Small molecule compound, water-soluble fluorescent probe and preparation method and application of water-soluble fluorescent probe

Abstract

The invention discloses a small molecule compound, a water-soluble fluorescent probe as well as a preparation method and application of the water-soluble fluorescent probe, and belongs to the technical field of medicines. According to the invention, a small molecule compound NQL containing a naphthalimide fluorophore is synthesized, and then a water-soluble fluorescent probe CS-NQL is prepared by forming Schiff base and coupling the Schiff base with chitosan oligosaccharide. The CS-NQL has environmental viscosity responsiveness and lysosome targeting specificity, so that tumor and inflammatory cell imaging can be carried out in a visible light range. The CS-NQL and DNA are subjected to electrostatic self-assembly to form nanoparticles CS-NQL coated DNA NPs, and the nanoparticles CS-NQL coated DNA NPs can generate active oxygen under an anoxic condition; under the irradiation of 660 nm laser, HeLa cell apoptosis can be induced, and an antibacterial effect is shown. An in-vivo experiment shows that CS-NQL-coated DNA NPs shows a strong anti-tumor immune response under the irradiation of 660 nm laser, so that the tumor is completely eradicated.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and particularly relates to a small molecule compound, a water-soluble fluorescent probe, and their preparation methods and applications. Background Art

[0002] Traditional oncology methods face substantial limitations in both diagnosis and treatment, including inaccurate early diagnosis, severe side effects, multi-drug resistance, limited therapeutic efficacy, and low efficacy against metastatic tumors. The development of biological probes has opened up new fields for cancer treatment and is expected to improve the accuracy of diagnosis and treatment. Near-infrared fluorescent probes have become a good candidate in the treatment field due to their superior optical properties, including deep tissue penetration and low background self-interference. Currently, the development of near-infrared probes mainly relies on chemical synthesis methods. These methods involve designing complex organic molecular structures, such as cyanine and BODIPY derivatives, to adjust their absorption and emission wavelengths to the near-infrared region. However, the synthesis of these probes often encounters multiple challenges. On the one hand, the long synthesis process leads to low yields and high costs. On the other hand, the photostability, biocompatibility, and targeting of the synthesized products still need to be further optimized. Summary of the Invention

[0003] In order to solve the above-mentioned deficiencies existing in the prior art, the object of the present invention is to provide a small molecule compound, a water-soluble fluorescent probe, and their preparation methods and applications to improve the absorption range and targeting of near-infrared probes.

[0004] The technical solution of the present invention to solve the above technical problems is as follows: Provide a small molecule compound containing a naphthalimide fluorescent group, and the molecular structural formula of the small molecule compound is shown in formula (Ⅰ):

[0005] (Ⅰ).

[0006] The present invention provides a water-soluble fluorescent probe, including the above-mentioned small molecule compound, and its molecular structural formula is shown in (Ⅱ):

[0007] (Ⅱ) Wherein, m and n are degrees of polymerization, m is 1000 - 5000, and n is 100 - 500.

[0008] The present invention provides a preparation method of the above water-soluble fluorescent probe, including the following steps: (1) Prepare the above-mentioned small molecule compound; (2) React chitosan with the small molecule compound in step (1) at 80 - 120 °C to obtain it.

[0009] Furthermore, the preparation of the above-mentioned small molecule compound in step (1) includes the following steps: (1) After adding absolute ethanol to 4-bromo-1,8-naphthalic anhydride and 4-aminoquinoline, reflux at 80-100 °C, then filter to collect the precipitate to obtain Compound 1; (2) Dissolve Compound 1, 4-(diphenylamino)phenylboronic acid, Pd(PPh3)4 and K2CO3 in a solvent and reflux, then dissolve the obtained product in dichloromethane to obtain Compound 2; (3) React Compound 2, 1-(4-(bromomethyl)phenyl)ethanone and dimethylformamide at 60-100 °C to obtain the product.

[0010] Furthermore, the solvent in step (2) is toluene and water.

[0011] The present invention provides an application of the above-mentioned water-soluble fluorescent probe in the preparation of tumor in vivo imaging reagents and nanoparticles.

[0012] The present invention provides a nanoparticle, which includes the above-mentioned water-soluble fluorescent probe, and the nanoparticle is formed by electrostatic self-assembly of the water-soluble fluorescent probe and DNA.

