ERK2-siRNA nano delivery system, preparation and application of ERK2-siRNA nano delivery system in inhibition of vascular intimal hyperplasia

Abstract

The invention belongs to the technical field of siRNA drugs, and more particularly relates to an ERK2-siRNA nano delivery system, preparation and application of the ERK2-siRNA nano delivery system in inhibition of vascular intimal hyperplasia. Cellular uptake and early endosome escape are enhanced through dual regulation of charge reversal and targeting peptide functions, so that the purpose of high-performance siRNA nano-delivery is achieved. The polyanion can be assembled to the surface of the ERK2-siRNA compound (initial RCs) through electrostatic adsorption, so that a diversified siRNA delivery nano system is formed. According to the technical scheme, powerful support is provided for a siRNA delivery system, the epsilon-PL derivative with the membrane destroying capacity and the initial RCs with the membrane penetrating function jointly destroy an early endosome, efficient ERK2 silencing is achieved in VSMCs, and powerful support is provided for inhibition of vascular intimal hyperplasia and anti-hepatic fibrosis and anti-pulmonary fibrosis treatment.

Description

Technical Field

[0001] The present invention belongs to the technical field of siRNA drugs, and more specifically relates to an ERK2-siRNA nano delivery system, its preparation and its application in inhibiting vascular intimal hyperplasia. Background Art

[0002] With the rapid development of biotechnology, siRNA (Small interfering RNA) delivery nanosystems have shown great potential in the field of biomedicine, especially in the treatment of cardiovascular and cerebrovascular diseases. Inhibiting the excessive proliferation and migration of vascular smooth muscle cells (VSMCs) through siRNA technology provides a new strategy for the treatment of vascular diseases such as intimal hyperplasia. However, this delivery system still faces many problems and challenges.

[0003] First, the delivery efficiency of siRNA across cell membranes and endosomal membranes is low, especially the efficiency of early endosomal escape, which limits the RNAi effect.

[0004] Secondly, although non-viral vectors have become the core of efficient siRNA delivery, their delivery efficiency is still insufficient to meet clinical needs. High doses of siRNA are required to achieve a certain transfection efficiency, which not only increases the cost of treatment but also may bring potential side effects.

[0005] In addition, the efficiency of existing delivery systems in the three processes of long blood circulation, cellular uptake and endosomal escape still needs to be improved. Long circulation time requires the carrier to have "hidden" surface properties, but this often leads to low target cell uptake rate. The design of carriers that enhance early endosomal escape faces the problem of balancing narrow time windows and cytotoxicity.

[0006] Therefore, there is an urgent need to further coordinate and improve the siRNA delivery nanosystem in terms of improving cell delivery efficiency, optimizing carrier design, enhancing early endosomal escape, and balancing cytotoxicity. Summary of the invention

[0007] In view of the above technical problems, the present invention provides an ERK2-siRNA nano-delivery system, its preparation and its application in inhibiting vascular intimal hyperplasia, so as to solve the problems in the prior art of long blood circulation, low efficiency of cellular uptake and early endosomal escape of siRNA nano-delivery systems, and conflict between the "hidden" surface property requirements of the carrier and the target cell uptake rate, so as to achieve a balance between blood circulation, cellular uptake and early endosomal escape, so as to realize the purpose of optimizing the ERK2-siRNA delivery system to fully exert the therapeutic effect.

[0008] To achieve the above objectives, in a first aspect, the present invention provides an ERK2-siRNA nano-delivery system, comprising a cationic carrier, ERK2-siRNA and a polyanion, wherein the polyanion is obtained by grafting a VSMCs targeting peptide and a maleic anhydride derivative onto a polylysine-3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester graft copolymer.

[0009] The ERK2-siRNA nano delivery system provided by the present invention enhances cell uptake and early endosomal escape through dual regulation of charge reversal and targeting peptide function to achieve the purpose of high-performance siRNA nano delivery. Polyanions can be assembled onto the surface of ERK2-siRNA complexes (initial RCs) by electrostatic adsorption, thereby forming a diversified siRNA delivery nano system. They exhibit a neutral zeta potential and extremely low hemolysis rate, supporting long-term blood circulation. The ERK2-siRNA nano delivery system of the present invention can act on targeted VSMCs through VAPG-receptors and exhibit a moderate increase in charge, thereby improving internalization efficiency. Subsequently, they are transported to early endosomes, where the absorbed polyanions undergo charge reversal in a short time, regenerating ε-PL derivatives and initial RCs with membrane destruction ability. Ultimately, the ε-PL derivatives with membrane destruction ability and the initial RCs with membrane penetration function jointly destroy the early endosomes, achieving efficient ERK2 silencing in VSMCs. The technical solution of the present invention provides strong support for the design of an "integrated" siRNA delivery system, and is particularly important in enhancing early endosomal escape, improving blood circulation and increasing cellular uptake.

[0010] In some preferred embodiments of the present invention, the amino acid sequence of the VSMCs targeting peptide is: Cys-Gly-Gly-Val-Ala-Pro-Gly-Trp.

[0011] In some preferred embodiments of the present invention, the maleic anhydride derivative is selected from citraconic anhydride CAA, aconitic anhydride and / or 2,3-dimethyl maleic anhydride DMMA. DMMA modification and CAA modified polyanions have a positive effect on the charge reversal rate of the ERK2-siRNA nano delivery system, and the synergistic effect of such charge regulation and VSMCs targeting strategy enhances the cell uptake performance of the ERK2-siRNA nano delivery system. In some more preferred embodiments, the maleic anhydride derivative is 2,3-dimethyl maleic anhydride DMMA. DMMA modification can further increase the mean fluorescence intensity MFI value, achieve a more optimized design of targeting ligands and charge regulation, and further enhance the uptake effect on cells. The DMMA-modified ERK2-siRNA nano delivery system can achieve rapid charge conversion, regenerate positively charged ε-PL derivatives, and disintegrate the initial RCs and ε-PL derivatives due to electrostatic repulsion. This dynamic process mainly occurs within 30 minutes and at a pH of about 6.7, which is considered to be the duration of early endosomes.

[0012] In some preferred embodiments of the present invention, the cationic carrier is selected from one of cationic octaarginine, polylysine, linear or branched polyethyleneimine, dendritic polyamidoamine and poly(β-amino ester) and cationic liposomes.

