Polymeric chromophores, compositions comprising the same, and methods of preparing and using the same

Abstract

To provide a polymeric chromophore that enables the use of an organic chromophore which is hydrophobic or slightly polar in an aqueous solution.SOLUTION: Described herein is a polymeric chromophore that includes a dye, a polymer, and optionally a bioconjugate group, wherein the polymeric chromophore may have a structure represented by: A-B-C or C-A-B, where A is a dye, B is a polymer comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s), and C is optional, and when present, C comprises a bioconjugate group. Also described herein are a composition comprising the polymeric chromophore and methods of preparing and using the polymeric chromophore.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Government Support Statement This invention was made with government support under grant number DE-SC0001035 awarded by the Department of Energy. The government has certain rights in this invention.

[0002] The present invention generally relates to polymeric chromophores that include a dye, a polymer segment, and optionally a bioconjugate group. The present invention also relates to compositions that include the polymeric chromophores, as well as methods of preparing and using the same.

Background Art

[0003] Many applications of chromophores are carried out in aqueous solutions, but most organic chromophores are hydrophobic or slightly polar. There are many approaches for encapsulating chromophores, and none yet meet the criteria of simplicity of synthesis, lack of fluorophore-fluorophore quenching, and the presence of a single bioconjugatable group.

Summary of the Invention

Means for Solving the Problems

[0004] A first aspect of the present invention is directed to a compound having a structure represented by: A-B-C, or C-A-B where: A is a dye (e.g., a fluorophore), where optionally the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da, B is a polymer that includes one or more hydrophobic units and one or more hydrophilic units, where optionally the polymer has a molecular weight in the range from about 1,000 Da, 5,000 Da or 10,000 Da to about 175,000 Da, and C is optional, where C comprises a bioconjugate group.

[0005] Another aspect of the invention is directed to a composition comprising a compound of the invention and optionally water. Also, an aspect of the invention is directed to a composition comprising particles (e.g., particles comprising a core and a shell), where the particles comprise a compound having a structure represented by: A-B-C, or C-A-B where A is a dye (e.g., a fluorophore), B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, C is optional, where C comprises a bioconjugate group, and water.

[0006] A further aspect of the invention is a method of preparing a compound, comprising polymerizing a hydrophobic monomer and a hydrophilic monomer to provide a copolymer, attaching a dye to a first portion (e.g., an end or terminus) of the copolymer, thereby providing the compound, and optionally attaching a bioconjugate group to a second portion (e.g., another end or terminus) of the copolymer and / or optionally crosslinking the compound. Polymerizing the hydrophobic monomer and the hydrophilic monomer can include polymerizing via living radical polymerization in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally a co-catalyst, to provide the copolymer.

[0007] Another aspect of the invention is directed to a compound prepared according to the method of the invention.

[0008] Also, according to an embodiment of the invention, a method of using a compound of the invention and / or a method of using a composition of the invention are provided, for example, in flow cytometry and the like.

[0009] A further aspect of the present invention is directed to a method for detecting cells and / or particles using flow cytometry, the method comprising labeling the cells and / or particles with a compound of the present invention and detecting the compound by flow cytometry, thereby detecting the cells and / or particles.

[0010] Another aspect of the present invention is directed to a method for detecting a tissue and / or an agent (e.g., a cell, an infecting agent, etc.), the method comprising administering to a subject a compound or a composition of the present invention, wherein optionally the compound associates with the tissue and / or the agent, and detecting the compound within the subject, thereby detecting the cell and / or the particle.

[0011] A further aspect of the present invention is directed to biomolecules (e.g., cells, antibodies, etc.) comprising one or more (e.g., 1, 2, 3, 4, 5, 6 or more) compounds of the present invention.

