Preparation method and application of a nanoplatform encapsulating NIR-II photodiagnostic agent and transcription factor EB agonist

Abstract

The present invention provides a preparation method and application of a nano-platform encapsulating a NIR-II photodiagnostic agent and a transcription factor EB agonist, and belongs to the field of biopharmaceutical technology. The present invention provides a lipid nanoparticle encapsulating a near-infrared molecule and a transcription factor EB agonist, comprising an active oxygen responsive lipid carrier and a near-infrared molecule IRFEM and a transcription factor EB agonist encapsulated in the active oxygen responsive lipid carrier; the mass ratio of the near-infrared molecule IRFEM, the transcription factor EB agonist and the active oxygen responsive lipid carrier is 1:(5-8):(15-20). The transcription factor EB agonist and IRFEM are encapsulated in the active oxygen responsive lipid carrier, which can achieve precise delivery and release of the drug in the liver. The transcription factor EB agonist and IRFEM synergistically promote cell autophagy, activate the lysosomal degradation pathway, promote lipid metabolism, and effectively treat non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and specifically relates to a preparation method and application of a nano-platform encapsulating a NIR-II optical diagnostic and therapeutic agent and a transcription factor EB agonist. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) results from a complex interaction of genetic, metabolic, and environmental factors and, if left untreated, can progress to severe liver diseases such as non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and even hepatocellular carcinoma. The prevalence of NAFLD has become a pressing global health issue, further exacerbated by the increasing incidence of obesity, metabolic syndrome, and a sedentary lifestyle.

[0003] Traditional treatment for non-alcoholic fatty liver disease focuses on lifestyle changes, including dietary adjustments, increased physical exercise, and weight management. There are no specific medications for non-alcoholic fatty liver disease, and the disease's progression is primarily controlled through liver-protective medications such as polyene phosphatidylcholine, diammonium glycyrrhizate, and silymarin, which have limited therapeutic effects.

[0004] Recent studies have shown that TFEB agonists have the potential to enhance autophagy and improve lipid metabolism. However, TFEB can be toxic in the process of promoting the degradation of damaged organelles and excess lipids, leading to cell damage. Due to the toxicity and complex mechanism of action of TFEB, its clinical application has been hindered. Summary of the Invention

[0005] In view of this, the present invention provides a new type of lipid nanoparticle, which encapsulates the transcription factor EB agonist and IRFEM in a reactive oxygen species responsive lipid carrier to achieve precise delivery and release of the drug. The transcription factor EB agonist and IRFEM synergistically promote lipid metabolism, thereby effectively treating non-alcoholic fatty liver disease.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a lipid nanoparticle encapsulating a near-infrared molecule and a transcription factor EB agonist, characterized in that it comprises an active oxygen responsive lipid carrier and a near-infrared molecule IRFEM and a transcription factor EB agonist encapsulated in the active oxygen responsive lipid carrier;

[0008] The mass ratio of the near-infrared molecule IRFEM, the transcription factor EB agonist and the active oxygen responsive lipid carrier is 1:(5-8):(15-20).

[0009] Preferably, the reactive oxygen species responsive lipid carrier comprises phospholipid-thiolketal-polyethylene glycol.

[0010] Preferably, the transcription factor EB agonist comprises Torin 1.

[0011] The present invention provides a method for preparing the lipid nanoparticles, comprising the following steps:

[0012] The near-infrared molecule IRFEM, the transcription factor EB agonist and the reactive oxygen species-responsive lipid carrier were mixed in water to obtain lipid nanoparticles.

[0013] Preferably, the mixing time is 8 to 12 minutes.

[0014] Preferably, the mixing is accompanied by stirring; the stirring rate is 400-600 rpm.

[0015] The present invention provides use of the lipid nanoparticles or the lipid nanoparticles prepared by the preparation method in preparing a drug for preventing and / or treating fatty liver disease.

[0016] Preferably, the fatty liver disease includes at least one of the following: non-alcoholic fatty liver disease, abnormal liver lipid metabolism and liver damage.

[0017] Preferably, the symptoms of fatty liver disease are lipid droplet accumulation and / or fatty degeneration.

[0018] Preferably, the dosage form of the drug includes injection solution and / or injection powder.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] The present invention provides a lipid nanoparticle encapsulating a near-infrared molecule and a transcription factor EB agonist, comprising an active oxygen-responsive lipid carrier and a near-infrared molecule IRFEM and a transcription factor EB agonist encapsulated in the active oxygen-responsive lipid carrier; the mass ratio of the near-infrared molecule IRFEM, the transcription factor EB agonist, and the active oxygen-responsive lipid carrier is 1:(5-8):(15-20). In the present invention, the near-infrared molecule IRFEM is a lysosome-targeting SDADS-type near-infrared II photothermal therapy molecule that can specifically target lysosomes and simultaneously generate heat and ROS under light irradiation, thereby promoting autophagy. The transcription factor EB agonist can promote cellular autophagy. IRFEM and the transcription factor EB agonist are encapsulated in the active oxygen-responsive lipid carrier and can be delivered to the liver. Under laser irradiation, the near-infrared molecule IRFEM generates active oxygen, and the active oxygen-responsive lipid carrier structure in the lipid nanoparticle disintegrates, accurately releasing the drug. The transcription factor EB agonist and IRFEM synergistically promote cellular autophagy, activate the lysosomal degradation pathway, and thus promote lipid metabolism, achieving the purpose of effectively treating NAFLD.

