A lipid-gold nanoparticle complex for the treatment of sepsis and its preparation method

Abstract

This invention discloses a lipid-gold nanoparticle complex for treating sepsis. The preparation method includes the following steps: Sodium dodecyl sulfate (SDS) is added to a dispersion of AuNPs and dispersed evenly; β-MEA is added, and stirring is continued for 4–10 hours. SDS is then removed by filtration, and the remaining liquid is concentrated by centrifugation. The concentrate is purified by dialysis. After purification, the pH is adjusted to obtain a cationic AuNPs solution; Claudin-5 overexpression plasmid is added, and the reaction is stirred to obtain an AC solution; soybean lecithin, cholesterol, and DOTAP are added to a round-bottom flask, and methanol solution is added. The mixture is heated to dissolve the liposomes, and the methanol is removed by cooling to obtain liposomes; the AC solution is added to the round-bottom flask, and the mixture is shaken to hydrate. The liposomes are then ultrasonically broken up, purified by centrifugation, and filtered to obtain LAC. A "post-interpolation method" is used to add REDVC-PEG. 2000 DSPE is modified onto the surface of the liposome complex to obtain the RLAC complex that targets vascular endothelial cells.

Description

Technical Field

[0001] This invention belongs to the field of sepsis treatment drug technology, specifically relating to a lipid-gold nanoparticle complex for treating sepsis and its preparation method. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Sepsis is a disordered response of the host to infection, resulting in life-threatening organ dysfunction. It is a major contributor to the global disease burden, characterized by long hospital stays, high treatment costs, and high mortality rates. Despite recent advancements in intensive care, including antibiotic therapy and supportive care, significant knowledge gaps remain regarding many aspects, including the unclear underlying mechanisms of sepsis pathogenesis. In-depth research into the pathogenesis of sepsis and the search for new prevention and treatment approaches have become urgent tasks in the international field of critical care medicine.

[0004] Clinically, sepsis patients often present with progressive subcutaneous edema and body cavity effusions, indicating a general increase in vascular permeability. Increased vascular permeability allows water and large protein molecules to enter tissues uncontrollably, causing tissue edema and further leading to tissue hypoxia. Simultaneously, elevated interstitial pressure impairs microvascular perfusion, potentially causing organ dysfunction. Loss of endothelial integrity is closely associated with sepsis-related organ damage and death. Therefore, improving endothelial barrier function and maintaining vascular endothelial integrity may become a novel therapeutic target for sepsis, possessing significant clinical implications.

[0005] The vascular endothelial barrier depends on the integrity of the connections between adjacent vascular endothelial cells. Tight junctions (TJs) are one of the main types of connections between vascular endothelial cells. Claudin-5 protein is a major component of tight junctions and plays a crucial role in maintaining endothelial cell permeability. Studies have shown that upregulating Claudin-5 protein expression can repair damaged tight junctions and effectively reduce vascular permeability.

[0006] Gene therapy refers to the use of gene vectors to deliver target genes into target cells, thereby treating many currently intractable diseases, such as cancer, by silencing pathogenic genes or increasing the expression of therapeutic genes. Therefore, directly upregulating the expression of Claudin-5 protein through gene therapy is a potential new strategy for repairing vascular endothelial cell junction damage. However, nucleic acids are degraded by nucleases in vivo, resulting in low nucleic acid concentrations reaching the target site and less than ideal therapeutic effects, which greatly limits the clinical application of nucleic acid drugs. To address the problem of nucleic acid molecules being easily degraded by nucleases in vivo, scientists have designed a series of gene vectors to protect nucleic acid molecules and improve delivery efficiency. Currently, gene vectors used in gene therapy are mainly divided into two categories: viral vectors and non-viral vectors. Viral vectors have high transfection efficiency, but they are immunogenic and easily trigger immune responses after entering the body, greatly limiting their clinical application. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a lipid-gold nanoparticle complex RLAC for the treatment of sepsis and its preparation method. This invention employs gene therapy, increasing Claudin-5 expression levels by delivering a Claudin-5 overexpression plasmid into endothelial cells, thereby rebuilding the vascular endothelial barrier, reducing vascular permeability, and ultimately treating sepsis.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution:

[0009] A lipid-gold nanoparticle complex for the treatment of sepsis, the preparation method of which includes the following steps:

[0010] Sodium dodecyl sulfate (SDS) was added to the dispersion of gold nanoparticles (AuNPs) and dispersed evenly.

