A small molecule compound, a water-soluble fluorescent probe and a preparation method and application thereof

Abstract

The application discloses a small-molecule compound, a water-soluble fluorescent probe and a preparation method and application thereof, and belongs to the technical field of medicines. A small-molecule compound NQL containing a naphthalimide fluorescent group is synthesized, and then a water-soluble fluorescent probe CS-NQL is prepared by forming a Schiff base and coupling with oligomeric chitosan. The CS-NQL has environmental viscosity responsiveness and lysosome targeting specificity, and thus can be used for tumor and inflammatory cell imaging in the visible light range. The CS-NQL is electrostatically self-assembled with DNA to form nanoparticles CS-NQL@DNA NPs, which can generate reactive oxygen under hypoxic conditions; under 660nm laser irradiation, the CS-NQL@DNA NPs can induce HeLa cell apoptosis and exhibit antibacterial effect. In vivo experiments show that the CS-NQL@DNA NPs exhibit a strong anti-tumor immune response under 660nm laser irradiation, resulting in complete eradication of tumors.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine, in particular to a small molecule compound, a water-soluble fluorescent probe and a preparation method and application thereof. BACKGROUND

[0002] Traditional oncology methods face substantial limitations in both diagnosis and treatment, including inaccurate early diagnosis, severe side effects, multidrug resistance, limited treatment efficacy and low efficacy on metastatic tumors. The development of biological probes opens up a new field for cancer treatment, which is expected to improve the accuracy of diagnosis and treatment. Near-infrared fluorescent probes become a good candidate in the treatment field due to their superior optical properties, including deep tissue penetration and low background self-interference. At present, the development of near-infrared probes mainly depends 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 yield and high cost. On the other hand, the light stability, biocompatibility and targeting of the synthesized products still need to be further optimized. SUMMARY

[0003] In order to solve the above-mentioned deficiencies existing in the prior art, the purpose of the present application is to provide a small molecule compound, a water-soluble fluorescent probe and a preparation method and application thereof, so as to improve the absorption range and targeting of the near-infrared probe.

[0004] The technical scheme for solving the above technical problems of the present application is as follows: a small molecule compound containing a naphthalimide fluorescent group is provided, and the molecular structure formula of the small molecule compound is shown as formula (I):

[0005]

[0006] (I).

[0007] The present application provides a water-soluble fluorescent probe, which comprises the above-mentioned small molecule compound, and the molecular structure formula is shown as (II):

[0008]

[0009] (II)

[0010] Wherein, m and n are the degree of polymerization, m is 1000-5000, and n is 100-500.

[0011] The present application provides a preparation method of the above-mentioned water-soluble fluorescent probe, comprising the following steps:

[0012] (1) preparing the above-mentioned small molecule compound;

[0013] (2) reacting the chitosan and the small molecule compound of step (1) at 80-120°C to obtain.

[0014] Further, the small molecule compound of step (1) is prepared by the following steps:

[0015] (1) adding anhydrous ethanol to 4-bromo-1,8-naphthalic anhydride and 4-aminoquinoline, refluxing at 80-100°C, then collecting the precipitate by filtration to obtain compound 1;

[0016] (2) dissolving compound 1, 4-(diphenylamino)phenylboronic acid, Pd(PPh3)4 and K2CO3 in a solvent, refluxing, then dissolving the obtained product in dichloromethane to obtain compound 2;

[0017] (3) reacting compound 2, 1-(4-(bromomethyl)phenyl)ethanone and dimethylformamide at 60-100°C to obtain.

[0018] Further, the solvent in step (2) is toluene and water.

[0019] The application provides application of the water-soluble fluorescent probe in preparation of a tumor in-vivo imaging reagent and nanoparticles.

[0020] The application provides a kind of nanoparticles, including the water-soluble fluorescent probe described above, and the nanoparticles are formed by electrostatic self-assembly of water-soluble fluorescent probe and DNA.

[0021] The application provides application of the nanoparticles described above in preparation of a tumor treatment drug.

[0022] Further, the tumor includes ovarian cancer.