[0021] The present invention provides a method for preparing the lipid nanoparticles, comprising the steps of mixing a near-infrared molecule (IRFEM), a transcription factor EB agonist, and a reactive oxygen species-responsive lipid carrier in water to produce the lipid nanoparticles. The lipid nanoparticles (IMTTNPs) prepared using the present method have a diameter of 76.1±19.3 nm, a negative surface charge, and a zeta potential of -10.86 mV. They can be rapidly engulfed by cells, exhibit good safety, and can enter cells to exert therapeutic effects.

[0022] The present invention provides a use of the lipid nanoparticles or lipid nanoparticles prepared by the preparation method in the preparation of a drug for preventing and / or treating fatty liver disease. The present invention evaluated the intracellular behavior and therapeutic potential of IMTTNPs using cell experiments. The results showed that IMTTNPs have efficient lysosomal targeting, can specifically accumulate in the liver, and have excellent biocompatibility, making them useful for the treatment of liver diseases. The present invention further compared the therapeutic effects of different treatment regimens on non-alcoholic fatty liver disease (NAFLD) using a mouse model. During treatment, the photothermal effect showed that the IMTTNPs+NIR group had a faster and higher temperature rise, and the temperature rise process could be precisely controlled by laser, which increased the safety of the treatment. Weight monitoring results showed that there was no significant difference in weight among the groups throughout the treatment period, demonstrating the tolerability of the lipid nanoparticle administration and photothermal therapy regimen. The therapeutic effect of each group was evaluated after treatment. Tissue staining results showed that the IMTTNPs+NIR group showed a significant reduction in lipid droplet accumulation and macrovesicular steatosis, and the liver morphology was similar to that of normal mice. Serum biochemical analysis and liver weight measurements showed that lipid metabolism and liver function were restored in the IMTTNPs+NIR group, with liver weight comparable to that of mice fed a normal diet (ND) diet. Pathological staining of vital organs and body weight monitoring of treated mice demonstrated a favorable safety profile for lipid nanoparticle administration and laser irradiation. Overall, these results demonstrate that IMTTNPs combined with phototherapy have superior therapeutic efficacy, providing a potential alternative for the treatment of NAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of lipid nanoparticles for treating fatty liver disease;

[0024] Figure 2 This is the electron microscopy result of lipid nanoparticles prepared in Comparative Example 2;

[0025] Figure 3Figure 3 shows the physicochemical properties of different lipid nanoparticles in Example 3; A and B are the particle size test results of IMTTNPs; C is the stability test result; D is the UV-visible absorption spectrum; E is the linear relationship between IRFEM and Torin 1 concentration and absorption intensity; F is the absorption spectrum and excitation spectrum of IMTT NPs; G is the absorption spectrum of IMTT NPs under laser irradiation (0.33w / cm 2 ) under the temperature spectrum; H is the DCFH-DA method quantitative analysis of the reactive oxygen species (ROS) generated by IMTTNPs at different concentrations; I is the result of the reactive oxygen species (ROS) generated by IMTTNPs after laser irradiation for different times;

[0026] Figure 4 Figures 2 and 3 are the results of the cell experiment in Example 4; a is a fluorescence imaging diagram of the co-localization of IMTT NPs and lysosomes in AML-12 cells; b is a quantitative analysis of the co-localization of IMTTNPs and lysosomes in AML-12 cells; c is the cellular uptake of IMTTNPs in AML-12 cells at different time points observed by fluorescence microscopy; d is a quantitative evaluation of the uptake of IMTTNPs by AML-12 cells at different incubation times; e is the cytotoxicity evaluation results of AML-12 cells treated with different concentrations of IMTTNPs for 4 hours;

[0027] Figure 5 The diagrams of the treatment scheme and effect evaluation of the animal model in Example 5 are shown in FIG. a is a schematic diagram of the IMTTNPs treatment scheme in the animal model; b is a laser (0.33w / cm 2 ) irradiation for 4 minutes; c is the comparative analysis of the weight changes of mice in different treatment groups before and after treatment; d is the temperature changes of mice in different treatment groups after laser (0.33w / cm 2 ) Quantitative evaluation of temperature changes during 4 min of irradiation;

[0028] Figure 6 Figures 2 and 3 are the results of liver tissue pathology staining and serum biochemical analysis of mice in different treatment groups in Example 5; a is a hematoxylin and eosin (HE) and oil red O (ORO) staining of liver tissue, and b is the result of serum biochemical analysis;