[0013] The present invention provides an application of the above-mentioned nanoparticle in the preparation of anti-tumor drugs.

[0014] Furthermore, the tumor includes ovarian cancer.

[0015] The present invention has the following beneficial effects: The present invention synthesizes a small molecule NQL containing a naphthalimide fluorescent group. In order to reduce cytotoxicity and achieve DNA regulation function, chitosan is introduced into NQL to obtain a water-soluble fluorescent probe CS-NQL. When excited by wavelengths in the visible light range, CS-NQL can detect the high-viscosity environment inside cells. Experimental results show that the probe has good lysosome targeting ability and can perform lysosome imaging in tumor cells. In vivo experiments show that CS-NQL can successfully target tumor tissues. This DNA-mediated regulation endows the water-soluble fluorescent probe with near-infrared photosensitivity. Through the interaction between chitosan and DNA, CS-NQL@DNA NPs are formed. CS-NQL@DNA NPs generate ROS through the type I mechanism, indicating that these nanoparticles can promote PDT even in the hypoxic tumor microenvironment. In vivo experiments show that under 660 nm laser irradiation (0.4 W / cm²), CS-NQL@DNA NPs can induce a strong anti-tumor immune response, resulting in complete eradication of tumors. At the same time, CS-NQL@DNA NPs show low toxicity and minimal damage to other biological tissues. Description of the Drawings

[0016] Figure 1 The 1 1H NMR spectrum of CS-NQL; Figure 2 The FTIR spectra of CS and CS-NQL; Figure 3 The spectral response diagram of CS-NQL to viscosity; Figure 4 The fluorescence images of various cells stained with CS-NQL for 60 min; Figure 5 The confocal images of live HeLa cells stained with CS-NQL; Figure 6 The fluorescence imaging diagram of the mouse model; Figure 7 The images of CS-NQL@DNA nanoparticles and the maps of their photosensitization properties; Figure 8 The absorption spectrum diagram of NQL when DNA is added to water; Figure 9 The fluorescence change diagram of SOSG and APF containing CS-NQL@DNA NPs under 660 nm laser irradiation for 360 s; Figure 10 The fluorescence images of the intracellular ROS generation ability of CS-NQL@DNA NPs in HeLa cells; Figure 11 The photodynamic antibacterial diagram of CS-NQL@DNA NPs; Figure 12 The treatment diagram of the HeLa tumor model; Figure 13 The in vivo treatment diagram of HeLa tumor-bearing mice at different times; Figure 14 The HE staining diagrams of the main organs of mice in different treatment groups 16 days after treatment. Detailed implementation manners

[0017] The following examples are only used to explain the present invention and are not intended to limit the scope of the present invention. For those not specified in the examples, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not indicated, they are all conventional products that can be obtained through commercial purchase.

[0018] Experimental methods: (1) Cytotoxicity experiment: The cell viability was repeatedly measured using the standard MTT assay. The cells were seeded into 96-well plates containing 100 μL of growth medium (1×10 per well 4In cells and incubated for 24 h to allow attachment. Dilute the probe solution to the desired concentration with the medium and then incubate for 24 h in the darkroom. Dispense 50 µL of 2.5 mg / mL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide solution (dissolved in PBS solution) into each well and incubate for 3 h in the dark. Then, aspirate the medium from all wells and add 100 µL of dimethyl sulfoxide (DMSO). Evaluate the optical density at 570 nm in each well using a Molecular Devices MaxF5 multimode microplate reader with an appropriate wavelength filter. Adjust all absorbance readings according to the background absorbance. Calculate the cell viability, and the calculation formula is as follows: Viability (%) = (number of live cells / total number of cells) × 100%.

