A multifunctional composition for treating SARS-CoV-2 and its variants infection and preparation method thereof

Abstract

The present invention provides a multifunctional composition for treating SARS-CoV-2 and its variant infection and a preparation method thereof, belonging to the field of biomedical technology. The multifunctional composition of the present invention comprises the following components by weight according to a final volume of 6 ml: 1 to 100 OD of a nucleic acid drug for a fixed sequence of a SARS-CoV-2 variant, 0.5 to 2.5 ml of a cell membrane expressing an ACE2 protein and / or 0.5 to 2.5 ml of a cell membrane expressing a cytokine receptor, 20 to 100 mg of a phospholipid material, 25 to 100 μg of a cationic material, and 0.5 to 10 mg of cholesterol. The multifunctional composition of the present invention has obvious advantages in terms of long circulation in the body, neutralization of free SARS-CoV-2 and its variants, viral inhibition in cells infected with SARS-CoV-2 and its variants, and neutralization of inflammatory factors.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a multifunctional composition for treating SARS-CoV-2 and its variant infections and a preparation method thereof. Background Art

[0002] The novel coronavirus mainly infects the respiratory system, causing a flu-like illness, with the main clinical manifestations being cough, fever, chills, myalgia, diarrhea, mental symptoms, and dyspnea. At the same time, the virus may cause overactivation of immune cells, which produces a large number of inflammatory factors, which will recruit and activate more immune cells, thereby producing more inflammatory factors, and forming an inflammatory factor storm through a positive feedback loop mechanism. Taking the lungs as an example, since lung tissue highly expresses ACE2 and is directly exposed to the outside world, it has become the main target of invasion by the novel coronavirus. Overactivated lung immune cells will release a large number of inflammatory factors, causing immune cells and tissue fluid to accumulate in the lungs, blocking gas exchange between alveoli and capillaries, and thus causing acute respiratory distress syndrome. Once a cytokine storm occurs, the immune system will indiscriminately attack normal lung cells and viruses, causing severe damage to the lung's ventilation function, causing severe lung damage and respiratory failure, and large white areas will appear on lung CT. Patients are prone to hypoxia and death. In addition, since all organs that express ACE2 may become battlefields between the novel coronavirus and the immune system, infection may lead to multiple organ failure and endanger life.

[0003] At present, there are many mutant strains of the new coronavirus, and it is imperative to develop an anti-new coronavirus drug that is effective against all mutant strains. To this end, some researchers have designed nanocarriers based on ACE2 cell membranes in order to achieve effective neutralization of SARS-CoV-2, including mutant strains, and have achieved satisfactory results; other researchers have designed nanocarriers based on macrophage (such as THP-1) membrane modification in order to achieve effective neutralization of inflammatory factors, and have achieved a relatively satisfactory inflammation inhibition effect. However, this type of nanocarrier only targets free viruses and inflammatory factors, and has no inhibitory effect on viruses in infected cells. There is currently no drug that is effective in neutralizing free viruses, neutralizing inflammatory factors, and inhibiting viruses in infected cells. Summary of the invention

[0004] In order to obtain a universal antiviral effect against SARS-CoV-2 and its variants, and to simultaneously solve the problems of neutralization of free viruses, neutralization of inflammatory factors, and inhibition of viruses in infected cells, the present invention provides a multifunctional composition for treating SARS-CoV-2 and its variants infection and a preparation method thereof, wherein the multifunctional composition has the potential to completely eliminate SARS-CoV-2 and its variants.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The present invention provides a multifunctional composition for treating infection with SARS-CoV-2 and its variants. The composition comprises the following components in parts by weight, based on a total volume of 6 ml: 1 to 100 OD of a nucleic acid drug targeting a fixed sequence of a SARS-CoV-2 variant, 0.5 to 2.5 ml of a cell membrane expressing an ACE2 protein and / or 0.5 to 2.5 ml of a cell membrane expressing a cytokine receptor, 20 to 100 mg of a phospholipid material, 25 to 100 μg of a cationic material, and 0.5 to 10 mg of cholesterol.

[0007] Preferably, the cell membrane is used at a concentration of 1×10 5 ~1×10 10 Cell membrane of cells / ml.

[0008] Preferably, the nucleic acid drug with a fixed sequence targeting SARS-CoV-2 and its variants comprises one or more of siRNA, mRNA, miRNA, and aptamers.

