CRGDyk-mitochondria composite delivery system of targeted hepatic stellate cells as well as preparation method and application of cRGDyk-mitochondria composite delivery system

Abstract

The invention discloses a cRGDyk-mitochondria composite delivery system of targeted hepatic stellate cells as well as a preparation method and application of the cRGDyk-mitochondria composite delivery system. According to the compound system, cRGDyk targeting peptide and exogenous mitochondria are used as main functional units, covalent linkage is performed through a thiol-maleimide click chemical reaction, and cerium oxide nanoparticles with mimic enzyme activity are introduced. According to the system, efficient targeting of activated hepatic stellate cells is achieved through cRGDyk peptide, exogenous mitochondria is used for repairing the cell metabolism function, cerium oxide nanoparticles provide auxiliary ROS removal capacity, the three components have a synergistic effect, the obvious hepatic fibrosis reversing effect is shown in an in-vitro cell model and an in-vivo animal model, biocompatibility is good, and the system is suitable for clinical application. Wide clinical application prospects are realized.

Description

Technical Field

[0001] This invention belongs to the fields of biomedical technology and nanomedicine, and specifically relates to a novel targeted delivery system for treating liver fibrosis, particularly a composite delivery system modified with cRGDyk targeting peptides, with exogenous components as the treatment core and cerium oxide nanoparticles as auxiliary antioxidant units. Background Technology

[0002] Liver fibrosis is a repair response following chronic liver injury, characterized by the activation of hepatic stellate cells (HSCs) and their transformation into myofibroblasts, leading to excessive extracellular matrix deposition. Currently, there are no effective drugs that can specifically target and reverse activated HSCs (aHSCs). Conventional nanomedicines face challenges such as low targeting efficiency, easy clearance by normal cells, and limited therapeutic mechanisms.

[0003] cRGDyk peptide is a cyclic arginine-glycine-aspartic acid peptide that specifically recognizes integrin αvβ3, which is highly expressed on the surface of activated hepatic stellate cells, exhibiting excellent active targeting capabilities, but lacking therapeutic function itself. Mitochondria, as the cell's energy factories, are closely related to hepatic stellate cell activation when their function is impaired. Exogenous mitochondrial transplantation, as an emerging organelle replacement therapy, can effectively repair the energy metabolism of damaged cells, but its targeting and stability in vivo are insufficient. Cerium oxide nanoparticles possess antioxidant enzyme mimicry activity and can effectively scavenge reactive oxygen species, but in existing systems they are mostly used as the primary therapeutic unit; their single antioxidant mechanism is insufficient to achieve metabolic repair of the root causes of fibrosis.

[0004] Therefore, this invention proposes a novel composite system with cRGDyk-mitochondria as the main component and cerium oxide as an auxiliary functional module, aiming to achieve a triple synergistic treatment of targeted delivery, metabolic repair and antioxidation. Summary of the Invention

[0005] Purpose The purpose of this invention is to provide a cRGDyk-mitochondrial complex delivery system for targeting hepatic stellate cells. This system can actively target aHSCs through cRGDyk peptides, repair cellular metabolic function using exogenous mitochondria, and scavenge reactive oxygen species with the help of cerium oxide nanoparticles. The three work synergistically to efficiently reverse the process of liver fibrosis.

[0006] Technical solution This invention first provides a mitochondrial-nanozyme composite delivery system targeting hepatic stellate cells, characterized in that it comprises: a thiol-modified cRGDyk targeting peptide; maleimide-modified cerium oxide nanoparticles; and thiol-modified exogenous mitochondria; wherein the cRGDyk targeting peptide and the exogenous mitochondria are covalently linked to the maleimide groups on the surface of the cerium oxide nanoparticles through thiol groups on their respective surfaces.

[0007] Preferably, the molar ratio of the cRGDyk targeting peptide to cerium oxide nanoparticles is 10:1 to 50:1.

[0008] Preferably, the mass ratio of the exogenous mitochondria to cerium oxide nanoparticles is 5:1 to 20:1.