[0012] It should be noted that aspects of the present invention described with respect to one embodiment, although not specifically described in relation thereto, may be incorporated into different embodiments. That is, all embodiments and / or features of any embodiment can be combined in any method and / or combination. The applicant reserves the right to amend the originally filed claims and / or to file new claims accordingly, including the right to correct the originally filed claims in order to depend on and / or incorporate features of other claims or claim clauses that were not originally so recited in the claims. These and other objects and / or aspects of the present invention are described in detail in the specification set forth below. Further features, advantages and details of the present invention will be understood by those skilled in the art from reading the drawings and the detailed description of the following preferred embodiments. And such description is merely illustrative of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013]

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[0014] The present invention will be fully described hereinafter with reference to the accompanying drawings in which embodiments of the invention are shown. However, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0015] The terms used in the detailed description of the invention herein are for the purpose of describing particular embodiments only and are not intended to be limiting of the invention. As used in the detailed description of the invention and the appended claims, the singular forms "a", "an", and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0016] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Further, terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and the relevant art, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terms used in the detailed description of the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification will control.

[0017] Also, as used herein, "and / or" shall be construed to mean any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0018] It is specifically intended that, unless the context otherwise indicates, the various features of the invention described herein can be used in any combination. Further, the invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. For purposes of illustration, if the present specification states that a complex contains components A, B, and C, it is specifically intended that any one or combination of A, B, or C can be omitted and disclaimed.

[0019] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be construed to include materials or steps that do not materially affect the "basic and novel one or more characteristics" of the invention recited in the claims. In re Herz , 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976), see (emphasis in original); see also MPEP §2111.03. Accordingly, as used herein, the term "consisting essentially of" should not be construed as equivalent to "comprising".

[0020] As used herein, "exemplary", "exemplification" and their grammatical variations are intended to refer to non-limiting examples and / or alternative embodiments discussed herein, and are not intended to indicate preference for one or more of the embodiments discussed herein over one or more other embodiments.

[0021] When the term "about" is used in this specification in cases where a measurable value, such as an amount or a concentration, etc., is mentioned, it is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified value and the specified value itself. For example, when X is a measurable value, "about X" is meant to include X and variations of ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of X. The ranges provided in this specification for measurable values may include any other ranges and / or individual values therein.

[0022] As used in this specification in connection with chemical molecules, "derivative" refers to a chemical molecule having one or more atoms (e.g., hydrogen atoms), functional groups and / or bonds that are modified (e.g., removed, substituted, etc.) compared to the parent molecular entity. For example, a derivative of a dye can refer to a parent dye compound having one or more atoms (e.g., hydrogen atoms) and / or functional groups that are modified (e.g., removed) to facilitate covalent bonding to another group or residue (e.g., to facilitate covalent bonding to a polymer). In some embodiments, the derivative may include functional groups (e.g., substituents and / or auxochromes) that change the absorption spectrum of the parent molecular entity.

[0023] As used herein, alone or as part of another group, "alkyl" refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms, which can be referred to as C1-C20 alkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. As used herein, "lower alkyl" is a subset of alkyl and, in some embodiments, refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like.The term "alkyl" or the term "lower alkyl" is intended to include both substituted and unsubstituted alkyl or lower alkyl, unless otherwise indicated, and these groups may be substituted with groups selected from the following: halogen atom, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, hydroxyl, alkoxy (thereby generating polyalkoxy, such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S(O)m, haloalkyl-S(O)m, alkenyl-S(O)m, alkynyl-S(O)m, cycloalkyl-S(O)m, cycloalkylalkyl-S(O)m, aryl-S(O)m, arylalkyl-S(O)m, heterocycle-S(O)m, heterocycloalkyl-S(O)m, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, where m = 0, 1, 2 or 3.

[0024] As used herein, either alone or as part of another group, "alkenyl" refers to a straight-chain or branched-chain hydrocarbon containing 1 to 20 carbon atoms (or 1 to 4 carbon atoms in lower alkenyl) which can contain 1 to 8 double bonds in the straight chain, and can be referred to as C1-C20 alkenyl. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. The term "alkenyl" or the term "lower alkenyl" is intended to include both substituted and unsubstituted alkenyl or lower alkenyl, unless otherwise indicated, and these groups can be substituted with the same groups as described in connection with alkyl and lower alkyl above.

