stained with different concentrations of lipid droplet probes (0.5uM and 1uM) for 30 minutes and with DAPI (1g / ml) for 10 minutes, and fluorescence imaging was performed using a Nikon Ni-E multiphoton laser scanning confocal microscope with an emission wavelength of 719nm.

[0085] Its S10 for clinical sample verification specifically includes:

[0086] Sample collection, five and six SN–ADC and GGN–ADC samples were collected, respectively, and hematoxylin and eosin (H&E) staining was performed to confirm the diagnosis of LUAD and identify the malignant tumor area;

[0087] In the synthesis of lipid droplet probes, dicyanomethylenebenzopyran and N, N-dimethylaniline were selected as the classic electron-withdrawing group and strong electron-donating group, respectively;

[0088] The spectral response of lipid droplet probe to polarity was used to evaluate the fluorescence performance of lipid droplet probe, which confirmed the typical ICT effect and high sensitivity to polarity.

[0089] Subcellular co-localization experiments were performed to evaluate the specificity of lipid droplet probes in living cells based on their superior performance;

[0090] Visualization of the fluorescence changes of lipid droplet probes in lung cancer cell lines. A549 and HBE cells were treated with lipid droplet probes at concentrations of 0.5 μM and 1 μM, confirming the specificity and robust targeting ability of lipid droplet probes to LDs.

[0091] Visualization of lipid droplet probe fluorescence changes in mouse tumor models, where lipid droplet probes (100uM) were directly administered into subcutaneous tumors in mice, including direct injection of lipid droplet probes (100uM) into normal mice and mice bearing tumors;

[0092] Visualization of lipid droplet probe fluorescence changes in early lung adenocarcinoma. Tissues of SN-ADC, GGN-ADC, and adjacent normal lung tissue (normal) were further stained. The tissues were cut into 10-micron sections and incubated with lipid droplet probes.

[0093] Example 2

[0094] In order to better describe the method of the present invention, more specifically, the experimental process is as follows:

[0095] Collection and processing of samples from lung cancer patients: In this example, lung adenocarcinoma tissue specimens from 11 patients undergoing thoracic surgery were collected. All patients were pathologically diagnosed with early lung adenocarcinoma in situ. According to the CT imaging characteristics, the samples were divided into two groups: solid nodules (SN-ADC) and ground glass nodules (GGN-ADC), which provided a basis for further research on tumor subtypes. All patients signed written informed consent forms, and the obtained tumor tissues were cooled to 4°C immediately after surgery and rinsed with isotonic saline to remove surface impurities. Each sample was divided into two parts, one for histological staining to analyze the pathological type, and the other for proteomic analysis to study the expression differences of key molecules.

[0096] Sample processing and preliminary analysis: In this study, samples of 5 SN-ADC and 6 GGN-ADC were collected, and the diagnosis of lung adenocarcinoma was confirmed by hematoxylin-eosin (H&E) staining, and the malignant tumor area was identified ( Figure 1 A and Figure 2A–D). Laser microdissection: Formalin-fixed paraffin-embedded (FFPE) specimens were cut into 10-μm-thick sections using a microtome and mounted on slides coated with Leica membrane (2-μm thickness). The sections were dewaxed in xylene and rehydrated in different concentrations of alcohol and water, and the cytoskeleton was stained with hematoxylin. After staining, the sections were dehydrated again and treated with graded alcohol and xylene. Subsequently, the Leica LMD 6500 laser microdissection system was used to precisely separate the malignant and normal tissue areas, and the collected tissues were placed in 0.5-mL tubes for subsequent analysis.

