WO2016137236A1 - Method for detecting virus by using liposome or liposome-polymer hybrid - Google Patents

Abstract

The present invention relates to a method for detecting a virus by using a liposome or a liposome-polymer hybrid and, more specifically, to a method for detecting a virus by making a liposome or a liposome-polymer hybrid, both of which contain a chromogenic substrate, come into contact with a sample assumed to contain a virus, and then detecting whether there is a virus, through color development according to whether there is an emission of the chromogenic substrate, which is contained in the liposome or the liposome-polymer hybrid, by the contact made therebetween. According to the present invention, the method for detecting a virus by using a liposome or a liposome-polymer hybrid allows a virus to be easily detected with the naked eye. Particularly, when using a liposome or a liposome-polymer hybrid, both of which contain hydrophobic 3,3',5,5'-tetramethyl bezidine (TMB) or iodine, which are chromogenic substrates, the stability to natural color development by oxygen or a chemical reaction is high since TMB or iodine does not leak to the outside of the liposome or the liposome-polymer hybrid. Additionally, it was shown that TMB or iodine contained in the liposome or the liposome-polymer hybrid is discharged and a virus detection signal to be generated by a reaction with HRP or starch, both of which are color development inducer materials, is remarkably high when a virus binds to the liposome or the liposome-polymer hybrid through a viral membrane protein.

Description

Virus detection method using liposomes or liposome-polymer hybrids

The present invention relates to a virus detection method using a liposome or a liposome-polymer hybrid, and more particularly, a liposome or a liposome-polymer hybrid containing a chromogenic substrate is contacted with a sample presumed to contain a virus. Next, the present invention relates to a virus detection method characterized by detecting the presence or absence of a virus by coloration according to the presence or absence of a chromophore substrate contained in a liposome or a liposome-polymer hybrid by the contact.

Liposomes are W / O / W type emulsions proposed by Bangham in the 1960s ( J. Mol . Biol., 13: 238, 1965), in which amphiphilic phospholipids are self-arranged by hydrophobic forces in the water phase. The phospholipid membrane constituting the liposome has the same structure as the cell, and is easily used for cell introduction, and has a large space for supporting a water-soluble substance therein compared to other structures, so that it is used as a carrier and carrier for the water-soluble substance ( Eur . J. Pharm . Biopham, 62: 110, 2006 ; Nat Rev. Drug Discov, 145:... 4, 1979).

In general, liposomes are nano-sized (1 μm or less) capsules as phospholipid delivery and mediators, and they can contain both lipophilic and hydrophilic functional materials, so they are suitable for living organisms similar to human skin cells. It is a substance that, when added to a hydrophilic formulation, remains suspended and has surface stability.

Liposomes are spherical vesicles in which the phospholipid bilayer surrounds the water phase. The lipid membrane is an amphiphilic phospholipid consisting of two hydrophobic fatty acid groups and a hydrophilic phosphate group, which forms a double membrane in aqueous solution, which forms closed vesicles like artificial cells. In the double membrane structure, the non-polar fatty acid tail faces the inside of the membrane and the polar head faces outward. Incorporating drugs into such liposomes has been attracting attention as a structure of particle bodies prepared by assembling with polymers, drugs, and antigens, as it can enhance the therapy by reducing the toxicity of drugs and increasing their effects.

Liposomes are fully enclosed structures that include a lipid bilayer membrane containing encapsulated aqueous medium. Liposomes may comprise many concentric lipid bilayers (multilamellar vesicles or MLVs) or single membrane bilayers (unilamellar vesicles) separated by an aqueous phase. The lipid bilayer consists of two lipid monolayers with hydrophobic "tail" and hydrophilic "head" regions. In the membrane bilayer, the hydrophobic “tails” in the lipid monolayer are arranged towards the center of the bilayer and the hydrophilic “heads” are arranged towards the aqueous phase.

The basic structure of liposomes can be prepared by known methods. For example, lipid molecules suspended in an organic solvent are evaporated under reduced pressure to form a dry film in a vessel, and an appropriate amount of aqueous phase is added to the vessel and the mixture is stirred. The mixture can then be prepared by standing without shaking for a time sufficient to form a multilamellar vesicle. Unilamella vesicles can also be prepared by known techniques (eg, US Pat.

Liposome-polymer hybrids are biofilm mimetic amphiphilic structures composed of low amounts of lipids (eg, phospholipids) and high molecular weight polymers (eg, amphiphilic block copolymers). The liposome-polymer hybrid is composed of a lipid component having a biological function (receptor, molecular recognition, etc.) and a polymer having a structural function (structural stability, etc.) can be prepared to bind to the target material (Olubummo A1 et al. , Langmuir, 30 (1): . 259-67, 2014; Schulz M et al, Angew Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem . Soc ., 81: 303-311, 1986; Binder WH et al., Angew Chem ., 115 (47): 5980-6007, 2003; Binder WH et al., Angew Chem Int Ed Engl. , 42 (47): 5802-27, 2003).

Protein conjugated liposomes or liposome-polymer hybrids can be designed for diagnostic purposes. Liposomes or liposome-polymer hybrids can be covalently bound to proteins, antibodies and immunoglobulins. For example, thiolated IgG or Protein A can be covalently bound to lipid vesicles and the thiolated antibody or Fab 'fragment can be bound to liposomes or liposome-polymer hybrids.

Biosensor systems can provide easier and more useful information for analyzing data by detecting signals using the properties of fluorescent nanomaterials at the cellular or in vivo level. Chemical and biosensors are materials or devices that detect and measure information from an object to be measured and change the measurable amount into a usable signal. When the sensor acquires information from the target, the sensor converts the signals into recognizable signals such as color, fluorescence, and electrical signals to assist human judgment. When the sensor recognizes the target material, it sends a signal through a signal converter for human recognition. In particular, sensors used in biosensors require high selectivity and sensitivity to target materials to be detected. Enzymes and antibodies have excellent substrate specificity and high binding capacity, but have the disadvantage of low stability and high price when immobilized in a sensor device.

Nanobiosensors are biosensors that are improved by advanced nanotechnology, which converts reactions by binding to biocognitive materials into signals, and refers to sensors that can quickly test specific materials. This is the same principle as the enzyme-substrate complex of the biological concept, in which one ligand is only reactive with one substance having a specific component for the ligand and measures the degree of reactivity. Miniaturized biosensor using nanotechnology minimizes human injury and enables painless human diagnosis and has the advantage of directly analyzing single cells. In addition, biosensors with improved operating characteristics such as high stability, fast response time, high sensitivity, and high selectivity using nanotechnology enable continuous measurement of human diagnosis and single-molecule analysis.