[0025] As used herein, either alone or as part of another group, "alkynyl" refers to a straight-chain or branched-chain hydrocarbon containing 1 to 20 carbon atoms (or 1 to 4 carbon atoms in lower alkynyl) which contains one triple bond in the straight chain, and can be referred to as C1-C20 alkynyl. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. The term "alkynyl" or the term "lower alkynyl" is intended to include both substituted and unsubstituted alkynyl or lower alkynyl, unless otherwise indicated, and these groups can be substituted with the same groups as described in connection with alkyl and lower alkyl above.

[0026] As used herein, "halogen atom" refers to any suitable halogen atom, for example, a halogen atom including -F, -Cl, -Br, and -I.

[0027] As used herein, "mercapto" refers to the -SH group.

[0028] As used herein, "azido" refers to the -N3 group.

[0029] As used herein, "cyano" refers to the -CN group.

[0030] As used herein, "hydroxyl" refers to the -OH group.

[0031] As used herein, "nitro" refers to the -NO2 group.

[0032] As used herein, alone or as part of another group, "alkoxy" refers to an alkyl group or lower alkyl group as defined herein attached to a parent molecular residue through an oxygen atom group, -O- (thus including substituted versions, such as polyalkoxy). Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0033] As used herein, alone or as part of another group, "acyl" refers to a -C(O)R group, where R is any suitable substitution, such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl, or other suitable substituents described herein.

[0034] As used herein, alone or as part of another group, "haloalkyl" refers to at least one halogen atom as defined herein attached to a parent molecular residue through an alkyl group as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.

[0035] As used herein, either alone or as part of another group, "alkylthio" refers to an alkyl group as defined herein attached to a parent molecular residue through a thio residue as defined herein. Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

[0036] As used herein, either alone or as part of another group, "cycloalkyl" refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 1 to 20 carbon atoms (optionally having a carbon atom substituted in a heterocyclic group discussed below). The cycloalkyl group may contain 0, 1, 2, or more double bonds or triple bonds. Representative examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclododecyl. These rings may optionally be substituted with additional substituents as described herein, such as halogen atoms or lower alkyl. The term "cycloalkyl" is intended to include heterocyclic groups discussed below, unless otherwise specified or the context clearly indicates otherwise.

[0037] As used herein, alone or as part of another group, the term "heterocyclic group" or "heterocycle" refers to an aliphatic (e.g., fully or partially saturated heterocycle), or aromatic (e.g., heteroaryl) monocyclic or bicyclic system. The monocyclic system is exemplified by any 5- or 6-membered ring containing 1, 2, 3 or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5-membered ring has 0 to 2 double bonds, and the 6-membered ring has 0 to 3 double bonds. Representative examples of monocyclic systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, etc. The bicyclic system is exemplified by any of the above monocyclic systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein or another monocyclic system as defined herein.Typical examples of bicyclic systems include, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxin, 1,3-benzodioxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolidine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, etc., but are not limited thereto. These rings include their quaternized derivatives and may be optionally substituted with groups selected from the following: halogen atom, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocycle, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S(O). m , haloalkyl-S(O) m , alkenyl-S(O) m , alkynyl-S(O) m , cycloalkyl-S(O) m , cycloalkylalkyl-S(O) m , aryl-S(O) m , arylalkyl-S(O) m , heterocycle-S(O) m , heterocycloalkyl-S(O) m , amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, where m = 0, 1, 2 or 3.

[0038] As used herein, alone or as part of another group, "aryl" refers to a monocyclic, carbocyclic, or bicyclic, carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryl include, but are not limited to, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, etc. The term "aryl" is intended to include both substituted and unsubstituted aryl unless otherwise indicated, and these groups can be substituted with the same groups described in connection with alkyl and lower alkyl above.

[0039] As used herein, alone or as part of another group, "arylalkyl" refers to an aryl group as defined herein attached to a parent molecular residue through an alkyl group as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, etc.

[0040] As used herein, "amino" means the -NH2 group.

[0041] As used herein, alone or as part of another group, "alkylamino" means -NHR, where R is an alkyl group.

