Compositions and methods using modified liposomes

Abstract

Disclosed herein are compositions and methods of using a modified liposome comprising (i) an encapsulated hydrophilic acridinium ester (AE), and (ii) a first agent encapsulated by the liposome and / or (iii) a second agent on the surface of the liposome. In particular, the disclosed methods provide methods of labeling a target of interest, determining a target antigen in a biological sample, and detecting a target antigen in a biological sample. Further disclosed herein are methods for improving signal strength detected by imaging modalities.

Description

[0001] This application is a divisional application of the invention patent application filed on February 1, 2021, with application number 202180017972.X and invention title "Compositions and methods using modified liposomes". Technical Field

[0002] This article discloses compositions and methods for preparing liposomes encapsulating hydrophilic acridinium esters and for detecting and labeling target antigens in biological samples. Background Technology

[0003] Immunoassays remain the preferred method in clinical laboratories for analyzing a wide range of analytes, particularly complex and heterogeneous molecules. However, the lack of immunoassay signals and sensitivity can be a major obstacle to accurate diagnosis and disease prediction. There has been a persistent need in the field for improved immunoassay labeling methods that provide rapid and reliable results, which would benefit both patients and healthcare providers. Summary of the Invention

[0004] This article discloses modified liposomes comprising (i) an encapsulated hydrophilic acridine ester (AE), and (ii) a first reagent encapsulated by the liposome and / or (iii) a second reagent on the surface of the liposome.

[0005] In some implementations, the first reagent includes nucleic acids, hydrophobic drugs, or hydrophilic drugs.

[0006] In some embodiments, the second reagent comprises a peptide, antibody, carbohydrate, polyethylene glycol (PEG), PEGylated peptide, small molecule, or drug. In some embodiments, the peptide is biotin, avidin, streptoavidin, or luciferin. In some embodiments, the PEGylated peptide comprises a PEGylated antibody or PEGylated biotin. In other embodiments, the drug is a hydrophobic drug or a drug conjugated to the surface of a liposome.

[0007] According to some implementation schemes, the encapsulated hydrophilic AE has a content of at least 1 x 10⁻⁶. -8 mol / L to at least 1 x 10 - 6 Concentrations in the mol / L range. In other embodiments, the encapsulated hydrophilic AE comprises at least 1,000 to at least 100,000,000,000 hydrophilic AE molecules.

[0008] In a further embodiment, the liposomes have a diameter of about 200 nm to about 1000 nm. In some embodiments, the liposomes have a diameter of about 30 nm to about 100 nm.

[0009] This paper also discloses a method for labeling targets of interest. The method includes conjugating the target to a modified liposome disclosed herein.

[0010] On the one hand, this document provides a method for determining target antigens in biological samples. The method includes (a) combining a biological sample with a modified liposome of the present disclosure in a culture medium; and examining the culture medium for target antigens bound to the modified liposomes.

[0011] This document also provides a method for detecting a target antigen in a biological sample. The method includes (a) contacting the biological sample with a modified liposome of the present disclosure, wherein the modified liposome is specifically conjugated to a target antigen; (b) imaging a signal generated by the conjugated target antigen; and (c) detecting the signal generated in step (b) thereby detecting the target antigen.

[0012] This document further provides a method for enhancing the signal intensity detected by imaging modality. The method includes (a) labeling the target by conjugating the target to a modified liposome of the present disclosure; (b) imaging the signal generated by the labeled target; and (c) detecting the signal generated in step (b).

[0013] A method for improving the sensitivity of an immunoassay is also provided. The method includes (a) labeling a target by conjugating the target to a modified liposome of the present disclosure; (b) imaging the signal generated by the labeled target; and (c) detecting the signal generated in step (b). Attached Figure Description

[0014] The overview and the following detailed description will be further understood when read in conjunction with the accompanying drawings. Exemplary embodiments of the disclosed devices, systems, and methods are shown in the drawings for the purpose of illustrating the devices, systems, and methods; however, the devices, systems, and methods are not limited to the specific embodiments disclosed. In the figures: Figure 1 These are schematic diagrams illustrating various examples of liposome modification using various materials.

[0015] Figure 2A-2C These are a series of schematic diagrams illustrating the collection of acridine esters (AE) into liposomes. Figure 2A and 2B Liposomes containing different concentrations of AE were shown. The biochemical and biophysical properties of liposomes can be modified, such as... Figure 2C As shown. In Figure 2C In liposomes, the surface carries a negative charge, while... Figure 2A and 2B In this case, the net charge is neutral.

[0016] Figures 3A-3B For example, liposomes containing or encapsulating AEs with additional modifications can be prepared ("liposomes encapsulated with AEs"). Figure 3A Liposomes encapsulating AE can be further modified with various functional groups, including but not limited to biotin, fluorescein and / or proteins on the surface of the liposomes. Figure 3B The permeability and hydrophilicity of liposome membranes can be enhanced by adding polyethylene glycol (PEG) to the surface of liposomes.

