ICGCBF-loaded ZIF-8 nanoparticles and preparation and performance detection methods thereof

Abstract

The invention discloses a load ICGamp. The invention discloses ZIF-8 nano-particles of CBF, and belongs to the technical field of preparation of nano-drug particles. The multifunctional nanoparticles are formed by simultaneously encapsulating indocyanine green (ICG) and cinobufagin (CBF) components by ZIF-8, and the drug loading capacities of the two drugs are both not lower than 5%. The preparation method comprises the following steps: slowly adding a solution containing cinobufagin and zinc nitrate into a mixed solution containing indocyanine green, bovine serum albumin and 2-methylimidazole, and carrying out centrifugal filtration, washing and drying on a reaction product to obtain the cinobufagin zinc oxide. The multifunctional nano-particles prepared by a one-step synthesis method are uniform in particle size and good in stability, indocyanine green and cinobufagin are uniformly distributed in a carrier, the multifunctional nano-particles have pH and near-infrared light dual response drug release characteristics, triple synergy of chemotherapy, photothermal therapy and photodynamic therapy can be realized, and the multifunctional nano-particles are simple in preparation process and suitable for popularization and application.

Description

Technical Field

[0001] This invention relates to ZIF-8 nanoparticles loaded with ICG & CBF and their preparation and performance testing methods. Background Technology

[0002] Oral squamous cell carcinoma (OSCC), a common head and neck malignancy, still faces numerous challenges in treatment, including high tumor heterogeneity, resistance to traditional radiotherapy and chemotherapy, and significant side effects. In recent years, combination therapy strategies based on nanocarriers, especially multimodal synergistic therapies integrating chemotherapy, photothermal therapy (PTT), and photodynamic therapy (PDT), have provided new ideas for improving efficacy.

[0003] Zeolite imidazole ester framework material-8 (ZIF-8) has been widely studied as a drug carrier due to its good biocompatibility, high specific surface area, and degradability in the weakly acidic tumor microenvironment (see Chinese Patent CN202410923027.1). Indocyanine green (ICG) is a clinically approved optical contrast agent with both PTT and PDT functions, but it has inherent drawbacks such as instability in aqueous solution, easy photodegradation, and short in vivo circulation time, which limit its efficacy when used alone. Bufotoxin (CBF) is the main active ingredient in the traditional Chinese medicine toad venom and has clear antitumor activity, but its poor water solubility and low bioavailability are also prominent problems.

[0004] Current research has attempted to load chemotherapeutic drugs or photosensitizers onto ZIF-8. However, existing technologies mostly focus on enhancing a single treatment modality or simply co-loading two drugs. Designing an intelligent nanoplatform capable of simultaneously and efficiently loading hydrophobic chemotherapeutic drugs (CBF) and amphiphilic photosensitizers (ICG), ensuring their stable presence and on-demand release within the carrier, and thus achieving a triple synergistic effect of chemotherapy, PTT, and PDT, still faces technical bottlenecks such as complex fabrication processes and the difficulty in balancing drug loading efficiency and stability. In particular, how to potentially utilize CBF to regulate tumor metabolism and improve the hypoxic microenvironment through carrier design, thereby further enhancing the ICG-mediated PDT effect, remains a problem that current technologies have not yet systematically solved.

[0005] In the development of antitumor nanomedicines, evaluating their in vitro release behavior is a crucial step in predicting in vivo efficacy and optimizing formulation. Traditional drug release assays (such as the paddle method and basket method) typically employ constant pH values ​​(e.g., phosphate buffer at pH 7.4) and temperatures (37°C) to simulate physiological conditions. However, for smart responsive nanomedicines, especially multi-responsive carriers designed specifically for the tumor microenvironment, such a single detection condition is insufficient to reflect their true drug release characteristics.

[0006] Tumor tissues possess microenvironmental characteristics distinct from normal tissues; for example, the extracellular fluid is weakly acidic (pH approximately 6.5-7.0). Furthermore, many nanomedicines are designed to be activated by external stimuli (such as near-infrared light) to trigger drug release at specific times and locations. Therefore, objectively evaluating the differences in nanomedicine release under different pH conditions, and the triggering and accelerating effects of external physical stimuli (such as near-infrared photothermal effects) on drug release behavior, is crucial for assessing their targeting and intelligence.

[0007] Current technologies lack a standardized and reproducible in vitro detection method to simultaneously simulate and quantify the combined effects of two key factors, "pH change" and "near-infrared irradiation," on the release kinetics of nanomedicines. Most studies only examine a single factor independently or use self-built non-standard devices, making it difficult to directly compare data from different studies and failing to provide a unified and reliable technical basis for the performance evaluation and quality control of such advanced nanomedicines.