[0019] (2) Cell culture and imaging: Culture L929 cells, HeLa cells, and RAW264 cells in DMEM medium enriched with 10 wt% fetal bovine serum, sodium pyruvate, L-glutamine, and 4.5 g / L glucose in a humidified incubator at 37 °C with 95% air and 5% CO2. When the cell confluence reaches 80%, passage the cells and culture them in DMEM containing 10 wt% fetal bovine serum (FBS) and 1 wt% streptomycin / penicillin antibiotics. After culturing in the culture dish for 24 h, wash the HeLa cells with PBS, then treat them with the CS-NQL probe (10 μg / mL), and further incubate in an incubator at 37 °C for 1 h. To confirm the localization of the CS-NQL probe in organelles, HeLa cells are co-localized with the CS-NQL probe (10 μg / mL) and a lysosome tracer (0.5 μM) or a mitochondrial tracer (0.5 μM). Treat HeLa cells with the CS-NQL probe together with LysoTracker Red DN-99 or MitoTracker DeepRed FM for 1 h, then wash with PBS before imaging, and measure the co-localization coefficient of the green and red channels.

[0020] (3) Reactive oxygen species detection: Mix the CS-NQL probe (20 μg / mL) with DCFH (2 μM), SOSG (2 μM), APF (2 μM), and DHR123 (2 μM) respectively, and irradiate the solution with white light (20 mm / cm 2 ), and collect fluorescence data. Add the CS-NQL probe (20 μg / mL) and DHR123 (2 μM) to a confocal culture dish containing HeLa cells, then incubate for 30 min, and then irradiate with white light (20 mm / cm 2 ), and record the data using a confocal laser scanning microscope.

[0021] (4) Tumor model and in vivo imaging: Nude mice, 4 - 5 weeks old, were from the Experimental Animal Center of North Sichuan Medical College in Nanchong City, China. All animal procedures were conducted according to the protocols approved by the Institutional Animal Care and Use Committee of North Sichuan Medical College. Under sterile conditions, 1×10 6 HeLa cells were injected subcutaneously into the anterior part of the right hind limb of these mice. Then the mice were individually housed in a sterile environment with free access to food and water. When the volume of the HeLa tumor reached approximately 150 mm 3 , 100 μL of CS - NQL NPs (10 mg / mL) was injected intratumorally, and fluorescence imaging was performed using an IVIS imaging system at the following time points: 0, 30, 60, 90, 120, 180, and 240 min.

[0022] (5) In vivo photodynamic therapy: When the tumor volume reached approximately 50 mm 3 , all nude mice bearing subcutaneous HeLa tumors were randomly divided into four groups (n = 3): PBS, PBS + Light, CS - NQL NPs, and CS - NQL NPs + Light groups. The intratumoral injection volume of PBS and CS - NQL NPs (1 mg / mL) was 25 μL per 50 mm³ of tumor. After intratumoral injection, the mice in the PBS + light and CS - NQL NPS + light groups were irradiated with a 660 nm (0.4 W / cm 2 ) laser for 30 min, while the other groups were not irradiated. During the treatment, the tumor volume was measured daily with a vernier caliper, and the calculation formula was: tumor volume = (width × width × length) / 2. After 16 days of treatment, the tumors and major organs (including the heart, liver, spleen, lungs, and kidneys) of the mice in all groups were harvested, and histopathological analysis was performed by hematoxylin - eosin (HE) staining, and histopathological changes were observed with an optical microscope.

[0023] (7) Statistical analysis: One - way analysis of variance (ANOVA) was used for statistical analysis. The data were expressed as mean ± standard deviation (SD). P ≤ 0.05 was considered statistically significant (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

[0024] Example 1: Synthesis of water - soluble fluorescent probe (CS - NQL) The synthesis route is as follows:

[0025] ① Synthesis of Compound 1: 4-Bromo-1,8-naphthalic anhydride (2.00 g, 7.22 mmol) and 4-aminoquinoline (1.44 g, 10.00 mmol) were mixed in a 100 mL two-necked flask. Then, 25 mL of anhydrous ethanol was added, and the mixture was refluxed at 90 °C for 12 h. After the reaction was completed, the mixture was cooled to room temperature and the precipitate was collected by filtration. Then, the precipitate was recrystallized from ethanol to obtain 1.89 g of a off-white solid product with a yield of 64.97%.

[0026] 1 H NMR(400MHz,DMSO)δ 8.97(DD,J=4.1,1.6Hz,1H),8.74-8.56(M,2H),8.43(D,J=8.0Hz,1H),8.38(D,J=7.9Hz,1H),8.31(D,J=7.9Hz,1H),8.19(D,J=8.5Hz,1H),8.08(dd,J=8.4,7.4Hz,1H),7.93(dd,J=8.5,7.4Hz,1H),7.76(dd,J=7.3,1.0Hz,1H),7.49(DD,J=8.6,4.1Hz,1H).