[0017] Figure 4 This is a series of schematic diagrams showing the diameters of liposomes encapsulating AEs that can be generated to collect AEs of different sizes. Liposomes collecting AEs with diameters of 30 nm, 50 nm, and 100 nm were prepared. Detailed Implementation

[0018] The disclosed methods can be more readily understood by taking into account the accompanying drawings, which form part of this disclosure, and the following detailed description. It should be understood that the disclosed methods are not limited to the specific methods described and / or shown herein, and the terminology used herein is for illustrative purposes only and is not intended to limit the claimed methods.

[0019] It should be understood that, for clarity, certain features of the disclosed methods described herein in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the disclosed methods described in the context of a single embodiment may also be provided separately or in any sub-combination.

[0020] definition Unless otherwise defined, 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 pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice of testing the invention, preferred materials and methods are described herein. The following terminology will be used in describing and claiming protection for this invention.

[0021] It should also be understood that the terminology used in this document is for the purpose of describing specific implementation schemes only and is not intended to be restrictive.

[0022] As used herein, the singular forms “a” ("a", "an") and “the” ("the") include plural indicators unless the context explicitly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context explicitly indicates otherwise.

[0023] As used herein, the term “about” will be understood by those skilled in the art and will vary to some extent in the context in which it is used. As used herein, when referring to measurable values ​​such as amount, concentration, duration, etc., the term “about” is intended to cover variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1%, as such variations are suitable for implementing the disclosed methods.

[0024] As disclosed herein, the term acridine ester refers to any acridine ester that can be encapsulated in liposomes and can generate a chemiluminescent signal.

[0025] As used herein, the term "analyte" is a broad term and is used in its usual sense, including but not limited to, referring to a detectable component or target of interest in a sample, such as a substance or chemical component in biological fluids (e.g., blood, interstitial fluid, cerebrospinal fluid, lymph, or urine). Analytes can include naturally occurring substances, man-made substances, metabolites, and / or reaction products. Examples of analytes include, but are not limited to, single or multiple epitopes, antigenic or haptenic ligands, or nucleic acids such as DNA or RNA.

[0026] As used herein, the terms “solid support,” “support structure,” and “substrate” are used interchangeably and refer to a material or group of materials having one or more rigid or semi-rigid surfaces. There are no limitations on the shape or size of the support structure. In many embodiments, the solid support will take the form of beads (e.g., silica beads, magnetic beads, paramagnetic beads, etc.), resin, gel, microspheres, or other geometries.

[0027] As used herein, a "functional group" refers to a chemical group within a molecule that is responsible for a characteristic chemical reaction. Exemplary functional groups include, but are not limited to, those containing oxygen, nitrogen, phosphorus, or sulfur atoms, such as primary amines, carboxyl groups, carbonyl groups, aldehydes, mercapto groups, hydroxyl groups, and esters. As used herein, a functional group is reactive to another group if two groups can react to form a covalent bond.

[0028] A "connector" is a molecule that connects two other molecules by covalent bonds or by ionic bonds, van der Waals bonds or hydrogen bonds. For example, a nucleic acid molecule that hybridizes with a complementary sequence at the 5' end and another complementary sequence at the 3' end, thereby connecting two non-complementary sequences.

[0029] A "crosslinking agent" is a connector that covalently links two other molecules.

[0030] As used herein, the term "liposome" refers to an artificially formed vesicle or capsule consisting of a membrane comprising at least one lipid bilayer. This term is understood to exclude naturally occurring vesicles or other naturally occurring membrane substances isolated from cells or biological samples containing cells. The terms "vesicle" and "liposome" may be used herein as synonyms for an artificially formed capsule comprising at least one lipid bilayer membrane. For example, a large, artificially formed monolayer liposome vesicle or "LUV" is referred to as a vesicle, but for the purposes of this patent application, it is also referred to as a liposome.

[0031] Poly(ethylene glycol), commonly known as PEG, refers to oligomers that form straight chains of ethylene oxide. PEG molecules can be straight-chain or branched, wherein each molecule has at least two, and usually three or more, PEG branches or arms derived from a central core group.

[0032] The term "antibody" refers to an immunoglobulin molecule capable of specifically binding to a specific epitope on an antigen. Antibodies are typically tetramers of immunoglobulin molecules. The antibodies of this invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabody"), Fv, Fab, and F(ab)2, as well as single-chain antibodies (scFv), camel antibodies, and humanized antibodies. As envisioned herein, antibodies conjugated to quantum dots and support structures can specifically or non-specifically recognize and / or bind to analytes, thereby enabling qualitative and quantitative analysis of the analytes.

[0033] As used herein, the terms "comprising," "including," "containing," and "characterized in" are interchangeable, inclusive, open-ended, and do not exclude additional, unlisted elements or method steps. Any reference to the term "comprising" herein, particularly in the description of the components of a composition or the elements of an apparatus, should be understood to cover compositions and methods that are substantially composed of the listed components or elements, as well as compositions and methods composed of the listed components or elements.

[0034] As used herein, the term "composed of" excludes any element, step, or component not specified in the claims.

[0035] "Disease" refers to any symptom or lesion that impairs or interferes with the normal function of cells, tissues, or organs.

[0036] "Detection" refers to identifying the presence, absence, or quantity of a target (e.g., an analyte to be detected).

