Liposome nano lysosome degradation agent for targeted degradation of LRG1 protein as well as preparation method and application of liposome nano lysosome degradation agent

Abstract

The invention belongs to the technical field of biological medicine, and provides a liposome nano lysosome degradation agent for targeting LRG1 as well as a preparation method and application thereof, and the liposome nano lysosome degradation agent is composed of egg yolk lecithin / cholesterol nano liposome, entrapped GYY4137, LRG1 targeting ET polypeptide with the surface subjected to click chemical modification and a lysosome targeting M6P ligand. A microfluidic technology is adopted for preparation, the molar ratio of DSPE-PEG2000-ET to DSPE-PEG2000-M6P is 1: 3, and the mass ratio of lipid to a medicine is 2: 1. The degradation agent can be used for accurately activating hepatic stellate cells in a targeted manner, guiding LRG1 to enter lysosome for degradation and cooperating with GYY4137 for release, so that a dual anti-hepatic fibrosis effect is realized, and the degradation agent is used for preparing medicines for treating hepatic diseases such as hepatic fibrosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a liposomal nanolysosome degrading agent for targeted degradation of LRG1 protein, its preparation method, and its application. Background Technology

[0002] Liver fibrosis, a common chronic liver disease, is characterized by the persistent activation of hepatic stellate cells and abnormal deposition of extracellular matrix. Currently, there is a lack of highly effective treatments with low side effects, making the development of novel targeted anti-fibrotic drugs of significant clinical importance. Studies have shown that the secretory glycoprotein LRG1 is significantly upregulated in the progression of liver fibrosis and directly promotes hepatic stellate cell activation and extracellular matrix production by activating the TGF-β / Smad signaling pathway, thus becoming a highly promising therapeutic target.

[0003] In recent years, lysosomal targeted degradation technology, especially lysosomal targeted chimera technology, has attracted widespread attention as an emerging strategy for degrading extracellular and membrane proteins. These molecules bind to the target protein at one end and a lysosomal membrane receptor at the other, guiding the target protein into the lysosomal degradation pathway. This not only expands the degradation range of traditionally "undruggable" targets but also provides new ideas for the treatment of liver fibrosis. Among existing technologies, there have been attempts to construct LYTAC molecules using N-acetylgalactosamine (GalNAC), which targets the desialyl glycoprotein receptor (ASGPR), as a ligand to achieve liver-specific protein degradation. However, ASGPR is mainly highly expressed in hepatocytes, and its targeting specificity to activated hepatic stellate cells—the core effector cells of liver fibrosis—is limited, potentially leading to insufficient drug concentration accumulation in lesion cells and affecting treatment efficacy. In contrast, the cation-independent mannose-6-phosphate receptor (CI-M6PR) has been shown to be specifically highly expressed on the surface of activated hepatic stellate cells. Therefore, using mannose-6-phosphate (M6P) instead of GalNAC as the lysosomal targeting ligand can more precisely deliver the degrading agent to the core driver cells of the disease, potentially upgrading from "liver tissue targeting" to "precise targeting of liver diseased cells," thereby significantly improving drug utilization and therapeutic efficiency. However, traditional LYTAC technology still faces limitations such as complex synthesis and poor tissue permeability. To overcome these problems, nanotechnology has been introduced to construct liposomal LYTAC systems. These systems not only possess good biocompatibility and stability but also achieve tissue targeting and controlled drug release through surface functionalization, significantly improving drug accumulation and retention at the lesion site. For example, encapsulating the H2S donor-type antifibrotic drug GYY4137 in targeted-modified lipid nanoparticles can enhance its targeting and acid-responsive release behavior, and synergize with the LRG1 degradation strategy to achieve a synergistic enhancement of antifibrotic efficacy, opening a new path for the precision treatment of liver fibrosis. Summary of the Invention

[0004] The present invention aims to provide a liposomal nanolysosome degrading agent that targets and degrades LRG1 protein, as well as its preparation method and application, which can effectively delay or prevent the progression of liver fibrosis, thus providing a new approach for the treatment of liver fibrosis.

[0005] To achieve the above-mentioned technical objectives and effects, the present invention provides the following technical solution:

[0006] A liposomal nanolysosome degrading agent for degrading leucine-rich α-2 glycoprotein 1 (LRG1) is characterized by comprising nanoliposomes, an antifibrotic drug loaded inside the nanoliposomes, an LRG1 protein targeting unit modified on the surface of the nanoliposomes, and a lysosome targeting unit modified on the surface of the nanoliposomes; the liposomal nanolysosome degrading agent is prepared by microfluidic technology.

[0007] Furthermore, the nanoliposomes are composed of egg yolk lecithin and cholesterol; the LRG1 protein targeting unit is an ET polypeptide with the amino acid sequence ESYSAKHRIMLT, as shown in SEQ ID NO.1; the lysosomal targeting unit is mannose-6-phosphate ligand (M6P); and the antifibrotic drug is hydrogen sulfide donor GYY4137, chemically named morpholine-4-methoxyphenyl (morpholine)phosphine dithiophosphate.