[0009] Preferably, the cell membrane expressing ACE2 protein includes a cell membrane extracted and purified from cells that can express ACE2 protein on their surface.

[0010] Preferably, the cell membrane expressing cytokine receptors comprises a cell membrane extracted and purified from cells expressing cytokine receptors on their surfaces.

[0011] Preferably, the phospholipid material includes one or more of hydrogenated soybean lecithin, egg yolk lecithin, dioleoyl lecithin, dipalmitoyl lecithin, distearoyl phosphatidylcholine, 2-palmitoyl phosphatidylglycerol, distearoyl phosphatidylethanolamine-polyethylene glycol and their derivatives.

[0012] Preferably, the cationic material includes a material that can assist the nucleic acid drug in achieving the endosomal / lysosomal escape function.

[0013] The invention provides a preparation method of the multifunctional composition, comprising the following steps: diluting a cationic material with a HEPES solution and adding the diluent to a HEPES solution containing siRNA, incubating at room temperature to obtain a cationic material / siRNA complex; dissolving a phospholipid material and cholesterol in chloroform, performing vacuum rotary evaporation to form a film, drying the phospholipid film, hydrating the dried phospholipid film with a HEPES solution containing the cationic material / siRNA complex, and then ultrasonically treating the prepared lipid nanoparticles to obtain lipid nanoparticles encapsulating the cationic material / siRNA complex; mixing a cell membrane expressing an ACE2 protein with the lipid nanoparticles encapsulating the cationic material / siRNA complex, and then sequentially extruding the mixed solution through a liposome of a polycarbonate membrane. The method comprises the steps of: passing the cell membrane expressing the ACE2 protein through a liposome extruder and repeatedly extruding to obtain the cell membrane expressing the ACE2 protein-siRNA / LP; mixing the cell membrane expressing the cytokine receptor with the lipid nanoparticles encapsulating the cationic material / siRNA complex, and then passing the mixed solution through a liposome extruder with a polycarbonate membrane and repeatedly extruding to obtain the cell membrane expressing the cytokine receptor-siRNA / LP; mixing the cell membrane expressing the ACE2 protein-siRNA / LP with the cell membrane expressing the cytokine receptor-siRNA / LP, ultrasonicating, and then mixing with the lipid nanoparticles encapsulating the cationic material / siRNA complex, and then passing the mixed solution through a liposome extruder with a polycarbonate membrane and repeatedly extruding to obtain the cell membrane expressing the ACE2 protein / cell membrane expressing the cytokine receptor-siRNA / LP.

[0014] The present invention provides the use of the multifunctional composition or the multifunctional composition obtained by the preparation method in preparing a product for treating infection with SARS-CoV-2 and its variants.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] On the one hand, the present invention utilizes the characteristic that the self-recognition molecule CD47 is simultaneously expressed on the surface of cells in vivo to realize the function of the constructed composition / carrier to evade the uptake and clearance of mononuclear macrophages in the body, thereby achieving a long circulation effect. On the other hand, the natural target characteristics of SARS-CoV-2 and its variant viruses of the cell membrane with high expression of ACE2 and the natural target characteristics of inflammatory factors of the THP-1 cell membrane are utilized, and therapeutic siRNA targeting the fixed nucleic acid sequence of SARS-CoV-2 and its variant viruses is simultaneously introduced to jointly construct a composition / carrier, so that the surface of the composition / carrier simultaneously carries ACE2 and inflammatory factor neutralizing receptors, and the therapeutic siRNA targeting the fixed nucleic acid sequence of SARS-CoV-2 and its variant viruses can be accurately delivered to the infected cells, thereby realizing multi-angle treatment of free virus neutralization, inflammatory factor neutralization and virus inhibition in infected cells, which can not only ensure the stable treatment ability of SARS-CoV-2 and its variant viruses, but also significantly improve the treatment effect of patients with new coronavirus, greatly reduce the dosage of the drug, and reduce its toxic and side effects.

[0017] The multifunctional composition proposed in the present invention is mainly composed of a genetically engineered 293T cell membrane with high expression of ACE2, a THP-1 cell membrane rich in cytokine receptors, siRNA with fixed sequences for SARS-CoV-2 and its variants, a phospholipid material, and a cationic material. Compared with a hybrid membrane composed of a genetically engineered 293T cell membrane with high expression of ACE2, a THP-1 cell membrane rich in cytokine receptors, and a genetically engineered 293T cell membrane with high expression of ACE2 and a THP-1 cell membrane rich in cytokine receptors, the composition has self-recognition function, free virus neutralization function, and inflammatory factor neutralization function due to the introduction of the cell membrane, and also has the function of inhibiting SARS-CoV-2 and its variants in infected cells, so it has the potential to completely eliminate SARS-CoV-2 and its variants. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the inhibition rate of 6 typical novel coronavirus strains.

