A nano-drug for treating renal fibrosis and a preparation method thereof

Abstract

The application belongs to the technical field of biological medicine, and provides a nano drug for treating renal fibrosis and a preparation method thereof. The preparation method comprises the following steps: dissolving 1,6-hexane diisocyanate, 2,2'-(propane-2,2-diylbis(thio diyl))bis(ethane-1-ol) and 2,2-bis(bromomethyl)propane-1,3-diol in a high-polarity aprotic solvent to obtain a first solution through first constant-temperature stirring; adding polyethylene glycol 2000 monomethyl ether, and performing second constant-temperature stirring; and performing dialysis and drying to obtain a polymer PHPDM; dissolving the polymer PHPDM and triphenylphosphine in a high-polarity aprotic solvent to obtain a second solution, performing third constant-temperature stirring, and performing dialysis and drying to obtain a polymer PHPDM-PPh3; and self-assembling sinomenine and the PHPDM-PPh3 into nano sinomenine through nano precipitation. The nano drug provided by the application can improve the targeting of the drug, is used for treating renal fibrosis, and can also reduce the side effects of sinomenine.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a nanomedicine for treating renal fibrosis and its preparation method. Background Technology

[0002] Renal fibrosis is a crucial pathological process in chronic kidney disease (CKD), and it is the ultimate common outcome of various chronic kidney diseases leading to chronic renal failure. It is divided into three parts: glomerular sclerosis, tubulointerstitial fibrosis, and renal vascular fibrosis. Among these, the epithelial-interstitial transition (EMT) of renal tubular epithelial cells is considered one of the important mechanisms in the development and progression of renal fibrosis. TGF-β1 can promote the formation of reactive oxygen species (ROS) through the Smad2 / 3 signaling pathway; simultaneously, ROS can promote the transcription of downstream signaling factors of TGF-β1, activating the proliferation, migration, and differentiation of fibroblasts, and inducing excessive deposition of EMT and ECM (extracellular matrix). Therefore, inhibiting EMT by reducing inflammation and oxidative stress may significantly delay tubulointerstitial damage and the progression of renal fibrosis. Mitochondria, as the core organelle regulating energy metabolism, oxidative stress, and inflammatory responses within cells, are involved in various renal pathological processes, especially the development and progression of EMT and renal fibrosis, due to their abnormal function, which mediates increased oxygen free radicals and dysregulation of inflammatory responses. Mitophagy, a highly selective form of autophagy, plays a crucial role in maintaining mitochondrial stability, intracellular oxidative stress, and energy metabolism balance by encapsulating and degrading damaged mitochondria. The PINK1 / Parkin signaling pathway is a classic pathway for mitophagy. Therefore, activating PINK1 / Parkin-mediated mitophagy is an important intervention target for improving renal fibrosis; however, currently there are no methods to precisely regulate mitophagy to intervene in the progression of renal fibrosis. Consequently, there are still no targeted drugs for the treatment of renal fibrosis.

[0003] Sinomenine (SIN) is an alkaloid monomer from the plant *Sinomenium acutum*, belonging to the Menispermaceae family. SIN is bitter, pungent, and neutral in nature, and enters the liver and spleen meridians. It was first recorded in the Song Dynasty's *Illustrated Materia Medica*, and possesses effects such as dispelling wind and dampness, clearing the meridians, and relieving pain. Modern pharmacological studies have shown that SIN has anti-inflammatory, immunosuppressive, immunomodulatory, and anti-tumor effects. In recent years, Sinomenine has been widely used to treat rheumatoid arthritis; however, it has skin and vascular toxic side effects, thus limiting its effectiveness in other disease areas. Nanomaterials can improve the shortcomings of traditional drugs, enhancing efficacy while reducing toxic side effects.