[0027] 13 C NMR(101MHz,DMSO)δ 164.07,151.33,148.60,133.42,132.22,131.91, 131.58,130.69,130.49,129.77,129.39,128.08,126.22,124.08,123.30,122.58. ESI-HRMS(m / Z):[m] Calculated for ([C 21 H 11 N2O2Br]+H)+( m / e,e=1): 403.0004, found 403.0072.

[0028] ② Synthesis of Compound 2: Compound 1 (403 mg, 1 mmol), 4-(diphenylamino)phenylboronic acid (289 mg, 1 mmol), Pd(PPh3)4 (23 mg, 0.02 mmol) and K2CO3 (138 mg, 1 mmol) were dissolved in a mixed solvent of 30 mL of toluene and 10 mL of water, and then refluxed under nitrogen for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed, and the residue was dissolved in dichloromethane and washed with saturated NaCl solution. The product was purified by column chromatography (PE:DCM = 8:1, V / V) to obtain a yellow solid (381 mg, 67.12%).

[0029] 1 1H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 4.2, 1.6 Hz, 1H), 8.77 - 8.71 (m, 2H), 8.58 (dd, J = 8.5, 1.0 Hz, 1H), 8.32 (d, J = 8.6 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.93 (dd, J = 8.6, 7.4 Hz, 1H), 7.87 - 7.79 (m, 2H), 7.62 (dd, J = 7.3, 0.9 Hz, 1H), 7.48 - 7.32 (m, 7H), 7.27 - 7.24 (m, 4H), 7.14 (t, J = 7.3 Hz, 2H).

[0030] 13 13C NMR (101 MHz, CDCl3) δ 164.63, 164.41, 150.68, 148.95, 148.59, 147.69, 147.29, 133.73, 132.29, 131.99, 131.74, 131.61, 130.86, 130.82, 130.35, 129.62, 129.56, 129.22, 127.92, 127.52, 126.93, 125.74, 125.16, 123.79, 122.72, 122.43, 121.97, 120.98, 120.21, 115.45. ESI - HRMS (m / z): [m] calculated for ([C 39 H 25 N3O2]+H)+(m / e, e = 1): 568.1947, found 568.2013.

[0031] ③ Synthesis of compound NQL: Compound 2 (284 mg, 0.5 mmol), 1-(4-(bromomethyl)phenyl)ethanone (106 mg, 0.75 mmol) and 5 mL of dimethylformamide (DMF) were placed in a 20 mL round-bottom flask and reacted at 80 °C for 24 h. The solvent was removed. The red product was recrystallized from a mixture of methanol and dichloromethane (DCM) to give 323 mg (82.73%).

[0032] 1 1H NMR (400 MHz, CD3OD_SPE) δ 9.33 (d, J = 6.9 Hz, 2H), 8.70 (d, J = 7.8 Hz, 2H), 8.62–8.57 (m, 1H), 8.40 (d, J = 6.9 Hz, 2H), 8.15 (d, J = 8.3 Hz, 2H), 7.90 (dd, J = 15.0, 7.5 Hz, 2H), 7.73 (t, J = 7.2 Hz, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.42–7.32 (m, 4H), 7.18 (ddd, J = 21.3, 14.0, 8.0 Hz, 7H), 6.79 (dd, J = 16.4, 8.0 Hz, 1H), 6.04 (d, J = 8.5 Hz, 2H), 2.66 (s, 3H).

[0033] 13 C NMR (101 MHz, CDCl3) δ 167.25, 151.29, 134.62, 133.53, 133.20, 132.99, 131.65, 128.81, 127.53, 126.04, 124.16, 67.58, 29.38. ESI-HRMS (m / Z): [m] calculated for ([C 48 H 34 N3O3]) + (m / e, e = 1): 700.8175, found 700.2571.

[0034] ④ Synthesis of compound CS-NQL: Under nitrogen atmosphere, chitosan (100 mg, 0.62 mmol) and NQL (434 mg, 0.62 mmol) were dissolved in a mixed solvent of DMSO (30 mL) and water (3 mL), and then the mixture was stirred at 100 °C for 24 h. After the reaction was completed, the product was washed repeatedly with DCM and methanol until small molecules were removed. The final product was dried in a vacuum oven to obtain a black solid powder, identified as CS-NQL (the detection results are shown in Figure 1-2 ).