[0019] Figure 2 The inhibition rate of each preparation group on WT and OmicronBA.4 / 5 (****P≤0.0001, *P≤0.05).

[0020] Figure 3 The results show the binding of each group of preparations with different inflammatory factors (n=3, ****P≤0.0001, *P≤0.05).

[0021] Figure 4The results of lysosomal escape are shown in Figure 2 (red: LysoTrackerRed; green: Cy5; blue: Hoechst 33258; scale bar: 10 μm). DETAILED DESCRIPTION

[0022] The present invention provides a multifunctional composition for treating infection with SARS-CoV-2 and its variants. The composition comprises the following components by weight according to a volume of 6 ml: 1 to 100 OD of a nucleic acid drug targeting a fixed sequence of a SARS-CoV-2 variant, 0.5 to 2.5 ml of a cell membrane expressing an ACE2 protein and / or 0.5 to 2.5 ml of a cell membrane expressing a cytokine receptor, 20 to 100 mg of a phospholipid material, 25 to 100 μg of a cationic material, and 0.5 to 10 mg of cholesterol.

[0023] In the present invention, the concentration of the cell membrane used is 1×10 5 ~1×10 10 Cell membranes of cells / ml, preferably 9.5×10 6 ~9×10 8 Cell membranes of cells / ml, more preferably 9.5×10 7 ~5.5×10 8 The concentration of the cell membrane expressing ACE2 protein in the present invention is preferably 1×10 8 The cell membrane of cells / ml, the cell membrane expressing cytokine receptors is preferably used at a concentration of 1×10 8 Cell membrane of cells / ml.

[0024] In the present invention, the nucleic acid drug with a fixed sequence for SARS-CoV-2 and its variants includes one or more of siRNA, mRNA, miRNA, and aptamers, preferably C6G25S siRNA. The C6G25S siRNA described in the present invention was purchased from Shanghai Jima Co., Ltd., batch number 192370. The present invention requires that the purchased C6G25S siRNA be dissolved in a HEPES solution into a C6G25S siRNA solution of a certain concentration, and then a certain volume is taken to obtain a C6G25S siRNA solution of the required OD amount. The OD unit definition of the siRNA quantification described in the present invention is: in a 1mL optical path standard cuvette, a wavelength of 260nm at an absorbance of 1A 260 The siRNA solution is defined as 1OD 260 Unit, according to this definition, 1OD 260This is approximately equivalent to 33 μg of siRNA, and the molar concentration of the solution can be calculated based on this and the molecular weight of siRNA (molecular weight is approximately 13,300). The sense strand of the C6G25S siRNA sequence of the present invention is: 5′-mCsmUsmGmUfCmAfAfAfCfCmCmGmGmUmAmAmUsmUsmU-3′, and the antisense strand is: 5′-mAsfAsmAfUfUmAmCfCfGmGmGmUmUfUmGfAmCfAmGsmUsmU-3′, the letter m represents methylation, the letter f represents fluorine modification, and the letter s represents phosphorothioate. The sequence without the letters m, f and s is the sense strand: 5′-CUGUCAAACCCGGUAAUUU-3′ (5′-SEQ ID No.1-3′), and the antisense strand is: 5′-AAAUUACCGGGUUUUGACAGUU-3′ (5′-SEQ ID No.2-3′).

[0025] In the present invention, the cell membrane expressing ACE2 protein includes a cell membrane extracted and purified from cells expressing ACE2 protein on the surface. The cells expressing ACE2 protein of the present invention may be cells of natural origin or cells modified by genetic engineering, such as 293T cells with high expression of ACE2. The cell membrane expressing ACE2 protein of the present invention is preferably an ACE2-HEK293T cell membrane.

[0026] In the present invention, the cell membrane expressing cytokine receptors includes a cell membrane extracted and purified from cells that can express cytokine receptors on their surfaces. The cells expressing cytokine receptors of the present invention can be cells of natural origin or cells that have been genetically engineered, such as macrophages, and further such as THP-1 cells. The cell membrane expressing cytokine receptors of the present invention is preferably a THP-1 cell membrane.

