Triterpenoid-siRNA / lipid nanovesicle complex, preparation method and application thereof

Abstract

The application discloses a triterpenoid-siRNA / lipid nanovesicle complex and a preparation method and application thereof, and relates to the technical field of biological medicines.The preparation method comprises the following steps: coupling a triterpenoid and siRNA to obtain a water-soluble triterpenoid-siRNA conjugate; and carrying out light-proof incubation reaction on the water-soluble triterpenoid-siRNA conjugate and a saccharina japonica lipid nanovesicle to obtain the triterpenoid-siRNA / lipid nanovesicle complex; the nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO. 23, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 24.The triterpenoid-siRNA / lipid nanovesicle complex can inhibit the progression of pulmonary fibrosis, provides a double regulation strategy for the treatment of pulmonary fibrosis, and realizes the breakthrough of treatment efficiency while guaranteeing the biological safety.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a triterpenoid compound-siRNA / lipid nanovesicle complex and a preparation method and application thereof. BACKGROUND

[0002] In the field of biological medicine, small interfering RNA (siRNA) has become an important tool for treating various diseases due to its potential to specifically silence the expression of target genes. However, despite the significant advantages of siRNA technology, its clinical application still faces many challenges, mainly focusing on the defects of the delivery system. First, naked siRNA molecules are easily degraded by nucleases in the serum, resulting in poor stability and difficulty in maintaining activity for a long time. Second, siRNA itself is negatively charged and difficult to penetrate the cell membrane to enter the cell interior, which further limits its bioavailability. In addition, traditional delivery systems such as liposomes or polymer nanoparticles can solve the above problems to some extent, but also have obvious shortcomings. For example, these artificially synthesized carriers often show low targeting, which can easily cause non-specific distribution and off-target effects, thereby reducing the therapeutic effect and increasing potential toxicity. At the same time, some artificially synthesized materials may trigger an immune response, limiting their long-term use.

[0003] In view of these problems, researchers have gradually turned their attention to natural source delivery carriers in recent years, such as exosomes, plant-derived nanovesicles, etc. These natural carriers exhibit great application potential due to their excellent biocompatibility and low immunogenicity. However, the current siRNA delivery system based on natural carriers is still in the initial development stage, and the conventional siRNA delivery into vesicles often uses electroporation technology, which has limited carrying efficiency and is difficult to achieve large-scale preparation process, so the delivery efficiency needs to be further improved. In addition, how to achieve large-scale preparation of vesicles carrying siRNA is still one of the key scientific problems to be solved in this field.

[0004] Meanwhile, in terms of disease treatment, pulmonary fibrosis as a complex chronic disease, its pathogenesis involves the fibrosis process mediated by TGF-β1 signaling pathway, which has a serious impact on the quality of life and survival time of patients. At present, the drugs used in the treatment of pulmonary fibrosis in the clinic mainly include pirfenidone and nintedanib, but these drugs can only delay the progression of the disease and cannot fundamentally reverse the fibrosis process. Therefore, the development of a new type of therapeutic drug that can accurately target and effectively inhibit the TGF-β1 signaling pathway is of great significance for improving the prognosis of patients with pulmonary fibrosis. In this context, siRNA therapy provides a new idea for the treatment of pulmonary fibrosis with its high specificity and designability. However, the limitations of existing siRNA delivery systems still constrain their application in the field of pulmonary fibrosis. In order to overcome these problems, it is urgent to develop an efficient and safe siRNA delivery platform, which can significantly improve the treatment efficiency while ensuring biological safety and provide new possibilities for the treatment of related diseases. SUMMARY

[0005] The purpose of the present application is to provide a triterpenoid compound-siRNA / lipid nanovesicle complex and its preparation method and application, in order to solve the problems existing in the prior art. The triterpenoid compound-siRNA / lipid nanovesicle complex can inhibit the progression of pulmonary fibrosis and provide a double regulation strategy for the treatment of pulmonary fibrosis, while ensuring biological safety and achieving a breakthrough in treatment efficiency.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The present application provides a preparation method of a complex for treating pulmonary fibrosis, comprising the following steps:

[0008] coupling a triterpenoid compound with siRNA to obtain a water-soluble triterpenoid compound-siRNA conjugate;

[0009] light-protected incubation reaction of the water-soluble triterpenoid compound-siRNA conjugate with Sargassum fusiforme lipid nanovesicles to obtain the triterpenoid compound-siRNA / lipid nanovesicle complex;

[0010] The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO. 23, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 24.

[0011] Further, the water-soluble triterpenoid compound-siRNA conjugate is prepared by EDC-NHS coupling reaction.

[0012] Further, the triterpenoid compound is ganoderic acid.

[0013] Further, the Sargassum fusiforme lipid nano vesicles are extracted by differential centrifugation.

[0014] Further, the mass ratio of the water-soluble triterpenoid-siRNA conjugate to the Sargassum fusiforme lipid nano vesicles is 1:16-20.

[0015] Further, the temperature of the light-proof incubation reaction is 4-8 DEG C, and the time is 1-4 hours.

[0016] The application further provides a triterpenoid-siRNA / lipid nano vesicle complex prepared by the preparation method.

[0017] The application further provides application of the triterpenoid-siRNA / lipid nano vesicle complex in preparation of a drug for treating pulmonary fibrosis.

[0018] The application further provides a drug for treating pulmonary fibrosis, and the active ingredient comprises the triterpenoid-siRNA / lipid nano vesicle complex.

[0019] Further, the drug further comprises a pharmaceutically acceptable excipient.