[0013] In some preferred embodiments of the present invention, the ratio of the amount of the cationic carrier, ERK2-siRNA and polyanion used is 6:1:(0.5-20) based on the mass ratio used.

[0014] In a second aspect, the present invention provides a method for preparing an ERK2-siRNA nano delivery system, comprising the following steps:

[0015] The poly-lysine-3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester graft copolymer and the VSMCs targeting peptide are mixed for the first time to obtain the targeting peptide-ε-poly-lysine;

[0016] The targeting peptide-ε-polylysine is added to the maleic anhydride derivative to perform a second mixing operation, dialyze, and freeze-dry to obtain a polyanion;

[0017] The cationic carrier, ERK2-siRNA and polyanion are assembled to obtain the ERK2-siRNA nano delivery system.

[0018] In some preferred embodiments of the present invention, the first mixing operation, based on the mass ratio used, has an amount relationship of 1:1 between the polylysine-3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester graft copolymer and the VSMCs targeting peptide, and comprises the steps of first dissolving the polylysine-3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester graft copolymer and the VSMCs targeting peptide in PBS and adjusting the pH value to 7.4.

[0019] In some preferred embodiments of the present invention, the second mixing operation further comprises the steps of first adding the targeting peptide-ε-polylysine to the mixed solution of the maleic anhydride derivative, cooling it to 4° C., and adjusting the pH value to 8.5.

[0020] In some preferred embodiments of the present invention, the amino acid sequence of the VSMCs targeting peptide is: Cys-Gly-Gly-Val-Ala-Pro-Gly-Trp, and the usage ratio of the cationic carrier, ERK2-siRNA and polyanion is 6:1:(0.5-20) based on the mass ratio used.

[0021] In a third aspect, the present invention further provides the use of the ERK2-siRNA nano-delivery system of the first aspect or the preparation method of the second aspect in the preparation of drugs for inhibiting vascular intimal hyperplasia, anti-liver fibrosis and anti-pulmonary fibrosis.

[0022] Different from the prior art, the preparation method of the ERK2-siRNA nano-delivery system in the above technical scheme consists of a cationic peptide carrier (Arg-Arg-Arg-Arg-Arg-Arg-Arg, R8), ERK2-siRNA and a multifunctional polyanion. For the polyanion, PL-SPDP was first synthesized by an amidation reaction between the amino group in ε-PL and the activated ester in 3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide SPDP. Subsequently, the polyanion was synthesized by a one-pot method, and the targeting peptide CGGVAPGW and maleic anhydride derivatives (such as CAA and DMMA) were sequentially grafted onto PL-SPDP to generate four types of polyanions. After that, continuous electrostatic self-assembly was performed to prepare the siRNA delivery nanosystem. First, the R8 carrier was used to electrostatically bind and load ERK2-siRNA at a weight ratio of R8 to ERK2-siRNA of 6 / 1 to prepare the initial RNAi chimeras (RCs). Then, the polyanion was assembled onto the surface of the initial RCs by electrostatic adsorption at a specific weight ratio of R8, ERK2-siRNA and polyanion to form an ERK2-siRNA delivery nanosystem, also known as a ternary siRNA complex (TRCs). After electrostatic coating with polyanions, the obtained ERK2-siRNA delivery nanosystem has excellent compatibility with blood and cells, and the superior design of targeting ligands and charge regulation enhances the uptake of cells. This rapid charge conversion property is crucial for improving early endosomal escape and ERK2 silencing, which makes the ERK2-siRNA nanodelivery system prepared by the present invention show great advantages in RNAi therapy.

[0023] The above-mentioned records related to the invention content are only an overview of the technical solution of the present application. In order to enable ordinary technicians in the field to more clearly understand the technical solution of the present application, and then implement it according to the text of the specification and the contents recorded in the drawings, and to make the above-mentioned purpose and other purposes, features and advantages of the present application easier to understand, the following is an explanation in combination with the specific implementation mode and drawings of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The drawings are only used to illustrate the principles, implementation methods, applications, characteristics and effects of the specific embodiments of the present application and other related contents, and shall not be considered as limitations of the present application.

[0025] In the drawings of the specification:

[0026] Figure 1 The synthetic route of the polyanion in the specific embodiment includes PL-CAA-PG, PL-CAA-GP, PL-CAA-PG / GP, and PL-DMMA-PG;

[0027] Figure 2 1H-NMR spectrum of PL-CAA-PG polyanion in a specific embodiment;

[0028] Figure 3 1H-NMR spectrum of PL-CAA-GP polyanion in a specific embodiment;

[0029] Figure 4 1H-NMR spectrum of PL-CAA-PG / GP polyanion in a specific embodiment;

[0030] Figure 5 1H-NMR spectrum of PL-DMMA-PG polyanion in a specific embodiment;

[0031] Figure 6 The size (bar graph) and zeta potential (scatter plot) of initial RCs of siRNA complexes are shown in Figure 1 ;

[0032] Figure 7 To illustrate the proposed process of forming TRCs through electrostatic self-assembly between initial RCs and multifunctional polyanions;

[0033] Figure 8 For a specific embodiment, the time-dependent zeta potential curves of TRCs-CAA-PG, TRCs-DMMA-PG and TRCs-(CAA-PG) / (DMMA-PG) within 4 hours under the environment of pH=7.4;

[0034] Fig. 9 For the specific implementation, the zeta potential of TRCs-CAA-PG, TRCs-DMMA-PG and TRCs-(CAA-PG) / (DMMA-PG) was subjected to another 4 h (time points were recorded from 4 to 8 h) continuous detection time-dependent zeta potential curves under pH = 7.4;

[0035] Fig.10 For the specific implementation, the zeta potential of TRCs-CAA-PG, TRCs-DMMA-PG and TRCs-(CAA-PG) / (DMMA-PG) was subjected to another 4 h (time points were recorded from 4 to 8 h) continuous detection time-dependent zeta potential curves under pH = 6.7;

[0036] Fig.11 For the specific implementation, the statistical results of mean fluorescence intensity were used to reflect the cellular uptake of the ERK2-siRNA nano-delivery system;

[0037] Fig.12For the specific implementation, the statistical results of cell uptake rate are used to reflect the cell uptake of ERK2-siRNA nano delivery system;

[0038] Fig.13 To illustrate the hemolysis rate of various ERK2-siRNA nanodelivery systems;