[0029] Figure 7 Figure 5 is a comparative analysis of the examination results of different treatment groups after treatment in Example 5; a is the result of a routine blood test; b is the result of staining of heart, spleen, lung and kidney tissues; c is the result of a body weight test; d is the result of a liver weight test;

[0030] Figure 8 Figures 1 and 2 are the immunohistochemical staining results of liver tissues of mice in different treatment groups in Example 5; a is an immunohistochemical staining image; b is an immunohistochemical staining statistical graph;

[0031] Figure 9 Figures 1 and 2 are the expression level detection results of autophagy-related proteins in liver tissues of different treatment groups after treatment in Example 5; a is a protein immunoblotting detection diagram; b is a statistical diagram of the expression level of autophagy-related proteins;

[0032] Figure 4 and Figures 6 to 9 *** indicates p < 0.001, ** indicates p < 0.01, and * indicates p < 0.05. DETAILED DESCRIPTION

[0033] The present invention provides a lipid nanoparticle encapsulating a near-infrared molecule and a transcription factor EB agonist, comprising an active oxygen responsive lipid carrier and a near-infrared molecule IRFEM and a transcription factor EB agonist encapsulated in the active oxygen responsive lipid carrier; the mass ratio of the near-infrared molecule IRFEM, the transcription factor EB agonist and the active oxygen responsive lipid carrier is 1:(5-8):(15-20).

[0034] In the present invention, the near-infrared molecule IRFEM is a SDADS-type near-infrared II photothermal therapy molecule targeting lysosomes. The IRFEM has a morpholino group that can specifically target lysosomes, while generating heat and ROS under light irradiation to promote autophagy. In an embodiment of the present invention, IRFEM is synthesized with reference to the prior art (Li Z, Yang S, Xiao H, et al. Lysosome-Targeted and pH-Activatable Phototheranostics for NIR-II Fluorescence Imaging-Guided Nasopharyngeal Carcinoma Phototherapy [J]. Bioconjugate Chemistry, 2024.). The characteristic absorption peak of the IRFEM is located at 731 nm, and its successful synthesis is confirmed by ultraviolet-visible spectrophotometry. The transcription factor EB agonist preferably includes Torin 1. Torin 1 is an effective mTORC1 / 2 inhibitor that can inhibit the expression of rapamycin target protein (mTOR) and effectively induce autophagy. In an embodiment of the present invention, Torin1 was purchased from Shanghai Titan Technology Co., Ltd. The transcription factor EB agonist has the function of promoting cell autophagy. The components of the reactive oxygen species responsive lipid carrier preferably include phospholipid-ketal-thiol-polyethylene glycol. Phospholipid-ketal-thiol-polyethylene glycol is an amphiphilic copolymer that responds to reactive oxygen species, and its encapsulation gives lipid nanoparticles water solubility and oxidative environment responsiveness. Phospholipid-ketal-thiol-polyethylene glycol can deliver drugs to hepatocytes. Under the stimulation of ROS, the ketal-thiol bond breaks and the reactive oxygen species responsive lipid carrier structure disintegrates, thereby accurately releasing the drug transcription factor EB agonist and IRFEM, improving the utilization rate of the drug, reducing the toxicity of the drug, and improving the therapeutic effect. Phospholipid-ketal-thiol-polyethylene glycol preferably includes DSPE-TK-PEG2000 and / or DSPE-TK-PEG5000. In an embodiment of the present invention, DSPE-TK-PEG2000 is purchased from Shanghai Titan Technology Co., Ltd. By encapsulating IRFEM and a transcription factor EB agonist with phospholipid-thiolketal-polyethylene glycol, the drugs are delivered to liver cells, reducing drug toxicity. The transcription factor EB agonist and IRFEM synergistically promote cellular autophagy and activate the lysosomal degradation pathway, thereby promoting lipid metabolism and effectively treating NAFLD. The mass ratio of the near-infrared molecule IRFEM, transcription factor EB agonist, and reactive oxygen species-responsive lipid carrier is preferably 1:(6-7):(16-19), and more preferably 1:6.5:18.In this embodiment of the present invention, the mass ratio of the near-infrared molecule IRFEM, transcription factor EB agonist, and reactive oxygen species-responsive lipid carrier is 1:5:18. This defined mass ratio effectively leverages the interactions between the components, allowing the lipid nanoparticles to quickly enter cells. The IRFEM precisely controls the temperature and ROS production via laser light, effectively promoting the disintegration of the reactive oxygen species-responsive lipid carrier. The released IRFEM synergizes with the transcription factor EB agonist to promote autophagy while preventing excessive temperature rise, which could damage lysosomal function.

[0035] The present invention provides a method for preparing the lipid nanoparticles, comprising the following steps:

[0036] The near-infrared molecule IRFEM, the transcription factor EB agonist and the reactive oxygen species-responsive lipid carrier were mixed in water to obtain lipid nanoparticles.