[0037] When these terms are used interchangeably herein, “individual,” “patient,” or “subject” includes members of any animal species, including but not limited to birds, humans, and other primates, as well as other mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs. Preferably, a subject is a human.

[0038] As used herein, the terms "treatment" and "treating" refer to methods used to obtain a beneficial or desired outcome, including but not limited to therapeutic and / or preventative benefits. For example, the term treatment includes administering a medication before or after the onset of a disease or lesion to prevent or eliminate all symptoms of the disease or lesion. As another example, administering a medication after the clinical manifestation of a disease to combat its symptoms constitutes "treatment" of the disease.

[0039] Therapeutic benefit refers to the eradication or improvement of the underlying lesion being treated, or the improvement of one or more physiological symptoms associated with the underlying lesion, thereby observed improvement in a patient, even though the patient may still have the underlying lesion. For preventative benefit, the composition may be administered to patients at risk of developing a specific disease, or to patients who have reported one or more physiological symptoms of a disease, even if a diagnosis of the disease may not yet have been made.

[0040] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the range format description is merely for convenience and brevity and should not be construed as a rigid limitation on the scope of the invention. Therefore, the range description should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges, and, where appropriate, partial integers of the numerical values ​​within the ranges. For example, a description of a range such as 1 to 6 should be considered as having specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and the individual numbers within those ranges, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0041] Various terms are used throughout the specification and claims to relate to the aspects described. Unless otherwise stated, these terms shall be given their ordinary meaning in the art. Other specifically defined terms shall be interpreted in a manner consistent with the definitions provided herein.

[0042] Detailed description This article provides modified liposomes comprising (i) an encapsulated hydrophilic acridine ester (AE), and (ii) a first reagent encapsulated by the liposome and / or (iii) a second reagent on the surface of the liposome.

[0043] Acetate esters (AEs) are stable compounds that offer superior immunoassay performance in the form of increased sensitivity compared to radioisotopes. The use of AEs can benefit a variety of applications, such as labeling ligands or analytes (e.g., antigens); labeling specific binding partners of ligands or analytes (e.g., corresponding antibodies); or labeling nucleic acids and molecules containing nucleic acids.

[0044] The hydrophilic properties of the AEs used in the modified liposomes of this disclosure make them suitable for encapsulation within liposomes without leakage through the liposome wall. Detailed descriptions of hydrophilic AEs can be found in the art, for example in U.S. Patent No. 5,656,426 A, the disclosure of which is incorporated herein by reference in its entirety.

[0045] In some embodiments, the concentration of hydrophilic AE encapsulated by the liposomes of this disclosure is at least 1.10. -10 mol / L to at least 1.10 -9 mol / L, at least 1.10 -9 mol / L to at least 1.10 -8 mol / L, at least 1.10 -8 mol / L to at least 1.10 - 7 mol / L, at least 1.10 -7 mol / L to at least 1.10 -6 mol / L, at least 1.10 -6 mol / L to at least 1.10 -5 mol / L, at least 1.10 -5 mol / L to at least 1.10 -4 mol / L, at least 1.10 -4 mol / L to at least 1.10 -3 mol / L, at least 1.10 -3 mol / L to at least 1.10 -2 mol / L, and at least 1.10 -2 mol / L to at least 1.10 -1 mol / L. In other embodiments, the hydrophilic AE has a range of at least 1.10 mol / L. -8 mol / L to at least 1.10 -6 Concentration in mol / L.

[0046] In some embodiments, the modified liposomes comprise at least 10 to at least 100 hydrophilic AE molecules, at least 100 to at least 1,000 hydrophilic AE molecules, at least 1,000 to at least 10,000 hydrophilic AE molecules, at least 10,000 to at least 100,000 hydrophilic AE molecules, at least 100,000 to at least 1,000,000 hydrophilic AE molecules, at least 1,000,000 to at least 10,000,000 hydrophilic AE molecules, at least 10,000,000 to at least 100,000,000 hydrophilic AE molecules, at least 100,000,000 to at least 1,000,000,000 hydrophilic AE molecules, at least 1,000,000,000 to at least 10,000,000,000 hydrophilic AE molecules, at least 10,000,000,000 to at least 100,000,000,000 hydrophilic AE molecules, and at least 100,000,000,000 to at least 1,000,000,000,000 hydrophilic AE molecules. In other embodiments, the modified liposomes comprise at least 1,000 to at least 100,000,000,000 hydrophilic AE molecules.

