CTX-TPD NPs fluorescent nano-probe, preparation method thereof and application of CTX-TPD NPs fluorescent nano-probe in targeting oral and maxillofacial tumor

Abstract

The invention discloses a fluorescent nano-probe and a preparation method and application thereof in targeting oral and maxillofacial tumors, the CTX-TPD NPs fluorescent nano-probe comprises an amphiphilic polymer, TPD entrapped by the amphiphilic polymer and cetuximab modified outside the amphiphilic polymer, the amphiphilic polymer is a polymer composed of DSPE-PEG2000 and DSPE-PEG2000-COOH, the amphiphilic polymer is an amphiphilic polymer, the amphiphilic polymer is an amphiphilic polymer, the amphiphilic polymer is an amphiphilic polymer, and the amphiphilic polymer is an amphiphilic polymer. A DSPE part of the amphiphilic polymer is combined with TPD through hydrophobic interaction to form a hydrophobic core of the nanoparticle, a PEG chain segment of the amphiphilic polymer is distributed on the outer layer of the nanoparticle through hydrophilic interaction, and cetuximab is coupled to carboxyl of the PEG chain segment of the outer layer of the nanoparticle. The TPD molecule has breakthrough imaging performance, the AIE characteristic of the TPD molecule is enhanced in fluorescence in an aggregation state, the ACQ problem of a traditional dye is solved, and the imaging sensitivity is improved.

Description

Technical Field

[0001] This invention relates to the field of fluorescence detection technology, and in particular to a CTX-TPD NPs fluorescent nanoprobe, its preparation method, and its application in targeting oral and maxillofacial tumors. Background Technology

[0002] In recent years, the incidence and mortality rates of malignant tumors have shown a significant upward trend, becoming a serious threat to people's health. Statistics show that oral cancer ranks sixth in incidence among all malignant tumors. Due to its highly invasive nature and tendency to metastasize and recur, its clinical diagnosis and treatment face severe challenges. Early detection, timely diagnosis, and precise treatment of oral cancer have become a consensus among oral and maxillofacial surgeons and head and neck surgeons. Currently, biopsy is the gold standard for diagnosing oral cancer; however, early screening of high-risk groups relies excessively on physician experience, and intraoperative margin determination is largely based on physical examination and traditional imaging methods, lacking real-time and objective assessment tools.

[0003] Fluorescence imaging technology, with its advantages of being non-invasive, radiation-free, and providing real-time high resolution, offers new insights for early screening and adjuvant treatment of oral cancer. As a core carrier, fluorescent nanoprobes are considered an ideal platform for detecting tumor molecular markers due to their high brightness, ease of functionalization, and high spatiotemporal resolution. However, current technologies still face significant bottlenecks: ① Inherent defects of traditional fluorescent materials: organic dyes are prone to fluorescence quenching (ACQ effect) due to aggregation; conjugated polymers lack photostability; inorganic quantum dots pose a risk of heavy metal toxicity, hindering their clinical translation; ② Limitations in probe performance: insufficient imaging resolution and low targeting accuracy make it difficult to meet the urgent needs of early diagnosis and real-time intraoperative navigation for oral and maxillofacial tumors.

[0004] To overcome the limitations of traditional fluorescent materials, aggregation-induced emission (AIE) materials have emerged. The representative molecule TPD (which overcomes the ACQ effect) exhibits weak fluorescence in the dispersed state but significantly enhanced luminescence in the aggregated state. This has greatly advanced the research progress of cell fluorescence imaging probes in applications such as cell activity monitoring, subcellular structure imaging, and tumor cell labeling.

[0005] Reactive oxygen species (ROS) play a crucial role in biological processes. High levels of ROS cause oxidative damage to cellular biomolecules such as lipids, proteins, and nucleic acids, leading to tumor cell death. Meanwhile, photodynamic therapy (PDT), based on photosensitizers, generates ROS through photoexcitation, inducing mitochondrial oxidative stress and immunogenic cell death, demonstrating synergistic therapeutic potential. At the targeting level, although the epidermal growth factor receptor (EGFR) monoclonal antibody cetuximab (C225) has been used in EGFR-positive tumors such as head and neck squamous cell carcinoma, its efficacy is limited and resistance is easily developed in tumors with abnormal RAS pathway activation due to KRAS mutations (such as some oral cancers). Traditional antibody-drug conjugates (ADCs), while improving targeting, face problems such as off-target toxicity and uncontrollable payload release. However, research and application of targeted fluorescent nanoprobes based on AIE molecule TPD conjugated with C225 in the integrated diagnosis and treatment of oral and maxillofacial tumors (especially the synergistic application of intraoperative navigation and photodynamic therapy) have not yet been systematically reported. Summary of the Invention

[0006] The purpose of this invention is to address the technical deficiencies in the existing technology by providing a CTX-TPD NPs fluorescent nanoprobe.