[0004] Therefore, this invention proposes a nanomedicine for treating renal fibrosis and its preparation method. Summary of the Invention

[0005] The purpose of this invention is to provide a nanomedicine for treating renal fibrosis and its preparation method. The proposed method involves constructing a PHPDM-PPh3 delivery system, utilizing the mitochondrial targeting effect of triphenylphosphine to target the inflammatory microenvironment of the kidney. Through its high binding affinity to renal tubular injury markers, it targets damaged renal tubular epithelial cells. A biomimetic delivery carrier with dual microenvironment-cell targeting is constructed to precisely deliver drug-loaded SIN nanoparticles to the renal tubular epithelial cells. In the high ROS environment within the cells, the SIN nanoparticles undergo ROS-responsive oxidative degradation, achieving a cascade release of sinomenine from its extracellular ligand to the intracellular microenvironment within the renal tubular epithelial cells. This provides a concept and feasibility for precise targeted therapy of renal fibrosis.

[0006] In a first aspect, the present invention provides a method for preparing a nanomedicine for treating renal fibrosis, comprising the following steps:

[0007] (1) Preparation of nanomaterial PHPDM-PPh3

[0008] 1) Dissolve 1,6-hexamethylene diisocyanate, 2,2'-(propane-2,2-diylbis(thiodiyl))bis(ethane-1-ol) and 2,2-bis(bromomethyl)propane-1,3-diol in a highly polar aprotic solvent and obtain a first solution by a first isothermal stirring; add polyethylene glycol 2000 monomethyl ether and perform a second isothermal stirring; dialyze and dry to obtain the polymer PHPDM;

[0009] 2) Dissolve the polymer PHPDM and triphenylphosphine from step 1) in a highly polar aprotic solvent to obtain a second solution. After a third constant-temperature stirring, dialyze and dry to obtain the polymer PHPDM-PPh3.

[0010] (2) Preparation of nanomedicine SIN@NP

[0011] The sinomenine and the polymer PHPDM-PPh3 from step 2) were self-assembled into nano-sinomenine through nanoprecipitation.

[0012] Preferably, the molar ratio of 1,6-hexanediisocyanate, 2,2'-(propane-2,2-diylbis(thiodiyl))bis(ethane-1-ol), 2,2-bis(bromomethyl)propane-1,3-diol and polyethylene glycol 2000 monomethyl ether is 1~1.2:0.5:0.5:1.

[0013] Preferably, the molar concentration of 1,6-hexamethylene diisocyanate in the first solution is 0.2~0.24 mmol / mL.

[0014] Preferably, the temperature of the first constant-temperature stirring is 32~38℃, and the stirring time is not less than 36 hours.

[0015] Preferably, the temperature of the second constant-temperature stirring is 32~38℃, and the stirring time is not less than 12h.

[0016] Preferably, the highly polar aprotic solvent is dimethyl sulfoxide, N-methylpyrrolidone, N,N-dimethylacetamide, or N,N-dimethylformamide.

[0017] Preferably, the highly polar aprotic solvent is N,N-dimethylformamide.

[0018] Preferably, the mass ratio of the polymer PHPDM to triphenylphosphine is 475~525:131.

[0019] Preferably, the mass concentration of polymer PHPDM in the second solution is 65~70 mg / mL.

[0020] Preferably, the temperature of the third constant-temperature stirring is 75~85℃, and the stirring time is not less than 36 hours.

[0021] Preferably, the drying method is freeze drying or vacuum drying.

[0022] Secondly, the present invention also provides a nanomedicine for treating renal fibrosis prepared by the above preparation method.

[0023] Thirdly, the present invention also provides the application of the above-mentioned nanomedicine for treating renal fibrosis in the preparation of a drug for treating renal fibrosis.

[0024] The beneficial effects of this invention are:

[0025] This invention provides a nanomedicine and its preparation method. The nanomedicine utilizes the mitochondrial targeting effect of triphenylphosphine to target the inflammatory microenvironment of the kidney. Through its high binding affinity to renal tubular injury markers, it targets damaged renal tubular epithelial cells. A biomimetic delivery carrier with dual microenvironment-cell targeting is constructed to precisely deliver drug-loaded SIN nanoparticles to the renal tubular epithelial cells. In vitro experiments have demonstrated that after entering the kidneys of mice, the nanoparticles rapidly accumulate in the high ROS environment post-surgery and have a long retention time, thus effectively exhibiting an anti-fibrotic effect. Furthermore, the nanomedicine contains mitochondrial targeting groups, making it more easily taken up by damaged renal tubules. It can rapidly target and specifically accumulate in the kidneys of UUO mice in a high ROS microenvironment, significantly improving renal tubular dilation and peritubular interstitial fibrosis. Attached Figure Description

[0026] Figure 1Characterization of nano-sized sinomenine (A is a transmission electron microscope image, B is a zeta potential image, C is a polydispersity index image, D is the distribution of dynamic light scattering particles before hydrogen peroxide incubation, E is the distribution of dynamic light scattering particles after hydrogen peroxide incubation, and F is a stability assessment image).