[0011] β-mercaptoethylamine (β-MEA) was added to the solution, where the final concentrations of AuNPs, SDS and β-MEA were (1-4 nM), (12-18 mM) and (55-60 mM) respectively. After stirring for 4-10 h, SDS was removed by filtration. The remaining solution was concentrated by centrifugation at 12000-18000 rpm / min. The concentrate was purified by dialysis. After purification, the pH was adjusted to 4-5 to obtain a positively charged AuNPs solution.

[0012] Add the Claudin-5 overexpression plasmid to the cationic AuNPs solution and stir overnight to obtain the AuNP-Claudin-5 complex (AC) solution;

[0013] Soybean lecithin, cholesterol, and 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride DOTAP were added to a flask in a mass ratio of 0.2–0.6:0.05–0.3:0.01–0.09, along with 150–200 mL of methanol solution. The mixture was heated to dissolve the liposomes, then cooled to room temperature. The methanol was removed by rotary evaporation, resulting in a liposome film adhering to the wall of the flask.

[0014] The AC complex solution was added to the eggplant-shaped flask, and the mixture was shaken and hydrated for 1–4 hours. The liposomes were then sonicated to break them up. After centrifugation, purification, and filtration, the Lipo-AuNP-Claudin-5 complex (LAC) was obtained.

[0015] REDVC-PEG 2000 -DSPE solution was added to the LAC complex solution, in which REDVC-PEG 2000 -DSPE is 5% to 20% of the phospholipid content. The solution is shaken for 4 to 8 hours to allow REDVC-PEG to react. 2000 -DSPE is fully inserted into the liposome layer of the LAC complex to obtain the RLAC complex.

[0016] When cationic AuNPs are electrostatically linked to the Claudin-5 overexpression plasmid, the AuNPs act as the core, significantly increasing the concentration of the nearby Claudin-5 overexpression plasmid. Subsequently, a cationic liposome is coated on the outermost layer, encapsulating the Claudin-5 overexpression plasmid and AuNPs within the liposome and protecting the Claudin-5 overexpression plasmid from nuclease degradation. Furthermore, the cationic liposome, due to its opposite charge to the cell membrane surface, enhances the gene vector's ability to enter cells and escape endosomes. The targeting peptide REDV is modified on the surface of the cationic liposomes. Through the specific interaction of the REDV peptide with integrin α4β1 on the endothelial cell membrane, the gene vector can specifically target vascular endothelial cells. After the gene vector delivers the Claudin-5 overexpression plasmid into vascular endothelial cells, the Claudin-5 overexpression plasmid is expressed, increasing Claudin-5 expression levels, rebuilding the vascular endothelial barrier, reducing vascular permeability, and achieving the therapeutic effect of sepsis.

[0017] In some embodiments, the AuNPs dispersion is prepared by adding a 20%–30% (w / w) gold tetrachloride solution and distilled water to a two-necked flask, wherein the volume ratio of the gold tetrachloride solution to the distilled water is 10:1. -3 ~5×10 -3 Heat to 4-8 degrees Celsius until boiling.

[0018] Then add a 0.8%–1.5% (w / w) trisodium citrate solution, with a volume ratio of trisodium citrate solution to distilled water of 6–10:40–80. Continue heating, boil for 15–30 minutes, then stop heating and cool down to obtain the AuNPs dispersion.

[0019] Preferably, the concentration of the trisodium citrate solution is 0.8% to 1.2%.

[0020] In some embodiments, the mass fraction of the gold tetrachloride solution is 24% to 28%.

[0021] Preferably, the prepared AuNPs dispersion is stored at 3–5°C in the dark.

[0022] In some embodiments, the filter membrane is 0.22 μm.

[0023] In some embodiments, the flow rate of the dialysis bag used for dialysis purification is 3000–4000 Da.

[0024] Preferably, the pH of the solution is adjusted to 4-5 using HCl.

[0025] In some embodiments, a Claudin-5 overexpression plasmid is added to a cationic AuNPs solution, wherein the mass ratio of cationic AuNPs to Claudin-5 overexpression plasmid is (35-40 ng): (6-10 mg).

[0026] The extraction method of the Claudin-5 overexpression plasmid is as follows:

[0027] Preparation: Clean the conical flask and prepare LB medium (5g tryptone + 5g sodium chloride + 2.5g yeast extract, diluted to 500mL with distilled water): seal and autoclave. After the medium cools to room temperature, add 1:1000 Kana antibiotic and 2mL Claudin-5 inoculum to the medium, and shake on a shaker at 150rpm / min and 37℃ for 12–16 hours.

[0028] Experimental steps:

[0029] 1) Transfer the shaken bacterial culture in batches to 50mL centrifuge tubes and centrifuge at 4000rpm / min at room temperature for 10min.

[0030] 2) After discarding the supernatant, add 10 mL of Solution I / Rnase A to the centrifuge tube, blow and aspirate evenly until the solution becomes turbid.