[0008] Furthermore, the LRG1 protein targeting unit ET peptide and lysosome targeting unit M6P ligand are modified on the surface of liposomes via a copper-catalyzed click chemistry reaction: the alkyne-modified ET peptide and M6P ligand undergo cycloaddition reactions with DSPE-PEG2000-N3 to generate functionalized lipid-polyethylene glycol copolymers DSPE-PEG2000-ET and DSPE-PEG2000-M6P.

[0009] Furthermore, the molar ratio of the raw materials is: lecithin: cholesterol: functionalized lipid-polyethylene glycol copolymer = 5:4:1; the functionalized lipid-polyethylene glycol copolymer is a mixture of DSPE-PEG2000-ET and DSPE-PEG2000-M6P.

[0010] Furthermore, the molar ratio of DSPE-PEG2000-ET to DSPE-PEG2000-M6P is 1:3.

[0011] Furthermore, the mass ratio of the nanoliposomes to the antifibrotic drug GYY4137 is 2:1.

[0012] On the other hand, the present invention also provides a method for preparing the above-mentioned liposome nanolysosome degrading agent, the method being based on microfluidic technology and comprising the following steps:

[0013] (1) Preparation of lipid organic phase and drug aqueous phase: Lecithin, cholesterol, DSPE-PEG2000-ET and DSPE-PEG2000-M6P are dissolved in an organic solvent to form lipid organic phase; anti-fibrotic drug GYY4137 is dissolved in an aqueous solvent to form drug aqueous phase;

[0014] (2) Microfluidic nanoparticle formation: The lipid organic phase obtained in step (1) and the drug aqueous phase are injected into the microfluidic device at a volume flow rate of 1:3 to mix, so that the lipid molecules self-assemble to form nanoparticles loaded with GYY4137.

[0015] (3) Purification and concentration: Collect the nanoparticle suspension obtained in step (2), remove the organic solvent and replace it with pure water or a buffer system to obtain the lipid nanolysosome degrading agent.

[0016] Further, in step (1), the organic solvent is methanol or ethanol; the aqueous solvent is phosphate buffer or physiological saline at pH 7.4.

[0017] Furthermore, in step (3), the organic solvent is removed by ultrafiltration and liquid replacement is performed. Specifically, an ultrafiltration tube with a molecular weight cutoff of 50 kDa is used, and the mixture is centrifuged at 4000 rpm for 30 minutes. This operation is repeated to complete the purification.

[0018] On the other hand, the present invention also provides the application of the above-mentioned liposome nanolysosome degrading agent in the preparation of drugs for the prevention or treatment of liver diseases, wherein preferred liver diseases include liver fibrosis, chronic liver disease, end-stage liver disease, etc.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. Achieved highly efficient degradation of traditionally "undruggable" targets: This invention innovatively employs lysosomal targeted chimeric technology, constructing a nano-degrader by combining an ET peptide targeting LRG1 protein with the lysosomal targeting ligand M6P. This nano-degrader guides cell membrane-associated LRG1 protein into the lysosomal degradation pathway. This technology overcomes the spatial limitations of traditional proteasome degradation technology, significantly expands the range of degradable targets, and achieves tissue-specific delivery through ligand engineering, providing a novel targeted degradation strategy for the treatment of liver fibrosis.

[0021] 2. Excellent liver targeting and therapeutic safety: The liposome carrier constructed using nanotechnology not only has good biocompatibility and stability, but its surface-modified ET and M6P ligands also accurately recognize the highly expressed LRG1 protein in the diseased liver and the M6PR receptor on the surface of activated hepatic stellate cells, respectively. This achieves efficient accumulation and cell-specific internalization of the drug at the lesion site, significantly improving the precision of treatment and effectively reducing toxic side effects on non-target tissues.

[0022] 3. Synergistic Enhancement of Anti-Hepatic Fibrosis Therapy: This degrading agent innovatively combines LRG1 protein degradation with anti-fibrotic drug therapy. On one hand, it effectively inhibits TGF-β / Smad signaling pathway activation and reduces extracellular matrix deposition by degrading LRG1; on the other hand, the encapsulated GYY4137 slowly releases H2S in the acidic environment of lysosomes, activating the Nrf2 pathway. These two components form a dual synergistic mechanism, working together on the core signaling pathways of liver fibrosis, thereby significantly enhancing the inhibitory effect on hepatic stellate cell activation and the anti-fibrotic therapeutic effect. Attached Figure Description

[0023] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the description of the specific embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 The synthesis, preparation, and characterization of the liposome nanolysosome degrading agent in this embodiment of the invention include: the synthesis and proportion screening of functionalized lipid-polyethylene glycol copolymers, a flowchart of liposome preparation, the encapsulation drug loading rate of GYY4137, particle size, zeta potential, and morphology.

[0025] Figure 2 The anti-liver fibrosis effect of drug GYY4137 in this embodiment of the invention and its in vitro pH-responsive release curve show its inhibitory effect on activated hepatic stellate cells and H2S release kinetics under different pH conditions.

[0026] Figure 3 To evaluate the degradation ability of the liposome nanolysosome degrading agent on LRG1 protein at the cellular level in this embodiment of the invention, its degradation efficiency was verified by Western blotting and quantitative analysis.

