CN115252555B - Membrane fusion liposome, preparation method and application thereof in protein delivery - Google Patents
Membrane fusion liposome, preparation method and application thereof in protein delivery Download PDFInfo
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- CN115252555B CN115252555B CN202210636337.6A CN202210636337A CN115252555B CN 115252555 B CN115252555 B CN 115252555B CN 202210636337 A CN202210636337 A CN 202210636337A CN 115252555 B CN115252555 B CN 115252555B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/10—Dispersions; Emulsions
- A61K9/127—Liposomes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
- A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Dispersion Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biomedical Technology (AREA)
- Medicinal Preparation (AREA)
Abstract
A membrane fusion liposome, a preparation method and application thereof in protein delivery belong to the technical field of protein delivery. The membrane fusion liposome comprises neutral lipid molecules, cationic lipid molecules and hydrophobic large conjugated pi structural molecules, wherein the number of electrons of pi is not less than 28. The mole ratio of the neutral lipid molecules, the cationic lipid molecules and the large conjugated pi structural molecules is 20-70:20-70:1-10. The membrane fusion liposome nano-carrier provided by the invention can be used for delivering protein drugs into cytoplasm in one step in a membrane fusion manner, and has the advantages of high delivery speed and high delivery efficiency compared with the way of mediating protein cytoplasmic delivery through an endocytic way. And the preparation process is simple, the cost is low, the use is convenient, and the protein does not need to be modified.
Description
Technical Field
The invention belongs to the technical field of protein delivery, and particularly relates to a membrane fusion liposome, a preparation method and application thereof in protein delivery.
Background
Most diseases are caused by protein dysfunction, which makes proteins potential candidates. For example, proteins such as insulin, monoclonal antibodies, cytokines, transcription factors, enzymes, and polypeptides have been developed as drugs for treating diabetes, cancer, cardiovascular diseases, bacterial infections, and the like. Protein therapy has higher specificity, lower adverse reactions, and shorter drug development cycles than traditional chemical drugs. Compared with genetic drugs, the protein acts on the target to regulate the biological process more directly and more specifically, so that permanent gene mutation, off-target effect caused by continuous gene expression and cancerogenic risk are avoided. However, all protein drugs on the market today are developed based on extracellular targets and do not act on intracellular targets. The main reason is that proteins are difficult to enter cells, and the cell uptake efficiency is low. At present, protein medicines mainly enter cells through cell uptake mediated by inclusion bodies, part of proteins are degraded in lysosomes, part of proteins are discharged from the cells through exocytosis, and a small amount of the remaining proteins can enter cytoplasm. Thus, protein delivery efficiency based on endosomal uptake is lower. While development of non-endosomal mediated delivery modes that bypass lysosomes is expected to increase the efficiency of protein delivery.
The liposome has wide application in the aspects of delivering small molecule drugs and nucleic acid drugs, and can effectively improve the cell delivery efficiency of the drugs. The membrane fusion liposome (fusogenic liposome, fuL) is a special liposome, can be fused with a cell membrane directly in theory, bypasses a lysosome, and directly conveys the medicine into cytoplasm, so that the degradation problem of the medicine in the lysosome is avoided. Currently, the most potential class of membrane-fusogenic liposomes is the class comprising hydrophobic pi molecules. Such membrane fusion liposomes are composed of neutral lipid molecules, cationic lipid molecules and hydrophobic pi-structure molecules. Neutral molecules stabilize the fusion intermediate state and cationic lipid molecules have a positive charge, which increases electrostatic attraction to cells. The hydrophobic pi structural molecules can promote membrane fusion of the liposome and the cell membrane. However, the existing membrane-fusogenic liposomes cannot meet the requirement of efficient protein delivery, and the fusion efficiency is still to be improved.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, it is an object of the present invention to devise a membrane-fusogenic liposome, a method of preparation and its use in protein delivery. The membrane fusion liposome provided by the invention is used as a nano carrier to deliver protein drugs into cytoplasm in one step in a membrane fusion mode, and has the advantages of high delivery speed and high delivery efficiency compared with the way of mediating protein cytoplasmic delivery through an endocytic pathway. The preparation process is simple, the cost is low, the use is convenient, and the protein does not need to be modified.
In order to achieve the above object, the present invention provides the following technical solutions:
a membrane-fusogenic liposome characterized in that the membrane-fusogenic liposome comprises a neutral lipid molecule, a cationic lipid molecule, and a hydrophobic large conjugated pi-structure molecule, wherein pi has an electron number of not less than 28.