[0023] The present application has the following beneficial effects: the present application synthesizes a small molecule NQL containing a naphthalimide fluorescent group. In order to reduce cytotoxicity and realize DNA regulation function, chitosan is introduced into NQL to prepare a water-soluble fluorescent probe CS-NQL. When excited by a wavelength in the visible light range, CS-NQL can detect the high viscosity environment in 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 of chitosan and DNA, CS-NQL@DNA NPs are formed. CS-NQL@DNA NPs produce ROS through type I mechanism, indicating that these nanoparticles can promote PDT even in the hypoxic tumor microenvironment. In vivo experiments show that under 660nm laser irradiation (0.4 W / cm²), CS-NQL@DNA NPs can induce a strong anti-tumor immune response, leading to complete eradication of tumors. At the same time, CS-NQL@DNA NPs show low toxicity and minimal damage to other biological tissues. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 FTIR spectra of CS and CS-NQL; 1 H NMR spectrum;

[0025] Figure 2 FTIR spectra of CS and CS-NQL;

[0026] Figure 3 Spectral response graph of CS-NQL to viscosity;

[0027] Figure 4 Fluorescence images of various cells stained with CS-NQL for 60 min;

[0028] Figure 5 Confocal images of live HeLa cells stained with CS-NQL;

[0029] Figure 6 Fluorescence imaging of mouse models;

[0030] Figure 7 CS-NQL@DNA nanoparticle images and their photosensitization characteristics;

[0031] Figure 8 Absorption spectrum of NQL when DNA is added in water;

[0032] Figure 9 Fluorescence change graph of SOSG and APF containing CS-NQL@DNA NPs under 660nm laser irradiation for 360s;

[0033] Figure 10 Fluorescent images of intracellular ROS generation ability of CS-NQL@DNA NPs in HeLa cells;

[0034] Figure 11 Photodynamic antibacterial images of CS-NQL@DNA NPs;

[0035] Figure 12 Treatment images of HeLa tumor model;

[0036] Figure 13 In vivo treatment images of HeLa tumor mice at different periods;

[0037] Figure 14 HE staining images of main organs of mice in different treatment groups after 16 days of treatment. DETAILED DESCRIPTION

[0038] The following examples are intended to illustrate but not limit the present application. Unless otherwise indicated, conventional conditions or manufacturer's recommended conditions are used in the examples. Unless otherwise indicated, reagents or instruments used are conventional products available commercially.

[0039] Experimental methods:

[0040] (1) Cytotoxicity experiment: Cell viability was repeatedly measured using the standard MTT assay. Cells were seeded into 96-well plates (1 x 10 4 cells per well) containing 100 µL of growth medium and incubated for 24 h to allow attachment. The probe solution was diluted to the desired concentration with medium and then incubated in the dark for 24 h. 50 µL of 2.5 mg / mL 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide solution (dissolved in PBS solution) was dispensed into each well and incubated in the dark for 3 h. Then, the medium was aspirated from all wells and 100 µL of dimethyl sulfoxide (DMSO) was added. The optical density at 570 nm in each well was evaluated using a Molecular Devices MaxF5 multi-mode microplate reader with appropriate wavelength filters. All absorbance readings were adjusted according to the background absorbance. The survival rate of cells was calculated according to the following formula: Survival rate (%) = (number of living cells / total number of cells) x 100%.

[0041] (2) Cell culture and imaging: L929 cells, HeLa cells and RAW264 cells were cultured in DMEM medium enriched with 10 wt% fetal bovine serum, sodium pyruvate, L-glutamine and 4.5 g / L glucose at 37 °C in a humidified incubator with 95% air and 5% CO2. When the cell confluency reached 80%, the cells were passaged and cultured in DMEM containing 10 wt% fetal bovine serum (FBS) and 1 wt% streptomycin / penicillin antibiotics. After 24 h of culture in a culture dish, HeLa cells were washed with PBS, then treated with CS-NQL probe (10 pg / mL) and further incubated in a 37 °C incubator for 1 h. To confirm the localization of CS-NQL probe in organelles, HeLa cells were co-localized with CS-NQL probe (10 pg / mL) and lysosome tracker (0.5 mM) or mitochondria tracker (0.5 mM). HeLa cells were treated with CS-NQL probe together with LysoTracker Red DN-99 or MitoTracker DeepRed FM for 1 h, then washed with PBS before imaging, and the co-localization coefficient of green and red channels was measured.

[0042] (3) Reactive oxygen species detection: CS-NQL probe (20 pg / mL) was mixed with DCFH (2 mM), SOSG (2 mM), APF (2 mM) and DHR123 (2 mM), respectively, and the solution was irradiated with white light (20 mm / cm 2 ) and the fluorescence data was collected. CS-NQL probe (20 pg / mL) and DHR123 (2 mM) were added to the confocal dish containing HeLa cells, then incubated for 30 min, followed by irradiation with white light (20 mm / cm 2 ) and the data was recorded using a confocal laser scanning microscope.

