Triterpenoid-siRNA / lipid nano-vesicle compound as well as preparation method and application thereof

Abstract

The invention discloses a triterpenoid-siRNA / lipid nano-vesicle compound as well as 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 with siRNA (small interfering Ribonucleic Acid) to obtain a water-soluble triterpenoid-siRNA conjugate; the water-soluble triterpenoid-siRNA conjugate and the sargassum fusiforme lipid nano-vesicle are subjected to a dark incubation reaction, and the triterpenoid-siRNA / lipid nano-vesicle compound is obtained; the nucleotide sequence of a positive-sense strand of the siRNA is as shown in SEQ ID NO.23, and the nucleotide sequence of an antisense strand of the siRNA is as shown in SEQ ID NO.24. The triterpenoid-siRNA / lipid nano-vesicle compound can inhibit the pulmonary fibrosis process, provides a dual regulation strategy for pulmonary fibrosis treatment, and realizes a breakthrough in treatment efficiency while ensuring biological safety.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a triterpene compound-siRNA / lipid nanovesicle complex and a preparation method and application thereof. Background Art

[0002] In the biopharmaceutical field, small interfering RNA (siRNA) has become an important tool for treating a wide range of diseases due to its potential to specifically silence target gene expression. However, despite the significant advantages of siRNA technology, its clinical application faces numerous challenges, primarily due to shortcomings in the delivery system. First, naked siRNA molecules are easily degraded by nucleases in serum, resulting in poor stability and difficulty maintaining long-term activity. Second, siRNA is inherently negatively charged, making it difficult to penetrate cell membranes and enter cells, further limiting its bioavailability. Furthermore, while traditional delivery systems such as liposomes and polymer nanoparticles can address these issues to some extent, they also have significant shortcomings. For example, these synthetic carriers often exhibit low targeting, which can lead to nonspecific distribution and off-target effects, reducing therapeutic efficacy and increasing potential toxicity. Furthermore, some synthetic materials may trigger immune responses, limiting their long-term use.

[0003] In response to these problems, researchers have gradually turned their attention to natural delivery vehicles, such as exosomes and plant-derived nanovesicles, in recent years. These natural vehicles have shown great application potential due to their excellent biocompatibility and low immunogenicity. However, the siRNA delivery system based on natural vehicles is still in its early stages of development. Conventional siRNA delivery into vesicles mostly uses electroporation technology, which has limited carrying efficiency and is difficult to achieve large-scale preparation processes. Therefore, the delivery efficiency needs to be further improved. In addition, how to achieve large-scale preparation of vesicles carrying siRNA remains one of the key scientific issues that need to be solved urgently in this field.

[0004] At the same time, regarding disease treatment, pulmonary fibrosis is a complex chronic disease whose pathogenesis involves the fibrotic process mediated by the TGF-β1 signaling pathway, severely impacting patients' quality of life and survival. Currently, the main drugs used clinically for the treatment of pulmonary fibrosis include pirfenidone and nintedanib, but these drugs only delay disease progression and cannot fundamentally reverse the fibrotic process. Therefore, the development of a new therapeutic agent that can precisely 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, with its high specificity and designability, offers a new approach for the treatment of pulmonary fibrosis. However, the limitations of existing siRNA delivery systems still restrict their application in the field of pulmonary fibrosis. To overcome these issues, there is an urgent need to develop an efficient and safe siRNA delivery platform that can significantly improve therapeutic efficiency while ensuring biosafety and provide new possibilities for the treatment of related diseases. Summary of the Invention

[0005] The present invention aims to provide a triterpene compound-siRNA / lipid nanovesicle complex, its preparation method, and its application to address the aforementioned problems of the prior art. This triterpene compound-siRNA / lipid nanovesicle complex can inhibit the progression of pulmonary fibrosis, providing a dual regulatory strategy for pulmonary fibrosis treatment, achieving a breakthrough in therapeutic efficiency while ensuring biosafety.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for preparing a composite for treating pulmonary fibrosis, comprising the following steps:

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

[0009] 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;

[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] Furthermore, the water-soluble triterpenoid compound-siRNA conjugate is prepared by using an EDC-NHS coupling reaction.