[0007] Another objective of this invention is to provide a method for preparing CTX-TPD NPs fluorescent nanoprobes.

[0008] The present invention also provides the application of CTX-TPD NPs fluorescent nanoprobes in the preparation of targeted detection agents for oral and maxillofacial tumors and in the preparation of targeted drugs for anti-oral and maxillofacial tumors.

[0009] The technical solution adopted to achieve the purpose of this invention is:

[0010] A CTX-TPD NPs fluorescent nanoprobe comprises an amphiphilic polymer, TPD encapsulated thereon, and cetuximab modified on the exterior of the amphiphilic polymer, wherein the amphiphilic polymer is DSPE-PEG. 2000 and DSPE-PEG 2000 The amphiphilic polymer is composed of -COOH. The DSPE portion of the amphiphilic polymer binds to TPD through hydrophobic interactions to form the hydrophobic core of the nanoparticle. The PEG segments of the amphiphilic polymer are distributed on the outer layer of the nanoparticle through hydrophilic interactions. The cetuximab is coupled to the carboxyl group of the PEG segments on the outer layer of the nanoparticle.

[0011] The above technical solution includes the following steps:

[0012] Step 1 involves reducing 4-bromobenzophenone and 4,4'-dimethoxybenzophenone with zinc powder and titanium tetrachloride to obtain compound 1; then reacting compound 1 with 4-formylphenylboronic acid via a palladium-catalyzed Suzuki coupling reaction to obtain compound 2; finally, condensing compound 2 with 2-(2-methyl-4H-chromium-4-ylidene)malonitrile under piperidine catalysis to obtain compound 3TPD; the reaction process of step 1 is as follows:

[0013]

[0014] Step 2, mix the TPD and DSPE-PEG from Step 1. 2000 and DSPE-PEG 2000 The mixed solution of -COOH was sonicated to remove organic solvent. After filtration and dilution, a nanoparticle suspension was obtained. The nanoparticle suspension was dialyzed in MES buffer, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution and N-hydroxysuccinimide sodium sulfonate aqueous solution were added. After the light-protected activation reaction, fluorescent nanoparticles TPD NPs were obtained.

[0015] Step 3: After adjusting the pH of the TPD NPs obtained in Step 2 in PBS buffer, react them with cetuximab C225 in the dark with shaking. After the reaction, BSA is added for blocking treatment, and then the mixture is concentrated by dialyzing to obtain CTX-TPD NPs fluorescent nanoprobes.

[0016] In the above technical solution, the molar ratio of 4-bromobenzophenone and 4,4'-dimethoxybenzophenone in step 1 is (1.25~1.35):1, the molar ratio of compound 1 and 4-formylphenylboronic acid is 1:(1.15~1.25), and the palladium catalyst is tetrakis(triphenylphosphine)palladium, with an amount of 0.1~1 mol% of the molar amount of compound 1.

[0017] In the above technical solution, the reduction reaction in step 1 is carried out by reflux at -75 to -80°C to 20 to 25°C.

[0018] In the above technical solution, the molar ratio of compound 2 and 2-(2-methyl-4H-chromium-4-ylidene)malononitrile in step 1 is 1:(1-2), and the amount of piperidine catalyst added is 1-5 mol% of the reaction system.

[0019] In the above technical solution, the TPD and DSPE-PEG mentioned in step 2 2000 and DSPE-PEG 2000 The mass ratio of -COOH is 1:(1~2):(1~2); the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt is 1:(1~2).

[0020] In the above technical solution, the activation reaction temperature in step 2 is 20-25℃, and the reaction time is 30-60 min.

[0021] In the above technical solution, the mass ratio of cetuximab C225 to TPD in step 3 is (0.5-1):1, and the concentration of BSA is 1%.

[0022] In the above technical solution, the pH value of the light-shielding oscillation reaction in step 3 is 7.0-7.4, and the reaction time is 1-2 hours; the BSA sealing treatment time is 30-60 minutes.

[0023] Another aspect of the present invention is the application of a CTX-TPD NPs fluorescent nanoprobe in the preparation of a targeted detection agent for oral and maxillofacial tumors and in the preparation of a targeted drug for anti-oral and maxillofacial tumors.