[0027] Figure 4 To evaluate the anti-liver fibrosis ability of the liposome nanolysosome degrading agent at the cellular level in this embodiment of the invention: the anti-liver fibrosis effect was verified by Western blotting and quantitative analysis;

[0028] Figure 5 To illustrate the anti-liver fibrosis effect of the liposome nanolysosome degrading agent in this embodiment of the invention: the pathway through which it achieves a dual synergistic effect by "degrading LRG1" and "activating Nrf2" is explained;

[0029] Figure 6 In the CCl4-induced liver fibrosis model of this invention, the targeting effect of different treatment groups on the liver, the gross appearance of the liver, the mouse body weight, the change in liver weight ratio, and the in vitro targeting situation are shown: intuitively demonstrating the targeting effect of the degradation agent on the liver, the protective effect on liver morphology, the effect on weight gain, and the targeting effect on the liver.

[0030] Figure 7 The inhibitory effect of the liposome nanolysosome degrading agent on the expression of liver fibrosis-related proteins in vivo in this embodiment of the invention includes staining results of collagen deposition in liver tissue sections and immunohistochemical analysis of key proteins;

[0031] Figure 8 To investigate the molecular mechanism of the liposome nanolysosome degrading agent in vivo against liver fibrosis in this invention, the changes in key proteins of the TGF-β / Smad pathway and Nrf2 nuclear translocation in mouse liver tissue were examined.

[0032] Figure 9 The in vivo safety evaluation of the liposome nanolysosome degrading agent in this embodiment of the invention includes histopathological observation of major organs and analysis of serum biochemical indicators. Detailed Implementation

[0033] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0034] This invention provides a lipid nano-lysosome degrading agent for degrading leucine-rich α-2 glycoprotein 1 (LRG1). The core of this degrading agent lies in a multi-level targeted delivery and synergistic therapeutic system. Specifically, the degrading agent comprises nanoliposomes prepared using microfluidic technology, an antifibrotic drug encapsulated within the nanoliposomes, LRG1 protein targeting units modified on the surface of the nanoliposomes, and lysosomal targeting units modified on the surface of the nanoliposomes.

[0035] In a preferred embodiment:

[0036] The nanoliposomes are composed of biocompatible egg yolk lecithin and cholesterol to provide a stable bilayer structure.

[0037] The LRG1 protein targeting unit is an ET polypeptide, the amino acid sequence of which is ESYSAKHRIMLT, as shown in SEQ ID NO.1 below, and is used to specifically recognize and bind to LRG1 protein that is highly expressed under pathological conditions.

[0038]

[0039] The lysosomal targeting unit is a mannose-6-phosphate (M6P) ligand, which binds to the M6P receptor highly expressed on the surface of activated hepatic stellate cells, mediating the endocytosis of the entire degrading agent and its eventual entry into the lysosome.

[0040] The anti-fibrotic drug is a hydrogen sulfide donor, GYY4137 (chemical name: morpholine-4-methoxyphenyl(morpholine)phosphine dithiophosphate), which can accelerate the release of hydrogen sulfide in the acidic environment of lysosomes and exert an anti-fibrotic effect.

[0041] Regarding the component ratio, the molar ratio of the raw materials used in the preparation of the lipid nano-lysosome degrading agent is: lecithin: cholesterol: functionalized lipid-polyethylene glycol copolymer = 5:4:1. The functionalized lipid-polyethylene glycol copolymer is a mixture of DSPE-PEG2000-ET and DSPE-PEG2000-M6P, and their molar ratio is preferably 1:3 to achieve an optimized balance of targeted function. The mass ratio (drug-lipid ratio) of the antifibrotic drug GYY4137 to the total lipids is preferably 1:2 to ensure a high drug loading and therapeutic effect.

[0042] The LRG1 protein targeting unit and lysosome targeting unit are modified onto the liposome surface using an advanced click chemistry strategy. Specifically, an alkyne-modified ET peptide and an alkyne-modified M6P ligand are coupled to DSPE-PEG2000-N3 via a copper-catalyzed click chemistry reaction to generate well-defined triazole ring linkers, namely, functionalized lipid-polyethylene glycol copolymers DSPE-PEG2000-ET and DSPE-PEG2000-M6P.

[0043] The degradative agent designed in this invention can accumulate in liver fibrosis areas through the EPR effect and active targeting. Its mechanism of action is as follows: firstly, it captures LRG1 protein via ET peptides, then is efficiently internalized by activated hepatic stellate cells through M6P-M6PR interactions, thereby carrying the LRG1 protein into lysosomes for degradation. Simultaneously, the encapsulated GYY4137 accelerates the release of H2S in acidic lysosomes, activating the Nrf2 pathway by promoting Keap1 thioylation, promoting Nrf2 nuclear translocation, and synergistically inhibiting the TGF-β / Smad signaling pathway with LRG1 degradation, jointly reducing extracellular matrix deposition, thus achieving highly effective treatment for liver fibrosis.