[0012] Furthermore, the triterpene compound is ganoderic acid.

[0013] Furthermore, the Sargassum fusiformis lipid nanovesicles are extracted by differential centrifugation.

[0014] Furthermore, the mass ratio of the water-soluble triterpenoid compound-siRNA conjugate to the Sargassum fusiformis lipid nanovesicles is 1:16-20.

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

[0016] The present invention also provides a triterpene compound-siRNA / lipid nanovesicle complex prepared according to the above preparation method.

[0017] The present invention also provides use of the triterpene compound-siRNA / lipid nanovesicle complex in preparing a drug for treating pulmonary fibrosis.

[0018] The present invention also provides a medicine for treating pulmonary fibrosis, wherein the active ingredient includes the above-mentioned triterpene compound-siRNA / lipid nanovesicle complex.

[0019] Furthermore, the medicine also includes pharmaceutically acceptable excipients.

[0020] The present invention discloses the following technical effects:

[0021] This invention provides a triterpene compound-siRNA / lipid nanovesicle complex (G-siRNA / SF-NVs) for the treatment of pulmonary fibrosis. This complex significantly enhances the stability of siRNA through covalent modification with triterpene compounds. Furthermore, using Sargassum fusiformis-derived lipid nanovesicles (SF-NVs) as a delivery vehicle overcomes the limitations of traditional delivery systems, such as limited tropism and poor biocompatibility. Studies have demonstrated that the G-siRNA / SF-NVs complex prepared in this invention effectively maintains the structural integrity of pulmonary vesicles, while triterpene modification significantly enhances the intracellular delivery efficiency of siRNA. This complex exhibits excellent gene silencing and lysosomal escape properties at the cellular level, providing key support for the functional realization of siRNA. In the treatment of pulmonary fibrosis, this complex achieved targeted lung accumulation via intraperitoneal injection and successfully inhibited the fibrotic process mediated by the TGF-β1 signaling pathway. Experimental results demonstrate that G-siRNA / SF-NVs not only effectively improves the structural and pathological features of lung tissue but also significantly reduces the expression of pro-fibrotic and inflammatory factors. In vitro studies further revealed its inhibitory effects on abnormal epithelial cell migration and pro-fibrotic factors. Compared to existing siRNA delivery systems, this invention combines plant-derived nanovesicles with natural active ingredient modification technology for the first time. Leveraging the anti-inflammatory properties of SF-NVs and the gene silencing effect of siTGF-β1, it synergistically inhibits the progression of pulmonary fibrosis, providing a dual regulatory strategy for pulmonary fibrosis treatment, achieving breakthroughs in treatment efficiency while ensuring biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Schematic diagram of the G-siRNA preparation process;

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

[0025] Figure 3 HPLC analysis diagrams of different samples;

[0026] Figure 4 Figure 2 is the result of stability test of siRNA and G-siRNA;

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

[0028] Figure 6 The figure shows the Zeta potential measurement results of SF-NVs;

[0029] Figure 7 This is the electrophoresis diagram after RNA extraction from SF-NVs;

[0030] Figure 8 The TME electron micrograph of SF-NVs (A) and the results of TME electron microscopic observation of the morphology of G-siRNA / SF-NVs synthesized by electroporation (B) and co-incubation (C);

[0031] Figure 9 Fluorescence co-localization analysis of siRNA / SF-NVs and G-siRNA / SF-NVs; scale bar: 10 μm;

[0032] Figure 10 Figure 2 is a stability analysis diagram of siRNA / SF-NVs and G-siRNA / SF-NVs;

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

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

[0035] Figure 13 Figure 1 shows the functional validation results of G-siRNA. A shows confocal images of HCC827, Huh-7, and HEK293t cells, with a scale of 40 μm. B shows the expression levels of GAPDH detected by RT-qPCR in A549, HCC827, Huh-7, and HEK293t cell lines after G-siRNA transfection (using β-Actin as an internal reference, n=3, "*" indicates p < 0.05, and "ns" indicates no significant difference). C shows the relative expression levels of GAPDH determined by RT-qPCR in different tissues (using β-Actin as an internal reference, n=3, "**" indicates p < 0.01, and "ns" indicates no significant difference).