Currently, most virus detection methods use materials harmful to the human body, such as metals and polymers, and there is always the inconvenience of purifying virus-derived proteins or nucleic acids and then using antibodies or probes for accurate molecular biological diagnosis. The whole process takes as little as a day, as much as a week. In addition, most of the analysis process has a characteristic to rely on expensive analysis equipment, there is a limit in analyzing the detection of viruses in real time in the field (onset area).

Regarding virus detection, Korean Patent No. 1561395 treats a hemagglutinin-specific degrading enzyme to activate a virus and contacts the activated virus with amphiphilic particles under acidic conditions of pH 4-8. Disclosed is a virus detection method for detecting the presence or absence of a virus by detecting a change in fluorescence intensity emitted by self-quenching dye present in sex particles by dequenching. Such detection methods essentially include viral hemagglutinin degrading enzymes and require conditions for bringing the activated virus into contact with the amphiphilic particles under acidic conditions. Therefore, the additional use of a reagent such as hemagglutinin degrading enzyme is inexpensive in terms of cost, and the process of forming and maintaining conditions suitable for detection is complicated and inefficient.

Under these technical backgrounds, the present inventors have made intensive efforts to develop a method for easily detecting a target virus in a short time in a field (onset area), and thus embedding a chromogenic substrate inside a liposome or a liposome-polymer hybrid, and then the liposome or When the virus binds to the liposome-polymer hybrid through the viral membrane protein, the chromosomal substrate contained in the liposome or liposome-polymer hybrid is released, and the color can be easily detected by reacting with the chromophore-inducing substance to visually detect the virus. It confirmed and completed this invention.

Summary of the Invention

SUMMARY OF THE INVENTION An object of the present invention is to provide a virus detection composition, virus detection kit, virus detection strip, and virus detection method using the same for easily detecting a virus.

In order to achieve the above object, the present invention comprises a liposome or liposome-polymer hybrid (chromosome) containing a chromogenic substrate, and a chromosome inducing substance, when the virus binds to the liposome or liposome-polymer hybrid, It provides a composition for detecting a virus, characterized in that the chromogenic substrate contained in the liposome or liposome-polymer hybrid is released while reacting with the chromophore-inducing substance.

The present invention also provides a virus detection kit comprising the composition and a virus detection strip with a pad coated with the composition.

The present invention also provides a method for preparing a liposomal cell containing (a) contacting a liposome or a liposome-polymer hybrid containing a chromophore substrate with an estimated virus-containing sample; And (b) provides a method for detecting a virus comprising the step of adding a coloring inducing substance, to determine whether the color.

1 shows a process of releasing TMB contained in a liposome or a liposome-polymer hybrid when the liposome or liposome-polymer hybrid containing TMB, which is a chromogenic substrate, is contacted (bound) with a virus.

2 shows a process of inducing color development by oxidizing HMB, which is a coloring enzyme, oxidizing TMB.

Figure 3 is to determine whether the virus (influenza virus) detection using the liposomes containing the color substrate TMB.

Figure 4 shows a virus detection strip containing a reagent pad containing liposomes containing the color substrate TMB.

FIG. 5 confirms whether a virus (influenza virus) is detected using the virus detecting strip of FIG. 4.

FIG. 6 shows whether a virus (influenza virus) is detected by iodine starch reaction using liposomes containing iodine, which is a chromogenic substrate.

Figure 7 shows the absorbance measured at 405nm during the color reaction using liposomes containing the color substrate pNPP for virus detection (influenza virus).

FIG. 8 shows whether virus (influenza virus) is detected using liposomes containing pNPP.

Figure 9 shows the absorbance measured at 405nm during the color reaction using liposomes containing the chromogenic substrate pNPP for the detection of virus (influenza virus) after trypsin treatment.

FIG. 10 shows whether a virus (influenza virus) is detected according to the presence or absence of trypsin using liposomes containing pNPP as a chromogenic substrate.

Detailed description of the invention and preferred Embodiment

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention relates to a virus detection method using a liposome or a liposome-polymer hybrid, and more particularly, a liposome or a liposome-polymer hybrid containing a chromogenic substrate is contacted with a sample presumed to contain a virus. Next, the present invention relates to a virus detection composition, and a virus detection method using the same, which detect the presence or absence of a virus by checking color development according to the release of a chromogenic substrate contained in a liposome or a liposome-polymer hybrid (see FIG. 1). .

In one embodiment of the present invention, using a liposome containing a color substrate (TMB) was confirmed whether the virus (influenza virus) in the solution. As a result, as shown in Figures 2 and 3, the sample containing the virus is green, the color does not appear in the sample containing no virus, and the presence of the virus was visually confirmed (see Example 2).

In another embodiment of the present invention, using a liposome containing a chromogenic substrate (iodine) was confirmed whether the virus (influenza virus) detected in solution. As a result, as shown in Figure 6, the sample containing the virus was found in blue-purple, the color does not appear in the sample containing no virus, it was confirmed by the naked eye (see Example 6).

In another embodiment of the present invention, using a liposome containing a color substrate (pNPP) was confirmed whether the virus (influenza virus) in the solution. As a result, as shown in Figures 7 and 8, the sample containing the virus was yellow, the color does not appear in the sample containing no virus, and the presence of the virus was visually confirmed (see Example 8).

In another embodiment of the present invention, using a liposome containing a chromophore substrate (pNPP) was confirmed whether the virus (influenza virus) detected by trypsin treatment in solution. As a result, as shown in Figures 9 and 10, irrespective of the trypsin treatment, the sample containing the virus turned yellow, and the sample containing no virus did not show a change in color. 9).

Accordingly, the present invention includes liposomes or liposome-polymer hybrids containing a chromophore substrate, and a chromosome inducing substance, and when the virus binds to the liposomes or liposome-polymer hybrids, the liposomes Or it relates to a virus detection composition characterized in that the color is generated by reacting with the color-inducing substance while the color substrate contained in the liposome-polymer hybrid is released.

In the present invention, the virus may bind to liposomes or liposome-polymer hybrids through viral lipid membranes or membrane proteins. In this case, the viral membrane protein may be characterized as being HA (hemagglutinin). The lipid membrane may be characterized in that the PC (Phosphatidylcholine), PI (Phosphoinositides), PS (Phosphatidylserine), PE (Phosphatidylethanolamine), SM (Sphingomyelin).

In the present invention, the liposome-polymer hybrid is a biomembrane-like amphiphilic structure composed of a low molecular weight lipid (eg, phospholipid) and a high molecular weight polymer (eg, an amphiphilic block copolymer). The liposome-polymer hybrid may be made of a lipid component having a biological function (receptor, molecular recognition, etc.) and a polymer having a structural function (structural stability, etc.) and may be manufactured to bind to a target material (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem . Soc ., 81: 303-311, 1986; Binder WH et al., Angew Chem ., 115 (47): 5980-6007, 2003; Binder WH et al., Angew Chem Int Ed Engl ., 42 (47): 5802-27, 2003).

