Lipid nanoparticles carrying material for detecting target nucleic acids, and membrane fusion-based method for detecting target nucleic acids by using same

Abstract

The present invention relates to lipid nanoparticles carrying a material for detecting target nucleic acids, and a use thereof.

Description

Lipid nanoparticles loaded with target nucleic acid detection substances and a membrane fusion-based target nucleic acid detection method using the same

[0001] The present invention relates to a lipid nanoparticle loaded with a target nucleic acid detection substance and a membrane fusion-based target nucleic acid detection method using the same.

[0002] Rapid, user-friendly point-of-care diagnostic tests enable frequent testing for early disease detection, allowing appropriate measures such as isolating infected individuals or providing prompt treatment, thereby contributing to preventing the spread of disease and increasing the effectiveness of treatment.

[0003] Antigen tests are currently the most widely used point-of-care diagnostic test because they are rapid and easy to use. However, compared to molecular diagnostic technologies targeting nucleic acids, they have lower sensitivity and result in more frequent false-negative results.

[0004] Additionally, antigen tests have the problem of reduced sensitivity when detecting RNA viruses that mutate frequently, such as coronaviruses, due to changes in the protein (antigen) structure caused by mutations.

[0005] Therefore, to effectively screen infected individuals and establish quarantine measures, it is necessary to develop a rapid and user-friendly field diagnostic technology based on molecular diagnostic technology that is more sensitive than antigen testing.

[0006] However, existing molecular diagnostic technologies are difficult to use for point-of-care diagnosis because they require sample pretreatment, including extraction and separation of nucleic acids, even if the diagnostic reaction is complex (e.g., quantitative reverse transcription polymerase chain reaction) or the reaction itself is relatively simple (e.g., isothermal-rapid amplification method or amplification-free detection method).

[0007] Recently, field-based molecular diagnostic technologies have been developed that minimize sample preparation steps or automate sample preparation using auxiliary devices. However, they still require additional testing time due to sample preparation and incur additional costs due to auxiliary devices, which significantly limits their use in field diagnosis.

[0008]

[0009] Accordingly, the inventors of the present invention continued research to solve the above-mentioned problems, developed lipid nanoparticles that do not require pretreatment and sample amplification processes, and reached the present invention.

[0010]

[0011] [Non-patent literature]

[0012] Xihui Gao et al. Rapid Detection of Exosomal microRNAs Using Virus-Mimicking Fusogenic Vesicles, Angewandte Chemie International Edition 2019

[0013] An object of the present invention is to provide a lipid nanoparticle loaded with a detection substance for detecting a target nucleic acid.

[0014] Another object of the present invention is to provide a method for manufacturing lipid nanoparticles loaded with a detection substance for detecting a target nucleic acid.

[0015] Another object of the present invention is to provide a membrane fusion-based target nucleic acid detection composition and detection kit including a lipid nanoparticle loaded with a detection substance for detecting a target nucleic acid.

[0016] Another object of the present invention is to provide a membrane fusion-based target nucleic acid detection method.

[0017]

[0018] However, the technical problem to be achieved by the present invention is not limited to the purpose mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0019] To solve the above problem, the present invention provides a lipid nanoparticle loaded with a detection substance for detecting a target nucleic acid.

[0020] Specifically, the lipid nanoparticles are prepared as follows:

[0021] Lipid nanoparticles are manufactured by independently passing an ethanol feeding solution containing a lipid mixture and a water feeding solution containing the detection substance through a microfluidic device to mix and undergo a self-assembly process.

[0022] The above lipid mixture is,

[0023] i) phosphocholine 0-40 mol%;

[0024] ii) 0-40 mol% of ionic lipids;

[0025] iii) 0-40 mol% of helper lipids;

[0026] iv) cholesterol 0-40mol%; and

[0027] v) Composed of 0-5 mol% lipids bound to water-soluble polymers.

[0028] The lipid nanoparticle may further comprise 1 mol % or less of a lipid for bioconjugation of a receptor that binds to a substance containing the target nucleic acid on the surface of the lipid nanoparticle.

[0029] The above bioconjugation means conjugating a receptor that binds to the surface of a material containing the target nucleic acid to the surface of the lipid nanoparticle.

[0030] The receptor may be, but is not limited to, ACE2 (angiotensin converting enzyme 2), and any receptor used in this technical field is included. The lipid for the bioconjugation may be, but is not limited to, maleimide-PEG-PE, and any receptor used in this technical field is included.

[0031] The detection material for detecting the target nucleic acid may be a) a nucleic acid probe linked to a fluorescent material; or b) a guide RNA, a CRISPR-associated endonuclease, and a reporter nucleic acid; or c) a lipid nanoparticle that is a LAMP (Loop-mediated Isothermal Amplification) reactant.

[0032] The above detection substances may be included in an amount of 0.00001-4 wt% or less, respectively, with respect to the total weight of the lipid nanoparticles.

[0033] The above phosphocholine may be DPhyPC (1,2-Diphytanoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine)) or (DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine).

[0034] 상기 이온성 지질은 양이온성 지질 또는 음이온성 지질일 수 있다. 구체적으로, 상기 양이온성 지질은, DDAB(dimethyldioctadecylammonium bromide); DOTAP(1,2-dioleoyloxy-3-(trimethyl ammonium)propane); DODAC(dimethyldioleylammonium chloride); N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; DOTMA(N-[1-[2,3-bis(oleoyloxy)]propyl]-N,N,N-trimethylammonium chloride); DOGS(di-octadecyl-amido-glycyl-spermine); DC-cholesterol(3β-[N―(N′,N′'-dimethyl amino ethane)carbamoyl] cholesterol); DOSPA(2,3-dioleoyloxy-N-(2(spermine carboxamido)-ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate); 1,2-diacyl-sn-glycero-3-ethyl phosphocholines; DODMA(1,2-dioleyloxy-3-dimethylaminopropane); MVL5(N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide); β-alanyl cholesterol; CTAB(cetyltrimethylammonium bromide); DOSPER(1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide); N,N,N′,N′'-tetramethyl-N,N′'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butane diammonium iodide;and mixtures thereof, and any cationic lipid usable in the art may be used.

[0035] Specifically, the anionic lipid is DMPA (dimyristyl glycerophosphate); DPPA (dipalmitoyl glycerophosphate); DMPG (dimyristyl glycerophosphate); disteroyl glycerophosphate (DSPA); DSPG (distearoyl-sn-glycero-3-phosphoglycerol); DPPG (dipalmitoyl glycerophosphoraglycerol); MPS (dimyristylyl glycerophosphoseline); DPPS (dipalmitoyl glycerophosphoseline);

[0036] Any anionic lipid usable in this technical field may be selected from the group consisting of DSPS (disteroyl glycerophosphoseline) and mixtures thereof, but is not necessarily limited thereto.

[0037] The above helper lipid may be selected from the group consisting of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-bis(diphenylphosphino)ethane), DMPE (1,2-bis(dimethylphosphino)ethane), and mixtures thereof, but is not necessarily limited thereto, and any helper lipid having a phosphoethanolamine head usable in this technical field or a lipid molecule packing parameter of 1 or more may be used.

[0038] The above cholesterol is used to ensure lipid membrane stability.

[0039] The water-soluble polymer may be selected from the group consisting of polyethylene glycol, polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polyethylene oxide, polysaccharide, and mixtures thereof, but is not necessarily limited thereto, and any water-soluble polymer usable in this technical field may be used.

[0040] In the lipid to which the water-soluble polymer is bound, the molecular weight of the water-soluble polymer may be 2000 to 5000 Da, and the lipid may be a phospholipid.

[0041] The lipid bound to the water-soluble polymer may be DOPE-PEG2000.

[0042] The above lipid nanoparticles may be conjugated to a receptor capable of binding to a substance containing a target nucleic acid on their surface. However, the conjugation is optional and not necessarily required.

[0043] The target nucleic acid may be DNA or RNA.

