Arginine nanoparticles, their preparation method and applications

Abstract

This invention provides arginine nanoparticles, their preparation method, and applications. Arginine is dissolved in DMSO and allowed to stand to obtain an arginine solution. Water is then added to the arginine solution to dilute it, resulting in a mixed solution. The mixture is then repeatedly agitated several times, aged at room temperature, and dialyzed to obtain arginine nanoparticles. This invention uses arginine as a substrate and leverages its hydrophobicity to combine with molecular self-assembly technology to prepare arginine nanoparticles. The operation is simple and low-cost.

Description

Technical Field

[0001] This invention relates to the field of arginine technology, specifically to arginine nanoparticles, their preparation method, and their applications. Background Technology

[0002] Liver fibrosis is a key pathological stage in the progression of chronic liver disease to cirrhosis and liver cancer, for which there are currently no reliable drugs. Arginine, one of the twenty essential amino acids, has long been proven to have a positive regulatory function on liver function. However, arginine is a water-soluble amino acid, and traditional administration methods allow it to easily travel through the bloodstream to various organs throughout the body. When administering arginine to liver disease patients, it is difficult to determine the dosage absorbed by the liver, and it can easily have negative effects on other healthy organs. Therefore, a method is needed to allow arginine to be delivered to the liver in a targeted manner, thereby improving drug specificity and efficacy. Summary of the Invention

[0003] The technical problem to be solved by this invention is how to target arginine to the liver for drug delivery and improve drug specificity.

[0004] The present invention solves the above-mentioned technical problems through the following technical means:

[0005] The first aspect of the present invention provides a method for preparing arginine nanoparticles, wherein arginine is dissolved in DMSO and allowed to stand to obtain an arginine solution, water is added to the arginine solution to dilute it to obtain a mixed solution, the mixed solution is repeatedly blown and stirred several times, aged at room temperature and dialyzed to obtain arginine nanoparticles.

[0006] Beneficial effects: This invention uses arginine as a substrate and utilizes its hydrophobicity to combine with molecular self-assembly technology to prepare arginine nanoparticles. The operation is simple and the cost is low.

[0007] Preferably, the settling time is 2 to 3 hours.

[0008] Preferably, the arginine is Fmoc-L-Arginine, with CAS number 91000-69-0.

[0009] Preferably, the concentration of the arginine solution is 10 mg / ml to 20 mg / ml.

[0010] Preferably, the volume of the arginine solution is 20ul to 40ul.

[0011] Preferably, the amount of water added is to bring the mixture to 1 mL.

[0012] Preferably, the aging time is 2 days.

[0013] Preferably, dialysis is performed using a dialysis bag, and the dialysis time is 5 to 6 hours.

[0014] Preferably, the mixture is repeatedly blown and agitated several times using a pipette.

[0015] The second aspect of the present invention provides a method for preparing the above-mentioned arginine nanoparticles to obtain arginine nanoparticles.

[0016] Beneficial effects: The arginine nanoparticles prepared by this invention have good stability, excellent passive liver targeting, and a long liver retention time.

[0017] A third aspect of the present invention provides a nano-targeted liver drug, wherein the effective component of the drug comprises arginine nanoparticles prepared by the above-mentioned method for preparing arginine nanoparticles or the above-mentioned arginine nanoparticles.

[0018] The fourth aspect of this invention provides the application of the above-mentioned arginine nanoparticles or the above-mentioned nano-targeted liver drugs in the treatment of liver fibrosis.

[0019] Preferably, the concentration of arginine nanoparticles is 4-16 mg / kg.

[0020] More preferably, the concentration of arginine nanoparticles is 8 mg / kg.