[0020] The application discloses the following technical effects:

[0021] The application provides a triterpenoid-siRNA / lipid nanovesicle complex (G-siRNA / SF-NVs) for treating pulmonary fibrosis, the stability of siRNA is significantly enhanced through a triterpenoid covalent modification strategy, and the limitations of poor tropism and poor biocompatibility of a traditional delivery system are overcome by using a lipid nanovesicle (SF-NVs) derived from Sargassum fusiforme as a delivery carrier. Research proves that the G-siRNA / SF-NVs complex prepared in the application can effectively maintain the structural integrity of lung vesicles, and the intracellular delivery efficiency of siRNA can be significantly improved through triterpenoid modification. The complex exhibits excellent gene silencing ability and lysosome escape characteristics at the cell level, which provides a key guarantee for the function realization of siRNA. In terms of pulmonary fibrosis disease treatment, the complex achieves lung targeting accumulation through intraperitoneal injection, and successfully inhibits the fibrosis process mediated by the TGF-beta 1 signaling pathway. Experimental results show that the G-siRNA / SF-NVs can not only effectively improve the pathological characteristics of lung tissue structure, but also significantly reduce the expression levels of fibrosis-promoting factors and inflammatory factors. In vitro research further reveals its inhibitory effect on abnormal migration of epithelial cells and fibrosis-promoting factors. Compared with existing siRNA delivery systems, the application first combines plant-derived nanovesicles with natural active ingredient modification technology, and by virtue of the anti-inflammatory properties of SF-NVs and the gene silencing effect of siTGF-beta 1, the lung fibrosis process is synergistically inhibited, providing a double regulation strategy for the treatment of pulmonary fibrosis, ensuring biological safety while achieving a breakthrough in treatment efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below only constitute some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0023] Figure 1 It is a schematic diagram for the preparation process of G-siRNA;

[0024] Figure 2 It is a PAGE electrophoresis diagram of siRNA and G-siRNA;

[0025] Figure 3 It is a high performance liquid chromatogram of different samples;

[0026] Figure 4 It is a result diagram of stability detection of siRNA and G-siRNA;

[0027] Figure 5 It is a particle size analysis diagram of SF-NVs;

[0028] Figure 6 Figure for Zeta potential determination results of SF-NVs;

[0029] Figure 7 Figure for electrophoresis map after RNA extraction in SF-NVs;

[0030] Figure 8 Figure for TME electron microscope map of SF-NVs (A) and G-siRNA / SF-NVs morphological results of TME electron microscope observation electroporation method (B) and co-incubation method (C);

[0031] Figure 9 Figure for fluorescence co-localization analysis of siRNA / SF-NVs and G-siRNA / SF-NVs; the scale is 10 μm;

[0032] Figure 10 Figure for stability analysis of siRNA / SF-NVs and G-siRNA / SF-NVs;

[0033] Figure 11 Figure for particle size analysis of SF-NVs (A), siRNA / SF-NVs (B) and G-siRNA / SF-NVs (C);

[0034] Figure 12 Figure for Zeta potential determination results of SF-NVs, siRNA / SF-NVs and G-siRNA / SF-NVs;

[0035] Figure 13 Figure for functional verification results of G-siRNA; wherein, A is the confocal imaging map of HCC827, Huh-7 and HEK293t cells, the scale is 40 μm; B is the result of detecting the expression level of GAPDH after G-siRNA transfection in A549, HCC827, Huh-7 and HEK293t cell lines using RT-qPCR technology (β-Actin as internal reference, n=3, “*” indicates p < 0.05, “ns” indicates no significant difference); C is the result of determining the relative expression level of GAPDH in different tissues by RT-qPCR technology (β-Actin as internal reference, n=3, “**” indicates p < 0.01, “ns” indicates no significant difference);

[0036] Figure 14 Figure for siRNA / SF-NVs and G-siRNA / SF-NVs synthesized by using Cy3 modified siRNA, observing the distribution in mice after intraperitoneal injection;

[0037] Figure 15Figure showing the results of lung distribution test after intraperitoneal injection and inhalation of Cy3-G-siRNA / SF-NVs complex in mice; the scale is 20 μm;

[0038] Figure 16 Figure showing the results of G-siRNA / SF-NVs knockdown effect analysis in A549 cells (A), BEAS-2B cells (B) and HCC827 cells (C);

[0039] Figure 17 Figure showing the results of laser confocal observation of co-localization of lysosome fluorescence (green) and G-siRNA fluorescence (red) after G-siRNA / SF-NVs and G-siRNA transfection for 12, 24, 36 and 48 hours in A549 cells (A), BEAS-2B cells (B) and HCC827 cells (C); the scale is 10 μm;

[0040] Figure 18 Figure showing the schematic diagram of mouse modeling;

[0041] Figure 19 Figure showing the statistical graph of mouse body weight (A) and lung weight / body weight ratio (B);

[0042] Figure 20 Figure showing the lung tissue morphology of mice in each group;

[0043] Figure 21 Figure showing the results of RT-qPCR detection of lung homogenate lung fibrosis related indicators TGF-β1 (A), Collagen I (B), Fibronectin (C) and α-SMA (D) expression in mice; GAPDH was used as an internal reference, n=5, "ns" means no statistical significance, "*" means p < 0.05, "**" means p < 0.01, "***" means p < 0.001, and "****" means p < 0.0001;

[0044] Figure 22 Figure showing the results of Western Blot detection of lung homogenate lung fibrosis related protein expression level in mice;

[0045] Figure 23 Figure showing the results of total protein content (A) and MPO activity (B) detection in bronchoalveolar lavage fluid (BALF);

[0046] Figure 24 Figure showing the H&E staining and Masson staining of lung tissue in mice in each group; the scale is 100 μm;

[0047] Figure 25Figure showing the results of detection of the content of hydroxyproline (HYP) in the lung tissue of mice in each group; "*" indicates p < 0.05, "**" indicates p < 0.01;

[0048] Figure 26 Figure showing the results of detection of the concentrations of inflammatory factors IL-1β (A), IL-6 (B) and TNF-α (C) in the serum of mice in each group; "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001. DETAILED DESCRIPTION

[0049] The detailed description set forth below of certain inventive embodiments is not offered as a limitation of the present application. Rather, it is an attempt to provide a thorough description of certain aspects, features and embodiments of the application.