[0044] In another aspect, the present invention provides a method for manufacturing lipid nanoparticles loaded with a detection substance for detecting the following target nucleic acid:

[0045] An ethanol feeding solution containing a lipid mixture and a water feeding solution containing a target nucleic acid detection substance are each prepared;

[0046] The ethanol feeding solution containing the prepared lipid mixture and the water feeding solution containing the detection substance are each independently passed through a microfluidic device to mix the ethanol feeding solution containing the lipid mixture and the water feeding solution containing the target nucleic acid detection substance; and

[0047] A method for manufacturing lipid nanoparticles loaded with a detection substance for detecting target nucleic acids through a self-assembly process of the above mixed solution,

[0048] The above lipid mixture is composed as follows:

[0049] i) phosphocholine 0-40 mol%;

[0050] ii) 0-40 mol% of ionic lipids;

[0051] iii) 0-40 mol% of helper lipids;

[0052] iv) cholesterol 0-40mol%; and

[0053] v) 0-5 mol% of lipids bound to water-soluble polymers.

[0054] The lipid nanoparticle may contain 1 mol % or less of a lipid for bioconjugation of a receptor that binds to a substance containing the target nucleic acid on the surface of the lipid nanoparticle.

[0055] In another aspect, the present invention provides a composition for detecting a target nucleic acid, comprising a lipid nanoparticle loaded with a detection substance for detecting the target nucleic acid.

[0056] In another aspect, the present invention provides a diagnostic kit comprising the composition for detecting the target nucleic acid.

[0057] In another aspect, the present invention provides a membrane fusion-based target nucleic acid detection method as follows:

[0058] By mixing a sample containing a substance in which a target nucleic acid exists inside a lipid bilayer and the above-mentioned lipid nanoparticle or composition for detecting a target nucleic acid, membrane fusion of the substance containing the target nucleic acid and the lipid nanoparticle carrying a detection substance for detecting the target nucleic acid is caused; and

[0059] The target nucleic acid detection material within the lipid nanoparticle reacts with the target nucleic acid to generate a detection signal.

[0060] The sample is a DNA virus or RNA virus obtained from a subject or a cell or lipid vesicle (exosome, etc.), and the membrane fusion of the substance containing the target nucleic acid with the lipid nanoparticle can be fusion between the lipid layer membrane of the virus or the lipid layer membrane of the cell membrane or lipid-based vesicle and the lipid nanoparticle membrane.

[0061] The above target nucleic acid detection material is a) a nucleic acid probe linked to a fluorescent material; or b) a guide RNA, a CRISPR-associated endonuclease, and a reporter nucleic acid; or c) a LAMP (Loop-Mediated Isothermal Amplification) reactant, and when the detection material reacts with the target nucleic acid, fluorescence or a color change can be generated as a detection signal.

[0062] The above method does not require pretreatment or nucleic acid amplification process for the target nucleic acid and can be performed at room temperature or isothermal temperature.

[0063] Additionally, the above method can be performed regardless of whether the target nucleic acid is of wild type or mutant origin.

[0064] Additionally, a sample containing a substance in which a target nucleic acid exists within the lipid bilayer can be obtained from a mask worn by the subject and / or the surface of an object on which the subject stays or uses, without a separate sample collection process.

[0065] Additionally, target nucleic acids can be detected by mixing the lipid nanoparticles or the detection composition on the surface of the mask or object to cause membrane fusion.

[0066] Additionally, the above detection signal occurs within 30 minutes, enabling rapid diagnosis.

[0067] Membrane fusion-based target nucleic acid detection is possible by using lipid nanoparticles loaded with a detection substance for detecting target nucleic acids according to the present invention.

[0068] The membrane fusion-based target nucleic acid detection method according to the present invention does not require preprocessing and nucleic acid amplification processes for the target nucleic acid, is performed at room temperature, and allows rapid confirmation of test results.

[0069] The membrane fusion-based target nucleic acid detection method using lipid nanoparticles of the present invention provides rapid diagnosis of infectious diseases.

[0070] Additionally, using the membrane fusion-based target nucleic acid detection method according to the present invention, detection of lipid vesicles (exosomes, extracellular vesicles, etc.) is also possible.

[0071] In addition, the lipid nanoparticles of the present invention can be stored in a refrigerator for a long period of time.

[0072] Figure 1(a) is a schematic diagram showing a liposome manufacturing method according to one embodiment of the present invention.

[0073] Figure 1(b) is a schematic diagram showing a bioconjugation method according to one embodiment of the present invention.

[0074] Figure 2 is a schematic diagram showing a membrane fusion-based target nucleic acid detection process according to one embodiment of the present invention.

[0075] Figure 3 shows a target nucleic acid detection process of CRISPR Cas13a according to one embodiment of a detection material loaded on a liposome.

[0076] Figure 4 shows the results of SAXS and cryo-TEM confirming the formation of a lipid bilayer of liposomes.

[0077] Figure 5 shows the capture rate of reporter RNA and Cas13a protein-guide RNA complexes within liposomes.

[0078] Figure 6 is a graph showing changes in liposome size and surface zeta potential according to loading and bioconjugation of a detection substance for detecting a target nucleic acid.

[0079] Figure 7 shows FRET-based fluorescence results showing membrane fusion of liposomes and SARS-CoV-2 virus according to one embodiment of the present invention.

[0080] Figure 8 is a cryogenic transmission electron micrograph showing membrane fusion of liposomes and the SARS-CoV-2 virus.

[0081] Figure 9(a) shows the SARS-CoV-2 RNA analysis sensitivity results of CRISPR Cas13a according to one example of a detection substance loaded on a liposome.

[0082] Figure 9(b) shows the specificity results of SARS-CoV-2 RNA analysis of CRISPR Cas13a according to one example of a detection substance loaded on a liposome.

[0083] Figure 10(a) shows the results of the analysis sensitivity of liposomes to SARS-CoV-2 virus according to one embodiment of the present invention.

[0084] Figure 10(b) shows the results of analysis specificity of liposomes for SARS-CoV-2 virus according to one embodiment of the present invention.

[0085] FIG. 11 shows that membrane fusion with the SARS-CoV-2 virus occurs only by electrical attraction by cationic lipids even when ACE2 is not present on the liposome surface according to one embodiment of the present invention, and that bioconjugation of ACE2 to the liposome surface further increases the detection signal.

[0086] Figure 12 shows the detection of SARS-CoV-2 virus mutation-independent liposomes according to one embodiment of the present invention.

[0087] Figure 13 shows that liposomes according to one embodiment of the present invention exhibit a visually distinguishable color change through membrane fusion with the SARS-CoV-2 virus.

[0088] Figure 14(a) shows that detection of the SARS-CoV-2 virus by liposomes according to an embodiment of the present invention is possible even on the surface of a mask.

[0089] Figure 14(b) shows that the liposome mask-based SARS-CoV-2 virus detection according to an embodiment of the present invention can detect up to 3 aM with a detection time of only 10 minutes.

[0090] Figure 15 shows that the detection of SARS-CoV-2 virus by liposomes according to an embodiment of the present invention is possible on various surfaces, and that detection of up to 3 aM is possible with a detection time of only 10 minutes.

[0091] Figure 16(a) shows that CRISPR Cas13a according to one embodiment of a detection substance loaded on a liposome is capable of detecting influenza A (H1N1) RNA.

[0092] Figure 16(b) shows that the membrane fusion-based detection method of liposomes according to an embodiment of the present invention is also possible for influenza A (H1N1) virus.

[0093] Figure 16(c) shows that the membrane fusion-based detection method of liposomes according to an embodiment of the present invention is a target nucleic acid-specific reaction.

[0094] Figure 17 shows a liposome according to an embodiment of the present invention, 4 times with light blocked. o When stored in a C refrigerator, the particle size continued to increase for up to 6 months, showing some particle agglomeration, but the SARS-CoV-2 virus detection performance was maintained.

[0095] Figure 18 shows the stability of liposomes against RNase according to one embodiment of the present invention.

[0096] Figure 19 shows that detection is performed in the same manner by simply adding a liposome solution to a SARS-CoV-2 virus sample without physically mixing the liposome solution and the SARS-CoV-2 virus sample according to one embodiment of the present invention.

[0097] Figure 20 shows that both saliva and nasopharynx are detectable samples using the liposome-based detection method according to one embodiment of the present invention.

[0098] Figure 21(a) shows that CRISPR Cas13a according to one embodiment of a detection substance loaded on a liposome is also capable of detecting Respiratory Syncytial Virus (RSV) RNA.

[0099] Figure 21(b) shows that the membrane fusion-based detection method of liposomes according to an embodiment of the present invention is also possible for the Respiratory Syncytial Virus (RSV) virus.