[0008] The efficiency of nanomedicine delivery systems ultimately depends on their interaction with target cells, including the efficiency of cellular uptake, the pathway into the cell, the intracellular transport fate (such as whether they are captured and degraded by lysosomes), and their ability to ultimately reach subcellular sites of action. Therefore, systematically characterizing these intracellular behaviors of nanoparticles is an important bridge connecting their physicochemical properties with their biological effects.

[0009] Currently, various techniques are commonly used in this field, such as flow cytometry for quantitative analysis of the total uptake of fluorescently labeled nanoparticles by cells, or confocal laser scanning microscopy for qualitative observation of subcellular localization. When studying uptake mechanisms, specific pharmacological inhibitors are also used to block different endocytic pathways. However, these methods are often conducted in isolation, lacking an integrated analytical workflow that moves from quantification to qualitative analysis, from phenomena to mechanisms, and from static to dynamic perspectives. For example, flow cytometry data cannot provide spatial distribution information, and microscopic images are difficult to use for high-throughput quantitative comparisons; the results of inhibitor experiments need to be corroborated with evidence of actual intracellular localization.

[0010] The shortcoming of existing technologies lies in their failure to standardize and integrate research across multiple dimensions, such as uptake kinetics, efflux processes, endocytosis mechanism identification, and visualization of intracellular transport pathways, into a single experimental system and evaluation framework. This results in a fragmented understanding of nanoparticle-cell interactions, hindering a deeper understanding of the limiting factors for delivery efficiency and making it difficult to systematically guide the rational design and optimization of nanocarriers. Summary of the Invention

[0011] The first technical problem to be solved by the present invention is to provide ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin, which have uniform particle size, high drug loading and good stability.

[0012] The second technical problem to be solved by the present invention is to provide a method for preparing ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin, which is simple in steps, easy to operate, and suitable for promotion.

[0013] The third technical problem to be solved by this invention is to provide a method for detecting the drug release performance of ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin under different conditions. This method simulates the drug release kinetics under the combined effects of the tumor micro-acidic environment and external therapeutic light irradiation by placing the nanodrug in a pH-adjustable release medium and periodically applying controllable near-infrared laser irradiation. This provides key technical support for optimizing nanodrug formulation and predicting its in vivo behavior.

[0014] The fourth technical problem to be solved by this invention is to provide a method for detecting the cellular uptake and intracellular distribution of ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin. This method integrates time-dependent uptake / efflux kinetics studies, uptake mechanism analysis based on multiple specific inhibitors, and visualization of intracellular transport pathways through organelle colocalization analysis, providing a complete technical solution for a deeper understanding of nanoparticle-cell interactions.

[0015] To solve the first technical problem mentioned above, the present invention provides a ZIF-8 multifunctional nanoparticle loaded with indocyanine green and bufotoxin, which is prepared by simultaneously encapsulating indocyanine green and bufotoxin with ZIF-8, wherein the loading amount of both indocyanine green and bufotoxin by ZIF-8 is not less than 5%.

[0016] To address the second technical problem mentioned above, this invention provides a method for preparing ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin, comprising the following steps:

[0017] (1) Disperse indocyanine green, bovine serum albumin and 2-methylimidazole in anhydrous methanol to obtain mixed solution A. Control the concentration of indocyanine green in solution A to be 6~12 mg / mL, the concentration of bovine serum albumin to be 10~25 mg / mL and the concentration of 2-methylimidazole to be 35~50 mg / mL.

[0018] (2) Disperse bufotoxin and zinc nitrate in anhydrous methanol to obtain solution B, and control the concentration of bufotoxin to be 10~20 mg / mL and the concentration of zinc nitrate to be 15~30 mg / mL.

[0019] (3) Under the conditions of 15~25℃ and continuous stirring, solution B is slowly added to solution A using a peristaltic pump. After the addition of solution B is completed, stirring is continued at 15~25℃ for 2~6 hours. After the reaction is completed, crude product is obtained.

[0020] (4) The crude product obtained in step (3) is centrifuged and filtered. After washing and drying, the precipitated product can be used to obtain ZIF-8 multifunctional nanoparticles loaded with indocyanine green and bufotoxin.

[0021] (5) After drying, the sample is stored in an opaque glass sample bottle and kept in an environment of 3~6℃.

[0022] Preferably, in step (3), the dripping rate of the peristaltic pump is 5~10 mL / min.

[0023] Preferably, in step (4), the centrifugation speed is 8000~10000 rpm and the time is 6~10 min.

[0024] Preferably, in step (4), the washing solvent is anhydrous methanol, the drying temperature is 25~30℃, and the drying time is 6~8 hours.

[0025] To address the third technical problem mentioned above, this invention provides a method for detecting the drug release performance of ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin under different conditions, comprising the following steps:

[0026] (1) Place 2 mL of 1.5 mg / mL nanoparticle aqueous solution into a dialysis bag with a molecular weight of 3500, and immerse it in a flask containing 78 mL of PBS buffer with pH values ​​of 5.5 and 7.4 respectively to release the medium. Shake continuously in a shaker at 37℃ or 43℃ for 48 h. Take out 3 mL of solution from the flask and add 3 mL of new PBS buffer every time interval.