[0031] 3) Add 10 mL of Solution II to the solution and gently invert it 8 to 10 times to make the solution completely clear. This operation should be completed within 2 to 3 minutes.

[0032] 4) Then add 5 mL of pre-cooled N3 buffer to the solution, and immediately invert the bottle to mix the solution thoroughly. At this time, a white flocculent precipitate will appear in the bottle.

[0033] 5) Open the stopper of the impurity removal filter, quickly transfer the lysate from the previous step into the filter, and gently transfer the lysate into a new 50 mL centrifuge tube using the stopper.

[0034] 6) Add 0.1 times the volume of ERT Solution to the centrifuge tube above, and gently invert the solution. The solution will become cloudy. Incubate the solution on ice for 10 minutes, inverting it several times every 2-3 minutes. The solution will become clear after the ice bath.

[0035] 7) Incubate the solution in a 42°C water bath for 5 minutes. The solution will become cloudy again. Centrifuge the solution at 4000 rpm / min at room temperature for 5 minutes. At this time, a blue precipitate will form at the bottom of the tube for ERT Solution.

[0036] 8) Carefully aspirate the upper aqueous phase into a new 50mL centrifuge tube, add 0.5 times the volume of anhydrous ethanol, and gently invert 6-7 times.

[0037] 9) Load the HiBind DNA Maxi Column into a centrifuge tube, add 3 mL of GPS buffer to the column, incubate at room temperature for 4 min, centrifuge at 4000 rpm for 3 min, and discard the filtrate.

[0038] 10) Take 20 mL of the supernatant from step 8 and add it to the column. Centrifuge at 4000 rpm for 3 min and discard the filtrate. Repeat this step until all the solution has been centrifuged.

[0039] 11) Add 10 mL of HBC buffer diluted with isopropanol to the column, centrifuge at 4000 rpm for 3 min, and discard the filtrate.

[0040] 12) Add 15 mL of DNA wash buffer pre-diluted with anhydrous ethanol to the column, centrifuge at 4000 rpm for 3 min, and discard the filtrate. Add another 10 mL of DNA wash buffer to the column, centrifuge at 4000 rpm for 3 min, and discard the filtrate. Centrifuge the column at 4000 rpm for 10 min to remove the anhydrous ethanol from the column.

[0041] 13) Transfer the column to a new 50 mL enzyme-free centrifuge tube. Add 500 μL of Endo-Free Elution buffer evenly to the column membrane and incubate at room temperature for 5 min. Centrifuge at 4000 rpm for 5 min. Add the filtrate back to the column membrane and centrifuge a second time. Collect the centrifuged liquid, which is the plasmid DNA, and store it at -80℃ for long-term storage.

[0042] In some embodiments, REDVC-PEG 2000 The preparation method of -DSPE is as follows: The amphiphilic compound DSPE-PEG is prepared by... 2000 -MAL and the targeting peptide REDVC are dissolved in water at a molar ratio of 1.3:1 to 1.7:1 and reacted at 3 to 5 °C for 8 to 15 hours to obtain the product.

[0043] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows:

[0044] The lipid-gold nanoparticle complex RLAC serves as a gene vector for Claudin-5 overexpression plasmids, exhibiting good biocompatibility and high delivery efficiency. Upon uptake by endothelial cells, RLAC is released and expressed within the cells, thereby increasing the expression level of Claudin-5 protein and potentially contributing to the repair of the vascular endothelial barrier and the treatment of sepsis.

[0045] Using liposomes to deliver plasmid DNA, encapsulating the plasmid DNA within liposomes protects it from nuclease degradation. Cationic liposomes, carrying an opposite charge to the cell membrane surface, significantly enhance their ability to enter cells. When cationic AuNPs are electrostatically adsorbed onto the plasmid, the cationic AuNPs act as the nucleus, greatly increasing the surrounding plasmid concentration. Therefore, this invention utilizes both cationic liposomes and AuNPs as gene vectors. First, functional AuNPs are used to concentrate the plasmid concentration, and then cationic liposomes are used to encapsulate the AuNPs and plasmid. This strategy not only improves plasmid DNA delivery efficiency by concentrating plasmid concentration and protecting plasmid DNA from degradation, but also uses cationic liposomes to increase the ability to enter cells and escape endosomes.

[0046] To improve the targeting specificity of the gene vector, this invention also utilizes the peptide REDV. REDV (Arg-Glu-Asp-Val) is a tetrapeptide with specific targeting function, derived from fibronectin, capable of efficiently and specifically binding to α4β1 integrin. This integrin is abundant in endothelial cell membranes but scarce on the surface of smooth muscle cells, enabling the REDV peptide to specifically adhere to endothelial cells, thus facilitating the selective adhesion of the gene vector to endothelial cells.