[0047] In further embodiments, the modified liposomes disclosed herein include various sizes. In some embodiments, the diameter of the liposomes is from about 20 nm to about 1000 nm. In some embodiments, the diameter of the liposomes is about 20 nm to about 30 nm; about 30 nm to about 40 nm; about 40 nm to about 50 nm; about 50 nm to about 60 nm; about 60 nm to about 70 nm; about 70 nm to about 80 nm; about 80 nm to about 90 nm; about 90 nm to about 100 nm; about 100 nm to about 110 nm; about 110 nm to about 120 nm; about 120 nm to about 130 nm; about 130 nm to about 140 nm; about 140 nm to about 150 nm; about 150 nm to about 160 nm; about 160 nm to about 170 nm; about 170 nm to about 180 nm; about 180 nm to about 190 nm; about 190 nm to about 200 nm; about 200 nm to about 250 nm; about 250 nm to about 300 nm; about 350 nm to about 400 nm; about 400 nm to about 450 nm; about 450 nm to about 500 nm. nm; about 500 nm to about 550 nm; about 550 nm to about 600 nm; about 600 nm to about 650 nm; about 650 nm to about 700 nm; about 700 nm to about 750 nm; about 750 nm to about 800 nm; about 800 nm to about 850 nm; about 850 nm to about 900 nm; about 900 nm to about 950 nm; about 950 nm to about 1000 nm. In other embodiments, the diameter of the liposomes is about 20 nm to about 500 nm. In still other embodiments, the diameter of the liposomes is about 30 nm to about 100 nm.

[0048] In some embodiments, the first reagent encapsulated by the modified liposomes of this disclosure may be a nucleic acid, a hydrophobic drug, or a hydrophilic drug. Examples of hydrophobic drugs include, but are not limited to, amphotericin B, silymarin, docetaxel, simvastatin, haloperidol, and albendazole. Examples of hydrophilic drugs include, but are not limited to, doxorubicin hydrochloride, cytosine-arabinoside, ethinylcytidine, and 5-fluorodeoxyuridine.

[0049] In some embodiments, the second agent modifying the liposome comprises a peptide, an antibody or its antigen-binding fragment, an aptamer, an affinity compound, an affinity polymer, a carbohydrate, polyethylene glycol (PEG), a PEGylated peptide, a small molecule, or a drug. In some embodiments, the peptide is biotin, avidin, or an avidin derivative (e.g., neutral avidin), streptavidin, or luciferin. In some embodiments, the PEGylated peptide comprises a PEGylated antibody or PEGylated biotin. In some embodiments, the drug is a hydrophobic drug or a drug conjugated to the surface of the liposome.

[0050] The second reagent may be a recombinant, chimeric, genetically engineered, or conjugated protein. In some embodiments, the second reagent may be a recombinant antibody or antibody fragment. The second reagent may include epitopes, antigens, or other modifications that can be used to molecularly or immunogenically label, express, or purify the second reagent. For example, in some embodiments, the second reagent may be conjugated to a 6-His tag (6-histidine), Myc tag, HA tag, FLAG tag, or similar epitope tag to facilitate the separation, isolation, or extraction of the second reagent from a reaction mixture containing the composition described herein and, for example, a biological sample. The second reagent of the modified liposomes described herein can be extracted from the liposomes by using a suitable detergent or by mechanical force that disrupts the membrane. Thus, the second reagent can be separated from the liposomes after a biochemical assay to determine the amount of interfering substances associated with the second reagent.

[0051] In some embodiments, the modified liposomes described in this application include multilayered liposome vesicles (MLVs), small monolayered liposome vesicles (SUVs), large monolayered liposome vesicles (LUVs), and giant monolayered liposome vesicles (GUVs). In some embodiments, the lipid bilayer may comprise sphingolipids, glycerophospholipids, sterols, and sterol derivatives. The sphingolipids to be used may include sphingomyelin and ceramides containing saturated, monounsaturated, and / or polyunsaturated acyl chains of varying lengths. Phospholipids having various head group structures may be used, including phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin, and phosphatidylserine (PS), containing saturated, monounsaturated, and / or polyunsaturated acyl chains of varying lengths. The sterols and sterol derivatives to be used may include cholesterol, brassosterol, allocholesterol, cholesterol methyl ether, campestanol, campesterol, cholesterol acetate, coprostanol, chain sterol, dehydrochain sterol, dihydrocholesterol, dihydrolanosterol, epicholesterol, lathosterol, lanosterol, sitostanol, sitosterol, stigmasterol, zymostenol, and zymosterol.

[0052] Lipids that can be used to form the liposomes of this disclosure may include natural or synthetic sphingolipids, glycerophospholipids, sterols, and sterol derivatives. Sphingolipids that can be used include sphingomyelin and ceramides, which contain saturated, monounsaturated, and / or polyunsaturated acyl chains of varying lengths. Phospholipids that can be used with various head group structures include phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin, and phosphatidylserine (PS), which contain saturated, monounsaturated, and / or polyunsaturated acyl chains of varying lengths. The sterols and sterol derivatives that may be used include, but are not limited to, cholesterol, brassosterol, allocholesterol, cholesterol methyl ether, campesterol, campesterol, cholesterol acetate, colistanol, chain sterol, dehydrochain sterol, dihydrocholesterol, dihydrolanosterol, epicholesterol, enolanol, lanosterol, sitosterol, sitosterol, stigmasterol, zimoenol, and yeast sterol.