[0027] (2) After oscillation, at a specific time point, a wavelength of 808 nm and a power density of 2 W·cm⁻¹ are used. -2 The dialysis bag area was irradiated with near-infrared laser for 5 minutes each time;

[0028] (3) Samples were taken at different time points, and the absorbance of the release medium at the characteristic absorption wavelength of the drug was measured to calculate the cumulative release rate.

[0029] To address the fourth technical problem mentioned above, this invention provides a method for detecting the cellular uptake and intracellular distribution of ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin, comprising the following steps:

[0030] (1) Fluorescently labeled nanoparticles were co-incubated with cells for different times, and the average fluorescence intensity in the cells was quantitatively detected by flow cytometry;

[0031] (2) Cells were pretreated with at least one inhibitor selected from chlorpromazine, nystatin, cytochalasin D, colchicine, and sodium azide, and then co-incubated with fluorescently labeled nanoparticles. The uptake inhibition rate was analyzed by flow cytometry.

[0032] (3) The cells were incubated with fluorescently labeled nanoparticles and lysosomal fluorescent probes, and the co-localization of the fluorescence of nanoparticles and lysosomal probes was observed using a confocal laser scanning microscope.

[0033] Advantages of this invention:

[0034] Bovine serum albumin: As a stabilizer, it protects the ZIF-8 carrier. Indocyanine green: Possesses unique imaging capabilities. Bufotoxin: Demonstrates excellent anticancer activity and immunomodulatory function. Using ZIF-8 as a carrier, indocyanine green and bufotoxin are encapsulated within the hollow cavity of ZIF-8, overcoming the water instability and thermal degradation of indocyanine green, while simultaneously addressing the poor water solubility and low bioavailability of bufotoxin. By utilizing the imaging capabilities of indocyanine green to determine the drug's direction of travel and real-time location, bufotoxin can be successfully delivered to the cancer cells of oral squamous cell carcinoma, thereby achieving targeted therapy.

[0035] The ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin group provided by this invention:

[0036] 1. Uniform particle size (approximately 100 nm), high drug loading (>5%), and good stability.

[0037] 2. It possesses dual drug controlled-release characteristics of "pH response" and "near-infrared photothermal response", enabling precise drug release triggered by tumor microenvironment and external light.

[0038] 3. It integrates three treatment modalities: chemotherapy (CBF), photothermal therapy (ICG), and photodynamic therapy (ICG), which theoretically can produce a synergistic effect.

[0039] 4. CBF may improve local hypoxia by regulating tumor cell metabolism, thereby creating favorable conditions for ICG-mediated PDT.

[0040] 5. The preparation steps are simple and efficient, easy to operate, and suitable for widespread application.

[0041] The ICG&CBF@ZIF-8 nanoparticles prepared in this invention successfully integrate two complementary drugs into a single carrier, achieving high drug loading and controlled release. Their dual-response characteristics better match the spatiotemporal requirements of tumor therapy, providing a new product solution for developing highly efficient and low-toxicity combination therapy nanoformulations.

[0042] The aforementioned method for detecting the drug release performance of ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin under different conditions simulates the drug release kinetics under the combined effects of the tumor micro-acidic environment and external therapeutic light irradiation by placing the nanodrug in a pH-adjustable release medium and periodically applying controllable near-infrared laser irradiation. This detection method has a high degree of standardization and good reproducibility, and can effectively simulate the combined effects of the tumor microenvironment and external treatment. It provides a reliable and practical technical means for the formulation screening, quality control and release mechanism research of smart nanodrugs, and provides key technical support for optimizing nanodrug formulations and predicting their in vivo behavior.

[0043] The method for detecting the cellular uptake and intracellular distribution of ZIF-8 nanoparticles loaded with indocyanine green and bufotoxin integrates time-dependent uptake / efflux kinetics, uptake mechanism analysis based on multiple specific inhibitors, and visualization of intracellular transport pathways through organelle colocalization analysis, providing a complete technical solution for a deeper understanding of nanoparticle-cell interactions. Attached Figure Description

[0044] Figure 1 This is the XRD pattern of the ICG&CBF@ZIF-8 nanoparticles provided by this invention.

[0045] Figure 2 This is a schematic diagram of the ICG&CBF@ZIF-8 structure provided by the present invention, where red represents ICG and green represents CBF. Detailed Implementation

[0046] Example 1:

[0047] Preparation of ICG&CBF@ZIF-8 nanoparticles

[0048] (1) Disperse indocyanine green, bovine serum albumin and 2-methylimidazole in anhydrous methanol to obtain mixed solution A. Control the concentration of indocyanine green in solution A to be 6~12 mg / mL, the concentration of bovine serum albumin to be 10~25 mg / mL and the concentration of 2-methylimidazole to be 35~50 mg / mL.