[0047] To address the problem of impaired vascular endothelial cell junctions caused by sepsis, a lipid-gold nanoparticle complex (REDVC-Lipo-AuNP-Claudin-5, RLAC) was designed to target vascular endothelial cells and deliver Claudin-5 overexpression plasmids, thereby increasing Claudin-5 protein expression and reconstructing tight cell junctions. AuNPs, after β-MEA modification, carry a positively charged surface, acting as a "core" to attract the negatively charged Claudin-5 overexpression plasmid, increasing the plasmid concentration near the AuNPs and effectively improving delivery efficiency. To protect the Claudin-5 overexpression plasmid from nuclease degradation, a cationic liposome membrane was coated on the outside of the plasmid, isolating it from nucleases. Furthermore, due to the negatively charged cell membrane surface, the cationic liposomes facilitate endocytosis and endosome escape. Finally, to enable the synthesized complex to target vascular endothelial cells, the outermost layer of the liposomes was modified with the REDV peptide. The REDV peptide can bind efficiently and specifically to α4β1 integrin, which is highly expressed on the surface of vascular endothelial cells, and has the ability to actively target vascular endothelial cells. Adding a cysteine ​​residue to the tail of the REDV peptide yields the REDVC peptide, which utilizes the sulfhydryl group of the cysteine ​​to react with DSPE-PEG. 2000 - An addition reaction occurs at the double bond site of maleimide in -MAL to obtain REDVC-PEG. 2000 -DSPE. REDVC-PEG is inserted using a "back-interpolation" method. 2000 -DSPE is inserted into the surface of liposomes, endowing the lipid complex with the ability to target vascular endothelial cells. In summary, the authors designed and synthesized the REDVC peptide-modified lipid-gold nanoparticle complex RLAC for targeted delivery of Claudin-5 overexpression plasmids into vascular endothelial cells. The expression of Claudin-5 protein is increased through expression of the Claudin-5 overexpression plasmid in cells, thereby repairing damaged tight junctions and treating sepsis. Attached Figure Description

[0048] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0049] Figure 1 This is a schematic diagram of the synthesis process of the lipid-gold nanoparticle complex RLAC of the present invention.

[0050] Figure 2In the figure, (a) is the particle size distribution of AuNPs; (b) is the particle size distribution of the complex RLAC; (c) is the change of Zeta potential during the synthesis of the complex RLAC; (d) is the TEM image of AuNPs; (e) is the TEM image of the complex RLAC; and (f) is the characterization of the encapsulation efficiency and release rate of the Claudin-5 overexpression plasmid by the complex RLAC.

[0051] Figure 3 This is a comparison of the cytotoxicity of different concentrations of the RLAC complex.

[0052] Figure 4 In the figure, (a) is a schematic diagram showing the effect of the complex RLAC on the expression level of Claudin-5 protein in vascular endothelial cells after TNF-α treatment of HUVEC cells; (b) is a schematic diagram showing the effect of the complex RLAC on the expression level of Claudin-5 mRNA in vascular endothelial cells after TNF-α treatment of HUVEC cells.

[0053] Figure 5 In the figure, (a) is a schematic diagram of the effect of the complex RLAC on the permeability of monolayer vascular endothelial cells in the Transwell-FITC-Dexran experiment after TNF-α treatment of HUVEC cells (n=9, ***P<0.001); (b) is a comparative diagram of the effect of the complex RLAC on the transmembrane resistance of monolayer vascular endothelial cells after TNF-α treatment of HUVEC cells (n=9, ***P<0.001).

[0054] Figure 6 In the figure, (a) is a schematic diagram of the effect of RLAC complex treatment on the survival rate of septic mice constructed by LPS method; (b) is a diagram of the effect of RLAC complex on the expression level of Claudin-5 in pulmonary vascular endothelial cells in septic mouse model constructed by LPS method.

[0055] Figure 7 In the figure, (a) is a schematic diagram of the effect of RLAC complex treatment on the survival rate of septic mice constructed by CLP method; (b) is a diagram of the effect of RLAC complex on the expression level of Claudin-5 in pulmonary vascular endothelial cells in septic mouse model constructed by CLP method. Detailed Implementation

[0056] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0057] The present invention will be further described below with reference to the embodiments.