[0027] Figure 2 This is a confocal microscope image of the intracellular uptake of nano-sinomenine;

[0028] Figure 3 These are flow cytometry peak diagrams and semi-quantitative fluorescence diagrams of cellular uptake of nano-sinomenine.

[0029] Figure 4 This is a mitochondrial colocalization staining image of nano-singed styracin in TCMK-1 cells;

[0030] Figure 5 This is a mitochondrial colocalization staining image of nano-singed senna in HK-2 cells;

[0031] Figure 6 These are a statistical graph of cytotoxicity of SIN detected by MTT assay and a confocal graph of cell proliferation detected by Edu assay (A is the statistical graph, B is the confocal graph).

[0032] Figure 7 The results are quantitative analysis of the fluorescence expression of TGF-β1, α-SMA, E-cadherin, Collagen and Fibronectin proteins in HK-2 cells after different drug treatments (PBS is the blank control group, SIN is the sinomenine group, TGF-β1 is the model group, and NP is the nano-sinomenine group).

[0033] Figure 8 The expression of fibrosis-related proteins (E-Cadherin, TGF-β1, and α-SMA) was detected by Western blot (GAPDH was used as an internal reference protein).

[0034] Figure 9 The effects of different drug treatments on the levels of ATP, ROS, and calcium ions in HK-2 cells are shown in Figure A (ATP production was quantitatively detected by microplate reader, ROS fluorescence expression was semi-quantitatively statistically analyzed by FCM, and calcium ion production was semi-quantitatively detected by FCM).

[0035] Figure 10 This is a confocal image of mitochondrial JC-1 cells in HK-2 cells after treatment with different drugs;

[0036] Figure 11 This is a confocal image of the mitochondrial membrane permeability transition pore in HK-2 cells after treatment with different drugs;

[0037] Figure 12It is a microplate reader used to detect the level of autophagy fluorescence in cells after treatment with different drugs;

[0038] Figure 13 The expression of mitophagy-related proteins (p62, PINK1, Parkin, and LC3B) was detected by Western blot.

[0039] Figure 14 The results show mouse body weight, cardiac, hepatic, and renal function as detected by ELISA, and plasma TGF-β1 expression (PBS represents the sham-operated group, UUO represents the model group, SIN represents the sinomenine group, SIN@NP represents the nano-sinomenine group, and Col represents the colchicine group).

[0040] Figure 15 The H&E staining of heart, liver, spleen, and lung tissues in UUO mice;

[0041] Figure 16 The in vivo fluorescence imaging statistics of normal mice and UUO mice after intravenous injection of Cy5.5 are shown in the figure (PBS-NP represents the control group, i.e., normal mice targeted by nano-singletonine, and UUO-NP represents the experimental group, i.e., model mice targeted by nano-singletonine).

[0042] Figure 17 This is a statistical analysis of organ fluorescence imaging after intravenous injection of Cy5.5 in normal mice and UUO mice;

[0043] Figure 18 This is a laser confocal microscopy observation of organ fluorescence imaging 48 hours after UUO mice were injected with NP@Cy5.5 and SIN@NP@Cy5.5 via the tail vein. Detailed Implementation

[0044] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0045] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0046] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0047] Example 1: Preparation of Nanomedicines

[0048] (1) Preparation of nanomaterials

[0049] like Figure 1As shown, a ROS-sensitive polymer, Poly-(HD-co-PSDE-co-Bbp)-mPEG, abbreviated as PHPDM, was first synthesized. This polymer was then bonded to triphenylphosphine to form the PHPDM-PPh3 complex, as detailed below:

[0050] 1,6-Hexamethylene diisocyanate (185 mg, 1.1 mmol), 2,2'-(propane-2,2-diylbis(thiodiyl))bis(ethane-1-ol) (98 mg, 0.5 mmol), and 2,2-bis(bromomethyl)propane-1,3-diol (131 mg, 0.5 mmol) were dissolved in 5 mL of ultra-dry N,N-dimethylformamide (DMF) and stirred continuously at 35 °C for 36 h. Polyethylene glycol 2000 monomethyl ether (2 g, 1 mmol) was added, and the mixture was stirred continuously at 35 °C for 12 h. Finally, the polymer PHPDM was obtained by dialysis and vacuum drying.