In the present invention, the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) , Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) Soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidylinositol (SPI), dipalmitoyl phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycer Rho-3-phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), hexadecyl Spokolin (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine ( PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), polyethylene glycol distea Roylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS) , Distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dypalmi Ilphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoyl phosphatidyl inositol (DMPI), distearoyl phosphatidyl inositol (DSPI), POPE ( 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac -glycero) and combinations thereof It may include one or more selected from.

In the present invention, the liposome-polymer hybrid is, for example, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI) ), Egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC) Soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidyl inositol (SPI), dipalmitoylphosphatidyl choline (DPPC), 1,2- Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoyl force Thidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE ), Palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3- Phosphatidylcholine (POPC), polyethylene glycol distearoylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), Dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA) ), Dimyristoyl phosphatidic acid (DMPA), distearo Phosphatidic acid (DSPA), dipalmitoylphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoylphosphatidyl inositol (DMPI), distearo Ilphosphatidylinositol (DSPI), POPE (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac- liposomes comprising one or more selected from the group consisting of glycero) and combinations thereof; And polymers such as polyisobutylene-block-polyethylene oxide copolymer, polybutadiene-b-polyethylene oxide copolymer, polydimethylsiloxane-g-polyethyleneoxide copolymer, poly (2-methyloxazoline) -b- It may be a mixture of a polymer comprising at least one selected from the group consisting of a copolymer of polymethylsiloxane-b-poly (2-methyloxazoline) and combinations thereof.

Specifically, a mixture of a lipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine) and an amphiphilic block polymer polyisobutylene-block-polyethyleneoxide copolymer or a lipid but POPC and an amphiphilic block polymer polybutadiene-b- mixture of polyethylene oxide, mixture of DPPC and poly (dimethylsiloxane) -g-poly (ethylene oxide), and mixture of DPPC or PE and poly (2-methyloxazolene) -b-poly (dimethylsiloxane) -b-poly (2-methyloxazoline) At least one selected from the group consisting of a mixture, but is not limited thereto.

In the present invention, the color development may be carried out through an enzymatic reaction or a non-enzymatic reaction, the color development substrate used in the enzymatic reaction is DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbasole), 5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium (BCIP / NBT), para-Nitrophenyl phosphate (pNPP), naphthol AS-TR phosphate, BCIP / INT (5-bromo 4-chloro-3-indolyl phosphate / iodonitrotetrazolium (NF), new fuchsin (NF), fast red TR salt (FRT), phenylenediamine, 3,3 ', 5,5'-tetramethylbenzidine (3 , 3 ', 5,5'-tetra methylbenzidine, dianisidine, amino-salicylic acid, 3,3'-diaminobenzidine, 3 3-amino-9-ethylcarbazole, 4-chloro-1-naphthol, TMB (3,3 ', 5,5'-tetramethyl bezidine) , ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] and OPD (ophenylenediamine) It is selected from the group consisting of, and the chromophore is a chromophore, HRP (Horseradish peroxidase), basic dephosphatase (Alkaline phosphatase), glucose oxidase (Glucose Oxidase), luciferase, beta-di-gal It may be characterized in that it is selected from the group consisting of lactosidase (β-D-galactosidase), malate dehydrogenase (MDH) and acetylcholinesterase (acetylcholinesterase).

In the present invention, the color substrate used in the non-enzymatic reaction is from the group consisting of iodine, calcium (calcium), copper (copper), iron (iron), amino acid (amino acid) and creatinine (creatinine) The chromophore-inducing substance is selected from starch, o-cresolphthalein complexone, bathocuproin disulfonate, and bashophenanthroline disulfonate. It may be characterized in that it is selected from the group consisting of ninhydrin (ninhydrin), o-phthalaldehyde (o-phthalaldehyde: OPA) and picrate (picrate).

In the present invention, the color development may be characterized by visual or enzyme-linked immunosorbent assay (ELISA), characterized in that the virus may be characterized in that the influenza virus, the viral membrane protein is It may be characterized as being HA (hemagglutinin). The lipid membrane may be characterized in that the PC (Phosphatidylcholine), PI (Phosphoinositides), PS (Phosphatidylserine), PE (Phosphatidylethanolamine), SM (Sphingomyelin).

In the present invention, the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) , Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) Soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidylinositol (SPI), dipalmitoyl phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycer Rho-3-phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), hexadecyl Spokolin (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine ( PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), polyethylene glycol distea Roylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS) , Distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dypalmi Ilphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoyl phosphatidyl inositol (DMPI), distearoyl phosphatidyl inositol (DSPI), POPE ( 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac -glycero) and combinations thereof It may include one or more selected from.

In the present invention, the liposome-polymer hybrid is, for example, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI) ), Egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC) Soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidyl inositol (SPI), dipalmitoylphosphatidyl choline (DPPC), 1,2- Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoyl force Thidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE ), Palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3- Phosphatidylcholine (POPC), polyethylene glycol distearoylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), Dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA) ), Dimyristoyl phosphatidic acid (DMPA), distearo Phosphatidic acid (DSPA), dipalmitoylphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoylphosphatidyl inositol (DMPI), distearo Ilphosphatidylinositol (DSPI), POPE (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac- liposomes comprising one or more selected from the group consisting of glycero) and combinations thereof; And polymers such as polyisobutylene-block-polyethylene oxide copolymer, polybutadiene-b-polyethylene oxide copolymer, polydimethylsiloxane-g-polyethyleneoxide copolymer, poly (2-methyloxazoline) -b- It may be a mixture of a polymer comprising at least one selected from the group consisting of a copolymer of polymethylsiloxane-b-poly (2-methyloxazoline) and combinations thereof.

Specifically, a mixture of a lipid DPPC (1,2-dipalmitoyl- sn -glycero-3-phosphatidylcholine) and an amphiphilic block polymer polyisobutylene-block-polyethyleneoxide copolymer or a lipid but POPC and an amphiphilic block polymer polybutadiene-b- mixture of polyethylene oxide, mixture of DPPC and poly (dimethylsiloxane) -g-poly (ethylene oxide), and mixture of DPPC or PE and poly (2-methyloxazolene) -b-poly (dimethylsiloxane) -b-poly (2-methyloxazoline) At least one selected from the group consisting of a mixture, but is not limited thereto.

In the present invention, the liposome or liposome-polymer hybrid may be characterized by having a negative charge.

Influenza viruses are inserted into host cells through receptor-mediated endocytosis, in which the virus converts HA ₀ to HA ₁ / HA ₂ through trypsin-mediated proteolytic cleavage at low pH, resulting in a host cell membrane. Enable fusion (Skehel, JJ et al., Annu . Rev. Biochem ., 69: 531-569, 2000; White, J. et al., J. Cell. Biol ., 89: 674-679, 1981 ).

