Preparation method of hijiki-derived lipid nanovesicles and its products and applications

Abstract

The present invention discloses a preparation method of Sargassum fusiformis-derived lipid nanovesicles, as well as its products and applications, and belongs to the field of cell biology technology. Sargassum fusiformis-derived lipid nanovesicles are extracted from Sargassum fusiformis by combining differential centrifugation, ultracentrifugation and gradient density centrifugation. The Sargassum fusiformis-derived lipid nanovesicles can reduce the release of LPS-induced inflammatory cytokines IL-6, IL-1β, and TNF-α, and can load siRNA into cells. They can be used in the preparation of anti-inflammatory drugs and can also be used as drug carriers, providing a new strategy for the development of anti-inflammatory drugs and drug carriers.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and in particular to a preparation method of Sargassum fusiformis-derived lipid nanovesicles, and a product and application thereof. Background Art

[0002] Inflammation is a defensive response of living tissues with vascular systems to damaging factors. It involves three aspects: damage caused by inflammatory factors, the body's defensive response, and tissue damage recovery. A moderate inflammatory response is an immune response that protects the body. Excessive inflammation can lead to immune damage and induce various diseases, such as rheumatoid arthritis, systemic lupus erythematosus, autoimmune hemolytic anemia, acute gouty arthritis, atherosclerosis, and diabetic nephropathy. A large number of inflammatory-related factors (such as TNF-α, IL-1β, IL-6, iNOS, and COX-2) are detected in the blood of diseased organisms. These factors infiltrate the body, triggering various adverse reactions and seriously threatening the body's health. Therefore, the prevention and adjuvant treatment of inflammation are particularly important.

[0003] In the research of treating and preventing inflammation, many anti-inflammatory drugs have been developed, such as adrenocortical hormone preparations, cyclophosphamide, methotrexate, etc., but the therapeutic effects are unsatisfactory. Therefore, it is very necessary to develop new ways or new drugs to treat inflammation.

[0004] Small interfering RNA (siRNA) is an initiator of RNA interference, silencing its complementary target mRNA. This approach holds important implications for gene regulation and disease treatment. As a therapeutic agent, siRNA must overcome the vascular barrier, achieve endocytosis and escape into lysosomes, while also avoiding degradation by nucleases. Therefore, designing suitable vectors to facilitate successful siRNA delivery into cells and its efficacy is a key goal in the development of siRNA drugs.

[0005] In summary, if a substance can be developed that has both anti-inflammatory effects, can be used to prepare anti-inflammatory drugs, and can serve as a carrier of siRNA, it will have very broad application prospects. Summary of the Invention

[0006] The purpose of the present invention is to provide a preparation method of lipid nanovesicles derived from Sargassum fusiformis, and its products and applications, so as to solve the above-mentioned problems existing in the prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] One of the technical solutions of the present invention is a method for preparing lipid nanovesicles derived from Sargassum fusiformis, comprising the following steps:

[0009] S1. Crushing and grinding: Wash the Sargassum fusiformis, mix the washed Sargassum fusiformis with PBS, and crush and grind;

[0010] S2, differential centrifugation: subjecting the crushed and ground grinding liquid to differential centrifugation to obtain a supernatant after differential centrifugation;

[0011] S3. Ultracentrifugation: Ultracentrifuge the supernatant after differential centrifugation at 4°C, 100,000-200,000 g for 60-120 min. After ultracentrifugation, discard the supernatant and retain the pellet.

[0012] S4. Gradient density centrifugation: The precipitate after ultracentrifugation in step S3 was resuspended in PBS and gently added to the top layer of a sucrose gradient solution with a concentration gradient of 60 wt.%, 45 wt.%, 30 wt.%, and 8 wt.% from bottom to top. The solution was then ultracentrifuged at 4°C, 100,000-200,000 g for 60-120 min. After centrifugation, the visible band between the 8 wt.% and 30 wt.% sucrose layers was aspirated, resuspended in PBS, and then centrifuged again at 4°C, 100,000-200,000 g for 60-120 min to obtain lipid nanovesicles derived from Sargassum fusiformis.