[0100] Figure 22(a) shows that CRISPR Cas13a according to one embodiment of a detection material loaded on a liposome is capable of detecting miRNA-21.

[0101] Figure 22(b) shows that the membrane fusion-based detection method of liposomes according to an embodiment of the present invention is also possible for extracellular vesicles (exosomes).

[0102] Figure 23(a) shows that CRISPR Cas12a according to one embodiment of a detection material loaded on a liposome can also detect HSV (Herpes Simplex virus) DNA.

[0103] Figure 23(b) shows that the membrane fusion-based detection method of liposomes according to an embodiment of the present invention is also possible for the Herpes Simplex virus (HSV).

[0104] Figure 24(a) shows that a nucleic acid probe linked to a fluorescent material according to one embodiment of a detection material loaded on a liposome is capable of detecting SARS-CoV-2 RNA.

[0105] Figure 24(b) shows that the membrane fusion-based detection method of liposomes according to an embodiment of the present invention is also possible for the SARS-CoV-2 virus.

[0106] The present invention provides lipid nanoparticles loaded with a detection substance for detecting a target nucleic acid, manufactured by the following method:

[0107] Lipid nanoparticles are manufactured by independently passing an ethanol feeding solution containing a lipid mixture and a water feeding solution containing the detection substance through a microfluidic device to mix and undergo a self-assembly process.

[0108] The above lipid mixture is,

[0109] i) phosphocholine 0-40 mol%;

[0110] ii) 0-40 mol% of ionic lipids;

[0111] iii) 0-40 mol% of helper lipids;

[0112] iv) cholesterol 0-40mol%; and

[0113] v) Composed of 0-5 mol% lipids bound to water-soluble polymers.

[0114]

[0115] The present invention provides a one-step method for producing lipid nanoparticles, which enables the formation of lipid nanoparticles while simultaneously loading a target nucleic acid detection substance (pre-encapsulation) by optimizing the composition of a lipid mixture included in the ethanol feeding solution.

[0116] The above “lipid nanoparticles” is a term commonly used in this technical field, and refers to nanoparticles composed of a lipid bilayer including a liposome.

[0117] The above “microfluidic device” is a widely known technology in this field, so a detailed description is omitted.

[0118] In the present invention, a microfluidic device having two or more inlets; a material mixing portion in which the inlets are combined into one; and a single outlet connected to the material mixing portion can be used (see Fig. 1(a)).

[0119] The above material mixing unit may include a SHM (staggered herringbone mixer) known in the art (Belliveau, NMet. al., Microfluidic Synthesis of Highly Potent Limit-Size Lipid Nanoparticles for in vivo Delivery of siRNA. Mol. Ther. Nucleic Acids 2012, 1, 1-9.).

[0120] The above-described multiple injection units may be, for example, two injection units. An ethanol feeding solution containing a lipid mixture and a water feeding solution containing a detection substance may be independently injected into each of the two injection units. The injected ethanol feeding solution containing a lipid mixture and the water feeding solution meet each other, and the lipids are monodispersely mixed in the material mixing unit. Thereafter, the lipid mixture solution undergoes a self-assembly process to form lipid crystal nanoparticles. This self-assembly process may be performed in a rotary evaporator.

[0121] The self-assembly conditions in the above rotary evaporator can be performed under conditions known in the art.

[0122]

[0123] The lipid nanoparticle may further comprise 1 mol % or less of a lipid for bioconjugation of a receptor that binds to a substance containing the target nucleic acid on the surface of the nanoparticle.

[0124] In the present invention, the term "bioconjugation" refers to linking a receptor capable of binding to a substance (e.g., a surface protein) present on the surface of a substance (e.g., a virus) containing a target nucleic acid to a binding functional group of the lipid nanoparticle (see Fig. 1(b)).

[0125] The lipid for bioconjugation may be maleimide-PEG-PE, but is not necessarily limited thereto, and any lipid used as a lipid for bioconjugation in this technical field may be included without limitation.

[0126] By combining a substance containing a target nucleic acid with a lipid nanoparticle through the above bioconjugation, membrane fusion can be promoted. For example, if the substance containing the target nucleic acid is SARS-CoV-2, this virus binds to ACE2 (angiotensin converting enzyme-2) of the host cell, and therefore, bioconjugating ACE2 to the surface of the lipid nanoparticle of the present invention can promote membrane fusion with SARS-CoV-2 (see FIGS. 2 and 11). In this case, the lipid used can be further included in an amount of 1 mol % or less to manufacture the lipid nanoparticle.

[0127] If the lipid for bioconjugation is used in an amount exceeding 1 mol%, problems such as disadvantageous membrane fusion or reduced loading efficiency may occur.

[0128]

[0129] The detection material for detecting the target nucleic acid may be a) a nucleic acid probe linked to a fluorescent material; or b) a guide RNA, a CRISPR-associated endonuclease, and a reporter nucleic acid; or c) a LAMP (Loop-Mediated Isothermal Amplification) reactant.

[0130] The above a) nucleic acid probe linked to a fluorescent substance may include a linker region in which a fluorescent substance and a quencher are labeled at each end, respectively, and the fluorescent substance and the quencher are bound to a nucleic acid; and a region capable of hybridizing with a target nucleic acid.

[0131]

[0132] The types of fluorescent substances and linkers are widely known in this technical field, so a detailed description is omitted.

[0133] The detection material comprising the above b) guide RNA, CRISPR-associated endonuclease, and reporter nucleic acid is a detection material based on CRIPSR (Clusters of Regularly Interspaced Palindromic Repeats).

[0134] The CRISPR system is an adaptive immune mechanism found in bacteria and other organisms, and is currently known as a genome editing technology that uses CRISPR-associated endonucleases.

[0135] The above CRISPR-associated endonucleases are Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Cas12a, Cas12b, Cas12c, Cas12d, Cas12e, Cas12g, Cas12h, Cas12i, Cas13a, Cas13b, Cas13c, Cas13d, Cas14, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, CsMT2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, It may be selected from the group consisting of, but is not necessarily limited to, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3 and Csf4.

[0136] The above guide RNA serves to recognize the target nucleic acid and may be a crRNA; or an sgRNA containing a crRNA.

[0137] The above CRISPR-associated endonuclease is an enzyme protein that plays a role in delivering target nucleic acids or cleaving external nucleic acids.

[0138] The reporter nucleic acid may be RNA or DNA, and is labeled with a fluorophore and a quencher at each end, respectively, and is cleaved when the CRISPR-associated endonuclease is activated, thereby emitting a fluorescent signal (see Fig. 3).

[0139] The above LAMP (Loop-Mediated Isothermal Amplification) is one of the DNA amplification methods performed using four or six primer sets and DNA polymerase under isothermal temperature conditions. The LAMP detector recognizes the target nucleic acid and causes a color change from red to yellow that can be seen with the naked eye through the LAMP amplification reaction (see Figure 13).

[0140] The above detection substances may be included in an amount of 0.00001-4 wt% or less, respectively, relative to the total weight of the lipid nanoparticles. If the amount exceeds the above range, the lipid nanoparticle structure may not be well maintained and the loading efficiency may decrease.

[0141] The above phosphocholine may be DPhyPC (1,2-Diphytanoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine)) or (DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine).

[0142] The above ionic lipid may be a cationic lipid or an anionic lipid.

[0143] The terms "cationic lipid" or "anionic lipid" are used herein to encompass any lipid of the present invention that is either cationic or anionic. A lipid will be determined to be cationic when it possesses a positive charge (at physiological pH) as measured by the instrument used for the measurement.

[0144] 구체적으로, 상기 양이온성 지질은, DDAB(dimethyldioctadecylammonium bromide); DOGS(N,N-di octadecyl amido glycyl spermine); DOTAP(1,2-dioleoyloxy-3-(trimethyl ammonium)propane); DODAC(dimethyldioleylammonium chloride); N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; DOTMA(N-[1-[2,3-bis(oleoyloxy)]propyl]-N,N,N-trimethylammonium chloride); DOGS(di-octadecyl-amido-glycyl-spermine); DC-cholesterol(3β-[N―(N′,N′'-dimethyl amino ethane)carbamoyl] cholesterol); DOSPA(2,3-dioleoyloxy-N-(2(spermine carboxamido)-ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate); 1,2-diacyl-sn-glycero-3-ethyl phosphocholines; β-alanyl cholesterol; CTAB(cetyl trimethylammonium bromide); DODMA(1,2-dioleyloxy-3-dimethylaminopropane); MVL5(N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide); DOSPER(1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide); N,N,N′,N′'-tetramethyl-N,N′'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butane diammonium iodide;and mixtures thereof, and any cationic lipid usable in the art may be used.