[0024] Compared with the prior art, the beneficial effects of the present invention are:

[0025] 1. The TPD molecule of the present invention has breakthrough imaging performance. Its AIE characteristic enhances fluorescence in the aggregated state, which overcomes the ACQ problem of traditional dyes and improves imaging sensitivity.

[0026] 2. The TPD molecule of the present invention has near-infrared emission properties (660nm), which can reduce the interference of autofluorescence in biological tissues and is suitable for deep tissue imaging.

[0027] 3. The present invention uses a PEG polymer nanoparticle drug delivery system to encapsulate TPD, which, upon entering the cell and being stimulated by light of a specific wavelength, can promote the production of a large amount of reactive oxygen species in tumor cells, leading to tumor cell death.

[0028] 4. This invention achieves efficient coupling between antibodies and nanoparticles through the EDC / Sulfo-NHS coupling method, and the prepared CTX-TPD NPs fluorescent probe combines the targeting activity of antibodies with the optical properties of TPD.

[0029] 5. The CTX-TPD NPs fluorescent probe prepared by this invention has excellent photostability and biocompatibility, and can be used for long-term live cell imaging and in vivo real-time fluorescence detection.

[0030] 6. Compared with traditional C225 therapy, the CTX-TPD NPs fluorescent probe prepared in this invention can enhance EGFR endocytosis, endow the probe with active targeting, making it easier for tumor cells to take up, while inhibiting downstream EGFR signaling pathways to exert synergistic anti-tumor effects, significantly enhancing the killing effect of PDT, and providing a new strategy for the treatment of EGFR-positive and drug-resistant tumors. Attached Figure Description

[0031] Figure 1 The diagram shown illustrates the synthesis process of the TPD of this invention.

[0032] Figure 2 The image shown is a high-resolution mass spectrum of the TPD synthesized in this invention.

[0033] Figure 3 The image shows the 1H NMR spectrum of the TPD synthesized in this invention.

[0034] Figure 4 The figure shows the carbon NMR spectrum of the TPD synthesized in this invention.

[0035] Figure 5 The image shows the CTX-TPD NPs fluorescent probe of the present invention.

[0036] Figure 6 The image shows a comparison of the fluorescence intensities of TDT NPs, DT NPs, and TPD NPs.

[0037] Figure 7 The UV absorption and fluorescence emission spectra of TPD NPs and CTX-TPD NPs are shown.

[0038] Figure 8a The diagram shows the particle size distribution of TPD NPs and CTX-TPD NPs.

[0039] Figure 8b The image shown is a transmission electron microscope image of TPD NPs and CTX-TPD NPs.

[0040] Figure 9 The figure shows a comparison of ROS yields for TPD NPs and CTX-TPD NPs.

[0041] Figure 10a The image shows the in vitro targeting patterns of TPD NPs and CTX-TPD NPs.

[0042] Figure 10b The results show the statistical results of fluorescence intensity of TPD NPs and CTX-TPD NPs (*p<0.05, **p<0.01).

[0043] Figure 11 The figure shows the effect of TPD NPs and CTX-TPD NPs on the viability of SCC-7 cells with or without light treatment (*p<0.05****p<0.0001).

[0044] Figure 12aThe image shows the in vivo targeting patterns of TPD NPs and CTX-TPD NPs.

[0045] Figure 12b The results show the statistical results of fluorescence intensity of TPD NPs and CTX-TPD NPs (**p<0.01).

[0046] Figure 13a The in vivo organ distribution of TPD NPs and CTX-TPD NPs is shown.

[0047] Figure 13b The results show the statistical results of fluorescence intensity of TPD NPs and CTX-TPD NPs (****p<0.0001). Detailed Implementation

[0048] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0049] Example 1

[0050] A method for preparing CTX-TPD NPs fluorescent nanoprobes includes the following steps:

[0051] Step 1, Synthesis of TPD (TPE-Ph-DCM) molecules: 4-bromobenzophenone (5.3 g, 20.3 mmol) and 4,4'-dimethoxybenzophenone (3.8 g, 15.7 mmol) were dissolved in 80 mL of tetrahydrofuran to obtain a mixed solution. Zinc powder (5.9 g, 90.8 mmol) was added to the mixed solution to obtain a suspension. 5.0 mL of titanium tetrachloride was added dropwise to the suspension at -78 °C. Subsequently, the reaction system was slowly raised to room temperature and heated under reflux with stirring for 8 hours. After the reaction was completed, the reaction mixture was obtained. The reaction mixture was cooled in an ice-water bath, and 50 mL of saturated sodium bicarbonate aqueous solution was slowly added to quench the reaction. The mixture was then extracted three times with 100 mL of ethyl acetate to obtain the synthesized organic phase. The combined organic phases were washed twice with 100 mL of saturated brine, and then dried with anhydrous sodium sulfate. After filtering with anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure. The residue was purified by column chromatography (petroleum ether / ethyl acetate = 100-30:1) to obtain the target product compound 1, which was a white solid (3.1 g, yield 41.8%).