When the liposomes or liposome-polymer hybrids of the present invention have a negative charge, the influenza virus contacts and releases the chromophore substrate contained without contacting the enzymes that degrade HA of the influenza virus.

Here, the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by color development, for example, to maintain the negative charge in the sample state for detecting color development, or to carry out the negative charge under various experimental conditions possible before color detection. It is enough to maintain, but is not limited thereto.

In another embodiment of the present invention, whether a virus (influenza virus) was detected using a strip attached to a reagent pad containing liposomes containing a chromosome substrate (TMB) was detected. As a result, as shown in Figure 5, in the sample containing the virus, the reagent pad showed a color reaction, and in the sample containing no virus, the reagent pad did not show a color reaction.

Therefore, in another aspect, the present invention relates to a virus detection kit comprising the composition and a virus detection strip with a pad coated with the composition.

In still another aspect, the present invention provides a method for preparing a liposomal cell containing (a) contacting a liposome or a liposome-polymer hybrid containing a chromogenic substrate with a putative virus-containing sample; And (b) by adding a coloring inducing substance, relates to a virus detection method comprising the step of confirming the color development.

In the present invention, the color development of step (b) is when the virus binds to the liposomes or liposome-polymer hybrids through the viral membrane protein, while the chromophore-containing substances contained in the liposomes or liposome-polymer hybrids are released, It may be characterized in that the color is expressed by the reaction, it can be characterized in that the color development of step (b) is characterized in that it is carried out through an enzyme reaction or a non-enzymatic reaction.

In the present invention, the color development of step (b) is when the virus binds to the liposomes or liposome-polymer hybrids through the viral membrane protein, while the chromophore-containing substances contained in the liposomes or liposome-polymer hybrids are released, It may be characterized in that the color is expressed by the reaction, it can be characterized in that the color development of step (b) is characterized in that it is carried out through an enzyme reaction or a non-enzymatic reaction.

In the present invention, the chromogenic substrate used for the enzyme reaction is DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbasole), BCIP / NBT (5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium ), para-Nitrophenyl phosphate (pNPP), naphthol AS-TR phosphate, BCIP / INT (5-bromo-4-chloro-3-indolyl phosphate / iodonitrotetrazolium), NF (new fuchsin), FRT (Fast Red TR Salt), phenylenediamine, 3,3 ', 5,5'-tetramethylbenzidine, 3,3', 5,5'-tetra methylbenzidine, dianisidine, Amino-salicylic acid, 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole, 4- 4-chloro-1-naphthol, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid )] And OPD (ophenylenediamine), and the chromophore is a chromophore, HRP (Horseradish peroxidase), Alkaline phosphatase, Glucose Oxidase, luciferase, beta-di-galactosidase, β-D-galactosidase, malate dehydrogenase and MDH It may be characterized in that it is selected from the group consisting of acetylcholinesterase (acetylcholinesterase).

In the present invention, the color substrate used in the non-enzymatic reaction is from the group consisting of iodine, calcium (calcium), copper (copper), iron (iron), amino acid (amino acid) and creatinine (creatinine) The chromophore-inducing substance is selected from starch, o-cresolphthalein complexone, bathocuproin disulfonate, and bashophenanthroline disulfonate. It may be characterized in that it is selected from the group consisting of ninhydrin (ninhydrin), o-phthalaldehyde (o-phthalaldehyde: OPA) and picrate (picrate).

In the present invention, the color development of step (b) may be characterized by visual or enzyme-linked immunosorbent assay (ELISA), the virus may be characterized in that the influenza virus, The viral membrane protein may be characterized as being HA (hemagglutinin).

In the present invention, the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), egg phosphatidylcholine (EPC) , Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) Soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidylinositol (SPI), dipalmitoyl phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glycer Rho-3-phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoyl phosphatidylglycerol (DMPG), hexadecyl Spokolin (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine ( PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), polyethylene glycol distea Roylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS) , Distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dypalmi Ilphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoyl phosphatidyl inositol (DMPI), distearoyl phosphatidyl inositol (DSPI), POPE ( 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac -glycero) and combinations thereof It may include one or more selected from.

In the present invention, the liposome-polymer hybrid is, for example, phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI) ), Egg phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC) Soy phosphatidylglycerol (SPG), soy phosphatidylethanolamine (SPE), soy phosphatidylserine (SPS), soy phosphatidyl acid (SPA), soy phosphatidyl inositol (SPI), dipalmitoylphosphatidyl choline (DPPC), 1,2- Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC), dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoyl force Thidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE ), Palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3- Phosphatidylcholine (POPC), polyethylene glycol distearoylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), Dimyristoylphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA) ), Dimyristoyl phosphatidic acid (DMPA), distearo Phosphatidic acid (DSPA), dipalmitoylphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoylphosphatidyl inositol (DMPI), distearo Ilphosphatidylinositol (DSPI), POPE (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac- liposomes comprising one or more selected from the group consisting of glycero) and combinations thereof; And polymers such as polyisobutylene-block-polyethylene oxide copolymer, polybutadiene-b-polyethylene oxide copolymer, polydimethylsiloxane-g-polyethyleneoxide copolymer, poly (2-methyloxazoline) -b- It may be a mixture of a polymer comprising at least one selected from the group consisting of a copolymer of polymethylsiloxane-b-poly (2-methyloxazoline) and combinations thereof.

Specifically, a mixture of a lipid DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine) and an amphiphilic block polymer polyisobutylene-block-polyethyleneoxide copolymer or a lipid but POPC and an amphiphilic block polymer polybutadiene-b- mixture of polyethylene oxide, mixture of DPPC and poly (dimethylsiloxane) -g-poly (ethylene oxide), and mixture of DPPC or PE and poly (2-methyloxazolene) -b-poly (dimethylsiloxane) -b-poly (2-methyloxazoline) At least one selected from the group consisting of a mixture, but is not limited thereto.

In the present invention, the liposome or liposome-polymer hybrid may be characterized by having a negative charge.

Influenza viruses are inserted into host cells through receptor-mediated endocytosis, in which the virus converts HA ₀ to HA ₁ / HA ₂ through trypsin-mediated proteolytic cleavage at low pH, resulting in a host cell membrane. Enable fusion (Skehel, JJ et al., Annu . Rev. Biochem . , 69: 531-569, 2000; White, J. et al., J. Cell. Biol ., 89: 674-679, 1981 ).

When the liposomes or liposome-polymer hybrids of the present invention have a negative charge, the influenza virus can be contacted to release the chromophore substrate contained therein without including an enzyme that degrades HA of the influenza virus.