[0039] Fig.14 To implement the present invention, the relative cell viability of VSMCs of various ERK2-siRNA nano-delivery systems was investigated;

[0040] Fig.15 To implement the present invention, the statistical results of the siRNA and endosome colocalization ratio (CLR) reflect the early endosomal escape of the ERK2-siRNA nanodelivery system;

[0041] Fig.16 For the specific implementation, the mRNA level of ERK2 in VSMCs after treatment with the ERK2-siRNA nanodelivery system was detected by RT-PCR analysis;

[0042] Fig.17 To detect the protein level of ERK2 in VSMCs after treatment with ERK2-siRNA nanodelivery system by Western blot analysis;

[0043] Fig.18 To evaluate the effect of siRNA delivery nanosystem on VSMCs migration and proliferation by Transwell assay;

[0044] Fig.19 The CCK-8 assay was used to evaluate the effects of the siRNA delivery nanosystem on the migration and proliferation of VSMCs. DETAILED DESCRIPTION

[0045] In order to explain in detail the possible application scenarios, technical principles, specific schemes that can be implemented, and the purposes and effects that can be achieved, the following is a detailed description of the specific embodiments listed in conjunction with the accompanying drawings. The embodiments described herein are only used to more clearly illustrate the technical solutions of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0046] Reference to "embodiment" herein means that the specific features, structures or characteristics described in conjunction with the embodiment may be included in at least one embodiment of the present application. The term "embodiment" appearing in various places in the specification does not necessarily refer to the same embodiment, nor does it particularly limit its independence or association with other embodiments. In principle, in the present application, as long as there is no technical contradiction or conflict, the various technical features mentioned in the embodiments can be combined in any way to form a corresponding implementable technical solution.

[0047] Unless otherwise defined, the technical terms used in this document have the same meanings as those generally understood by those skilled in the art to which this application belongs; the use of relevant terms in this document is only for describing specific embodiments and is not intended to limit this application.

[0048] In the description of this application, the term "and / or" is an expression used to describe the logical relationship between objects, indicating that three relationships may exist, for example, A and / or B, which means: A exists, B exists, and A and B exist at the same time. In addition, the character " / " in this article generally indicates that the objects before and after are in an "or" logical relationship.

[0049] In the present application, terms such as “first” and “second” are merely used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship of quantity, priority or sequence between these entities or operations.

[0050] Without further limitations, in this application, the words "include", "comprises", "has" or other similar expressions used in the sentences are intended to cover non-exclusive inclusion. These expressions do not exclude the presence of additional elements in the process, method or product including the elements, so that the process, method or product including a series of elements may include not only those limited elements, but also other elements not explicitly listed, or also include elements inherent to such process, method or product.

[0051] Similar to the understanding in the Examination Guidelines, in this application, expressions such as "greater than", "less than", "exceed" and the like are understood to exclude the number itself; expressions such as "above", "below", "within" and the like are understood to include the number itself. In addition, in the description of the embodiments of this application, "multiple" means more than two (including two), and similar expressions related to "multiple" are also understood in this way, such as "multiple groups", "multiple times", etc., unless otherwise clearly and specifically limited.

[0052] In the description of the embodiments of the present application, space-related expressions used, such as "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "vertical", "top", "bottom", "inside", "outside", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicate the orientation or position relationship based on the orientation or position relationship shown in the specific embodiments or drawings, and are only for the convenience of describing the specific embodiments of the present application or facilitating the reader's understanding, and do not indicate or imply that the referred device or component must have a specific position, a specific orientation, or be constructed or operated in a specific orientation, and therefore cannot be understood as a limitation on the embodiments of the present application.

[0053] Unless otherwise expressly specified or limited, in the description of the embodiments of the present application, the terms such as "install", "connect", "connect", "fix", "set", etc. used should be understood in a broad sense. For example, the "connection" can be a fixed connection, a detachable connection, or an integrated setting; it can be a mechanical connection, an electrical connection, or a communication connection; it can be a direct connection, or it can be indirectly connected through an intermediate medium; it can be the internal connection of two elements or the interaction relationship between two elements. For technicians in the technical field to which the present application belongs, the specific meanings of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0054] Small interfering RNA (siRNA) has become a promising drug for the treatment of various serious diseases due to its unique ability to specifically downregulate target genes in cells. In this context, non-viral vectors have become the core of efficient siRNA delivery. Therefore, a variety of siRNA vectors have been designed and manufactured over the past few decades. Recently, siRNA delivery in clinical trials has shown high patient safety and significant therapeutic effects. However, it cannot be ignored that siRNA therapeutic methods are still severely limited. Studies have shown that only 2000 copies of siRNA per cell are required to induce maximum silencing of target genes, but due to the low delivery efficiency of non-viral vectors, higher doses of siRNA are required to achieve a certain transfection efficiency. Therefore, there is an urgent need to develop efficient and safe vectors to promote the development of siRNA therapy.

[0055] Cytoplasmic siRNA delivery includes three processes in sequence, namely blood circulation, cellular uptake, and endosomal escape. In order to improve the quality of siRNA therapy, the efficiency of each process must be maximized. Therefore, the ideal siRNA carrier should have the characteristics of long blood circulation, high target cell uptake rate, and rapid endosomal escape.