[0043] (4) Tumor model and in vivo imaging: Nude mice, 4-5 weeks old, were from the Experimental Animal Center of Chuanbei Medical College in Nanchong, China. All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee of Chuanbei Medical College. 1 x 10 6 HeLa cells were injected subcutaneously into the right hind leg of these mice under sterile conditions. Then the mice were individually caged in a sterile environment with free access to food and water. When the HeLa tumor volume reached about 150 mm 3 , 100 pL 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.

[0044] (5) In vivo photodynamic therapy: When the tumor volume reached about 50 mm 3All nude mice carrying subcutaneous HeLa tumors were randomly divided into four groups (n=3): PBS, PBS+Light, CS-NQL NPs, and CS-NQL NPs+Light. The intratumoral injection volume of PBS and CS-NQL NPs (1 mg / mL) was 25 μL per 50 mm³ tumor. After intratumoral injection, mice in the PBS+Light and CS-NQL NPs+Light groups were exposed to light at 660 nm (0.4 W / cm²). 2 The mice in the first group were irradiated with laser for 30 minutes, while the other groups were not irradiated. During the treatment period, the tumor volume was measured daily using calipers, and the calculation formula was: Tumor volume = (width × width × length) / 2. After 16 days of treatment, tumors and major organs (including heart, liver, spleen, lung, and kidney) were harvested from all groups of mice, and histopathological analysis was performed by hematoxylin-eosin (HE) staining. Histopathological changes were observed using an optical microscope.

[0045] (7) Statistical analysis: One-way ANOVA was used for statistical analysis. Data are expressed as mean ± standard deviation (SD). P ≤ 0.05 was considered statistically significant (*: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001).

[0046] Example 1: Synthesis of a water-soluble fluorescent probe (CS-NQL)

[0047] The synthesis route is as follows:

[0048]

[0049] ① Synthesis of Compound 1: 4-Bromo-1,8-naphthalenedicarboxylic anhydride (2.00 g, 7.22 mmol) and 4-aminoquinoline (1.44 g, 10.00 mmol) were mixed in a 100 mL double-necked flask, and then 25 mL of anhydrous ethanol was added. The mixture was refluxed at 90 °C for 12 h. After the reaction was complete, the mixture was cooled to room temperature and the precipitate was collected by filtration. The precipitate was then recrystallized from ethanol to give 1.89 g of off-white solid product, with a yield of 64.97%.

[0050] 1H NMR (400 MHz, DMSO) δ 8.97 (DD, J = 4.1, 1.6 Hz, 1H), 8.74 - 8.56 (M, 2H), 8.43 (D, J = 8.0 Hz, 1H), 8.38 (D, J = 7.9 Hz, 1H), 8.31 (D, J = 7.9 Hz, 1H), 8.19 (D, J = 8.5 Hz, 1H), 8.08 (dd, J = 8.4, 7.4 Hz, 1H), 7.93 (dd, J = 8.5, 7.4 Hz, 1H), 7.76 (dd, J = 7.3, 1.0 Hz, 1H), 7.49 (DD, J = 8.6, 4.1 Hz, 1H).

[0051] 13 C NMR (101 MHz, 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.

[0052] ESI-HRMS (m / Z): [M] calcd for (C 21 H 11 N2O2Br]+H)+( m / e,e=1) : 403.0004, found 403.0072.

[0053] 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 30 mL of toluene and 10 mL of water mixed solvent, then refluxed under nitrogen for 24 h. The reaction progress was monitored by TLC. After the reaction was completed, the solvent was removed, then 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%).

[0054] 1 H NMR (400 MHz, CDCl3) δ 8.99 (dd, J = 4.2, 1.6 Hz, 1H), 8.77 - 8.71 (m, 2H),

[0055] 8.58(dd,J=8.5,1.0Hz,1H),8.32(d,J=8.6Hz,1H),8.03(d,J=7.9Hz,1H),7.93(dd,J=8.6,7.4Hz,1H),7. 87-7.79(m,2H),7.62(dd,J=7.3,0.9Hz,1H),7.48-7.32(m,7H),7.27-7.24(m,4h),7.14(T,J=7.3Hz,2h).