[0052] 1.18 g of compound 1 (2.50 mmol) and 450 mg of 4-formylphenylboronic acid (3.0 mmol) were dissolved in 12 mL of tetrahydrofuran, and 3 mL of 2 M potassium carbonate aqueous solution was added. After stirring for 0.5 hours under argon protection, 10 mg of tetra(triphenyl)phosphine palladium was added, and the reaction was carried out at 90 °C for 12 hours. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was poured into 15 mL of water. The organic matter was extracted with ethyl acetate (10 mL × 3). The organic phases were combined, dried with anhydrous sodium sulfate, filtered to remove the drying agent, and the solvent was removed under reduced pressure. The residue was purified by column chromatography (ethyl acetate / petroleum ether = 5:95) to give the target product compound 2 (870 mg, yield 70%).

[0053] Under an argon atmosphere, 500 mg of compound 2 (1.0 mmol) and 210 mg of 2-(2-methyl-4H-chromium-4-ylidene) malononitrile (10 mg, 1.0 mmol) were dissolved in anhydrous acetonitrile. Piperidine (catalytic amount 4.26 mg-8.52 mg) was added, and the mixture was heated under reflux with stirring for 12 hours. After the reaction was completed, the reaction mixture was filtered, and the resulting solid residue was washed with acetonitrile and dried under reduced pressure to obtain the target product compound 3 (273 mg, 39.8% yield), denoted as TPD. The preparation process of TPD described in step 1 is as follows. Figure 1 .

[0054] Step 2, Preparation of fluorescent nanoparticles: 1 mg TPD, 2 mg DSPE-PEG 2000 and DSPE-PEG 2000After equilibrating the -COOH material in a desiccator for 2 hours to eliminate the effects of moisture absorption, it was dissolved in 1 mL of tetrahydrofuran to obtain a mixed solution. This mixed solution was stored at low temperature and protected from light for later use. The mixed solution was placed in an ultrasonic oscillator and oscillated for 10 minutes to mix thoroughly. A 15 mL centrifuge tube was then used to add the mixed solution dropwise to 9 mL of distilled water under ultrasonic conditions (note that the distilled water should be added slowly, perhaps along the ultrasonic probe). The power was set to 60-75 W, the temperature to 25°C, and the mixture was sonicated for 10 minutes. The tetrahydrofuran in the mixed solution was removed using an air pump. The solution was then placed in a dialysis bag and dialyzed in distilled water for 24 hours (changing the solution every 4 hours) to obtain a nanoparticle suspension. The nanoparticle suspension was centrifuged in an ultrafiltration tube to concentrate the nanoparticle solution to 2 mL. The 2 mL nanoparticle suspension was then diluted with ultrapure water to 8 mL. The solution was dialyzed at room temperature for 24 hours (changing the buffer every 4 hours) in 0.1 M, pH 6.0 2-(N-morpholino)ethanesulfonic acid buffer (MES buffer). Then, 20 μL of 0.2 mmol / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide aqueous solution and 20 μL of 0.2 mmol / mL N-hydroxysuccinimide sodium sulfonate aqueous solution were added, and the solution was activated in the dark to obtain TPD NPs and DSPE-PEG. 2000 As an amphiphilic polymer, the DSPE segment binds to TPD through hydrophobic interactions, forming the hydrophobic core of the nanoparticles; the PEG segments are distributed on the outer layer of the particles through hydrophilic interactions, improving water solubility and biocompatibility. Carboxylated DSPE-PEG 2000 -COOH provides a reaction site for subsequent antibody conjugation.

[0055] Step 3: Adjust the pH of the TPD NPs prepared in Step 2 to 7.0-7.4 in phosphate-buffered saline (PBS). Incubate with 100 μL of 5 mg / mL cetuximab (C225) at room temperature with light-protected shaking for 2 hours. Use the carbodiimide method (EDC / Sulfo-NHS) to react the antibody (cetuximab) with the carboxyl groups on the surface of the TPD NPs to form stable amide bonds. Add 1% BSA (bovine serum albumin) for blocking treatment and react at room temperature for 30 min. Centrifuge three times using an Amicon Ultra-4 centrifuge filter (100 kDa), 15 minutes each time. Then concentrate the solution to 1 mL to obtain 1 mg / mL of fluorescent nanoprobe CTX-TPD NPs. (See details below.) Figure 5 .