[0013] Furthermore, in step S1, the mass ratio of the sea lettuce to PBS is 1:5, the crushing and grinding is performed 3-10 times, each time for 1-5 minutes, and the crushing and grinding are refrigerated for 5-60 minutes between the two times (heat is generated during the grinding process); the crushing and grinding speed is 15000-28000 rpm, and the refrigeration temperature is 4°C.

[0014] Furthermore, the specific operation of the differential centrifugation in step S2 is: first centrifuge at 1000-2500g for 10-30min, discard the precipitate, retain the supernatant, then centrifuge at 3000-8000g for 10-30min, discard the precipitate, retain the supernatant, and finally centrifuge at 10000-30000g for 30-60min, discard the precipitate, and obtain the supernatant after differential centrifugation.

[0015] Furthermore, the method for preparing the sucrose gradient solution with a concentration gradient of 60 wt.%, 45 wt.%, 30 wt.%, and 8 wt.% from bottom to top in step S3 is as follows: first prepare sucrose solutions with concentrations of 8 wt.%, 30 wt.%, 45 wt.%, and 60 wt.%, respectively, then prepare a clean ultracentrifuge tube, and add 8 ml of each of 60 wt.%, 45 wt.%, 30 wt.%, and 8 wt.% sucrose solutions in sequence to form a gradient to obtain the sucrose gradient solution.

[0016] Furthermore, the method further includes S5, storage of the Sargassum fusiformis-derived lipid nanovesicles: resuspending the Sargassum fusiformis-derived lipid nanovesicle precipitate obtained in step S4 with PBS, filtering with a 220 nm filter membrane to obtain a Sargassum fusiformis-derived lipid nanovesicle suspension, and storing the suspension at -80°C.

[0017] The second technical solution of the present invention: a lipid nanovesicle derived from Sargassum fusiformis prepared according to the above method.

[0018] The third technical solution of the present invention: application of the above-mentioned Sargassum fusiformis-derived nanovesicles in the preparation of anti-inflammatory drugs.

[0019] Furthermore, the inflammation is LPS-induced inflammation.

[0020] Technical solution 4 of the present invention: Application of the above-mentioned Sargassum fusiformis-derived nanovesicles as drug carriers.

[0021] Furthermore, the drug is a siRNA drug.

[0022] The Sargassum fusiformis-derived nanovesicles are used as nucleic acid carriers to carry siRNA into cells and exert the effect of inhibiting gene levels.

[0023] The present invention discloses the following technical effects:

[0024] (1) The present invention discloses a method for extracting Sargassum fusiformis-derived lipid nanovesicles from Sargassum fusiformis by combining differential centrifugation, ultracentrifugation, and gradient density centrifugation. The Sargassum fusiformis-derived lipid nanovesicles extracted by this method can reduce the release of LPS-induced inflammatory cytokines IL-6, IL-1β, and TNF-α, and can load siRNA into cells. They can be used in the preparation of anti-inflammatory drugs and can also be used as drug carriers, providing a new strategy for the development of anti-inflammatory drugs and drug carriers.

[0025] (2) The present invention extracts nanoparticles from Sargassum fusiformis by differential centrifugation, ultracentrifugation combined with gradient density centrifugation (1000-2500g in differential centrifugation removes larger residues, 3000-8000g removes medium particles, and 10000-30000g removes cell debris. Ultracentrifugation can separate vesicles, and sucrose gradient centrifugation is used for purification). It is characterized by TEM and nanoparticle size analysis and found to be a nanovesicle-like structure. The nanovesicle structure can be taken up by most cells, including inflammatory cells, and plays an inhibitory role in inflammation. In addition, when used as a nucleic acid carrier, it can successfully carry siRNA into cells and play an inhibitory role at the gene level. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] 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.

[0027] Figure 1 These are pictures of the extraction process of lipid nanovesicles from Sargassum fusiformis and the characterization after extraction, where: A is the sucrose density gradient centrifugation during the extraction process of lipid nanovesicles from Sargassum fusiformis; B is the TEM image of lipid nanovesicles from Sargassum fusiformis; C is the particle size analysis diagram of lipid nanovesicles from Sargassum fusiformis.