[0042] As used herein, alone or as part of another group, "ester" refers to -C(O)OR, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0043] As used herein, "formyl" means the -C(O)H group.

[0044] As used herein, "carboxylic acid" means the -C(O)OH group.

[0045] As used herein, "sulfoxyl" refers to a compound of the formula -S(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0046] As used herein, "sulfonyl" refers to a compound of the formula -S(O)(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0047] As used herein, "sulfonate" refers to a salt of sulfonic acid (e.g., sodium (Na) salt) and / or a compound of the formula -S(O)(O)OR, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0048] As used herein, "sulfonic acid" refers to a compound of the formula -S(O)(O)OH.

[0049] As used herein, when used alone or as part of another group, "amide" refers to -C(O)NR a R b group, where R a and R b are any suitable substituents, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0050] As used herein, when used alone or as part of another group, "sulfonamide" refers to -S(O)2NR a R b group, where R a and R b are any suitable substituents, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.

[0051] The compounds of the present invention have a polymeric fluorophore. The compounds of the present invention have a single (i.e., one) polymer bound to a single (i.e., one) dye and optionally have a single (i.e., one) bioconjugate group, which may have a single binding site for a biomolecule. Exemplary compounds are shown in Figure 1. In some embodiments, the one polymer is bound to both the dye and the bioconjugate group (if present). In some embodiments, the one dye is bound to both the polymer and the bioconjugate group (if present). In some embodiments, the compositions of the present invention comprise the compounds of the present invention in solution, such as in water, aqueous solutions, and / or hydrophobic solvents, etc.

[0052] While the compounds of the present invention are bound to a single biomolecule via a bioconjugate group, the biomolecule may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) compounds of the present invention. Thus, in some embodiments, the biomolecule and / or a portion thereof may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) compounds of the present invention.

[0053] In some embodiments, the compounds of the present invention have a structure represented by: A - B - C, or C - A - B where A is a dye (e.g., a fluorophore), B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C, if present, comprises a bioconjugate group.

[0054] The terms "dye" and "chromophore" are used interchangeably herein and refer to a lumophore (e.g., a fluorescent molecular entity and / or a phosphorescent molecular entity) and / or a non-luminescent molecular entity (e.g., a non-fluorescent molecular entity and / or a non-phosphorescent molecular entity). As used herein, the term "non-luminescent molecular entity" refers to a molecular entity that does not have or has negligible luminescence. In some embodiments, the non-luminescent molecular entity does not form an excited state with any significant lifetime and / or relaxes rapidly and essentially quantitatively to the ground state. In some embodiments, the non-luminescent molecular entity has an excited state lifetime of less than about 100 picoseconds, less than about 75 picoseconds, less than about 50 picoseconds, less than about 25 picoseconds, less than about 10 picoseconds, less than about 5 picoseconds, less than about 1 picosecond, less than about 0.5 picoseconds or less than about 0.1 picoseconds. In some embodiments, the non-luminescent molecular entity has an internal conversion quantum yield of greater than about 0.8, greater than about 0.85, greater than about 0.9, greater than about 0.95, greater than about 0.99, greater than about 0.999, greater than about 0.9999 or greater than about 0.99999, where a quantum yield of 1.0 corresponds to 100%. In some embodiments, the non-luminescent molecular entity has a luminescence quantum yield of less than about 0.2, less than about 0.15, less than about 0.1, less than about 0.05, less than about 0.01, less than about 0.001, less than about 0.0001 or less than about 0.00001, where a quantum yield of 1.0 corresponds to 100%. The luminescence quantum yield is known to be obtained from the competing process of radiative decay relative to the sum of all processes for depolarizing the excited state manifold. Such compounds are often referred to as "non-luminescent", but sensitive detection techniques can often detect small amounts of residual luminescence as predicted by such low luminescence quantum yields. Maximum possible conversion from optical input to thermal output is desired, but small amounts of luminescence may not adversely affect some applications, such as photoacoustic imaging methods. Thus, the term "non-luminescent" is used herein to denote a molecular entity that does not have or has negligible luminescence. In some embodiments, the compounds of the invention include a dye, where the dye is a non-luminescent molecular entity (e.g., a non-fluorescent molecular entity and / or a non-phosphorescent molecular entity). In some embodiments, the compounds of the invention include a dye, where the dye is a lumophore (e.g., a fluorescent molecular entity and / or a phosphorescent molecular entity)."Fluorescent molecular entity" and "fluorophore" are used interchangeably herein and refer to a molecular entity that emits fluorescence.