[0037] In the present invention, the mixing is preferably accompanied by stirring; the stirring rate is preferably 400-600 rpm, more preferably 450-550 rpm, and most preferably 500 rpm. The mixing time is preferably 8-12 minutes, more preferably 9-11 minutes, and most preferably 10 minutes. The mixing temperature is preferably room temperature, more preferably 25-35°C, further preferably 28-32°C, and most preferably 30°C.

[0038] The lipid nanoparticles (IMTTNPs) prepared by the preparation method of the present invention have a diameter of 76.1±19.3nm, a negative surface charge, and a zeta potential of -10.86mv. Under laser irradiation, the near-infrared molecules IRFEM in the IMTTNPs generate reactive oxygen species, which respond to the disintegration of the lipid carrier structure and have good ROS responsiveness. In addition, the near-infrared molecules IRFEM in the IMTTNPs have concentration-dependent photothermal effects and fluorescence properties, and are suitable for photodynamic therapy applications. Cell experiments have shown that IMTTNPs can enter cells and exert therapeutic effects in a short period of time. Cell localization experiments have shown that IMTT NPs have the ability to target lysosomes and can be efficiently localized in lysosomes.

[0039] The present invention provides use of the lipid nanoparticles or the lipid nanoparticles prepared by the preparation method in preparing a drug for preventing and / or treating fatty liver disease.

[0040] In the present invention, the fatty liver disease preferably includes at least one of the following: non-alcoholic fatty liver disease, abnormal liver lipid metabolism, and liver damage. The symptoms of the fatty liver disease preferably manifest as lipid droplet accumulation and / or steatosis.

[0041] In the present invention, the dosage form of the drug preferably includes an injection solution and / or an injection powder. The drug is preferably administered by intravenous injection. The working concentration of the drug is preferably 40 to 100 μmol / L, more preferably 45 to 60 μmol / L, and most preferably 50 μmol / L.

[0042] In one embodiment of the present invention, cell experiments were used to evaluate the intracellular behavior and therapeutic potential of IMTT NPs. Cell experiments showed that IMTTNPs have the ability to target lysosomes, can be efficiently located in lysosomes, and can be phagocytosed by cells in a short period of time and quickly and effectively internalized. In addition, IMTTNPs have good safety and do not cause obvious toxicity to cells. The biodistribution and therapeutic potential of IMTT NPs were evaluated using animal injection experiments. Fluorescence imaging results 2 hours after injection showed that IMTTNPs had obvious specific accumulation in the liver, indicating that IMTTNPs can achieve effective delivery and activation of IRFEM and Torin 1 in the liver. In summary, the results show that IMTTNPs have efficient lysosomal targeting, can accumulate specifically in the liver, and have excellent biocompatibility, and can be used to treat liver diseases.

[0043] The present invention further compared the therapeutic effects of different treatment regimens of lipid nanoparticles combined with laser irradiation on non-alcoholic fatty liver disease (NAFLD) through a mouse model. The photothermal effect during the treatment showed that the livers of the IMTT NPs+NIR group (IMTTNPs refers to lipid nanoparticles formed by encapsulating IRFEM and Torin 1 inside DSPE-TK-PEG2000) and the IMCTNPs+NIR group (IMCTNPs refers to lipid nanoparticles formed by encapsulating IRFEM and CQ inside DSPE-TK-PEG2000) heated up quickly and highly, and the heating process could be precisely controlled by laser, and the treatment was highly safe. In the NS+NIR group (NS refers to normal saline), the liver temperature heated up slowly and only heated up to a low level. The results of weight monitoring showed that there was no significant difference in weight among the groups during the entire treatment period, proving the tolerability of the lipid nanoparticle administration and photothermal therapy regimen. After treatment, the therapeutic effect of each group was evaluated by pathological section staining and biochemical analysis. Tissue staining results showed that the IMTT NPs+NIR group showed a significant reduction in lipid droplet accumulation and macrovesicular steatosis, and liver morphology was similar to that of normal mice, while the IMCT NPs+NIR group exhibited extensive liver inflammation and increased lipid deposition. Mice in the NS+NIR group showed persistent steatosis with no significant improvement, indicating the key role of ROS-mediated activation in achieving therapeutic benefits. Serum biochemical analysis results were consistent with the histological results. Lipid metabolism and liver function were restored in the IMTT NPs+NIR group, while lipid metabolism was impaired and inflammation was exacerbated in the IMCT NPs+NIR group. Routine blood test results showed that the IMTTNPs+NIR group showed a transient increase in white blood cell count, indicating enhanced autophagic response and immune regulation. This then returned to normal within a week, indicating that the inflammatory response was effectively controlled. Further liver mass was weighed, and the results showed that the liver weight of the IMTTNPs+NIR group was comparable to that of mice fed a normal diet (ND). Pathological staining of the mice's vital organs and weight monitoring after treatment revealed no significant abnormalities in vital organs or weight between groups, confirming the safety of lipid nanoparticle administration and laser irradiation. Overall, these results suggest that the IMTTNPs + NIR group has a superior therapeutic effect on fatty liver disease by promoting cellular autophagy to clear lipids.