[0044] II. Preparation Method

[0045] Based on the same inventive concept, this invention also provides a method for preparing the aforementioned lipid nanolysosome degrading agent. The key to this method lies in utilizing microfluidic technology to achieve uniform, controllable, and reproducible preparation of nanoparticles.

[0046] The method includes the following steps:

[0047] (1) Synthesis of functionalized lipid materials:

[0048] Synthesis of DSPE-PEG-ET and DSPE-PEG-M6P: Both functionalized lipid-polyethylene glycol copolymers were prepared via copper-catalyzed click chemistry. DSPE-PEG-ET was synthesized by dissolving DSPE-PEG-N3 and excess ET-alkyne in methanol, reacting at room temperature for 5 hours under the catalysis of copper sulfate pentahydrate and sodium ascorbate. DSPE-PEG-M6P was synthesized using DMF as solvent, with DSPE-PEG-N3 and an equimolar amount of M6P-alkyne reacted under the same catalytic system and reaction conditions. After the reaction, the reaction solutions were dialyzed for two days using a dialysis bag with a molecular weight cutoff of 500 Da to completely remove copper ions. Finally, the solutions were freeze-dried to obtain white flocculent products DSPE-PEG-ET and DSPE-PEG-M6P, respectively.

[0049] (2) Preparation and purification of lipid nanolysosome degrading agent:

[0050] Solution preparation: Dissolve lecithin, cholesterol, DSPE-PEG2000-ET and DSPE-PEG2000-M6P in an organic solvent (such as methanol or ethanol) at a predetermined molar ratio to form a lipid organic phase; dissolve GYY4137 in an aqueous solvent (such as phosphate buffer or physiological saline at pH 7.4) at a predetermined drug-lipid ratio to form a drug aqueous phase.

[0051] Microfluidic nanoassembly: The lipid organic phase and drug aqueous phase prepared in step (2) are injected into the two inlets of the microfluidic chip, respectively. The two phases are instantaneously and efficiently mixed in the microchannel at a volumetric flow rate ratio of 1:3 by precise pump control. The solubility of lipid molecules in the aqueous phase decreases sharply and they self-assemble into nanoparticles. At the same time, GYY4137 in the aqueous phase is encapsulated in the internal water cavity. This process can be repeated until the organic phase is exhausted.

[0052] Purification and collection: The crude product obtained in step (2) was collected, and the organic solvent was removed by ultrafiltration and replaced with a pure buffer system. Specifically, an ultrafiltration tube with a molecular weight cutoff of 50 kDa was used, and the mixture was centrifuged at 4000 rpm for 30 minutes. Pure water or buffer was added repeatedly for liquid replacement, and finally, lipid nanolysosome degrading agents with uniform particle size and stable dispersion were obtained.

[0053] The invention will now be further described with reference to the accompanying drawings.

[0054] Example 1

[0055] The preparation, component screening, and characterization of lipid nano-lysosomal degrading agents are as follows:

[0056] As described in preparation method (I), we synthesized two functionalized lipid-polyethylene glycol copolymers, DSPE-PEG-ET and DSPE-PEG-M6P, via click chemistry, and identified their molecular weights using time-of-flight mass spectrometry (MALDI-TOF MS). The results are as follows... Figure 1 As shown in A, B, and C, the characteristic peak positions in the spectra are consistent with the expected molecular weights, proving the successful synthesis of these two functionalized lipids. Based on this, we used microfluidic technology to prepare lipid nanolysosome degradative agents, the specific process of which is illustrated in the diagram. Figure 1 As shown in Figure D: Two functionalized lipids, DSPE-PEG2000-M6PR and DSPE-PEG2000-ET, were mixed with phospholipids and cholesterol and dissolved in an organic phase. Simultaneously, the H2S donor drug GYY4137 was dissolved in an aqueous phase. The flow rates and mixing conditions of the two phases were precisely controlled using a microfluidic device, allowing the organic and aqueous phases to mix efficiently within microchannels and self-assemble into liposomes. This process endows the liposomes with targeting functionality. DSPE-PEG2000-M6PR and DSPE-PEG2000-ET act as targeting ligands, recognizing specific cell surface receptors, thereby improving the active targeting of the drug to diseased tissues and achieving efficient encapsulation of GYY4137.