[0028] Figure 2 These are the results of the inhibition of cellular inflammation by lipid nanovesicles derived from Sargassum fusiformis, where: A is a laser confocal image of Kupffer cells taking up lipid nanovesicles derived from Sargassum fusiformis; B is the relative mRNA expression levels of IL-6, IL-1β, and TNF-α in Kupffer cells detected by RT-qPCR; C is the relative mRNA expression levels of IL-6, IL-1β, and TNF-α in RAW cells detected by RT-qPCR, * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001.

[0029] Figure 3 Figure 3 shows the situation of siRNA carried by lipid nanovesicles derived from Sargassum fusiformis. A is a laser confocal microscopy image of siRNA-loaded lipid nanovesicles from Sargassum fusiformis entering A549 cells; B is the relative mRNA expression level of lnc070974 in A549 cells detected by RT-qPCR. * indicates P < 0.05. DETAILED DESCRIPTION

[0030] 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.

[0031] 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. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] 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 materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the present invention. The present description and examples are intended to be illustrative only.

[0034] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0035] The harvest period of the Sargassum fusiformis used in the following examples is from March to May. Studies have shown that the anti-inflammatory effects and drug carrier effects of lipid nanovesicles extracted from Sargassum fusiformis raw materials in different periods are basically the same.

[0036] Example 1 Extraction of Sargassum fusiformis-derived lipid nanovesicles

[0037] (1) Crushing and grinding: Wash and drain the seaweed, weigh 120 g of the washed and drained seaweed, then add the seaweed to a Blendtec blender and add 1×PBS (0.01 M) 5 times the mass of the seaweed, crush and grind it, grind it at 20,000 rpm 5 times, each time for 1 min, and place it in a cold storage at 4°C for 10 min between the two crushing and grinding.

[0038] (2) Differential centrifugation: Pour the grinding solution after 5 times of crushing and grinding into a clean 50ml Eppendorf tube, and then perform differential centrifugation: centrifugation at 1000g for 10 minutes (discard the precipitate and keep the supernatant), centrifugation at 3000g for 20 minutes (discard the precipitate and keep the supernatant), and centrifugation at 10000g for 40 minutes (discard the precipitate and keep the supernatant).

[0039] (3) Ultracentrifugation: The final supernatant was accurately balanced in an ultracentrifuge tube and centrifuged at 4°C and 150,000 g for 90 min. During this time, a sucrose gradient solution was prepared using 1× PBS as the solvent to prepare sucrose solutions with concentrations of 8 wt.%, 30 wt.%, 45 wt.%, and 60 wt.%. Then, 8 ml of each of the 60 wt.%, 45 wt.%, 30 wt.%, and 8 wt.% sucrose solutions were added to a clean ultracentrifuge tube to form a gradient.

[0040] (4) Gradient density centrifugation: After 90 minutes of ultracentrifugation, resuspend the pellet in 2 ml of 1×PBS and gently add it to the top layer of the sucrose gradient solution. Centrifuge at 4°C, 150,000 g for 120 minutes. After centrifugation, three bands will appear (between 8 wt.% and 30 wt.%, between 30 wt.% and 45 wt.%, and between 45 wt.% and 60 wt.%). Carefully aspirate the visible band between the 8 wt.% and 30 wt.% sucrose layers into another clean ultracentrifuge tube (bands as shown in Figure 2). Figure 1 The suspension was resuspended in 1 ml of 1× PBS and accurately balanced, and then centrifuged again at 150,000 g for 90 min at 4°C to remove residual sucrose.

[0041] (5) After centrifugation, the supernatant was discarded and the residual liquid on the wall of the supernatant was wiped dry with a paper towel. The precipitate obtained was the desired Sargassum-derived lipid nanovesicles.

[0042] (6) The precipitate was resuspended in 10 ml of 1× PBS and filtered through a 220 nm filter membrane to obtain a suspension of lipid nanovesicles derived from Sargassum fusiformis. The protein concentration was determined to be 3 μg / μl using a BCA assay kit and the suspension was stored at −80°C.

[0043] Effect verification

[0044] 1. Characterization of Sargassum fusiforme-derived lipid nanovesicles

[0045] (1) The lipid nanovesicle suspension derived from Sargassum fusiformis obtained in step (6) of Example 1 was negatively stained with phosphomolybdic acid and observed under an electron microscope. The TEM image was as follows: Figure 1 As shown in B, the results showed that the extract was a nanoscale vesicle-like structure.