[0044] To further illustrate the present invention, the preparation method and application of a nanoplatform for encapsulating NIR-II phototherapy agents and transcription factor EB agonists provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of protection of the present invention.

[0045] Example 1

[0046] Preparation method of lipid nanoparticles encapsulating IRFEM and Torin 1

[0047] 1 mg IRFEM, 5 mg Torin 1 and 18 mg DSPE-TK-PEG2000 were dissolved in water and stirred at 30°C for 10 min at a stirring rate of 500 rpm to obtain lipid nanoparticles (IMTTNPs) loaded with IRFEM and Torin 1.

[0048] Example 2

[0049] Preparation method of lipid nanoparticles encapsulating IRFEM and Torin 1

[0050] 1 mg IRFEM, 7 mg Torin 1 and 20 mg DSPE-TK-PEG2000 were dissolved in water and stirred at 30°C for 10 min at a stirring rate of 500 rpm to obtain lipid nanoparticles encapsulating IRFEM and Torin 1.

[0051] Comparative Example 1

[0052] 1 mg of IRFEM, 5 mg of chloroquine (CQ), and 18 mg of DSPE-TK-PEG2000 were dissolved in water and stirred at 30°C for 10 min at a stirring rate of 500 rpm to obtain lipid nanoparticles (IMCTNPs) loaded with IRFEM and Torin 1. Chloroquine is an autophagy inhibitor.

[0053] Comparative Example 2

[0054] 1 mg of IRFEM, 4 mg of Torin 1, and 25 mg of DSPE-TK-PEG2000 were dissolved in water and stirred at 30°C for 10 minutes at a stirring rate of 500 rpm. After stirring, the supernatant was discarded and the precipitate was resuspended in PBS. Electron microscopy revealed that complete lipid nanoparticles encapsulating IRFEM and Torin 1 could not be obtained ( Figure 2 ).

[0055] Example 3

[0056] Physical and chemical characteristics detection

[0057] The absorption spectrum of the lipid nanoparticles (IMTTNPs) prepared in Example 1 in aqueous phase was measured using a spectrometer.

[0058] The results showed that IMTTNPs had an obvious absorption peak at 305nm and IMCTNPs had an obvious absorption peak at 336nm, which confirmed that Torin 1 was encapsulated in IMTT NPs and CQ was encapsulated in IMCT NPs. Figure 3 Middle D).

[0059] Dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used to observe the structure of lipid nanoparticles. The results showed that IMTTNPs were spherical with an average diameter of 76.1±19.3nm ( Figure 3 A and B). Zeta potential measurements showed that the IMTTNPs had a value of approximately -10.86 mV, indicating that the IMTTNPs had a negative surface charge and good colloidal stability. Stability test results showed that the IMTTNPs did not spontaneously rupture within 14 days and had good stability ( Figure 3 UV-visible spectroscopy confirmed that IMTT NPs contained IRFEM and Torin 1 ( Figure 3 E), the absorption and excitation spectra of IMTTNPs are shown in Figure 3 Middle F.

[0060] ROS responsive decomposition detection: 808 nm laser irradiation (0.33 w / cm 2 After adding a 50 μmol / L IMTTNP aqueous solution (4 min), the IMTTNPs underwent substantial structural collapse, with bubbles forming in the aqueous solution. The hydrodynamic diameter increased to 1032 nm, and the size distribution became uneven. These changes indicate that the DSPE-TK-PEG2000 in the IMTTNPs decomposed in response to ROS under laser activation.

[0061] Fluorescence Performance Testing: IMTTNPs exhibited a strong fluorescence signal under 808 nm laser excitation, confirming their suitability for photodynamic therapy applications. The linear relationship between IRFEM and Torin 1 concentration and absorption intensity allowed for precise determination of drug loading and encapsulation efficiency.

[0062] Photothermal performance test: aqueous solutions of IMTTNPs and IMCTNPs with different concentrations were irradiated with 808 nm laser (0.33 w / cm 2 ) for 10 minutes, and the temperature of the solution was recorded using an infrared thermal imager. Temperature monitoring showed a concentration-dependent photothermal effect, with a higher temperature being reached at 50 μmol / L, which was determined to be the optimal concentration for treatment ( Figure 3 Middle G).

[0063] ROS production capacity detection: 2′,7′-dichlorofluorescein diacetate (DCFH-DA) probe can be sensitively converted into fluorescent dichlorofluorescein (DCF) by ROS. The DCFH-DA probe was used to evaluate the ROS production capacity of IMTTNPs. The results showed that under 808nm laser irradiation (0.33w / cm 2 ), the generation of ROS increased in a concentration-dependent manner ( Figure 3H), time-dependent analysis showed that the generation of ROS increased with the increase of laser irradiation time ( Figure 3 (I), indicating that IMTTNPs have excellent photodynamic properties.