[0097] After precisely extracting the malignant area from each specimen, protein levels were measured using quantitative mass spectrometry ( Figure 1 C). LC-MS / MS analysis: Samples were analyzed by liquid chromatography on an Ultimate 3000RSLCnano system coupled to an Orbitrap Elite mass spectrometer (ThermoScientific). 15 μL of peptide sample was injected into the trapping column and pre-concentrated in 0.1% trifluoroacetic acid (TFA) solution for 7 minutes (Acclaim ProMap 100, C18 column, flow rate 30 μL / min). Subsequently, gradient separation (2% to 60%) was completed in 250 minutes in an HPLC system with buffer B (80% acetonitrile, 0.5% formic acid) as the matrix using a flow rate of 200 nL / min. The mass spectrometer scan used a resolution of 70,000 m / z (60,000 m / z for QExactive HF) and a scan range of 300 to 1650 m / z. The three most abundant MS1 features with charge greater than 2 were selected for high-energy collisional dissociation fragmentation with a fragmentation resolution of 17,500 m / z (15,000 m / z for Q Exactive HF). The dynamic exclusion time was set to 45 s. The ion injection times were 20 ms for survey scans and 220 ms for MS / MS scans, respectively, and the ion target value was 3 × 10 6 and 1×10 5 Data acquisition was managed by Xcalibur software (Thermo Scientific).

[0098] HPLC separation: Tryptic peptides were separated by a high pH reversed phase HPLC system using a Thermo Betasil C18 column (5 μm particle diameter, 10 mm inner diameter, 250 mm column length). The peptide sample was separated in a gradient of 8% to 32% acetonitrile (pH 9.0) over 60 minutes to generate 60 fractions. Subsequently, these fractions were combined into 10 samples and dried by vacuum centrifugation for further mass spectrometry analysis.

[0099] Principal component analysis and heat map showed significant differences in protein expression patterns between SN-ADC and GGN-ADC ( Figure 1 D and Figure 1 E), these differentially expressed proteins are mainly involved in metabolic regulation and cell biological processes ( Figure 1 F).

[0100] Experimental design and data visualization: According to the experimental design, Figure 1 A shows the classification method of SN-ADC and GGN-ADC samples based on CT images and H&E staining. Figure 1 BC), laser microdissection was used to precisely label and collect protein fragments in the malignant area. These fragments were then digested with trypsin and analyzed by mass spectrometry. Figure 1 D shows the partial least squares discriminant analysis (PLS-DA) score plots of SN-ADC and GGN-ADC samples, revealing significant clustering differences between the two groups of samples and showing the explanatory differences of the two principal components of supervised PLS-DA.

[0101] Differential protein and function analysis: Figure 1 E shows the differentially expressed proteins through a heat map, among which the up-regulated proteins enriched in SN-ADC samples are mainly related to cell structure, biological process and molecular function ( Figure 1 F). Further analysis showed that the expression of lipid metabolism-related molecules was significantly upregulated in SN-ADC samples, and the results were verified by Western blotting ( Figure 1 HI). In addition, Figure 1 JK showed representative electron microscopy images of lipid droplet accumulation in normal tissue, GGN-ADC, and SN-ADC samples, further revealing potential differences in lipid metabolism among different subtypes of lung adenocarcinoma.

[0102] Through the above methods and analysis, this study systematically revealed the differences in molecular characteristics between SN-ADC and GGN-ADC, providing key data support for the precise diagnosis and targeted treatment of lung adenocarcinoma subtypes.

[0103] Lipid droplet molecular analysis and electron microscopy: In SN-ADC, the expression of lipid droplet-related molecules was significantly upregulated. Western blot analysis results showed that the expression levels of CD36, SREBP, FABP5 and PLIN2 in SN-ADC were significantly higher than those in GGN-ADC. For TEM analysis, cell or tissue samples were first fixed with 0.1 M cacodylate buffer (pH 7.4) containing 2.5% glutaraldehyde at 4 °C for 2 h, followed by washing and further fixation with 1% osmium tetroxide. The samples were dehydrated with a series of gradient ethanol solutions (30%, 50%, 70%, 85%, 100%). After dehydration, the samples were embedded in Epon resin LX112 and cut into ultrathin sections using Ultrotome III. Finally, the FEI Tecnai 10 transmission electron microscope (Philips) was used for observation at 80 keV, and the sections were fixed on electron microscope grids coated with non-porous formvar. Electron microscopy results showed that the number of lipid droplets in the cytoplasm of tumor cells in SN-ADC samples was significantly higher than that in GGN-ADC.