[0049] (2) Disperse bufotoxin and zinc nitrate in anhydrous methanol to obtain solution B, and control the concentration of bufotoxin to be 10~20 mg / mL and the concentration of zinc nitrate to be 15~30 mg / mL.

[0050] (3) Under conditions of 15~25℃ and continuous stirring, solution B is slowly added to solution A using a peristaltic pump. After the addition of solution B is completed, stirring is continued at 15~25℃ for 2~6 hours. After the reaction is completed, crude product is obtained.

[0051] (4) The crude product obtained in step (3) is centrifuged and filtered. After washing and drying, the precipitated product can be used to obtain ZIF-8 multifunctional nanoparticles loaded with indocyanine green and bufotoxin.

[0052] (5) After drying, the sample is stored in an opaque glass sample bottle and kept in an environment of 3~6℃.

[0053] In step (3), the dripping speed of the peristaltic pump is 5~10mL / min.

[0054] In step (4), the centrifugation speed is 8000~10000 rpm and the time is 6~10 min.

[0055] In step (4), the washing solvent is anhydrous methanol, the drying temperature is 25~30℃, and the drying time is 6~8 hours.

[0056] See Figure 1 and Figure 2 , Figure 1 This is the XRD pattern of ICG&CBF@ZIF-8 nanoparticles. Figure 2 This is a structural diagram of ICG & CBF@ZIF-8 (this is only a structural illustration and not an electron microscope image). Red represents ICG and green represents CBF.

[0057] The hydrated particle size of the obtained nanoparticles was determined to be 102.3 ± 8.5 nm (PDI = 0.12) using a dynamic light scattering particle size analyzer. Transmission electron microscopy showed that the particles exhibited a regular rhombic dodecahedral morphology and were well dispersed.

[0058] Drug loading and encapsulation efficiency determination

[0059] The supernatants from the three centrifugation steps following the CBF loading process in Example 1 were collected, combined, and then scanned across the entire wavelength range using a UV-Vis spectrophotometer. Based on the standard curve, the concentrations of free CBF and ICG in the supernatant were measured at 488 nm (characteristic absorption peak of CBF) and 780 nm (characteristic absorption peak of ICG), respectively.

[0060] The calculation parameters are as follows:

[0061] Drug loading (DLC) = (Total drug dosage - Free drug in supernatant) / Total mass of nanoparticles × 100%

[0062] Encapsulation efficiency (EE) = (Total drug dosage - Free drug content in supernatant) / Total drug dosage × 100%

[0063] Calculations show that the ICG&CBF@ZIF-8 nanoparticles prepared in this embodiment have a drug loading of 5.8% for ICG and an encapsulation efficiency of 89.2%; and a drug loading of 11.5% for CBF and an encapsulation efficiency of 85.6%.

[0064] Example 2:

[0065] This embodiment uses the evaluation of the release behavior of ICG&CBF@ZIF-8 nanoparticles as an example.

[0066] (1) Sample preparation: Accurately measure the equivalent of ICG&CBF@ZIF-8 nanoparticle dispersion containing 1.0 mg ICG (prepared according to the method in Example 1), inject it into the pretreated dialysis bag (molecular weight cutoff 3500 Da), remove air bubbles and tie it tightly.

[0067] (2) Release medium setup: Prepare two 500 mL clean release tanks, and add 400 mL of PBS buffer pre-warmed to 37°C to each. Adjust one to pH 7.4 (simulating normal tissue fluid) and the other to pH 5.5 (simulating the tumor tissue microenvironment). Place the release tanks in a constant temperature magnetically stirred water bath, maintain (37.0 ± 0.5)°C, and set the stir bar speed to 100 rpm.

[0068] (3) Illumination parameter settings: Use a semiconductor laser with an output wavelength of 808 nm. Fix the laser fiber output end 1 cm away from the surface of the dialysis bag, and adjust the spot diameter to completely cover the sample area inside the dialysis bag. Set the laser power density to 2.0 W / cm². 2 The irradiation mode is as follows: continuous irradiation for 5 minutes before each sampling time point.

[0069] (4) Experimental grouping and operation:

[0070] Group 1 (pH 7.4, no light): Dialysis bags were immersed in a pH 7.4 release tank.

[0071] Group 2 (pH 5.5, no light): Dialysis bags were immersed in a pH 5.5 release tank.

[0072] Group 3 (pH 7.4, NIR irradiation): The dialysis bag was immersed in the pH 7.4 release tank and irradiated according to the set mode.

[0073] Group 4 (pH 5.5, NIR irradiation): The dialysis bag was immersed in the pH 5.5 release tank and irradiated according to the set mode.

[0074] Each group has 3 parallel samples.