Here, the negative charge of the liposome or liposome-polymer hybrid is sufficient to maintain the negative charge before detection by color development, for example, to maintain the negative charge in the sample state for detecting color development, or to carry out the negative charge under various experimental conditions possible before color detection. It is enough to maintain, but is not limited thereto.

In the present invention, liposomes or liposome-polymer hybrids may contain (capture) various molecules such as absorbers, fluorescent materials, or chemiluminescent materials in the inner aqueous phase. Amplified signals and moments expressed by chromogenic inducer / chromatogenic properties in addition to time-dependent chromogenic reactions using enzymes / substrates, as they can alter the ability to capture large numbers of substances and the composition of liposomes or liposome-polymer hybrids. Because it is possible to acquire a specific signal, it is possible to detect a small amount of virus in a short time.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Example 1: Preparation of liposomes or liposome-polymer hybrids containing TMB

80 mol% POPC (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine, Avanti Polar Lipids Inc., USA), 20 mol% POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac- glycero), Avanti Polar Lipids Inc., USA) was dissolved in chloroform, and then the chloroform was evaporated for at least 1 hour under reduced pressure, and the chloroform evaporated complex was dried in a 25 ° C. vacuum oven for one day. To form a thin lipid film. A liposome suspension was prepared by adding 0-50 μM TMB solution (Sigma-Aldrich, USA) to the lipid membrane and dispersing it for 10 minutes with an ultrasonicator (Jeiotech, Korea). Here, the process of freezing and thawing for the preparation of liposomes of a single lipid layer was repeated five times. In addition, liposomes of the same size were prepared using a Mini-Extruder (Avanti Polar Lipids Inc., USA) to prepare liposomes of uniform size. Liposomes containing the TMB prepared above were removed using a desalting column (GE Healthcare, UK) and stored at 4 ° C. in cold storage. The size of liposomes containing TMB was measured using DLS (dynamic light scattering, Otsuka, Japan).

As a result, liposomes containing TMB and having a diameter of about 50 to 200 nm were prepared.

Meanwhile, a method for preparing a liposome-polymer hybrid is prepared by dissolving an appropriate amount of lipid components (PC, PE, PS, etc.) in chloroform-methanol, depositing it on an anode, then applying an electric field and adding distilled water. Using liposome electroformation (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew) Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem . Soc ., 81: 303-311, 1986; Binder WH et al., Angew Chem ., 115 (47): 5980-6007, 2003; Binder WH et al., Angew Chem Int Ed Engl ., 42 (47): 5802-27, 2003), polymers were prepared in thin lipid membranes by dissolving appropriate amounts of lipid components (PC, PE, PS, etc.) in chloroform-methanol and evaporating the solvent under reduced pressure. Two-step liposome preparation (Two-step method) was prepared by putting in an aqueous polymeric nanoparticle suspension. (Kunn Hadinoto et al., European Journal of Pharmaceutics and Biopharmaceutics , 85 (3): 427-43,2013)

Example 2: Virus Detection Method (Enzyme Reaction)

Liposomes and influenza viruses containing TMB (3,3 ', 5,5'-tetramethyl bezidine) were prepared by conventional methods in the art, and used at appropriate concentrations and exposure times. Herein, the liposomes containing the TMB can be replaced with liposome-polymer hybrids containing the TMB.

Reaction conditions

50 μl of the liposome containing TMB prepared by the method of Example 1, 10 μl of influenza virus (A / California / 04/2009 (H1N1)) (1 * 10 ⁷ TCID / ml), 100 mM CP buffer (citrate-phosphate buffer, pH) 5.0) 10 μl was mixed and reacted at 37 ° C. for 30 minutes to allow virus and liposomes to contact (couple). Then, 25 μl of hydrogen peroxide (Hig, Horseradish peroxidase, Sigma-Aldrich, USA) and 1 U (unit) of hydrogen peroxide (hydrogen peroxide, Sigma-Aldrich, USA) were added to the reaction to perform a color reaction. After the reaction, the reaction stop solution (H ₂ SO ₄ , Sigma-Aldrich, USA) was added to stop the reaction and the absorbance at 450 nm was measured by ELISA Reader (SpectraMax, Molecular Devices).

As a result, as shown in Figures 2 and 3, the virus (influenza virus) was detected using liposomes containing the chromophore substrate (TMB). Here, the sample containing the virus was green, and the sample containing no virus did not show a color reaction, and the presence of the virus was visually confirmed.

Example 3: Preparation of Diagnostic Strips

The TMB encapsulated liposome (100 μl) prepared by the method of Example 1 and the peroxidase (2.5 units / μL), which is a color developing enzyme, were diluted with a phosphate buffer solution and used as a coating solution of the pad. 20 μl of the coating solution was applied to a pad to dry the reaction solution so as to be absorbed well, thereby preparing a reagent pad (see FIG. 4). As the material of the pad, polyester, glass fiber, cellulose, or the like was used. The reagent pads to which the coating solution was adsorbed were attached to the upper strips cut into widths of 1-4 mm × length of 10-40 mm to prepare detection (diagnosis) strips. Herein, the liposomes containing the TMB can be replaced with liposome-polymer hybrids containing the TMB.

Example 4 Virus Detection Using Strips (Enzyme Reaction)

Influenza detection test was carried out using a strip coated with the TMB inclusion liposome and peroxidase mixture prepared by the method of Example 3. The color of the pad changes when the strip is immersed in the virus solution and a reaction occurs between the virus and the mixed solution on the strip pad. That is, when the virus contacts (binds) the liposomes on the pads, the TMB released from the liposomes and the peroxidase react with the color to develop a color change of the pads on the strip to detect the presence of the virus. Herein, the liposomes containing the TMB can be replaced with liposome-polymer hybrids containing the TMB.

As a result, as shown in Fig. 2 and 5, the virus (influenza virus) was detected using a strip attached to the reagent pad containing liposomes containing the chromosome substrate (TMB). Here, the reagent pad showed a color reaction in the sample containing the virus, and the reagent pad did not show the color reaction in the sample containing no virus.

Example 5: Preparation of liposomes or liposome-polymer hybrids containing iodine

When the iodine solution was added to the starch solution, the iodine-starch complex was formed to detect the virus by using the iodine starch reaction which turns blue violet. To prepare liposomes containing iodine, iodine (Sigma-Aldrich, USA) was dissolved in an aqueous solution of potassium iodide (Potassium Iodide, Sigma-Aldrich, USA) to obtain a 10% iodine-potassium iodide solution. It was used to prepare the liposomes to prepare a liposome containing iodine. Liposomes containing iodine include POPC (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine, Avanti Polar Lipids Inc., USA) and POPG (1-palmitoyl-2-oleoyl- sn -glycero-3- It consists of phospho- (1'- rac- glycero), Avanti Polar Lipids Inc., USA, and used Mini-Extruder (Avanti Polar Lipids Inc., USA) to prepare liposomes of uniform size. Liposomes containing the prepared iodine were removed using a desalting column (GE Healthcare, UK) to remove residual iodine and stored at 4 ° C. The size of liposomes containing iodine was measured using DLS (dynamic light scattering, Otsuka, Japan).