[0035] Example 2: Spectral properties of the water-soluble fluorescent probe The present invention designed a water-soluble fluorescent probe CS-NQL based on chitosan with lysosome targeting ability and viscosity sensing function. The probe exhibits a twisted intramolecular charge transfer (TICT) state, indicating its sensitivity to environmental viscosity. The viscosity sensitivity of CS-NQL at different water-glycerol ratios was studied using fluorescence spectroscopy. By Figure 3As can be seen from Figure 3 Figure (b)),

[0036] Considering the complexity of biological systems, in order to accurately detect viscosity changes under complex conditions, the following interference experiments were carried out: First, the fluorescence changes in different solvents were detected. The results are shown in Figure 3 Figure (c). Compared with other solvents, it was found that the viscosity of glycerol significantly enhanced the fluorescence response, indicating that the interference of these solvents was minimal. Subsequently, interference experiments were carried out using interfering substances (LPA, GSH, CYS, AMP, ADP, ATP, K + , Na + , Ca 2+ , Mg 2+ , H2S, HClO, H2O2, ·OH, 1 O2 and ONOO−) to evaluate the selectivity of the probe. The results showed that the fluorescence of CS-NQL remained basically unchanged under various interfering substances. These findings indicate that the probe has great potential as a fluorescent probe for detecting cell viscosity and can be further used to monitor viscosity changes in the physiological environment. The density functional theory (DFT) was used to study the response mechanism of CS-NQL to viscosity. Since probes with lower oscillator strengths are less likely to undergo transitions, and the oscillator strength of CS-NQL is 0.42 ( Figure 3 Figure (d)), it was confirmed that the fluorescence enhancement of the probe was due to twisted intramolecular charge transfer.

[0037] Example 3: Cell imaging Considering the excellent selectivity and high sensitivity of CS-NQL, the present invention evaluated its potential use for cell imaging. Before imaging, the cytotoxicity of CS-NQL was evaluated. The results showed that CS-NQL exhibited negligible cytotoxicity, which may be attributed to the biocompatibility of chitosan. Studies have shown that cancer cells and inflammatory cells have higher viscosities than normal cells. Confocal microscopy fluorescence imaging was performed on L929, HeLa, and Raw 264.7 cells. After incubation for 60 min, HeLa and Raw 264.7 cells showed stronger fluorescence intensities than L929 cells (see Figure 4 ). These findings indicate that CS-NQL can distinguish abnormal cells from normal cells based on viscosity differences, indicating its potential for further use in in vivo tumor imaging.

[0038] Mitochondria and lysosomes were stained with MitoTracker Deep Red FM (MDRF) and LysoTracker Red DND-99, respectively. As shown in Figure 5 (a)-(d), the fluorescence imaging of CS-NQL did not show significant overlap with that of the mitochondrial probe, and the Pearson colocalization coefficient was 0.56, indicating that CS-NQL did not target mitochondria (see Figure 5 (e)). In contrast, the fluorescence signal of CS-NQL was closely aligned with that of the lysosome probe, resulting in an ideal Pearson correlation coefficient of 0.93 ( Figure 5 (f)-(j)). This high correlation indicated that CS-NQL could effectively target lysosomes within cells. This targeting ability was beneficial for lysosomal localization. Therefore, this probe could be used as an effective tool for detecting lysosomal viscosity.

[0039] Example 4: Fluorescence imaging in a mouse model To explore the ability of CS-NQL to detect high viscosity, fluorescence imaging was performed in tumor-bearing mice using CS-NQL. As shown in Figure 6 (a), the tumor-bearing mouse model was initially established by injecting HeLa cells into mice. Subsequently, CS-NQL was administered to the tumor-bearing mice via tail vein injection. Obvious fluorescence signals were observed at the tumor sites of the mice. In addition, as shown in Figure 6 (b)-(c), at the tumor sites of the tumor-bearing mice, the fluorescence signal of the CS-NQL probe gradually increased over time and reached a peak at 3 h after injection. These findings indicated that CS-NQL could be used as a valuable tool for early tumor diagnosis by virtue of its ability to image tumors by taking advantage of the high viscosity within cells.