[0057] To optimize liposome performance, we screened the molar ratio of targeting units and the mass ratio of drug carriers. In the targeting unit ratio screening, we used a human hepatic stellate cell (LX-2) model, storing cells at 3 × 10⁶ cells per well. 5 LRG1 protein was seeded at a density of 1000 mg / m³ in 6-well plates and induced to activate by treatment with medium containing 5 ng / m³ LGF-β for 24 hours. Then, the medium was replaced with medium containing different molar ratios (DSPE-PEG-ET:DSPE-PEG-M6P = 1:1, 1:2, 1:3, 1:4, 1:5) of lipid nanolysosome degrading agent and incubated for another 24 hours. LRG1 protein expression levels were detected by Western blot. Results are as follows: Figure 1 As shown in Figures E and F, the degradation effect on LRG1 protein was most significant when the molar ratio of DSPE-PEG-ET to DSPE-PEG-M6P was 1:3; therefore, this ratio was used in subsequent experiments. In the screening of drug carrier mass ratios, we fixed the total mass of the liposome carrier and set different mass ratios (1:2, 1:4, 1:8, 1:12, 1:16) with GYY4137 drug. The absorbance was measured spectrophotometrically at 670 nm, and the encapsulation efficiency (EE%) and drug loading rate (LE%) were calculated based on the GYY4137 standard curve using the formulas EE (%) = (Wtotal - Wfree) / Wtotal and LE (%) = (Wtotal - Wfree) / WLNP. The results are as follows: Figure 1 As shown in Figure G, the encapsulation efficiency gradually decreased with increasing GYY4137 dosage. After comprehensive comparison, it was determined that the encapsulation efficiency was 55.5% and the drug loading rate was 18.4% when the drug-to-lipid ratio was 1:2, indicating the optimal overall performance. Based on the above optimal conditions (ET:M6P molar ratio 1:3, drug-to-lipid ratio 1:2), we prepared a lipid nano-lysosome degrader encapsulating GYY4137 using microfluidic technology and characterized it: the particle size distribution was measured using a Zeta potential and particle size analyzer, and the results are shown in Figures H and I. The hydrodynamic particle size range of this lipid nano-lysosome degrader was 97-120 nm, and the polydispersity index (PDI) was approximately 0.2, indicating that the particles were uniform in size and narrowly distributed.

[0058] Example 2

[0059] Verification of the anti-hepatic fibrosis effect of drug GYY4137 and determination of its pH-responsive hydrogen release behavior

[0060] This embodiment first verified the anti-hepatic fibrosis bioactivity of the encapsulated drug GYY4137, and then examined its hydrogen release behavior under different pH conditions to demonstrate its rationale as a drug in a lysosomal targeted delivery system. We used a human hepatic stellate cell (LX-2) model, induced cell activation with 5 ng / mL TGF-β to simulate the pathological environment of liver fibrosis, and then treated the cells with different concentrations (0-50 μM) of GYY4137. Western blot results are shown below. Figure 2 A and Figure 2 Quantitative analysis showed that GYY4137 significantly and in a concentration-dependent manner reduced the expression level of α-smooth muscle actin (α-SMA), a key marker of liver fibrosis, fully demonstrating its good anti-liver fibrosis potential. To further elucidate how this drug achieves precise efficacy in practical applications, we simulated different physiological environments in vivo and used a modified methylene blue method to detect the H2S release kinetics of GYY4137. The release curves are shown below. Figure 2 As shown in Figure C, GYY4137 exhibits a slow and sustained H2S release characteristic under simulated lysosomal acidic environment (pH 5.0), while its release rate is significantly slowed down under physiological neutral conditions (pH 7.4). This result strongly demonstrates that GYY4137 possesses ideal pH-responsive drug release behavior, indicating that it can be specifically activated in acidic environments to efficiently release therapeutic H2S molecules, thereby achieving precise intracellular drug delivery.

[0061] Example 3

[0062] To systematically evaluate the degradation ability of the lipid nanolysosome degrader (Lipo-LYTAC) on LRG1 protein, we first treated HSC cells with a medium containing 5 ng / mL TGF-β for 24 hours to induce their activation, and then treated them with different concentrations (0–50 μM) of Lipo-LYTAC. Figure 3 As shown in Figure A, Lipo-LYTAC significantly reduced the level of LRG1 protein in the culture medium, and the degradation effect was concentration-dependent; Figure 3 B, further quantitative analysis yielded its half-degradation concentration (DC). 50 The concentration was 19.26 μM, and the maximum degradation rate was approximately 80%, indicating its highly efficient targeted degradation ability. In the kinetic study, TGF-β-induced LX-2 cells were treated with Lipo-LYTAC for different durations (0–24 hours), and changes after drug elution (0–24 hours) were observed. The results are as follows: Figure 3 C showed that LRG1 degradation increased over time, reaching a peak at 24 hours, indicating that the drug degradation effect is time-dependent. Figure 3LRG1 protein levels gradually recovered within 24 hours after drug removal, indicating that the degradation was reversible. To clarify whether the degradation mechanism depended on the lysosomal pathway, we set up a PBS control group, a Lipo-LYTAC-only treatment group, and a Lipo-LYTAC-chloroquine co-treatment group. LRG1 expression was measured after 12 hours of treatment in TGF-β-induced LX-2 cells. The results are as follows: Figure 3 E and F show that, compared with Lipo-LYTAC alone, the combination with chloroquine significantly inhibited the degradation of LRG1, confirming that the degradation process depends on intact lysosomal activity and thus confirming that the degradation is mediated by the lysosomal pathway.