[0009] Preferably, the hydration kinetic diameter of the cerium oxide nanoparticles is 2nm-10nm.

[0010] Preferably, the exogenous mitochondria are derived from the heart tissue of c57BL / 6 mice.

[0011] Preferably, the hydration dynamic diameter of the composite delivery system is 600nm-900nm.

[0012] The present invention further provides a method for preparing the composite delivery system, characterized by comprising the following steps: S1: Preparation of thiol-modified cRGDyk targeting peptide (SH-cRGDyk). S2: Preparation of cerium oxide nanoparticles (Mal-Ce NPs) with maleimide groups on their surface. S3: Mitochondria were extracted from the heart tissue of c57BL / 6 mice and thiolized using Traut's reagent to obtain thiol-modified exogenous mitochondria (SH-Mito). S4: Incubate the product obtained in step S1 with the product obtained in step S2 to obtain cRGDyk-cerium oxide nanoparticle composite (cRGDyk-Ce). S5: Incubate the complex obtained in step S4 and the product obtained in step S3 on ice to allow the remaining maleimide groups on the surface of the complex to undergo a coupling reaction with the thiol groups on the surface of the mitochondria. After purification, the cRGDyk-mitochondrial complex delivery system is obtained.

[0013] Finally, this invention provides the use of the above-described composite system in the preparation of medicaments for the prevention and / or treatment of liver fibrosis.

[0014] Preferably, the drug is administered via intravenous injection.

[0015] Compared with the prior art, this application has the following beneficial effects: 1. Structural Innovation and Synergistic Treatment: This invention is the first to design and construct a "three-in-one" composite system with "maleimide cerium oxide" as the connecting core. Through precise chemical coupling, it integrates three major functional modules: targeting, anti-oxidation, and metabolic repair, achieving synergistic treatment of the core pathological links of liver fibrosis, with significantly better results than single therapies.

[0016] 2. Precise and active targeting: By specifically recognizing the integrin αvβ3 highly expressed on the surface of aHSCs through the cRGDyk peptide, this system achieves efficient enrichment of drugs in diseased cells, significantly improving treatment efficiency and greatly reducing off-target toxicity.

[0017] 3. Stable and reliable preparation process: The stepwise click chemical assembly strategy is adopted, which has mild reaction conditions, high connection efficiency, and few by-products. It can well maintain the biological activity of mitochondria and the catalytic activity of nanozymes, with good reproducibility and easy scale-up production.

[0018] 4. Proven efficacy and high safety: As shown in the following examples, this composite system has demonstrated excellent anti-fibrotic efficacy in both in vivo and in vitro models. It can significantly reduce fibrosis markers, reduce collagen deposition, and improve liver function, while having no significant toxicity to major organs, and has great potential for clinical translation. Attached Figure Description

[0019] Figure 1 A schematic diagram (A) of the synthesis of the cRGDyk-mitochondrial complex delivery system of the present invention and a schematic diagram (B) of its targeted therapy for liver fibrosis. Figure 2 The figures show the characterization results of the composite system of the present invention, where (A) is a transmission electron microscope image, (B) is a hydration dynamics diameter distribution diagram, (C, D) are fluorescence colocalization images, (E) is a zeta potential diagram, and (F) is a diagram of SOD and (G) CAT mimic enzyme activity assays. Figure 3 The images show the in vitro anti-fibrotic effect of the composite system in this invention. (A, B) are images of intracellular ROS level detection, (CE) are immunofluorescence images of liver fibrosis markers (TGF-β1, α-SMA and Collagen I), and (F) is an image of collagen gel shrinkage experiment.

[0020] Figure 4 The diagram shows the in vivo targeting and anti-fibrotic effects of the composite system of the present invention, wherein (A) is an in vivo fibrosis pattern diagram, (B) is a live imaging diagram, (C) is a liver tissue section staining diagram, and (D) is a statistical diagram of serum liver function indicators. Detailed Implementation

[0021] The present invention will be further illustrated below through specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Test methods not specifically specified shall be performed under conventional conditions in the art or as recommended by the manufacturer.