[0145] Specifically, the anionic lipid is DMPA (dimyristyl glycerophosphate); DPPA (dipalmitoyl glycerophosphate); DMPG (dimyristyl glycerophosphate); disteroyl glycerophosphate (DSPA); DSPG (distearoyl-sn-glycero-3-phosphoglycerol); DPPG (dipalmitoyl glycerophosphoraglycerol); MPS (dimyristylyl glycerophosphoseline); DPPS (dipalmitoyl glycerophosphoseline);

[0146] Any anionic lipid usable in this technical field may be selected from the group consisting of DSPS (disteroyl glycerophosphoseline) and mixtures thereof, but is not necessarily limited thereto.

[0147] The above helper lipid may be selected from the group consisting of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-bis(diphenylphosphino)ethane), DMPE (1,2-bis(dimethylphosphino)ethane), and mixtures thereof, but is not necessarily limited thereto, and any helper lipid having a phosphoethanolamine head usable in this technical field or a lipid molecule packing parameter of 1 or more may be used.

[0148] The above cholesterol is used to ensure lipid membrane stability.

[0149] The lipid bound to the above water-soluble polymer is used as a stabilizer for lipid crystal nanoparticles.

[0150] The water-soluble polymer may be selected from the group consisting of polyethylene glycol, polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polyethylene oxide, polysaccharide, and mixtures thereof, but is not necessarily limited thereto, and any water-soluble polymer usable in this technical field may be used.

[0151] In the lipid to which the above water-soluble polymer is bound, the lipid may be a phospholipid.

[0152] The lipid bound to the water-soluble polymer may be DOPE-PEG2000.

[0153] In the present invention, a lipid can be used in which the length of a water-soluble polymer is shortened (2000 to 5000 Da) and covalently bonded to the head portion of a lipid.

[0154] By using short, water-soluble polymers, micelle formation within the polymer itself can be prevented, and uniform particles can be formed using microfluidic devices. Furthermore, by separating the fusion and self-assembly processes of lipid nanoparticles from the mixing process, lipid nanoparticles of uniform size and structure can be formed.

[0155] That is, since the polymer and lipid are dissolved in the same solvent, ethanol, they are always mixed uniformly when mixed within the microfluidic device, and thus homogeneous particles can be formed.

[0156] The present invention does not use any oil or emulsifier, but oil and emulsifier may be mixed in if necessary.

[0157] The lipid nanoparticles according to the present invention may be conjugated to their surface with a substance containing a target nucleic acid, for example, a receptor capable of binding to a virus. This is to effectively facilitate membrane fusion with the virus, as previously mentioned.

[0158] The target nucleic acid may be DNA or RNA, and may include ssDNA, dsDNA, ssRNA, and microRNA.

[0159] The substance containing the detectable target nucleic acid includes, but is not necessarily limited to, RNA viruses, DNA viruses, cells, lipid-based vesicles (exosomes) containing nucleic acids secreted from the target organism, such as cells.

[0160] In another aspect, the present invention provides a method for producing lipid crystal nanoparticles, comprising:

[0161] The ethanol feeding solution containing the prepared lipid mixture and the water feeding solution containing the detection substance are each independently passed through a microfluidic device to mix the ethanol feeding solution containing the lipid mixture and the water feeding solution containing the target nucleic acid detection substance; and

[0162] A method for manufacturing lipid nanoparticles loaded with a detection substance for detecting target nucleic acids through a self-assembly process of the above mixed solution,

[0163] The above lipid mixture is composed as follows: a method for manufacturing lipid nanoparticles loaded with a detection substance for detecting a target nucleic acid:

[0164] i) phosphocholine 0-40 mol%;

[0165] ii) 0-40 mol% of ionic lipids;

[0166] iii) 0-40 mol% of helper lipids;

[0167] iv) cholesterol 0-40mol%; and

[0168] v) 0-5 mol% of lipids bound to water-soluble polymers.

[0169]

[0170] The lipid nanoparticle may further comprise 1 mol % or less of a lipid for bioconjugation of a receptor that binds to a substance containing the target nucleic acid on the surface of the lipid nanoparticle.

[0171] The definition of the above bioconjugation is as described above.

[0172] The contents for each material are the same as those mentioned above.

[0173]

[0174] In another aspect, the present invention provides a composition for detecting a target nucleic acid, comprising a lipid nanoparticle loaded with a detection substance for detecting the target nucleic acid.

[0175] The above composition may further include distilled water or a buffer solution that maintains the structure of the lipid nanoparticles stable.

[0176] In another aspect, the present invention provides a diagnostic kit comprising the composition for detecting the target nucleic acid.

[0177] The kit of the present invention may further comprise one or more other component compositions, solutions, or devices suitable for the detection method. For example, the kit may comprise a test tube or other suitable container, a reaction buffer (with varying pH and magnesium concentrations), sterile water, and the like.

[0178] In another aspect, the present invention provides a membrane fusion-based target nucleic acid detection method as follows:

[0179] By mixing a sample containing a substance in which a target nucleic acid exists inside a lipid bilayer and the above-mentioned lipid nanoparticle or composition for detecting a target nucleic acid, membrane fusion of the substance containing the target nucleic acid and the lipid nanoparticle carrying a detection substance for detecting the target nucleic acid is caused; and

[0180] The target nucleic acid detection material within the lipid nanoparticle reacts with the target nucleic acid to generate a detection signal.

[0181] The sample is a DNA virus or RNA virus, cell or lipid-based vesicle (exosome, extracellular vesicle, etc.) obtained from a subject, and the membrane fusion of the substance containing the target nucleic acid with the lipid nanoparticle may be a fusion of a viral lipid layer membrane or a cell membrane or a lipid-based vesicle lipid layer membrane with the lipid nanoparticle membrane.

[0182] As shown in Fig. 2, a viral material containing a target nucleic acid and a lipid nanoparticle of the present invention are combined through membrane fusion, and the target nucleic acid is detected by the target nucleic acid detection material of the lipid nanoparticle, thereby generating a fluorescent signal.

[0183] The term "subject" may include mammals, such as primates or humans. It may also include farmed animals (e.g., cats, dogs, etc.), livestock (e.g., cows, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mice, rabbits, rats, guinea pigs, etc.), and birds.

[0184] The term "sample" above refers to a sample obtained from a subject. For example, it can be obtained from a supernatant from the nasopharynx, saliva, sputum, blood, tissue, serum, feces, urine, and cell lysates.

[0185] The above target nucleic acid detection material is a) a nucleic acid probe linked to a fluorescent material; or b) a guide RNA, a CRISPR-associated endonuclease, and a reporter nucleic acid; or c) a LAMP (Loop-Mediated Isothermal Amplification) reactant, and when the detection material reacts with the target nucleic acid, fluorescence or a color change can be generated as a detection signal.

[0186] The membrane fusion-based target nucleic acid detection method according to the present invention does not require pretreatment or nucleic acid amplification processes for the target nucleic acid and can be performed at room temperature.

[0187] Additionally, the detection time is very fast, within 30 minutes. Preferably within 15 minutes, more preferably within 10 minutes, even more preferably within 5 minutes, and even more preferably within 2 minutes.

[0188] Additionally, the above method can be performed regardless of whether the target nucleic acid is of wild type or mutant origin.

[0189] Additionally, a sample containing a substance in which a target nucleic acid exists within the lipid bilayer can be obtained from a mask worn by the subject and / or the surface of an object on which the subject stays or uses, without a separate sample collection process.

[0190] In the object where the subject stays or uses, the subject is not limited to a human, and all animals other than humans are also included if the subject is suspected of having a virus, for example, a substance containing the target nucleic acid to be detected.

[0191] Additionally, target nucleic acids can be detected by mixing the lipid nanoparticles or the detection composition on the surface of the mask or object to cause membrane fusion.