[0064] IMTTNPs exhibited excellent photothermal properties and ROS responsiveness under laser irradiation, suggesting that IMTT NPs could be used for photodynamic therapy.

[0065] The absorption spectrum, photothermal properties and ROS responsiveness of the lipid nanoparticles prepared in Example 2 were detected using the same detection method as that of IMTTNPs. The results showed that the lipid nanoparticles prepared in Example 2 had a complete structure, excellent photothermal properties and ROS responsiveness.

[0066] Example 4

[0067] Cellular localization and biodistribution experiments

[0068] 1. Cell localization experiment: AML-12 cells were treated with IMTTNPs prepared in Example 1 (final concentration of 50 μmol / L) for 2 h, and co-localization with lysosomal red fluorescent probe (LyseTracker Red LTR) was detected by confocal laser scanning microscopy (CLSM). The results showed that IRFEM in IMTTNPs has the ability to target lysosomes after release and can be efficiently localized in lysosomes ( Figure 4 a and b).

[0069] 2. Evaluation of cell uptake of IMTT NPs: Confocal laser scanning microscopy (CLSM) was used to evaluate the uptake of IMTT NPs by AML-12 cells at different incubation times. The results showed that the uptake of IMTT NPs by cells increased rapidly within the first 2 hours and stabilized between 2 hours and 6 hours. Figure 4 Figures (c and d) show that IMTTNPs can be rapidly internalized within 2 hours and reach saturation between 2 and 6 hours. Rapid and effective internalization of IMTT NPs is a prerequisite for achieving therapeutic outcomes.

[0070] 3. Cytotoxicity and biocompatibility evaluation: After AML-12 cells were treated with different concentrations of IMTTNPs for 4 hours, the cell viability was detected. The results showed that the cell viability of cells exposed to different concentrations of IMTTNPs remained above 90%, indicating that IMTT NPs have low cytotoxicity and excellent biocompatibility ( Figure 4 Such a good safety profile is crucial for potential clinical applications.

[0071] In vivo bioassays: Two hours after intravenous injection of IMTT and IMCT NPs into normal mice, fluorescence imaging revealed significant and specific accumulation of IMTT and IMCT NPs in the liver. This is attributed to the optimized design and physicochemical properties of the lipid nanoparticles. This liver-specific accumulation facilitates the effective delivery and activation of IRFEM and Torin 1 / CQ in the liver.

[0072] The above test results show that IMTT NPs have efficient lysosomal targeting, can accumulate specifically in the liver, and have excellent biocompatibility, which provides a solid foundation and important guidance for further studying the therapeutic potential of IMTT NPs in liver-related diseases and optimizing IMTTNPs concentration, dosing regimen and laser irradiation time in subsequent treatment evaluation.

[0073] The cellular localization and biodistribution of the lipid nanoparticles prepared in Example 2 were detected using the same detection method as that of IMTTNPs. The results showed that the lipid nanoparticles prepared in Example 2 had efficient lysosomal targeting, could accumulate specifically in the liver, and had excellent biocompatibility.

[0074] Example 5

[0075] Animal model experiments

[0076] 1. Establishment of a Non-alcoholic Fatty Liver Disease (NAFLD) Mouse Model

[0077] Male C57BL / 6 mice were used as experimental mice. High-fat diet (HFD) mice were fed a high-fat diet (HFD, 60 kcal% fat) for 16 weeks to induce hepatic steatosis; normal diet (ND) mice were maintained on a normal diet.

[0078] Sixteen weeks later, the mice were examined. Hematoxylin-eosin (HE) and Oil Red O (ORO) staining of liver tissue revealed extensive lipid droplet accumulation and macrovesicular steatosis in the liver tissue of mice fed a high-fat diet, consistent with hallmarks of NAFLD. Biochemical analysis revealed significantly elevated serum levels of total cholesterol (TC), triglycerides (TG), direct bilirubin (DBIL), and total bilirubin (TBIL) in HFD mice compared with mice fed a normal diet (ND). These results confirm the successful establishment of a NAFLD mouse model and provide a platform for subsequent studies of IMTTNPs in the treatment of liver-related diseases.