[0027] In the present invention, the extraction and purification method of the ACE2-HEK293T cell membrane or the cell membrane expressing the cytokine receptor can be one or more of the following methods: hypotonic method, ultrasonic disruption method, repeated freeze-thaw method and ultra-high-speed centrifugation method.

[0028] In the present invention, the phospholipid material includes one or more of hydrogenated soybean lecithin (HSPC), egg yolk lecithin (EPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 2-palmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-MPEG2000) and their derivatives; preferably one or more of egg yolk lecithin (EPC), dioleoyl phosphatidylcholine (DOPC), dipalmitoyl phosphatidylcholine (DPPC), distearoyl phosphatidylcholine (DSPC), 2-palmitoyl phosphatidylglycerol (DPPG), distearoyl phosphatidylethanolamine-polyethylene glycol (DSPE-MPEG2000) and their derivatives; further preferably dioleoyl phosphatidylcholine (DOPC).

[0029] In the present invention, the cationic material includes a material that can assist the nucleic acid drug in achieving the endosomal / lysosomal escape function, preferably one or more of protein cationic materials, polypeptide cationic materials, and chemical cationic materials, further preferably one or more of protamine and its derivatives, arginine polymers, 1,2-dioctadeceneoxy-3-methylammonium propane (DOTMA), and polyethyleneimine, and further preferably protamine and DOTMA.

[0030] The present invention provides a preparation method of the multifunctional composition, comprising the following steps: (1) diluting a cationic material with a HEPES solution and adding the diluent to a HEPES solution containing siRNA, and incubating at room temperature to obtain a cationic material / siRNA complex; (2) dissolving a phospholipid material and cholesterol in chloroform, performing vacuum rotary evaporation to form a film, drying the phospholipid film, hydrating the dried phospholipid film with a HEPES solution containing a cationic material / siRNA complex, and then ultrasonically treating the prepared lipid nanoparticles to obtain lipid nanoparticles encapsulating the cationic material / siRNA complex; and (3) mixing a cell membrane expressing an ACE2 protein with the lipid nanoparticles encapsulating the cationic material / siRNA complex, and then sequentially passing the mixed solution through a liposome containing a polycarbonate membrane. Extruder, repeatedly extruding to obtain cell membrane expressing ACE2 protein-siRNA / LP; (4) mixing the cell membrane expressing cytokine receptor with lipid nanoparticles encapsulating cationic material / siRNA complex, and then passing the mixed solution through a liposome extruder with a polycarbonate membrane, and repeatedly extruding to obtain cell membrane expressing cytokine receptor-siRNA / LP; (5) mixing the cell membrane expressing ACE2 protein-siRNA / LP with the cell membrane expressing cytokine receptor-siRNA / LP, ultrasonicating, and then mixing with lipid nanoparticles encapsulating cationic material / siRNA complex, and then passing the mixed solution through a liposome extruder with a polycarbonate membrane, and repeatedly extruding to obtain cell membrane expressing ACE2 protein / cell membrane expressing cytokine receptor-siRNA / LP.

[0031] The present invention also provides the use of the multifunctional composition or the multifunctional composition obtained by the preparation method in the preparation of products for treating SARS-CoV-2 and its variants. The products of the present invention include medicines. The pharmaceutical dosage forms of the present invention include suspensions, tablets, granules, capsules, pills, and injections.

[0032] In the present invention, unless otherwise specified, all components or reagents are commercially available products well known to those skilled in the art.

[0033] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1

[0035] This embodiment provides a multifunctional composition for treating infection with SARS-CoV-2 and its variants, wherein the total volume of the liquid is 6 ml and the components are as follows in parts by weight, as shown in Table 1. Among them, the sense chain and antisense chain sequences of the C6G25S siRNA sequence are shown in SEQ ID No.1-2, respectively, purchased from Shanghai Jima Pharmaceutical Co., Ltd., batch number 192370; protamine was purchased from Sigma-Aldrich, batch number 111M7405V; HEPES was purchased from Sigma-Aldrich, batch number: SLBM7880V; DOPC was purchased from Avite Shanghai Pharmaceutical Technology Co., Ltd., batch number C00257; DOTMA was purchased from Avite Shanghai Pharmaceutical Technology Co., Ltd., batch number ZKY20220412; cholesterol was purchased from Sinopharm Chemical Reagent Co., Ltd., batch number 20201112; chloroform was purchased from Beijing Tongguang Fine Chemical Company Sanhe Branch, batch number 20211216. 293T cells were purchased from Yisheng Bio, batch number: H0125121; THP-1 cells were purchased from the Cell Resource Center of the Basic College of Peking Union Medical College.