[0046] (2) 50 μl (protein content 150 μg) of the lipid nanovesicle suspension derived from Sargassum fusiformis (prepared in step (6) of Example 1) was tested for its particle size (e.g. Figure 1 The results showed that the average particle size was 122.5±51.8 nm.

[0047] 2. Kupffer Cell Uptake of Sargassum Fusiforme Lipid Nanovesicles

[0048] 1. PKH26 fluorescent dye labeling of lipid nanovesicles derived from Sargassum fusiformis

[0049] (1) 1 μl of PKH26 was mixed with 20 μg of Sargassum fusiformis-derived lipid nanovesicle suspension and allowed to stand at room temperature for 10 min to allow the dye to bind to the vesicles. 37.5 ml of 1× PBS was added to the 1 cm opening of the centrifuge tube. Unbound PKH26 dye was removed by centrifugation at 150,000 g for 90 min. The final pellet was resuspended in 50 μl of 1× PBS to obtain the dyed lipid nanovesicle suspension.

[0050] (2) Place a slide in a 24-well plate, plate 50,000 Kupffer cells per well, and place the plate in an incubator to allow the cells to adhere for 6 hours.

[0051] (3) The stained Sargassum fusiformis-derived lipid nanovesicle suspension was added to the attached cells and incubated for 24 h.

[0052] (4) Collect the sample, aspirate the culture medium, wash twice with 1×PBS, add 300 μl of 4% paraformaldehyde, and fix for 0.5 h.

[0053] (5) Remove formaldehyde by aspiration, wash twice with 1× PBS, add 300 μl of DAPI staining solution, stain for 10 min, recover DAPI, and wash twice with 1× PBS.

[0054] (6) Prepare a glass slide, add 5 μl of anti-fluorescence quenching sealing solution, take out the slide, place it cell side down, and place it on the area where the sealing solution was added.

[0055] (7) Confocal photography (such as Figure 2 The results showed that the lipid nanovesicles derived from Sargassum fusiformis could be taken up by Kupffer cells.

[0056] Effects of Sargassum fusiforme-derived lipid nanovesicles on inflammatory factors in Kupffer and RAW cells

[0057] (1) Grouping: NC, LPS stimulation group, LPS stimulation + Sargassum fusiformis lipid-derived nanovesicles (C1, C2, C3). Cells were plated according to the grouping, with 50,000 cells plated per well in a 24-well plate.

[0058] (2) After 6 hours of cell attachment, 30 μg, 15 μg, and 7.5 μg of the Sargassum-derived lipid nanovesicle suspension (prepared in step (6) of Example 1) were added to the corresponding wells, respectively, from high to low concentration. After incubation with the cells for 18 hours, LPS (10 μg / ml) was added to the wells of the LPS stimulation group and the LPS stimulation + Sargassum lipid nanovesicle group.

[0059] (3) After 6 h of stimulation, observe that there is no cell death, collect samples to extract RNA, aspirate the cell culture medium, wash twice with 1× PBS, add 300 μl of Trizol reagent to each well, and pipette the cells into the corresponding EP tube.

[0060] (4) The precipitate was completely lysed on ice for 5 min, 60 μl of chloroform was added, and the mixture was immediately vortexed for 15 s to mix thoroughly.

[0061] (5) Precipitate on ice for 5 min, then centrifuge at 4°C, 14,000 rpm, for 15 min.

[0062] (6) Pre-cool isopropanol and prepare a corresponding number of 1.5 ml enzyme-free EP tubes and label them.

[0063] (7) Use a pipette to transfer an appropriate volume of supernatant (120 μl) to the enzyme-free EP tube labeled above.

[0064] (8) Add an equal volume of isopropanol to precipitate RNA, mix thoroughly by inverting, and precipitate on ice for 10 min.