As a result, liposomes containing iodine were prepared to have a diameter of about 50 to 200 nm.

Meanwhile, a method for preparing a liposome-polymer hybrid is prepared by dissolving an appropriate amount of lipid components (PC, PE, PS, etc.) in chloroform-methanol, depositing it on an anode, then applying an electric field and adding distilled water. Using liposome electroformation (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew) Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem . Soc ., 81: 303-311, 1986; Binder WH et al., Angew Chem ., 115 (47): 5980-6007, 2003; Binder WH et al., Angew Chem Int Ed Engl ., 42 (47): 5802-27, 2003), polymers were prepared in thin lipid membranes by dissolving appropriate amounts of lipid components (PC, PE, PS, etc.) in chloroform-methanol and evaporating the solvent under reduced pressure. Two-step liposome preparation (Two-step method) was prepared by putting in an aqueous polymeric nanoparticle suspension. (Kunn Hadinoto et al., European Journal of Pharmaceutics and Biopharmaceutics , 85 (3): 427-43,2013)

Example 6 Virus Detection Method Using Iodine Starch Reaction (Non-Enzyme Reaction)

Liposomes and influenza viruses containing iodine were prepared by routine methods in the art and used at appropriate concentrations and exposure times. Non-enzymatic assays used conventional methods in the art (Sarkar BC et al., Anal Biochem . , 20 (1): 155-66, 1967; Zak B., Clin Chim Acta ., 3 (4): 328-34, 1958; Hawk, Philip B. et al., Practical Physiological Chemistry , Churchill, London, pp 839-844, 1947). Herein, the liposomes containing iodine can be replaced with liposome-polymer hybrids containing iodine.

Reaction conditions

50 μl of the liposome containing iodine prepared in Example 5, 10 μl of influenza virus (A / California / 04/2009 (H1N1)) (1 × 10 ⁷ TCID / ml), 100 mM CP buffer (citrate-phosphate buffer, pH 5.0) ) 10μl was mixed and reacted at 37 ° C. for 30 minutes to allow virus and liposomes to contact (couple). Then, starch solution (Starch, Acros organics, USA) was added to the reaction to iodine starch reaction to confirm the presence of virus.

As a result, as shown in FIG. 6, the virus (influenza virus) was detected using liposomes containing the chromogenic substrate (iodine). Here, the sample containing the virus was blue violet, and the sample containing no virus did not show color change, and the presence of the virus was visually confirmed.

Example 7: Preparation of liposomes or liposome-polymer hybrids containing pNPP

40 mol% POPC (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphocholine, Avanti Polar Lipids Inc., USA), 60 mol% POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- ( 1- rac -glycerol), Avanti Polar Lipids Inc., USA) was dissolved in chloroform, the chloroform was evaporated for at least 1 hour under reduced pressure, and the chloroform evaporated complex was dried in a 25 ° C. vacuum oven for 1 day to thin. A lipid membrane was formed. A liposome suspension was prepared by dispersing 270 mM pNPP solution (4-Nitrophenyl phosphate disodium salt hexahydrate, Sigma-Aldrich, USA) for 10 minutes with an ultrasonicator (Jeiotech, Korea). Here, the process of freezing and thawing for the preparation of liposomes of a single lipid layer was repeated five times. In addition, liposomes of the same size were prepared using a Mini-Extruder (Avanti Polar Lipids Inc., USA) to prepare liposomes of uniform size. The prepared liposomes containing pNPP were removed using residual Sephacryl S-1000 column (sephacryl S-1000 column, 1.5 x 12 cm, GE Healthcare, UK) and refrigerated at 4 ℃. The size of liposomes containing pNPP was measured using DLS (dynamic light scattering, Otsuka, Japan).

As a result, liposomes containing pNPP and having a diameter of about 50 to 200 nm were prepared.

Meanwhile, a method for preparing a liposome-polymer hybrid is prepared by dissolving an appropriate amount of lipid components (PC, PE, PS, etc.) in chloroform-methanol, depositing it on an anode, then applying an electric field and adding distilled water. Using liposome electroformation (Olubummo A1 et al., Langmuir , 30 (1): 259-67, 2014; Schulz M et al., Angew) Chem Int Ed Engl ., 52 (6): 1829-33, 2013; Miglena I et al., Faraday Discuss. Chem . Soc ., 81: 303-311, 1986; Binder WH et al., Angew Chem ., 115 (47): 5980-6007, 2003; Binder WH et al., Angew Chem Int Ed Engl ., 42 (47): 5802-27, 2003), polymers were prepared in thin lipid membranes by dissolving appropriate amounts of lipid components (PC, PE, PS, etc.) in chloroform-methanol and evaporating the solvent under reduced pressure. Two-step liposome preparation (Two-step method) was prepared by putting in an aqueous polymeric nanoparticle suspension. (Kunn Hadinoto et al., European Journal of Pharmaceutics and Biopharmaceutics , 85 (3): 427-43,2013)

Example 8: Virus Detection Method (Enzyme Reaction)

Influenza viruses were prepared by routine methods in the art and used at appropriate concentrations and exposure times.

Reaction conditions

50 μl of liposomes containing pNPP prepared by the method of Example 7, 10 μl of influenza virus (A / chicken / korea / S1 / 2003 (H9N2)) (1 * 10 ⁷ TCID ₅₀ / ml), 100 mM CP buffer (citrate-phosphate) buffer, pH 4.0) 10μl was mixed and reacted for 30 minutes at 37 ℃ to contact (bind) virus and liposomes. Next, 1.5 U (unit) of chromatase phosphatase (phosphatase, Sigma-Aldrich, USA) was added to the reaction, followed by coloring by adjusting the appropriate pH with 0.1 M glycine buffer containing 1 mM ZnCl ₂ and 1 mM MgCl _2. The reaction proceeded. Absorbance at 405 nm during color development was measured by ELISA Reader (SpectraMax, Molecular Devices) (FIG. 7). Herein, the liposomes containing pNPP are replaceable with liposome-polymer hybrids containing pNPP.

As a result, as shown in Figures 7 and 8, the virus (influenza virus) was detected using liposomes containing the chromophore substrate (pNPP). Here, in the sample containing the virus, the presence of the virus was visually confirmed in yellow.

Example 9 Virus Detection Method (Enzyme Reaction)

Influenza viruses were prepared by routine methods in the art and used at appropriate concentrations and exposure times.