[0058] Example 1

[0059] The specific steps for preparing the lipid-gold nanoparticle complex RLAC are as follows:

[0060] 1) Preparation of AuNPs

[0061] All glassware used was soaked in aqua regia, then rinsed three times with distilled water and dried for later use. In a 100 mL two-necked flask, 30 μL of 25.6% gold tetrachloride solution and 60 mL of distilled water were added and heated to boiling. Then, 8 mL of 1% trisodium citrate solution was added, and heating continued. The solution changed from pale yellow to wine red. After boiling for 20 min, heating was stopped and the solution was cooled to room temperature to obtain AuNPs with a wavelength of 13 ± 2 nm. These were stored at 4 °C protected from light.

[0062] 2) Preparation of cationic AuNPs

[0063] 0.3 g SDS was added to the synthesized AuNPs solution and stirred for 2 h. Then, 0.45 g β-MEA was added and stirring continued for 10 h. The solution was filtered three times through a 0.22 μm filter membrane to remove the precipitated SDS. The solution was concentrated by centrifugation at 16800 rpm / min, and the concentrate was purified by dialysis in a dialysis bag with a capacity cutoff of 3500 Da. The pH was adjusted to 4 with HCl to obtain the cationic AuNPs solution, which was stored at 4 °C protected from light.

[0064] 3) Preparation of AuNP-Claudin-5 complex (AC)

[0065] Add 8 mg of Claudin-5 overexpression plasmid to the cationic AuNPs solution and stir overnight to obtain the AuNP-Claudin-5 solution.

[0066] 4) Preparation of REDVC-Lipo-AuNP-Claudin-5 complex (RLAC)

[0067] First, DSPE-PEG 2000 MAL and REDVC were dissolved in water at a molar ratio of 1.5:1 and reacted overnight at 4°C to obtain REDVC-PEG. 2000 -DSPE.

[0068] 0.2 g of soybean lecithin, 0.05 g of cholesterol, and 0.025 g of 2-dioleoylhydroxypropyl-3-N,N,N-trimethylammonium chloride (DOTAP) were placed in a 500 mL round-bottom flask, and 200 mL of methanol solution was added. The mixture was heated to 80 °C to dissolve the three components, and stirred until homogeneous. After cooling to room temperature, the methanol was removed using a rotary evaporator, forming a uniform layer of liposomes on the flask wall. The AC complex solution was added to the round-bottom flask and hydrated by shaking for 2 h. Then, the large liposomes were disrupted using an ultrasonic homogenizer (200 W, 5 s working time, 5 s interval, 10 min total). The mixture was then centrifuged at 12000 rpm for 10 min. After filtration three times through a 0.22 μm filter membrane, the REDVC-Lipo-AuNP-Claudin-5 complex (RLAC) was obtained.

[0069] Example 2

[0070] The specific steps for preparing the lipid-gold nanoparticle complex RLAC are as follows:

[0071] 1) Preparation of AuNPs

[0072] All glassware used was soaked in aqua regia, then rinsed three times with distilled water and dried for later use. In a 100 mL two-necked flask, 10 μL of 25.6% gold tetrachloride solution and 40 mL of distilled water were added, and the mixture was heated to boiling. Then, 3 mL of 1% trisodium citrate solution was added, and heating continued. The solution changed from pale yellow to wine red. After boiling for 15 minutes, heating was stopped, and the mixture was cooled to room temperature to obtain AuNPs with a wavelength of 13 ± 2 nm. These were stored at 4 °C protected from light.

[0073] 2) Preparation of cationic AuNPs

[0074] Add 0.5 g SDS to the synthesized AuNPs solution and stir for 3 h. Then add 0.3 g β-MEA and continue stirring for 4 h. Filter the solution three times through a 0.22 μm filter membrane to remove precipitated SDS. Centrifuge at 12000 rpm / min to concentrate the solution, and then dialyze it into a dialysis bag with a capacity cutoff of 4000 Da for purification. Adjust the pH to 5 with HCl to obtain the cationic AuNPs solution, and store it at 4 °C protected from light.

[0075] 3) Preparation of AuNPs-Claudin-5 complex (AC)

[0076] Add 5 mg of Claudin-5 overexpression plasmid to the cationic AuNPs solution and stir overnight to obtain AC solution.

[0077] 4) Preparation of REDVC-Lipo-AuNP-Claudin-5 complex (RLAC)

[0078] First, DSPE-PEG 2000 MAL and REDVC were dissolved in water at a molar ratio of 1.3:1 and reacted overnight at 4°C to obtain REDVC-PEG. 2000 -DSPE.