The membrane fusion liposome is characterized in that the head group of the neutral lipid molecule is phosphatidylcholine or phosphatidylethanolamine, the phase transition temperature is below 37 ℃, and the neutral lipid molecule comprises dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-oleoyl phosphatidylcholine, L-alpha-phosphatidylethanolamine, L-alpha-phosphatidylcholine and the like.
The membrane fusion liposome is characterized in that the phase transition temperature of the cationic lipid molecules is below 37 ℃, and the cationic lipid molecules comprise (2, 3-dioleoyl-propyl) -trimethylamine, 1, 2-dioleoyl-3-dimethylaminopropane, didodecyl trimethylammonium bromide, 1, 2-dilauroyl-sn-glycerol-3-ethylphosphocholine, dioleoyl ethyl phosphatidylcholine and the like.
The membrane fusion liposome is characterized in that the hydrophobic large conjugated pi structural molecule comprises CyBI7, cyBI5 and the like, wherein the chemical structural formula of CyBI7 (Nano Research,2021,14 (7), 2432-2440) is shown in the following formula I, and the chemical structural formula of CyBI5 (Colloids Surf B Biointerfaces,2021, 199:111537) is shown in the following formula II. Wherein CyBI7 is near infrared fluorescent dye, and the hydrophobic large conjugated pi structural molecule is synthesized by a structural symmetrical synthesis method or an asymmetrical synthesis method.
The membrane fusion liposome is characterized in that the molar ratio of the neutral lipid molecules to the cationic lipid molecules to the hydrophobic large conjugated pi structural molecules is 20-70:20-70:1-10, preferably 45-55:45-55:5-10, most preferably 50:50:5, and the particle size of the membrane fusion liposome is 50-200 nm.
A method for preparing a membrane-fusogenic liposome according to any one of the preceding claims, comprising the steps of:
(1) Weighing neutral lipid molecules, cationic lipid molecules and hydrophobic large conjugated pi structural molecules, dissolving in a solvent, and evaporating to remove the solvent to form a lipid membrane;
(2) Adding pure water into the lipid membrane formed in the step (1), and carrying out vortex rotation in a water bath for hydration to obtain a liposome solution;
(3) Extruding the liposome solution obtained in the step (2) by a liposome extruder under the atmosphere of inert gas to obtain liposome dispersion liquid with uniform particle size distribution;
(4) And (3) adding the liposome dispersion liquid obtained in the step (3) into pure water for dialysis to obtain the membrane fusion liposome with the hydrophobic interlayer wrapping pi structural molecules.
The preparation method is characterized in that the solvent in the step (1) is a halogenated hydrocarbon solvent or an alcohol solvent, and the molar ratio of the neutral lipid molecules to the cationic lipid molecules to the hydrophobic large conjugated pi structural molecules is 20-70:20-70:1-10, preferably 45-55:45-55:5-10, and most preferably 50:50:5.
The preparation method is characterized in that pure water is added to the liposome solution in the step (2) to ensure that the concentration of phospholipid in the liposome solution is 1-50mM.
The preparation method is characterized in that the inert gas in the step (3) is nitrogen, and the extrusion times are 5-20 times;
the number of times of dialysis in the step (4) is 3 to 5.
The use of any of the membrane-fusion liposomes as nanocarriers for intracellular delivery of proteins or gene drugs;
the proteins include electronegative model proteins, protein drugs, vaccines, and the gene drugs include antisense DNA, antisense RNA, micrornas, small interfering RNAs, messenger RNAs, and plasmids.
Compared with the prior art, the invention has the following beneficial effects:
1) The invention adopts a film hydration method to entrap hydrophobic large conjugated pi structural molecules in a liposome interlayer. The hydrophobic large conjugated pi structural molecule is loaded in the interlayer of the liposome, which is beneficial to accelerating the fusion process of the liposome and the cell membrane. CyBI7 is an infrared fluorescent dye, and can be directly observed under a fluorescence microscope to take up fusogenic liposomes.
3) The preparation method and the using equipment of the membrane fusion liposome are simple, the operation is easy to control, the cost is low, the use is convenient, the in vitro protein delivery effect is good, and a novel carrier is provided for intracellular delivery of protein medicines.