[0104] Cell culture and molecular detection: To investigate the molecular mechanisms controlling the role of lipid droplets in tumor cell proliferation, Western blot experiments were performed using normal epithelial cells and lung cancer cell lines. The accumulation of lipid droplets in tumor cells is controlled by complex regulatory mechanisms, and CD36, PLIN2, and SREBP are essential for lipid uptake and droplet formation, such as Fig. 9 As shown in D and 9E, the expression levels of CD36, SREBP, and PLIN2 were significantly increased in lung cancer cells compared with normal epithelial cells. In addition, electron microscopy confirmed the significant accumulation of lipid droplets in the cytoplasm of tumor cells compared with normal epithelial cells ( Fig. 9 F and 9G). Thus, these results emphasize the importance of lipid droplets for lipid metabolism and tumor cell proliferation.

[0105] Chemical structure design of fluorescent probe: In order to develop a highly sensitive fluorescent probe for detecting the distribution and dynamic changes of lipid droplets, this example selects dicyanomethylenebenzopyran and N,N-dimethylaniline as key electron-withdrawing and electron-donating groups. This strong electron push-pull structure triggers a significant intramolecular charge transfer effect (ICT), which shifts the optical properties of the probe to the near-infrared region. Generally, such molecules with ICT effect have good lipid droplet targeting in a lipophilic environment, and their large two-photon absorption cross-section also makes them suitable for deep tissue imaging. The chemical structure of the lipid droplet probe was confirmed by 1H NMR, 13C NMR and high-resolution mass spectrometry to ensure the accuracy of its molecular design and synthesis.

[0106] Verification of the physicochemical properties of the fluorescent probe: This example comprehensively evaluated the fluorescence performance of the probe. The experiment showed that the fluorescence intensity of the probe in an oil environment was much higher than that in an aqueous solution, indicating that it is significantly lipophilic. Under different viscosity conditions, the fluorescence performance of the probe varies. For example, in methanol (MeOH) and tetrahydrofuran (THF) with different polarities but similar viscosities, the probe exhibits stronger fluorescence in THF, indicating the dominant role of polarity in fluorescence changes. Further studies found that the maximum fluorescence emission wavelength of the probe significantly red-shifted from 625nm to 780nm, reflecting its high sensitivity to polarity changes. This property originates from the typical ICT effect in the molecular structure (Table 1, Figure 3 C).

[0107] Table 1

[0108]

[0109]

[0110] Photostability and environmental adaptability test of fluorescent probes: In the photostability test, the fluorescence intensity of the probe still showed stability even after continuous exposure to THF for 900 seconds, in contrast to its performance in PBS. This difference further demonstrates the excellent adaptability of the probe in oily environments. In addition, the fluorescence intensity of the probe at different concentrations was positively correlated, indicating that it has good concentration dependence. The experiment also verified that metal ions or small molecules have little effect on the fluorescence of the probe, except when the environmental polarity is significantly reduced, the probe will show an enhanced fluorescence response. Combining these characteristics, the lipid droplet probe exhibits good stability and high sensitivity, and can accurately monitor the dynamic changes of lipid droplets in biological systems, providing a solid foundation for further research on its application in lipid metabolism-related diseases.

[0111] Cell culture and fluorescence staining verification: In this experiment, we first evaluated the specificity of the lipid droplet probe and verified its superior performance in living cells. HBE, A549, NCI-H1299 and NCI-H1975 cells were cultured in DMEM medium containing 10% fetal bovine serum and maintained at 37°C, 5% CO2 for 24 hours. By analyzing the distribution of lipid droplets in these cell lines, it was found that the expression level of PLIN2 in A549 and NCI-H1299 cells was significantly increased, indicating that the concentration of lipid droplets in these cell lines was high. Figure 5 As shown, RT-PCR results showed that the mRNA levels of PLIN2 were different in HBE, A549, NCI-H1299, NCI-H1975, NCI-H1650, and NCI-H522 cells.