[0051] The polymer PHPDM (1 g) and triphenylphosphine (262 mg, 1 mmol) were dissolved in 15 mL of ultradry N,N-dimethylformamide (DMF) and stirred continuously at 80 °C for 36 h. Finally, the polymer PHPDM-PPh3 was obtained by dialysis and vacuum drying.

[0052] (2) Preparation of nanomedicines

[0053] SIN and PHPDM-PPh3 were self-assembled into nano-Sinogenine (SIN@NP, or NP for short) via nanoprecipitation. This SIN@NP can undergo cleavage under high ROS microenvironment conditions.

[0054] Example 2 Morphology and Characterization of SIN@NP

[0055] To investigate the morphology and characterization of the SIN@NPs prepared in Example 1, this example utilizes transmission electron microscopy (TEM) to characterize the surface microstructure, structure, size, and the dispersion and quantitative analysis of elemental composition (S, N, P, etc.) in the micro-areas of the SIN@NPs. TEM observation revealed that the synthesized SIN@NPs exhibited uniform and complete spherical characteristics, with a particle size of approximately 120 nm and a polydispersity index (PDI) of 0.1344 (see...). Figure 1 Further measurements using dynamic light scattering (DLS) technology showed that the average particle size of SIN@NP was 108 nm. These results indicate that SIN@NP exhibits good dispersibility in solution.

[0056] Since the cell membrane is composed of a negatively charged phospholipid bilayer, positively charged liposomes and lipid nanoparticles exhibit significant cell membrane penetration capabilities. This embodiment utilizes a nanoparticle size zeta potential analyzer to characterize the surface charge properties, particle size, and distribution characteristics of SIN@NPs. Electrophoresis was used to measure the migration velocity and direction of SIN@NPs in an electric field, and the zeta potential (charge and potential magnitude) was calculated to determine the charge properties on the SIN@NP surface, thereby optimizing the interaction between sinomenine and the receptor. The measured zeta potential of SIN@NPs was 0.25 ± 1.59 mV (see...). Figure 1 This further confirms that the SIN@NP prepared in Example 1 has good biocompatibility.

[0057] Because the PHPDM polymer backbone contains disulfide bonds, it can be activated by excess ROS in the inflammatory state of chronic kidney disease, leading to the degradation of the polymer carrier, the release of nanoparticles, and the release of the drug. To further investigate this process, SIN@NP was incubated in 10 mM hydrogen peroxide solution for 24 hours. TEM results showed that SIN@NP degraded into smaller fragments (see...). Figure 1 Further analysis by DLS showed that the SIN@NP particle size curve changed from a single peak to a double peak after incubation with hydrogen peroxide (see...). Figure 1 This indicates that SIN@NP did indeed undergo fragmentation. Particle size measurements of SIN@NP were performed every other day, and the results showed that the particle sizes of the seven measurements were relatively consistent, indicating that SIN@NP has good stability.

[0058] In summary, SIN@NP possesses drug release characteristics responsive to reactive oxygen species.

[0059] Example 3 Cellular uptake of SIN@NP

[0060] Nanoparticles can only effectively exert their drug effects after being absorbed by cells or in vivo. To investigate whether SIN@NP can enhance the accumulation of small molecule SIN in cells, this example first analyzed the uptake of SIN@NP by renal tubular cells (HK-2). SIN@NP was labeled with Cy5.5 dye (red), named SIN@NP@Cy5.5, and applied to HK-2 cells. Using confocal laser scanning microscopy (CLSM), the changes in the intensity of intracellular red fluorescence in HK-2 cells were observed directly at 0, 1, 4, and 7 hours after treatment with SIN@NP@Cy5.5. Cell nuclei were stained blue with DAPI, and actin filaments (F-actin) were stained with Actin-Tracker Green-488 (a microfilament green fluorescent probe). The results showed that after 4 hours of SIN@NP drug treatment, a significant red fluorescence signal was visible in HK-2 cells, while after 7 hours, the intracellular red fluorescence signal became extremely prominent. Furthermore, after entering the cells, SIN@NP is mainly distributed in the cytoplasm (…). Figure 2 ).