[0036] Table 1 Multifunctional composition prescription

[0037]

[0038] Note: The amount of protamine in Table 1 is calculated by rounding off.

[0039] Example 2

[0040] The method for preparing the multifunctional composition for treating SARS-CoV-2 and its variants infection by the prescription described in Example 1 comprises the following steps:

[0041] 1. Extraction of ACE2-HEK293T cell membrane: Resuspend the 293T cell pellet with PBS at pH 7.4 (containing 1% protease inhibitor PMSF), freeze at -80°C at room temperature and thaw at room temperature; repeat freezing and thawing three times. The broken cell homogenate was centrifuged at 4°C, 6,000×g for 10 minutes, and the pellet was washed with cold PBS containing protease inhibitors, and the crude membrane was further broken with ice bath probe ultrasound (power 120W, work 2s stop 5s, total 5min). The broken cell suspension was centrifuged again at 4°C, 100,000×g for 10 minutes using an ultrahigh speed centrifuge, and the collected pellet was the CE2-HEK293T cell membrane. The cell membrane was washed once with ice-cold PBS and centrifuged again, and the amount of extracted and purified membrane protein was determined according to the instructions of the BCA protein quantification kit. The above-collected cell membrane was prepared with HEPES solution (HEPES solution described in step 2 below) to 5×10 7 ACE2-HEK293T cell membrane solution with a concentration of 1 cell / mL was used for subsequent experiments.

[0042] Extraction of THP-1 cell membrane: Collect THP-1 cells whose cell number reaches the required dosage or whose cell fusion degree reaches about 80% and disperse them in PBS (containing 1% protease inhibitor PMSF) at pH 7.4. Place the cell suspension in a homogenizer and squeeze it 15-25 times. After disruption, centrifuge the mixture at 4°C, 800×g for 5 minutes to collect the supernatant. Centrifuge the supernatant at 4°C, 10,000×g for 25 minutes to collect the supernatant again. Centrifuge the collected supernatant at 4°C, 150,000×g for 50 minutes, discard the supernatant and collect the cell membrane, wash the cell membrane with ice-cold PBS and centrifuge again to collect. Determine the amount of extracted and purified membrane protein according to the instructions of the BCA protein quantification kit. Prepare 5×10 7 THP-1 cell membrane solution with a concentration of 1 cell / mL was used for subsequent experiments.

[0043] 2. Preparation of siRNA / protamine complex

[0044] Preparation of HEPES solution: Use the purchased HEPES solid to prepare a solution with a concentration of 0.02M and pH 7.0 using DEPC-treated water.

[0045] Preparation of C6G25S siRNA solution: Take 3OD of purchased C6G25S siRNA solid, add 1.5mL of HEPES solution to prepare a 2OD / mL C6G25S siRNA solution.

[0046] Preparation of protamine solution: Accurately weigh 10 mg of purchased protamine, add 9 mL of HEPES solution to dissolve, then take 1 mL and dilute to 50 mL with HEPES solution to prepare a protamine solution with a concentration of 22 μg / mL.

[0047] Preparation of protamine / siRNA complex suspension: Take 1.5 mL of 2OD / mL C6G25S siRNA solution and 1.5 mL of 22 μg / mL protamine solution respectively, mix them well to obtain the protamine / siRNA complex, which can be directly used to hydrate the phospholipid film.

[0048] 3. Preparation of lipid nanoparticles (siRNA / LP) encapsulating protamine / siRNA complexes: Precisely weigh the prescribed amount of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioctadeceneoxy-3-methylammonium propane (DOTMA) and cholesterol, dissolve them in chloroform, and form a thin film by vacuum rotary evaporation at 45°C and 75rpm for 40min. The phospholipid film is placed in a vacuum drying oven and dried overnight to further remove trace amounts of organic solvents. The dried phospholipid film is hydrated with the HEPES solution (pH 7.0) containing protamine / siRNA complexes prepared above at 55°C and 100rpm for 40min to mix them evenly. Then, the obtained lipid nanoparticles are subjected to probe ultrasound in a 55°C water bath (power 45%, total 2min) to obtain lipid nanoparticles encapsulating protamine / siRNA complexes.