[0075] (5) Sampling and Measurement: At preset time points (0.5, 1, 2, 4, 6, 8, 12, 24, 36, 48 h), accurately remove 3.0 mL of medium from the release cell and immediately replenish with the corresponding fresh medium at the same temperature and volume. Avoid sampling during periods of light exposure. Use a UV-Vis spectrophotometer to measure the absorbance of the sample at the maximum absorption wavelengths of ICG and CBF.

[0076] (6) Data analysis: Convert absorbance to drug concentration based on the standard curve, calculate the cumulative release rate (Q%), and plot the release curve (Q% ~ t). Compare the differences in release behavior under different conditions.

[0077] Application and Result Analysis of Detection Methods

[0078] Three batches of ICG&CBF@ZIF-8 nanoparticles were tested using the method described in Example 1.

[0079] Results: Group 1 (pH 7.4, no light) showed the slowest release, with a cumulative release rate of <20% after 48 h, indicating good stability of the nanoparticles under physiological conditions. Group 2 (pH 5.5, no light) showed a significantly faster release, with a release rate of ~50% after 48 h, verifying its pH responsiveness. Group 3 (pH 7.4, NIR irradiation) exhibited a steep release peak after each irradiation, indicating that the photothermal effect effectively triggered drug release. Group 4 (pH 5.5, NIR irradiation) showed the fastest release rate and the highest plateau release rate (>85%), demonstrating the synergistic enhancement effect of pH and NIR stimulation. The release curves among the three batches of samples showed good reproducibility.

[0080] Conclusion: This detection method can clearly distinguish and quantify the release characteristics of nanoparticles under different single-factor and dual-factor combined stimulation, providing accurate and comprehensive in vitro evaluation data for the response performance of formulations.

[0081] Example 3:

[0082] 1. Dynamics of Nanoparticle Uptake and Efflux in Cells

[0083] This embodiment uses human oral squamous cell carcinoma HSC3 cells and ICG&CBF@ZIF-8 nanoparticles (prepared according to Example 1, with ICG fluorescence as a tracer signal) as the research objects.

[0084] (1) Uptake kinetics:

[0085] HSC3 cells were seeded at a density of 5 × 10^5 cells per well in 6-well plates (for FCM) and confocal culture dishes (for CLSM) and cultured overnight at 37°C in a 5% CO2 incubator until adherence.

[0086] Replace with fresh culture medium containing ICG&CBF@ZIF-8 (equivalent ICG concentration of 10 μg / mL) and incubate for 0.5, 1, 2, 4, 6 and 8 hours respectively.

[0087] FCM Analysis: At the predetermined time point, cells were gently washed three times with pre-cooled PBS, digested with trypsin, and resuspended in PBS. Immediately, flow cytometry was used for detection, with excitation light at 785 nm, collecting ICG fluorescence signals in the 820-850 nm range. At least 10,000 cellular events were collected per sample. Cells without nanoparticles were used as a negative control for gating. Uptake kinetics were plotted by graphing mean fluorescence intensity (MFI) against incubation time.

[0088] CLSM observation: At the predetermined time point, cells were washed with PBS, fixed with 4% paraformaldehyde for 15 minutes, and the nuclei were stained with DAPI (1 μg / mL) for 5 minutes. Observation was performed using a confocal microscope. The DAPI channel (Ex 405 nm) showed the cell nucleus (blue), and the ICG channel (Ex 785 nm) showed the nanoparticles. The distribution of nanoparticles in the cytoplasm was observed by Z-axis tomography and 3D reconstruction.

[0089] (2) Exhaustion dynamics:

[0090] HSC3 cells were co-incubated with ICG&CBF@ZIF-8 (10 μg / mL ICG equivalent) for 5 hours to allow for full cell uptake.

[0091] Wash thoroughly three times with pre-cooled PBS, then replace with fresh complete culture medium without nanoparticles, and continue culturing for 0, 1, 2, 4, 8, and 12 hours.

[0092] At predetermined time points, cells were treated and analyzed using the FCM and CLSM methods described above, and the residual fluorescence intensity within the cells was measured. The efflux rate and efflux half-life were calculated.

[0093] 2. Research on inhibitors of cellular uptake mechanisms

[0094] To investigate the main pathways by which HSC3 cells take up ICG & CBF@ZIF-8, a series of inhibitors with known mechanisms of action were used for pretreatment.

[0095] (1) Inhibitor pretreatment: HSC3 cells were seeded in 24-well plates and cultured overnight. The cells were pretreated with the following inhibitors at 37°C for 1 hour:

[0096] Chlorpromazine (10 μg / mL): Inhibits clathrin-mediated endocytosis.

[0097] Nystatin (50 μM): Inhibits caveolin-mediated endocytosis.

[0098] Cytochalasin D (5 μM): Inhibits macropinocytosis (by disrupting microfilaments).

[0099] Colchicine (10 μM): Inhibits microtubule-dependent endocytosis / transport.

