Compositions and methods using modified liposomes

Abstract

Disclosed herein are compositions and methods for using modified liposomes, comprising (i) encapsulated hydrophilic acridinium ester (AE), and (ii) a first drug encapsulated in the liposome and / or (iii) a second drug on the surface of the liposome.In particular, the disclosed methods provide a method for labeling a target of interest, a method for assaying a biological sample for a target antigen, and a method for detecting a target antigen in a biological sample.Disclosed herein are further methods for increasing the intensity of the signal detected by an imaging modality.

Description

[Technical field]

[0001] Disclosed herein are compositions and methods for preparing liposomes that encapsulate hydrophilic acridinium esters, and for detecting and labeling target antigens in biological samples. [Background technology]

[0002] Immunoassays remain the method of choice in clinical laboratories for the analysis of many analytes, especially complex, heterogeneous molecules. Summary of the Invention [Problem to be solved by the invention]

[0003] Lack of immunoassay signal and sensitivity can be a major obstacle to accurately diagnosing and prognosing disease. There is a constant need in the art for improved immunoassay labeling methods that provide rapid and reliable results that are beneficial to both patients and health care providers. [Means for solving the problem]

[0004] Disclosed herein are modified liposomes comprising (i) an encapsulated hydrophilic acridinium ester (AE), and (ii) a first drug encapsulated in the liposome and / or (iii) a second drug on the surface of the liposome.

[0005] In some embodiments, the first agent comprises a nucleic acid, a hydrophobic drug, or a hydrophilic drug.

[0006] In some embodiments, the second agent comprises a polypeptide, an antibody, a carbohydrate, a polyethylene glycol (PEG), a pegylated polypeptide, a small molecule, or a drug. In some embodiments, the polypeptide is biotin, avidin, streptavidin, or fluorescein. In some embodiments, the pegylated polypeptide comprises a pegylated antibody or a 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 embodiments, the encapsulated hydrophilic AE is at least 1×10 -8 mol / L ~ at least 1 x 10 -6 In another embodiment, the encapsulated hydrophilic AE comprises at least 1000 to at least 100,000,000,000 hydrophilic AE molecules.

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

[0009] Also disclosed herein is a method of labeling a target of interest, comprising conjugating the target to the modified liposomes disclosed herein.

[0010] In one aspect, provided herein is a method of assaying a biological sample for a target antigen, the method comprising: (a) combining the biological sample with modified liposomes of the present disclosure in a medium; and examining the medium for the target antigen bound to the modified liposomes.

[0011] Also disclosed herein is a method for detecting a target antigen in a biological sample, the method comprising: (a) contacting the biological sample with the disclosed modified liposome that is specifically conjugated with the target antigen; (b) imaging the signal generated by the conjugated target antigen; and (c) detecting the signal generated in step (b), thereby detecting the target antigen.

[0012] Further provided herein is a method for increasing the intensity of a signal detected by an imaging modality, comprising: (a) labeling a target by conjugating the target to the disclosed modified liposome; (b) imaging the signal generated by the labeled target; and (c) detecting the signal generated in step (b).

[0013] Also provided is a method for increasing the sensitivity of an immunoassay, comprising: (a) labeling a target by conjugating the target to a disclosed modified liposome; (b) imaging a signal produced by the labeled target; and (c) detecting the signal produced in step (b).

[0014] The summary, as well as the following detailed description, will be better understood when read in conjunction with the appended drawings. For the purposes of illustrating the disclosed devices, systems, and methods, there are shown in the drawings exemplary embodiments of the devices, systems, and methods; however, the devices, systems, and methods are not limited to the particular embodiments disclosed. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 is a schematic diagram illustrating various examples of liposome modification using various materials. [Diagram 2] Figures 2A-C are a series of schematic diagrams illustrating liposomes entrapped with acridinium esters (AE). Figures 2A and 2B show liposomes containing various concentrations of AE. The biochemical and biophysical properties of the liposomes can be modified as shown in Figure 2C. The surface of the liposome is negatively charged in Figure 2C, whereas the net charge is neutral in Figures 2A and 2B. [Diagram 3]3A-B are diagrams illustrating that liposomes containing or encapsulating AE ("AE-encapsulating liposomes") can be made with further modifications. Figure 3A: AE-encapsulating liposomes are further modified with various functional groups on the liposome surface, including but not limited to biotin, fluorescein, and / or proteins. Figure 3B: Permeability and hydrophilicity of the liposome membrane is enhanced by adding polyethylene glycol (PEG) to the surface of the liposome. [Figure 4] A series of schematic diagrams showing that the diameter of AE-encapsulated liposomes can be produced in various sizes. AE-entrapped liposomes of 30 nm, 50 nm, and 100 nm were prepared. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The disclosed methods may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying drawings, which form a part of this disclosure: It will be understood that the disclosed methods are not limited to the specific methods described and / or illustrated herein, and that the terminology used herein is for the purpose of describing specific embodiments by example only, and is not intended to be limiting of the claimed methods.