[0056] Long-circulation siRNA carriers need to have "stealth" surface properties to avoid protein deposition and rapid clearance by the mononuclear phagocyte system (MPS), which is usually achieved through surface functionalization, mainly including polyethylene glycolization (PEGylation), red blood cell (RBC) membrane camouflage, zwitterionic modification, and electrostatic coating. However, these strategies also lead to low target cell uptake rates due to lack of cell specificity and weak interaction with cell membranes. To address this challenge, strategies combining stealth surfaces with targeting ligand modification have been shown to be feasible. For example, polyanionic electrostatic coatings modified with targeting peptides were used to simultaneously improve blood compatibility and uptake rates by endothelial cells (ECs). In addition, matrix metalloproteinases (MMPs)-triggered PEG shedding and targeting ligand exposure have been reported to improve the efficiency of siRNA delivery from blood circulation to cellular uptake. Co-assembly of RBC membranes and cRGD-functionalized lipids onto siRNA complexes increased their retention time in the blood, targeting ability, and uptake rates of melanoma cancer cells. Furthermore, internalized siRNA is usually trapped in subcellular endosomal / lysosomal compartments, where low pH and a large number of hydrolases have a great destructive effect on the efficacy of siRNA. Therefore, endosomal / lysosomal escape has become the biggest obstacle to efficient siRNA delivery. Through a large number of theoretical studies, we noticed that only a small part of siRNA (<4% of internalized siRNA) can successfully escape from endosomes / lysosomes and enter the cytoplasm, while most siRNA is trapped in late endosomes and lysosomes, thereby obtaining little therapeutic activity. More interestingly, there is a narrow time window for siRNA escape and cytoplasmic release in early endosomes. These evidences suggest that rational carrier design should focus on enhancing early endosomal escape, but the narrow time window needs to be overcome. At the same time, it is equally important to balance high cell membrane disruption ability and low cytotoxicity. To resolve this contradiction, a siRNA carrier with ultra-pH sensitivity and rapid response was developed using a narrow pH gradient from plasma (pH = 7.4) to early endosomes (pH = 6.5) as a biological stimulus. For example, by finely tuning the ratio of cationic and hydrophobic monomers in random copolymers, a rapid response to pH = 6.8 was achieved, thereby enhancing early endosomal escape. Unfortunately, there are no reports on combining long blood circulation, high cellular uptake, and strong early endosomal escape to achieve optimized siRNA delivery.

[0057] The therapeutic potential of siRNA is another important consideration. The occurrence of cardiovascular and cerebrovascular diseases (such as intimal hyperplasia) is closely related to the excessive migration and proliferation of vascular smooth muscle cells (VSMCs). Therefore, inhibiting VSMCs proliferation through siRNA technology will be a promising therapeutic strategy. Extracellular regulated protein kinase (ERK) can transmit biological signals from plasma membrane receptors to the cell nucleus, thereby playing a central role in cell growth. Previous studies have shown that ERK2 (an ERK isoform) is involved in the abnormal growth of VSMCs induced by platelet-derived growth factor (PDGF). In addition, the present study demonstrated that the ERK2-siRNA delivery system can inhibit the migration and proliferation of VSMCs in vitro.

[0058] To better optimize the delivery and therapeutic effect of siRNA, we carefully designed an ERK2-siRNA delivery nanosystem. First, cationic octaarginine (Arg-Arg-Arg-Arg-Arg-Arg-Arg, referred to as R8) was used as a peptide carrier to generate the initial ERK2-siRNA complexes (RCs) due to its positive charge and membrane penetration ability. However, its application is still severely limited by poor blood compatibility, lack of VSMCs targeting ability, and low efficiency of early endosomal escape. To address these problems, the present invention designed a series of negatively charged polymers with VSMCs targeting function and endowed them with different pH-induced charge reversal properties. These polymers were synthesized by co-grafting VSMCs targeting peptides (Val-Ala-Pro-Gly, VAPG) and maleic anhydride derivatives with different substituents onto ε-polylysine (ε-PL). These anionic polymers can be assembled onto the surface of the initial RCs by electrostatic adsorption, thus forming a diversified siRNA delivery nanosystem. They exhibit neutral zeta potential and extremely low hemolysis rate, supporting long-term blood circulation. During in vitro transfection, these nanosystems were able to target VSMCs via the VSMCs targeting peptide VAPG-receptor interaction and exhibited a moderate charge increase, thereby improving internalization efficiency. Subsequently, they were transported to early endosomes, where the absorbed polyanions underwent charge reversal in a short period of time, regenerating ε-PL derivatives with membrane-disrupting ability and initial RCs. Ultimately, the ε-PL derivatives with membrane-disrupting ability and the initial RCs with membrane-penetrating function jointly destroyed early endosomes, achieving efficient ERK2 silencing in VSMCs. The results of the present invention will provide guidance for the design of an "all-in-one" siRNA delivery system, especially in enhancing early endosomal escape.

[0059] The materials and reagents involved in the following implementation of the present invention are as follows:

[0060] R8 peptide, CGGVAPGW peptide, and CGGVAGPW peptide were purchased from Shanghai GL Biochem Co., Ltd. Citraconic anhydride (CAA) and 2,3-dimethylmaleic anhydride (DMMA) were purchased from Alfa Aesar Chemical Co., Ltd., China. ε-Polylysine (ε-PL) (molecular weight = 4200 Da) was purchased from Shanghai Yuanye Biotechnology Co., Ltd. ERK2-small interfering RNA (ERK2-siRNA) and Cy5-labeled ERK2-small interfering RNA (Cy5-siRNA) were purchased from Guangzhou Ruibo Biotechnology Co., Ltd. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen Life Sciences Inc. (Carlsbad, CA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. LysoTracker Green (DND-26, LifeTechnologies) and Hoechst 33342 were purchased from Shanghai Yingjie Life Science Co., Ltd. (Shanghai, China). ERK2 primers were purchased from Shanghai Sangon Biotechnology Co., Ltd. Rat aortic vascular smooth muscle cells (VSMCs) were obtained from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences (Shanghai, China).

[0061] All experiments in the following examples of the present invention were conducted in accordance with the Animal Care Guidelines of Fujian Medical University, which have also been approved by the Fujian Animal Experiment Ethics Review Center and are in accordance with the "Guide for the Care and Use of Laboratory Animals" of the National Institutes of Health of the United States.

[0062] Unless otherwise specified, the reagents, instruments and methods used in the following examples of the present invention are generally known in the art.

[0063] Unless otherwise specified, the data of the following examples of the present invention are expressed in the form of mean ± standard deviation (mean ± SD). The statistical analysis results were analyzed by one-way analysis of variance (ANOVA) or Student's t-test when applicable. When p < 0.05, p < 0.01, p < 0.001, there was a significant difference between the data.

[0064] Example 1

[0065] Preparation of ERK2-siRNA complexes (initial RCs)

[0066] The R8 peptide was fully dissolved in PBS (10 mM, pH = 7.4) at a concentration of 0.2 mg / mL by ultrasound, and then, ERK2-siRNA solution (66.50 μg / mL) was added, in which the weight ratio of R8 to ERK2-siRNA was 6:1, to prepare R8 / ERK2-siRNA nanocomplexes (initial siRNA complexes, referred to as initial RCs).