[0022] Example 1: Preparation of cRGDyk-mitochondrial complex delivery system 1. Dissolve the cRGDyk targeting peptide in PBS buffer, add 20 times the molar amount of Traut's reagent to thiolate the cRGDyk peptide, incubate in the dark for 1 hour, and remove excess reagent by desalting column to obtain the thiolated cRGDyk targeting peptide (SH-cRGDyk).

[0023] 2. Cerium oxide nanoparticles were synthesized using the sol-gel method. The cerium oxide nanoparticles were then modified with maleimide reagent and purified by centrifugation to obtain maleimide-modified cerium oxide nanoparticles (Mal-Ce NPs). 3. Mitochondria were extracted from the heart tissue of C57BL / 6 mice. The mitochondria were thiolated using Traut's reagent on ice and incubated in the dark for 30 minutes. Fresh, thiolated exogenous mitochondria (SH-Mito) were obtained.

[0024] 4. SH-cRGDyk and Mal-Ce NPs were mixed at a molar ratio of 30:1 and incubated at room temperature for 2 hours to obtain the cRGDyk-Ce complex; then the cRGDyk-Ce complex was incubated with SH-Mito at a mass ratio of 10:1 on ice for 1 hour, and coupling was achieved through click chemistry to obtain the cRGDyk-mitochondrial complex delivery system.

[0025] Example 2: Characterization of the composite delivery system The composite delivery system prepared in Example 1 was characterized as follows: Transmission electron microscopy (TEM): Multiple nanoparticles were observed attached to the mitochondrial surface. Figure 2 A) confirms successful assembly.

[0026] Dynamic light scattering (DLS): The measured hydration dynamic diameter of the composite system was approximately 715.60 ± 98.42 nm. Figure 2 B).

[0027] Fluorescent colocalization: After conjugating the FITC-labeled cRGDyk-Ce complex with MitoTracker Deep Red-labeled mitochondria, high overlap of green and red fluorescence was observed under a confocal microscope. Figure 2 (C) This demonstrates that the cRGDyk-Ce complex was successfully anchored on the mitochondrial surface.

[0028] Zeta potential: The zeta potential of the composite delivery system is approximately -15 mV ( Figure 2 D), which is beneficial for its stable circulation in the body.

[0029] Enzyme activity assay: Using a commercially available kit, the results showed that the cRGDyk-mitochondrial complex delivery system maintained SOD and CAT mimic enzyme activities comparable to those of free cerium oxide nanoparticles. Figure 2 E), proving that the assembly process did not affect the activity and function of each part.

[0030] Example 3: In vitro anti-fibrotic function verification In a TGF-β1 (10 ng / mL, 24 h)-induced human hepatic stellate cell (LX2) fibrosis model, the cells were treated with the cRGDyk-mitochondrial complex delivery system (80 μg / mL, based on CeO2).

[0031] ROS clearance efficacy: Detected using DCFH-DA probes and flow cytometry. For example... Figure 3 As shown in Figure A, compared with the model group, the intracellular ROS level in the cRGDyk-mitochondrial composite delivery system treatment group was significantly reduced, showing better results than either the cerium oxide nanoparticles alone or the mitochondrial group alone.

[0032] Downregulation of fibrosis markers: by Western Blot analysis ( Figure 3 B) shows that treatment with the cRGDyk-mitochondrial complex delivery system significantly downregulated the protein expression levels of α-smooth muscle actin (α-SMA) and type I collagen (Collagen I) in activated HSCs.

[0033] Cell function inhibition: Collagen gel contraction assay ( Figure 3 C) indicates that the cRGDyk-mitochondrial complex delivery system can effectively inhibit the contractile ability of aHSCs, which is an important function of restoring their resting state.

[0034] This embodiment demonstrates that the composite delivery system of the present invention has excellent anti-fibrotic function at the cellular level.

[0035] Example 4: Evaluation of in vivo targeting and anti-fibrotic efficacy A CCl4-induced mouse model of liver fibrosis was established (6 weeks). The cRGDyk-mitochondrial complex delivery system was administered via tail vein injection (twice a week for 2 weeks).