[0079] 2. Treatment options

[0080] After the NAFLD model was successfully established, mice fed a high-fat diet (HFD) for 16 weeks were randomly divided into four treatment groups. Two hours after intravenous injection of the drug, the liver area was irradiated with 808 nm laser (0.33 w / cm 2 To ensure the therapeutic effect and tolerance, the treatment was performed twice, with an interval of one week between the two times ( Figure 5 (a) The specific groups are as follows:

[0081] IMTTNPs+NIR group (I): injection of 200 μL IMTTNPs (50 μmol / L) solution combined with laser irradiation;

[0082] IMCTNPs+NIR group (II): injection of 200 μL IMCTNPs (50 μmol / L) solution combined with laser irradiation;

[0083] IMTNPs+NIR group (III): injection of 200 μL IRFEM (50 μmol / L) solution combined with laser irradiation;

[0084] The preparation method of IMT NPs is as follows: 1 mg IRFEM and 20 mg DSPE-TK-PEG2000 are dissolved in water, stirred at 30°C for 10 min at a stirring rate of 500 rpm to obtain IRFEM-loaded lipid nanoparticles (IMTNPs);

[0085] NS+NIR group (IV): injection of 200 μL normal saline combined with laser irradiation.

[0086] Mice fed a normal diet (ND) were set up as the control ND group (V). 。

[0087] During the experiment, mice on a high-fat diet (HFD) maintained a high-fat diet, while mice on a normal diet (ND) maintained a standard diet to avoid confounding dietary effects.

[0088] 3. Photothermal effect during treatment

[0089] During laser irradiation, photothermal imaging revealed distinct temperature changes between the groups ( Figure 5 (B) In the IMTTNPs+NIR, IMCTNPs+NIR, and IMTNPs+NIR groups, liver temperature rapidly increased by approximately 6°C within a short period of time, reaching above 40°C, reaching the photothermal activation threshold required for effective treatment. This temperature quickly returned to baseline after laser irradiation was discontinued, demonstrating that the temperature increase can be precisely controlled by laser parameters, ensuring treatment safety. In contrast, the liver temperature in the NS+NIR group increased more slowly, by only 3°C.

[0090] Additionally, weekly body weight measurements were recorded throughout treatment to monitor overall health and system safety ( Figure 5 Quantitative analysis of liver temperature dynamics confirmed consistent results during treatment ( Figure 5 (middle d). No significant differences in body weight trends were observed across all treatment groups, with only minor fluctuations attributable to external factors, such as photothermal hair removal. The stability of body weight further supports the tolerability of the lipid nanoparticle delivery and photothermal therapy regimen.

[0091] This treatment regimen, which combines lipid nanoparticle delivery with controlled laser irradiation, demonstrated precise temperature dynamics, excellent safety, and feasibility. These results provide a solid foundation for further evaluation of the efficacy of lipid nanoparticles in alleviating NAFLD-related liver pathology.

[0092] 4. Histological and Biochemical Analysis after Treatment

[0093] Histological analysis and pathological evaluation were performed using hematoxylin-eosin (HE) and oil red O (ORO) staining.

[0094] The results showed that there were significant differences in liver morphology among the groups ( Figure 6 (a) The NS+NIR group showed persistent steatosis; the IMT NPs+NIR group showed some improvement in steatosis, indicating that IRFEM-generated ROS-mediated lysosomal activation has a therapeutic effect. The IMTT NPs+NIR group showed a significant decrease in lipid droplet accumulation and macrovesicular steatosis, and liver morphology was similar to that of mice fed a normal diet (ND), indicating a synergistic effect between IRFEM and Torin 1. IRFEM-generated ROS triggered Torin 1 release, and IRFEM and Torin 1 promoted autophagy activation and alleviated fatty liver disease. The IMCTNPs+NIR group showed extensive liver inflammation and increased lipid deposition.

[0095] Serum biochemical analysis was consistent with histological findings ( Figure 6 (B) In the IMTTNPs+NIR group, total cholesterol (TC), triglycerides (TG), direct bilirubin (DBIL), and total bilirubin (TBIL) were close to normal levels, indicating recovery of lipid metabolism and liver function. In contrast, in the IMCTNPs+NIR group, TC, TG, and bile acid (TBA) levels were significantly elevated, indicating impaired lipid metabolism and increased inflammation.

[0096] Routine blood tests of mice after treatment showed a transient increase in white blood cell (WBC) counts, particularly neutrophils and monocytes, in the IMTTNPs+NIR group, indicating enhanced autophagy and immune regulation. Routine blood tests were performed again one week after treatment, and these indicators returned to normal within a week, indicating effective control of the inflammatory response.

[0097] Pathological sections of the important organs (heart, spleen, lung and kidney) of the treated mice were stained. The results showed that no obvious abnormalities were found among the groups ( Figure 7 (b) The safety of lipid nanoparticle delivery and laser irradiation was confirmed.

[0098] Body weight analysis showed no significant differences between the groups, and the body weight of the IMTT NPs+NIR group tended to be normal, which was very similar to that of the normal diet (ND) group mice ( Figure 7 c). The liver weight was further weighed, and the results showed that the liver weight of the IMTT NPs+NIR group was comparable to that of mice fed a normal diet (ND), while the other groups, including the IMCTNPs+NIR group and the NS+NIR group, showed no significant improvement ( Figure 7 (d)

[0099] In summary, these results demonstrate superior therapeutic efficacy in the IMTT NPs+NIR group, while the IMCT NPs+NIR group showed poor efficacy, highlighting the importance of precision medicine. These analyses provide a comprehensive understanding of the histological, biochemical, and immunological responses underlying the efficacy of lipid nanoparticle-based interventions in NAFLD. Notably, the dose of Torin 1 in this study was significantly lower than that used for direct oral gavage, yet the treatment was well tolerated and effective.