[0050] It is to be understood that the terminology used in the present application is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. Additionally, for a range of values of a parameter, unless otherwise indicated, each intervening value by each intervening value, as well as any other stated or intervening value in that stated range is encompassed. In addition, any combination of the above ranges, as well as any other stated or intervening value in that stated range, is encompassed. Unless otherwise stated, the above ranges and parameters are approximate, meaning further deviations are allowed. Other embodiments of the present application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. Various embodiments of the present application will be described in detail with reference to drawings, which are illustrated by way of example and not limitation.

[0051] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, the preferred methods and materials are described. All documents mentioned herein are incorporated by reference to disclose and describe the methods and / or materials in connection with which the documents are cited. In case of conflict, the content of the specification will control.

[0052] Various modifications and changes can be made to the specific embodiments of the present application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the application disclosed herein. Each of the referenced publications and each of the priority documents cited herein are expressly incorporated herein by reference. The specification is to be regarded as exemplary, rather than limiting, of the present application.

[0053] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having", "contains", "containing", or variations thereof, are intended to be open-ended, meaning that they include but are not limited to the stated elements.

[0054] The primer sequences involved in the present application are shown in Table 1, and the sequences of the siRNAs interference strands are shown in Table 2.

[0055] Table 1 Primer sequences

[0056]

[0057] Note: Mus stands for mouse; Hsa stands for human.

[0058] Table 2 Sequences of siRNAs interference strands

[0059]

[0060] Example 1

[0061] 1. Ganoderic acid modification of siRNA (see schematic diagram of preparation process Figure 1 )

[0062] EDC reaction principle: carboxyl (-COOH) can react with N-hydroxysulfosuccinimide (Sulfo-NHS) in the presence of carbodiimide to obtain a semi-stable state of Sulfo-NHS ester, and then react with primary amine (-NH2) to form amide crosslinking. The activation reaction of EDC and Sulfo-NHS is most effective at pH 4.5~7.2, and the EDC reaction is usually carried out in a MES buffer at pH 4.7~6.0. The reaction of Sulfo-NHS activated molecules with primary amines is most effective at pH 7.0~8.0, and the reaction of Sulfo-NHS ester is usually carried out in a phosphate buffer (PBS) at pH 7.2~7.5.

[0063] (1) Solution preparation

[0064] Ganoderic acid mother liquor preparation: dissolve ganoderic acid (GHA) in dimethyl sulfoxide (DMSO) to prepare a 10 mmol / L ganoderic acid mother liquor.

[0065] Coupling activator solution preparation: prepare 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) aqueous solution of 10 mg / mL and N-hydroxysuccinimide (NHS) aqueous solution of 20 mg / mL, respectively;

[0066] MES buffer preparation: accurately weigh 0.64 g 2-(N-morpholino) ethanesulfonic acid (MES) and 0.175 g sodium chloride (NaCl), add 30 mL double distilled water to dissolve thoroughly, and adjust the pH to 5.5 for standby use.

[0067] (2) Construction of activation reaction system

[0068] Take 1 mL MES buffer (pH 5.5) as the reaction medium, add 1 μL ganoderic acid mother liquor (the final concentration of ganoderic acid in the reaction system is 10 μmol / L), 20 μL EDC aqueous solution (the final concentration of EDC in the reaction system is 0.2 mg / mL) and 20 μL NHS aqueous solution (the final concentration of NHS in the reaction system is 0.4 mg / mL) in turn, and place the obtained reaction system in a constant temperature oscillator, activate at 37 ℃, 200 rpm for 30 minutes.

[0069] (3) Covalent coupling reaction

[0070] The activated mixed system is adjusted to pH 7.0 with 20×PBS buffer, and 30 μL of 3' end amino-modified siGAPDH solution with a concentration of 20 μmol / L is added. Maintain the constant temperature condition of 37℃ for continuous oscillation reaction for 120 minutes, detect the pH value of the system every 30 minutes during the period and make necessary adjustment, maintain the pH value between 7.0-7.3.

[0071] (4) Product purification

[0072] The mixed solution obtained after the reaction is transferred to an ultrafiltration centrifuge tube with a molecular weight cut-off of 10 kDa, and centrifuged at 8000×g at 4℃ for 45 minutes. During the centrifugation process, PBS buffer (pH 7.4) is supplemented every 15 minutes for elution replacement, and the elution step is repeated for 3 times. Finally, the cut-off component is collected, that is, the water-soluble triterpenoid compound-siRNA conjugate (G-siRNA) is obtained.

[0073] 2. G-siRNA quality control

[0074] To verify whether the triterpenoid compound is successfully coupled to siRNA by EDC two-step method, the present application uses high performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE) method to observe the molecular weight difference between G-siRNA and siRNA. The PAGE analysis result shows that the electrophoretic speed of G-siRNA is obviously smaller than that of single siRNA, which shows that the molecular weight is increased after coupling with ganoderic acid (G-siRNA has a higher molecular weight than siRNA). Figure 2 The HPLC analysis result shows that the peak time of pure siRNA is 23 min, the peak time of ganoderic acid is 33 min, and the peak time of the conjugate is 31 min. The shift of peak time shows that G-siRNAs is successfully coupled (G-siRNAs has a different peak time than siRNA). Figure 3 ).

[0075] 3. G-siRNA stability research

[0076] Freshly prepared G-siRNA samples were mixed with fetal bovine serum (FBS) at a volume ratio of 9:1, then were aliquoted into 1.5 mL EP tubes and incubated in a constant temperature incubator (37°C) for 0, 12, 24 and 48 hours. The reaction was terminated immediately after reaching the preset time point, and the stability of G-siRNA in the 10% FBS system was evaluated by agarose gel electrophoresis.