[0021] Beneficial effects: The present invention uses arginine nanoparticles to treat liver fibrosis, which can significantly reduce the degree of collagen deposition in liver tissue, reduce fibrous septa, basically restore the structure of liver lobules, and significantly repair liver cell damage. Attached Figure Description

[0022] Figure 1 The images show the DLS distribution diagram (A) and particle size stability statistics diagram (B) of the FLA-NPs obtained in Example 1 of this invention. Figure 2 This is a statistical chart of the PDI stability of FLA-NPs obtained in Example 1 of this invention; Figure 3 This is a statistical graph of the zeta potential stability of FLA-NPs obtained in Example 1 of this invention; Figure 4 These are TEM images of FLA-NPs obtained in Example 1 of this invention; Figure 5 This is the UV-vis spectrum of the FLA-NPs obtained in Example 1 of this invention; Figure 6 This is an HPLC chromatogram of FLA-NPs and FLA obtained in Example 1 of the present invention; Figure 7 This is a DLS distribution map of FLA-NPs obtained in Example 2 of the present invention; Figure 8 This is a physical image of the product obtained in Comparative Example 3 of this invention; Figure 9This is a physical image of the product obtained in Comparative Example 4 of this invention; Figure 10 This is the standard curve graph of FLA; Figure 11 This is a laser confocal image of the uptake of DiD-FLA-NPs by SK-Hep-1 cells in the normal group and model group in Example 2 of this invention; Figure 12 This is Example 2 of the present invention, which uses flow cytometry to analyze the qualitative and quantitative uptake of DiD-FLA-NPs by SK-Hep-1 in the normal group and the model group; where B represents the qualitative uptake and C represents the quantitative uptake. Figure 13 This refers to the targeting ability of small animal in vivo imaging for detecting FLA-NPs in vivo in Example 2 of the present invention; where A represents the fluorescence imaging of FLA-NPs aggregation results in mice at different time points; and B represents the fluorescence imaging of FLA-NPs tissue distribution results after 72 hours. Figure 14 This is an immunofluorescence colocalization map of FLA-NPs (red) and LYVE1 (green) in liver tissues of the normal group and the model group in Example 2 of the present invention; Figure 15 This is a Western Blot analysis of the treatment effects in different groups in Embodiment 2 of the present invention; Figure 16 This is a graph showing the therapeutic effects of different groups analyzed by qRT-PCR in Example 2 of this invention; Figure 17 This is a diagram illustrating the process of establishing a mouse model of liver fibrosis in Example 2 of this invention. Figure 18 These are images of mouse livers from different groups in Example 2 of this invention; Figure 19 These are images showing the H&E, Masson, and SR staining results of liver sections from different groups of mice in Example 2 of this invention. Figure 20 This is a Western Blot analysis of the treatment effects in different groups in Embodiment 2 of the present invention; Figure 21 This is a graph showing the therapeutic effects of different groups analyzed by qRT-PCR in Example 2 of this invention. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0025] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0026] The arginine used in the examples and comparative examples was Fmoc-L-Arginine (Fmoc-L-Arg, abbreviated as FLA), CAS number 91000-69-0, purchased from Aladdin Reagent Company. The DMSO was purchased from Shanghai Husheng Laboratory Equipment Co., Ltd., and the purity of the DMSO was HPLC grade.

[0027] Example 1 This embodiment provides a method for preparing arginine nanoparticles, specifically including the following steps: S1. Place 10 mg of Fmoc-L-Arginine in a 1.5 mL EP tube, add 1 mL of DMSO using a pipette, tighten the cap, and dissolve in an 80% power sonicator for about 3 minutes. After dissolution, let it stand at room temperature for 2 hours to obtain the arginine solution.

[0028] S2. Use a pipette to draw 40 μL of arginine solution into a new 1.5 mL EP tube. Then use a pipette to draw 960 μL of ultrapure water and add it to the EP tube to dilute the arginine solution and obtain a mixture. Use a 1 mL pipette to repeatedly blow the mixture about 50 times. Tighten the cap and let it stand at room temperature for 1 hour, then place it in a 4℃ refrigerator for 2 days to age.

[0029] S3. Transfer the aged solution into a dialysis bag and seal both ends. Then place a 1 L beaker on a magnetic stirrer, add approximately 800 mL of ultrapure water and a stir bar. Place the dialysis bag containing the solution into the beaker, turn on the magnetic stirrer, and adjust the speed to allow the dialysis bag to rotate slowly. Maintain this for 5 hours to obtain arginine nanoparticles (FLA-NPs).

[0030] The arginine nanoparticles were characterized as follows: according to Figure 1 It can be seen that the average particle size of FLA-NPs is 274.96±2.36nm.