[0112] Next, the cytotoxicity of lipid droplet probes to normal epithelial cells (HBE) and lung cancer cells (A549, NCI-H1299, NCI-H1975) was evaluated at different concentrations and time. The experimental results showed that the LD probe had extremely low cytotoxicity, indicating its good biocompatibility (e.g. Figure 6 and Figure 7 To further verify the specificity of the lipid droplet probe, A549 cells were co-stained with the lipid droplet-specific dye LD-Tracker Dark Green. The results showed that the lipid droplet probe and LD-Tracker Dark Green had excellent co-localization effects in A549 cells, with a Pearson correlation coefficient of 0.909 (as shown in Figure 2A). Figure 8 (A–C). Figure 8 The co-localization images of lipid droplet probe (1 μM, excitation wavelength 561 nm, emission wavelength 570-620 nm) and LD-Tracker Dark Green (1 μM, excitation wavelength 488 nm, emission wavelength 500-550 nm) in NCI-H1299 cells are shown.

[0113] To further measure the fluorescence intensity in cells, we placed the cell slides in a 6-well plate and seeded 5 × 10 5 After 24 hours of culture, the cells were fixed with acetone and washed with PBS. Subsequently, they were incubated with different concentrations of LD probe (0, 0.5uM, 1uM, 5uM, 10uM, 20uM) for 1 hour, washed with PBS for 5 minutes after incubation, and stained with DAPI. Finally, fluorescence imaging was performed using a Nikon Ni-E multiphoton laser scanning confocal microscope.

[0114] Example 3

[0115] To further explore the fluorescence changes of lipid droplet probes in lung cancer cell lines, we treated A549 and HBE cells at concentrations of 0.5 μM and 1 μM, respectively. The results showed that A549 cells showed obvious fluorescence enhancement, while HBE cells had almost no fluorescence signal (e.g. Fig. 9 As the concentration of lipid droplet probes increases, the fluorescence intensity in A549 cells gradually increases (as shown in Fig. 9 B). Flow cytometry analysis further confirmed these observations ( Fig. 9 C). In addition, we also studied the effect of low concentrations of lipid droplet probes on staining and imaging in A549 cells. After A549 cells were treated with oleic acid for 2 hours, lipid droplet probes formed obvious fluorescent signals even at the lowest concentration (as shown in Figure 2A). Fig.10 shown). Fig.10A shows the fluorescence intensity of lipid droplet probes at different concentrations in HBE cells treated with oleic acid (excitation wavelength 561 nm, emission wavelength 570-620 nm). In summary, the lipid droplet probes exhibited specific binding to lipid droplets and robust targeting capabilities.

[0116] Example 4 Visualization of Fluorescence Changes of Lipid Droplet Probes in Mouse Tumor Model

[0117] After initially verifying the targeted imaging ability of lipid droplet probes in lung cancer cells, this study was further extended to mouse tumor models. 6 A549 cells were subcutaneously injected into the right side of 5-week-old nude mice, and imaging began when the tumor volume reached about 1 cubic centimeter. Subsequently, the lipid droplet probe (100 μM) was directly injected into the subcutaneous tumor of the mouse. After 30 minutes, significant fluorescence signals were observed in the tumor site through the in vivo imaging system, while no obvious fluorescence was observed in the PBS control group ( Fig.11 A and 11B). Then, the lipid droplet probe (50 μM) was injected through the tail vein. After circulating for 1 hour, real-time imaging showed that strong fluorescent signals also appeared in the tumor area ( Fig.11 C). These results confirm the specificity and effectiveness of lipid droplet probes in in vivo imaging of lung cancer. The experiment was further conducted to collect major organs (heart, liver, spleen, lung, kidney) and tumor tissues for imaging. Fig.11 D–F show that the fluorescence signals of major organs are weak, while tumor tissues show obvious strong fluorescence signals, further demonstrating the targeting ability of the lipid droplet probe.