[0053] Lipid mixtures can also be used in the modified liposomes of this disclosure, including mixtures of sphingolipids, glycerophospholipids, sterols, and sterol derivatives. Sterols and sterol derivatives should not be used alone; that is, sterols and sterol derivatives should be included in a mixture having liposomes containing sphingolipids or glycerophospholipids ranging from about 0% to about 50% of the total lipids. In some embodiments, sphingolipids may include porcine brain sphingomyelin, egg sphingomyelin, and milk sphingomyelin. In some embodiments, glycerophospholipids may comprise phospholipids having various head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin, phosphatidylserine (PS) having two saturated acyl chains of different lengths (e.g., 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearate-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine). 1,2-Distearyl-sn-glycerol-3-phosphate ethanolamine, 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-distearyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycerol-3-phosphate-(1'-inositol), 1,2-distearyl-sn-glycerol-3-phosphate inositol, 1',3'-bis[1,2-dipalmitoyl- [sn-glycerol-3-phosphate]-glycerol, 1',3'-bis[1,2-distearyl-sn-glycerol-3-phosphate]-glycerol, 1,2-dispalmitoyl-sn-glycerol-3-phosphate-L-serine, 1,2-distearyl-sn-glycerol-3-phosphate-L-serine), and a saturated acyl chain of varying lengths and a monounsaturated acyl chain of varying lengths (e.g., 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-sn-glycerol-3-phosphate-L-serine), as well as saturated acyl chains of varying lengths and monounsaturated acyl chains of varying lengths (e.g., 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-sn-glycerol-3-phosphate choline). -2-Oleoyl-sn-glycerol-3-phosphate choline, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate inositol, 1',3'-Bis[1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate]-glycerol, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine), and a saturated acyl chain of varying lengths and a polyunsaturated acyl chain of varying lengths (e.g., 1-palmitoyl-2-linoleoyl-sn-glycerol-3-phosphate choline, 1-stearoyl-2-linoleoyl-sn-glycerol-3-phosphate choline, 1-palmito ... Acyl-2-linoleyl-sn-glycerol-3-phosphate ethanolamine, 1-stearoyl-2-linoleyl-sn-glycerol-3-phosphate ethanolamine, 1-palmitoyl-2-linoleyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-stearoyl-2-linoleyl-sn-glycerol-3-phosphate-(1'-rac-glycerol), 1-palmitoyl-2-linoleyl-sn-glycerol-3-phosphate-L-serine, 1-stearoyl-2-linoleyl-sn-glycerol-3-phosphate-L-serine. In some embodiments, each fatty acid acyl chain has about 16 to 20 carbon atoms. In other embodiments, each fatty acid acyl chain has 16, 18, or 20 carbon atoms. In some embodiments, the number of double bonds in each fatty acid acyl chain ranges from 0 to 2. Sterols and sterol derivatives used with liposomes containing sphingolipids or glycerophospholipids may include cholesterol, dihydrocholesterol, epicholesterol, sitosterol, and encholestanol. Sphingolipids and glycerophospholipids may be used alone or as a mixture of sphingolipids and glycerophospholipids in the presence of sterols and sterol derivatives to form the modified liposomes described in this application. For example, in some embodiments, the liposomes may contain 100% porcine brain sphingomyelin or a mixture of porcine brain sphingomyelin and cholesterol. In some non-limiting examples, sterols and sterol derivatives may range from about 0% to about 50% of the total lipids, most preferably about 30%.

[0054] The modified liposomes disclosed herein may comprise modified phospholipids. For example, sphingolipids and glycerophospholipids may be modified with small molecules, polyethylene glycol (PEG), fluorescent molecules, fluorescent PEG, and / or bromine. Modified forms of sphingolipids and glycerophospholipids, sterols, sterol derivatives, and lipids are readily available commercially from various sources, such as Sigma-Aldrich (St. Louis, MO); Invitrogen (Carlsbad, CA); Avanti Polar Lipids (Alabaster, AL); Fisher Scientific (Pittsburgh, PA); and Steraloids (Newport, RI).

[0055] This paper also discloses a method for labeling targets of interest. The method includes conjugating the target to a modified liposome disclosed herein.

[0056] In some embodiments, the target of interest may be an antigen, hapten, DNA probe, RNA probe, or antibody. In other embodiments, the target of interest may be present in a biological sample. In some embodiments, the biological sample may be, but is not limited to, whole blood, serum, plasma, urine, saliva, semen, or cerebrospinal fluid.

[0057] On the one hand, this document provides a method for determining target antigens in biological samples. The method includes (a) combining a biological sample with a modified liposome of the present disclosure in a culture medium; and examining the culture medium for target antigens bound to the modified liposomes.

[0058] In some embodiments, the assay includes a biochemical assay, such as an immunoassay, a clinical chemistry assay, or other medical or diagnostic test. In some embodiments, the assay may include a sandwich assay or an in situ hybridization assay.

[0059] In some embodiments, in addition to biological samples, the modified liposomes of this disclosure can be added to or incubated with reagents used for biochemical assays. For example, the modified liposome composition can be incubated with a buffer solution provided as a component of a biochemical assay. Furthermore, the modified liposomes can be incubated with a reaction mixture for a biochemical assay, the mixture comprising one or more reagents for the assay and a biological sample. In some embodiments, the modified liposome composition is added in suspension form to a biological sample, reagent, or reaction mixture for a biochemical assay. In some embodiments, the modified liposome composition is reconstituted from a "dry form" in the biological sample, reagent, or reaction mixture, or in one or more components of the reaction mixture used for a biochemical assay.