[0056] Reference Figure 2-4 The structure of the TPD (compound 3) synthesized in step 1 was characterized by high-resolution mass spectrometry (HRMS) and proton nuclear magnetic resonance (NMR) spectroscopy. 1 H NMR and carbon spectroscopy (H NMR) and carbon spectroscopy13 C10 NMR analysis was used to determine whether the structure of the synthesized TPD molecule was correct. The figure shows... 1 H NMR (400MHz, CDCl3): The characteristic signal δ6.84 (d, J=16Hz, 1H) confirms the formation of a trans olefin bond (-CH=CH-); δ3.75 (s, 6H) is assigned to a dimethoxy group (-OCH3), which is consistent with the designed structure. 13 C10 NMR (101 MHz, CDCl3): Key carbon signals δ 158.17 / 158.10 (CO), 144.12 / 142.77 (C=C), and 55.10 (-OCH3), consistent with the molecular skeleton. HRMS (MALDI-TOF): Measured m / z 686.2565 [M], consistent with the theoretical value (C101 MHz, CDCl3). 48 H 34 N2O3: 686.2569) with an error of 0.58 ppm, confirming the molecular formula. The above data verify the correctness of the TPD molecular structure and molecular weight, confirming the successful synthesis of TPD.

[0057] Comparative Example 1

[0058] The commonly used laboratory AIE molecules DPA-TCyP(DT) and TPE-DPA-TCyP(TDT), as well as TPE-Ph-DCM(TPD) prepared in Example 1, were selected and utilized with the amphiphilic copolymer DSPE-PEG. 2000 AIE molecules were encapsulated to prepare TPD NPs nanoparticles, DT NPs nanoparticles, and TDT NPs nanoparticles. The fluorescence intensity of the three nanoparticles was measured and compared using a fluorescence imaging system at a concentration of 50 μg / mL (excitation wavelength: 430 nm). (Reference) Figure 6 Compared to DT NPs and TDT NPs, TPDNPs exhibited the strongest fluorescence brightness, indicating that they possess excellent fluorescence imaging performance.

[0059] Test Example 1

[0060] 1. Photophysical properties testing of CTX-TPD NPs and TPD NPs: Refer to Figure 7The UV absorption and fluorescence emission spectra of the TPD NPs prepared in step 2 and the CTX-TPD NPs prepared in step 3 of Example 1 are shown. The UV absorption spectra show that both TPD NPs and CTX-TPD NPs exhibit a maximum absorption peak around 450 nm in aqueous solution. In the fluorescence spectra, both TPD NPs and CTX-TPD NPs exhibit a strong emission peak around 650 nm in aqueous solution. The UV absorption and fluorescence emission curves of the prepared CTX-TPD NPs and TPD NPs are almost identical, indicating that the surface modification of CTX does not alter the photophysical properties of the TPD molecules. This also verifies that CTX-TPD NPs and TPD NPs possess good tissue penetration capabilities.

[0061] 2. Size and morphology analysis of TPD NPs and CTX-NPD NPs: Refer to Figure 8a The sizes of TPD NPs and CTX-NPD NPs were analyzed using dynamic light scattering (DLS). Specific results are detailed in [reference needed]. Figure 8a As shown in the figure, before TPD was coupled with C225, the average diameter of TPD NPs was 119.5 nm, and the polydispersity index (PDI) was 0.228. After coupling with C225, the average diameter of CTX-TPD NPs was observed to be 139.3 nm, and the PDI was 0.2, indicating that approximately 19.8 nm of antibody molecules were coated on the TPD NPs. The morphology of TPD NPS and CTX-TPD NPs was further verified by transmission electron microscopy (TEM), referring to... Figure 8b The spherical structure of TPD NPs and CTX-TPD NPs indicates that they can be well dispersed in aqueous solution as individual entities without forming indivisible aggregates.