[0040] Example 5: Formation of CS-NQL@DNA nanoparticles and their photosensitive properties The interaction between DNA and CS-NQL was designed with the expectation of achieving absorption in the near-infrared region. As shown in Figure 7 (a), with the addition of DNA, the UV absorption at 660 nm gradually increased. In contrast, NQL that was not conjugated to chitosan showed minimal change in UV absorption when interacting with DNA ( Figure 8 (a)). The NQL after DNA addition was attributed to the polymerization of chitosan and DNA through electrostatic interaction, ultimately forming nanoparticles. Further studies using dynamic light scattering (DLS) showed that the size of the nanoparticles increased with the increase in DNA concentration, thus confirming that DNA could interact with chitosan to form aggregates (see Figure 7(b)). Subsequent experiments were carried out using nanoparticles (CS-NQL@DNA NPs) formed by the interaction of CS-NQL (10 μg / mL) and DNA (6 μg / mL). Transmission electron microscopy (TEM) images of CS-NQL were obtained using water as a dispersant. As can be seen from Figure 7 (c), CS-NQL can be uniformly dispersed in water. CS-NQL@DNA NPs, CS-NQL, and NQL generate reactive oxygen species (ROS) under 660 nm laser irradiation (0.4 W / cm 2 ) and were evaluated using the commercially available fluorescent indicator 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The ROS generated by CS-NQL@DNA NPs under 660 nm laser irradiation highlights their photosensitizing ability; in contrast, CS-NQL and NQL cannot generate ROS under the same irradiation conditions due to their negligible absorption at 660 nm (see Figure 7 (d) and Figure 8 (b)-(d)). To determine the pathway by which CS-NQL@DNA NPs generate ROS, commercially available singlet oxygen sensor green (SOSG), dihydrorhodamine 123 (DHR123), and aminophenyl fluorescein (APF) were used as 1 O2, O2 ·− and ·OH indicators, respectively, to identify the types of reactive oxygen species (ROS) generated by the nanoparticles. Only DHR 123 showed a significant increase in fluorescence at 530 nm (see Figure 7 (e) and Figure 9 ). This indicates that CS-NQL@DNA NPs generate O2 ·− through a type I photodynamic mechanism. This observation implies an oxygen-dependent reduction in the photosensitivity of CS-NQL@DNA NPs. To confirm the generation of ROS under hypoxic conditions, the ability of CS-NQL to generate ROS under such conditions was evaluated using DCFH-DA. The results showed that the fluorescence intensity increased with prolonged light exposure, thus confirming the generation of ROS under hypoxic conditions ( Figure 7 (f)). This indicates that CS-NQL@DNA NPs can promote photodynamic therapy even in the hypoxic microenvironment of tumors.

[0041] Example 6: Photodynamic therapy using CS-NQL-NP in HeLa cells To investigate the photodynamic therapy ability of CS-NQL@DNA NPs intracellularly, the ability of these nanoparticles to generate ROS intracellularly was evaluated using confocal microscopy. HeLa cells were treated with the nanoparticles and DCFH-DA for 1 h and irradiated with a 660 nm laser (0.4 W / cm 2), and the fluorescence change was observed from below. The fluorescence intensity increased with the prolongation of the exposure time, indicating that CS-NQL@DNA NPs could generate ROS intracellularly (see Figure 10 ). After irradiating HeLa cells loaded with the probe for 20 min and then incubating them with the fluorescent marker AV647 for 30 min, most cells showed apoptosis. The apoptotic effect of CS-NQL@DNA NPs on cells after irradiation (0.4 W / cm 2 ) for 20 min was further evaluated using the MTT assay. More than 90% of HeLa cells were killed by CS-NQL@DNA nanoparticles after irradiation, confirming that the nanoparticles could induce apoptosis of tumor cells through photodynamic therapy (PDT).