[0049] 4. Preparation of ACE2-HEK293T cell membrane-siRNA / LP (PS-AMLN): Mix the prescribed amount of ACE2-HEK293T cell membrane with the lipid nanoparticles encapsulating the protamine / siRNA complex prepared in step 3, and then pass the mixed solution through a liposome extruder with a polycarbonate membrane with a pore size of 400 nm and 200 nm in sequence, and repeat the extrusion 20 times to obtain;

[0050] Preparation of THP-1 cell membrane-siRNA / LP (PS-TMLN): Mix the prescribed amount of THP-1 cell membrane with the lipid nanoparticles encapsulating the protamine / siRNA complex prepared in step 3, and then pass the mixed solution through a liposome extruder with a polycarbonate membrane with a pore size of 400 nm and 200 nm in sequence, and repeat the extrusion 20 times to obtain.

[0051] 5. Preparation of ACE2-HEK293T cell membrane / THP-1 cell membrane-siRNA / LP (PS-[A&T]MLN): The prescribed amount of ACE2-HEK293T cell membrane and THP-1 cell membrane were mixed, sonicated under ice bath conditions, and then mixed with the lipid nanoparticles encapsulating the protamine / siRNA complex prepared in step 3. The mixed solution was then passed through a liposome extruder with a polycarbonate membrane with a pore size of 400 nm and 200 nm in sequence, and extruded repeatedly 20 times to obtain.

[0052] Example 3

[0053] The prescription of the multifunctional composition is shown in Table 2. Except for the inconsistent prescription dosage and some preparation methods, the rest of the preparation process is the same as Example 2. The inconsistent preparation methods are as follows:

[0054] Preparation of C6G25S siRNA solution: Take 60 OD of purchased C6G25S siRNA solid, add 1.5 mL of HEPES solution to prepare a 40 OD / mL C6G25S siRNA solution.

[0055] Preparation of protamine solution: Accurately weigh 10 mg of purchased protamine, add 3 mL of HEPES solution to dissolve, then take 1 mL and dilute to 50 mL with HEPES solution to prepare a protamine solution with a concentration of 66.67 μg / mL (this value is rounded off).

[0056] Preparation of protamine / siRNA complex suspension: Take 1.5 mL of 40 OD / mL C6G25S siRNA solution and 1.5 mL of 66.67 μg / mL protamine solution respectively, mix them well to obtain the protamine / siRNA complex, which can be directly used to hydrate the phospholipid film.

[0057] Table 2 Multifunctional composition prescription

[0058] Prescription composition Dosage C6G25S siRNA 60OD Protamine 100 μg DOPC 87mg DOTMA 3mg cholesterol 10mg Chloroform 3mL HEPES solution 3mL <![CDATA[ACE2-HEK293T cell membrane (1×10 8 cells / mL)]]> 1.5mL <![CDATA[and / or THP-1 cell membrane (1×10 8 cells / mL)]]> 1.5mL

[0059] Note: The amount of protamine in Table 2 is calculated by rounding off.

[0060] Example 4

[0061] The prescription of the multifunctional composition is shown in Table 3. Except for the inconsistent prescription dosage and some preparation methods, the rest of the preparation process is the same as Example 2. The inconsistent preparation methods are as follows:

[0062] Preparation of C6G25S siRNA solution: Take 100 OD of purchased C6G25S siRNA solid, add 1.5 mL of HEPES solution, and prepare a C6G25S siRNA solution with a concentration of 66.67 OD / mL (this value is rounded off).

[0063] Preparation of nona-arginine solution: accurately weigh 10 mg of purchased nona-arginine, add 3 mL of HEPES solution to dissolve, then take 1 mL and dilute to 50 mL with HEPES solution to prepare a protamine solution with a concentration of 66.67 μg / mL (this value is rounded off).

[0064] Preparation of nona-arginine / siRNA complex suspension: Take 1.5 mL of 66.67 OD / mL C6G25S siRNA solution and 1.5 mL of 66.67 μg / mL nona-arginine solution respectively, mix them well to obtain nona-arginine / siRNA complex, which can be directly used to hydrate phospholipid film.

[0065] Table 3 Multifunctional composition prescription

[0066]

[0067]

[0068] Note: The amounts of C6G25S siRNA and protamine in Table 3 are rounded off.