[0061] Then, flow cytometry was used to perform a semi-quantitative analysis of the changes in intracellular fluorescence intensity in HK-2 cells under different drug treatment times. The results showed that after 7 hours of treatment with SIN@NP@Cy5.5, the mean fluorescence intensity (MFI) of Cy5.5 in HK-2 cells was 1.37 times and 2.95 times that after 4 hours and 1 hour of treatment with SIN@NP@Cy5.5, respectively (P < 0.001). Figure 3 ).

[0062] In summary, SIN@NP can enhance the accumulation of small molecule SIN in cells.

[0063] Example 4: Mitochondrial targeting of SIN@NP

[0064] Given that SIN@NPs are modified with mitochondrial targeting groups, this embodiment uses a mitochondrial probe to study co-localization with SIN@NPs labeled with Cy5.5 (referred to as SIN@NP@Cy5.5). TCMK-1 and HK-2 renal tubular cells were treated with SIN@NPs labeled with Cy5.5 dye (red), and the co-localization within the mitochondria was verified by MitoTracker Green staining. This embodiment uses confocal laser scanning microscopy (CLSM) to visually observe the red fluorescence intensity and the fluorescence of the green mitochondrial probe in TCMK-1 and HK-2 cells at 1, 2, 4, 6, and 7 hours after treatment with SIN@NP@Cy5.5. The results show that a weak yellow fluorescence signal is visible in TCMK-1 and HK-2 cells 1 hour after SIN@NP@Cy5.5 treatment, while from 2 to 7 hours after treatment, the yellow fluorescence signal gradually increases and remains at a strong level.

[0065] In summary, after entering the cell, SIN@NP drugs are mainly distributed in the mitochondria of the cytoplasm and can remain there for a long time. Figure 4 and Figure 5 ).

[0066] Example 5: Effects of SIN@NP on renal cell toxicity and cell proliferation

[0067] First, this embodiment used the cytotoxicity MTT assay to compare the cytotoxicity of sinomenine (SIN) against three different kidney cell lines (TCMK-1, HK-2, and NRK-52E). The results showed that SIN had good safety. The IC50 of SIN against HK-2 cells was [not specified in the original text]. 50 The value was 488.7 μM, and the IC50 value for TCMK-1 cells was [missing value]. 50 The value was 497 μM, and the IC50 value for NRK-52E cells was [value missing]. 50 The value is 328.6 μM ( Figure 6 (A in the middle).

[0068] Subsequently, we used the EdU assay to detect HK-2 cell proliferation. In normal HK-2 cells, DNA synthesis was normal, while DNA synthesis was reduced in fibrotic renal tubular cells induced by TGF-β1. After treatment with SIN and SIN@NP, DNA synthesis in fibrotic renal tubular cells recovered. Figure 6 (B in the middle).

[0069] Example 6: Effects of SIN@NP on TGF-β1-induced HK-2 fibrotic cells and its influence on the mitophagy pathway.

[0070] To investigate the effects of SIN@NP on TGF-β1-induced HK-2 fibrotic cells and the mitophagy pathway, this study grouped HK-2 cells as follows:

[0071] Blank control group (PBS): HK-2 cells were cultured normally in MEM medium containing 10% fetal bovine serum and 1% penicillin / streptomycin for 24h and 48h.

[0072] TGF-β1 induction group (model group, TGF): After HK-2 cells adhered to the culture vessel under normal conditions, they were cultured in a medium containing 10 ng / ml TGF-β1 for 24 h and 48 h.

[0073] Sinensis alkaloid group (SIN): After HK-2 cells adhered to the culture medium under normal conditions, they were cultured for 24 h and 48 h in a medium containing 10 ng / ml TGF-β1 and 60 μM drug.