[0017] It will be appreciated that certain features of the disclosed methods that are, for clarity, described herein in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, can also be provided separately or in any subcombination.

[0018] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein that can be used in carrying out the testing of the present invention can be used, but the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0019] It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0020] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise.

[0021] As used herein, the term "about" will be understood by those of ordinary skill in the art and will vary to some extent with the context in which it is used. As used herein, when referring to a measurable value, such as an amount, concentration, time, etc., the term "about" is intended to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the specified value, as such variations are appropriate for carrying out the disclosed method.

[0022] The term acridinium ester, as disclosed herein, refers to any acridinium ester that can be encapsulated within a liposome and produce a chemiluminescent signal.

[0023] The term "analyte," as used herein, is a broad term and is used in its ordinary sense, including, without limitation, to refer to a detectable component or target of interest in a sample, such as a substance or chemical component in a biological fluid (e.g., blood, interstitial fluid, cerebrospinal fluid, lymphatic fluid, or urine). Analytes can include naturally occurring substances, man-made substances, metabolic products, and / or reaction products. Examples of analytes include, but are not limited to, ligands that are mono- or polyepitopes, antigenic, or haptens, or nucleic acids such as DNA or RNA.

[0024] The terms "solid support", "support structure" and "substrate" as used herein are used interchangeably and refer to a material or group of materials having one or more surfaces that are rigid or semi-rigid. There is no limit to the shape or size of the support structure. In many embodiments, the solid support takes the form of beads (e.g., silica beads, magnetic beads, paramagnetic beads, etc.), resins, gels, microspheres, or other geometric configurations.

[0025] As used herein, "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 that contain oxygen, nitrogen, phosphorus, or sulfur atoms, such as primary amines, carboxyls, carbonyls, aldehydes, sulfhydryls, hydroxyls, and esters. As used herein, a functional group is reactive with another group if it can react with that group to form a covalent bond.

[0026] "Linker" refers to a molecule that links two other molecules by covalent, or ionic, van der Waals, or hydrogen bonds, e.g., a nucleic acid molecule that hybridizes to one complementary sequence at its 5' end and to another complementary sequence at its 3' end, thus linking two non-complementary sequences.

[0027] "Crosslinker" refers to a linker that covalently joins two other molecules.

[0028] The term "liposome" as used herein refers to an artificially formed vesicle or sac composed of a membrane that comprises at least one lipid bilayer. The term is understood to exclude naturally occurring vesicles or other naturally occurring membranous materials isolated from cells or biological samples that contain cells. The terms "vesicle" and "liposome" may be synonymous when used herein with reference to an artificially formed sac that comprises at least one lipid bilayer membrane. For example, artificially formed large unilamellar liposomal vesicles, or "LUV", are referred to as vesicles, but for the purposes of this patent application, they are also referred to as liposomes.

[0029] Poly(ethylene glycol), commonly known as PEG, refers to oligomers of ethylene oxide that form linear chains. PEG molecules can be linear or branched, with each molecule having at least two, and typically three or more, PEG branches, or arms, emanating from a central core group.

[0030] The term "antibody" refers to an immunoglobulin molecule that can specifically bind to a particular epitope on an antigen. An antibody is typically a tetramer of immunoglobulin molecules. The antibody in the present invention can exist in various forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)2, as well as single-chain antibodies (scFv), camelid antibodies and humanized antibodies. As contemplated herein, the antibody conjugated to quantum dots and supporting structures can specifically or nonspecifically recognize and / or bind to an analyte, so that the analyte can be analyzed qualitatively and quantitatively.

[0031] As used herein, the terms "comprising," "including," "containing," and "characterized by" are interchangeable, inclusive, open-ended, and do not exclude additional, unrecited elements or method steps. Any reference herein to the term "comprising," particularly in describing a component of a composition or describing an element of a device, is understood to encompass compositions and methods consisting essentially of, and consisting of, the recited component or element.