[0100] Sodium azide (0.1%): Inhibits energy-dependent processes (consuming ATP).

[0101] Control group: Pretreated with an equal volume of culture medium only.

[0102] (2) Intake experiment: After pretreatment, without changing the culture medium, ICG&CBF@ZIF-8 (final concentration 10 μg / mL ICG equivalent) was added directly to each well and incubated for 2 hours.

[0103] (3) Detection and Analysis: After incubation, cells were washed with ice-cold PBS, digested with trypsin, and their fluorescence microfluidic index (MFI) was detected by FCM. The uptake inhibition rate of each inhibitor group was calculated:

[0104] Inhibition rate (%) = [1 - (MFI in inhibitor group / MFI in control group)] × 100%

[0105] (4) Results and Mechanism Inference: If chlorpromazine significantly inhibited uptake (>60%), it suggests that the clathrin pathway is dominant; if nystatin significantly inhibited uptake, it suggests the caveolin pathway; if cytochalasin D significantly inhibited uptake, macropinocytosis is important; if sodium azide strongly inhibited uptake, it indicates that uptake is an active, energy-dependent process. The results of this example show that the chlorpromazine and sodium azide treatment groups had the highest inhibition rates (75% and 85%, respectively), indicating that the uptake of ICG&CBF@ZIF-8 by HSC3 cells mainly depends on the clathrin-mediated, energy-dependent endocytosis pathway.

[0106] 3. Observation of intracellular transport of nanoparticles and lysosomal escape

[0107] (1) Organelle colocalization experiment:

[0108] HSC3 cells were seeded in confocal culture dishes and incubated with ICG&CBF@ZIF-8 (10 μg / mL ICG equivalent) for 4 hours.

[0109] Staining was performed using commercially available organelle-specific fluorescent probes according to the instructions:

[0110] Lysosomes: LysoTracker Red DND-99 (50 nM, incubated for 45 minutes).

[0111] Mitochondria: MitoTracker Deep Red FM (100 nM, incubated for 30 minutes).

[0112] Endoplasmic reticulum: ER-Tracker Red (1 μM, incubated for 30 minutes).

[0113] After staining, the cells were washed with PBS, and the nuclei were stained with DAPI. The cells were observed using a CLSM assay immediately after staining or in a viable state or after slight fixation. Images were acquired for the DAPI (blue), ICG (green), and organelle probe (red) channels, respectively.

[0114] (2) Image analysis and determination of lysosomal escape:

[0115] Image processing software was used to calculate the colocalization coefficients (Pearson correlation coefficients or Manders coefficients) between ICG fluorescence and the fluorescence signals of each organelle.

[0116] Key observation point: In the early stage of incubation (2 hours), the ICG signal (from nanoparticles) and the LysoTracker signal (lysosomes) highly overlapped (yellow spots), indicating that the nanoparticles entered the lysosomal pathway after endocytosis.

[0117] After prolonged incubation (6 or 8 hours), a large amount of green ICG signal was observed distributed outside the red lysosomal signal region, particularly diffusing into the cytoplasm surrounding the cell nucleus. This suggests that the ZIF-8 carrier degrades in the acidic environment of lysosomes, achieving "lysosomal escape" of the drug (especially CBF), which is beneficial for its action on targets such as the cell nucleus. In this example, after 6 hours of incubation, the colocalization coefficient between ICG and lysosomes decreased from 0.82 after 4 hours to 0.35, visually confirming effective lysosomal escape.

[0118] ICG&CBF@ZIF-8 related evaluation tests:

[0119] I. Biocompatibility evaluation of ICG & CBF@ZIF-8:

[0120] (1) Cytotoxicity assay (MTT method):

[0121] The toxicity of the composite nanoparticles to normal cells was evaluated using normal human oral epithelial cells (HOEC) and mouse fibroblasts (L929).

[0122] Cells were spaced at 1×10⁶ cells per well. 4 The cells were seeded at a density of 100 cells per well in a 96-well plate and incubated at 37°C in a 5% CO2 incubator for 12 hours until adherence to the plate.

[0123] Discard the old culture medium and add fresh complete culture medium containing different concentration gradients (80, 160, 320, 640, 1280 μg / mL) of ICG&CBF@ZIF-8 nanoparticles (based on ZIF-8 mass). Set up 5 replicates for each concentration and set up a control group without nanoparticles.

[0124] After culturing for another 24 hours, add 20 μL of MTT solution (5 mg / mL) to each well and continue incubation for another 4 hours.

[0125] Carefully aspirate the liquid from the wells, add 150 μL of dimethyl sulfoxide (DMSO) to each well, and shake at low speed for 10 minutes to fully dissolve the purple crystals.

[0126] The absorbance (OD490) of each well was measured at 490 nm using a microplate reader. Cell viability (%) = (OD490 of experimental group / OD490 of control group) × 100%.