[0192] The above objects include all objects in public spaces such as residential and office spaces as well as public facilities, for example, desks, furniture, stair railings, escalator handrails, elevator buttons, toilets and sinks in public restrooms, etc.

[0193] In addition, the lipid nanoparticles of the present invention can be stored in a refrigerator at 4°C for a long period of time.

[0194]

[0195] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0196]

[0197] [Example]

[0198] Hereinafter, the present invention will be described in detail by examples.

[0199] However, the following examples are only illustrative of the present invention, and the content of the present invention is not limited by the following examples.

[0200] <Example 1>

[0201] 1-1: Materials

[0202] DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), Cholesterol, DOTAP(Cholesterol, 1,2-dioleoyl-3-trimethylammonium-propane), DOPE-PEG2k, DSPE-PEG2k-maleimide, 18:1 NBD PE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl), and Liss Rhod PE(1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) were purchased from Avanti Polar Lipids (Alabaster, AL, USA) and were purified without purification. ACE2 (Human angiotensin converting) was used. Enzyme-2) was purchased from Sino Biological (Beijing, China). α2,6-linked sialic acid (Neu5Acα(2-6)Galβ(1-4)GlcNAc-β-ethylamine) was purchased from TCI Chemicals (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). Murine RNase Inhibitor was purchased from New England Biolabs (Ipswich, MA, USA). HEPES solution, potassium chloride solution, magnesium chloride solution, glycerol, ethanol, chloroform, and DTT (1,4-Dithiothreitol) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure TMDiethylpyrocarbonate (DEPC)-treated water was purchased from Thermo-Fisher (Waltham, MA, USA). Single-donor human saliva was purchased from Innovative Research, Inc. (Novi, MI, USA). Exosomes were extracted from in vitro cell (HeLa) culture medium using ExoQuick-TC (System Bioscience, Palo Alto, CA, USA).

[0203] Leptotrichia buccalis (Lbu) Cas13a was purchased from SignalChem Biotech Inc. (Richmond, BC, Canada). NEBuffer TM r2.1, SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit were purchased from New England Biolabs (Ipswich, MA, USA). Lachnospiraceae bacteriumCas12a, crRNA, and reporter nucleic acids were all purchased from Integrated DNA Technologies (Coralville, IA, USA), and the base sequences are listed in Table 1.

[0204]

[0205]

[0206] All lipids were dissolved in chloroform at a concentration of 25 mg / mL and used as a stock solution. After mixing at the desired ratio, the solvent was evaporated with nitrogen gas. After that, ethanol was added to make the concentration 10 mM and used as an ethanol feeding solution. The solvents and lipids used here were used without further purification.

[0207]

[0208] 1-2: Small-Angle X-ray Scattering

[0209] Synchrotron small-angle X-ray (S-X) scans were performed at beamline 4C (PLS-II) of the Pohang Accelerator Laboratory (PAL, Republic of Korea). The synchrotron X-ray source had an average photon energy of 16.9 keV with an average beam size of 100 μm Х 300 μm (V Х H). The sample-to-detector distance was approximately 1 m, and was compensated for using silver behenate.

[0210] Scattered photons were recorded using a Rayonix 2D SX 165 CCD detector (Rayonix, USA) and radially integrated using custom software provided at the beamline (beamline 4C). Information such as phospholipid bilayer thickness was analyzed using the Sasfit program.

[0211]

[0212] 1-3: Cryogenic transmission electron microscopy

[0213] The detailed morphology and structure of individual lipid nanoparticles, viruses, and membrane fusion intermediates between them were characterized by cryo-TEM (FEI Tecnai F20 G2 at 200 kV). Lipid nanoparticles were loaded onto lacey carbon-coated copper grids (Structure Probe Incorporation, PA, USA) using a Vitrobot (Vitrobot Mark IV, FEI). Briefly, 5 μL of synthesized lipid nanoparticles were dropped onto the carbon grids at 24°C and 100% relative humidity. The carbon grids were then rapidly quenched in liquid ethane after two blotting sessions (1 s blotting time) to form amorphous ice films less than 500 nm thick. The samples were then transferred to the cryo-EM using a cryo-transfer holder (Gatan, CA, USA). To prevent ice recrystallization, the sample temperature was maintained below -170°C until successfully transferred to the cryo-EM. Images were acquired underfocus at ~4000 nm. Higher magnification images were obtained using a smaller aperture to minimize electron beam damage and sample drift.

[0214]

[0215] 1-4: Particle size and zeta potential analysis

[0216] The size and zeta potential of individual lipid nanoparticles were characterized by Zetasizer Ultra (Malvern Panalytical Ltd., Malvern, UK).

[0217] 1-5: Analysis of the entrapment rate of reporter RNA and Cas13a protein-guide RNA complexes in liposomes

[0218] To evaluate the entrapment efficiency of reporter RNA and Cas13a protein-guide RNA complexes within liposomes, we first characterized the concentration of unentrapped reporter RNA and Cas13a protein-guide RNA complexes, and calculated the entrapment efficiency by assuming that all reporter RNA and Cas13a protein-guide RNA complexes were entrapped, minus the initial amount of reporter RNA and Cas13a protein-guide RNA added during liposome preparation.

[0219] To quantify the uncaptured Cas13a-crRNA complexes at this time, a calibration curve was obtained using the fluorescence values ​​of cleavage reactions performed for 1 h for two-fold diluted Cas13a-crRNA complexes in the presence of 395 nM short target 1 (see Table 1) and 1975 nM reporter RNA. Nonlinear regression was then obtained using the Boltzmann function as follows, and the amount of uncaptured Cas13a-crRNA complexes was quantified.

[0220] [Mathematical Formula 1]

[0221]

[0222]

[0223] To calculate the amount of uncaptured reporter RNA, RNA quantification was achieved using the Quant-iT RiboGreen RNA kit. A linear regression was then obtained from the calibration curve, as follows. Based on this, the amount of uncaptured reporter RNA was quantified.

[0224] [Equation 2]

[0225]

[0226]

[0227] 1-6: Long-term storage stability analysis of liposomes

[0228] The liposome solution loaded with Cas13a protein-crRNA complex and reporter RNA was aliquoted, protected from light, stored at 4°C, and then used immediately for stability analysis tests. The size of the liposomes was measured using a DLS device at 3 and 6 months after storage, and the membrane fusion-based assay performance of the present invention was characterized using a microplate reader. In the assay, a 384-well plate was loaded with a mixture of 10 μL of the liposome solution and 10 μL of a SARS-CoV-2 virus-negative sample or a mixture of 10 μL of the liposome solution and 10 μL of a 1.5 pM SARS-CoV-2 virus-positive sample, and the fluorescence intensity values ​​at 520 nm were acquired at 30-second intervals using a microplate reader (SpectraMax M2, Molecular Devices, CA, USA) excited at 485 nm.

[0229]

[0230] <Example 2> Liposome synthesis

[0231] 2-1: Liposome preparation

[0232] A schematic diagram of a liposome preparation method according to the present invention is shown in Fig. 1(a). A microfluidic device having a staggered herringbone mixer (SHM) was fabricated by a known method (Belliveau, NMet. al., Microfluidic Synthesis of Highly Potent Limit-Size Lipid Nanoparticles for in vivo Delivery of siRNA. Mol. Ther. Nucleic Acids 2012, 1, 1-9.). A lipid mixture (DOPC 40 mol%, DOPE 40 mol%, DOTAP 20 mol%) was prepared. The lipid mixture (DOPC 40 mol%, DOPE 40 mol%, DOTAP 20 mol%) dissolved in chloroform was dried in a vial and then dissolved again in 1 mL of an ethanol feeding solution (15 mM total lipid concentration).

[0233] Next, 3 mL of water feeding solution and 1 mL of ethanol feeding solution were injected into the inlet of a microfluidic device with a staggered herringbone mixer (SHM) to monodispersely mix the lipids. Specifically, the flow conditions of the ethanol and water feeding solutions were controlled using a syringe pump (NE-400, New Era Pump Systems, NY, USA). The flow conditions and flow rate ratio were fixed at 0.05 ml / min and 3, respectively. The emulsion obtained by mixing was collected in a 50 ml round-bottomed glass flask. The collected emulsion solution (total lipid concentration: 3.75 mM) was transferred to a rotary evaporator (RV 10, IKA, NC, USA) operating at P = 75 mbar and T = 57 °C for approximately 15 min. By evaporating the solvent, a final 0.15 M 100 μl solution was obtained, which exhibited a color change from transparent to white opaque but no macroscopic precipitates. The generated lipid nanoparticle solution was filtered through a centrifugal filter (Nanosep 100K MWCO, Sigma Aldrich, MO, USA) as needed to remove unreacted substances.