[0032] As used herein, the term "consisting of" excludes any element, step, or ingredient not specified in the claim element.

[0033] By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

[0034] "Detecting" refers to identifying the presence, absence, or amount of a target (eg, the analyte being detected).

[0035] An "individual," "patient," or "subject," as these terms are used interchangeably herein, includes a member of any animal species, including, but not limited to, birds, humans and other primates, and other mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, cats, and dogs. Preferably, the subject is a human.

[0036] As used herein, the terms "treatment" and "treating" refer to an approach to obtain beneficial or desired results, including but not limited to therapeutic and / or prophylactic effects. For example, the term treatment includes administering an agent before or after the onset of a disease or disorder, thereby preventing or eliminating all signs of the disease or disorder. As another example, administration of an agent after clinical signs of a disease that is effective against the symptoms of the disease includes "treatment" of the disease.

[0037] By therapeutic effect, it is meant the eradication or amelioration of the underlying disorder being treated, or the amelioration of one or more of the physiological symptoms associated with the underlying disorder, such that improvement is observed in the patient, although the patient may still be afflicted by the underlying disorder.For prophylactic effect, the composition can be administered to a patient who is at risk of developing a particular disease, or who complains of one or more of the physiological symptoms of the disease, even if the diagnosis of the disease has not been made.

[0038] Throughout this disclosure, various aspects of the invention are expressed in range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to specifically disclose all possible subranges as well as individual numerical values ​​within that range, and, where appropriate, partial integers of numerical values ​​within the range. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0039] Various terms relating to the described embodiments are used throughout the specification and claims. Such terms are given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms are to be interpreted in a manner consistent with the definitions provided herein.

[0040] Detailed Description Provided herein are modified liposomes comprising (i) an encapsulated hydrophilic acridinium ester (AE), and (ii) a first agent encapsulated in the liposome and / or (iii) a second agent on the surface of the liposome.

[0041] AEs are stable compounds that provide superior immunoassay performance in the form of increased sensitivity compared to radioisotopes. The use of AEs can be advantageous in a variety of applications, such as labeling ligands or analytes (such as antigens); labeling specific binding partners of ligands or analytes (such as corresponding antibodies); or labeling nucleic acids and molecules that contain nucleic acids.

[0042] The hydrophilicity of the AEs used in the disclosed modified liposomes makes them suitable for inclusion within liposomes without leakage through the liposome wall. A detailed description of hydrophilic AEs can be found in the art, such as in U.S. Patent No. 5,656,426(A), the disclosure of which is incorporated herein by reference in its entirety.

[0043] In some embodiments, the concentration of hydrophilic AE encapsulated in the disclosed modified liposomes is at least 1.10 -10 mol / L ~ at least 1.10 -9 mol / L, at least 1.10 -9 mol / L ~ at least 1.10 -8 mol / L, at least 1.10 -8 mol / L ~ at least 1.10 -7 mol / L, at least 1.10 -7 mol / L ~ at least 1.10 -6 mol / L, at least 1.10 -6 mol / L ~ at least 1.10 -5 mol / L, at least 1.10 -5 mol / L ~ at least 1.10 -4 mol / L, at least 1.10 -4 mol / L ~ at least 1.10 -3 mol / L, at least 1.10 -3 mol / L ~ at least 1.10 -2 mol / L, and at least 1.10 -2 mol / L ~ at least 1.10 -1 In another embodiment, the hydrophilic AE is at least 1.10 -8 mol / L ~ at least 1.10 -6 It has a concentration in the range of mol / L.

[0044] In some embodiments, the modified liposomes comprise at least 10 to at least 100 hydrophilic AE molecules, at least 100 to at least 1000 hydrophilic AE molecules, at least 1000 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 hydrophilic AE molecules. In other embodiments, the modified liposome comprises at least 1000 to at least 100,000,000 hydrophilic AE molecules.

[0045] In further embodiments, the modified liposomes disclosed herein include a variety of sizes. In some embodiments, the diameter of the liposomes is 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; 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; and about 950 nm to about 1000 nm. In other embodiments, the diameter of the liposome is about 20 to about 500 nm. In still other embodiments, the diameter of the liposome is about 30 nm to about 100 nm.