[0067] Example 2

[0068] Synthesis of R8 peptide co-grafted with maleic anhydride derivatives and polyanions

[0069] The peptide part includes CGGVAPGW (peptide targeting vascular smooth muscle cells VSMCs; amino acid sequence: Cys-Gly-Gly-Val-Ala-Pro-Gly-Trp). In another comparative example, a non-targeting peptide CGGVAGPW (non-targeting peptide; amino acid sequence: Cys-Gly-Gly-Val-Ala-Gly-Pro-Trp) is used. The detailed synthesis route is as follows Figure 1 shown.

[0070] First, ε-polylysine-grafted-3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide (PL-SPDP, the preparation method of PL-SPDP is shown in Example 13) with a grafting rate of 21% was synthesized according to our previous work. Then, 10 mg PL-SPDP and 10 mg peptide (in three different experiments, 10 mg of targeting peptide CGGVAPGW; 10 mg of non-targeting peptide CGGVAGPW; 5 mg of targeting peptide CGGVAPGW + 5 mg of non-targeting peptide CGGVAGPW) were dissolved in 8 mL PBS (0.1 M, pH = 7.4) and stirred for 12 hours at room temperature in the dark. Then, the solution was cooled to 4 ° C and the pH was adjusted to 8.5 using sodium hydroxide. Subsequently, the corresponding maleic anhydride derivatives (in different experiments, 13 mg of citraconic anhydride (CAA); 15 mg of 2,3-dimethylmaleic anhydride (DMMA)) were added respectively under the condition of pH control between 8 and 9. The reaction was continued with stirring at 4 °C for 12 h. Finally, four polyanion samples were obtained by dialysis and freeze drying, including ε-polylysine-g-citraconic anhydride-g-CGGVAPGW (PL-CAA-PG), ε-polylysine-g-citraconic anhydride-g-CGGVAGPW (PL-CAA-GP), ε-polylysine-g-citraconic anhydride-g-CGGVAPGW / CGGVAGPW (PL-CAA-PG / GP), and ε-polylysine-g-2,3-dimethylmaleic anhydride-g-CGGVAPGW (PL-DMMA-PG).

[0071] From the 1H-NMR results ( Figure 2-5 ), the successful grafting of the peptide and maleic anhydride derivative was confirmed. In addition, since the pyridyldithiol in PL-SPDP was completely replaced by the peptide during the reaction, the grafting rate of the peptide part was calculated to be about 21%.

[0072] Example 3

[0073] Preparation of multifunctional siRNA delivery nanosystem

[0074] First, polyanions were dissolved in PBS (10 mM, pH = 7.4) to obtain five polyanion solutions (0.1 mg / mL) according to the formula (100 wt% PL-CAA-PG; 100 wt% PL-CAA-GP; 100 wt% PL-CAA-PG / GP; 100 wt% PL-DMMA-PG; 50 wt% PL-CAA-PG and 50 wt% PL-DMMA-PG). Then, these polyanion solutions were added to the pre-prepared initial RCs at a mass ratio of 6:1:3 of R8, ERK2-siRNA and polyanion. Thus, five ternary siRNA complexes (TRCs, also known as siRNA nanodelivery systems) were prepared by the electrostatic assembly strategy. They were labeled as TRCs-CAA-PG, TRCs-CAA-GP, TRCs-CAA-PG / GP, TRCs-DMMA-PG and TRCs-(CAA-PG) / (DMMA-PG), respectively.

[0075] In order to verify the successful synthesis of the ERK2-siRNA nano-delivery system, the particle size and zeta potential of the TRCs samples prepared above in PBS (10 mM, pH=7.4) were measured using Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK).

[0076] like Figure 6 As shown, the initial RCs exhibited nanoscale size (125 nm) and highly positive zeta potential (+26 mV). After adding a predetermined dose of polyanion, a slight increase in size (25-35 nm) and a significant decrease in zeta potential (19-22 mV) were clearly visible.

[0077] like Figure 7As shown, this indicates that various polyanions are adsorbed on the surface of the initial RCs through electrostatic interaction, forming a multifunctional coating with a thickness of about 25-35 nm. At the same time, due to electrostatic neutralization, the surface charge properties are greatly changed, thereby reducing the zeta potential. These results reflect the successful preparation of the above-mentioned siRNA nanodelivery systems. In addition, their size (150nm) and neutral zeta potential (+5mV) are beneficial for blood compatibility and cytocompatibility.

[0078] Example 4

[0079] pH-induced charge switching of TRCs

[0080] The zeta potential of TRCs at pH 7.4 was measured using a Zeta sizer Nano ZS at the following time points (0 hours, 1 hour, 2 hours, and 4 hours). At 4 hours, the pH of the TRCs samples was immediately adjusted to 7.4 (no change) and 6.7, respectively. Next, these samples were incubated at room temperature for an additional 15 minutes, 30 minutes, 1 hour, 2 hours, and 4 hours (time points were recorded at 4.25 hours, 4.5 hours, 5 hours, 6 hours, and 8 hours), after which their zeta potential was monitored again.

[0081] like Figure 8 As shown in the figure, under pH = 7.4, the ζ potential of TRCs-CAA-PG remained basically stable within 4 hours, while the ζ potential of TRCs-DMMA-PG gradually increased from +6.8mV to +11.5mV due to the small amount of hydrolysis of 2,3-dimethylmaleic acid amide bonds. For TRCs-(CAA-PG) / (DMMA-PG), the ζ potential increased slightly from +6.2mV to +8.5mV. These data indicate that at pH = 7.4, the mixing ratio of DMMA-modified and CAA-modified polyanions has an effect on the charge reversal rate of TRCs, and there is a positive correlation. Therefore, the combined use of this charge regulation strategy and VSMCs targeting strategy can enhance cellular uptake.

[0082] We further analyzed the cellular uptake of TRCs by flow cytometry after 4 h of incubation. Fig.11 and Fig.12As shown, the siRNA delivery nanosystem achieved almost 100% cellular uptake, indicating that they were effectively taken up by all VSMCs. However, their mean fluorescence intensities (MFI) were significantly different for VSMCs, which was attributed to their different abilities to enter cells. Obviously, the MFI values ​​of TRCs-CAA-GP, TRCs-CAA-PG / GP, and TRCs-CAA-PG were enhanced in turn. This fully demonstrated that VAPG modification enhanced the uptake of VSMCs through specific ligand-receptor interactions. In addition, we observed that DMMA modification could further increase the MFI value compared with CAA modification and DMMA / CAA co-modification, which was mainly due to its rapid increase in zeta potential within 4 hours. From the above results, we confirmed the superior design of targeting ligands and charge regulation, as well as their enhanced effects on cellular uptake.