[0060] This document also provides a method for detecting a target antigen in a biological sample. The method includes (a) contacting the biological sample with a modified liposome of the present disclosure, wherein the modified liposome is specifically conjugated to a target antigen; (b) imaging a signal generated by the conjugated target antigen; and (c) detecting the signal generated in step (b) thereby detecting the target antigen.

[0061] This document further provides a method for improving the signal intensity detected by an imaging modality. The method includes (a) labeling the target by conjugating the target to a modified liposome of the present disclosure; (b) imaging the signal generated by the labeled target; and (c) detecting the signal generated in step (b).

[0062] A method for improving the sensitivity of an immunoassay is also provided. The method includes (a) labeling a target by conjugating the target to a modified liposome of the present disclosure; (b) imaging the signal generated by the labeled target; and (c) detecting the signal generated in step (b).

[0063] In some embodiments, the modified liposomes of this disclosure are ruptured, and the amount of signal generated by the encapsulated hydrophilic AE is measured.

[0064] In some embodiments, the modified liposomes of this disclosure are used to detect peptides and / or nucleic acids. For example, DNA or RNA probes are labeled using ligands, such as haptens or biotinylated modified nucleotides. The DNA or RNA probe is allowed to hybridize with complementary DNA or RNA and immobilized on a solid support. The immobilized probe then reacts with a modified liposome containing a receptor (e.g., an antibody; or, if the probe is biotinylated, an avidin). The liposome is ruptured, and the amount of signal generated by the encapsulated acridinium ester is measured.

[0065] Other aspects of this disclosure include determining the presence or amount of a target of interest (e.g., a peptide or polypeptide) or detecting its biological activity using methods known in the art. These methods include immunoassay devices and methods that can utilize labeled molecules in various sandwich, competitive, or other assay formats. Such assays will produce a signal indicating the presence or absence of the peptide or polypeptide. Furthermore, the signal intensity can preferably be directly or indirectly correlated (e.g., inversely proportional) with the amount of polypeptide present in the sample. Further suitable methods include measuring peptide- or polypeptide-specific physical or chemical properties, such as its precise molecular weight or NMR spectrum. These methods include, for example, biosensors, optical devices coupled to immunoassays, biochips, analytical devices such as mass spectrometers, NMR analyzers, or chromatographic devices. Further, methods include microplate ELISA-based methods, fully automated or robotic immunoassays (e.g., Siemens platforms such as ADVIA Centaur). ® XPT, ADVIA Centaur ® XP, ADVIA Centaur ® CP, IMMULITE ® 1000, IMMULITE ® 2000 XPi and Atellaca ® ), enzyme-catalyzed cobalt binding assay (CBA) and latex agglutination assay.

[0066] Specific hybridization can be performed under highly stringent or moderately stringent conditions, depending on the circumstances. In a preferred embodiment, the hybridization conditions for specific hybridization are highly stringent. Specific hybridization is then detected using standard methods, if present. If specific hybridization occurs between the nucleic acid probe and a gene in the test sample, the sequence present in the nucleic acid probe is also present in the subject's mRNA. More than one nucleic acid probe may also be used.

[0067] Explanatory implementation plan This document provides illustrative embodiments of the disclosed technology. These embodiments are illustrative only and do not limit the scope of this disclosure or the appended claims.

[0068] Implementation Scheme 1. A modified liposome comprising (i) an encapsulated hydrophilic acridine ester (AE), and (ii) a first reagent encapsulated by the liposome and / or (iii) a second reagent on the surface of the liposome.

[0069] Implementation Scheme 2. The modified liposome of Implementation Scheme 1, wherein the first reagent comprises at least one selected from the group consisting of nucleic acids, hydrophobic drugs, and hydrophilic drugs.

[0070] Implementation Scheme 3. The modified liposome of Implementation Scheme 1, wherein the second reagent comprises a peptide, antibody, carbohydrate, polyethylene glycol (PEG), PEGylated peptide, small molecule or drug.

[0071] Implementation Scheme 4. The modified liposome of Implementation Scheme 3, wherein the polypeptide is biotin, avidin, streptavidin, or fluorescein.

[0072] Implementation Scheme 5. The modified liposome of Implementation Scheme 3, wherein the polyethylene glycol-modified polypeptide comprises a polyethylene glycol-modified antibody or a polyethylene glycol-modified biotin.

[0073] Implementation Scheme 6. The modified liposome of Implementation Scheme 3, wherein the drug is a hydrophobic drug or a drug conjugated to the surface of the liposome.

[0074] Implementation Scheme 7. The modified liposomes of Implementation Scheme 1, wherein the encapsulated hydrophilic AE has a molecular weight of at least 1 x 10⁻⁶. -8 mol / L to at least 1 x 10 -6 Concentration in the range of mol / L.

[0075] Implementation Scheme 8. The modified liposome of Implementation Scheme 1, wherein the encapsulated hydrophilic AE contains at least 1,000 to at least 100,000,000,000 hydrophilic AE molecules.