[0063] Example 4

[0064] Evaluation of the anti-fibrotic effect of lipid nanolysosome degrading agents

[0065] To evaluate the anti-fibrotic effect and synergistic effect of Lipo-LYTAC@GYY, we divided TGF-β-induced LX-2 cells into PBS control group, TGF-β model group, TGF-β + blank liposome group, TGF-β + Lipo@GYY group, TGF-β + Lipo-LYTAC group, and TGF-β + Lipo-LYTAC@GYY group, and treated them accordingly for 24 hours. Subsequently, the expression of fibrosis marker proteins α-SMA, Collagen I, and Fibronectin was detected by Western blot. Figure 4 As shown in Figure A, TGF-β stimulation significantly upregulated the expression of three fibrosis-related proteins, indicating that the cell activation model was successfully established. Figure 4 Further quantitative analysis by BD showed that, compared with the control group, the expression of α-SMA, Collagen I, and Fibronectin in the TGF-β model was upregulated by 4.64-fold, 4.2-fold, and 6.2-fold, respectively. The Lipo@GYY group reduced these three proteins by 44.5%, 51%, and 43%, respectively, while the Lipo-LYTAC group reduced them by 42.2%, 48%, and 43%, respectively. The combined treatment group (Lipo-LYTAC@GYY) showed the most significant inhibitory effect, with reductions of 67.5%, 70.2%, and 71.3%, respectively. These results indicate that both Lipo@GYY and Lipo-LYTAC can downregulate the expression of fibrotic proteins to some extent, and their combined use exhibits a significant synergistic anti-fibrotic effect, effectively reversing the activation state of hepatic stellate cells and inhibiting the synthesis of extracellular matrix.

[0066] Implementing Regulations 5

[0067] To elucidate the molecular mechanism by which the lipid nanolysosome degrader (Lipo-LYTAC@GYY) alleviates liver fibrosis at the cellular level, this study used human hepatic stellate cells (LX-2) as a model and set up PBS control group, TGF-β model group, TGF-β+ blank liposome group, TGF-β+Lipo@GYY group, TGF-β+Lipo-LYTAC group, and TGF-β+Lipo-LYTAC@GYY group. Western blotting was used to analyze the expression of relevant proteins. Figure 5 As shown in Figure A, TGF-β stimulation significantly upregulated LRG1 protein expression, while treatment with Lipo-LYTAC and Lipo-LYTAC@GYY significantly reversed LRG1 levels, indicating that this degrader can effectively intervene in TGF-β-induced increases in LRG1 expression. Further analysis of the phosphorylation levels of Smad2 / 3, a key molecule in the TGF-β / Smad signaling pathway, yielded the following results: Figure 5 C10 Western blot showed that TGF-β significantly enhanced the phosphorylation of Smad2 and Smad3; quantitative analysis was as follows: Figure 5 Figures D and E show that p-Smad2 and p-Smad3 in the TGF-β group were 3.0 times and 3.5 times higher than those in the control group, respectively. All nanomedicine groups effectively inhibited the abnormal activation of this pathway. Lipo@GYY reduced p-Smad2 and p-Smad3 by 40% and 53%, respectively, while Lipo-LYTAC reduced them by 41% and 52%, respectively. The combination therapy of Lipo-LYTAC@GYY showed the most significant effect, with reductions of 68.8% and 81.5%, respectively. Lipo-GYY alone also showed a certain inhibitory effect on the pathway. To elucidate the mechanism of GYY4137's anti-liver fibrosis effect, we extracted nuclear and cytoplasmic proteins from LX-2 cells using a nuclear and cytoplasmic protein extraction kit. The results are as follows: Figure 5 As shown in Figure FH, GYY4137 promotes Nrf2 nuclear translocation. In summary, Lipo-LYTAC@GYY promotes Nrf2 nuclear translocation through LRG1 targeted degradation mediated by surface ET peptides, synergistically releasing H2S from encapsulated GYY4137, thereby jointly inhibiting the overactivation of the TGF-β–Smad2 / 3 signaling pathway. This, in turn, inhibits hepatic stellate cell activation at the molecular level, providing a key in vitro mechanistic basis for its anti-hepatic fibrosis effect.

[0068] Implementing Regulations 6

[0069] This study systematically evaluated the liver targeting and in vivo therapeutic effects of lipid nanolysosomal degraders using a CCl4-induced mouse liver fibrosis model. Six-week-old wild-type male C57BL / 6 mice were administered a CCl4 solution (1:3 volume ratio to soybean oil) twice weekly by gavage for four weeks to establish a liver fibrosis model. From week 3 onwards, different formulations were administered via tail vein according to experimental groups (once every two days for two weeks), and liver tissue was collected for analysis at the end of week 4. To investigate the liver targeting of the nanomedicines, we used Cy5-labeled nanomedicines modified with different ligands (Cy5-Lipo, Cy5-Lipo-E, Cy5-Lipo-M, Cy5-Lipo-(E+M)) via tail vein injection. Figure 6 As shown in Figure A, 24 hours after injection, in vitro tissue fluorescence imaging revealed that all nanomedicines exhibited the strongest fluorescence intensity in the liver, while signals were weaker in other organs. Among them, ligand-free Cy5-Lipo showed a passive enrichment effect, while the single-ligand group showed enhanced targeting. Cy5-Lipo-(E+M), possessing both ET peptide and M6P dual ligands, exhibited the strongest liver-targeting ability. This targeting ability stems from the fact that the approximately 120 nm particle size facilitates enrichment at the hepatic sinusoidal pores, and that LRG1 secreted by fibrotic liver and CI-M6PR on the surface of activated hepatic stellate cells can be actively recognized by the ligands on the nanomedicine surface. In therapeutic experiments, such as... Figure 6 As shown in Figure C, compared with the Sham group, the CCl4 model group and the blank liposome group showed pale liver color, rough surface, and obvious nodules; Lipo@GYY and Lipo-LYTAC monotherapy made the liver surface smoother, while the Lipo-LYTAC@GYY combination therapy group further restored the liver's reddish color and smooth morphology, approaching the normal state. Figure 6 As shown in D, the weight statistics indicate that CCl4 modeling led to weight loss in mice, and Lipo-LYTAC@GYY treatment restored their weight to near-normal levels; Figure 6 E-hepatic weight ratio analysis showed that monotherapy reduced the liver weight ratio, while combination therapy restored it to a level similar to that of the Sham group. These results demonstrate that Lipo-LYTAC@GYY can effectively alleviate CCl4-induced liver injury and fibrosis, with significantly better therapeutic effects than single-component therapy. Furthermore, the active targeting capability conferred by dual-ligand modification provides a crucial in vivo targeting basis for its synergistic anti-fibrotic effect.