[0062] 3. Determination of ROS generation capacity of TPD NPs and CTX-TPD NPs fluorescent probes:

[0063] To verify whether TPD NPs and CTX-TPD NPs can generate ROS, 9,10-anthracene dimethyl dipropionic acid (ABDA) was used as an ROS indicator, and the commercially available photosensitizer Rose Bengal was used as a control. The ROS were generated under white light irradiation (0.25 W / cm²). 2 The decay curves of characteristic absorption peaks of ABDA in TPD NPs and CTX-TPD NPs solutions were monitored at different time points after [the initial treatment]. First, the UV absorption spectra of TPD NPs, CTX-TPD NPs, and Rose bengal (all at 10 μM) were acquired. Then, 50 μM ABDA was added, and UV absorption spectra were acquired again. Next, the solutions were irradiated with white light (0.25 W / cm²). 2Under these conditions, UV absorption spectra were collected every 60 seconds. The absorbance of ABDA at 378 nm before and after white light irradiation was obtained from the UV absorption decrease curve of ABDA. A graph was plotted based on the linear relationship between ABDA absorbance and irradiation time to compare the ability of different molecules to generate excited-state oxygen molecules. (Refer to...) Figure 9 It can be seen that the decomposition rate constants of Rose Bengal, TPD NPs, and CTX-TPD NPs are 0.00122 s⁻¹. -1 0.00472s -1 0.00509s -1 Compared to TPD NPs and Rose bengal, CTX-TPD NPs exhibited the best ROS production capacity. In tumor cells, photosensitizers, upon irradiation, generate large amounts of ROS, which can effectively induce mitochondrial oxidative stress in cancer cells, directly killing tumor cells through oxidative damage.

[0064] 4. In vitro targeting experiments of CTX-TPD NP and TPD NPS:

[0065] The tumor-targeting ability of CTX-TPD NPs was investigated using SCC-7 (mouse-derived) oral cancer cells, with TPD NPs serving as a control. SCC-7 cells in logarithmic growth phase were injected at a dose of 1×10⁻⁶. 5 SCC7 cells were seeded at a density of 1 / mL onto confocal microplates and incubated overnight. Serum-free medium containing CTX-TPD NPs and TPD NPs nanoparticles (25 μg / mL each) was then added, and incubation continued at 37°C for 2 h, 4 h, and 6 h. After washing three times with PBS, cells were fixed with 4% paraformaldehyde for 30 min. Finally, cell nuclei were stained with DAPI for 5 min, washed three times with PBS, and fluorescence imaging of SCC7 cells after incubation with TPD NPs and CTX-TPD NPs for different time periods was observed using a laser scanning confocal microscope. (Reference) Figure 10a Red fluorescence was observed in the cytoplasm of all groups, and the fluorescence intensity gradually increased with prolonged incubation time, indicating that CTX-TPD NPs and TPD NPs could be taken up by tumor cells in a time-dependent manner. (See reference...) Figure 10bAfter incubation in serum-free medium containing CTX-TPD NPs and TPD NPs for different times (2h, 4h, 6h), the fluorescence intensity of SCC7 oral cancer cells was statistically analyzed. As shown in the figure, the fluorescence intensity of SCC7 cells incubated with CTX-TPD NPs was significantly higher than that of the TPD NPs group at all time points, and the difference in fluorescence intensity was statistically significant. This suggests that CTX-TPD NPs has a stronger cellular uptake capacity, indicating that CTX significantly enhances the active uptake efficiency of the fluorescent probe CTX-TPD NPs by tumor cells through high affinity with EGFR overexpressed in SCC7 oral cancer cells.

[0066] 5. In vitro cytotoxicity and photodynamic killing effects of CTX-TPD NPs and TPD NPs:

[0067] The effects of CTX-TPD NPs and TPD NPs on the cell viability of SCC7 cells under no light and after light exposure were examined using the CCK-8 assay. SCC7 cells in the logarithmic growth phase were subjected to an induction reaction at 8 × 10⁻⁶ cells / year. 4 Cells were seeded at a density of 1 / mL in 96-well plates. After overnight culture, the medium was replaced with 100 μL of serum-free medium containing CTX-TPD NPs and TPD NPs (concentrations of 0, 6, 13, 25, 50, and 100 μg / mL, respectively) and incubated for 4 h. The medium was discarded, and the cells were washed three times with PBS. Cells without light were added to an appropriate amount of fresh medium and incubated in the dark for 12 h, while SCC7 cells were incubated at 0.25 W / cm². 2 Under white light irradiation, each cell was exposed to light for 10 min, followed by incubation in the dark for 12 h. After incubation, 10 μL of CCK-8 detection solution and 100 μL of culture medium were added to each well, and the cells were incubated for another 2 h. CCK-8 medium was used as the blank group, and CCK-8 medium containing only SCC7 cells was used as the control group. The absorbance (OD) of each group was then measured at 450 nm using a microplate reader.