[0036] Figure 14 The results show the distribution of siRNA / SF-NVs and G-siRNA / SF-NVs synthesized using Cy3-modified siRNA in mice after intraperitoneal injection.

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

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

[0039] Figure 17 Figure 3: Colocalization of lysosomal fluorescence (green) and G-siRNA fluorescence (red) observed by laser confocal microscopy in A549 cells (A), BEAS-2B cells (B), and HCC827 cells (C) 12, 24, 36, and 48 hours after transfection with G-siRNA / SF-NVs and G-siRNA, respectively. Scale bars are 10 μm.

[0040] Figure 18 Schematic diagram of mouse modeling;

[0041] Figure 19 Statistical graphs of mouse body weight (A) and the ratio of lung weight to body weight (B);

[0042] Figure 20 Figure 3 is the morphology of lung tissue of mice in each group;

[0043] Figure 21 Figure 2: RT-qPCR detection of the expression levels of pulmonary fibrosis-related indicators TGF-β1 (A), Collagen Ⅰ (B), Fibronectin (C), and α-SMA (D) in mouse lung homogenates; GAPDH was used as the internal reference, n = 5, "ns" indicates no statistical significance, "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, and "****" indicates p < 0.0001;

[0044] Figure 22 The figure shows the results of Western Blot detection of the expression levels of pulmonary fibrosis-related indicator proteins in mouse lung homogenate;

[0045] Figure 23 Figure 2 shows the test results of total protein content (A) and MPO activity (B) in bronchoalveolar lavage fluid (BALF);

[0046] Figure 24 H&E staining and Masson staining images of lung tissues of mice in each group; scale bars are 100 μm;

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

[0048] Figure 26 Figure 2 shows the detection results 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] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0050] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0051] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0052] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0053] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0054] The primer sequences involved in the present invention are shown in Table 1, and the sequences of the siRNAs interfering 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 interfering strands of siRNAs

[0059]

[0060] Example 1

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

[0062] EDC reaction principle: Carboxyl groups (-COOH) react with N-hydroxysulfosuccinimide (Sulfo-NHS) in the presence of carbodiimides to form semi-stable Sulfo-NHS esters, which then react with primary amines (-NH2) to form amide crosslinks. The activation reaction between EDC and Sulfo-NHS is most efficient at pH 4.5-7.2, and EDC reactions are typically performed in MES buffer at pH 4.7-6.0. The reaction of Sulfo-NHS-activated molecules with primary amines is most efficient at pH 7.0-8.0, and Sulfo-NHS ester reactions are typically performed in phosphate buffered saline (PBS) at pH 7.2-7.5.

[0063] (1) Solution preparation

[0064] Preparation of ganoderic acid stock solution: dissolve ganoderic acid (GHA) in dimethyl sulfoxide (DMSO) to prepare 10 mmol / L ganoderic acid stock solution.

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

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

[0067] (2) Construction of activation reaction system

[0068] Take 1 mL of MES buffer (pH 5.5) as the reaction medium, and add 1 μL of ganoderic acid mother solution (the final concentration of ganoderic acid in the reaction system is 10 μmol / L), 20 μL of EDC aqueous solution (the final concentration of EDC in the reaction system is 0.2 mg / mL), and 20 μL of NHS aqueous solution (the final concentration of NHS in the reaction system is 0.4 mg / mL) in sequence. The resulting reaction system is placed in a constant temperature oscillator and activated at 37°C and 200 rpm for 30 min.