Drawings
FIG. 1 is a particle size distribution of membrane-fusogenic liposomes after two months of fresh preparation and placement;
FIG. 2 is a graph showing in vitro membrane fusion efficiency of membrane-fusion liposomes over time;
FIG. 3 shows uptake of membrane-fusion liposomes on gastric carcinoma cell 823;
FIG. 4 is a graph showing transfection efficiency and enzymatic activity of membrane-fusogenic liposome delivery model protein β -galactosidase;
FIG. 5 is a cellular uptake mechanism of the membrane-fusogenic liposome delivery model protein β -Gal;
FIG. 6 is a mechanism of intracellular delivery of a membrane-fusogenic liposome.
Detailed Description
The technical scheme of the invention will be further described with reference to the accompanying drawings and examples. It is to be understood that the following examples are illustrative only and are not to be construed as limiting the scope of the invention. All techniques implemented based on the above description of the invention are intended to be included within the scope of the invention.
Unless otherwise indicated, the starting materials and reagents used in the following examples were either commercially available or may be prepared by known methods.
In the following examples, dioleoyl phosphatidylethanolamine (DOPE), (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (7-nitro-2, 1, 3-benzoxadiazol-4-yl) (DPPE-NBD), and 1, 2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (Lissamine rhodamine B) (DPPE-Rh) were all purchased from Avanti Polar Lipids Biol; 1,1 '-dioctadecyl-3, 3' -tetramethylindole tricarbocyanine iodide (DiR) was purchased from AAT Bioquest; cholesterol (Chol) was purchased from beggar's carboline technologies. CyBI7 was synthesized by a symmetrical synthesis (Nano Research,2021,14 (7), 2432-2440). Basal medium (RPMI-1640), fetal Bovine Serum (FBS) was purchased from Gibco. D-anhydrous glucose and regenerated cellulose dialysis bags (3500D) were purchased from Shanghai Source leaf organisms Co., ltd; beta-galactosidase (beta-Gal), beta-galactosidase reporter gene staining kit, beta-glucosidase activity detection kit was purchased from Beijing Soy Corp. Lipofectamine 3000 (LPF 3K) was purchased from Sieimer.
Example 1: preparation and stability characterization of CyBI7-FuL
The preparation method of the CyBI7-FuL comprises the following steps:
di-oleoyl phosphatidylethanolamine (DOPE), (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP), cyBI7 in a molar ratio of 50:50:5 (total lipid molar concentration 2 mM) were dissolved in chloroform, and then chloroform was removed by rotary evaporation to form a lipid film;
the pure water solution was added to the lipid membrane, and the lipid membrane was hydrated by spinning in a water bath at 30 ℃. After the formed lipid film is hydrated to become emulsion-like liposome suspension, the suspension is extruded on a 100nm polycarbonate film for 10 times by an extruder;
the liposome solution after extrusion was dialyzed 3 times against 100mM pure water to obtain CyBI7-FuL.
The membrane-fusion liposome DiR-FuL reported in the literature was also prepared as a control study group, and the preparation method thereof was as follows:
di-oleoyl phosphatidylethanolamine (DOPE), (2, 3-dioleoyl-propyl) -trimethylamine (DOTAP), diR in a molar ratio of 50:50:5 (total lipid molar concentration 2 mM) were dissolved in chloroform, and then chloroform was removed by rotary evaporation to form a lipid film;
the pure water solution was added to the lipid membrane, and the lipid membrane was hydrated by spinning in a water bath at 30 ℃. After the formed lipid film is hydrated to become emulsion-like liposome suspension, the suspension is extruded on a 100nm polycarbonate film for 10 times by an extruder;
the extruded liposome solution was dialyzed 3 times against pure water to obtain DiR-FuL.
The particle size distribution of CyBI7-FuL when freshly prepared and after two months of standing was determined by dilution in pure water, the results are shown in FIG. 1; as can be seen from FIG. 1, the particle size of CyBI7-FuL was about 140nm, and the particle size of the liposome was not substantially changed after two months, so that the stability of the membrane-fusogenic liposome prepared by the present invention was good.