[0118] In addition, the lipid droplet probe (100 μM) was directly injected into normal mice and tumor mice. After 30 minutes, the tumor mice showed strong fluorescence signals at the tumor site, while normal mice showed no obvious subcutaneous fluorescence (Figure S11A). Subsequently, samples of major organs from tumor mice and normal mice were collected, including heart, liver, spleen, lung, kidney, intestine and tumor tissue. The imaging results showed that the tumor tissue had a strong fluorescence signal, while other organs did not show fluorescence (Figure S11B). In addition, the experimental results of using the lipid droplet probe to stain normal lung tissue and lung cancer tissue showed that only the tumor tissue showed a strong fluorescence signal ( Fig.11 G–H). These data further indicate that the lipid droplet probe has high specificity and effectiveness in the detection of lung cancer tissues, and exhibits more significant targeting compared to normal tissues.

[0119] Visualization of lipid droplet probe fluorescence changes in early lung adenocarcinoma: Based on the above results, we further studied the lipid droplet probe staining performance of SN-ADC, GGN-ADC and adjacent normal lung tissue (normal). After tissue sections (10 μm) were incubated with LD probes, the imaging results showed that the fluorescence intensity of SN-ADC tissue was significantly higher than that of GGN-ADC tissue ( Figure 6 AB). In addition, only weak fluorescence signals were detected in normal lung tissue. H&E staining further verified the diagnosis and pathological type of the tumor ( Figure 6 A). These findings indicate that the lipid droplet probe can effectively distinguish early-stage SN-ADC from GGN-ADC tissues, highlighting its potential clinical value in the early detection of lung cancer.

[0120] This study successfully achieved accurate identification of early lung cancer pathological types by targeting changes in lipid droplets in tumor cells and tissues, providing an important theoretical basis and new diagnostic tool for the clinical diagnosis of early lung cancer. Therefore, we developed a multi-mode two-photon fluorescent probe to monitor changes in lipid droplets in early lung cancer, thereby supporting accurate diagnosis of different types of early lung cancer.

[0121] It is to be understood that the present invention is described by some embodiments, and it is known to those skilled in the art that various changes or equivalent substitutions may be made to these features and embodiments without departing from the spirit and scope of the present invention. In addition, under the teachings of the present invention, these features and embodiments may be modified to adapt to specific circumstances and materials without departing from the spirit and scope of the present invention. Therefore, the present invention is not limited by the specific embodiments disclosed herein, and all embodiments falling within the scope of the claims of this application are within the scope of protection of the present invention.