[0069] (3) Covalent coupling reaction

[0070] Adjust the activated mixture to pH 7.0 with 20× PBS buffer and add 30 μL of 20 μmol / L 3'-amino-modified siGAPDH. Maintain the mixture at 37°C with continuous shaking for 120 minutes. Check the pH every 30 minutes and adjust as necessary to maintain a pH between 7.0 and 7.3.

[0071] (4) Product purification

[0072] After the reaction, the resulting mixture was transferred to an ultrafiltration centrifuge tube with a 10 kDa molecular weight cutoff and centrifuged at 8000 × g for 45 minutes at 4°C. During the centrifugation process, PBS buffer (pH 7.4) was added every 15 minutes for elution. This elution step was repeated three times, and the retained fraction was collected to obtain the water-soluble triterpene compound-siRNA conjugate (G-siRNA).

[0073] 2. G-siRNA Quality Control

[0074] To verify whether the triterpenoid compound was successfully coupled to siRNA via the two-step EDC method, the present invention used high performance liquid chromatography (HPLC) and polyacrylamide gel electrophoresis (PAGE) to observe the molecular weight difference between G-siRNA and siRNA. The PAGE analysis results showed that the electrophoretic velocity of G-siRNA was significantly lower than that of single siRNA, indicating that the molecular weight increased after coupling with ganoderic acid ( Figure 2 HPLC analysis results showed that the peak time of pure siRNA was 23 min, the peak time of ganoderic acid was 33 min, and the peak time after coupling was 31 min. The shift of the peak time indicated that G-siRNAs were successfully coupled ( Figure 3 ).

[0075] 3. G-siRNA Stability Study

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

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

[0078] 4. Extraction of Sargassum fusiformis lipid nanovesicles

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

[0080] (1) Take out the sea lettuce from the -30℃ freezer, thaw it, wash it, and weigh an appropriate amount of sea lettuce.

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

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

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

[0084] (5) After centrifugation, take the supernatant, discard the precipitate, and centrifuge the supernatant at 10,000 g for 40 minutes.

[0085] (6) After differential centrifugation, remove the supernatant and perform ultracentrifugation at 4°C, 150,000 g, and 90 minutes. Prepare in advance a sucrose solution with a concentration gradient of 60%, 45%, 30%, and 8% from bottom to top, with each gradient of 8 mL.

[0086] (7) After ultracentrifugation, discard the supernatant, resuspend the precipitate with an appropriate amount of PBS, add the resuspension to the top layer of sucrose solution, and then ultracentrifuge at 150,000 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). The nanoparticles in the band between 8% and 30% were then collected for subsequent analysis. The sucrose was then removed by ultracentrifugation at 150,000 g for 1.5 hours at 4°C.

[0088] (9) Resuspend the pellet in PBS for subsequent characterization and store at -80°C.

[0089] 5. Characterization of Sargassum fusiformis lipid nanovesicles

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

[0091] Take 100 μg of Sargassum fusiformis lipid nanovesicles and mix them evenly with 900 μL of PBS. The thoroughly mixed sample solution is added to the nanoparticle tracking analyzer. The Stokes-Einstein equation is used to measure the average velocity of the particles, thereby calculating and analyzing the size and number of nanoparticles. Figure 5 As shown in the figure, 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 Fusiforme Lipid Nanovesicles

[0093] Take 100 μg of Sargassum fusiformis lipid nanovesicles and mix them evenly with 900 μL of PBS. Load the sample onto the instrument and slowly inject 1 mL of the thoroughly mixed sample solution into the potential cup to measure the surface charge of the Sargassum fusiformis lipid nanovesicles and detect the Zeta potential. Figure 6 As shown, the surface charge of SF-NVs is -28.43 ± 0.34 mV.

[0094] 5.3 Analysis of RNA Content in Sargassum Fusiformis Lipid Nanovesicles

[0095] Take 100 μg of Sargassum fusiformis lipid nanovesicles and add 200 μL of RNAiso Plus lysis buffer to extract RNA, and then use 1% agarose gel electrophoresis, as shown in Figure 2. Figure 7 As shown, the vesicles contain almost no RNA.