[0070] Implementing Regulations 7

[0071] Based on liver tissue samples obtained after treatment in a CCl4-induced mouse liver fibrosis model, this study systematically evaluated the in vivo anti-fibrotic effect of lipid nanolysosome degrading agents at the molecular and tissue levels using Western blotting and immunohistochemical experiments. Figure 7As shown in Figure A, Western Blot results showed that, compared with the Sham group, the expression of key liver fibrosis proteins α-SMA, Collagen I, and Fibronectin was significantly upregulated in the CCl4 model group. Figure 7 BD quantitative analysis further showed that the expression levels of the three proteins were upregulated by 4.60-fold, 3.13-fold, and 3.80-fold, respectively. After drug intervention, the Lipo@GYY group showed a decrease of 52.0%, 48.2%, and 47.4% in the three proteins, respectively, while the Lipo-LYTAC group showed a decrease of 54.1%, 49.7%, and 48.8%, respectively. The Lipo-LYTAC@GYY combination therapy group showed the most significant inhibitory effect, with decreases of 67.2%, 62.5%, and 56.0%, respectively, indicating that it has superior anti-fibrotic ability at the protein level compared to single drugs. Figure 7 As shown in Figure E, immunohistochemical staining further validated the above conclusions in situ: the positive staining areas of the three proteins were significantly increased in the CCl4 model group, and all drug treatment groups reduced the positive signal area to varying degrees, with the Lipo-LYTAC@GYY combination group showing the most significant improvement. In summary, the results from both in vivo protein and tissue layers jointly confirm that Lipo-LYTAC@GYY can effectively inhibit the expression and tissue deposition of fibrosis-related proteins, and its efficacy is significantly better than that of a single component, fully demonstrating the synergistic effect of LRG1 targeted degradation and GYY4137 drug intervention in anti-liver fibrosis.

[0072] Implementing Regulations 8

[0073] This study investigates the mechanism by which lipid nanolysosome degradative agents alleviate fibrosis in vivo.

[0074] To further investigate the mechanism of action of lipid nanolysosome degraders in alleviating liver fibrosis in vivo, we analyzed the phosphorylation levels of key TGF-β signaling pathway proteins LRG1, Smad2, and Smad3 in mouse liver tissue using Western blotting. Figure 8 As shown in Figure A, the CCl4 model significantly upregulated LRG1 protein expression, while treatment with Lipo-LYTAC and Lipo-LYTAC@GYY significantly reversed LRG1 levels, indicating that this degrader can effectively intervene in CCl4-induced increases in LRG1 expression. Figure 8 As shown in Figure C, compared with the Sham group, the levels of p-Smad2 and p-Smad3 in the liver of mice in the CCl4-induced fibrosis model group were significantly increased. Figure 8Quantitative analysis of D and E further revealed that the levels of p-Smad2 and p-Smad3 in the CCl4 group were 6.8 times and 5.52 times higher than those in the control group, respectively. After nanomedicine treatment, all experimental groups effectively reversed this abnormal activation: the Lipo@GYY group reduced p-Smad2 and p-Smad3 levels by 40% and 41.6%, respectively; the Lipo-LYTAC group reduced them by 55.3% and 63.2%, respectively; and the Lipo-LYTAC@GYY combined treatment group showed the most significant effect, with reductions of 63.5% and 80.7%, respectively. To elucidate the in vivo mechanism of GYY4137's anti-liver fibrosis effect, we used a nuclear and cytoplasmic protein extraction kit to extract nuclear and cytoplasmic proteins from LX-2 cells, and the results are as follows: Figure 8 As shown in FH, GYY4137 promoted nuclear translocation of Nrf2. These results indicate that all nanomedicines inhibited Smad2 / 3 phosphorylation to varying degrees, with combination therapy exhibiting the strongest pathway inhibition. Therefore, it is inferred that lipid nanolysosomal degraders may exert their anti-liver fibrosis therapeutic effect in vivo by effectively inhibiting the overactivation of the TGF-β-Smad2 / 3 signaling pathway.