[0031] according to Figure 2 The PDI is 0.10±0.03, indicating that the FLA-NPs have a uniform particle size distribution.

[0032] according to Figure 3The zeta potential is -30.43m ± 0.31V, and the absolute value of the potential is greater than 20mV, indicating that there is a strong electrostatic repulsion between particles, which can effectively inhibit particle aggregation.

[0033] according to Figures 1-3 It can be seen that, under storage conditions at 4℃, the particle size and zeta potential of FLA-NPs did not fluctuate significantly within 0 to 7 days, and the PDI remained below 0.20. Within 14 to 28 days, the particle size increased slightly (331.58±7.68nm) and the zeta potential decreased slightly (-27.90±2.37mV), but neither of these changes was significant and still met the application standards for nanomaterials, indicating that FLA-NPs have good stability and storage potential.

[0034] according to Figure 4 It can be seen that FLA-NPs have a regular spherical structure, uniform particle morphology, no obvious aggregation, and good dispersibility; the particle size of FLA-NPs measured by TEM is about 300 nm, which is basically consistent with the hydrated particle size detected by DLS, further verifying the successful preparation of arginine nanoparticles.

[0035] according to Figure 5 It can be seen that FLA-NPs exhibit a strong absorption peak at 255 nm, which is a characteristic absorption peak of the Fmoc group, indicating that FLA molecules have successfully self-assembled to form nanoparticles, and the structure of the Fmoc group remains intact.

[0036] according to Figure 6 It can be seen that under the same chromatographic conditions, the elution time (retention time of about 8.5 min) and chromatographic waveform of FLA and FLA-NPs are completely consistent, indicating that during the self-assembly of FLA molecules to form nanoparticles, only the physical morphology changes, without the breaking or formation of chemical bonds, and its chemical composition remains stable.

[0037] Using FLA as a standard, a standard curve was plotted at a wavelength of 255nm as follows: Figure 10 As shown, the regression equation is y = 47210x - 119848 (R² = 0.9989), indicating that FLA has a good linear relationship in the concentration range of 0~200 μg / mL. Based on the standard curve, the concentration of FLA-NPs in this example is 0.29 mg / mL.

[0038] Example 2 The difference between this embodiment and Example 1 is that in S1, 10 mg of Fmoc-L-Arginine was replaced with 20 mg, while all other aspects were the same as in Example 1, and arginine nanoparticles were obtained.

[0039] Example 3 The difference between this embodiment and Example 1 is that in S1, 10 mg of Fmoc-L-Arginine is replaced with 15 mg, while all other aspects are the same as in Example 1, and arginine nanoparticles are obtained.

[0040] Example 4 The difference between this embodiment and Example 2 is that in S2, 40 μL of arginine solution was replaced with 20 μL, and 980 μL of ultrapure water was added. Everything else was the same as in Example 2, and arginine nanoparticles were obtained.

[0041] Comparative Example 1 The difference between this comparative example and Example 1 is that in S1, 10 mg of Fmoc-L-Arginine was replaced with 5 mg, while everything else was the same as in Example 1. Arginine nanoparticles could not be obtained.

[0042] Comparative Example 2 The difference between this comparative example and Example 1 is that in S1, 10 mg of Fmoc-L-Arginine was replaced with 25 mg, while everything else was the same as in Example 1. Arginine nanoparticles could not be obtained.

[0043] Comparative Example 3 The difference between this comparative example and Example 1 is that DMSO was replaced with methanol, and solid particles precipitated after adding ultrapure water for 3 hours, making it impossible to obtain arginine nanoparticles.

[0044] Comparative Example 4 The difference between this comparative example and Example 1 is that DMSO was replaced with ethanol, FLA could not be dissolved, and arginine nanoparticles could not be obtained.

[0045] Experimental Example The arginine nanoparticles prepared in Example 1 were subjected to performance testing, and the specific details are as follows: Model group: SK-Hep-1 cells in logarithmic growth phase were seeded into six-well plates, cultured in 2 mL of complete culture medium for 24 hours, washed twice with 1 mL of PBS, then 2 mL of complete culture medium was added, followed by 4 μL of CoCl2·6H2O (0.1 mol / mL), and the incubation time was extended for another 24 hours to induce cell capillary formation.