[0096] 5.4 Electron Microscopy of Sargassum fusiformis Lipid Nanovesicles

[0097] 10 μL of the resuspended Sargassum fusiformis lipid nanovesicles was dropped onto the copper grid and allowed to stand for 1 minute. The sample was carefully removed by aspiration and 10 μL of 2% phosphotungstic acid staining solution was added for staining. The staining was continued for 1 minute, the dye was removed by aspiration, and the sample was photographed under an electron microscope after natural drying. Figure 8As shown in middle A, the extracted SF-NVs have a diameter of approximately 150 nm under transmission electron microscopy (TEM).

[0098] 5.5 Electroporation of siRNAs and Analysis of Their Morphology

[0099] 10 μL of the resuspension of Sargassum fusiformis lipid nanovesicles was added dropwise onto the copper grid and allowed to stand for 1 minute. The sample was carefully removed by aspiration and 10 μL of 2% phosphotungstic acid staining solution was added for staining. The staining was continued for 1 minute, the dye was removed by aspiration, and the sample was naturally dried before being photographed under an electron microscope.

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

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

[0102] SF-NVs and G-siRNA were mixed at a mass ratio of 16:1 (final concentration of SF-NVs 80 μg / mL, siRNA 5 μg / mL) in pH 7.4 PBS buffer (the mass ratio of SF-NVs to G-siRNA can be expanded to 16-20:1). Incubate in a shaker at 4°C (up to 8°C) in the dark for 2 hours (the incubation time can be controlled to 1-4 hours). After incubation, remove unencapsulated siRNA by ultrafiltration centrifugation (100 kDa molecular weight cutoff, 10,000 × g at 4°C for 30 minutes). The retained fraction is collected to obtain G-siRNA / SF-NVs.

[0103] Following the same method as above, G-siRNA was replaced with siRNA to prepare siRNA / SF-NVs.

[0104] 7. Fluorescence Colocalization Analysis of siRNA / SF-NVs and G-siRNA / SF-NVs Complexes

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

[0106] (2) Preparation of G-siRNA: Using Cy3 fluorescently labeled siRNA as raw material, water-soluble triterpene compound-siRNA conjugate (G-siRNA) was synthesized.

[0107] (3) According to the experimental steps in section “6. Preparation of Sargassum fusiformis lipid nanovesicle-siRNA complexes”, 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 and observed under a laser confocal microscope 24 hours later.

[0109] like Figure 9 As shown in the figure, 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 co-localization characteristics. The results showed that the ganoderic acid modification strategy can significantly increase the loading of vesicles on siRNAs.

[0110] 8. Stability Study of siRNA / SF-NVs and G-siRNA / SF-NVs Complexes

[0111] To assess the stability of the complex in a physiological environment, it was added to a final concentration of 10% fetal bovine serum (FBS) and incubated at 37°C. After 12, 24, 36, and 48 hours of incubation, samples were incubated in IP lysis buffer for 15 minutes and analyzed by agarose gel electrophoresis to detect changes in the stability of the complex.

[0112] like Figure 10 As shown in the figure, 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 analysis showed that the particle sizes of SF-NVs, siRNA / SF-NVs and G-siRNA / SF-NVs were 132.5 ± 53.6 nm, 134.4 ± 63.8 nm and 130.9 ± 61.6 nm, respectively. Figure 11 ); their potentials are -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 cells were inoculated in Ham's F-12K complete 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, gently wash the cells twice with preheated 1× PBS (1 mL / time); add 0.25% trypsin digestion solution (1 mL / well), incubate at 37°C for 2 minutes, and terminate the digestion after observing the expansion of the intercellular gap under a microscope. Add an equal volume of complete culture medium to terminate the reaction, gently pipette to form a single-cell suspension, transfer to a 1.5 mL centrifuge tube, centrifuge at 300×g for 5 minutes, and discard the supernatant.