[0083] Furthermore, ingested TRCs are often transferred to the endosomal / lysosomal dilemma. Here, efficient escape of siRNA from early endosomes is considered to be a crucial process. In this process, the charge conversion of the polyanion coating plays a key role. Therefore, Fig. 9 and 10As shown in Figure 2, we continuously measured the zeta potential of TRCs under different pH conditions for another 4 h (time points were recorded from 4 to 8 h). At pH = 7.4, the zeta potential of all TRCs groups did not change significantly from 4 to 8 h. In contrast, under stimulation at pH = 6.7 (similar to early endosomes), the zeta potential of all TRCs increased with time, but at different rates. Specifically, DMMA modification caused the zeta potential to increase dramatically from +11.5 mV to +18.5 mV in just 15 min, and to +22.8 mV (comparable to the initial RCs) within 30 min. Its zeta potential remained stable for the next 3.5 h. These data indicate that the TRCs-DMMA-PG coating enables rapid charge conversion, regeneration of positively charged ε-PL derivatives, and disintegration of the initial RCs and ε-PL derivatives due to electrostatic repulsion. This dynamic process mainly occurred within 30 min and under the condition of pH = 6.7, which is considered to be the duration of early endosomes. In contrast, CAA modification showed a slow increase in ζ potential from +5.3 mV to +7.8 mV within 30 min, making it difficult to achieve complete charge conversion in the early endosome. Meanwhile, the ζ potential continued to increase to +15.5 mV within 4 h, indicating that the charge reversal and disintegration processes may occur in the late endosome or lysosome; DMMA / CAA co-modification had a moderate charge reversal rate, increasing from +8.5 mV to +14.5 mV within 30 min. Then, the ζ potential rose to +19.6 mV within 4 h. This suggests that partial charge conversion can be performed in the early endosome, while the remaining hydrolysis reactions are mainly performed in the late endosome or lysosome. Based on the fact that enhanced escape at the early stage is beneficial for target gene knockout, we believe that the burst charge conversion property is crucial for improving early endosomal escape and ERK2 silencing.

[0084] Example 5

[0085] Cellular uptake studies

[0086] Various TRCs were prepared using fluorescent dye Cy5-labeled ERK2-siRNA (referred to as Cy5-siRNA) for cellular uptake studies. Specifically, VSMCs (2×10 5 10 cells / well) were seeded in 6-well plates and transfected. After 4 hours, VSMCs were washed three times with PBS (10 mM, pH = 7.4) and then treated with 0.25% trypsin. Next, VSMCs were centrifuged and resuspended in PBS for further flow cytometry analysis.

[0087] Example 6

[0088] Hemolysis test

[0089] Biocompatibility is a basic requirement for safe siRNA administration. Therefore, we characterized the blood compatibility and cytocompatibility of siRNA delivery nanosystems. The blood samples were centrifuged and washed until the supernatant was clear. The obtained red blood cells (RBCs) were resuspended in PBS (pH = 7.4). Next, the red blood cell suspension was added to the TRCs (ternary siRNA complex) solution prepared in Example 3, and the mixture was incubated at 37°C for 24 hours. Subsequently, the absorbance of the supernatant was measured at a wavelength of 540nm. The percentage of hemolysis was calculated according to the following formula:

[0090]

[0091] Among them, OD sample, OD negative control and OD positive control represent the absorbance of the sample group, negative control group and positive control group solutions, respectively.

[0092] First, their blood compatibility with respect to their red cell membrane-damaging activity was investigated by hemolytic assay. Fig.13 As shown, the quantitative data of hemolysis rate of the TRCs group (3.2%) was even lower than the medical standard (5%), indicating its excellent blood compatibility.

[0093] Example 7

[0094] MTT colorimetric assay

[0095] In this example, the methylthiazolyl tetrazolium (MTT) test was used to study the cell compatibility of different concentrations of siRNA delivery nanosystems to evaluate the survival rate of vascular smooth muscle cells (VSMCs). First, MTT powder was dissolved in phosphate buffered saline (PBS, 10mM, pH=7.4) to prepare a 5mg / mL solution, which was then filtered through a sterile syringe filter (pore size 0.22µm). The prepared MTT solution was stored at 4°C for future use. Vascular smooth muscle cells (8×10 3 cells / well) were seeded in a 96-well plate and cultured in a high glucose medium (DMEM) containing 10% fetal bovine serum (FBS) for 24 hours until the cells reached 80% confluence. Then, the medium was replaced with a serum-free medium and incubated for another 12 hours. Thereafter, VSMCs were treated with the therapeutic small molecule compounds (TRCs) prepared in Example 3 and incubated for another 48 hours. Subsequently, 100µL of serum-free medium containing MTT (0.5mg / mL) was added and incubated for 4 hours until purple appeared. The medium was discarded and the VSMCs were washed with PBS to remove residual MTT. Finally, 100µL of dimethyl sulfoxide (DMSO) was added to dissolve the formazan salt. The absorbance (OD) of each well was measured at a wavelength of 490nm. The formula for calculating the relative cell survival rate is as follows:

[0096]

[0097] Among them, ODsample, ODDMSO and ODcontrol group represent the absorbance of the sample group, DMSO group and control group solution, respectively.

[0098] exist Fig.14 In the experiment, even at a high concentration of 500 nM, the relative cell viability of vascular smooth muscle cells (VSMCs) in all TRCs groups exceeded 80%, indicating that the TRCs samples were cell-friendly.

[0099] Based on Examples 6 and 7, after electrostatic coating with polyanions, the obtained ERK2-siRNA delivery nanosystem has excellent compatibility with blood and cells, which shows great advantages in RNAi therapy. In addition, if TRCs can overcome intracellular delivery barriers and achieve optimized knockdown of ERK2, it will be more attractive.