[0074] Nano-Sinomenine group (SIN@NP): After HK-2 cells adhered to the culture medium under normal conditions, they were cultured for 24 h and 48 h in a medium containing 10 ng / ml TGF-β1 and 40 μM of drug.

[0075] 1. Effects of SIN@NP on TGF-β1-induced HK-2 fibrotic cells

[0076] First, we investigated whether SIN and SIN@NP could inhibit the protein expression of TGF-β1, α-SMA, E-cadherin, Collagen, and Fibronectin in fibrotic HK-2 cells, and then performed a semi-quantitative analysis of these proteins in HK-2 cells.

[0077] Immunofluorescence staining (red) was performed on TGF-β1, α-SMA, E-cadherin, Collagen, and Fibronectin proteins in the four groups of HK-2 cells. Then, the cytoskeleton was stained using Alexa Fluor 488 (green), and finally observed using CLSM. Quantitative analysis showed that the levels of TGF-β1, α-SMA, E-cadherin, Collagen, and Fibronectin in HK-2 cells treated with SIN@NP and SIN were 1.56-fold and 1.24-fold, 1.54-fold and 1.30-fold, 1.96-fold and 1.56-fold, 1.95-fold and 1.59-fold, and 1.55-fold and 1.42-fold lower, respectively, than those in the model group. Figure 7 ).

[0078] The expression of fibrosis-related proteins in the four groups of HK-2 cells was analyzed by Western blotting. The results showed that compared with HK-2 cells treated with SIN, cells treated with SIN@NP showed significantly increased E-cadherin protein expression, while the expression of model histones was significantly decreased. Fibrosis proteins TGF-β1 and α-SMA were significantly reduced after drug treatment. These results indicate that SIN@NP and SIN can inhibit the expression of fibrosis proteins. Figure 8 ).

[0079] Furthermore, the effects of SIN and SIN@NP on intracellular ATP content, ROS fluorescence expression, and calcium ion content in HK-2 cells were investigated.

[0080] In this embodiment, the ATP, ROS, and calcium ion contents in the four groups of HK-2 cells were measured and analyzed using an enzyme-linked immunosorbent assay (ELISA) reader and FCM, respectively. The results showed that the ATP content in HK-2 cells treated with SIN@NP and SIN was 2.68 times and 1.27 times higher than that in the model group, respectively. Figure 9 (A in the text); The ROS content in HK-2 cells treated with SIN@NP and SIN was 1.49 times and 1.19 times lower than that in the model group, respectively. Figure 9 (B in the text); The calcium ion content in HK-2 cells treated with SIN@NP and SIN was 1.3 times and 1.09 times higher than that in the model group, respectively. Figure 9 (C in the middle).

[0081] Furthermore, the effects of SIN and SIN@NP on changes in mitochondrial membrane potential and mitochondrial membrane permeability transition pore (MPTP) in HK-2 cells were investigated.

[0082] The four groups of HK-2 cells were subjected to immunofluorescence staining according to the JC-1 detection kit and the mitochondrial membrane permeability detection kit, and then the cell nuclei were stained with DAPI. Finally, the cells were observed using CLSM.

[0083] In JC-1 assays, stronger red fluorescence in the cytoplasm indicated a more stable mitochondrial membrane potential. After depolarization, the mitochondrial membrane potential was altered, resulting in green fluorescence. The results showed that the red fluorescence intensity in HK-2 cells treated with SIN and SIN@NP was significantly higher than that in the model group. Figure 10 In MPTP detection, stronger green fluorescence indicates lower MPTP opening; weaker green fluorescence indicates higher MPTP opening. Experimental results showed that after SIN and SIN@NP treatment, the green fluorescence intensity in HK-2 cells was significantly lower than that in the model group. Figure 11 ).

[0084] 2. Effects of SIN@NP on TGF-β1-induced mitochondrial autophagy pathway in HK-2 fibrotic cells

[0085] Autophagy occurring in the four groups of HK-2 cells was measured and analyzed using an ELISA reader. The results showed that the autophagy fluorescence in HK-2 cells treated with SIN@NP and SIN was 2.32 times and 1.7 times higher than that in the model group (TGF-β1), respectively. Figure 12 ).