[0046] NC group: SK-Hep-1 cells in the logarithmic growth phase were seeded into six-well plates, cultured in 2 mL of complete culture medium for 24 hours, washed twice with 1 mL of PBS, and then cultured in 2 mL of complete culture medium.

[0047] 1. In vivo and in vitro uptake of FLA-NPs Preparation of DiD-FLA-NPs: 10 mg of FLA was dissolved in 1 mL of DMSO by sonication. After standing for 2 hours, 40 μL was transferred to an EP tube. 2 μL of DiD fluorescent dye (10 mg / mL, dissolved in DMSO) was added under light-protected conditions, followed by 960 μL of ultrapure water. The mixture was repeatedly stirred until homogeneous and then aged under light-protected conditions for 2 days. DiD-FLA-NPs were then obtained by dialyzing with ultrapure water for 5 hours under light-protected conditions.

[0048] (1) When the cells in the Model group and NC group reached 70% confluence, 20 μL of DiD-FLA-NPs were added to the Model group and NC group, and incubated in the dark for 3 h, followed by incubation with DAPI for 5 min in the dark. Then, laser confocal microscopy was used to photograph the cells at blue (for DAPI detection) and red (for DiD-FLA-NPs detection) wavelengths. The results are as follows: Figure 11 As shown.

[0049] according to Figure 11 It can be seen that obvious DiD red fluorescence can be observed in both normal SK-Hep-1 cells and SK-Hep-1 cells in a capillary state induced by 6H2O·CoCl2, indicating that FLA-NPs can be efficiently taken up by human LSEC.

[0050] (2) When the cells in the Model and NC groups reached 70% confluence, 20 μL of DiD-FLA-NPs were added to both groups and incubated in the dark for 3 hours. The cells were then digested with trypsin and detected by flow cytometry at a red wavelength (for detecting DiD-FLA-NPs), followed by quantitative analysis. The results are as follows: Figure 12 As shown.

[0051] according to Figure 12 It can be seen that both normal SK-Hep-1 cells and SK-Hep-1 cells in a capillary-induced state induced by 6H2O·CoCl2 have excellent FLA-NP uptake capacity.

[0052] Model mice: CCl4 was dissolved in olive oil to obtain a CCl4 solution (the volume ratio of CCl4 to olive oil was 1:9). The CCl4 solution was injected intraperitoneally into C57BL / 6 mice twice a week to obtain model mice. The model mice were mice with liver fibrosis.

[0053] NC mice: Normal C57BL / 6 mice.

[0054] Starting from week 5, mice in the NC group and Model group were injected intravenously with DiD-FLA-NPs and free DiD dye, respectively, followed by fluorescence detection. Results are as follows: Figure 13As shown.

[0055] The preparation of free DiD dye: 2 μL of DiD fluorescent dye (10 mg / mL, dissolved in DMSO) was added to 1 mL of ultrapure water under light-protected conditions. After being blown evenly and allowed to stand for 2 h, the dye was dialyzed with ultrapure water for 1 h under light-protected conditions to obtain free DiD dye.

[0056] according to Figure 13 It was found that the fluorescence signal of the free DiD dye group was rapidly distributed throughout the mouse body, with no obvious fluorescence enrichment in the liver, and the fluorescence signal decayed rapidly 4 hours after injection; while the fluorescence signal of the DiD-FLA-NPs group was mainly specifically concentrated in the liver, with the liver fluorescence intensity reaching its peak 4 hours after injection, and a strong fluorescence signal could still be detected within 24 hours. After 48 hours, the fluorescence intensity gradually decreased, indicating that FLA-NPs have excellent passive liver targeting and a long liver retention time.

[0057] 72 hours after injection, mice were sacrificed, and major organs such as the heart, liver, spleen, lungs, and kidneys were harvested for fluorescence imaging. The results showed that fluorescence signals were detected in the liver and kidneys in both the DiD-FLA-NPs group and the free DiD group, while almost no fluorescence was observed in the heart, spleen, and lungs. It is speculated that the fluorescence signal after 72 hours is related to the metabolic characteristics of the DiD dye itself; after being absorbed by liver and kidney cells, it easily produces residual fluorescence.