Claims

1. A method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe targeting lipid droplets, which achieves sensitive monitoring of early lung cancer by detecting the dynamic changes of lipid droplets in tumor cells and tissues, characterized in that: The following steps are involved: S1. Clinical sample collection: Clinical samples were collected from lung cancer centers, including normal lung tissue and cancerous lung tissue of patients with early lung cancer. The samples were pathologically confirmed to include two subtypes: solid lung adenocarcinoma and ground-glass lung adenocarcinoma. S2. Laser microdissection: Laser microdissection is used to separate malignant cells and tumor areas from tissue samples, providing an accurate sample source for subsequent analysis; S3, mass spectrometry detection and proteomic analysis: extract tumor tissue proteins, use mass spectrometry technology to analyze the differential proteins and signaling pathways between the two cancer subtypes, and combine bioinformatics methods to analyze the expression of key molecules; S4. Lipid droplet molecular analysis and detection: Western blotting and scanning electron microscopy were used to detect the expression levels of lipid droplet-related molecules, and the distribution and aggregation characteristics of lipid droplets in solid lung adenocarcinoma and ground-glass lung adenocarcinoma were compared; S5. Cell culture and preliminary validation: Normal epithelial cells and lung cancer cell lines were cultured in a 5% CO2 cell culture incubator at 37°C, and the expression differences of lipid droplet-related molecules in the two were detected; S6. Chemical structure design of fluorescent probe: A two-photon fluorescent probe targeting lipid droplet polarity change was designed based on cyanomethylene-benzopyran and N,N-dimethylaniline. The chemical structure of the probe was confirmed by nuclear magnetic resonance and high-resolution mass spectrometry, and its fluorescence properties in different solvents and environmental conditions were further analyzed. S7. Verification of the characteristics of fluorescent probes: Detect the fluorescence properties of fluorescent probes, analyze the extremely weak fluorescence intensity of fluorescent probes in oil and water and under high viscosity conditions, analyze the photophysical properties of fluorescent probes in various solvents with different polarities, including toluene (TOL), 1,4-dioxane (DIO), ethyl acetate (EA), THF and acetone, and analyze the changes in fluorescence intensity of fluorescent probes at different concentrations in neutral, acidic and alkaline environments; S8. Cell-level validation: Add fluorescent probes to normal epithelial cells and lung cancer cell lines, and verify their sensitivity and specificity for detecting early lung cancer through changes in fluorescence intensity; S9. Animal model experiments: Establish subcutaneous and tail vein tumor mouse models to evaluate the distribution of fluorescent probes in tumor tissues and the intensity of fluorescent signals, and analyze their safety and background signals in other tissues and organs; S10. Clinical sample verification: For clinical samples of different types of early lung cancer, detect the changes in the signal intensity of the fluorescent probe in lung cancer tissue to further confirm its potential in actual clinical applications.

2. According to claim 1, a method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe targeting lipid droplets is characterized in that: After the sample tissues in S1 were collected, the tumor tissues were immediately cooled to 4°C and rinsed with isotonic saline, and then each sample was divided into two parts: one part for histological staining and the other part for mass spectrometry and proteomics analysis.

3. The method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe targeting lipid droplets according to claim 1, characterized in that: The laser microdissection in S2 includes using a slicer to cut the formalin-fixed paraffin-embedded (FFPE) specimen into 10uM thickness and fix it on a Leica pen film slide (2uM), then the slide is dewaxed with xylene, rehydrated through different gradients of ethanol and water, and stained with hematoxylin; after staining, the slices are dehydrated again through different concentrations of alcohol and xylene, and the malignant and normal areas of the tumor are selected using Leica's LMD 6500 laser microdissection system and collected in 0.5 ml test tubes; the total area collected is 1-5×10 6 mm 2 , equivalent to 5,000 to 25,000 cells.

4. A method for accurately diagnosing early lung cancer using a two-photon near-infrared fluorescent probe targeting lipid droplets according to claim 1, characterized in that ,The specific process of mass spectrometry detection analysis in S3 is as follows: first, a 15L volume of peptides were injected and pre-concentrated on a trapping column with 0.1% TFA for 7 min (Acclaim ProMap100, 300m x 5mm, C18, 5m, 100; flow rate 30 L / min), and then they were separated using a 250-min HPLC gradient from 2% to 60% at a flow rate of 200 nL / min in buffer B (80% acetonitrile, 0.5% formic acid); using 70000 m / z Survey scans were performed at a resolution of 200 (60,000 for Q accurate HF) and the scan range was set from 300 to 1650 m / z; the three most abundant MS1 features (charge > 2) were selected for high-energy collisional dissociation fragmentation at a resolution of 17,500 m / z (15,000 for Q accurate HF), dynamic exclusion of sequenced peptides was set to 45 s, ion injection time and ion target value were set to 20 ms and 3 × 106 for survey scans, and ion injection time and ion target value were set to 220 ms and 1 × 10^5 for MS / MS scans, respectively; data acquisition was managed using Xcalibur software (Thermo Scientific).