[0076] Implementation Scheme 9. The modified liposomes of Implementation Scheme 1, wherein the diameter of the liposomes is about 20 nm to about 1000 nm.

[0077] Implementation Scheme 10. The modified liposomes of Implementation Scheme 9, wherein the diameter of the liposomes is about 30 nm to about 100 nm.

[0078] Implementation Scheme 11. A method for labeling a target of interest, the method comprising conjugating the target to a modified liposome according to any one of the preceding embodiments.

[0079] Implementation Scheme 12. A method for determining a target antigen in a biological sample, the method comprising: a. In a culture medium, the biological sample is combined with the modified liposomes according to any one of embodiments 1-11; b. Examine the culture medium for target antigens that bind to the modified liposomes.

[0080] Implementation Scheme 13. A method for detecting a target antigen in a biological sample, the method comprising: c. Contact the biological sample with a modified liposome according to any one of embodiments 1-11, wherein the modified liposome is specifically conjugated to the target antigen; d. Imaging the signals generated by the conjugated target antigen; and e. Detect the signal generated in step (b) to detect the target antigen.

[0081] Implementation Scheme 14. A method for increasing the signal intensity detected by an imaging mode, the method comprising: f. Labeling the target by conjugating the target with a modified liposome according to any one of embodiments 1-11; g. Imaging the signal generated by the marked target; and h. Detect the signal generated in step (b).

[0082] Implementation Scheme 15. A method for improving the sensitivity of an immunoassay, the method comprising: i. Labeling the target by conjugating the target with a modified liposome according to any one of embodiments 1-11; j. Imaging the signal generated by the marked target; and k. Detect the signal generated in step (b). Example

[0083] The following examples are provided to further describe some embodiments disclosed herein. These examples are intended to illustrate, and not limit, the disclosed embodiments.

[0084] Materials and methods reagents 1,2-Dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC); 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC); 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-L-serine (POPS); porcine brain sphingomyelin (SM); cholesterol (CHOL); 1,2-distearatel-sn-glycerol-3-phosphate ethanolamine-N-[Biotinyl (PEG)-2000](PEG 2000) Biotin-DSPE); 1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(Biotinyl) (Biotin-DPPE); 1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(Rhodamine-DPPE); N-(Fluorescence-5-thiocarbamoyl)-1,2-Dipalmitoyl-sn-glycerol-3-phosphate ethanolamine (Fluorescence-DHPE); N-sulfopropyl-dimethylacridinium ester-N-hydroxysuccinimide (NSP-DMAE) and trimethylsilylpropionate DMAE (TSP-DMAE).

[0085] Lipids are stored at -20°C. Various polycarbonate membrane filters with pore sizes of 30, 50, 100, 200, and 400 nanometers are used.

[0086] Example 1: Preparation and purification of liposomes encapsulating acridinium esters This article presents liposomes of various sizes (e.g., 30–1000 nm) that encapsulate a large number of signal-generating molecules (e.g., hundreds of millions), such as acridinium esters (AE).

[0087] To prepare 4 mM large monolayer liposome vesicles (LUVs) encapsulating acridinium esters, lipids (SM, DPPC, POPC, DOPC, SM / POPC 1 / 1, DPPC / POPC 1 / 2, or POPC / POPS 3 / 1) were mixed and dried under nitrogen, followed by drying under high vacuum for at least 2 hours. The amount of cholesterol used in the liposomes varied between 0 and 50 mol% depending on the specific experiment. All lipid mixtures contained 0.025 mol% rhodamine PE to track the final concentration of the liposomes. The dried lipid membranes were dispersed at 70 °C in phosphate-buffered saline (PBS, 137 mM NaCl, pH 7.4) containing NSP-DMAE or TSP-DMAE and then cooled to room temperature before use. Figure 2A-2CAs shown, the concentration of AE can be changed ( Figure 2A-2B ), or by altering the biochemical and biophysical properties of liposomes (e.g., negatively charged liposomes, Figure 2C Various liposomes encapsulating acridine esters (AEs) were designed. The concentrations of NSP-DMAE and TSP-DMAE varied between 0 and 15 mg / mL. The lipid mixtures were subjected to 10 freeze-thaw cycles and then extruded through polycarbonate filters with specific pore sizes (e.g., 30 nm, 50 nm, 100 nm, 200 nm, and 400 nm) to obtain uniform liposome sizes. Figure 4 A NAP-5 (Sephadex G-25) column was used to remove uncollected NSP-DMAE or TSP-DMAE. Dynamic light scattering (DLS) measurements were performed before and after NAP-5 column purification. The particle size distribution of the resulting liposomes showed that the average diameter of the liposomes was maintained after the purification step.