[0058] NC mice and Model mice were injected with DiD-FLA-NPs via the tail vein. Four hours later, liver tissue was harvested, frozen sections were prepared, and immunofluorescence co-localization was performed. Results are as follows: Figure 14 The results showed that LYVE1 (an LSEC-specific marker, green fluorescence) and DiD red fluorescence could be effectively co-localized, indicating that FLA-NPs are mainly taken up by LSEC in mouse liver.

[0059] 2. In vitro therapeutic effects of FLA-NPs Capillary formation of human liver sinusoidal endothelial cells SK-Hep-1 were regulated in vitro using 6H2O·CoCl2, followed by administration of different drug doses to assess the therapeutic effect. Results are as follows: Figure 15 and Figure 16 As shown.

[0060] Western blotting was used to detect the expression of relevant proteins at the cellular level. The cells in the six-well plates were washed, and 200 μL of protein lysis buffer (PMSF:RIPA = 1:100) was added and incubated on ice for 10 min. Cells were then scraped off and transferred to EP tubes, and centrifuged at 12000 rpm and 4°C for 30 min. The bottom precipitate was removed, and 4 volumes of 5×SDS-PAGE protein loading buffer were added. The plates were then incubated in a metal bath at 100°C for 10 min. The expression of relevant proteins was then detected following the steps of electrophoresis, transfer, blocking, incubation with primary antibody, washing, incubation with secondary antibody, washing, and development.

[0061] Detection of relevant mRNA expression at the cellular level using real-time quantitative PCR: Cells in six-well plates were washed, 200 μL of TRIzol reagent was added, and cells were repeatedly pipetted and transferred to EP tubes. 1 / 4 volume of chloroform was added, and the cells were centrifuged at 12000 rpm and 4°C for 30 min. The supernatant was collected, incubated overnight at -20°C, and then centrifuged at 12000 rpm and 4°C for 30 min. The cells were washed three times with anhydrous ethanol, and then an appropriate amount of enzyme-free water was added. After quantification, reverse transcription was performed, and the corresponding primers were added according to the programmed sequence for mRNA content detection.

[0062] according to Figure 15 and Figure 16 It was found that, compared with the model group, the 25, 50, and 100 μM FLA-NPs treatment groups significantly downregulated the protein and mRNA expression levels of CD31 in cells, while upregulating the protein and mRNA expression levels of LYVE1. Furthermore, this regulatory effect gradually increased with increasing FLA-NPs concentration, exhibiting a clear dose-dependent effect. Among these, the 100 μM FLA-NPs group showed the greatest downregulation of CD31 and the greatest upregulation of LYVE1.

[0063] 3. In vitro therapeutic effects Free L-Arg served as the control group, and its molar mass was consistent with that of arginine in 50 μM FLA-NPs. It was prepared by dissolving L-Arg in ultrapure water.

[0064] A liver fibrosis model was established in C57BL / 6 mice using CCl4, as follows: Figure 17 As shown, solvent-based olive oil was used as a control, administered via intraperitoneal injection twice weekly. Starting from week 5, different concentrations of FLA-NPs, free L-Arg, and saline were administered to different groups every two days via tail vein, simultaneously with model establishment. The therapeutic effects of different concentrations of FLA-NPs and free L-Arg on the mouse model were investigated. After model establishment, serum and liver tissue were collected for the detection of liver fibrosis-related indicators, and direct observation was performed. Figure 18 Irregular grooves were observed in the liver of the model group, and this phenomenon was improved by treatment with 8 mg / kg of FLA-NPs.

[0065] Liver histopathological staining: Mouse liver tissue was obtained and prepared into sections through dehydration, permeabilization, embedding, dewaxing, hydration, staining, and slide preparation in paraformaldehyde. The sections were then scanned using a slide scanner. Results are as follows: Figure 19 As shown, the normal group of mice exhibited intact liver tissue structure, regular arrangement of liver lobules, and no collagen fiber deposition. The model group of mice showed significant pathological changes of liver fibrosis, characterized by abnormal collagen fiber deposition, fibrous septum formation, and destruction of liver lobule structure, accompanied by hepatocyte degeneration, necrosis, and inflammatory cell infiltration. The 8 mg / kg FLA-NPs treatment group showed a significant reduction in liver collagen deposition, a decrease in fibrous septum, and near-complete restoration of liver lobule structure, with significant repair of hepatocyte damage. The 16 mg / kg FLA-NPs treatment group and the free L-Arg control group showed no significant improvement in liver tissue pathological changes, with collagen deposition remaining severe and showing no significant difference from the model group.