[0100] 5. Immunofluorescence (IF) and Western blot (WB) analysis after treatment

[0101] To elucidate the molecular mechanisms of IMTTNPs+NIR and IMCTNPs+NIR in the treatment of NAFLD, immunofluorescence (IF) and Western blot (WB) analyses were performed. These analyses focused on the expression and localization of autophagy-related proteins and transcription factors to illustrate the activation of the autophagy pathway.

[0102] Quantitative analysis of IF data confirmed that the expression of TFEB was significantly increased in the IMTTNPs+NIR group. TFEB is a key marker for lysosomal autophagy activation, indicating that Torin 1 and ROS have a synergistic effect in activating TFEB and can upregulate the lysosomal autophagy pathway, which is associated with reduced lipid accumulation and improved hepatic steatosis ( Figure 8In contrast, in the IMCTNPs+NIR group, the overall expression of TFEB was significantly decreased, approaching that of the normal diet (ND) group, indicating that CQ has an inhibitory effect on TFEB activation, which may hinder the induction of lysosomal autophagy and reduce the therapeutic effects observed in histological analysis.

[0103] Western blotting was used to detect the expression of autophagy-related proteins in each group after treatment. The results of WB analysis showed that there were significant differences in the expression of key autophagy-related proteins in each treatment group ( Figure 9 Among them, LC3-II, TFEB, lysosomal associated membrane protein 1-(LAMP1) and cathepsin d-(CTSD) were significantly upregulated in the IMTT NPs+NIR group, indicating that autophagy flux was enhanced and lysosomal degradation pathway was activated. IMTT NPs combined with phototherapy can effectively promote autophagy, which is crucial for lipid droplet clearance and mitigation of lipotoxicity ( Figure 9 (middle b). In contrast, the IMCT NPs + NIR group showed elevated levels of the autophagy inhibition-related markers p62 and mammalian target of rapamycin (mTOR). Increased mTOR expression indicates suppressed autophagy initiation, and high p62 levels indicate impaired autophagic degradation, further suggesting a detrimental effect of CQ on the autophagic process.

[0104] Combined IF and WB results highlight the critical role of TFEB in mediating lysosomal autophagy and its direct impact on NAFLD pathology. IMTTNPs+NIR treatment effectively enhanced autophagic flux by promoting TFEB expression and lysosomal activation, leading to significant improvements in liver histology and lipid metabolism function, indicating that IMTTNPs combined with phototherapy is a promising therapeutic strategy for NAFLD, which can effectively reduce the toxicity of lipid accumulation to hepatocytes by activating TFEB and enhancing autophagy through the synergistic effects of Torin 1 and ROS. In contrast, IMCTNPs+NIR treatment had limited efficacy, which was attributed to the inhibitory effect of CQ on TFEB and lysosomal function. The results of the IMCTNPs+NIR group emphasize the need for precise drug selection to avoid adverse interactions that impair the autophagic pathway and exacerbate disease progression.

[0105] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A lipid nanoparticle encapsulating a near-infrared molecule and a transcription factor EB agonist, characterized in that: The invention comprises an active oxygen responsive lipid carrier and a near infrared molecule IRFEM and a transcription factor EB agonist encapsulated in the active oxygen responsive lipid carrier; The mass ratio of the near-infrared molecule IRFEM, the transcription factor EB agonist and the reactive oxygen species responsive lipid carrier is 1: (5-8): (15-20); The active oxygen responsive lipid carrier is phospholipid-thiolketal-polyethylene glycol; The transcription factor EB agonist is Torin 1.

2. A method for preparing the lipid nanoparticles according to claim 1, characterized in that: The following steps are involved: The near-infrared molecule IRFEM, the transcription factor EB agonist and the reactive oxygen species-responsive lipid carrier were mixed in water to obtain lipid nanoparticles.

3. The preparation method according to claim 2, characterized in that: The mixing time is 8 to 12 minutes.

4. The preparation method according to claim 2 or 3, characterized in that The mixing is accompanied by stirring; the stirring rate is 400-600 rpm.

5. Use of the lipid nanoparticles according to claim 1 or the lipid nanoparticles prepared by the preparation method according to any one of claims 2 to 4 in the preparation of a medicament for preventing and / or treating fatty liver disease; The fatty liver disease is non-alcoholic fatty liver disease.

6. The application according to claim 5, characterized in that: The symptoms of fatty liver disease are manifested as lipid droplet accumulation and / or fatty degeneration.

7. The use according to claim 5 or 6, characterized in that The dosage form of the drug includes injection solution and / or injection powder.

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