[0127] Experimental results showed that at a concentration as high as 1280 μg / mL, the survival rate of ICG&CBF@ZIF-8 on HOEC and L929 cells was greater than 85%, indicating that it has good in vitro biocompatibility.

[0128] (2) Hemolysis test:

[0129] Fresh anticoagulated whole blood was collected from healthy mice, washed with PBS, and centrifuged three times to obtain purified red blood cells (RBCs).

[0130] RBCs were prepared into a 4% (v / v) suspension using PBS.

[0131] Take 0.5 mL of RBC suspension and mix it with 0.5 mL of PBS solution containing ICG&CBF@ZIF-8 nanoparticles at different concentrations (50, 100, 200, 400 μg / mL). Use 0.5 mL of PBS mixed with 0.5 mL of RBC suspension as a negative control (0% hemolysis) and 0.5 mL of deionized water mixed with 0.5 mL of RBC suspension as a positive control (100% hemolysis).

[0132] Each mixture was incubated at 37°C for 3 hours.

[0133] After incubation, centrifuge at 3000 rpm for 10 minutes, carefully aspirate the supernatant, and measure its absorbance value (OD570) at 570 nm using an ELISA reader.

[0134] Hemolysis rate (%) = (OD570 of sample group - OD570 of negative control group) / (OD570 of positive control group - OD570 of negative control group) × 100%.

[0135] The results showed that, within the experimental concentration range, the hemolysis rate of the composite nanoparticles was less than 5% (international standard safety limit), demonstrating its good blood compatibility.

[0136] II. Evaluation of the in vitro antitumor effects of ICG&CBF@ZIF-8:

[0137] The MTT assay was used to evaluate the killing effect and synergistic therapeutic effect of ICG&CBF@ZIF-8 nanoparticles on HSC3 cells.

[0138] HSC3 cells were seeded into 96-well plates and cultured overnight.

[0139] Set up the following experimental groups:

[0140] Group A: PBS (control)

[0141] Group B: Free ICG

[0142] Group C: Free CBF

[0143] Group D: ICG@ZIF-8

[0144] Group E: ICG&CBF@ZIF-8

[0145] A separate group was established, which, after 4 hours of incubation with the drug, received an 808 nm near-infrared laser (2 W / cm²). 2 Irradiate for 5 minutes (marked as "+L").

[0146] The final ICG concentration in all drug-containing groups was 16 μg / mL, and the final CBF concentration was 32 μg / mL (calculated based on drug loading).

[0147] After treatment with the drug (or PBS) and completion of light exposure (or protection from light), the cells were cultured for another 20 hours.

[0148] Then, following the MTT assay steps described in Example 3 (1), the cell viability of each group was determined.

[0149] The results are as follows: Drug administration alone (groups B, C, and D) or laser administration alone (group A+L) had little impact on cell survival. Free ICG under laser irradiation (group B+L) showed a certain killing effect due to PDT / PTT. ICG&CBF@ZIF-8 in the absence of laser (group E) showed stronger cell inhibition than free CBF (group C) due to the chemotherapeutic effect of CBF, which is attributed to the increased intracellular delivery efficiency of CBF by the nanocarrier. Most importantly, the ICG&CBF@ZIF-8 combined with laser irradiation group (group E+L) had the lowest cell survival rate, and its killing effect was significantly stronger than that of the ICG@ZIF-8+L group (D+L) and the free ICG+L group (B+L), demonstrating the synergistic antitumor effect of ICG (PTT / PDT) and CBF (chemotherapy) through the nanocarrier.

[0150] III. A Preliminary Exploration of the Mechanism of Influence of ICG&CBF@ZIF-8 on the Tumor Cell Microenvironment

[0151] (1) Detection of intracellular reactive oxygen species (ROS) levels:

[0152] After incubating HSC3 cells with ICG&CBF@ZIF-8 (ICG 10 μg / mL) for 4 hours, the culture medium was discarded and the cells were washed with PBS.

[0153] Add PBS working solution containing 10 μM DCFH-DA fluorescent probe and incubate at 37°C in the dark for 30 minutes.

[0154] Wash cells three times with PBS and observe green fluorescence (excitation / emission: 488 / 525nm) under a laser confocal microscope, or quantitatively analyze fluorescence intensity using flow cytometry.

[0155] The experimental setup included a control group with only probes, a free ICG+L group, an ICG&CBF@ZIF-8 (without laser) group, and an ICG&CBF@ZIF-8+L group.

[0156] The results showed that the ICG&CBF@ZIF-8+L group had the strongest green fluorescence intensity in its cells, indicating that this treatment could induce the highest level of ROS, which is one of the key mechanisms of PDT killing cells.

[0157] (2) Detection of intracellular catalase (CAT) activity and hypoxia-related protein (HIF-1α) expression:

[0158] HSC3 cells were treated with different concentrations of CBF or ICG&CBF@ZIF-8 for 48 hours, and then the cells were collected, lysed, and total protein was extracted.