[0066] Western blot analysis of relevant protein expression in liver tissue: Approximately 30 mg of dissected mouse liver tissue was collected, and 1 mL of protein lysis buffer (PMSF:RIPA = 1:100) and two grinding beads were added. After cryogenic grinding, the mixture was centrifuged at 12000 rpm and 4°C for 30 min. The supernatant was collected into a new 1.5 mL EP tube to obtain the extracted total protein. The protein sample was mixed with 5×SDS-PAGE protein loading buffer at a ratio of 4:1. The mixture was incubated in a metal bath at 100°C for 10 min. Subsequently, the expression of relevant proteins was detected by electrophoresis, transfer, blocking, incubation with primary antibody, washing, incubation with secondary antibody, washing, and development.

[0067] Real-time quantitative PCR detection of relevant mRNA expression in liver tissue: Approximately 30 mg of dissected mouse liver tissue was collected, and 1 mL of TRIzol reagent and 2 grinding beads were added. After cryogenic grinding, 1 / 4 volume of chloroform was added, and the mixture was centrifuged at 12,000 rpm and 4°C for 30 min. The supernatant was collected, incubated overnight at -20°C, and then centrifuged at 12,000 rpm and 4°C for 30 min. After washing three times with anhydrous ethanol, an appropriate amount of enzyme-free water was added. After quantification, reverse transcription was performed, and then the corresponding primers were added according to the program for mRNA content detection.

[0068] according to Figure 20-21 It was found that the treatment group with 8 mg / kg FLA-NPs significantly upregulated the protein and mRNA expression levels of LYVE1 in liver tissue, while downregulating the protein and mRNA expression levels of CD31, indicating that 8 mg / kg FLA-NPs can reverse the capillary formation of LSEC in vivo.

[0069] Regarding HSC activation biomarkers, the 8 mg / kg FLA-NPs treatment group significantly downregulated the protein and mRNA expression levels of Collagen1a and α-SMA in liver tissue, effectively inhibiting HSC activation and ECM synthesis. In contrast, the expression levels of the above biomarkers in the 16 mg / kg FLA-NPs group were not significantly different from those in the model group. The free L-Arg control group only slightly downregulated the expression of related biomarkers, and the effect was significantly weaker than that of the medium-dose group.

[0070] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing arginine nanoparticles, characterized in that, Arginine was dissolved in DMSO and allowed to stand to obtain an arginine solution. Water was added to the arginine solution to dilute it and obtain a mixture. The mixture was then repeatedly blown and stirred several times, aged at room temperature, and dialyzed to obtain arginine nanoparticles.

2. The method for preparing arginine nanoparticles according to claim 1, characterized in that, The settling time is 2 to 3 hours.

3. The method for preparing arginine nanoparticles according to claim 1, characterized in that, The concentration of the arginine solution is 10 mg / ml to 20 mg / ml.

4. The method for preparing arginine nanoparticles according to claim 3, characterized in that, The volume of the arginine solution is 20ul~40ul.

5. The method for preparing arginine nanoparticles according to claim 4, characterized in that, Add water to bring the mixture to 1 mL.

6. The method for preparing arginine nanoparticles according to claim 1, characterized in that, The aging time is 2 days.

7. The method for preparing arginine nanoparticles according to claim 1, characterized in that, Dialysis is performed using dialysis bags, and the dialysis time is 5 to 6 hours.

8. A method for preparing arginine nanoparticles according to any one of claims 1-7.

9. A nano-targeted liver drug, characterized in that, The active ingredient of the drug includes arginine nanoparticles prepared by the method described in any one of claims 1 to 7, or arginine nanoparticles as described in claim 8.

10. The use of arginine nanoparticles as described in claim 8 or nano-targeted liver drugs as described in claim 9 in the treatment of liver fibrosis.

Images