[0068] Cell viability is calculated as follows:

[0069] Cell viability (%) = ((OD sample - OD blank) / (OD control - OD blank)) × 100%

[0070] Reference Figure 11Even at a concentration of 100 μg / mL, the survival rate of TPD NPs exposed to light remained above 80% in SCC-7 cells, indicating that this nanosystem did not exhibit significant cytotoxicity. However, at the same concentration, the cell survival rate of the CTX-TPD NPs group significantly decreased to 69%, suggesting that CTX-TPD NPs can exert anti-tumor effects by targeting the EGFR pathway and synergistically enhancing cytotoxicity. Under light irradiation, both TPD NPs and CTX-TPD NPs groups showed dose-dependent cytotoxicity. When the concentration reached 100 μg / mL, the cell survival rates of the TPD NPs and CTX-TPD NPs groups decreased to 34% and 15%, respectively. These results fully demonstrate that light irradiation can stimulate the PDT effect of TPD, enhancing the cytotoxicity of nanoparticles. Due to its active targeting capability, CTX-TPD NPs are more easily taken up by tumor cells, exerting a synergistic anti-tumor effect and significantly enhancing the PDT killing effect. The monoclonal antibody cetuximab (C225) has proven to be an effective therapeutic agent by specifically targeting EGFR, which is overexpressed in a range of cancers that promote tumor growth and progression. However, C225 alone has drawbacks such as poor penetration, high drug resistance, and limited therapeutic function. CTX-TPD NPs, on the other hand, mediate EGFR-specific binding through C225, synergistically enhancing the EPR effect of the nanocarrier, resulting in a 10-fold increase in tumor enrichment. TPD molecules accumulate and emit light at the tumor foci, allowing for real-time monitoring of treatment efficacy. This overcomes the limitations of monotherapy and further promotes the development of precision oncology treatment through "therapeutic integration."

[0071] 6. Statistical analysis of TPD NPs and CTX-NPD NPs' in vivo targeting and fluorescence intensity in important organs:

[0072] C3H / HeN mice were used to investigate the tumor-specific enrichment ability of fluorescent probes by analyzing their fluorescence distribution in tumors and major organs in vivo. The concentration of SCC-7 cell suspension was adjusted to 2 × 10⁻⁶. 7 A mouse oral and maxillofacial tumor model was established by submucosal injection of 30 μL of cell suspension into the buccal mucosa of mice. The tumor was allowed to grow to 100 mm. 3 Around 10:00 AM, tumor-bearing mice were randomly divided into two groups, and each group received a tail vein injection of 200 μL of TPD NPs and CTX-TPD NPs (both at a concentration of 1 mg / mL). At time points of 1 h, 3 h, 5 h, 8 h, and 24 h, Xenogen was used to administer the drugs. The Lumina II small animal in vivo imaging system was used to perform fluorescence imaging in mice to evaluate their tumor-targeting performance, determine the optimal imaging time of the fluorescent probe in vivo, and examine the accuracy of tumor tissue imaging. (Refer to...) Figure 12aTPD NPs and CTX-TPD NPs showed significant fluorescence signals at the tumor site one hour after tail vein injection, clearly distinguishing tumors from normal tissue, indicating that they can accurately locate tumor tissue and visualize it in real time. (Reference) Figure 12b The fluorescence intensity at the tumor site gradually increased over time, exhibiting a time-dependent effect, and reached its peak 8 hours after injection, indicating a high enrichment of nanoparticles at the tumor site. Notably, the fluorescence intensity of CTX-TPD NPs was significantly higher than that of TPD NPs at all time points, which is attributed to the active targeting effect mediated by CTX.

[0073] In addition, mice were sacrificed at the optimal imaging time point (8 hours), and tumor tissues and major organs (heart, liver, spleen, lung, and kidney) from tumor-bearing mice were collected for fluorescence imaging to determine the tumor-specific enrichment degree and organ distribution of the nanosystem. (Refer to...) Figure 13a Strong fluorescence signals were observed in tumor tissue and liver, while less distribution was observed in other organs such as the heart, spleen, lungs, and kidneys, indicating that CTX-TPD NPs and TPD NPs were mainly distributed in tumors and liver. To quantitatively analyze the in vivo tumor-targeting ability of CTX-TPD NPS and TPD NPS, [further details are needed]. Figure 13b The accumulation of CTX-TPD NPs in tumor tissues was significantly higher than that of TPD NPs, further demonstrating its superior tumor-specific targeting ability.