[0065] (9) Centrifuge at 14000 rpm at 4°C for 10 min and carefully discard the supernatant;

[0066] (10) Add 75 wt.% ethanol (prepared with DEPC water) to wash the precipitate, centrifuge at 14000 rpm at 4°C for 5 min, repeat the wash twice, carefully discard the supernatant, centrifuge for 1 minute, and dry the precipitate at 42°C for about 3 min;

[0067] (11) Add 10 μl of DEPC water to dissolve RNA;

[0068] (12) The reverse transcription and qPCR steps were based on the Nanjing Novozymes kit (NO: R223-01) as follows:

[0069] I. Reverse transcription

[0070] ①Removal of genomic DNA

[0071] Table 1 Reagents for removing genomic DNA

[0072]

[0073] After gently pipetting to mix, place the PCR tube into the PCR instrument and set the program to run at 42°C for 2 minutes.

[0074] ②Prepare reverse transcription reaction system

[0075] Table 2 Reverse transcription reaction system

[0076]

[0077] Use a pipette to gently pipette to mix the mixture, then briefly centrifuge the PCR tube at 2000 rpm for 10 seconds, and place it in the PCR instrument to proceed to the next step.

[0078] ③Reverse transcription reaction

[0079] Table 3 Reverse transcription reaction conditions

[0080]

[0081] After the PCR process was completed, the cDNA samples were stored in a -20°C refrigerator.

[0082] II. Real-time quantitative PCR (qRT-PCR)

[0083] Refer to the Nanjing Novozymes kit (NO: Q712-02) and the steps are as follows:

[0084] Table 4 Primer sequences

[0085]

[0086] ①Prepare the reaction system

[0087] Table 5 Reaction system

[0088]

[0089] Mark the 96-well plate and add 20 μl of the above mixture to each well in sequence. Place the PCR plate in a centrifuge, level it, and centrifuge briefly to collect the reaction solution.

[0090] ②PCR reaction (operated on CFX96 Real-Time PCR System)

[0091] Table 6 PCR reaction program

[0092]

[0093] The relative expression level of the gene after the reaction was calculated according to 2 -△△Ct ( △ Ct value = Ct 目的 -Ct 内参 , △△ Ct value = △ Ct 实验组 - △ Ct 对照组 ) were calculated to obtain the relative expression levels of genes, and GAPDH gene was used as the control gene to detect the mRNA expression of IL-6, IL-1β, and TNF-α in cells (the results are shown in Figure 2RT-qPCR results showed that the relative mRNA expression levels of IL-6, IL-1β, and TNF-α in Kupffer and RAW cells in the LPS-stimulated + Sargassum-derived lipid nanovesicles group were significantly lower than those in the LPS-only stimulation group (P < 0.05). This suggests that Sargassum-derived lipid nanovesicles have an anti-inflammatory effect.

[0094] 4. Cellular Uptake Experiment of siRNA Loaded in Sargassum Fusiformis-Derived Lipid Nanovesicles

[0095] (1) 1 μl of PKH67 was mixed with 20 μg of Sargassum fusiformis-derived lipid nanovesicle suspension and incubated at room temperature for 10 min. Free dye was removed by incubation at 150,000 g at 4°C for 1.5 h, and the precipitate was resuspended in 40 μl of 1× PBS to obtain a Sargassum fusiformis-derived lipid nanovesicle suspension stained with PKH67.

[0096] (2) Place a slide in a 24-well plate, plate 100,000 A549 cells per well, and place the plate in an incubator to allow the cells to adhere for 6 hours.

[0097] (3) Before sample addition, a suspension of PKH67-stained Sargassum-derived lipid nanovesicles (40 μg) was incubated with 2.5 μl of Cy3-labeled siRNA at room temperature for 2 h.

[0098] (4) The plated cells were divided into NC, siRNA alone, and Sargassum fusiforme-derived lipid nanovesicles + siRNA groups, and samples were added according to the groups.

[0099] (5) Collect the sample, remove the culture medium, wash twice with 1×PBS, add 300 μl of 4% paraformaldehyde, and fix for half an hour.

[0100] (6) Remove formaldehyde, wash twice with 1× PBS, add 300 μl of DAPI staining solution, stain for 10 min, recover DAPI, and wash twice with 1× PBS.

[0101] (7) Prepare a glass slide, add 5 μl of anti-fluorescence quenching sealing solution, take out the slide, place it cell side down, and place it on the area where the sealing solution was added.