[0055] The dyes of the present invention may have certain spectroscopic features and / or properties, for example, spectroscopic features and / or properties suitable for use in the methods of the present invention. In some embodiments, the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da, about 400 Da to about 1100 Da, or about 300 Da to about 1,000 Da. In some embodiments, the dye has a molecular weight of about 150, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900, about 1000, about 1200, about 1300, about 1400, about 1500, about 1600, about 1700, about 1800, about 1900, about 2000, about 2200, about 2300, about 2400, about 2500, about 2600, about 2700, about 2800, about 2900 or about 3000 Da. Exemplary dyes include, but are not limited to, tetrapyrroles; rilenes, such as perylene, terrylene and quarternarylene; fluoresceins, such as TET (tetramethylfluorescein), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyfluorescein (HEX) and 5-carboxyfluorescein (5-FAM); phycoerythrin; resorufin dyes; coumarin dyes; rhodamine dyes, such as 6-carboxy-X-rhodamine (ROX), Texas Red and N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); cyanine dyes; phthalocyanines; boron-dipyrromethene (BODIPY) dyes; quinoline; pyrene; acridine; stilbene; and derivatives thereof. In some embodiments, the dye is a tetrapyrrole, which includes porphyrins, chlorins and bacteriochlorins, and derivatives thereof.Exemplary tetrapyrroles include, but are not limited to, the tetrapyrroles described below: U.S. Patent Nos. 6,272,038; 6,451,942; 6,420,648; 6,559,374; 6,765,092; 6,407,330; 6,642,376; 6,946,552; 6,603,070; 6,849,730; 7,005,237; 6,916,982; 6,944,047; 7,884,280; 7,332,599; 7,148,361; 7,022,862; 6,924,375; 7,501,507; 7,323,561; 7,153,975; 7,317,108; 7,501,508; 7,378,520; 7,534,807; 7,919,770; 7,799,910; 7,582,751; 8,097,609; 8,187,824; 8,207,329; 7,633,007; 7,745,618; 7,994,312; 8,278,340; 9,303,165; and 9,365,722; and International Patent Application Nos. PCT / US17 / 47266 and PCT / US17 / 63251. In some embodiments, the dye is hydrophobic. In some embodiments, the dye is attached and / or bound to a monomer that polymerizes with one or more different monomers (e.g., polymerizes with a hydrophobic monomer and / or a hydrophilic monomer). In some embodiments, the dye is a lumophore (i.e., a material and / or compound that can emit light and does not specify the properties of the original state (e.g., singlet state, triplet state, and / or other states)). Exemplary lumophores include, but are not limited to, phosphors and / or fluorophores that provide phosphorescence and / or fluorescence, respectively.