[0118] (3) Cell counting and plating: Add 1 mL of complete culture medium to each tube, pipette to mix, remove the cell counting plate, and aspirate 10 μL of cell suspension into the counting plate for counting. Counting method: Count the number of cells in the four large squares under a microscope, counting the cells on the top and not the bottom, and counting the cells on the left and not the right. The total number of cells in the four large squares is recorded as N, and the number of cells per mL of cell suspension = N / 4×10 4 .

[0119] (4) Cell plating: Adjust the HCC-827 cell density to 5×10 4 cells / mL, and the density of HEK-293T and Huh-7 cells was 10×10 4 The cells were seeded in 24-well plates with sterile cover slips in a final volume of 500 μL per well and cultured at 37°C for 12 h.

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

[0121] (6) Transfection operation: After the cells adhered to the wall, the original culture medium was discarded and the cells were washed twice with PBS. 500 μL of culture medium containing gradient concentrations of Cy3-G-siRNA was added to the experimental group, and the same amount of complete culture medium was added to the blank control group. The cells were cultured at 37°C for 24 hours.

[0122] (7) Sample fixation and staining: Fix with 4% paraformaldehyde at room temperature for 45 minutes, rinse with PBS three times (5 minutes / time, shaker 50 rpm); stain with DAPI solution in the dark for 10 minutes, rinse with PBS three times to remove residual dye.

[0123] (8) Preparation of microscopic samples: Pre-coat the slide with anti-fade mounting medium, invert the slide to attach the cells, seal the edges with neutral gum, and store in the dark.

[0124] (9) Confocal imaging.

[0125] like Figure 13 As shown in Figure A, the red fluorescence signal of Cy3 is mainly distributed in the cytoplasm and does not overlap with the cell nucleus labeled with DAPI (blue fluorescence), indicating that Cy3-G-siRNA successfully enters the cytoplasm.

[0126] Example 2

[0127] 1. G-siRNA transfection of A549, HCC827, HEK293t and Huh-7 cells to analyze the knockdown effect of GAPDH

[0128] (1) HCC827 cells and A549 cells were plated into 12-well plates, 1×10 5 HEK-293T and Huh-7 cells were plated at 1.5×10 5 The cells were cultured in a cell culture incubator for 12 hours.

[0129] (2) Preparation of ganoderic acid-siNC conjugate (G-siNC) and ganoderic acid-siGAPDH conjugate (G-siGAPDH) was carried out in the same manner as in Example 1. G-siNC and G-siGAPDH were added to complete culture medium and mixed evenly (final concentration of both was 20 μg / mL).

[0130] (3) Cells were treated differently according to the groups. 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, and the cells were treated for 36 hours.

[0131] After the culture was completed, RNA was extracted and analyzed. The results were as follows: Figure 13 As shown in Figure B, GAPDH expression remained unchanged in the G-siGAPDH-treated group compared to the G-siNC group in A549, HCC827, and Huh-7 cell lines. However, in the HEK293T cell line, GAPDH expression was significantly downregulated in the G-siGAPDH-treated group (0.815 ± 0.036) compared to the G-siNC group.

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

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

[0134] (2) Mouse sacrifice and tissue collection: Mice were sacrificed 48 hours later, and major tissues and organs, including heart, liver, spleen, lung, kidney, stomach, intestine, eye and muscle, were collected to provide samples for subsequent analysis. Figure 13 As shown in center C, in all tissues except liver tissue, there was no significant difference in GAPDH expression among all treatment groups (PBS, siGAPDH, and G-siGAPDH).