Example 2: in vitro Membrane fusion efficiency of CyBI7-FuL
The efficiency of membrane fusion was determined using the fluorescence resonance energy transfer effect between membrane fusion liposomes and membrane component-mimicking liposomes. DiR-FuL was set as a control group to compare the membrane fusion efficiencies of CyBI7-FuL and DiR-FuL. Specifically, liposomes of FRET-L (DOPC/Chol/NBD-PE/Rho-PE 10/1/0.1/0.05) and of CyBI7-F100 (DOPC/Chol/NBD-PE/Rho-PE/DiR 10/1/0.01/0.005/0.50) and DiR-F100 (DOPC/Chol/NBD-PE/Rho-PE/CyBI 710/1/0.01/0.005/0.50) mimicking complete membrane fusion were first synthesized. Next, cyBI7-FuL and DiR-FuL were mixed with FRET-L for various periods of time (0-60 min), and the fluorescence intensity of Rho-PE (final concentration of Rho-PE 150nM, excitation 475nM, emission 595 nM) was measured, divided by the fluorescence intensities of Rho-PE in CyBI7-F100 and DiR-F100, respectively, to obtain membrane fusion efficiencies for various periods of time. As shown in FIG. 2, after 10min incubation with FRET-L, the membrane fusion efficiency of CyBI7-FuL was 98.6%, much higher than that of DiR-FuL (28.9%); after 20min incubation, the membrane fusion efficiency of CyBI7-FuL has reached 100% saturation, which is far higher than that of DiR-FuL (32.6%); after 1h incubation, the membrane fusion efficiency of DiR-FuL was still not high, only up to 41.2%. These results demonstrate that CyBI7-FuL has a rapid and efficient membrane fusion capability.
Example 3: uptake of CyBI7-FuL in gastric carcinoma cell 823
BGC-823 cells were cultured in RPMI-1640 medium containing 10% FBS and 1% P/S (penicillin/streptavidin double antibody), and the flask was placed at 37℃in a 5% carbon dioxide incubator. 823 cells were seeded in 96-well plates (0.5-1X 10) 4 Individual/well) and at 37 ℃,5% co 2 Culturing in an incubator for 24 hours. Then 100. Mu.L of serum-free medium containing CyBI7-FuL (lipid concentration 80. Mu.M) was added, followed by incubation in an incubator for different times (5 min,10min,30min,60 min), followed by washing 3 times with PBS, adding 100. Mu.L of serum-containing medium, and finally, in a fluorescence microscope (Leica)Observation photographing (CyBI 7 detection using Cy5 channel) was performed under the condition, and fluorescence intensity was quantified by Image J, and the result is shown in FIG. 3. At 5min, cellular uptake of CyBI7-FuL has been observed; with the time, the cell uptake increased gradually, and at 30min, the cell uptake reached saturation, indicating that the cell uptake rate of CyBI7-FuL was extremely fast.
Example 4: transfection efficiency enzyme Activity of CyBI7-FuL to deliver beta-Gal
First, complexes of CyBI7-FuL and control DiR-FuL and LPF3K with β -Gal were prepared. Wherein the CyBI7-FuL and DiR-FuL and beta-Gal complexes were prepared as CyBI7-FuL or DiR-FuL (2 mM, 13. Mu.L) mixed with beta-Gal (1 mg/mL, 2.0. Mu.L) for 5min; LPF3K and beta-Gal complex was prepared as beta-Gal (1 mg/mL, 1.95. Mu.L) and mixed with serum-free medium (163. Mu.L) and then mixed with LPF3K (3. Mu.L) in serum-free medium for 10min.
Next, the transfection efficiency of each group delivering β -Gal was determined. Since β -Gal is a relatively high molecular weight (473 kDa), negatively charged (pi=5.1) enzyme that is difficult to permeate cell membranes by itself, and is capable of catalyzing the hydrolysis of X-Gal to insoluble blue dye and galactose, transfection efficiency can be determined by observing the blue material in the cell to determine CyBI7-FuL mediated β -Gal delivery under different processing conditions. Specifically, a density of 5000 cells/well was seeded in a 96-well plate into 823 cells, and when the cell density reached 80%, free β -Gal, LPF3K@β -Gal, β -Gal@DiR-FuL, and β -Gal@CyBI7-FuL (β -Gal: 6. Mu.g/mL) were added to the cells, and incubated for 60 minutes, followed by fixation of the cells and staining of β -Gal. The specific fixing and dyeing steps are as follows: the cell culture was aspirated and washed 3 times with PBS. Subsequently, 100. Mu.L of the beta-Gal staining fixative solution was added and the mixture was fixed at room temperature for 10min. The cell fixative was aspirated and washed three times with 3min each with PBS. The PBS was removed by pipetting, 100. Mu.L of staining solution was added to each well and incubated overnight at 37 ℃. Finally, the distribution of β -Gal in the cells in all treatment groups was photographed with bright field. Image J was used to count the percentage of cells containing blue material and the transfection efficiency of the different treatment groups was calculated and the results are shown in figure 4 a.