[0079] Take 0.1g of soybean lecithin, 0.025g of cholesterol, and 0.03g of DOTAP, and place them in a 500mL round-bottom flask. Add 100mL of methanol solution, heat to 60℃ to dissolve the three components, and stir until well mixed. After cooling to room temperature, remove the methanol using a rotary evaporator, forming a uniform layer of liposomes on the flask wall. Add AC solution to the round-bottom flask and hydrate by shaking for 4 hours. Then, use an ultrasonic homogenizer to break up the large liposomes (200W power, 3s working time, 3s interval, total 6 minutes). Purify by centrifugation at 8000rpm / min for 30 minutes. Filter three times through a 0.22μm filter membrane to obtain the RLAC complex.

[0080] Example 3

[0081] The specific steps for preparing the lipid-gold nanoparticle complex RLAC are as follows:

[0082] 1) Preparation of AuNPs

[0083] All glassware used was soaked in aqua regia, then rinsed three times with distilled water and dried for later use. In a 200 mL two-necked flask, 50 μL of 25.6% gold tetrachloride solution and 80 mL of distilled water were added and heated to boiling. Then, 10 mL of 1% trisodium citrate solution was added, and heating continued. The solution changed from pale yellow to wine red. After boiling for 30 minutes, heating was stopped and the solution was cooled to room temperature to obtain AuNPs with a wavelength of 13 ± 2 nm. These were stored at 4 °C protected from light.

[0084] 2) Preparation of cationic AuNPs

[0085] Add 0.5 g SDS to the synthesized AuNPs solution and stir for 1 h. Then add 0.7 g β-MEA and continue stirring for 10 h. Filter the solution three times through a 0.22 μm filter membrane to remove precipitated SDS. Centrifuge at 18000 rpm / min to concentrate the solution, and then dialyze it into a dialysis bag with a capacity cutoff of 3000 Da for purification. Adjust the pH to 4 with HCl to obtain the cationic AuNPs solution, and store at 4 °C protected from light.

[0086] 3) Preparation of AuNPs-Claudin-5 complex (AC)

[0087] Add 10 mg of Claudin-5 overexpression plasmid to the cationic AuNPs solution and stir overnight to obtain AC solution.

[0088] 4) Preparation of REDVC-Lipo-AuNP-Claudin-5 complex (RLAC)

[0089] First, DSPE-PEG 2000 MAL and REDVC were dissolved in water at a molar ratio of 1.7:1 and reacted overnight at 4°C to obtain REDVC-PEG. 2000 -DSPE.

[0090] Take 0.6g of soybean lecithin, 0.3g of cholesterol, and 0.05g of DOTAP, and place them in a 500mL round-bottom flask. Add 300mL of methanol solution, heat to 90℃ to dissolve the three components, and stir until well mixed. After cooling to room temperature, evaporate the methanol using a rotary evaporator, forming a uniform layer of liposomes on the flask wall. Add AC solution to the round-bottom flask and hydrate by shaking for 4 hours. Then, use an ultrasonic homogenizer to break up the large liposomes (200W power, 7s working time, 5s interval, total 12min). Purify by centrifugation at 13000rpm / min for 30min. Filter three times through a 0.22μm filter membrane to obtain the RLAC complex.

[0091] Example 4

[0092] The specific steps for preparing the RLAC complex are as follows:

[0093] 1) Preparation of AuNPs

[0094] All glassware used was soaked in aqua regia, then rinsed three times with distilled water and dried for later use. In a 100 mL two-necked flask, 40 μL of 25.6% gold tetrachloride solution and 60 mL of distilled water were added and heated to boiling. Then, 10 mL of 1% trisodium citrate solution was added, and heating continued. The solution changed from pale yellow to wine red. After boiling for 25 minutes, heating was stopped and the solution was cooled to room temperature to obtain AuNPs with a wavelength of 13 ± 2 nm. These were stored at 4 °C protected from light.

[0095] 2) Preparation of cationic AuNPs

[0096] 0.1 g SDS was added to the synthesized AuNPs solution and stirred for 2 h. Then 0.7 g β-MEA was added and stirring continued for 10 h. The solution was filtered three times through a 0.22 μm filter membrane to remove precipitated SDS. The solution was concentrated by centrifugation at 15000 rpm / min, and the concentrate was purified by dialysis in a dialysis bag with a capacity cutoff of 3500 Da. The pH was adjusted to 4.5 with HCl to obtain the cationic AuNPs solution, which was stored at 4 °C protected from light.

[0097] 3) Preparation of AuNPs-Claudin-5 complex (AC)

[0098] Add 6 mg of Claudin-5 overexpression plasmid to the cationic AuNPs solution and stir overnight to obtain the AuNP-Claudin-5 solution.

[0099] 4) Preparation of REDVC-Lipo-AuNP-Claudin-5 complex (RLAC)

[0100] First, DSPE-PEG 2000 MAL and REDVC were dissolved in water at a molar ratio of 1.5:1 and reacted overnight at 4°C to obtain REDVC-PEG. 2000 -DSPE.