[0135] 3. Distribution of Cy3-siRNA / SF-NVs and Cy3-G-siRNA / SF-NVs Complexes in Vivo

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

[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 grade) were selected and treated in the following groups:

[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: The drug was administered by intraperitoneal injection (injection volume 200 μL / mouse). The mice were allowed to eat and drink freely after the injection. The corresponding tissues were taken for imaging 24 hours later. Figure 14 As shown in the figure, the fluorescence of siRNA was mainly enriched in the lungs and kidneys, and the fluorescence enrichment of Cy3-G-siRNA / SF-NVs group in the lungs and kidneys was significantly stronger than that of Cy3-siRNA / SF-NVs.

[0141] 4. Evaluation of the Lung Distribution of Cy3-G-siRNA / SF-NVs Complexes in Mice after Intraperitoneal Injection and Aerosol Inhalation

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

[0143] (2) Experimental animal treatment: 6-week-old C57 male mice (SPF grade) were selected and treated in the following groups:

[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) Dosage regimen: The drug was administered to mice via intraperitoneal injection and nebulized inhalation, and the mice were allowed to eat and drink freely after treatment.

[0147] (4) Immediately after taking the lungs, cryosections were performed and mouse lung epithelial cells were labeled with Cytokeratin 7 and observed under a laser confocal microscope. Figure 15 As shown, Cy3 fluorescence is 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) G-siRNA / SF-NVs were synthesized according to the method in "6. Preparation of Sargassum fusiformis lipid nanovesicle-siRNA complex" of Example 1, and then co-incubated with A549, HCC827 and BEAS-2B cells, respectively. The specific groups are as follows:

[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: they were co-incubated with the cells for 36 hours, and then the GAPDH levels in the cells were detected by qPCR.

[0151] like Figure 16 As shown in the figure, the expression of GAPDH was significantly downregulated in both siGAPDH / SF-NVs and G-siGAPDH / SF-NVs treatment groups, and the G-siGAPDH / SF-NVs treatment group had a better silencing effect on GAPDH.

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

[0153] (1) A549, HCC827, and BEAS-2B cells were seeded in DMEM medium (Ham's F-12K medium) containing 10% fetal bovine serum, with 7.5×10 cells per well in a 24-well plate. 4 After 12 hours of cell culture, different Cy3-labeled siRNAs or siRNA-coupled complexes were added (final concentration was 20 μg / mL).

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

[0155] (3) Lysosome staining: After 12, 24, 36, and 48 hours, remove the 24-well plate, discard the culture medium in the clean bench, and add PBS to wash the cells. Repeat once. Prepare the lysosome staining working solution (prepare immediately): add 0.1 μL LysoTracker™ Green DND-26 (10000×) to each milliliter of complete culture medium. After the preparation is complete, add 500 μL of lysosome staining working solution to each well and return to the cell culture incubator to continue incubation for 2 hours.

[0156] (4) Fixation and staining: Fix with 4% paraformaldehyde at room temperature for 45 minutes, rinse with PBS three times (5 minutes / time, shaker 50 rpm); stain with DAPI solution in the dark for 10 minutes, rinse with PBS three times to remove residual dye.

[0157] (5) Pre-coat the slide with anti-fading mounting medium, invert the slide to attach the cells, seal the edges with neutral gum, and store in the dark.

[0158] (6) Confocal imaging such as Figure 17 As shown, both G-siRNA and G-siRNA / SF-NVs can enter cells. G-siRNA alone can co-localize with lysosomes well, while G-siRNA / SF-NVs can escape capture by lysosomes after entering cells.

[0159] Example 3

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

[0161] 1. Construction of a Bleomycin-induced Pulmonary Fibrosis Model in Mice

[0162] SPF C57 / B6 mice (6-8 weeks old, male) were acclimated for 1 week and then randomly divided into seven groups: NC, NC + G-siTGF-β1 / SF-NVs, BLM, BLM + G-siTGF-β1, BLM + SF-NVs, BLM + G-siNC / SF-NVs, and BLM + G-siTGF-β1 / SF-NVs, with 5 mice in each group. In the BLM model group, mice were anesthetized and then administered bleomycin using a nebulizer at a 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 each group of mice:

[0166] NC group: PBS was injected intraperitoneally every two days;

[0167] NC+G-siTGF-β1 / SF-NVs: G-siTGF-β1 / SF-NVs were injected intraperitoneally every two days;

[0168] BLM group: PBS was injected intraperitoneally every two days;

[0169] BLM+G-siTGF-β1 group: G-siTGF-β1 was injected intraperitoneally every two days after BLM model establishment;

[0170] BLM+SF-NVs group: SF-NVs were injected intraperitoneally every two days after BLM model establishment;

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

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

[0173] The above siRNA dosage is 2 mg / kg. For the specific process, see Figure 18 .