5. A two-photon near-infrared fluorescent probe targeting lipid droplets for accurate diagnosis of early lung cancer according to claim 1, characterized in that: In the S5, normal epithelial cells and lung cancer cell lines were cultured in a 5% CO2 cell culture incubator at 37°C. After 24 hours, the culture medium was removed and the cells were fixed with pre-cooled acetone for 20 minutes; the cells were washed three times with PBS, and then incubated with different concentrations of lipid droplet probes (0, 0.5uM, 1uM, 5uM, 10uM, 20uM) for 1 hour; after incubation, the cells were washed with PBS for 5 minutes and stained with DAPI for 10 minutes; finally, 20 μL was applied to a cell slide, the edges were sealed with nail polish, and fluorescence imaging was performed using a multiphoton laser scanning confocal microscope.

6. The method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe targeting lipid droplets according to claim 1, characterized in that: The cell level verification specifically includes the following steps: Step 1: The cytotoxicity of lipid droplet probes in normal epithelial cells and lung cancer cells was detected at different concentrations and time intervals; Step 2: Minimal cytotoxicity was observed, indicating the excellent biocompatibility of the lipid droplet probe; Step 2: A549 cells were stained with lipid droplet probe and a commercially available LD-specific dye, LD-Tracker Dark Green. The LD-probe and LD-Tracker Dark Green showed excellent co-localization.

7. The method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe targeting lipid droplets according to claim 1, characterized in that: The S9 animal model experiment specifically includes: To establish the mouse tumor model, a total of 2×10 6 A549 cells were injected subcutaneously into the right flank of 5-week-old nude mice, and imaging was performed once tumors reached approximately 1 cubic centimeter in size; For fluorescence measurements in the mouse model, LD-probes at a concentration of 100 μM were administered intratumorally at the tumor edge or via the tail vein. In vivo imaging was performed 30 minutes after injection using a PerkinElmer IVIS Spectrum with an emission wavelength of 719 nm. After imaging, mice were euthanized, and tumors as well as major organs (lungs, liver, kidneys, spleen, and heart) were quickly collected for ex vivo imaging. Fluorescence measurement in lung cancer tissues. First, frozen tissues from SN-ADC, GGN ADC, and adjacent normal tissues were embedded with OCT embedding medium and cut into 10-20 micron slices using a microtome; these slices were fixed on gelatin / potassium dichromate-coated slides and stored at -80°C. The slices were then stained with different concentrations of lipid droplet probes (0.5uM and 1uM) for 30 minutes and with DAPI (1g / ml) for 10 minutes, and fluorescence imaging was performed using a Nikon Ni-E multiphoton laser scanning confocal microscope with an emission wavelength of 719nm.

8. The method for accurately diagnosing early lung cancer by using a two-photon near-infrared fluorescent probe targeting lipid droplets according to claim 1, characterized in that: The S10 for clinical sample verification specifically includes: Sample collection, five and six SN–ADC and GGN–ADC samples were collected, respectively, and hematoxylin and eosin (H&E) staining was performed to confirm the diagnosis of LUAD and identify the malignant tumor area; In the synthesis of lipid droplet probes, dicyanomethylenebenzopyran and N, N-dimethylaniline were selected as the classic electron-withdrawing group and strong electron-donating group, respectively; The spectral response of lipid droplet probe to polarity was used to evaluate the fluorescence performance of lipid droplet probe, which confirmed the typical ICT effect and high sensitivity to polarity. Subcellular co-localization experiments were performed to evaluate the specificity of lipid droplet probes in living cells based on their superior performance; Visualization of the fluorescence changes of lipid droplet probes in lung cancer cell lines. A549 and HBE cells were treated with lipid droplet probes at concentrations of 0.5uM and 1uM, confirming the specificity and robust targeting ability of lipid droplet probes to LDs. Visualization of lipid droplet probe fluorescence changes in mouse tumor models, where lipid droplet probes (100uM) were directly administered into subcutaneous tumors in mice, including direct injection of lipid droplet probes (100uM) into normal mice and mice bearing tumors; Visualization of lipid droplet probe fluorescence changes in early lung adenocarcinoma. Tissues of SN-ADC, GGN-ADC, and adjacent normal lung tissue (normal) were further stained. The tissues were cut into 10-micron sections and incubated with lipid droplet probes.

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