[0046] In some embodiments, the first drug that is encapsulated in the disclosed modified liposome can be nucleic acid, hydrophobic drug, and hydrophilic drug.Examples of hydrophobic drugs include, but are not limited to, amphotericin, silybin, docetaxel, simvastatin, haloperidol, and albendazole.Examples of hydrophilic drugs include, but are not limited to, doxorubicin hydrochloride, cytosine arabinoside, ethynylcytidine, and 5-fluoro-deoxyuridine.

[0047] In some embodiments, the second agent of the modified liposome comprises a polypeptide, an antibody or its antigen-binding fragment, an aptamer, an affibody, an affimer, a carbohydrate, a polyethylene glycol (PEG), a pegylated polypeptide, a small molecule, or a drug. In some embodiments, the polypeptide is biotin, avidin or an avidin derivative (e.g., neutravidin), streptavidin, or fluorescein. In some embodiments, the pegylated polypeptide comprises a pegylated antibody or a pegylated biotin. In some embodiments, the drug is a hydrophobic drug, or a drug conjugated to the surface of the liposome.

[0048] The second agent may be a recombinant, chimeric, genetically modified, or conjugated protein. In some embodiments, the second agent may be a recombinant antibody or antibody fragment. The second agent may include an epitope, antigen, or other modification useful for molecularly or immunogenically labeling, expressing, or purifying the second agent. For example, in some embodiments, the second agent may be conjugated to a 6-his tag (6-histidine), Myc tag, HA tag, FLAG tag, or similar epitope tag to facilitate the isolation, separation, or extraction of the second agent from reaction mixtures containing the compositions described herein, and, for example, from biological samples. The second agent of the modified liposomes described herein is extracted from the liposomes by the use of appropriate detergents or membrane-disrupting mechanical forces. In this way, the second agent is separated from the liposomes after a biochemical assay is performed to determine the amount of interfering substances associated with the second agent.

[0049] In some embodiments, the modified liposomes described herein include multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs), large unilamellar vesicles (LUVs), and giant unilamellar vesicles (GUVs). In some embodiments, the lipid bilayer may include sphingolipids, glycerophospholipids, sterols, and sterol derivatives. The sphingolipids used may include sphingomyelins and ceramides containing saturated, monounsaturated, and / or polyunsaturated acyl chains of various lengths. Phospholipids with 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 various lengths. The sterols and sterol derivatives used may include cholesterol, brassicasterol, allocholesterol, cholesterol methyl ether, campestanol, campesterol, cholesteryl acetate, coprostanol, desmosterol, dehydrodesmosterol, dihydrocholesterol, dihydrolanosterol, epicholesterol, lathosterol, lanosterol, sitostanol, sitosterol, stigmasterol, zymostanol, and zymosterol.

[0050] The lipids that can be used to form the liposomes of the present disclosure can include sphingolipids, glycerophospholipids, sterols, and sterol derivatives, either natural or synthetic.The sphingolipids that can be used include sphingomyelin and ceramide, which contain saturated, monounsaturated, and / or polyunsaturated acyl chains of various lengths.The phospholipids with various head group structures that can be used include phosphatidic acid (PA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin, phosphatidylserine (PS), which contain saturated, monounsaturated, and / or polyunsaturated acyl chains of various lengths. Sterols and sterol derivatives that may be used include, but are not limited to, cholesterol, brassicasterol, allocholesterol, cholesterol methyl ether, campestanol, campesterol, cholesteryl acetate, coprostanol, desmosterol, dehydrodesmosterol, dihydrocholesterol, dihydrolanosterol, epicholesterol, lathosterol, lanosterol, sitostanol, sitosterol, stigmasterol, zymostanol, and zymosterol.