[0174] 4.Indicator detection

[0175] 4.1 Observation of mouse physical signs and monitoring of physiological indicators

[0176] During treatment, mice were weighed and their activity levels were checked every two days. At the end of the experiment, all mice were sacrificed, their lungs photographed, and their bronchoalveolar lavage fluid (BALF) collected. Lung wet weight was recorded to calculate the lung-to-body ratio. Cardiac blood and tissues including heart, liver, spleen, lung, kidney, stomach, and small intestine were collected using a 1mL syringe for subsequent experiments. Serum and BALF were stored at -80°C for subsequent experiments.

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

[0178] 4.2 Lung tissue total RNA, reverse transcription, and qPCR

[0179] Weigh approximately 0.05 g of lung tissue and place it in an enzyme-free EP tube. Add 500 μL of RNAiso Plus lysis buffer and two magnetic beads, then place the tube in a biological sample homogenizer for tissue grinding until the tissue chunks disappear. Then, lyse the tube on ice for 10 minutes. Then, add chloroform and isopropanol precipitation, ethanol washing, and DEPC water dissolution to obtain total RNA, which was then reverse transcribed and detected by qPCR.

[0180] The 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 elevated TGF-β1, Collagen 1, Fibronectin and α-SMA were significantly reduced after siTGF-β1 / SF-NVs intervention ( Figure 21 ).

[0181] 4.3 Immunoblotting

[0182] Total protein was extracted from mouse lung homogenate and immunoblotting was performed. The results showed that G-siTGF-β1 / SF-NVs could effectively inhibit the abnormal expression of key factors in the 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 BALF

[0184] (1) Thaw the mouse bronchoalveolar lavage fluid stored in a -80°C freezer on ice. Centrifuge the lavage fluid at 1000 × g for 10 minutes at 4°C to separate the supernatant and cell pellet. Reserve the supernatant for total protein detection.

[0185] (2) Use BCA method to measure the total protein of bronchoalveolar lavage fluid. Figure 23 As shown in Figure A, the protein concentration in the lung lavage fluid increased significantly after BLM treatment. G-siTGF-β1 / SF-NVs can effectively protect the alveolar epithelial barrier of mice and inhibit the exudation of plasma proteins, thereby significantly reducing the total protein content in BALF.

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

[0187] Thaw mouse bronchoalveolar lavage fluid stored at -80°C on ice. Centrifuge the lavage fluid at 1000 × g for 10 minutes at 4°C to separate the supernatant and cell pellet. Assay the supernatant for MPO activity using an MPO assay kit.

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

[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.

2. The preparation method according to claim 1, characterized in that The water-soluble triterpenoid compound-siRNA conjugate is prepared by adopting EDC-NHS coupling reaction.

3. The preparation method according to claim 1, characterized in that The triterpenoid compound is ganoderic acid.

4. The preparation method according to claim 1, characterized in that The Sargassum fusiformis lipid nanovesicles are extracted by utilizing a differential centrifugation method.

5. 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.

6. 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.

7. A triterpene compound-siRNA / lipid nanovesicle complex prepared according to the preparation method according to any one of claims 1 to 6.

8. Use of the triterpene compound-siRNA / lipid nanovesicle complex according to claim 7 in the preparation of a drug for treating pulmonary fibrosis.

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

10. The drug according to claim 9, characterized in that The drug also includes pharmaceutically acceptable excipients.

Images