[0056] 13 C NMR (101MHz, CDCl3) δ 164.63,164.41,150.68,148.95,148.59,147.69,

[0057] 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 values ​​for ([C 39 H 25 N3O2]+H)+(m / e,e=1):568.1947,Measured value 568.2013。

[0058] ③ Synthesis of compound NQL: Compound 2 (284 mg, 0.5 mmol), 1-(4-(bromomethyl)phenyl)ethyl ketone (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 then removed. Recrystallization from a mixture of methanol and dichloromethane (DCM) yielded a red product, weighing 323 mg (82.73%).

[0059] 1 H NMR(400MHz, CD3OD_SPE)δ 9.33(D,J=6.9Hz,2H),8.70(D,J=7.8Hz,

[0060] 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).

[0061] 13 C NMR (101 MHz, CDC13) 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] calcd for ( [C 48 H 34 N3O3] )+ (m / e, e = 1): 700.8175, found 700.2571.

[0062] (4) Synthesis of compound CS-NQL: 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) under nitrogen atmosphere, and then the mixture was stirred at 100 °C for 24 h. After the reaction was completed, the product was repeatedly washed with DCM and methanol until the small molecules were removed. The final product was dried in a vacuum oven to obtain a black solid powder identified as CS-NQL (the test results are shown in Table 1). Figures 1-2 ).

[0063] Example 2: Spectral properties of water-soluble fluorescent probe

[0064] The present application designs a water-soluble fluorescent probe CS-NQL based on chitosan with lysosome targeting ability and viscosity sensing function. The probe shows a twisted intramolecular charge transfer (TICT) state, showing its sensitivity to environmental viscosity. The viscosity sensitivity of CS-NQL under different water-glycerol ratios was studied using fluorescence spectrum. From the results of the experiment, it can be seen that the fluorescence intensity of CS-NQL is significantly reduced in the glycerol solution, and the fluorescence intensity of CS-NQL is significantly reduced in the glycerol solution. Figure 3(a) As can be seen, the fluorescence intensity of CS-NQL gradually increased with increasing glycerol ratio. In the viscosity range of 1.31 MPa-s (10 wt% glycerol) to 1150.0 MPa-s (90 wt% glycerol), the intensity (I) at the maximum fluorescence peak was measured and a scatter plot was generated with log η vs log I, and a linear equation was fitted in the linear range to obtain the standard curve. A good linear relationship between log I and log η was observed at 440 nm (R2= 0.9964) (see Figure 3 (b).

[0065] 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, and the results are shown in Figure 3 (c) It was found that the viscosity of glycerol significantly enhanced the fluorescence response compared to other solvents, 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, HCIO, H2O2, ·OH, 1 O2 and ONOO−) to evaluate the selectivity of the probe. The results showed that the fluorescence of CS-NQL was essentially unchanged under a variety of interfering substances. These findings indicate that the probe has great potential as a fluorescence probe for cell viscosity detection and can be further used to monitor viscosity changes in physiological environments. The response mechanism of CS-NQL to viscosity was studied using density functional theory (DFT), and since probes with low oscillator strength are less likely to undergo transition, the oscillator strength of CS-NQL is 0.42 ( Figure 3 (d)), confirming that the fluorescence enhancement of the probe is due to twisted intramolecular charge transfer.

[0066] Example 3: Cell imaging

[0067] Considering the excellent selectivity and high sensitivity of CS-NQL, the present application evaluated its potential use for cell imaging. Prior to imaging, the cytotoxicity of CS-NQL was evaluated. The results showed that CS-NQL exhibited negligible cytotoxicity, which can be attributed to the biocompatibility of chitosan. Studies have shown that cancer cells and inflammatory cells have higher viscosity than normal cells. Confocal fluorescence imaging of L929, HeLa and Raw 264.7 cells was performed. After 60 min of incubation, HeLa and Raw 264.7 cells showed stronger fluorescence intensity than L929 cells (see Figure 4). These findings suggest that CS-NQL can differentiate abnormal cells from normal cells based on viscosity differences, indicating its potential for further use in tumor imaging in vivo.