[0077] The results show that single siRNA is significantly degraded at 24h and almost completely degraded at 36h, while the stability of G-siRNA is significantly improved after coupling with ganoderic acid, and there is still a large amount of siRNA at 48h (see Figure 4 ).

[0078] 4. Extraction of ulva lactuca lipid nanovesicles

[0079] The extraction steps of ulva lactuca lipid nanovesicles (SF-NVs) are as follows:

[0080] (1) Take out the ulva lactuca from the -30°C refrigerator, thaw, wash, and weigh an appropriate amount of ulva lactuca.

[0081] (2) Put the ulva lactuca and the pre-cooled PBS into the grinder at a mass ratio of 1:5, and grind them by breaking and grinding. Grind at the maximum speed for 5 times, 1 minute each time. After each grinding, place it in the cold storage for 5-10 minutes.

[0082] (3) Pour the grinding liquid into the prepared 50 mL centrifuge tube and centrifuge at 1000 g for 10 minutes.

[0083] (4) After centrifugation, take the supernatant and discard the precipitate. Centrifuge the supernatant at 3000 g for 20 minutes.

[0084] (5) After centrifugation, take the supernatant and discard the precipitate. Centrifuge the supernatant at 10000 g for 40 minutes.

[0085] (6) After differential centrifugation, take the supernatant and perform ultracentrifugation at 4°C, 150000 g for 90 minutes. Prepare the sucrose solution with concentrations from bottom to top as 60%, 45%, 30%, and 8% in advance, 8 mL for each gradient.

[0086] (7) After ultracentrifugation, discard the supernatant, resuspend the precipitate with an appropriate amount of PBS, and add the resuspended liquid to the uppermost layer of the sucrose solution. Then perform ultracentrifugation at 150000 g, 4°C for 2 hours.

[0087] (8) After centrifugation, three distinct bands appeared in the sucrose gradient solution (between 8% and 30%, 30% and 45%, and 45% and 60%, respectively), and then the nanoparticles in the band between 8% and 30% were aspirated for subsequent study. Then 150000 g, 1.5 hours, 4°C ultracentrifugation to remove sucrose.

[0088] (9) PBS resuspended the precipitate for subsequent characterization, etc., and stored at -80°C.

[0089] 5. Characterization of Sargassum muticum lipid nanovesicles

[0090] 5.1 Particle size analysis of Sargassum muticum lipid nanovesicles

[0091] Take 100 μg of Sargassum muticum lipid nanovesicles and mix with 900 μL of PBS. Add the well-mixed sample solution to the nanoparticle tracking analyzer, use the Stokes-Einstein equation to measure the average speed of the particles therein, and thus calculate the size and number of nanoparticles. As shown in Figure 5 , the hydrated particle size of the nanovesicles showed a unimodal distribution, with an average hydrodynamic diameter of 132.5 ± 53.6 nm.

[0092] 5.2 Zeta potential analysis of Sargassum muticum lipid nanovesicles

[0093] Take 100 μg of Sargassum muticum lipid nanovesicles and mix with 900 μL of PBS. Sample on machine, slowly inject 1 mL of well-mixed sample solution into the potential cup, and measure the surface charge of Sargassum muticum lipid nanovesicles to detect Zeta potential. As shown in Figure 6 , the surface charge of SF-NVs was -28.43 ± 0.34 mV.

[0094] 5.3 RNA content analysis of Sargassum muticum lipid nanovesicles

[0095] Take 100 μg of Sargassum muticum lipid nanovesicles and add 200 μL of RNAiso Plus lysis solution for RNA extraction, and then perform electrophoresis using a 1% agarose gel, as shown in Figure 7 , the vesicles contained almost no RNA.

[0096] 5.4 Electron microscopy of Sargassum muticum lipid nanovesicles

[0097] Drop 10 μL of Sargassum muticum lipid nanovesicle resuspension on a copper grid, let stand for 1 minute, carefully aspirate the sample, drop 10 μL of 2% phosphotungstic acid staining solution for staining, stain for 1 min, aspirate the dye, and naturally dry, then take electron microscopy. As shown in Figure 8As shown in FIG. 8A, the extracted SF-NVs have a diameter of about 150 nm under a transmission electron microscope (TEM).

[0098] 5.5 Electroporation of siRNAs and analysis of their morphology

[0099] 10 μL of Sargassum siliquosum lipid nanovesicle resuspension was dropped on a copper mesh, and stood for 1 minute. The sample was carefully sucked off, and 10 μL of 2% phosphotungstic acid staining solution was added for staining. After 1 minute of staining, the dye was sucked off, and the copper mesh was naturally dried before being photographed under an electron microscope.

[0100] The results show that after electroporation, there is obvious vesicle fragmentation, and a large number of fragments, while the co-incubation method is relatively mild and does not damage the vesicles (see FIGS. 8B and 8C). Figure 8

[0101] 6. Preparation of Sargassum siliquosum lipid nanovesicle-siRNA complexes

[0102] SF-NVs and G-siRNA were mixed in a pH 7.4 PBS buffer at a mass ratio of 16:1 (final concentration of SF-NVs 80 μg / mL, siRNA 5 μg / mL) (the mass ratio of SF-NVs to G-siRNA can be extended to 16-20:1), and incubated in a constant-temperature shaker at 4°C (up to 8°C) for 2 hours in the dark (the incubation time in the dark can be controlled within 1-4 hours). After the incubation, the unencapsulated siRNA was removed by ultrafiltration centrifugation (100 kDa molecular weight cutoff, 10000 x g centrifugation at 4°C for 30 minutes), and the trapped component was collected to obtain G-siRNA / SF-NVs.

[0103] According to the same method described above, siRNA / SF-NVs were prepared by replacing G-siRNA with siRNA.