[0051] Mixtures of lipids, including mixtures of sphingolipids, glycerophospholipids, sterols, and sterol derivatives, may also be used in the disclosed modified liposomes. Sterols and sterol derivatives should not be used alone, i.e., sterols and sterol derivatives should be included in the mixture with sphingolipids or glycerophospholipid-containing liposomes in the range of about 0% to about 50% of the total lipids. In some embodiments, sphingolipids may include porcine brain sphingomyelin, chicken egg sphingomyelin, and bovine milk sphingomyelin. In some embodiments, the glycerophospholipids are composed of two saturated acyl chains of various lengths (e.g., 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dipalmito ... n-Glycero-3-phospho-(1'-rac-glycerol), 1,2-distearoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1'-myoinositol), 1,2-distearoyl-sn-glycero-3-phosphoinositol, 1',3'-bis[1,2-dipalmitoyl-sn-glycero-3-phospho] -glycerol, 1',3'-bis[1,2-distearoyl-sn-glycero-3-phospho]-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, 1,2-distearoyl-sn-glycero-3-phospho-L-serine), as well as one saturated acyl chain of various lengths and one monounsaturated acyl chain of various lengths (e.g., 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, 1',3'-bis[1,2-distearoyl-sn-glycero-3-phospho]-glycerol, 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine, 1,2-distearoyl-sn-glycero-3-phospho-L-serine). 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol),1-Stearoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol, 1',3'-bis[1-palmitoyl-2-oleoyl-sn-glycero-3-phospho]-glycerol, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine, 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine and one saturated acyl chain of various lengths and one polyunsaturated acyl chain of various lengths (e.g., 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine ... The lipid composition may include phospholipids having a variety of head group structures, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), cardiolipin, phosphatidylserine (PS), ... In some embodiments, each fatty acyl chain has a number of carbon atoms ranging from about 16 to 20. In other embodiments, each fatty acyl chain has 16, 18, or 20 carbon atoms. In some embodiments, the number of double bonds per each fatty acyl chain ranges from 0 to 2. Sterols and sterol derivatives for use with sphingolipid- or glycerophospholipid-containing liposomes may include cholesterol, dihydrocholesterol, epicholesterol, sitosterol, and lathosterol. Sphingolipids and glycerophospholipids may be used alone or in the presence of sterols and sterol derivatives.or as a mixture of sphingolipids and glycerophospholipids to form modified liposomes as described herein. 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%.

[0052] The disclosed modified liposome can include modified phospholipids. For example, sphingolipids and glycerophospholipids are modified with small molecules, polyethylene glycol (PEG), fluorescent molecules, fluorescent PEG, and / or bromine. Sphingolipids and glycerophospholipids, sterols, sterol derivatives, and modified types of lipids are readily commercially available from various sources, such as Sigma-Aldrich (St. Louis, MO); Invitrogen (Carlsbad, CA); Avanti Polar Lipids (Alabaster, AL); Fisher Scientific (Pittsburgh, PA); Steraloids (Newport, RI).

[0053] Also disclosed herein is a method of labeling a target of interest, comprising conjugating the target to the modified liposomes disclosed herein.

[0054] In some embodiments, the target of interest can be an antigen, a hapten, a DNA probe, an RNA probe, or an antibody.In other embodiments, the target of interest can be present in a biological sample.In some embodiments, the biological sample can be, but is not limited to, whole blood, serum, plasma, urine, saliva, semen, or cerebrospinal fluid.

[0055] In one aspect, provided herein is a method of assaying a biological sample for a target antigen, the method comprising: (a) combining the biological sample with modified liposomes of the present disclosure in a medium; and examining the medium for the target antigen bound to the modified liposomes.

[0056] In some embodiments, the assay comprises a biochemical assay, such as an immunoassay, a clinical chemistry assay or other medical or diagnostic test. In some embodiments, the assay may comprise a sandwich assay or an in situ hybridization assay.

[0057] In some embodiments, the disclosed modified liposomes are added to or incubated with a reagent for a biochemical assay in addition to a biological sample. For example, the modified liposome composition is incubated with a buffer that is provided as a component of a biochemical assay. In addition, the modified liposomes are incubated with a reaction mixture for a biochemical assay, which mixture includes one or more reagents for the assay and a biological sample. In some embodiments, the modified liposome composition is added in the form of a suspension to a biological sample, a reagent, or a reaction mixture for a biochemical assay. In some embodiments, the modified liposome composition is reconstituted from a "dry form" in a biological sample, a reagent, or a reaction mixture, or in one or more components that contribute to a reaction mixture for a biochemical assay.

[0058] Also provided herein is a method for detecting a target antigen in a biological sample, the method comprising: (a) contacting the biological sample with the disclosed modified liposome that is specifically conjugated with the target antigen; (b) imaging the signal generated by the conjugated target antigen; and (c) detecting the signal generated in step (b), thereby detecting the target antigen.

[0059] Further provided herein is a method for increasing the intensity of a signal detected by an imaging modality, comprising: (a) labeling a target by conjugating the target to the disclosed modified liposome; (b) imaging the signal generated by the labeled target; and (c) detecting the signal generated in step (b).