[0102] (8) Confocal photography (such as Figure 3 The results showed that the lipid nanovesicles derived from Sargassum fusiformis can carry siRNA into A549 cells.

[0103] 5. Cellular Interference Experiment with siRNA-loaded Lipid Nanovesicles from Sargassum Fusiformis

[0104] (1) 40 μg of Sargassum fusiforme-derived lipid nanovesicle suspension was incubated with 2.5 μl of siRNA (interfering with the lnc070974 gene) at room temperature for 2 h.

[0105] (2) Grouping: NC, RNAiMAX+siRNA group, Sargassum-derived lipid nanovesicles+siRNA group, siRNA alone group, and Sargassum-derived lipid nanovesicles alone group. Cells were plated according to the grouping, with 100,000 cells plated per well in a 24-well plate.

[0106] (3) After 6 hours, the cells adhered to the wall and samples were added according to the groups. In the RNAiMAX+siRNA group, 1 μl of RNAiMAX+2.5 μl of siRNA was added to the cells. In the Sargassum-derived lipid nanovesicle+siRNA group, the sample prepared in the first step was added to the cells. In the siRNA group alone, 2.5 μl of siRNA was added to the cells. In the Sargassum-derived lipid nanovesicle group alone, 40 μg of Sargassum-derived lipid nanovesicle suspension was added to the cells.

[0107] (4) After 24 hours of co-incubation, collect samples for RNA extraction. Aspirate the cell culture medium, wash twice with 1× PBS, and add 300 μl of Trizol reagent to each well. Pipet the cells into the corresponding EP tube.

[0108] (5) The precipitate was completely lysed on ice for 5 min, 60 μl of chloroform was added, and the mixture was immediately vortexed for 15 s to mix thoroughly.

[0109] (6) Precipitate on ice for 5 min, then centrifuge at 4°C, 14,000 rpm, for 15 min.

[0110] (7) Pre-cool isopropanol and prepare a corresponding number of 1.5 ml enzyme-free EP tubes and label them.

[0111] (8) Use a pipette to transfer an appropriate volume of supernatant (120 μl) into the enzyme-free EP tube labeled above.

[0112] (9) Add an equal volume of isopropanol to precipitate the RNA. Mix by inverting the tube and let it precipitate on ice for 10 minutes.

[0113] (10) Centrifuge at 14000 rpm at 4°C for 30 min and carefully discard the supernatant.

[0114] (11) Add 75 wt.% ethanol (prepared with DEPC water) to wash the precipitate, centrifuge at 14000 rpm and 4°C for 5 min, repeat the washing twice, carefully discard the supernatant, centrifuge for 1 minute, and dry the precipitate at 42°C for about 3 min.

[0115] (12) Add 10 μl of DEPC water to dissolve RNA.

[0116] (13) The reverse transcription and qPCR steps were based on the Nanjing Novozymes kit (NO: R223-01) as follows:

[0117] I. Reverse transcription

[0118] ①Removal of genomic DNA

[0119] Table 7 Reagents for removing genomic DNA

[0120]

[0121] After gently pipetting to mix, place the PCR tube into the PCR instrument and set the program to run at 42°C for 2 minutes.

[0122] ②Prepare reverse transcription reaction system

[0123] Table 8 Reverse transcription reaction system

[0124]

[0125] Use a pipette to gently pipette to mix the mixture, then briefly centrifuge the PCR tube at 2000 rpm for 10 seconds, and place it in the PCR instrument to proceed to the next step.

[0126] ③Reverse transcription reaction

[0127] Table 9 Reverse transcription reaction conditions

[0128]

[0129] After the PCR process was completed, the cDNA samples were stored in a -20°C refrigerator.

[0130] II. Real-time quantitative PCR (qRT-PCR)

[0131] Refer to the Nanjing Novozymes kit (NO: Q712-02) and the steps are as follows:

[0132] Table 10 Primer sequences

[0133]

[0134] ①Prepare the reaction system

[0135] Table 11 Reaction system

[0136]

[0137] Mark the wells of a 96-well PCR plate and add 20 μl of the above mixture to each well in sequence. Place the PCR plate in a centrifuge, level it, and briefly centrifuge to collect the reaction solution.