[0234] From the SAXS and cryo-TEM results in Fig. 4, it was confirmed that the liposome was well prepared by confirming the formation of a lipid bilayer.

[0235] A schematic diagram of the bioconjugation according to the present invention is shown in Fig. 1(b). When bioconjugating a receptor, 1 mol% of DOPC is replaced with 1 mol% of Maleimide-PEG-PE and the same method is used to prepare a final product (0.15 M 100 μl) with a protruding Maleimide functional group. Simultaneously, the receptor is immersed in a 10 mM Tris(2-corboxyethyl)phosphine hydrochloride solution to dissociate the dithiol bond. Then, a lipid nanoparticle solution is mixed with the receptor solution from which the dithiol bond has been dissociated and reacted at room temperature for 1 hour to prepare a lipid nanoparticle solution in which the receptor is bioconjugated. After that, the unreacted products are diluted with a Float-a-lyzer product and separated.

[0236]

[0237] 2-2: Preparation of liposomes loaded with Cas13a protein and reporter RNA

[0238] In the liposome manufacturing process of Example 2-1, 0.025 wt% and 0.055 wt% of Leptotrichia buccalis (Lbu) Cas13a protein-guide RNA complex and reporter RNA, respectively, were further added to the water feeding solution to manufacture liposomes loaded with detection substances.

[0239] Through Figure 5, it can be seen that the capture efficiency is 88-94% for RNA and ~75% for Cas13a protein-guide RNA complex.

[0240]

[0241] 2-3: Preparation of liposomes loaded with LAMP detection material

[0242] In the liposome manufacturing process of Example 2-1, 50 uL of WarmStart Colorimetric LAMP 2X Master Mix with UDG, 10 uL of SARS-CoV-2 LAMP Primer Mix (N / E), and 10 uL of Guanidine Hydrochloride from the SARS-CoV-2 Rapid Colorimetric LAMP Assay Kit solution were further added to the water feeding solution to manufacture liposomes loaded with a detection substance.

[0243]

[0244] 2-4: Preparation of liposomes loaded with Cas12a protein and reporter DNA

[0245] In the liposome manufacturing process of Example 2-1, 0.026 wt% and 0.05 wt% of Lachnospiraceae bacteriumCas12a protein-guide RNA complex and reporter DNA, respectively, were further added to the water feeding solution to manufacture liposomes loaded with detection substances.

[0246]

[0247] 2-5: Preparation of liposomes loaded with fluorescently linked nucleic acid probes

[0248] In the liposome manufacturing process of Example 2-1, 0.28 wt% and 0.296 wt% of fluorescent substance-linked nucleic acid probe 1 and fluorescent substance-linked nucleic acid probe 2 were further added to the water feeding solution, respectively, to manufacture liposomes loaded with detection substances.

[0249]

[0250] <Example 3> Confirmation of liposome characteristics

[0251] Figure 6 is a graph showing changes in liposome size and surface zeta potential according to loading of a detection agent for target nucleic acid detection and surface modification. It can be seen that liposome particle size increases with loading of the detection agent and surface modification, and that high zeta potential values ​​are observed even after introduction of ACE2 using cationic lipid molecules.

[0252]

[0253] <Example 4> Confirmation of membrane fusion properties between liposomes and coronaviruses

[0254] Figure 7 shows that liposomes were prepared by adding NBD-PE at 0.625 mol% of DOPC and Liss Rhod-PE at 0.625 mol% of DOPC to the lipid mixture of Example 2-1 based on 40 mol% of DOPC. When only liposomes were present, the distance between the fluorophore pairs (fluorescent donor NBD and fluorescence acceptor Liss Rhod) on the liposome surface was close, so FRET occurred and the fluorescence intensity ratio of the fluorescence donor / fluorescence acceptor was low at the level of 0.2. Next, as a result of observing the membrane fusion of the prepared liposome and the 30.73 pM SARS-CoV-2 virus, it was confirmed that membrane fusion had occurred by observing that the distance between the fluorophore pairs increased during membrane fusion and the fluorescence intensity ratio of the fluorescence donor / fluorescence acceptor increased. In addition, the fluorescence intensity ratio of the fluorescent donor / acceptor was confirmed by mixing the liposomes in lysis buffer (50 mM Tris(hydroxymethyl)-aminomethane (TRIS), 0.05% sodium azide) to completely disintegrate the liposomes, and comparing it with the membrane fusion results showed that membrane fusion occurred efficiently. The analysis was performed by loading the liposome and virus mixture into a 384-well plate and measuring the fluorescence change using a fluorescence microplate reader (SpectraMax M2, Molecular Devices, CA, USA) with excitation at 460 nm.

[0255] Figure 8 is a result of observing membrane fusion between liposomes and SARS-CoV-2 virus according to Example 2-2 using a transmission electron microscope, confirming that membrane fusion occurred by observing that the SARS-CoV-2 virus and liposomes were attached before membrane fusion and that the particle size increased after fusion.

[0256]

[0257] <Example 5> Confirmation of detection characteristics of liposomes

[0258] Figure 9(a) shows the analytical sensitivity results of CRISPR Cas13a, one of the detection substances loaded on the liposomes of Example 2-2. First, 2 M Lbu Cas13a and 2 M crRNA were mixed and reacted at room temperature for 30 minutes to prepare the Lbu Cas13a-crRNA complex. Next, Lbu Cas13a-crRNA was diluted to 100 nM in cleavage buffer (20 mM HEPES, 50 mM KCl, 5 mM MgCl2, 5% glycerol, and 1 U / mL Murine RNase Inhibitor) containing various concentrations of SARS-CoV-2 RNA and 400 nM reporter RNA. The solution was loaded at 20 μL in a 384-well plate and the fluorescence intensity value at 520 nm was obtained using a microplate reader (SpectraMax M2, Molecular Devices, CA, USA) excited at 485 nm. As a result, regardless of mutation, it was confirmed that SARS-CoV-2 RNA It can be seen that it shows sensitivity at the level of 10 fM.

[0259] Figure 9(b) shows the analytical specificity results of CRISPR Cas13a, one of the detection substances loaded onto liposomes in Example 2-2. The experiment was performed under the same experimental conditions as Figure 9(a), but instead of various concentrations of SARS-CoV-2 RNA, 100 fM of SARS-CoV-2 RNA, 100 fM of miR-21, or 100 fM of H1N1 RNA were included for analysis. As a result, a detection signal was observed only for SARS-CoV-2 RNA.

[0260] Figure 10(a) shows the analytical sensitivity of the liposome according to Example 2-2 to the SARS-CoV-2 virus, showing that even a virus of 2 aM can be detected with a detection time of only 2 minutes, and Figure 10(b) shows the analytical specificity of the liposome according to Example 2-2. 1.3 pM exosome and 1 10 7 TCID 50 Compared to H1N1 at / mL (~16.61 pM), only 3.8 pM SARS-CoV-2 virus showed a detectable signal. In the above assay, a mixture of 10 uL liposome solution and 10 uL SARS-CoV-2 virus-negative sample or 10 uL liposome solution and 10 uL SARS-CoV-2 virus-positive sample was loaded into a 384-well plate, and fluorescence intensity values ​​at 520 nm were obtained at 30-second intervals using a microplate reader (SpectraMax M2, Molecular Devices, CA, USA) excited at 485 nm.

[0261] Figure 11 shows that even when ACE2 is not present on the liposome surface according to Example 2-2, membrane fusion with the SARS-CoV-2 virus occurs only by the electrical attraction by cationic lipids, and that the detection signal further increases when ACE2 is introduced to the liposome surface. In the above analysis, a mixture of 10 uL of liposome solution and 10 uL of 30.4 pM inactivated SARS-CoV-2 virus was loaded into a 384-well plate, and the fluorescence intensity value at 520 nm was obtained using a microplate reader (SpectraMax M2, Molecular Devices, CA, USA) excited at 485 nm.