[0060] Also provided is a method for increasing the sensitivity of an immunoassay, comprising: (a) labeling a target by conjugating the target to a disclosed modified liposome; (b) imaging a signal produced by the labeled target; and (c) detecting the signal produced in step (b).

[0061] In some embodiments, the disclosed repair liposomes are ruptured and the amount of signal generated by the encapsulated hydrophilic AE is measured.

[0062] In some embodiments, peptides and / or nucleic acids are detected using the disclosed modified liposomes. For example, DNA or RNA probes are tagged with ligands such as haptens or biotinylated modified nucleotides. The DNA or RNA probes can hybridize with complementary DNA or RNA and are immobilized on a solid support. The immobilized probes are then reacted with modified liposomes that contain receptors for the ligands, such as antibodies, or avidin if the probes are biotinylated. The liposomes are ruptured and the amount of signal generated by the encapsulated acridinium ester is measured.

[0063] Other aspects of the present disclosure include determining the presence or amount of a target of interest (e.g., a peptide or polypeptide) or detecting the biological activity of a target of interest by means known in the art. These means include immunoassay devices and methods that may utilize labeling molecules in a variety of sandwich, competitive, or other assay formats. Such assays generate signals indicative of the presence or absence of a peptide or polypeptide. Furthermore, the signal strength may preferably be directly or indirectly correlated (e.g., inversely proportional) to the amount of polypeptide present in the sample. Further suitable methods include measuring physical or chemical properties specific to the peptide or polypeptide, such as accurate 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. Furthermore, the 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 Atellica®), enzymatic cobalt-binding assays (CBA), and latex agglutination assays.

[0064] Specific hybridization is carried out under high stringency or medium stringency conditions as required.In a preferred embodiment, the hybridization conditions for specific hybridization are high stringency.Specific hybridization, if present, is then detected using standard methods.If specific hybridization occurs between the nucleic acid probe and the gene in the test sample, the sequence present in the nucleic acid probe is also present in the mRNA of the subject.More than one nucleic acid probe can also be used.

[0065] Illustrative Embodiments Illustrative embodiments of the disclosed technology are now provided, which are merely illustrative and do not limit the scope of the disclosure or the claims appended hereto. Embodiment 1. A modified liposome comprising: (i) an encapsulated hydrophilic acridinium ester (AE); and (ii) a first agent encapsulated in the liposome and / or (iii) a second agent on the surface of the liposome. Embodiment 2. The modified liposome of embodiment 1, wherein the first agent comprises at least one selected from the group consisting of: a nucleic acid, a hydrophobic drug, and a hydrophilic drug. Embodiment 3. The modified liposome of embodiment 1, wherein the second agent comprises a polypeptide, an antibody, a carbohydrate, polyethylene glycol (PEG), a pegylated polypeptide, a small molecule, or a drug. Embodiment 4. The modified liposome of embodiment 3, wherein the polypeptide is biotin, avidin, streptavidin, or fluorescein. Embodiment 5. The modified liposome of embodiment 3, wherein the pegylated polypeptide comprises a pegylated antibody or a pegylated biotin. Embodiment 6. The modified liposome of embodiment 3, wherein the drug is a hydrophobic drug, or a drug conjugated to the surface of the liposome. Embodiment 7. The encapsulated hydrophilic AE is at least 1 × 10 -8 mol / L ~ at least 1 x 10 -6 2. The modified liposome of embodiment 1, having a concentration in the range of moles / L. Embodiment 8. The modified liposome of embodiment 1, wherein the encapsulated hydrophilic AE comprises at least 1000 to at least 100,000,000,000 hydrophilic AE molecules. Embodiment 9. The modified liposome of embodiment 1, wherein the liposome has a diameter of about 20 nm to about 1000 nm. Embodiment 10. The modified liposome of embodiment 9, wherein the liposome has a diameter of about 30 nm to about 100 nm. Embodiment 11. A method for labeling a target of interest, comprising conjugating the target to a modified liposome described in any one of embodiments 1 to 10. Embodiment 12. A method of assaying a biological sample for a target antigen, comprising: a. combining a biological sample with a modified liposome according to any one of the embodiments 1 to 11 in a culture medium; b. examining the medium for the target antigen bound to the modified liposomes; A method comprising: Embodiment 13. A method for detecting a target antigen in a biological sample, comprising: c. contacting the biological sample with a modified liposome according to any one of embodiments 1 to 11, which is specifically conjugated to a target antigen; d. imaging the signal generated by the conjugated target antigen; e. detecting the signal generated in step (b) thereby detecting the target antigen; A method comprising: Embodiment 14. A method for increasing the intensity of a signal detected by an imaging modality, comprising: f. labeling the target by conjugating the target to a modified liposome according to any one of embodiments 1 to 11; g. imaging the signal produced by the labeled targets; h. detecting the signal generated in step (b); A method comprising: Embodiment 15. A method for increasing the sensitivity of an immunoassay, comprising: i. labeling the target by conjugating the target to a modified liposome according to any one of embodiments 1 to 11; j. imaging the signal produced by the labeled target; k. detecting the signal generated in step (b); A method comprising: EXAMPLES