[0088] Example 2: Preparation and purification of functionalized liposomes encapsulating acridinium esters The aim was to determine whether liposomes capable of collecting AEs could be effectively prepared even when various functional groups were present on the surface of liposomes, and to confirm that the addition of functional groups did not affect the quality of the resulting liposomes. Figures 3A-3B To this end, biotin-DPPE, PEG 2000 biotin-DSPE, or fluorescein-DHPE were added to lipid mixtures, i.e., SM, DPPC, POPC, DOPC, SM / POPC 1 / 1, DPPC / POPC 1 / 2, or POPC / POPS 3 / 1, with or without cholesterol, as described above, followed by drying of the lipids under nitrogen. The amounts of biotin-DPPE, PEG 2000 biotin-DSPE, and fluorescein-DHPE used in the liposomes varied between 0 and 20 mol%. The addition of polyethylene glycol (PEG) particularly enhanced the permeability and hydrophilicity of the liposome surface. Dynamic light scattering (DLS) analysis of the resulting liposomes showed that functionalized liposomes with diameters of 30 nm, 50 nm, and 100 nm could be formed efficiently. The addition of biotin-DPPE, PEG 2000 biotin-DSPE, and fluorescein-DHPE did not affect the ability of the liposomes to collect acridine esters. To check the stability of liposomes containing functional groups, the size of the liposomes remains constant.

[0089] like Figures 3A-3B As shown, the liposomes encapsulating AE in this disclosure may include additional modifications. These modifications include, but are not limited to, the addition of various functional groups to the liposome surface, such as biotin, luciferin, and / or proteins. Figure 3ABy adding polyethylene glycol (PEG) to the surface of liposomes, the permeability and hydrophilicity of the liposome membrane can be enhanced. Figure 3B ).

[0090] Therefore, various functional groups, including but not limited to biotin, luciferin, and proteins, can be attached to the surface of the liposomes disclosed herein. These complexes can be used to detect targets of interest using immunoassay platforms, such as, but not limited to, Siemens platforms like ADVIA Centaur. ® XPT, ADVIA Centaur ® XP, ADVIA Centaur ® CP, IMMULITE ® 1000, IMMULITE ® 2000 XPi and Atellaca ® The vesicles encapsulating AEs disclosed in this invention significantly enhance the immunoassay signal or relative light units (RLUs) and optimize the sensitivity of the output results. Depending on the liposome surface modification, the functionalized AE liposomes can be used as lite reagents or signal amplifiers.

[0091] The publications of every patent, patent application, and publication cited in this article are incorporated herein in their entirety by reference.

[0092] Those skilled in the art will understand that many changes and modifications can be made to the preferred embodiments of the invention, and such changes and modifications can be made without departing from the spirit of the invention. Therefore, the appended claims are intended to cover all such equivalent variations that fall within the true spirit and scope of the invention.

Claims

1. A modified liposome comprising (i) an encapsulated hydrophilic acridine ester (AE), and (ii) a first reagent encapsulated by said liposome and / or (iii) a second reagent on the surface of said liposome.

2. The modified liposome of claim 1, wherein the first reagent comprises at least one selected from the group consisting of nucleic acids, hydrophobic drugs, and hydrophilic drugs.

3. The modified liposome of claim 1, wherein the second reagent comprises a polypeptide, antibody, carbohydrate, polyethylene glycol (PEG), PEGylated polypeptide, small molecule, or drug.

4. The modified liposome of claim 3, wherein the polypeptide is biotin, avidin, streptavidin, or fluorescein.

5. The modified liposome of claim 3, wherein the polyethylene glycol-modified polypeptide comprises a polyethylene glycol-modified antibody or a polyethylene glycol-modified biotin.

6. The modified liposome of claim 3, wherein the drug is a hydrophobic drug or a drug conjugated to the surface of the liposome.

7. The modified liposome of claim 1, wherein the encapsulated hydrophilic AE has a size of at least 1 x 10⁻⁶. -8 mol / L to at least 1 x 10 -6 Concentration in the range of mol / L.

8. The modified liposome of claim 1, wherein the encapsulated hydrophilic AE comprises at least 1,000 to at least 100,000,000,000 hydrophilic AE molecules.

9. The modified liposome of claim 1, wherein the diameter of the liposome is about 20 nm to about 500 nm.

10. The modified liposome of claim 9, wherein the diameter of the liposome is from about 30 nm to about 100 nm.

11. A method for labeling a target of interest, the method comprising conjugating the target to a modified liposome according to any one of the preceding claims.

12. A method for determining a target antigen in a biological sample, the method comprising: a. In a culture medium, the biological sample is combined with the modified liposomes according to any one of claims 1-11; b. Examine the culture medium for the target antigens that bind to the modified liposomes.

13. A method for detecting a target antigen in a biological sample, the method comprising: a. Contact the biological sample with the modified liposomes according to any one of claims 1-11, wherein the modified liposomes are specifically conjugated to the target antigen; b. Imaging the signals generated by the conjugated target antigen; and c. Detect the signal generated in step (b) to detect the target antigen.

14. A method for increasing the signal intensity detected by an imaging mode, the method comprising: a. Labeling the target by conjugating the target to a modified liposome according to any one of claims 1-11; b. Imaging the signals generated by the marked target; and c. Detect the signal generated in step (b).

15. A method for improving the sensitivity of an immunoassay, the method comprising: a. Labeling the target by conjugating the target to a modified liposome according to any one of claims 1-11; b. Imaging the signals generated by the marked target; and c. Detect the signal generated in step (b).

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