[0159] Protein concentration was determined using the BCA method.

[0160] The enzyme activity of CAT in cell lysate was detected according to the instructions of the commercial kit.

[0161] Meanwhile, the protein expression level of hypoxia-inducible factor-1α (HIF-1α) was detected by Western blotting.

[0162] The results showed that cells treated with CBF or ICG&CBF@ZIF-8 exhibited varying degrees of increased CAT activity, while HIF-1α protein expression was downregulated. This suggests that CBF may alleviate tumor cell hypoxia by enhancing CAT activity and catalyzing the decomposition of excess H2O2 within tumor cells to produce oxygen. This effect helps improve the oxygen environment dependent on PDT, creating more favorable conditions for ICG-mediated PDT, and mechanistically explains the synergistic effect of CBF and ICG.

Claims

1. A ZIF-8 multifunctional nanoparticle loaded with indocyanine green and bufotoxin, characterized in that: It was prepared by simultaneously encapsulating indocyanine green and bufotoxin with ZIF-8, wherein the loading of ZIF-8 on both indocyanine green and bufotoxin was not less than 5%.

2. The method for preparing ZIF-8 multifunctional nanoparticles loaded with indocyanine green and bufotoxin group according to claim 1, characterized in that, Includes the following steps: (1) Disperse indocyanine green, bovine serum albumin and 2-methylimidazole in anhydrous methanol to obtain mixed solution A. Control the concentration of indocyanine green in solution A to be 6~12 mg / mL, the concentration of bovine serum albumin to be 10~25 mg / mL and the concentration of 2-methylimidazole to be 35~50 mg / mL. (2) Disperse bufotoxin and zinc nitrate in anhydrous methanol to obtain solution B, and control the concentration of bufotoxin to be 10~20 mg / mL and the concentration of zinc nitrate to be 15~30 mg / mL. (3) Under the conditions of 15~25℃ and continuous stirring, solution B is slowly added to solution A using a peristaltic pump. After the addition of solution B is completed, stirring is continued at 15~25℃ for 2~6 hours. After the reaction is completed, crude product is obtained. (4) The crude product obtained in step (3) is centrifuged and filtered. After washing and drying, the precipitated product can be used to obtain ZIF-8 multifunctional nanoparticles loaded with indocyanine green and bufotoxin. (5) After drying, the sample is stored in an opaque glass sample bottle and kept in an environment of 3~6℃.

3. The method for preparing ZIF-8 functional nanoparticles loaded with indocyanine green and bufotoxin group according to claim 2, characterized in that: In step (3), the dripping speed of the peristaltic pump is 5~10mL / min.

4. The method for preparing ZIF-8 multifunctional nanoparticles loaded with indocyanine green and bufotoxin group according to claim 2, characterized in that: In step (4), the centrifugation speed is 8000~10000 rpm and the time is 6~10 min.

5. The method for preparing ZIF-8 multifunctional nanoparticles loaded with indocyanine green and bufotoxin group according to claim 2, characterized in that: In step (4), the washing solvent is anhydrous methanol, the drying temperature is 25~30℃, and the drying time is 6~8 hours.

6. The method for detecting the drug release performance of nanoparticles under different conditions according to claim 1, characterized in that, Includes the following steps: (1) Place 2 mL of 1.5 mg / mL nanoparticle aqueous solution into a dialysis bag with a molecular weight of 3500, and immerse it in a flask containing 78 mL of PBS buffer with pH values ​​of 5.5 and 7.4 respectively to release the medium. Shake continuously in a shaker at 37℃ or 43℃ for 48 h. Take out 3 mL of solution from the flask and add 3 mL of new PBS buffer every time interval. (2) After oscillation, at a specific time point, a wavelength of 808 nm and a power density of 2 W·cm⁻¹ are used. -2 The dialysis bag area was irradiated with near-infrared laser for 5 minutes each time; (3) Samples were taken at different time points, and the absorbance of the release medium at the characteristic absorption wavelength of the drug was measured to calculate the cumulative release rate.

7. A method for detecting cellular uptake and intracellular distribution of nanoparticles as described in claim 1, characterized in that, Includes the following steps: (1) Fluorescently labeled nanoparticles were co-incubated with cells for different times, and the average fluorescence intensity in the cells was quantitatively detected by flow cytometry; (2) Cells were pretreated with at least one inhibitor selected from chlorpromazine, nystatin, cytochalasin D, colchicine, and sodium azide, and then co-incubated with fluorescently labeled nanoparticles. The uptake inhibition rate was analyzed by flow cytometry. (3) The cells were incubated with fluorescently labeled nanoparticles and lysosomal fluorescent probes, and the co-localization of the fluorescence of nanoparticles and lysosomal probes was observed using a confocal laser scanning microscope.

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