[0104] 7. Fluorescence co-localization analysis of siRNA / SF-NVs and G-siRNA / SF-NVs complexes

[0105] (1) Fluorescent labeling of SF-NVs: 40 μg of SF-NVs was mixed with 4 μL of PKH67 staining working solution (prepared by mixing mother liquor and diluent at a volume ratio of 1:9) uniformly, and incubated at room temperature in the dark for 15 minutes. The mixed solution after staining was transferred to an ultracentrifuge tube, and PBS buffer was added to the full volume, and centrifuged at 150,000 x g for 1.5 hours at 4°C. After discarding the supernatant, the precipitate was resuspended with appropriate PBS buffer to obtain a suspension of PKH67-labeled SF-NVs.

[0106] ​(2) Preparation of G-siRNA: The water-soluble triterpenoid-siRNA conjugate (G-siRNA) was synthesized using Cy3 fluorescently labeled siRNA as raw material.

[0107] (3) According to the experimental steps in the section "6. Preparation of Sargassum fusiforme lipid nanovesicles-siRNA complex", PKH67-labeled SF-NVs were complexed with Cy3-labeled siRNA and G-siRNA, respectively, to prepare SiRNA / SF-NVs and G-siRNA / SF-NVs complexes.

[0108] (4) The prepared complexes were transfected into A549 cells, respectively, and observed under a laser confocal microscope after 24 hours.

[0109] As shown in Figure 9 , the green fluorescence signal of SF-NVs (excitation wavelength 488 nm / emission wavelength 518 nm) and the red fluorescence of siRNA / G-siRNA (excitation wavelength 552 nm / emission wavelength 570 nm) showed significant spatial colocalization characteristics, indicating that the modification strategy of ganoderic acid can significantly increase the loading of siRNAs on vesicles.

[0110] 8. Stability study of siRNA / SF-NVs and G-siRNA / SF-NVs complexes

[0111] To evaluate the stability of the complexes in a physiological environment, they were added to a solution of fetal bovine serum (FBS) with a final concentration of 10% and incubated at 37°C. After 12, 24, 36, and 48 hours of incubation, samples were taken and incubated in IP lysis buffer for 15 min before agarose gel electrophoresis analysis to detect changes in the stability of the complexes.

[0112] As shown in Figure 10 , after 48 hours of incubation, the electrophoretic band intensity of G-siRNA / SF-NVs did not change significantly, while the electrophoretic band intensity of siRNA / SF-NVs decreased significantly over time, indicating that G-siRNA / SF-NVs can improve the stability of siRNAs.

[0113] 9. Nanoparticle size analysis and potential analysis of SF-NVs, siRNA / SF-NVs, and G-siRNA / SF-NVs

[0114] The particle size of SF-NVs, siRNA / SF-NVs and G-siRNA / SF-NVs was found to be 132.5 ± 53.6 nm, 134.4 ± 63.8 nm and 130.9 ± 61.6 nm, respectively, by particle size analysis. Figure 11 The potential size was -23.32 ± 3.507 mV, -37.103 ± 9.297 mV and -53.647 ± 4.101 mV, respectively. Figure 12

[0115] 10. G-siRNA cell uptake experiment

[0116] (1) Cell pretreatment: HEK-293T and Huh-7 cells were inoculated in DMEM medium containing 10% fetal bovine serum. HCC-827 was inoculated in Ham's F-12K complete culture medium containing 10% fetal bovine serum. The culture was terminated when the cell confluence rate reached 80%.

[0117] (2) Cell digestion and collection: after discarding the culture medium, the cells were gently washed twice with preheated 1xPBS (1 mL / time); 0.25% trypsin digestion solution (1 mL / well) was added, incubated at 37°C for 2 minutes, and the digestion was terminated after observing the cell gap under a microscope. An equal volume of complete culture medium was added to terminate the reaction, and a single cell suspension was formed by gentle blowing. It was transferred to a 1.5 mL centrifuge tube and centrifuged at 300xg for 5 minutes, and the supernatant was discarded.

[0118] (3) Cell counting and plating: 1 mL of complete culture medium was added to each tube, mixed well by pipette blowing, and a cell counting plate was taken out. 10 μL of cell suspension was taken and added to the counting plate for counting. The counting method: under a microscope, the number of cells in 4 large squares was counted according to the principle of not counting down and not counting left, and the total number of cells in 4 large squares was recorded as N. The number of cells in each mL of cell suspension was N / 4x10 4 .

[0119] (4) Cell plating: the cell density of HCC-827 was adjusted to 5x10 4 / mL, and the cell density of HEK-293T and Huh-7 was adjusted to 10x10 4 / mL, inoculated in a 24-well plate with pre-set sterile coverslips, and the final volume per well was 500 μL, and cultured at 37°C for 12 hours.

[0120] (5) Transfection complex preparation: Cy3-labeled G-siRNA complex was synthesized according to the previous method, and gradient concentration working solution was prepared with DMEM complete culture medium (final concentration was 20 μg / mL).

[0121] ​(6) Transfection operation: after cell adhesion, the original culture medium was discarded, and PBS was washed twice; the experimental group was added with 500 μL of culture medium containing gradient concentration of Cy3-G-siRNA, and the blank control group was added with the same amount of complete culture medium, and was cultured at 37°C for 24 hours.

[0122] (7) Sample fixation and staining: 4% paraformaldehyde was used for room temperature fixation for 45 minutes, PBS was washed for 3 times (5 minutes / time, 50 rpm on the shaking table); DAPI staining solution was used for dark staining for 10 minutes, and PBS was washed for 3 times to remove residual dye.

[0123] (8) Microscopic sample preparation: the glass slide was pre-coated with anti-quenching mounting medium, the inverted cell climbing slide was attached, the neutral gum edge was sealed, and was stored in the dark.

[0124] (9) Confocal imaging.