[0262] Figure 12 shows that the liposome according to Example 2-2 can detect the wild type SARS-CoV-2 virus and the XBB.1.5 mutant SARS-CoV-2 virus regardless of the mutation by showing similar detection signals.

[0263] Figure 13 shows that liposomes according to Example 2-3 exhibit a visually distinguishable color change through membrane fusion with the SARS-CoV-2 virus. In the above analysis, a PCR tube was loaded with a mixture of 10 uL of liposome solution and 10 uL of a SARS-CoV-2 virus-negative sample or a mixture of 10 uL of liposome solution and 10 uL of a 150 fM SARS-CoV-2 virus-positive sample, and the mixture was reacted at 65 degrees for 30 minutes.

[0264] Fig. 14(a) shows that the liposome according to Example 2-2 can detect the SARS-CoV-2 virus even on the surface of a mask. When the liposome solution according to the present invention was sprayed onto a mask contaminated with the SARS-CoV-2 virus, membrane fusion with the SARS-CoV-2 virus present on the mask occurred, and green fluorescence was obtained using an LED exciting at 465 nm. Fig. 14(b) shows that even a virus of 3 aM can be detected with a detection time of only 10 minutes. Therefore, using the membrane fusion-based detection method according to the present invention, target nucleic acids can be detected without a separate sample collection process.

[0265] Figure 15 demonstrates that liposomes according to Example 2-2 can detect SARS-CoV-2 viruses on various surfaces where the virus is present. The various surfaces included a wooden desk, a stainless steel surface, and a ceramic glass surface. When the liposome solution was sprayed, it fused with the SARS-CoV-2 virus present on the desk, and green fluorescence was observed using an LED excited at 465 nm. Consequently, detection of viruses as small as 3 aM was possible on all surfaces with a detection time of only 10 minutes.

[0266] Figure 16(a) shows that when the crRNA of Table 2 is used instead of the crRNA of Table 1 when preparing the Lbu Cas13a-crRNA complex among the detection substances loaded on the liposome of Example 2-2, the detection substance can also detect influenza A (H1N1) RNA.

[0267]

[0268]

[0269] Figure 16(b) shows that when crRNA4 and crRNA5 of Table 2 are used instead of crRNA of Table 1 in the liposome manufacturing according to Example 2-2, liposomes can detect influenza A (H1N1) virus. The detection time of only 2 minutes was 7.9 10 2 TCID 50 It can be seen that viruses can be detected even at / mL levels.

[0270] Figure 16(c) shows that when the crRNA of Table 1 is used in the liposome preparation according to Example 2-2, the liposome cannot be detected against the influenza A (H1N1) virus, demonstrating that the detection reaction is a target nucleic acid specific reaction.

[0271] Figure 17 shows the liposome prepared according to Example 2-2 at 4°C with light blocked. o When stored in a C refrigerator, the particle size continued to increase for up to 6 months, showing some particle agglomeration, but the detection performance was maintained by showing the same detection result for 750 fM SARS-CoV-2 virus.

[0272] Figure 18 shows that liposomes prepared according to Example 2-2 exhibit high RNase stability, as they show no change in fluorescence in the presence of 100 νg / mL of RNase A. In contrast, reporter RNA is degraded by RNase A, resulting in fluorescence.

[0273] Figure 19 shows that the same detection is achieved by simply adding the liposome solution to a 2 aM SARS-CoV-2 virus sample without physically mixing the liposome solution according to Example 2-2 with the SARS-CoV-2 virus sample. Here, the physical mixing was performed using a pipetting method.

[0274] Figure 20 demonstrates that the liposomal solution according to Example 2-2 can detect SARS-CoV-2 virus present in nasopharyngeal swabs and saliva. It can be seen that SARS-CoV-2 virus in saliva can be detected at concentrations of up to 2 aM with a detection time of only 2 minutes.

[0275] Figure 21(a) shows that when the crRNA of Table 3 is used instead of the crRNA of Table 1 when preparing the Lbu Cas13a-crRNA complex among the detection substances loaded on the liposome of Example 2-2, the detection substance can also detect RSV (Respiratory Syncytial Virus) RNA.

[0276]

[0277]

[0278] Figure 21(b) shows that liposomes can detect RSV virus when crRNA of Table 3 is used instead of crRNA of Table 1 in liposome preparation according to Example 2-2. The detection time of only 2 minutes was 1.25 10 4 TCID 50 It can be seen that viruses can be detected even at / mL levels.

[0279] Figure 22(a) shows that when the crRNA of Table 4 is used instead of the crRNA of Table 1 when preparing the complex of Lbu Cas13a-crRNA among the detection substances loaded on the liposome of Example 2-2, the detection substance can also detect miRNA-21.

[0280]

[0281]

[0282] Figure 22(b) shows that detection of exosomes is possible when using crRNA of Table 4 instead of crRNA of Table 1 during liposome preparation according to Example 2-2. It can be seen that liposomes can detect 1.3 pM of exosomes with a detection time of only 2 minutes.

[0283] Figure 23(a) shows that when the crRNA of Table 5 is used in the preparation of the Lb Cas12a-crRNA complex among the detection substances loaded on the liposomes of Example 2-4, the detection substance can also detect HSV (Herpes Simplex virus) DNA. First, the Lb Cas12a-crRNA complex was prepared by mixing 200 nM Lb Cas12a and 250 nM crRNA7 and reacting at room temperature for 30 minutes. Then, the Lb Cas12a-crRNA was diluted to 100 nM in 1X NEB buffer containing 12 pM of isolated HSV DNA and 750 nM of reporter DNA. 20 uL of the solution was loaded into a 384-well plate, and the fluorescence intensity value at 520 nm was obtained using a microplate reader (SpectraMax M2, Molecular Devices, CA, USA) that excites at 485 nm. At this time, the temperature inside the microplate reader is 37 o It was maintained as C.

[0284]

[0285]

[0286] Figure 23(b) shows that when the crRNA of Table 4 is used in the liposome preparation according to Example 2-4, the liposome can detect the HSV virus. A detection time of only 10 minutes is enough to achieve 7900 TCID. 50 It can be seen that viruses can be detected even at / mL levels.

[0287] Figure 24(a) shows that the nucleic acid probe linked to a fluorescent substance loaded on the liposome of Example 2-5 can detect SARS-CoV-2 RNA. First, the nucleic acid probe 1 linked to a fluorescent substance and the nucleic acid probe 2 linked to a fluorescent substance were diluted to 1 νM in 1X PBS buffer and 95 o Heat at C for 5 minutes and 4 o By cooling to C, a secondary structure in the shape of a hairpin was formed. Then, the fluorophore-linked nucleic acid probe 1 and the fluorophore-linked nucleic acid probe 2 were diluted to 500 nM in 1X PBS buffer containing 1.55 nM of isolated SARS-CoV-2 RNA. 20 μL of the solution was loaded into a 384-well plate, and the fluorescence intensity value at 520 nm was obtained using a microplate reader (SpectraMax M2, Molecular Devices, CA, USA) that excites at 485 nm. At this time, the internal temperature of the microplate reader was 37 o It was maintained as C.

[0288] Figure 24(b) shows that the liposome according to Example 2-5 can detect the SARS-CoV-2 virus. The detection time was only 10 minutes, which is 1.3 10 7 TCID 50 It can be seen that viruses can be detected even at / mL levels.

[0289]

[0290] As described above, the lipid nanoparticles loaded with the target nucleic acid detection agent according to the present invention can fuse with lipid bilayers and membranes, and thus can be used to detect internal nucleic acids of cells, exosomes, viruses, etc. comprised of lipid bilayers. In addition, the lipid nanoparticles loaded with the target nucleic acid detection agent according to the present invention can detect wild type or mutants without distinction, and do not require separate preprocessing or amplification processes, and thus can be used as field diagnostic kits. In addition, viruses present on the surfaces of masks and / or various objects can be detected without a separate sample collection process.

[0291]

[0292] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0293] The lipid nanoparticles according to the present invention can be used for membrane fusion-based target nucleic acid detection. The membrane fusion-based target nucleic acid detection method according to the present invention does not require preprocessing or nucleic acid amplification of the target nucleic acid, can be performed at room temperature, and allows for rapid confirmation of test results.