[0075] Implementing Regulations 9

[0076] This study evaluates the safety of the prepared liposomal nanomedicine in vivo.

[0077] To assess the biosafety of the prepared lipid nanolysosome degrading agent, after the completion of the CCl4-induced liver fibrosis mouse model experiment, we collected blood samples from the mice and euthanized them. Subsequently, we harvested major organs such as the heart, liver, spleen, lungs, and kidneys, fixed them in 4% paraformaldehyde, and performed paraffin sectioning and H&E staining for pathological analysis. In vitro hemolysis experiments were conducted as follows: Figure 9 The hemolysis results of experiments A and B showed that the prepared liposome nanomedicine had a low hemolysis rate, indicating good blood compatibility. Further observation of H&E stained sections of various major organs, such as... Figure 9 The results showed no obvious pathological damage or inflammatory infiltration in any tissues, indicating that the nanomedicine had no significant toxic side effects on major organs in in vivo experiments. These results demonstrate that the prepared lipid nanolysosomal degrading agent has good biocompatibility at therapeutic doses, providing important preclinical safety evidence for its potential as an anti-liver fibrosis drug candidate.

[0078] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0079] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Clearly, many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A liposomal nanolysosome degrading agent for degrading leucine-rich α-2 glycoprotein 1 (LRG1), characterized in that, The formulation includes nanoliposomes, an antifibrotic drug loaded inside the nanoliposomes, an LRG1 protein targeting unit modified on the surface of the nanoliposomes, and a lysosome targeting unit modified on the surface of the nanoliposomes; the liposome nanolysosome degrading agent is prepared by microfluidic technology.

2. The liposome nanolysosome degrading agent according to claim 1, characterized in that, The nanoliposomes are composed of egg yolk lecithin and cholesterol; the LRG1 protein targeting unit is an ET polypeptide with the amino acid sequence ESYSAKHRIMLT, as shown in SEQ ID NO.1; the lysosomal targeting unit is mannose-6-phosphate ligand (M6P); and the antifibrotic drug is hydrogen sulfide donor GYY4137, chemically named morpholine-4-methoxyphenyl (morpholine)phosphine dithiophosphate.

3. The liposome nanolysosome degrading agent according to claims 1-2, characterized in that, The LRG1 protein targeting unit ET peptide and lysosome targeting unit M6P ligand are modified on the surface of liposomes via a copper-catalyzed click chemistry reaction: the alkyne-modified ET peptide and M6P ligand undergo cycloaddition reactions with DSPE-PEG2000-N3 to generate functionalized lipid-polyethylene glycol copolymers DSPE-PEG2000-ET and DSPE-PEG2000-M6P.

4. The liposome nanolysosome degrading agent according to claims 1-3, characterized in that, The molar ratio of the raw materials used in the preparation is: lecithin: cholesterol: functionalized lipid-polyethylene glycol copolymer = 5:4:1; the functionalized lipid-polyethylene glycol copolymer is a mixture of DSPE-PEG2000-ET and DSPE-PEG2000-M6P.

5. The liposome nanolysosome degrading agent according to claim 4, characterized in that, The molar ratio of DSPE-PEG2000-ET to DSPE-PEG2000-M6P is 1:

3.

6. The liposome nanolysosome degrading agent according to claims 1-5, characterized in that, The mass ratio of the nanoliposomes to the antifibrotic drug GYY4137 is 2:

1.

7. A method for preparing a liposome nanolysosome degrading agent as described in any one of claims 1-6, characterized in that, The method is based on microfluidics technology and includes the following steps: (1) Preparation of lipid organic phase and drug aqueous phase: Lecithin, cholesterol, DSPE-PEG2000-ET and DSPE-PEG2000-M6P are dissolved in an organic solvent to form lipid organic phase; anti-fibrotic drug GYY4137 is dissolved in an aqueous solvent to form drug aqueous phase; (2) Microfluidic nanoparticle formation: The lipid organic phase obtained in step (1) and the drug aqueous phase are injected into the microfluidic device at a volume flow rate of 1:3 to mix, so that the lipid molecules self-assemble to form nanoparticles loaded with GYY4137. (3) Purification and concentration: Collect the nanoparticle suspension obtained in step (2), remove the organic solvent and replace it with pure water or a buffer system to obtain the lipid nanolysosome degrading agent.

8. The preparation method according to claim 7, characterized in that, In step (1), the organic solvent is methanol or ethanol; the aqueous solvent is phosphate buffer or physiological saline at pH 7.

4.

9. The preparation method according to claim 8, characterized in that, In step (3), the organic solvent is removed by ultrafiltration and liquid replacement is performed. Specifically, an ultrafiltration tube with a molecular weight cutoff of 50 kDa is used, and the mixture is centrifuged at 4000 rpm for 30 minutes. This operation is repeated to complete the purification.

10. The use of the liposome nanolysosome degrading agent according to any one of claims 1-6 in the preparation of drugs for the prevention or treatment of liver diseases, wherein the preferred liver diseases include liver fibrosis, chronic liver disease, end-stage liver disease, etc.

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