Under the same conditions, the protein transfection efficiencies of free β -Gal and LPF3K@β -Gal were negligible, whereas the average transfection efficiencies of the proteins of the DiR-FuL and CyBI7-FuL groups were 33.1% and 79.4%, respectively. The protein delivery efficiency of the membrane fusion liposome is far higher than that of the traditional endocytic liposome LPF3K, and the membrane fusion liposome CyBI7-FuL developed by the invention is far higher than that of the DiR-FuL reported in the literature.
Next, the enzyme activity of each group delivering β -Gal was determined. Specifically, 823 cells were inoculated in 96-well plates at a density of 5000 cells/well, and when the cell density reached 80%, free β -Gal, LPF3K@β -Gal, β -Gal@DiR-FuL, and β -Gal@CyBI7-FuL (β -Gal: 6. Mu.g/mL) were added to the cells, incubated for 60min, the cell culture broth was aspirated, and the cell lysate was added for 60min. Then adding the lysate and sample into another 96-well plate, adding detection reagent, incubating at 37deg.C for at least 3 hr, and adding 150 μL Na 2 CO 3 The reaction was terminated with the solution (1M). The optical density (420 nm) of the reaction solution was immediately measured. The enzymatic activity of β -Gal in each group was obtained by comparing the optical density value of each group with the optical density value of the enzymatic activity of free β -Gal, as shown in FIG. 4 b.
The enzyme activity of free β -Gal delivered into cells was only 6.5%; LPF3K entrapped beta-Gal achieves the effect of protecting part of enzyme, and the enzyme activity is improved to 21.9%; diR-FuL is a membrane fusion liposome reported in the literature, and the membrane fusion performance of the membrane fusion liposome enhances the protection effect of the membrane fusion liposome on enzyme compared with the traditional liposome, and the enzyme activity reaches 49.7%; the CyBI7-FuL has extremely strong protection capability to enzyme and enzyme activity reaching 92.5 percent due to the high-efficiency membrane fusion capability. These data demonstrate that CyBI7-FuL can effectively protect the enzymatic activity of the delivered protein when used for cytoplasmic delivery of the protein, far superior to DiR-FuL reported in the literature.
Example 5: cell uptake mechanism for CyBI7-FuL delivery-Gal
823 cells were individually treated at 5X 10 4 A density of/mL was plated in 24-well plates with 1mL of medium per well. When the cell density reached 80%, the old medium was removed, new medium was added, and inhibitor group, low temperature group, no inhibitor group was set. In the inhibitor group, various inhibitors (chlorpromazine (CPZ) 10. Mu.g/mL, genistein (Genistein) 150. Mu.M, wortmanni (Wortmanni) 5. Mu.M, and cell pine) were addedRelaxin (CB) 10. Mu.g/mL and methyl-cyclodextrin (MβCD) 5mg/mL were incubated in an incubator for 30min; then, cyBI7-FuL/-Gal was added and incubation was continued for 1.5h at 37 degrees, followed by cell fixation and β -Gal staining. In the low temperature group, cyBI7-FuL/β -Gal complex was added, incubated at 4℃for 1.5h, followed by cell fixation and β -Gal staining. In the inhibitor-free group, cyBI7-FuL/β -Gal complex was directly incubated with cells for 1.5h at 37℃followed by cell fixation and β -Gal staining. At the same time, the DiR-FuL/β -Gal complex was incubated with the cells for 1.5h under different inhibitor treatments, and cell fixation and β -Gal staining were performed. The cell fixation and beta-Gal staining and image processing method were the same as in example 4, and the results are shown in FIGS. 5a and 5 b.
The results indicate that the average transfection efficiency of CyBI7-FuL mediated beta-Gal is 86.5% in the inhibitor-free group; after 4 degrees of treatment, the average transfection efficiency was 70.6%; the average transfection efficiency after treatment with different endocytic inhibitors was: genistein (79.0%), cytochalasin beta (76.8%), methyl-beta-cyclodextrin (71.9%), chlorpromazine (75.9%), wortmannin (75.2%). Through statistical analysis, there was no significant difference between the protein transfection efficiency of the 4 degree treated and endocytic inhibitor treated groups and the non-inhibitor treated group, demonstrating that CyBI7-FuL delivered the protein into the cells in a non-endocytic cell uptake mode, the protein did not have lysosomal degradation problems, in concert with the high enzymatic activity of the delivered protein above. Whereas the average transfection efficiency of the control group DiR-FuL mediated beta-Gal is 39.8%, and after 4-degree treatment, the average transfection efficiency is 9.4%; the average transfection efficiency after treatment with different endocytic inhibitors was: genistein (22.1%), cytochalasin beta (11.0%), methyl-beta-cyclodextrin (8.5%), chlorpromazine (41.5%), wortmannin (28.9%). Through statistical analysis, the 4-degree group, the cytochalasin beta group and the methyl-beta-cyclodextrin group are significantly different from the group without inhibitor, which shows that DiR-FuL has membrane fusion capability, but part of liposome still enters cells through phagocytosis and lipid raft mediated endocytosis, so that the potential of the membrane fusion liposome cannot be fully exerted.