[0066] The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate, but not limit, the disclosed embodiments.

[0067] material and method reagent 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC); 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC); 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS); porcine brain sphingomyelin (SM); cholesterol (CHOL); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)-2000] (PEG2000 biotin-DSPE); 1 ,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl) (biotin-DPPE); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(rhodamine-DPPE); N-(fluorescein-5-thiocarbamoyl-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (fluorescein-DHPE); N-sulfo-propyl-dimethylacridinium ester-N-hydroxysuccinimide (NSP-DMAE) and trimethylsilylpropionate DMAE (TSP-DMAE).

[0068] Lipids were stored at −20° C. Various polycarbonate membrane filters with pore sizes of 30, 50, 100, 200, and 400 nanometers were used. EXAMPLES

[0069] Preparation and purification of liposomes encapsulating acridinium esters Provided herein are liposomal vesicles of various sizes (eg, 30-1000 nm) that encapsulate a large number (eg, hundreds of millions) of signal-generating molecules, such as acridinium esters (AEs).

[0070] To prepare 4 mM large unilamellar liposomal vesicles (LUVs) encapsulating acridinium esters, lipids (SM or DPPC or POPC or DOPC or SM / POPC 1 / 1 or DPPC / POPC 1 / 2 or POPC / POPS 3 / 1) were mixed and dried under nitrogen and then high vacuum for at least 2 h. The amount of cholesterol used in the liposomes was 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 liposomes. The dried lipid film was dispensed into NSP-DMAE or TSP-DMAE containing phosphate buffered saline (PBS, 137 mM NaCl, pH 7.4) at 70 °C and then cooled to room temperature before use. As shown in Figure 2A-C, various acridinium ester (AE)-encapsulated liposomes could be designed either by varying the concentration of AE (Figure 2A-B) or by modifying the biochemical and biophysical properties of the liposomes (e.g., negatively charged liposomes, Figure 2C). The concentrations of NSP-DMAE and TSP-DMAE were varied between 0 and 15 mg / mL. The lipid mixture was subjected to 10 cycles of freeze / thaw and then extruded through polycarbonate filters with constant 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 unentrapped NSP-DMAE or TSP-DMAE. Dynamic light scattering (DLS) measurements were performed both before and after NAP-5 column purification. The size distribution of the resulting liposomes indicated that the average liposome size was still maintained after the purification process. EXAMPLES

[0071] Preparation and purification of liposomes encapsulating functionalized acridinium esters. The aim was to define whether liposomes entrapped with AE could be efficiently prepared also in the presence of various functional groups on the surface of the liposomes and to verify that the addition of functional groups did not affect the resulting liposomes (Figure 3A-B). To that end, before drying the lipids under nitrogen, biotin-DPPE, PEG 2000 biotin-DSPE or fluorescein-DHPE were added to the lipid mixtures, i.e. SM or DPPC or POPC or DOPC or SM / POPC 1 / 1 or DPPC / POPC 1 / 2 or POPC / POPS 3 / 1, with or without cholesterol as described above. The amount of biotin-DPPE, PEG 2000 biotin-DSPE and fluorescein-DHPE used in the liposomes was varied between 0 and 20 mol%. The permeability and hydrophilicity of the surface of the liposomes was especially promoted by the addition of polyethylene glycol (PEG). Dynamic light scattering (DLS) analysis performed on the resulting liposomes showed that liposomes with diameters of 30 nm, 50 nm and 100 nm were efficiently formed with functional groups. The addition of biotin-DPPE, PEG 2000 biotin-DSPE and fluorescein-DHPE did not affect the ability of the liposomes to entrap acridinium ester. To examine the stability of functionalized liposomes, the size of the liposomes was maintained throughout.