[0036] In vivo targeting: In vivo imaging using a Cy5-labeled composite delivery system. For example... Figure 4As shown in Figure A, 6 hours after injection, the fluorescence signal of the cRGDyk-mitochondrial complex delivery system in fibrotic liver was significantly stronger than that in the non-targeted group, demonstrating that cRGDyk modification endows it with excellent liver targeting ability.

[0037] Histological improvement: After treatment, liver tissue was taken for Sirius red staining. (e.g.) Figure 4 As shown in Figure B, the model group had a large amount of collagen deposition in the liver (red), while the collagen area in the cRGDyk-mitochondrial complex delivery system treatment group was significantly reduced, approaching normal levels.

[0038] Liver function recovery: detection of serum biochemical indicators ( Figure 4 In the model group, the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were significantly elevated, while these indicators were significantly reduced in the cRGDyk-mitochondrial complex delivery system treatment group, indicating that liver function was improved.

[0039] Detection of fibrosis markers: Immunohistochemical staining ( Figure 4 D) showed that the expression level of α-SMA in liver tissue of the cRGDyk-mitochondrial complex delivery system treatment group was significantly lower than that of the model group.

[0040] This embodiment demonstrates that the composite system of the present invention can effectively target diseased liver in vivo and exert a significant therapeutic effect.

[0041] in conclusion This invention successfully constructs a novel composite delivery system with cRGDyk-mitochondria as the core and cerium oxide as an auxiliary antioxidant, achieving precise targeting of activated hepatic stellate cells, synergistic treatment of cell metabolism repair and antioxidant effects, providing a new and efficient solution for the treatment of liver fibrosis, and has significant clinical translational value and application prospects.

Claims

1. A cRGDyk-mitochondrial complex delivery system targeting hepatic stellate cells, characterized in that, include: Thiol-modified cRGDyk targeting peptide; Exogenous mitochondria modified with thiolation; Cerium oxide nanoparticles modified with maleimide serve as connecting units; wherein the cRGDyk targeting peptide and the exogenous mitochondria are covalently linked to the maleimide groups on the surface of the cerium oxide nanoparticles through thiol groups on their surfaces.

2. The cRGDyk-mitochondrial complex delivery system for targeting hepatic stellate cells according to claim 1, characterized in that, The molar ratio of the cRGDyk targeting peptide to cerium oxide nanoparticles is 10:1 to 50:1, and the mass ratio of exogenous mitochondria to cerium oxide nanoparticles is 5:1 to 20:

1. This ratio range can simultaneously satisfy the synergistic effect of targeting, antioxidant activity and metabolic repair function.

3. The cRGDyk-mitochondrial complex delivery system for targeting hepatic stellate cells according to claim 1, characterized in that, The hydration dynamic diameter of the composite system is 600nm-900nm, which can avoid rapid clearance by the reticuloendothelial system while ensuring targeted delivery efficiency.

4. A method for preparing a cRGDyk-mitochondrial complex delivery system for targeting hepatic stellate cells as described in any one of claims 1 to 3, characterized in that, Includes the following steps: S1: Preparation of thiol-modified cRGDyk targeting peptide; S2: Preparation of cerium oxide nanoparticles with maleimide groups on the surface; S3: Preparation of thiol-modified exogenous mitochondria; S4: Incubate the product obtained in step S1 with the product obtained in step S2 to obtain the cRGDyk / Ce complex; S5: Incubate the complex obtained in step S4 with the product obtained in step S3 to obtain the cRGDyk-mitochondrial complex delivery system.

5. The use of the cRGDyk-mitochondrial complex delivery system for targeting hepatic stellate cells as described in any one of claims 1 to 4 in the preparation of a medicament for the prevention and / or treatment of liver fibrosis, wherein the medicament can target and reverse hepatic stellate cell activation, reduce extracellular matrix deposition, and repair damaged cellular metabolic function.

6. The application according to claim 5, characterized in that, The drug is administered via intravenous injection.

Images