[0068] MitoTracker Deep Red FM (MDRF) and LysoTracker Red DND-99 were used to stain mitochondria and lysosomes, respectively. As shown in Figure 5 (a)-(d), the fluorescence imaging of CS-NQL did not significantly overlap with the fluorescence imaging of the mitochondrial probe, with a Pearson's colocalization coefficient of 0.56, indicating that CS-NQL is not mitochondria-targeted (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's correlation coefficient of 0.93 (see Figure 5 (f)-(j)). This high correlation indicates that CS-NQL can effectively target lysosomes within cells. This targeting ability is beneficial for lysosome localization. Therefore, this probe can serve as an effective tool for detecting lysosome viscosity.

[0069] Example 4: Fluorescence imaging in a mouse model

[0070] To explore the ability of CS-NQL to detect high viscosity, fluorescence imaging was performed in tumor mice using CS-NQL. As can be seen from Figure 6 (a), the tumor mouse model was initially established by injecting HeLa cells into mice. Subsequently, CS-NQL was administered to the tumor mice by tail vein injection. A distinct fluorescence signal was observed at the tumor site of the mice. In addition, as shown in Figure 6 (b)-(c), the fluorescence signal of the CS-NQL probe gradually increased over time at the tumor site of the tumor mice, reaching a peak at 3 h after injection. These findings suggest that CS-NQL can utilize its ability to image tumors by exploiting intracellular high viscosity as a valuable tool for early tumor diagnosis.

[0071] Example 5: Formation of CS-NQL@DNA nanoparticles and their photosensitive properties

[0072] The interaction between DNA and CS-NQL was designed to achieve 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 was not conjugated with chitosan, and the change in ultraviolet absorption was minimal when interacting with DNA (see Figure 8(a)). The NQL following DNA addition is attributed to the polymerization of chitosan and DNA through electrostatic interactions, ultimately forming nanoparticles. Further studies using dynamic light scattering (DLS) showed that the size of the nanoparticles increased with increasing DNA concentration, thus confirming that DNA can interact with chitosan to form aggregates (see [link to study]). Figure 7 (b) Subsequent experiments were conducted 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. Figure 7 (c) It can be seen that CS-NQL can be uniformly dispersed in water. CS-NQL@DNA NPs, CS-NQL, and NQL generate reactive oxygen species (ROS) (0.4 W / cm²) under 660 nm laser irradiation. 2 The evaluation was performed 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 highlighted their photosensitivity; conversely, CS-NQL and NQL failed to generate ROS under the same irradiation conditions due to their negligible absorption at 660 nm (see [link to relevant documentation]). Figure 7 (d) and Figure 8 (b)-(d)). To determine the pathway of ROS generation by CS-NQL@DNA NPs, commercially available singlet oxygen sensor green (SOSG), dihydrorhodamine 123 (DHR123), and aminophenylfluorescein (APF) were used as... 1 O2, O2 ·− Indicators of ·OH were used to identify the type of reactive oxygen species (ROS) generated by nanoparticles. Only DHR 123 showed a significant increase in fluorescence at 530 nm (see [link to DHR 123]). Figure 7 (e) and Figure 9 This indicates that CS-NQL@DNA NPs generate O2 through a type I photodynamic mechanism. ·− This observation implies a reduced oxygen dependence of the photosensitivity of CS-NQL@DNA NPs. To confirm ROS generation under hypoxic conditions, the ability of CS-NQL to generate ROS under such conditions was assessed using DCFH-DA. The results showed that fluorescence intensity increased with prolonged light exposure, thus confirming ROS generation under hypoxic conditions. Figure 7 (f) This indicates that CS-NQL@DNA NP can promote photodynamic therapy even in the hypoxic microenvironment of tumors.

[0073] Example 6: Photodynamic therapy in HeLa cells using CS-NQL-NP

[0074] To investigate the photodynamic therapy capabilities of CS-NQL@DNA NPs within cells, confocal microscopy was used to evaluate the ability of these nanoparticles to generate ROS intracellularly. HeLa cells were treated with the nanoparticles and DCFH-DA for 1 h and then irradiated with a 660 nm laser (0.4 W / cm²). 2 Fluorescence changes were observed under [the specified exposure time]. The fluorescence intensity increased with prolonged exposure time, indicating that CS-NQL@DNA NPs can generate ROS within cells (see [reference needed]). Figure 10 After irradiating HeLa cells carrying the probe for 20 min, followed by incubation with the fluorescent label AV647 for 30 min, most cells exhibited apoptosis. The irradiation (0.4 W / cm²) was further evaluated using an MTT assay. 2 The apoptotic effect of CS-NQL@DNA NPs on cells was observed 20 minutes later. Over 90% of HeLa cells were killed by CS-NQL@DNA nanoparticles after irradiation, confirming that nanoparticles can induce tumor cell apoptosis through photodynamic therapy (PDT).