[0069] Example 5

[0070] Pseudovirus neutralization experiment: ACE2-293T cells were plated in 96-well plates, 2×10 4 / well, 100 μL culture medium; when the cell growth density reaches about 80% on the second day, prepare for pseudovirus neutralization experiment. 50 μL of drugs of different concentrations (prepared according to the prescription process of Example 1 and Example 2) and an equal volume of 650 TCID 50 The six typical pseudovirus liquids were incubated in a 37°C incubator for 1 hour; the supernatant of ACE2-293T cells in the 96-well plate was discarded, and the pseudovirus-composition mixture was diluted 10 times with culture medium and gently added along the well wall, 100 μL / well, and placed in a cell culture incubator for 24 hours. At the same time, a positive control group and a negative control group were set up, in which the positive control group was a culture medium and pseudovirus mixed in equal volumes and then added to ACE2-293T cells; the negative control group was a group with only culture medium added. The Luciferase luminescence value of the samples in the 96-well plate was detected using a chemiluminometer and its IC 50 IC values ​​and inhibition rates of the multifunctional composition PS-[A&T]MLN against each pseudovirus subtype 50 (μg / ml) Specific results are as follows Figure 1 .

[0071] Pseudovirus inhibition experiment: Based on the results of the above PS-[A&T]MLN virus neutralization ability assay, a PS-[A&T]MLN concentration with a virus inhibition rate of approximately 80% was selected, and blank culture medium (Control) and Free siRNA, PS-AMLN, PS-TMLN, and PS-[A&T]MLN samples containing the same amount of siRNA were prepared, and incubated with 650TCID of WT virus and Omicron (BA.4 / 5) virus in the culture medium for 1 hour. Subsequently, according to the above pseudovirus neutralization experimental steps, the results of each group of preparations blocking the infection of the two viruses were obtained. The specific results of the inhibition rate of each preparation group on WT and OmicronBA.4 / 5 are shown as follows. Figure 2 .

[0072] Inhibition rate = 1-(reading of experimental group-reading of negative control group) / (reading of positive control group-reading of negative control group).

[0073] like Figure 1As shown in Figure 2, the multifunctional composition PS-[A&T]MLN has a strong inhibitory effect on six different subtypes of SARS-CoV-2 pseudoviruses, and the IC 50 Between 0.01124-0.08087mg / ml.

[0074] like Figure 2 As shown, the pseudovirus inhibition effects of different prescription compositions are different. The composition modified by cell membrane has obvious virus inhibition effect, and the composition modified by ACE2 high-expressing cell membrane has the strongest virus inhibition effect.

[0075] Example 6

[0076] Neutralization experiment of inflammatory factors: Each preparation containing 100 μL of different concentrations was mixed with PBS containing cytokines IL-6 (2000 pg / mL), IL-1β (1600 pg / mL), TNF-α (2500 pg / mL), and GM-CSF (500 pg / mL), and incubated at 37°C for 30 min. The sample was then centrifuged at 15,000 × g for 15 min to remove the nanoparticles, and the residual IL-6, IL-1β, TNF-α, and GM-CSF concentrations in the supernatant were measured using ELISA kits for IL-6, IL-1β, and GM-CSF to examine the neutralization of each group of vectors and IL-6, IL-1β, TNF-α, and GM-CSF. The binding of each group of preparations to different inflammatory factors is shown in Figure 3 .

[0077] like Figure 3 As shown, the cell membrane modified composition has a significant effect of neutralizing inflammatory factors.

[0078] Example 7

[0079] Intracellular siRNA delivery experiment: RAW cell suspension in logarithmic growth phase was cultured at 1.8×10 5The cells were planted at a density of 100 / well in a laser confocal culture dish and placed in a cell incubator for 24 hours. PS-[A&T]MLN (siRNAs are all labeled with Cy5, and the labeled siRNAs can be purchased from the corresponding company) was diluted with DMEM culture medium to a siRNA concentration of 300nM, and 2mL was added to the confocal dish containing cells, and placed in a cell culture incubator for incubation under light-proof conditions. After the incubation was terminated, the dish was removed, the culture medium was discarded, 2mL PBS was added for rinsing twice, 2mL 37°C preheated LysoTracker Red dye solution was added, and the dish was placed in a cell culture incubator for 30 minutes in the dark. After the culture was completed, the dish was removed, the culture medium was discarded, 2mL PBS was added for rinsing twice, 2mL 4% paraformaldehyde tissue fixative was added, and 2mL PBS was added for rinsing twice after fixation at room temperature for 15-20 minutes. Add 2 mL of 10 μg / mL Hoechst 33258 dye solution to stain the cell nucleus. After 10 minutes, discard the dye solution and add 2 mL PBS to rinse twice. Finally, add 1 mL of 50% glycerol (prepared in sterile PBS) to each Confocal dish for sealing and store in the dark. Use CLSM for observation and select appropriate fields for photography and analysis. The endosomal escape results of the multifunctional composition are shown in Figure 2. Figure 4 .