[0125] As shown in FIG. 1A, the red fluorescence signal of Cy3 was mainly distributed in the cytoplasmic region, and there was no overlap with the nucleus labeled by DAPI (blue fluorescence), indicating that Cy3-G-siRNA successfully entered the cytoplasm. Figure 13 Example 2

[0126] 1. G-siRNA transfection of A549, HCC827, HEK293t and Huh-7 cells, and analysis of knockdown effect on GAPDH

[0127] (1) HCC827 cells and A549 cells were respectively plated into 12-well plates at 1×10 5 cells / well, and HEK-293T and Huh-7 cells were plated at 1.5×10 5 cells / well, and the cell culture box was continued to be cultured for 12 hours.

[0128] (2) Ganoderma acid-siNC conjugate (G-siNC) and ganoderma acid-siGAPDH conjugate (G-siGAPDH) were prepared, and the method was the same as that in Example 1. G-siNC and G-siGAPDH were added into the complete culture medium and mixed (the final concentration was 20 μg / mL).

[0129] (3) The cells were treated according to the grouping. RNAiMAX+siNC group and RNAiMAX+siGAPDH group: 1 mL of culture medium containing G-siNC or G-siGAPDH was added, and the transfection reagent RNAiMAX was added, and the cells were treated for 36 hours; G-siNC group and G-siGAPDH group: 1 mL of culture medium containing G-siNC or G-siGAPDH was added for 36 hours.

[0130]

[0131] ​After the end of the culture, RNA was extracted for analysis, and the results are shown in Figure 13 As shown in FIG. 5C, in A549, HCC827 and Huh-7 cell lines, GAPDH expression in the G-siGAPDH treatment group did not change compared with the G-siNC group. However, in the HEK293T cell line, GAPDH expression in the G-siGAPDH treatment group (0.815 ± 0.036) was significantly down-regulated compared with the G-siNC group.

[0132] 2. Intraperitoneal injection of G-siGAPDH and siGAPDH to analyze the silencing effect of genes in different tissues of mice

[0133] (1) Experimental grouping and drug administration: The mice were randomly divided into three groups, two in each group: PBS group, siGAPDH group and G-siGAPDH group. The PBS group was injected with 300 μL of PBS; the siGAPDH group and the G-siGAPDH group were diluted with PBS to 300 μL, and the drug concentration was 2 mg / kg.

[0134] (2) Mouse sacrifice and tissue collection: The mice were sacrificed 48 hours later, and the main organs and tissues, including the heart, liver, spleen, lung, kidney, stomach, intestine, eye and muscle, were collected to provide samples for subsequent analysis. As shown in FIG. 6A, the main organs and tissues of the mice were collected. Figure 13 As shown in FIG. 6C, in all tissues except liver tissue, GAPDH expression showed no significant difference between all treatment groups (PBS, siGAPDH, G-siGAPDH).

[0135] 3. In vivo distribution of Cy3-siRNA / SF-NVs and Cy3-G-siRNA / SF-NVs complexes

[0136] (1) Cy3-labeled siRNA treatment: Cy3-fluorescent labeled siRNA was coupled with triterpenoid compounds to prepare Cy3-G-siRNA, which was stored in the dark after ultrafiltration purification.

[0137] (2) SF-NVs loading: The prepared SF-NVs were mixed with Cy3-siRNA / Cy3-G-siRNA at a mass ratio of 16:1 to prepare Cy3-siRNA / SF-NVs and Cy3-G-siRNA / SF-NVs complexes.

[0138] (3) Experimental animal treatment: 8-week-old c57 male mice (SPF level) were treated according to the following grouping

[0139] Blank control group (PBS), free Cy3-siRNA group (2 mg / kg), Cy3-siRNA / SF-NVs group (same siRNA 2 mg / kg), Cy3-G-siRNA / SF-NVs group (same siRNA 2 mg / kg)

[0140] (4) Administration method: intraperitoneal injection (injection volume 200 μL / each), and free feeding and drinking after injection. The corresponding tissues were taken for imaging 24 hours later. As shown in Figure 14 , the fluorescence of siRNA was mainly enriched in the lungs and kidneys, and the fluorescence enrichment degree of Cy3-G-siRNA / SF-NVs in the lungs and kidneys was significantly stronger than that of Cy3-siRNA / SF-NVs.

[0141] 4. Lung distribution evaluation after intraperitoneal injection and aerosol inhalation of Cy3-G-siRNA / SF-NVs complex in mice

[0142] (1) Preparation of Cy3-G-siRNA / SF-NVs complex

[0143] (2) Experimental animal treatment: 6-week-old c57 male mice (SPF level) were selected and treated according to the following grouping:

[0144] Cy3-G-siRNA / SF-NVs aerosol inhalation group (siRNA 2 mg / kg);

[0145] Cy3-G-siRNA / SF-NVs intraperitoneal injection group (siRNA 2 mg / kg).

[0146] (3) Administration scheme: intraperitoneal injection and aerosol inhalation administration in mice, and free feeding and drinking after treatment.

[0147] (4) Immediately after taking the lung, freeze sectioning was performed, and Cytokeratin 7 was used to label mouse lung epithelial cells for observation under a laser confocal microscope. As shown in Figure 15 , Cy3 fluorescence was mainly distributed in mouse epithelial cells.

[0148] 5. Analysis of the knockdown effect of G-siRNA / SF-NVs in A549, HCC827, and BEAS-2B cells

[0149] (1) According to the method in the "6. Preparation of Undaria pinnatifida lipid nanovesicle-siRNA complex" section of Example 1, G-siRNA / SF-NVs was synthesized, and then co-incubated with A549, HCC827, and BEAS-2B cells, respectively, with the following specific grouping:

[0150] RNAiMAX+siNC group, RNAiMAX+siGAPDH group, siRNA group, G-siRNA group, G-siNC / SF-NVs group, G-siRNA / SF-NVs group and siRNA / SF-NVs group: respectively incubated with cells for 36 hours, and then the GAPDH level in cells was detected by qPCR.