[0101] Take 0.4g of soybean lecithin, 0.1g of cholesterol, and 0.07g of DOTAP, and place them in a 500mL round-bottom flask. Add 200mL of methanol solution, heat to 70℃ to dissolve the three components, and stir until well mixed. After cooling to room temperature, evaporate the methanol using a rotary evaporator to form a uniform layer of liposomes on the flask wall. Add AC solution to the round-bottom flask and hydrate by shaking for 3 hours. Then, use an ultrasonic homogenizer to break up the large liposomes (150W power, 6s working time, 7s interval, total 13min). Purify by centrifugation at 10000rpm / min for 20min. Filter three times through a 0.22μm filter membrane to obtain the RLAC complex.

[0102] Experimental Example

[0103] After synthesizing the RLAC complex in Example 1, the present invention conducted in vivo and in vitro experiments to demonstrate the effects of the RLAC complex on repairing vascular endothelial cell junctions and alleviating sepsis.

[0104] This invention uses human umbilical vein endothelial cells (HUVECs) for cell experiments. First, the cytotoxicity of the RLAC complex was investigated. HUVECs were cultured and treated with 0–0.01 μg / μL of the RLAC complex, and cell viability was observed after 24 hours. The experimental results showed that the RLAC complex had no significant cytotoxicity. Figure 3 ).

[0105] Subsequently, this invention investigated the effect of adding 0.001 μg / μL of the RLAC complex on the expression level of Claudin-5 in cells using Western blot experiments. Figure 4 As shown in figure a, the expression level of Claudin-5 protein in cells significantly increased after the addition of the RLAC complex, indicating that the RLAC complex successfully delivered and expressed Claudin-5 in cells. The authors used qPCR to investigate the effect of the RLAC complex on Claudin-5 mRNA expression. Figure 4 b. Treatment with the RLAC complex significantly increased the expression level of Claudin-5 mRNA.

[0106] This invention also investigated the effect of the RLAC complex on cell permeability using a transmembrane cell barrier assay (Transwell-FITC-Dexran permeation method). Figure 5 As shown in figure a, compared with the control group treated with only TNF-α, the fluorescence intensity in the lower chamber of the experimental group treated with both TNF-α and the RLAC complex was significantly reduced, indicating that RLAC complex treatment significantly decreased vascular barrier permeability. In addition, the authors investigated the changes in transmembrane resistance of the vascular endothelial cell barrier after RLAC complex treatment. Figure 5 As shown in b, the transmembrane resistance (TEER) after treatment with the RLAC complex was significantly increased compared to the control group, further confirming that the vascular barrier permeability was significantly reduced after treatment with the RLAC complex.

[0107] After completing the in vitro experiments, this invention also conducted in vivo experiments. This invention employed two methods to construct a mouse model of sepsis: lipopolysaccharide injection (LPS) and cecal ligation and puncture (CLP).

[0108] The specific procedure for establishing a sepsis mouse model using the LPS method is as follows: Mice must be fasted for 12 hours before the experiment. A lethal dose of 20 mg / kg of LPS suspension is injected intraperitoneally to prepare a mouse sepsis model.

[0109] The specific procedures for constructing septic mice using the CLP method are as follows: Mice were fasted for 12 hours prior to the experiment. They were then anesthetized with an intraperitoneal injection of 5% chloral hydrate at a dose of 60 μg / 10g. The mice were then fixed on a surgical board, and the surgical area of ​​the abdomen was routinely disinfected and shaved. Under aseptic conditions, a 2 cm incision was made in the abdominal wall using a scalpel. The abdomen was then accessed through the incision, and the cecum was dissected distal to the ileocecal valve and ligated at one-third of its length with No. 3 silk suture. A small puncture was then made at the ligation point using an 18-gauge needle, and a small amount of feces was expelled. This step required careful handling to avoid damaging blood vessels. Finally, the peritoneum and skin were intermittently sutured with No. 4 silk suture. To prevent shock, 50 mL / kg of physiological saline was immediately injected subcutaneously. The systemic inflammatory response in the mice reached its peak 12 hours post-surgery.