[0075] Example 7: Photodynamic antibacterial potential of CS-NQL@DNA NPs

[0076] 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 validation. After co-incubation with CS-NQL@DNA NPs (100 μg / mL), Gram-negative and Gram-positive bacteria were subjected to 660 nm laser irradiation (0.4 W / cm²). 2 Significant growth inhibition was observed in all samples. In the absence of light, the addition of CS-NQL@DNA NPs alone achieved partial antibacterial effects, which may 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.

[0077] Example 8: In vivo anti-tumor therapy

[0078] Based on tumor targeting and in vitro photodynamic effects, the antitumor effects of CS-NQL@DNA NPs in an orthotopic HeLa tumor mouse model were investigated. Treatment regimens were as follows: Figure 12 (a) Schematic diagram. When the tumor volume reached approximately 50 mm³, 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 treated with a 660 nm laser (0.4 W / cm²).2 The tumor was irradiated for 30 minutes. Three days later, the same treatment regimen was administered, followed by monitoring of tumor growth. After a 16-day observation period, the treatment results were analyzed in detail. Compared with PBS, PBS+LIGHT, and CS-NQL@DNA NPs, the CS-NQL@DNA NPs+LIGHT group showed a significant inhibitory effect on rapid tumor growth (see...). Figure 12 (b) and Figure 13 The CS-NQL@DNA NPs+Light group showed a significant reduction in relative tumor volume, while the control group exhibited a rapid and sustained increase in tumor volume (see [link to relevant documentation]). Figure 12 (c)). Furthermore, light treatment with CS-NQL@DNA NPs did not result in significant changes in body weight (see [link]). Figure 12 (d) indicates that CS-NQL@DNA NPs have minimal toxicity to the organism. Sixteen days later, major organs (heart, liver, spleen, lung, and kidney) were removed from all mouse groups, and histopathological analysis was performed using HE staining. HE-stained sections of the major organs showed no abnormalities (see [link to article]). Figure 14 This further demonstrates that CS-NQL@DNA NPs not only have excellent photodynamic therapy efficiency, but also have minimal side effects on organisms.

[0079] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should 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 structure of the small molecule compound is shown in formula (Ⅰ): （Ⅰ）。 2. A water-soluble fluorescent probe, characterized in that, The small molecule compound according to claim 1 has the molecular structure shown in formula (II): （Ⅱ） Where m and n are the degree of polymerization, m is 1000-5000 and n is 100-500.

3. The method for preparing the water-soluble fluorescent probe according to claim 2, characterized in that, Includes the following steps: (1) Preparation of the small molecule compound according to claim 1; (2) Chitosan and the small molecule compound from step (1) are reacted at 80-120℃ to obtain the product.

4. The preparation method according to claim 3, characterized in that, Step (1) in preparing the small molecule compound of claim 1 includes the following steps: (1) After adding anhydrous ethanol to 4-bromo-1,8-naphthalenedicarboxylic anhydride and 4-aminoquinoline, the mixture was refluxed at 80-100℃, and then the precipitate was collected by filtration to obtain compound 1. (2) Compound 1, 4-(diphenylamino)phenylboronic acid, Pd(PPh3)4 and K2CO3 were dissolved in a solvent and refluxed. The resulting product was then dissolved in dichloromethane to prepare compound 2. (3) Compound 2, 1-(4-(bromomethyl)phenyl)ethyl ketone and dimethylformamide were reacted at 60-100℃ to prepare the product; The molecular structural formula of compound 1 is shown in formula (Ⅲ): （Ⅲ）； The molecular structure of compound 2 is shown in formula (Ⅳ): （Ⅳ）。 5. The preparation method according to claim 4, characterized in that, The solvents mentioned in step (2) are toluene and water.

6. The application of the water-soluble fluorescent probe according to claim 2 in the preparation of in vivo tumor imaging reagents and nanoparticles.

7. A nanoparticle, characterized in that, Includes the water-soluble fluorescent probe of claim 2, wherein the nanoparticles are formed by electrostatic self-assembly of the water-soluble fluorescent probe with DNA.

8. The application of the nanoparticles according to claim 7 in the preparation of antibacterial drugs, characterized in that, The antibacterial activity is against Escherichia coli and Staphylococcus aureus.

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