[0151] As shown in FIG. 6, the expression of GAPDH was significantly down-regulated after siGAPDH / SF-NVs and G-siGAPDH / SF-NVs treatment, and the silencing effect of G-siGAPDH / SF-NVs on GAPDH was better. Figure 16

[0152] 6. Lysosome escape analysis of G-siRNA / SF-NVs in A549, HCC827 and BEAS-2B cells

[0153] (1) A549, HCC827 and BEAS-2B cells were respectively inoculated in DMEM medium (Ham's F-12K medium) containing 10% fetal bovine serum, 7.5 x 10 4 cells were inoculated in each well of a 24-well plate, and after the cells were cultured for 12 hours, different Cy3-labeled siRNA or siRNA coupling complexes (final concentration of 20 μg / mL) were added.

[0154] (2) The experimental group was added with 500 μL of G-siRNA / SF-NVs-containing medium, and the control group was added with the same amount of G-siRNA complete medium, and was cultured at 37°C for 12, 24, 36 and 48 hours.

[0155] (3) Lysosome staining: the 24-well plate was taken out after 12, 24, 36 and 48 hours, and the culture medium was discarded in the clean bench and the cells were washed with PBS, and the washing was repeated once. Lysosome staining working solution (freshly prepared): 0.1 μL LysoTracker™ Green DND-26 (10000x) was added to each milliliter of complete medium. After preparation, 500 μL of lysosome staining working solution was added to each well, and the cells were cultured for another 2 hours in the cell culture box.

[0156] (4) Fixation and staining: 4% paraformaldehyde was used for room temperature fixation for 45 minutes, PBS was washed for 3 times (5 minutes / time, 50 rpm on a shaker); DAPI staining solution was stained for 10 minutes in the dark, and PBS was washed for 3 times to remove residual dye.

[0157] (5) The glass slide was pre-coated with anti-quenching mounting medium, the cell climbing slide was inverted and attached, the neutral gum edge was sealed, and the slide was stored in the dark.

[0158] (6) Confocal imaging​ Figure 17 As shown, both G-siRNA and G-siRNA / SF-NVs can enter cells, and the G-siRNA alone can be well co-localized with lysosomes, while the G-siRNA / SF-NVs can escape the capture of lysosomes after entering cells.

[0159] Example 3

[0160] Construction of a mouse bleomycin (BLM)-induced pulmonary fibrosis model and verification of the treatment effect of G-siRNA / SF-NVs:

[0161] 1. Construction of a bleomycin-induced mouse pulmonary fibrosis model

[0162] SPF-grade C57 / B6 mice (6-8 weeks old, male) were first adaptively fed for 1 week, and then randomly divided into 7 groups: NC group, NC+G-siTGF-β1 / SF-NVs group, BLM group, BLM+G-siTGF-β1 group, BLM+SF-NVs group, BLM+G-siNC / SF-NVs, and BLM+G-siTGF-β1 / SF-NVs, 5 mice in each group. The BLM modeling group was first anesthetized, and then administered using an aerosol device, with a bleomycin administration concentration of 1.5 U / kg.

[0163] 2. Preparation of experimental materials

[0164] SF-NVs, G-siTGF-β1, G-siTGF-β1 / SF-NVs, G-siNC, and G-siNC / SF-NVs were prepared according to the method of Example 1.

[0165] 3. Drug administration to mice in each group:

[0166] NC group: PBS was administered intraperitoneally every other day;

[0167] NC+G-siTGF-β1 / SF-NVs: G-siTGF-β1 / SF-NVs were administered intraperitoneally every other day;

[0168] BLM group: PBS was administered intraperitoneally every other day;

[0169] BLM+G-siTGF-β1 group: G-siTGF-β1 was administered intraperitoneally every other day after BLM modeling;

[0170] BLM+SF-NVs group: SF-NVs were administered intraperitoneally every other day after BLM modeling;

[0171] BLM+G-siNC / SF-NVs group: G-siNC / SF-NVs were injected intraperitoneally every two days after BLM modeling;

[0172] BLM+G-siTGF-β1 / SF-NVs group: G-siTGF-β1 / SF-NVs were injected intraperitoneally every two days after BLM modeling.

[0173] The above siRNA was used in a dose of 2 mg / kg, and the specific process was as follows Figure 18 .

[0174] 4. Index detection

[0175] 4.1 Observation of mouse signs and monitoring of physiological indexes

[0176] During the treatment of mice, the body weight of mice and the activity of mice were checked every two days. After the experiment, all mice were sacrificed, and the lung status of mice was recorded by taking pictures. The mouse alveolar lavage fluid was collected, and the lung wet weight was recorded to calculate the lung / body ratio. A 1 mL syringe was used to extract the heart blood and heart, liver, spleen, lung, kidney, stomach, small intestine and other tissues for later experiments. The serum and alveolar lavage fluid were stored in a-80°C refrigerator for subsequent experiments.

[0177] The results showed that the body weight of mice in the BLM treatment group decreased, and the lung weight / body weight ratio increased, while the body weight of mice after different interventions was slightly recovered, and the lung weight / body weight ratio slightly decreased, especially after siTGF-β1 / SF-NVs intervention, the lung weight / body weight ratio was significantly reduced (P<0.05) Figure 19 ). Compared with the healthy state of red lung in the NC group, extensive hemorrhagic necrotic foci appeared in the lungs of mice in the BLM group and the BLM+G-siTGF-β1 group, while the hemorrhagic necrosis of the lungs in the BLM+SF-NVs group and the BLM+siNC / SF-NVs group was reduced, and the necrotic area of the lungs in the BLM+G-siTGF-β1 / SF-NVs group was significantly reduced, and the treatment effect was better than that of the BLM+SF-NVs group and the BLM+siNC / SF-NVs group Figure 20 ).