[0072] As shown in Figures 3A-3B, the disclosed AE-encapsulated liposomes may include further modifications. These modifications include, but are not limited to, the addition of various functional groups, such as biotin, fluorescein, and / or proteins, on the liposome surface (Figure 3A). The permeability and hydrophilicity of the liposome membrane may be enhanced by adding polyethylene glycol (PEG) on the surface of the liposome (Figure 3B).

[0073] Thus, various functional groups, including but not limited to biotin, fluorescein, and proteins, can be attached to the surface of the disclosed liposomal vesicles. These conjugates are useful for detecting targets of interest using immunoassay platforms, such as, but not limited to, Siemens platforms, such as ADVIA Centaur® XPT, ADVIA Centaur® XP, ADVIA Centaur® CP, IMMULITE® 1000, IMMULITE® 2000 XPi, and Atellica®. The encapsulated AE vesicles of the present disclosure significantly enhance the immunoassay signal or relative light units (RLU), optimizing the sensitivity of the output results. Depending on the liposomal surface modification, the functionalized AE liposomes can be utilized as Lite reagents or as signal amplifiers.

[0074] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety.

[0075] Those skilled in the art will recognize that numerous changes and modifications may be made to the preferred embodiments of the invention and that such changes and modifications may be made without departing from the spirit of the invention, and it is therefore intended that the appended claims cover all such equivalent variations as fall within the true spirit and scope of the invention.

Claims

1. A modified liposome, comprising: (i) an encapsulated hydrophilic acridinium ester (AE), and (ii) a first drug encapsulated in the liposome; and (iii) a second agent on the surface of the liposome; the first drug comprises at least one selected from the group consisting of a nucleic acid, a hydrophobic drug, and a hydrophilic drug; the hydrophobic drug is selected from the group consisting of amphotericin, silybin, docetaxel, simvastatin, haloperidol and albendazole; the hydrophilic drug is selected from the group consisting of doxorubicin hydrochloride, cytosine arabinoside, ethynylcytidine, and 5-fluoro-deoxyuridine; the second agent comprises a polypeptide, an antibody, a carbohydrate, a polyethylene glycol (PEG), a pegylated polypeptide, a small molecule, or a drug; The diameter of the liposome is 30 nm to 100 nm. The modified liposome.

2. The modified liposome described in claim 1, wherein the second drug is biotin, avidin, streptavidin, or fluorescein.

3. The modified liposome described in claim 1, wherein the second drug comprises a pegylated antibody or a pegylated biotin.

4. The modified liposome of claim 1 , wherein the drug is a hydrophobic drug or a drug conjugated to the surface of the liposome.

5. The encapsulated hydrophilic AE was 1 × 10 -8 mol / L ~ 1 x 10 -6 The modified liposome of claim 1 having a concentration in the moles / L range.

6. The encapsulated hydrophilic AE is between 1000 and 100,000,000,000 hydrophilic A The modified liposome of claim 1 , comprising an E molecule.

7. A method for labeling a target of interest, comprising conjugating the target to a modified liposome according to any one of claims 1 to 6.

8. 1. A method for determining the presence or amount of a target antigen in a biological sample, or detecting a biological activity of a target antigen, comprising: a. combining a biological sample with the modified liposome of any one of claims 1 to 6 in a culture medium; b. detecting or measuring a signal generated by the target antigen bound to the modified liposome.

9. 1. A method for detecting a target antigen in a biological sample, comprising: a. contacting a biological sample with a modified liposome according to any one of claims 1 to 6, wherein the modified liposome specifically conjugates with a target antigen; b. Imaging the signal generated by the conjugated target antigen; c. detecting the signal generated in step (b) thereby detecting the target antigen; The method comprising:

10. 1. A method for increasing the intensity of a signal detected by an imaging modality, comprising: a. labeling a target by conjugating the target to a modified liposome according to any one of claims 1 to 6; b. imaging the signal produced by the labeled target; c. detecting the signal generated in step (b); The method comprising:

11. 1. A method for increasing the sensitivity of an immunoassay, comprising: a. labeling a target by conjugating the target to a modified liposome according to any one of claims 1 to 6; b. imaging the signal produced by the labeled target; c. detecting the signal generated in step (b); The method comprising:

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