[0100] Example 8

[0101] Subcellular localization studies

[0102] In this example, Cy5-labeled small interfering RNA (Cy5-siRNA) was used again. Vascular smooth muscle cells (VSMCs) were seeded into confocal culture dishes (8×10 4 cells / well) and cultured for 24 hours. Then, the therapeutic small molecule compound (TRCs) sample was added to the serum-free medium for 4 hours of transfection. Next, VSMCs were washed twice with phosphate buffered saline (PBS, 10 mM, pH=7.4). Pre-warmed medium containing lysosomal green fluorescent probe (Lyso TrackerGreen, 0.5mM) was added and incubated for 1 hour. After washing with pre-warmed PBS, VSMCs were treated with Hoechst 33342 (2μg / mL) at room temperature for 20 minutes and then washed thoroughly with PBS for subsequent confocal laser scanning microscopy (CLSM) observation. The excitation wavelengths were set to 649nm, 504nm, and 350nm to activate Cy5-siRNA (red), LysoTracker Green (green), and Hoechst 33342 (blue), respectively. Finally, the co-localization ratio (CLR) of siRNA to endosomes / lysosomes was calculated to reflect the early endosomal escape of siRNA.

[0103] like Fig.15As shown, the statistical results of the colocalization ratio (CLR) of siRNA and endosomes / lysosomes showed that the DMMA-modified group had the lowest CLR (31%), proving its strong ability to escape from early endosomes. For CAA modification and co-modification, the CLR values ​​were higher, 68% and 56%, respectively, which confirmed that their efficiency of escape from early endosomes was relatively low. The above results show that the charge reversal rate has a significant effect on the escape effect of TRCs. More importantly, TRCs with explosive charge conversion characteristics within 30 minutes significantly enhanced early endosome escape. Furthermore, we speculate that the rapid charge conversion in the early endosome induces the regeneration of initial RCs with membrane penetration function and ε-PL derivatives with membrane destruction ability, thereby destroying the endosomal membrane and enhancing escape.

[0104] Example 9

[0105] Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) analysis

[0106] In view of the satisfactory performance in biocompatibility and intracellular delivery, we continued to study the knockdown of ERK2 after treatment with the ERK2-siRNA delivery nanosystem.

[0107] First, the knockdown efficiency was quantified at the mRNA and protein levels by RT-PCR and Western blot analysis. Real-time fluorescence quantitative polymerase chain reaction (RT-PCR) analysis was used to determine the level of extracellular signal-regulated kinase 2 (ERK2) mRNA in transfected cells. Total RNA was extracted from transfected vascular smooth muscle cells (VSMCs) using TRIzol reagent and then reverse transcribed into cDNA using HiFiScript cDNA synthesis kit. The resulting cDNA was used as a template for quantification using Ultra SYBR Mixture (Low ROX) on a 7500 Real-Time PCR System.

[0108] Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalization reference gene for the target gene. The forward and reverse primer sequences for ERK2 were 5'-GACAAGGGCTCAGAGGACTG-3' and 5'-ACGGCTCAAAGGAGTCAAGA-3', respectively; the forward and reverse primer sequences for GAPDH were 5'-AGGTGAAGGTCGGAGTCAAC-3' and 5'-CGCTCCTGGAAGATGGTGAT-3', respectively.

[0109] like Fig.16As shown, all TRCs groups showed lower ERK2-mRNA levels than the PBS control group, indicating that the delivery material we designed solved the dilemma of naked siRNA. Among the TRCs groups, TRCs-DMMA-PG showed the best knockdown efficiency (relative ERK2-mRNA level of 37%), which was 0.54 times that of TRCs-CAA-PG and 0.44 times that of TRCs-CAA-GP. This may be due to the enhanced function of TRCs-DMMA-PG in VSMCs uptake and early endosomal escape. Generally, mRNA needs to be translated into the corresponding protein to exert its biological function. Therefore, it is also important to evaluate the knockdown efficiency by analyzing ERK2-protein expression.

[0110] Example 10

[0111] Western blot experiment

[0112] In this example, Western blotting was used to determine the level of extracellular signal-regulated kinase 2 (ERK2) protein in transfected cells. First, total protein was extracted from transfected vascular smooth muscle cells (VSMCs) using RIPA lysis buffer containing phenylmethylsulfonyl fluoride (PMSF, 1% by volume), and the protein was quantified using a BCA protein assay kit. Before the assay, the protein was denatured by adding 6 volumes of sodium dodecyl sulfate (SDS). Next, the proteins were separated by SDS-polyacrylamide gel electrophoresis, followed by Western blotting. The membrane was incubated overnight in a rabbit anti-ERK2 polyclonal antibody containing TBST (Tris buffered saline + Tween20), and then incubated for 2 hours with a horseradish peroxidase-labeled anti-rabbit secondary antibody. Finally, the membrane was scanned and analyzed using the Tanon-5200 fully automatic chemiluminescence image analysis system.

[0113] like Fig.17 As shown in the figure, the trend is similar to the RT-PCR results. The ERK2 protein silencing effect was the best in the TRCs-DMMA-PG group, and its relative ERK2 protein level was as low as 0.47 times that of the PBS control group. ERK2 silencing can regulate the behavior of VSMCs related to vascular intimal hyperplasia.

[0114] Embodiment 11

[0115] Transwell migration assay

[0116] ERK2-siRNA can inhibit the proliferation of VSMCs, so we further evaluated the effect of ERK2 silencing on the activity of VSMCs. The migration ability of transfected VSMCs was detected by Transwell assay. This example uses Transwell migration assay to evaluate the migration characteristics of transfected vascular smooth muscle cells (VSMCs). Specifically, VSMCs were centrifuged and resuspended after 24 hours of TRCs transfection. Subsequently, these cells were added to the upper chamber of the Transwell chamber (1×10 5 After 6 hours of incubation, VSMCs that passed through the chamber membrane were stained with crystal violet and photographed.

[0117] like Fig.18 As shown in the figure, the number of VSMCs that passed through the chamber in the TRCs group was significantly less than that in the PBS control group, indicating that the migration ability of VSMCs was weakened after TRCs transfection. For the TRCs group, the best migration inhibition effect was TRCs-DMMA-PG, and its migration rate was significantly lower than that of TRCs-CAA-PG.

[0118] Example 12

[0119] CCK-8 cell proliferation assay

[0120] This example uses a CCK-8 cell proliferation detection kit to evaluate the proliferation of vascular smooth muscle cells (VSMCs) transfected with TRCs. First, VSMCs were seeded into 96-well plates and cultured with complete DMEM medium for 24 hours. Then, transfection experiments were performed for 12 hours, 24 hours, and 48 hours. Next, the culture medium was replaced with a serum-free medium containing 10% CCK-8 reagent and cultured for another 4 hours at 37°C and 5% CO2. Finally, the absorbance was measured at a wavelength of 450nm using a microplate reader (ThermoScientific brand).