[0110] Sepsis model mice were randomly divided into two groups. The experimental group was injected with 0.1 mg / kg RLAC complex, while the control group was injected with physiological saline. Mice mortality was observed within 24 hours. Figure 6As shown in Figures a and 7a, the survival rate of septic mice treated with the RLAC complex significantly increased within 24 hours, indicating that RLAC complex treatment can effectively alleviate sepsis and improve the survival rate of septic mice. Eight hours after RLAC injection, mice were randomly selected and sacrificed. Western blot experiments were performed on pulmonary vascular endothelial cells from these mice to investigate the effect of the RLAC complex on the expression level of Claudin-5 in endothelial cells. Figure 6 As shown in b and 7b, the expression level of Claudin-5 protein in lung endothelial cells was increased in both LPS and CLP-constructed mouse models, indicating that the RLAC complex can reach lung endothelial cells in vivo and successfully deliver the Claudin-5 overexpression plasmid into them. The Claudin-5 overexpression plasmid was released and successfully expressed in the cells, significantly increasing the expression level of Claudin-5 protein. The experimental results show that the RLAC complex can reach the lungs through systemic blood circulation and successfully deliver the Claudin-5 overexpression plasmid into lung endothelial cells. The expression of the Claudin-5 overexpression plasmid in lung endothelial cells significantly increases the expression level of Claudin-5, effectively repairing pulmonary vascular endothelial cell junctions and alleviating sepsis.

[0111] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A lipid-gold nanoparticle complex RLAC for the treatment of sepsis, characterized in that: Its preparation method includes the following steps: Sodium dodecyl sulfate (SDS) was added to the dispersion of AuNPs and dispersed evenly. Then, β-MEA was added to the solution, with the concentrations of AuNPs, SDS, and β-MEA being 1–4 nM, 12–18 mM, and 55–60 mM, respectively. The mixture was stirred for 4–10 h, and SDS was removed by filtration. The remaining liquid was concentrated by centrifugation at 12,000–18,000 rpm / min. The concentrate was purified by dialysis. After purification, the pH was adjusted to 4–5 to obtain a cationic AuNPs solution. Add the Claudin-5 overexpression plasmid to the cationic AuNPs solution, stir overnight to obtain AC solution, wherein the mass ratio of cationic AuNPs to Claudin-5 overexpression plasmid is 35~40 ng: 6~10 mg; Soybean lecithin, cholesterol, and DOTAP were added to a round-bottom flask in a mass ratio of 0.2~0.6:0.05~0.3:0.01~0.09, along with a methanol solution. The mixture was heated to dissolve and mix the three components, then cooled to room temperature. The methanol was removed by rotary evaporation to obtain cationic liposomes. Add AC solution to the eggplant-shaped flask, shake to hydrate for 1-4 h, then sonicate to break up liposomes, centrifuge to purify, filter and obtain LAC complex. The REDVC-PEG 2000 -DSPE solution is added to the LAC complex solution, and shaken for 4-8 h to make the REDVC-PEG 2000 -DSPE completely inserted into the liposome layer of the LAC complex, and a lipid gold nanoparticle complex RLAC is obtained. wherein REDVC-PEG 2000 The preparation method of DSPE is as follows: the amphiphilic compound DSPE-PEG 2000 MAL and the targeting polypeptide REDVC are dissolved in water at a molar ratio of 1.3:1~1.7:1, and reacted at 3~5℃ for a set time, and the product is obtained.

2. The lipid-gold nanoparticle complex RLAC for treating sepsis according to claim 1, characterized in that: The preparation method of the AuNPs dispersion liquid is as follows: adding gold tetrachloride solution with a mass fraction of 20%-30% and distilled water in two-mouth bottles, the volume ratio of the gold tetrachloride solution and the distilled water is 10 -3 ~5×10 -3 :4-8, and heating to boiling. Then add a 0.8%~1.5% (w / w) trisodium citrate solution, with a volume ratio of trisodium citrate solution to distilled water of 6~10:40~80. Continue heating, boil for 15~30 min, then stop heating and cool down to obtain the AuNPs dispersion.

3. The lipid-gold nanoparticle complex RLAC for treating sepsis according to claim 2, characterized in that: The concentration of trisodium citrate solution is 0.8%~1.2%.

4. The lipid-gold nanoparticle complex RLAC for treating sepsis according to claim 2, characterized in that: The mass fraction of the gold tetrachloride solution is 24%~28%.

5. The lipid-gold nanoparticle complex RLAC for treating sepsis according to claim 2, characterized in that: The prepared AuNPs dispersion was stored at 3-5℃ in the dark.

6. The lipid-gold nanoparticle complex RLAC for treating sepsis according to claim 1, characterized in that: SDS is removed by filtration using a 0.22 μm filter membrane.

7. The lipid-gold nanoparticle complex RLAC for treating sepsis according to claim 1, characterized in that: The dialysis bag used for dialysis purification of the concentrate has a flow rate of 3000~4000 Da.

8. The lipid-gold nanoparticle complex RLAC for treating sepsis according to claim 1, characterized in that: Preparation of REDVC-PEG 2000 In the -DSPE method, the reaction time is 8-15 h at 3-5℃.

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