[0086] Western blotting (WB) was used to further analyze the expression of mitophagy-related proteins in the four groups of HK-2 cells. The results showed that compared with HK-2 cells treated with SIN, the levels of mitophagy proteins LC3, PINK1, and Parkin were significantly increased in cells treated with SIN@NP, while p62 protein was decreased. These results indicate that SIN@NP and SIN can enhance mitophagy (…). Figure 13 ).

[0087] Example 7 In vivo animal experiments

[0088] 1. Establishment of the left ureteroscopic ligation (UUO) model and experimental grouping

[0089] C57 / BL6J mice (6-8 weeks old, purchased from Spiefer Biotech) underwent left ureteral ligation surgery and were divided into 5 groups of 6-7 mice each: sham-operated group, UUO model group, SIN group, SIN@NP group, and colchicine (Col) group. Drug administration was performed continuously for 7 days. Specific model establishment and drug administration details are as follows:

[0090] The UUO model group refers to mice with renal interstitial fibrosis model, which are injected with physiological saline via the tail vein.

[0091] Healthy male C57BL / 6 mice were injected via the tail vein with saline, SIN, Col, and SIN@NP solutions, respectively.

[0092] 2. Detection of renal function and other indicators in UUO mice

[0093] After administration, the mice were in good condition, with no significant abnormalities in activity, mental state, or diet. There was no statistically significant difference in body weight between the drug-treated mice (SIN group, SIN@NP group, and Col group) and the control group (sham-operated group), indicating that SIN and related preparations have almost no systemic toxicity.

[0094] Renal function (BUN, CRE), liver function (AST), cardiac function (CK), and plasma TGF-β1 were evaluated in mice. Results showed that compared with the model group, AST levels in the SIN, Col, and SIN@NP groups were not significantly elevated, and the differences were not statistically significant. CK, BUN, CRE, and serum TGF-β1 levels in the model group were significantly higher than in other groups, indicating impaired cardiac and renal function in the model group. Serum ELISA in the model group showed significantly elevated TGF-β1 levels, indicating a higher level of inflammation in the model group (see...). Figure 14 ).

[0095] 3. H&E staining and pathological morphology observation of the heart, liver, spleen and lungs of UUO mice

[0096] Seven days after administration, H&E staining was performed on the major organs (heart, liver, spleen, and lungs) of mice. The results showed that, compared with the control group, there were no significant morphological changes in the major organs of the model group, mice treated with SIN, Col, and SIN@NP, indicating that the drug and formulation have low toxicity and good biocompatibility in vivo. Figure 15 ).

[0097] 4. In vivo biodistribution of SIN@NP in UUO mice

[0098] Next, we established a unilateral ureteral obstruction (UUO) mouse model to investigate the biodistribution of SIN@NP in vivo and its targeting effect on the kidney. Fluorescence signals in mice were monitored via tail vein injection of SIN@NP@Cy5.5 (labeled Cy5.5) carrying a mitochondrial targeting group using an IVIS imaging system. Results showed that fluorescence signals were detectable in the kidneys of both UUO mice and normal control mice one hour after UUO surgery, with the fluorescence intensity in UUO mice increasing by 1.18 times compared to the blank control group. Over time, the fluorescence intensity in the kidneys of the UUO-SIN@NP group mice remained at a stable level, peaking at approximately 48 hours. Figure 16 ).

[0099] Subsequently, the mice were euthanized, and their major organs were subjected to in vitro imaging analysis. The results showed that the liver, as a major metabolic organ, exhibited the highest fluorescence intensity (…). Figure 17The mean fluorescence intensity (MFI) of the kidney tissue was 19.67 × 10^9 p / s / cm² / sr. The kidney tissue ranked second in fluorescence intensity among all tissues, with an MFI of 11.93 × 10^9 p / s / cm² / sr, significantly higher than that of the heart, spleen, lung, and small intestine. In conclusion, after entering the mouse body, SIN@NP rapidly accumulates in the kidney under postoperative high reactive oxygen species (ROS) conditions and remains there for a relatively long time, thus effectively exhibiting an anti-fibrotic effect.