Unlike the membrane-fusion liposome DiR-FuL reported in the literature, the CyBI7-FuL developed by the invention enters cells in a full membrane fusion mode, so that the advantages of the membrane-fusion liposome can be fully exerted, a lysosome is bypassed, and the protein delivery efficiency and the protein activity are improved, as shown in figure 6.
The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. A membrane-fusogenic liposome characterized in that the membrane-fusogenic liposome comprises a neutral lipid molecule, a cationic lipid molecule, and CyBI7;
the head group of the neutral lipid molecule is phosphatidylcholine or phosphatidylethanolamine, the phase transition temperature is below 37 ℃, and the neutral lipid molecule comprises dioleoyl phosphatidylethanolamine, dioleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-oleoyl phosphatidylcholine, L-alpha-phosphatidylethanolamine and L-alpha-phosphatidylcholine;
the phase transition temperature of the cationic lipid molecules is below 37 degrees.
2. A membrane-fusogenic liposome according to claim 1, wherein the cationic lipid molecule comprises (2, 3-dioleoyl-propyl) -trimethylamine, 1, 2-dioleoyloxy-3-dimethylaminopropane, 1, 2-dilauroyl-sn-glycero-3-ethylphosphocholine, dioleoylethylphosphocholine.
3. The membrane-fusogenic liposome according to claim 1, wherein the molar ratio of the neutral lipid molecule to the cationic lipid molecule to CyBI7 is 20-70:20-70:1-10, and the particle size of the membrane-fusogenic liposome is 50-200 nm.
4. A membrane-fusogenic liposome according to claim 3, wherein the molar ratio of neutral lipid molecules, cationic lipid molecules and CyBI7 is 45-55:45-55:5-10.
5. A membrane-fusogenic liposome according to claim 4, wherein the molar ratio of neutral lipid molecules, cationic lipid molecules and CyBI7 is 50:50:5.
6. A method for preparing a membrane-fusogenic liposome according to any one of claims 1 to 5, comprising the steps of:
weighing neutral lipid molecules, cationic lipid molecules and CyBI7, dissolving in a solvent, and evaporating to remove the solvent to form a lipid membrane;
adding pure water into the lipid membrane formed in the step (1), and carrying out vortex rotation in a water bath for hydration to obtain a liposome solution;
extruding the liposome solution obtained in the step (2) by a liposome extruder under the atmosphere of inert gas to obtain liposome dispersion liquid with uniform particle size distribution;
and (3) adding the liposome dispersion liquid obtained in the step (3) into pure water for dialysis to obtain the membrane fusion liposome with the hydrophobic interlayer wrapping pi structural molecules.
7. The method according to claim 6, wherein the solvent in the step (1) is a halogenated hydrocarbon solvent or an alcohol solvent, and the molar ratio of the neutral lipid molecule, the cationic lipid molecule and CyBI7 is 20-70:20-70:1-10.
8. The method of claim 7, wherein the molar ratio of neutral lipid molecules, cationic lipid molecules and CyBI7 is 45-55:45-55:5-10.
9. The method of claim 8, wherein the molar ratio of neutral lipid molecules, cationic lipid molecules, and CyBI7 is 50:50:5.
10. The method of claim 6, wherein pure water is added to the liposome solution in step (2) to achieve a phospholipid concentration of 1 to 50mM.
11. The method according to claim 6, wherein the inert gas in the step (3) is nitrogen, and the number of times of extrusion is 5 to 20;
and (3) dialyzing for 3-5 times in the step (4).
12. Use of the membrane-fusion liposome according to any one of claims 1-5 as nanocarriers in the preparation of a medicament for intracellular delivery of a protein or a gene drug;
the proteins include electronegative model proteins, protein drugs, vaccines, and the gene drugs include antisense DNA, antisense RNA, micrornas, small interfering RNAs, messenger RNAs, and plasmids.
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