Claims

1. A lipid nanoparticle loaded with a detection substance for detecting a target nucleic acid, wherein the lipid nanoparticle is manufactured by the following method: Lipid nanoparticles are manufactured by independently passing an ethanol feeding solution containing a lipid mixture and a water feeding solution containing the detection substance through a microfluidic device to mix and undergo a self-assembly process. The above lipid mixture is, i) phosphocholine 0-40 mol%; ii) 0-40 mol% of ionic lipids; iii) 0-40 mol% of helper lipids; iv) cholesterol 0-40mol%; and v) Composed of 0-5 mol% lipids bound to water-soluble polymers.

2. In paragraph 1, A lipid nanoparticle further comprising 1 mol % or less of a lipid for bioconjugation of a receptor that binds to a substance containing the target nucleic acid on the surface of the lipid nanoparticle.

3. In paragraph 2, The above lipid is a lipid nanoparticle comprising maleimide-PEG-PE.

4. In paragraph 1, The detection material for detecting the target nucleic acid is a lipid nanoparticle comprising: a) a nucleic acid probe linked to a fluorescent material; or b) a guide RNA, a CRISPR-associated endonuclease, and a reporter nucleic acid; or c) a LAMP (Loop-Mediated Isothermal Amplification) reactant.

5. In paragraph 4, A lipid nanoparticle, wherein the above detection substances are contained in an amount of 0.00001-4 wt% or less, respectively, with respect to the total weight of the lipid nanoparticle.

6. In paragraph 1, A lipid nanoparticle, wherein the phosphocholine is selected from the group consisting of DPhyPC (1,2-Diphytanoyl-sn-glycero-3-phosphocholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine), (DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine)) and mixtures thereof.

7. In paragraph 1, Lipid nanoparticles wherein the ionic lipid is a cationic lipid or anionic lipid.

8. In paragraph 7, 상기 양이온성 지질은, DDAB(dimethyldioctadecylammonium bromide); DOTAP(1,2-dioleoyloxy-3-(trimethyl ammonium)propane); DODAC(dimethyldioleylammonium chloride); N-[1-(2,3-Dioloyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; DOTMA(N-[1-[2,3-bis(oleoyloxy)]propyl]-N,N,N-trimethylammonium chloride); DOGS(di-octadecyl-amido-glycyl-spermine); DC-cholesterol(3β-[N―(N′,N′-dimethyl amino ethane)carbamoyl] cholesterol); DOSPA(2,3-dioleoyloxy-N-(2(spermine carboxamido)-ethyl)-N,N-dimethyl-1-propanaminium trifluoroacetate); 1,2-diacyl-sn-glycero-3-ethyl phosphocholines; β-alanyl cholesterol; DODMA(1,2-dioleyloxy-3-dimethylaminopropane); MVL5(N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide); CTAB(cetyltrimethylammonium bromide); DOSPER(1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide); N,N,N′,N′'-tetramethyl-N,N′'-bis(2-hydroxylethyl)-2,3-dioleoyloxy-1,4-butane diammonium iodide; 및 이들의 혼합물로 이루어진 군으로부터 선택되는 것인, 지질 나노입자. 9.제7항에 있어서, The anionic lipid includes DMPA (dimyristyl glycerophosphate); DPPA (dipalmitoyl glycerophosphate); DMPG (dimyristyl glycerophosphate); disteroyl glycerophosphate (DSPA); DSPG (distearoyl-sn-glycero-3-phosphoglycerol); DPPG (dipalmitoyl glycerophosphoraglycerol); MPS (dimyristylyl glycerophosphoseline); DPPS (dipalmitoyl glycerophosphoseline); A lipid nanoparticle selected from the group consisting of DSPS (disteroyl glycerophosphoseline); DSPE (1,2-distearoyl-3-sn-phosphatidyl ethanolamine) and mixtures thereof.

10. In paragraph 1, A lipid nanoparticle, wherein the helper lipid is selected from the group consisting of DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-bis(diphenylphosphino)ethane), DMPE (1,2-bis(dimethylphosphino)ethane), and mixtures thereof.

11. In paragraph 1, A lipid nanoparticle, wherein the water-soluble polymer is selected from the group consisting of polyethylene glycol, polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polyethylene oxide, polysaccharide, and mixtures thereof.

12. In paragraph 1, A lipid nanoparticle in which the water-soluble polymer is bound to the lipid, wherein the molecular weight of the water-soluble polymer is 2000 to 5000 Da and the lipid is a phospholipid.

13. In paragraph 1, The lipid nanoparticle to which the above water-soluble polymer is bound is DOPE-PEG2000.

14. In paragraph 1, The above lipid nanoparticle is a lipid nanoparticle having a receptor conjugated to its surface that can bind to a substance containing a target nucleic acid.

15. In paragraph 1, The above target nucleic acid is a lipid nanoparticle of DNA or RNA.

16. Prepare an ethanol feeding solution containing a lipid mixture and a water feeding solution containing a target nucleic acid detection substance, respectively; The ethanol feeding solution containing the prepared lipid mixture and the water feeding solution containing the detection substance are each independently passed through a microfluidic device to mix the ethanol feeding solution containing the lipid mixture and the water feeding solution containing the target nucleic acid detection substance; and A method for manufacturing lipid nanoparticles loaded with a detection substance for detecting target nucleic acids through a self-assembly process of the above mixed solution, The above lipid mixture is composed as follows: a method for manufacturing lipid nanoparticles loaded with a detection substance for detecting a target nucleic acid: i) phosphocholine 0-40 mol%; ii) 0-40 mol% of ionic lipids; iii) 0-40 mol% of helper lipids; iv) cholesterol 0-40mol%; and v) 0-5 mol% of lipids bound to water-soluble polymers.

17. In paragraph 16, A method for manufacturing lipid nanoparticles, wherein the lipid nanoparticles further comprise 1 mol % or less of lipid for bioconjugation of a receptor that binds to a target on the surface of the nanoparticles.

18. A composition for detecting a target nucleic acid, comprising a lipid nanoparticle loaded with a detection substance for detecting a target nucleic acid according to any one of claims 1 to 15.

19. In paragraph 18, A composition for detecting a target nucleic acid, wherein the target nucleic acid is DNA or RNA.

20. A kit for detecting target nucleic acid comprising the composition of Article 18.

21. By mixing a sample containing a substance in which a target nucleic acid exists inside a lipid bilayer with a lipid nanoparticle of any one of claims 1 to 15 or a detection composition of claim 18, membrane fusion of the substance containing the target nucleic acid and the lipid nanoparticle carrying a detection substance for detecting the target nucleic acid is caused; and The target nucleic acid detection material in the lipid nanoparticle reacts with the target nucleic acid to generate a detection signal; A membrane fusion-based target nucleic acid detection method comprising steps.

22. In paragraph 21, The above sample is a DNA virus or RNA virus or a cell or lipid-based vesicle obtained from a subject, A membrane fusion-based target nucleic acid detection method, wherein the membrane fusion of the substance containing the target nucleic acid and the lipid nanoparticle is performed by fusion of the lipid nanoparticle membrane with a viral lipid layer membrane, a cell membrane, or a lipid-based vesicle lipid layer membrane.

23. In paragraph 21, The target nucleic acid detection material is a) a nucleic acid probe linked to a fluorescent material; or b) a guide RNA, a CRISPR-associated endonuclease, and a reporter nucleic acid; or c) a LAMP (Loop-Mediated Isothermal Amplification) reactant. A membrane fusion-based target nucleic acid detection method, wherein the detection material reacts with the target nucleic acid to generate fluorescence or color change as a detection signal.

24. In paragraph 21, The above method is a membrane fusion-based target nucleic acid detection method that does not require pretreatment and nucleic acid amplification or pretreatment process for the target nucleic acid and is performed at room temperature or isothermal temperature.

25. In paragraph 21, The above method is a membrane fusion-based target nucleic acid detection method, wherein the target nucleic acid is of wild type or mutant origin.

26. In paragraph 21, A sample containing a substance in which a target nucleic acid exists inside the lipid bilayer is obtained from a mask worn by the subject and / or the surface of an object on which the subject stays or uses without a separate sample collection process, and A membrane fusion-based target nucleic acid detection method, wherein the lipid nanoparticles or the detection composition are mixed on the surface of the mask or object to cause membrane fusion.

27. A membrane fusion-based target nucleic acid detection method according to claim 21, wherein the detection signal occurs within 30 minutes.

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