[0121] like Fig.19 As shown in the figure, within 48 hours, the relative cell viability of the TRCs group decreased significantly, reflecting their inhibitory effect on VSMCs proliferation. In addition, since TRCs-DMMA-PG achieved efficient ERK2 knockdown, it had the strongest inhibitory effect on VSMCs proliferation, with a relative cell viability of 45% at 48 hours. Relatively weak inhibitory effects were also detected for the other TRCs groups.

[0122] From the above results of ERK2 silencing, it can be seen that TRCs-DMMA-PG has a superior ability to deliver ERK2-siRNA to VSMCs. In addition, its powerful ERK2 knockdown effect greatly inhibited the migration and proliferation of VSMCs.

[0123] Based on regulating the charge reversal rate to match the narrow time and pH window of early endosomal escape, the weakness of "delayed escape" is further solved. Combined with ligand modification, this combined strategy effectively delivers siRNA to the target cell membrane and early endosomal membrane, thereby making the siRNA in the cytoplasm more active and achieving efficient gene silencing. The research results of the present invention will provide a powerful tool for the treatment of inhibiting vascular intimal hyperplasia, anti-liver fibrosis and anti-pulmonary fibrosis, including but not limited to its use as a preparation for drugs for inhibiting vascular intimal hyperplasia, anti-liver fibrosis and anti-pulmonary fibrosis.

[0124] In addition, the applicability issue needs to be addressed. The multifunctional polyanions used in this study coated the initial RCs via electrostatic interactions, aiming to endow the siRNA delivery system with excellent surface / interface properties, including VSMCs targeting and weak acid-driven burst charge conversion. These two benefits enhanced VSMCs uptake and efficient early endosomal escape during transfection. Given that the initial RCs were composed of other common polymer carriers, such as poly(L-lysine) (PLL), linear or branched polyethyleneimine (PEI), dendritic polyamidoamine (PAMAM), cationic liposomes, and poly(β-amino esters), we inferred that polyanion coatings might also be applicable to improve siRNA delivery efficiency, thanks to their unique functionality in surface / interface engineering.

[0125] Example 13

[0126] Preparation method of PL-SPDP with a grafting rate of 21%

[0127] Weigh 20 mg of PLL with a number average molecular weight of 4200 Da and 10 mg of a bifunctional coupling agent (SPDP), dissolve them in 15 mL of deionized water and 2 mL of methanol, respectively. Slowly add the SPDP solution to the PLL solution, stir for 4 h in the dark, place it in a dialysis bag with a molecular weight cutoff of 2000 Da, dialyze it with deionized water for 48 h, and freeze-dry it.

[0128] Finally, it should be noted that although the above embodiments have been described in the specification and drawings of this application, this does not limit the scope of patent protection of this application. All technical solutions generated by replacing or modifying equivalent structures or equivalent processes based on the essential concept of this application using the contents recorded in the specification and drawings of this application, as well as directly or indirectly implementing the technical solutions of the above embodiments in other related technical fields, are included in the scope of patent protection of this application.

Claims

1. An ERK2-siRNA nano delivery system, characterized in that: The invention comprises a cationic carrier, ERK2-siRNA and a polyanion. The polyanion is obtained by grafting a VSMCs targeting peptide and a maleic anhydride derivative onto a polylysine-3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester graft copolymer.

2. The ERK2-siRNA nano delivery system according to claim 1, characterized in that: The amino acid sequence of the VSMCs targeting peptide is: Cys-Gly-Gly-Val-Ala-Pro-Gly-Trp.

3. The ERK2-siRNA nano delivery system according to claim 1, characterized in that: The maleic anhydride derivative is selected from citraconic anhydride, aconitic anhydride and / or 2,3-dimethylmaleic anhydride. Preferably, the maleic anhydride derivative is 2,3-dimethylmaleic anhydride.

4. The ERK2-siRNA nano delivery system according to claim 3, characterized in that: The cationic carrier is selected from one of cationic octaarginine, polylysine, linear or branched polyethyleneimine, dendritic polyamidoamine, poly(β-amino ester) and cationic liposome.

5. The ERK2-siRNA nano delivery system according to claim 1, characterized in that: Calculated by the mass ratio used, the usage ratio of the cationic carrier, ERK2-siRNA and polyanion is 6:1:(0.5-20).

6. A method for preparing an ERK2-siRNA nano delivery system, characterized in that: The following steps are involved: The poly-lysine-3-(2-pyridyldithiol) propionic acid N-hydroxysuccinimide ester graft copolymer and the VSMCs targeting peptide are mixed for the first time to obtain the targeting peptide-ε-poly-lysine; The targeting peptide-ε-polylysine is added to the maleic anhydride derivative to perform a second mixing operation, dialyze, and freeze-dry to obtain a polyanion; The cationic carrier, ERK2-siRNA and polyanion are assembled to obtain the ERK2-siRNA nano delivery system.

7. The preparation method according to claim 6, characterized in that: The first mixing operation, based on the mass ratio used, has an amount relationship of PL-SPDP and VSMCs targeting peptide of 1:1, and includes the steps of first dissolving the PL-SPDP and VSMCs targeting peptide in PBS and adjusting the pH value to 7.

4.

8. The preparation method according to claim 6, characterized in that: The second mixing operation further includes the steps of first adding the targeting peptide-ε-polylysine to the mixed solution of the maleic anhydride derivative, cooling the solution to 4° C., and adjusting the pH value to 8.

5.

9. The preparation method according to claim 6, characterized in that: The amino acid sequence of the VSMCs targeting peptide is: Cys-Gly-Gly-Val-Ala-Pro-Gly-Trp. Calculated by the mass ratio used, the dosage relationship of the cationic carrier, ERK2-siRNA and polyanion is 6:1:(0.5-20).

10. Use of the ERK2-siRNA nano delivery system according to any one of claims 1 to 5 or the preparation method according to any one of claims 6 to 9 in the preparation of drugs for inhibiting vascular intimal hyperplasia, anti-liver fibrosis and anti-pulmonary fibrosis.

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