[0080] like Figure 4 As shown in the figure, the vector enters the cell for 4h, 6h, 8h and 12h, where the blue part is the cell nucleus labeled by Hoechst33258, the red part is the lysosome labeled by Lyso trackerred, the green part is Cy5siRNA, and the yellow part is due to the superposition of green and red, indicating that the vector is taken up by the lysosome. The results in the figure show that: at 4h, the green and red are separated, indicating that the vector has not entered the lysosome; at 6h, there is a small amount of yellow part, and the yellow part reaches the maximum at 8h, indicating that most of the vector is taken up by the lysosome 8h after entering the cell; at 12h, the yellow fluorescence signal decreases, the green fluorescence signal increases, and the red fluorescence weakens, indicating that the vector successfully escapes from the lysosome.

[0081] From the above experiments, it can be seen that the multifunctional composition prepared by the present invention can inhibit the SARS-CoV-2 virus and its variants in free and infected cells, while neutralizing cellular inflammatory factors. It is not only effective against the SARS-CoV-2 virus and its variants, but also can treat both the symptoms and the root causes. It is expected to achieve a complete cure for SARS-CoV-2 virus and its variants infection while reducing the patient's symptoms.

[0082] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A multifunctional composition for treating infection with SARS-CoV-2 and its variants, characterized in that: The composition comprises the following components by weight, based on a final volume of 6 ml: 1-100 OD C6G25S siRNA, 0.5-2.5 ml ACE2-HEK293T cell membrane and / or 0.5-2.5 ml THP-1 cell membrane, 20-100 mg phospholipid material, 25-100 μg cationic material, and 0.5-10 mg cholesterol; The phospholipid materials are dioleoyl phosphatidylcholine and 1,2-dioctadeceneoxy-3-methylammonium propane; The cationic material is protamine or nona-arginine; The sequences of the sense strand and antisense strand of the C6G25S siRNA are shown in SEQ ID No. 1-2 respectively; The preparation method of the multifunctional composition comprises the following steps: The cationic material was diluted with a HEPES solution and then added to a HEPES solution containing C6G25S siRNA, and incubated at room temperature to obtain a cationic material / siRNA complex; The phospholipid material and cholesterol are dissolved in chloroform, and vacuum rotary evaporation is performed to form a film, the phospholipid film is dried, and the dried phospholipid film is hydrated with a HEPES solution containing a cationic material / siRNA complex, and then the prepared lipid nanoparticles are subjected to ultrasonic treatment to obtain lipid nanoparticles encapsulating the cationic material / siRNA complex; The cell membrane expressing ACE2 protein is mixed with lipid nanoparticles encapsulating a cationic material / siRNA complex, and then the mixed solution is sequentially passed through a liposome extruder with a polycarbonate membrane and repeatedly extruded to obtain a cell membrane expressing ACE2 protein-siRNA / LP; The cell membrane expressing the cytokine receptor is mixed with lipid nanoparticles encapsulating the cationic material / siRNA complex, and then the mixed solution is passed through a liposome extruder with a polycarbonate membrane and repeatedly extruded to obtain the cell membrane expressing the cytokine receptor-siRNA / LP; The cell membrane expressing ACE2 protein-siRNA / LP and the cell membrane expressing cytokine receptor-siRNA / LP are mixed, ultrasonicated, and then mixed with lipid nanoparticles encapsulating a cationic material / siRNA complex, and then the mixed solution is passed through a liposome extruder with a polycarbonate membrane and repeatedly extruded to obtain the cell membrane expressing ACE2 protein / cell membrane expressing cytokine receptor-siRNA / LP; The cell membrane expressing the ACE2 protein is an ACE2-HEK293T cell membrane, and the cell membrane expressing the cytokine receptor is a THP-1 cell membrane.

2. The multifunctional composition according to claim 1, characterized in that The cell membrane was used at a concentration of 1×10 5 ~1×10 10 Cell membrane of cells / ml.

3. Use of the multifunctional composition according to claim 1 or 2 in the preparation of a product for treating infection with SARS-CoV-2 and its variants.

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