[0100] Forty-eight hours after in vivo imaging, the kidney was removed for paraffin-embedded mass preparation, sectioning, DAPI staining, and Cy5.5 fluorescence in the kidney tissue was observed under a 20x and 40x laser confocal microscope. The results showed that ( Figure 18 In normal kidneys, the red fluorescence in the renal tubules of the SIN@NP@Cy5.5 group was stronger than that of the NP@Cy5.5 group, indicating that the mitochondrial-targeting polymer material with an average particle size of 108 nm has renal tubule targeting properties. In the kidneys of the UUO model group, the red fluorescence in the renal tubules of the SIN@NP@Cy5.5 group was significantly enhanced than that of the NP@Cy5.5 group, and obvious renal tubular dilation was observed in both groups. Under the stimulation of inflammation, the ROS in the renal tissue microenvironment increased significantly. In a high ROS environment, SIN@NP@Cy5.5, due to its mitochondrial-targeting group, is more easily taken up by damaged renal tubules.

[0101] In contrast, in the negative control group injected via tail vein with NP@Cy5.5 solution, the renal fluorescence signal was weaker than that of SIN@NP@Cy5.5, but stronger than that of the non-surgical group. This indicates that NP@Cy5.5 is more easily accumulated in a ROS environment, while SIN@NP@Cy5.5, due to its mitochondrial targeting group, is more easily taken up by the kidneys. These results suggest that SIN@NP@Cy5.5 can rapidly target and specifically accumulate in the kidneys of UUO mice in a high ROS microenvironment.

[0102] The embodiments described above are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for preparing a nanomedicine for treating renal fibrosis, characterized in that, Includes the following steps: (1) Preparation of nanomaterial PHPDM-PPh3 1) 1,6-hexamethylene diisocyanate, 2,2'-(propane-2,2-diylbis(thiodiyl))bis(ethane-1-ol), and 2,2-bis(bromomethyl)propane-1,3-diol were dissolved in a highly polar aprotic solvent at a molar ratio of 1~1.2:0.5:0.

5. A first solution was obtained by a first isothermal stirring, wherein the molar concentration of 1,6-hexamethylene diisocyanate in the first solution was 0.2~0.24 mmol / mL. Polyethylene glycol 2000 monomethyl ether was added, and a second isothermal stirring was performed. The polymer PHPDM was obtained by dialyzing and drying, wherein the molar ratio of 2,2-bis(bromomethyl)propane-1,3-diol to polyethylene glycol 2000 monomethyl ether was 0.5:

1. 2) Dissolve the polymer PHPDM and triphenylphosphine from step 1) in a highly polar aprotic solvent at a mass ratio of 475~525:131 to obtain a second solution. The mass concentration of polymer PHPDM in the second solution is 65~70 mg / mL. After a third constant temperature stirring, the solution is dialyzed and dried to obtain polymer PHPDM-PPh3. (2) Preparation of nanomedicine SIN@NP The sinomenine and the polymer PHPDM-PPh3 from step 2) were self-assembled into nano-sinomenine through nanoprecipitation.

2. The preparation method according to claim 1, characterized in that, The temperature of the first constant-temperature stirring is 32~38℃, and the stirring time is not less than 36 hours.

3. The preparation method according to claim 1, characterized in that, The temperature for the second constant-temperature stirring is 32~38℃, and the stirring time is not less than 12 hours.

4. The preparation method according to claim 1, characterized in that, The highly polar aprotic solvent is dimethyl sulfoxide, N-methylpyrrolidone, N,N-dimethylacetamide, or N,N-dimethylformamide.

5. The preparation method according to claim 1, characterized in that, The highly polar aprotic solvent is N,N-dimethylformamide.

6. The preparation method according to claim 1, characterized in that, The temperature of the third constant-temperature stirring is 75~85℃, and the stirring time is not less than 36 hours.

7. The preparation method according to claim 1, characterized in that, The drying method is freeze drying or vacuum drying.

8. A nanomedicine for treating renal fibrosis prepared by the preparation method according to any one of claims 1-7.

9. The use of the nanomedicine as described in claim 8 in the preparation of a medicament for treating renal fibrosis.

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