[0138] ②PCR reaction (performed on CFX96 Real-Time PCR System)

[0139] Table 12 PCR reaction program

[0140]

[0141]

[0142] The relative expression level of the gene after the reaction was calculated according to 2 -△△Ct ( △ Ct value = Ct 目的 -Ct 内参 , △△ Ct value = △ Ct 实验组 - △ Ct 对照组 ) were calculated to obtain the relative expression level of the gene, and GAPDH gene was used as the control gene to detect the mRNA expression of lnc070974 in cells (the results are shown in Figure 3 (B) RT-qPCR results showed that the relative mRNA expression of lnc070974 in A549 cells in the Sargassum-derived lipid nanovesicles + siRNA group was significantly lower than that in the Sargassum-derived lipid nanovesicles alone group (P < 0.05). The relative mRNA expression of lnc070974 in A549 cells in the siRNA alone group was not significantly different from that in NC (P > 0.05). This suggests that Sargassum-derived lipid nanovesicles can serve as carriers for siRNA entry into cells and exert their effects.

[0143] 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 drug for treating LPS-induced inflammation by using a lipid nanovesicle derived from Sargassum fusiformis, characterized in that: The method for preparing the hijiki-derived lipid nanovesicles comprises the following steps: S1. Crushing and grinding: Wash the Sargassum fusiformis, mix the washed Sargassum fusiformis with PBS, and crush and grind; S2, differential centrifugation: subjecting the crushed and ground grinding liquid to differential centrifugation to obtain a supernatant after differential centrifugation; S3. Ultracentrifugation: Ultracentrifuge the supernatant after differential centrifugation at 4°C, 100,000-200,000 g for 60-120 min. After ultracentrifugation, discard the supernatant and retain the pellet. S4, gradient density centrifugation: The precipitate after ultracentrifugation in step S3 was resuspended in PBS and gently added to the top layer of a sucrose gradient solution with a concentration gradient of 60 wt.%, 45 wt.%, 30 wt.%, and 8 wt.% from bottom to top, followed by ultracentrifugation at 4°C, 100,000-200,000 g for 60-120 min. After centrifugation, the visible band between the 8 wt.% and 30 wt.% sucrose layers was aspirated, resuspended in PBS, and then centrifuged again at 4°C, 100,000-200,000 g for 60-120 min to obtain a precipitate of lipid nanovesicles derived from Sargassum fusiformis; S5. Preservation of Sargassum fusiformis-derived lipid nanovesicles: The Sargassum fusiformis-derived lipid nanovesicle precipitate obtained in step S4 was resuspended in PBS, filtered through a 220 nm filter membrane to obtain a Sargassum fusiformis-derived lipid nanovesicle suspension, and stored at -80°C.

2. The use according to claim 1, characterized in that In step S1, the mass ratio of the seaweed to PBS is 1:5, the crushing and grinding is performed 3-10 times, each time for 1-5 minutes, and refrigeration is performed for 5-60 minutes between two crushing and grinding; the crushing and grinding speed is 15000-28000 rpm, and the refrigeration temperature is 4°C.

3. The use according to claim 1, characterized in that The specific operation of the differential centrifugation in step S2 is: first centrifuge at 1000-2500g for 10-30min, discard the precipitate, and retain the supernatant, then centrifuge at 3000-8000g for 10-30min, discard the precipitate, and retain the supernatant, and finally centrifuge at 10000-30000g for 30-60min, discard the precipitate, and obtain the supernatant after differential centrifugation.

4. The use according to claim 1, characterized in that The method for preparing the sucrose gradient solution with a concentration gradient of 60 wt.%, 45 wt.%, 30 wt.%, and 8 wt.% from bottom to top in step S4 is as follows: first, sucrose solutions with concentrations of 8 wt.%, 30 wt.%, 45 wt.%, and 60 wt.% are prepared, and then, a clean ultracentrifuge tube is prepared, and 8 ml of each of the 60 wt.%, 45 wt.%, 30 wt.%, and 8 wt.% sucrose solutions are added in sequence to form a gradient to obtain the sucrose gradient solution.

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