[0074] The above description is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A CTX-TPD NPs fluorescent nanoprobe, characterized in that, It includes an amphiphilic polymer, a TPD encapsulated therein, and cetuximab modified on the exterior of the amphiphilic polymer, wherein the amphiphilic polymer is DSPE-PEG. 2000 and DSPE-PEG 2000 The amphiphilic polymer is composed of -COOH. The DSPE portion of the amphiphilic polymer binds to TPD through hydrophobic interactions to form the hydrophobic core of the nanoparticle. The PEG segments of the amphiphilic polymer are distributed on the outer layer of the nanoparticle through hydrophilic interactions. The cetuximab is coupled to the carboxyl group of the PEG segments on the outer layer of the nanoparticle.

2. A method for preparing the CTX-TPD NPs fluorescent nanoprobe as described in claim 1, characterized in that, Includes the following steps: Step 1 involves reducing 4-bromobenzophenone and 4,4'-dimethoxybenzophenone with zinc powder and titanium tetrachloride to obtain compound 1; then reacting compound 1 with 4-formylphenylboronic acid via a palladium-catalyzed Suzuki coupling reaction to obtain compound 2; finally, condensing compound 2 with 2-(2-methyl-4H-chromium-4-ylidene)malonitrile under piperidine catalysis to obtain compound 3TPD; the reaction process of step 1 is as follows: Step 2, mix the TPD and DSPE-PEG from Step 1. 2000 and DSPE-PEG 2000 The mixed solution of -COOH was sonicated to remove organic solvent. After filtration and dilution, a nanoparticle suspension was obtained. The nanoparticle suspension was dialyzed in MES buffer, and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride aqueous solution and N-hydroxysuccinimide sodium sulfonate aqueous solution were added. After the light-protected activation reaction, fluorescent nanoparticles TPD NPs were obtained. Step 3: After adjusting the pH of the TPD NPs obtained in Step 2 in PBS buffer, react them with cetuximab C225 in the dark with shaking. After the reaction, BSA is added for blocking treatment, and then the mixture is concentrated by dialyzing to obtain CTX-TPD NPs fluorescent nanoprobes.

3. The method for preparing CTX-TPD NPs fluorescent nanoprobes as described in claim 2, characterized in that, In step 1, the molar ratio of 4-bromobenzophenone to 4,4'-dimethoxybenzophenone is (1.25–1.35):1, the molar ratio of compound 1 to 4-formylphenylboronic acid is 1:(1.15–1.25), and the palladium catalyst is tetrakis(triphenylphosphine)palladium, used in an amount of 0.1–1 mol of compound 1.

4. The method for preparing CTX-TPD NPs fluorescent nanoprobes as described in claim 3, characterized in that, The reduction reaction described in step 1 is carried out by reflux at -75 to -80°C to 20 to 25°C.

5. The method for preparing CTX-TPD NPs fluorescent nanoprobes as described in claim 2, characterized in that, In step 1, the molar ratio of compound 2 and 2-(2-methyl-4H-chromium-4-ylidene)malononitrile is 1:(1-2), and the amount of piperidine catalyst added is 1-5 mol% of the reaction system.

6. The method for preparing CTX-TPD NPs fluorescent nanoprobes as described in claim 2, characterized in that, The TPD and DSPE-PEG mentioned in step 2 2000 and DSPE-PEG 2000 The mass ratio of -COOH is 1:(1~2):(1~2); the molar ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysulfosuccinimide sodium salt is 1:(1~2).

7. The method for preparing CTX-TPD NPs fluorescent nanoprobes as described in claim 2, characterized in that, The activation reaction temperature in step 2 is 20–25°C, and the reaction time is 30–60 min.

8. The method for preparing CTX-TPD NPs fluorescent nanoprobes as described in claim 2, characterized in that, In step 3, the mass ratio of cetuximab C225 to TPD is (0.5-1):1, and the concentration of BSA is 1%.

9. The method for preparing CTX-TPD NPs fluorescent nanoprobes as described in claim 2, characterized in that, The pH value of the light-shielded shaking reaction in step 3 is 7.0-7.4, and the reaction time is 1-2 hours; the BSA blocking treatment time is 30-60 minutes.

10. The application of the CTX-TPD NPs fluorescent nanoprobe as described in claim 1 in the preparation of a targeted detection agent for oral and maxillofacial tumors and in the preparation of a targeted drug for oral and maxillofacial tumors.

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