[0178] 4.2 Total RNA of lung tissue, reverse transcription and qPCR

[0179] About 0.05 g of lung tissue was weighed and placed in a deenzyme EP tube, 500 μL of RNAiso Plus lysis solution and 2 magnetic beads were added, and the tissue was ground in a biological sample homogenizer. The tissue was ground until the tissue pieces disappeared, and then lysed on ice for 10 minutes. Then chloroform, isopropanol precipitation, ethanol washing, and DEPC water dissolution were added respectively to obtain total RNA, and then reverse transcription and qPCR detection were performed.

[0180] Results showed that the expression levels of TGF-β1, Collagen 1, Fibronectin and α-SMA in lung tissue were significantly increased after BLM modeling, while the increased TGF-β1, Collagen 1, Fibronectin and α-SMA were significantly reduced after siTGF-β1 / SF-NVs intervention Figure 21 ).

[0181] 4.3 Western blotting experiment

[0182] The total protein of mouse lung homogenate was extracted and detected by Western blotting. The results showed that G-siTGF-β1 / SF-NVs could effectively inhibit the abnormal expression of key factors in TGF-β1 signaling pathway and its downstream fibrosis-related markers (TGF-β1, Collagen 1, Fibronectin and α-SMA) Figure 22 ).

[0183] 4.4 Total protein detection in bronchoalveolar lavage fluid (BALF)

[0184] (1) The mouse bronchoalveolar lavage fluid stored in the -80℃ refrigerator was thawed on ice. The lavage fluid was centrifuged at 1000xg for 10 minutes at 4℃ to separate the supernatant and cell precipitate. The supernatant was used for total protein detection.

[0185] (2) The total protein of bronchoalveolar lavage fluid was detected by BCA method. As shown in FIG. 4A, the protein concentration in the lung lavage fluid after BLM treatment increased significantly, and G-siTGF-β1 / SF-NVs could effectively protect the mouse alveolar epithelial barrier and inhibit the exudation of plasma protein, thereby significantly reducing the total protein content in BALF. Figure 23

[0186] 4.5 Myeloperoxidase (MPO) detection in bronchoalveolar lavage fluid

[0187] The mouse bronchoalveolar lavage fluid stored in the -80℃ refrigerator was thawed on ice. The lavage fluid was centrifuged at 1000xg for 10 minutes at 4℃ to separate the supernatant and cell precipitate. The supernatant was taken and MPO activity detection was performed using MPO detection kit.

[0188] As shown in FIG. 4B, BLM+SF-NVs group could effectively alleviate the increase of MPO activity, while BLM+G-siTGF-β1 / SF-NVs group showed the most significant therapeutic effect. Figure 23

[0189] 4.6 H&E staining and Masson staining of tissue sections

[0190] ​​Fresh lung tissues were dissected and paraffin sections were prepared. H&E and Masson staining were performed, and the sections were examined under a microscope. Images were collected and analyzed.

[0191] like Figure 24 As shown in the results, the G-siTGF-β1 / SF-NVs treatment group can effectively reduce the infiltration of inflammatory cells in the lungs of mice and prevent the destruction of alveolar structure; the G-siTGF-β1 / SF-NVs treatment group can effectively inhibit the deposition of collagen fibers and the proliferation of collagen.

[0192] 4.7 Detection of Hydroxyproline (HYP) in Mouse Lung Homogenate

[0193] The mouse lung tissue homogenate was taken and the hydroxyproline content was detected using a hydroxyproline content detection kit. Figure 25 The results showed that BLM treatment significantly increased the hydroxyproline content in lung tissue, while SF-NVs and G-siTGF-β1 / SF-NVs could effectively reduce the increase in HYP content, and the treatment effect of the BLM+G-siTGF-β1 / SF-NVs group was better.

[0194] 4.8 Detection of inflammatory factors in mouse serum by enzyme-linked immunosorbent assay (ELISA)

[0195] Take the mouse serum samples, return to room temperature, and use enzyme-linked immunosorbent assay to detect the expression of inflammatory factors. The results are shown in Figure 26 The results showed that SF-NVs and G-siTGF-β1 / SF-NVs could inhibit the expression of inflammatory factors (IL-6, IL-1β and TNF-α) induced by bleomycin in mice.

[0196] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A method for preparing a triterpene compound-siRNA / lipid nanovesicle complex, characterized in that: The following steps are involved: Conjugating the triterpenoid compound with siRNA to obtain a water-soluble triterpenoid compound-siRNA conjugate; Incubating the water-soluble triterpenoid compound-siRNA conjugate with the Sargassum fusiformis lipid nanovesicles in the dark to obtain the triterpenoid compound-siRNA / lipid nanovesicle complex; The nucleotide sequence of the sense strand of the siRNA is shown in SEQ ID NO. 23, and the nucleotide sequence of the antisense strand is shown in SEQ ID NO. 24; The water-soluble triterpenoid compound-siRNA conjugate is prepared by using an EDC-NHS coupling reaction; The triterpenoid compound is ganoderic acid; The structural formula of the ganoderic acid is as follows: 。 2. The preparation method according to claim 1, characterized in that The Sargassum fusiformis lipid nanovesicles are extracted by utilizing a differential centrifugation method.

3. The preparation method according to claim 1, characterized in that The mass ratio of the water-soluble triterpenoid compound-siRNA conjugate to the Sargassum fusiformis lipid nanovesicles is 1:16-20.

4. The preparation method according to claim 1, characterized in that The temperature of the light-proof incubation reaction is 4° C.-8° C., and the time is 1-4 hours.

5. A triterpene compound-siRNA / lipid nanovesicle complex prepared according to the preparation method according to any one of claims 1 to 4.

6. Use of the triterpene compound-siRNA / lipid nanovesicle complex according to claim 5 in the preparation of a drug for treating pulmonary fibrosis.

7. A drug for treating pulmonary fibrosis, characterized in that: The active ingredient comprises the triterpene compound-siRNA / lipid nanovesicle complex according to claim 5.

8. The drug according to claim 7, characterized in that The drug also includes pharmaceutically acceptable excipients.

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