Reaction conditions

10 μl of influenza virus (A / chicken / korea / S1 / 2003 (H9N2)) (1 * 10 ⁷ TCID ₅₀ / ml) was first treated with 50ng of trypsin (TPCK-trypsin, Thermo) and reacted at 37 ° C for 30 minutes. Next, 50 μl of the liposome containing pNPP prepared by the method of Example 7, 10 μl of influenza virus treated with trypsin (A / chicken / korea / S1 / 2003 (H9N2)) (1 * 10 ⁷ TCID ₅₀ / ml), 10 μl of 100 mM CP buffer (citrate-phosphate buffer, pH 4.0) was mixed and reacted at 37 ° C. for 30 minutes to allow virus and liposomes to contact (combine). Next, 1.5 U (unit) of chromatase phosphatase (phosphatase, Sigma-Aldrich, USA) was added to the reaction, and then titrated with 0.1M Glycine buffer containing 1 mM ZnCl ₂ and 1 mM MgCl ₂ . The color reaction was carried out. Absorbance at 405 nm during the color reaction was measured by ELISA Reader (SpectraMax, Molecular Devices) (Fig. 9). Herein, the liposomes containing pNPP are replaceable with liposome-polymer hybrids containing pNPP.

As a result, as shown in FIG. 9 and FIG. 10, the virus (influenza virus) was detected using liposomes containing the chromogenic substrate (pNPP). Here, two samples containing the trypsin-treated virus and the untreated virus appeared yellow to confirm the presence of the virus visually.

The virus detection method using liposomes or liposome-polymer hybrids according to the present invention is easily detectable by the naked eye, and in particular, hydrophobic TMB (3,3 ', 5,5'-tetramethyl), which is a chromogenic substrate. When using liposomes or liposome-polymer hybrids containing bezidine) or iodine, TMB or iodine does not leak out of the liposomes or liposome-polymer hybrids, and thus have high stability against natural coloration by oxygen or chemical reaction. When the virus binds to liposomes or liposome-polymer hybrids through viral membrane proteins, the TMB or iodine contained in the liposomes or liposome-polymer hybrids are released and reacted with HRP or starch, a color-inducing substance. The virus detection signal appearing was significantly higher.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (27)

1. Liposomes or liposome-polymer hybrids containing chromogenic substrates, and chromophore-inducing substances, and when the virus binds to the liposomes or liposome-polymer hybrids, the chromosomes contained in liposomes or liposome-polymer hybrids A composition for detecting a virus, characterized in that when the substrate is released, the color is reacted with the color inducing substance.
2. The composition of claim 1, wherein the virus binds to liposomes or liposome-polymer hybrids through viral lipid membranes or membrane proteins.
3. The composition of claim 1, wherein the color development is performed through an enzymatic reaction or a non-enzymatic reaction.
4. According to claim 3, wherein the chromogenic substrate used for the enzyme reaction is DAB (diaminobenzidine), AEC (3-amino-9-ethylcarbasole), BCIP / NBT (5-bromo-4-chloro-3-indolyl-phosphate / nitroblue tetrazolium), pNPP (para-Nitrophenyl phosphate), naphthol AS-TR phosphate, BCIP / INT (5-bromo-4-chloro-3-indolyl phosphate / iodonitrotetrazolium), NF (New fuchsin), Fast Red TR Salt (FRT), phenylenediamine, 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetra methylbenzidine), dianisidine , Amino-salicylic acid, 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole, 4 4-chloro-1-naphthol, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid)] and OPD (ophenylenediamine), and the chromophoric inducer is chromophore, HRP (Horseradish peroxidase), salt Alkaline phosphatase, Glucose Oxidase, luciferase, beta-di-galactosidase, β-D-galactosidase, malate dehydrogenase and MDH Acetylcholinesterase (acetylcholinesterase) A composition for detecting a virus, characterized in that selected from the group consisting of.
5. According to claim 3, wherein the color substrate used in the non-enzymatic reaction group consisting of iodine, calcium, copper, iron, amino acid and creatinine And the chromophore-inducing substance is starch, o-cresolphthalein complexone, bathocuproin disulfonate, bathophenanthroline disulfonate ), A ninhydrin, o-phthalaldehyde (o-phthalaldehyde: OPA) and a composition for detecting a virus, characterized in that selected from the group consisting of (picrate).
6. According to claim 1, wherein the color of the virus detection composition, characterized in that confirmed by the naked eye or enzyme-linked immunosorbent assay (ELISA).
7. The virus detection composition of claim 1, wherein the virus is an influenza virus.
8. The virus detection composition of claim 1, wherein the viral membrane protein is HA (hemagglutinin).
9. According to claim 1, wherein the viral lipid membrane is a virus detection composition, characterized in that the PC (Phosphatidylcholine), PI (Phosphoinositides), PS (Phosphatidylserine), PE (Phosphatidylethanolamine) or SM (Sphingomyelin).
10. The method of claim 1, wherein the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI), egg phosphatidylcholine (EPC) ), Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) ), Soy phosphatidylethanolamine (SPE), Soy phosphatidylserine (SPS), Soy phosphatidyl acid (SPA), Soy phosphatidylinositol (SPI), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn- Glycero-3-phosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoylphosphatidylglycerol (DMPG), hexadecylfo Pocholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine ( PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), polyethylene glycol distea Roylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS) , Distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dapalmito Phosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoyl phosphatidyl inositol (DMPI), distearoyl phosphatidyl inositol (DSPI), POPE (1 -palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac -glycero) and combinations thereof A composition for detecting a virus, characterized in that it comprises one or more selected.
11. The method of claim 1, wherein the liposome-polymer hybrid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), eggs Phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soybean phosphatidylcholine (SPC), soybean phosphatidylcholine (SPC) Glycerol (SPG), Soy phosphatidylethanolamine (SPE), Soy phosphatidylserine (SPS), Soy phosphatidyl acid (SPA), Soy phosphatidyl inositol (SPI), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl -sn-glycero-3-phosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoylphosphatidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE) Palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine ( POPC), polyethylene glycol distearoylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyri Stolesphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), Dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatide (DSPA), dipalmitoylphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoyl phosphatidyl inositol (DMPI), distearoyl phosphatidyl inositol (DSPI), POPE (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac -glycero) and Liposomes comprising one or more selected from the group consisting of combinations thereof; And amphiphilic block copolymers, polyisobutylene-block-polyethylene oxide copolymers, polybutadiene-b-polyethylene oxide copolymers, polydimethylsiloxane-g-polyethylene oxide copolymers, poly (2-methyloxazolines)- b-polymethylsiloxane-b-poly (2-methyloxazoline) copolymer and a combination of a polymer comprising at least one selected from the group consisting of a combination thereof.
12. The method of claim 1, wherein the liposome or liposome-polymer hybrid is a virus detection composition, characterized in that it has a negative charge.
13. A virus detection kit comprising the composition of any one of claims 1 to 12.
14. A virus detection strip with a pad coated with the composition of any one of claims 1 to 12.
15. Virus detection method comprising the following steps:

    (a) contacting a liposome or liposome-polymer hybrid containing a chromogenic substrate with a putative sample containing a virus; And

    (b) adding a color inducing substance, to determine whether color development.
16. 16. The method of claim 15, wherein the color development of step (b) is performed when the virus binds to the liposome or the liposome-polymer hybrid, and the color reaction substrate reacts with the color inducing substance while the color substrate contained in the liposome or the liposome-polymer hybrid is released. The detection method of the virus characterized by the above-mentioned.
17. The method of claim 15, wherein the virus binds to liposomes or liposome-polymer hybrids through viral lipid membranes or membrane proteins.
18. 17. The method of claim 16, wherein the identification of the color development of step (b) is performed by an enzymatic reaction or a non-enzymatic reaction.
19. 19. The method according to claim 18, wherein the chromogenic substrate used for the enzymatic reaction is diaminobenzidine (DAB), 3-amino-9-ethylcarbasole (AEC), BCIP / NBT (5-bromo-4-chloro-3-indolyl-phosphate / nitroblue). tetrazolium), pNPP (para-Nitrophenyl phosphate), naphthol AS-TR phosphate, BCIP / INT (5-bromo-4-chloro-3-indolyl phosphate / iodonitrotetrazolium), NF (New fuchsin), Fast Red TR Salt (FRT), phenylenediamine, 3,3 ', 5,5'-tetramethylbenzidine (3,3', 5,5'-tetra methylbenzidine), dianisidine , Amino-salicylic acid, 3,3'-diaminobenzidine, 3-amino-9-ethylcarbazole, 4 4-chloro-1-naphthol, TMB (3,3 ', 5,5'-tetramethyl bezidine), ABTS [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid)] and OPD (ophenylenediamine), and the chromophore-inducing substance is a chromophorase, HRP (Horseradish peroxidase), Alkaline phosphatase, Glucose Oxidase, luciferase, beta-di-galactosidase, β-D-galactosidase, malate dehydrogenase and MDH A method for detecting a virus, wherein the virus is selected from the group consisting of acetylcholinesterases.
20. 19. The group of claim 18, wherein the color substrate used for the non-enzymatic reaction is composed of iodine, calcium, copper, iron, amino acid, and creatinine. And the chromophore-inducing substance is starch, o-cresolphthalein complexone, bathocuproin disulfonate, bathophenanthroline disulfonate ), Ninhydrin, o-phthalaldehyde (OPA) and picrate (pirate) is selected from the group consisting of a virus detection method.
21. The method of claim 18, wherein the color development of step (b) is confirmed by visual or enzyme-linked immunosorbent assay (ELISA).
22. The method of claim 16, wherein the virus is an influenza virus.
23. 18. The method of claim 17, wherein the viral membrane protein is HA (hemagglutinin).
24. 18. The method of claim 17, wherein the viral lipid membrane is PC (Phosphatidylcholine), PI (Phosphoinositides), PS (Phosphatidylserine), PE (Phosphatidylethanolamine) or SM (Sphingomyelin).
25. The method of claim 15, wherein the liposome is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidyl inositol (PI), egg phosphatidylcholine (EPC) ), Egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soy phosphatidylcholine (SPC), soy phosphatidylglycerol (SPG) ), Soy phosphatidylethanolamine (SPE), Soy phosphatidylserine (SPS), Soy phosphatidyl acid (SPA), Soy phosphatidylinositol (SPI), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-sn- Glycero-3-phosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoylphosphatidylglycerol (DMPG), hexadecylfo Pocholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE), palmitoylstearoylphosphatidylcholine ( PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), polyethylene glycol distea Roylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyristoylphosphatidylserine (DMPS) , Distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), dimyristoylphosphatidic acid (DMPA), distearoylphosphatidic acid (DSPA), dapalmito Ilphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoyl phosphatidyl inositol (DMPI), distearoyl phosphatidyl inositol (DSPI), POPE ( 1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac -glycero) and combinations thereof Virus detection method comprising at least one selected from.
26. The method of claim 15, wherein the liposome-polymer hybrid is phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylinositol (PI), eggs Phosphatidylcholine (EPC), egg phosphatidylglycerol (EPG), egg phosphatidylethanolamine (EPE), egg phosphatidylserine (EPS), egg phosphatidyl acid (EPA), egg phosphatidyl inositol (EPI), soybean phosphatidylcholine (SPC), soybean phosphatidylcholine (SPC) Glycerol (SPG), Soy phosphatidylethanolamine (SPE), Soy phosphatidylserine (SPS), Soy phosphatidyl acid (SPA), Soy phosphatidyl inositol (SPI), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl -sn-glycero-3-phosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylglycerol (DPPG), diol phosphatidylglycerol (DOPG), dimyristoylphosphatidylglycerol (DMPG), hexadecylphosphocholine (HEPC), hydrogenated soybean phosphatidylcholine (HSPC), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylglycerol (DSPG), dioleylphosphatidylethanolamine (DOPE) Palmitoylstearoylphosphatidylcholine (PSPC), palmitoylstearoylphosphatidylglycerol (PSPG), monooleoylphosphatidylethanolamine (MOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine ( POPC), polyethylene glycol distearoylphosphatidylethanolamine (PEG-DSPE), dipalmitoylphosphatidylserine (DPPS), 1,2-dioleoyl-sn-glycero-3-phosphatidylserine (DOPS), dimyri Stolesphosphatidylserine (DMPS), distearoylphosphatidylserine (DSPS), dipalmitoylphosphatidic acid (DPPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), Dimyristoyl phosphatidic acid (DMPA), distearoyl phosphatide Acid (DSPA), dipalmitoylphosphatidyl inositol (DPPI), 1,2-dioleoyl-sn-glycero-3-phosphatidyl inositol (DOPI), dimyristoyl phosphatidyl inositol (DMPI), distearoyl phosphatidyl Inositol (DSPI), POPE (1-palmitoyl-2-oleoyl- sn -glycero-3-phosphoethanolamine), POPG (1-palmitoyl-2-oleoyl- sn -glycero-3-phospho- (1'- rac -glycero) Liposomes comprising one or more selected from the group consisting of a combination thereof; And polyisobutylene-block-polyethylene oxide copolymers, polybutadiene-b-polyethylene oxide copolymers, polydimethylsiloxane-g-polyethylene oxide copolymers, poly (2-methyloxazoline) -b-polymethylsiloxanes -b-poly (2-methyloxazoline) copolymers and a combination of a polymer comprising at least one selected from the group consisting of a combination thereof.
27. The method of claim 15, wherein the liposome or liposome-polymer hybrid has a negative charge.

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