[0042] Example 7: Photodynamic antibacterial potential of CS-NQL@DNA NPs PDT can induce apoptosis and also has antibacterial properties. To evaluate the photodynamic antibacterial potential of CS-NQL@DNA NPs, Gram-negative bacteria (Escherichia coli) and Gram-positive bacteria (Staphylococcus aureus) were used for verification. After co-incubation with CS-NQL@DNA NPs (100 μg / mL), both Gram-negative and Gram-positive bacteria showed significant growth inhibition under 660 nm laser irradiation (0.4 W / cm 2 ). In the absence of light, the addition of CS-NQL@DNA NPs alone could achieve partial antibacterial effects, which might be attributed to the inherent antibacterial properties of chitosan (see Figure 11 ). Therefore, CS-NQL NPs can achieve photodynamic antibacterial effects, thus broadening their application prospects in disease treatment.

[0043] Example 8: Antitumor therapy in vivo Based on tumor targeting and in vitro photodynamic effects, the antitumor effect of CS-NQL@DNA NPs in an orthotopic HeLa tumor mouse model was studied. The treatment protocol was carried out according to Figure 12 (a) The schematic diagram. When the tumor volume reached approximately 50 mm³, the animals were randomly divided into 4 groups (n = 3) for intravenous injection. The dose of CS-NQL@DNA NPs (1 mg / mL) was 100 μL. After injection, the tumor was irradiated with a 660 nm laser (0.4 W / cm 2 ) for 30 min. The same treatment protocol was used 3 days later, and then the tumor growth was monitored. After a 16-day observation period, a detailed analysis of the treatment results was performed. Compared with PBS, PBS + LIGHT, and CS-NQL@DNA NPs, the CS-NQL@DNA NPs + LIGHT group showed a significant ability to inhibit the rapid growth of tumors (see Figure 12 (b) and Figure 13), the relative tumor volume of the CS-NQL@DNA NPs + Light group was significantly reduced. While the control group showed a rapid and continuous increase in tumor volume (see Figure 12 (c)). In addition, treatment with CS-NQL@DNA NPs + light did not cause significant changes in body weight (see Figure 12 (d)), indicating that CS-NQL@DNA NPs have minimal toxicity to organisms. After 16 days, the major organs (heart, liver, spleen, lungs, and kidneys) were excised from all groups of mice and histopathological analysis was performed using HE staining. HE-stained sections of the major organs showed no abnormal changes (see Figure 14 ), further demonstrating that CS-NQL@DNA NPs not only have excellent photodynamic therapy efficiency but also minimal side effects on organisms.

[0044] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims

1. A small molecule compound containing a naphthalimide fluorescent group, characterized in that, The molecular structural formula of the small molecule compound is shown in Formula (I): （Ⅰ）。 2. A water-soluble fluorescent probe, characterized in that, It includes the small molecule compound described in Claim 1, and its molecular structural formula is shown in (II): （Ⅱ） Wherein, m and n are degrees of polymerization, m is 1000 - 5000, and n is 100 - 500.

3. The preparation method of the water-soluble fluorescent probe according to claim 2, characterized in that, It includes the following steps: (1) Prepare the small molecule compound described in Claim 1; (2) React chitosan with the small molecule compound in step (1) at 80 - 120 °C to obtain it.

4. The preparation method according to claim 3, characterized in that, The preparation of the small molecule compound described in Claim 1 in step (1) includes the following steps: (1) Add absolute ethanol to 4-bromo-1,8-naphthalic anhydride and 4-aminoquinoline, reflux at 80 - 100 °C, then filter and collect the precipitate to obtain Compound 1; (2) Dissolve Compound 1, 4-(diphenylamino)phenylboronic acid, Pd(PPh3)4 and K2CO3 in a solvent and reflux, then dissolve the obtained product in dichloromethane to obtain Compound 2; (3) React Compound 2, 1-(4-(bromomethyl)phenyl)ethanone and dimethylformamide at 60 - 100 °C to obtain it.

5. The preparation method according to claim 4, characterized in that, The solvent described in step (2) is toluene and water.

6. Use of the water-soluble fluorescent probe described in Claim 2 in the preparation of tumor in vivo imaging reagents and nanoparticles.

7. A nanoparticle, characterized in that, It includes the water-soluble fluorescent probe described in Claim 2, and the nanoparticles are formed by electrostatic self-assembly of the water-soluble fluorescent probe and DNA.

8. Use of the nanoparticles described in Claim 7 in the preparation of anti-tumor drugs.

9. The application according to claim 8, wherein The tumor includes ovarian cancer.

Images