[0056] In some embodiments, the compounds of the invention include a recognition motif. In some embodiments, the dyes of the invention may include a recognition motif, and the linker that attaches the dyes of the invention to the polymers of the invention may include a recognition motif. The recognition motif may be attached to the dye and / or the linker. As used herein, a "recognition motif" refers to a molecular entity that can bind to a binding entity, and such binding changes the absorption spectrum of the dye and / or turns on the fluorescence of the dye. Recognition motifs and binding entities known to those of skill in the art may be used in the compounds of the invention. Exemplary recognition motifs include, but are not limited to, crown ethers, cryptands, pincers, and / or chelating motifs. Examples of binding entities are metal ions (e.g., Hg, Cr, Li, etc.). The mechanism for changing the absorption spectrum of the dye and / or turning on the fluorescence of the dye can be achieved, for example, by various means such as: for example, (i) metal ion binding promotes ring opening of the ring that generates the conjugated chromophore; or, (ii) metal ion binding to an electron-rich group, which causes quenching of fluorescence when not bound, thereby stopping the quenching by the binding. In some embodiments, the compounds of the invention act and / or function as chromogenic sensors and / or fluorescent sensors. In some embodiments, the compounds of the invention provide and / or enable metal ion sensing in water, optionally without the addition and / or presence of an organic solvent. In some embodiments, the compounds of the invention are used in sensing applications and / or in sensors. For example, in some embodiments, the compounds of the invention are present within and / or on a sensor (e.g., embedded). The sensor can be an in vivo sensor and / or for in vivo sensing applications and / or an environmental sensor and / or for environmental sensing applications. The recognition motif can be at least partially solvent accessible and / or available to enable binding of the binding entity.In some embodiments, the compounds of the invention contain a recognition motif and can optionally be used in an aqueous solution for sensing applications and / or in photoacoustic imaging methods. In some embodiments, when the compounds of the invention are used in a photoacoustic imaging method and the compounds contain a recognition motif, the recognition motif can cause a shift in the absorption spectrum of the dye in response to binding to a binding entity.

[0057] The polymer of the compound of the invention can include one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophobic units and one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophilic units. The polymer can be prepared from one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophobic monomers and one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophilic monomers using any type of polymerization to provide a polymer that includes the one or more hydrophobic units and the one or more hydrophilic units. In some embodiments, the polymer can be prepared from two or more (e.g., 2, 3, 4, 5 or more) hydrophobic monomers that are different from each other and / or two or more (e.g., 2, 3, 4, 5 or more) hydrophilic monomers that are different from each other. For example, in some embodiments, the polymer of the compound of the invention can be prepared from at least one hydrophobic monomer, at least one first hydrophilic monomer, and at least one second hydrophilic monomer, where the first hydrophilic monomer and the second hydrophilic monomer are different from each other.

[0058] As used herein, "hydrophilic monomer" refers to a monomer containing a hydrophilicity (e.g., an ionic functional group and / or a polar functional group (e.g., a hydrophilic pendant functional group)), where optionally, the hydrophilic functional group is at a residue and / or the terminal portion of the monomer. As will be understood by those skilled in the art, a portion of a hydrophilic monomer, e.g., a portion that forms a polymer backbone when polymerized with other monomers and / or contains an ionic residue, but a portion of the functional group (e.g., a hydrocarbon chain) that is still referred to as a hydrophilic monomer when it contains a hydrophilic functional group can be hydrophobic. As used herein, "hydrophilic unit" refers to a section or unit of a polymer prepared from individual hydrophilic monomers. As used herein, "hydrophobic monomer" refers to a monomer containing a hydrophobic functional group (e.g., a hydrophobic pendant functional group), where optionally, the hydrophobic functional group is at a residue and / or the terminal portion of the monomer. In some embodiments, the hydrophobic functional group is a hydrocarbon residue (e.g., an alkyl). As used herein, "hydrophobic unit" refers to a section or unit of a polymer prepared from individual hydrophobic monomers.

[0059] In some embodiments, the polymer of the compounds of the invention may also be referred to as a polymer segment of the compounds of the invention. The one or more hydrophobic units and the one or more hydrophilic units may be randomly distributed in the polymer. In some embodiments, the polymer is a random copolymer. The polymer can be an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer. The one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10 (hydrophobic unit: hydrophilic unit), where the ratio of hydrophobic units to hydrophilic units is from about 1:4 to about 1:6. The length of the polymer can be varied and / or controlled. In some embodiments, the polymer has a molecular weight in the range of about 1,000 Da to about 175,000 Da, about 5,000 Da to about 175,000 Da, about 10,000 Da to about 175,000 Da, about 20,000 Da to about 175,000 Da, about 28,000 Da to about 175,000 Da, about 28,000 Da to about 35,000 Da, about 28,000 Da to about 50,000 Da, about 100,000 Da to about 150,000 Da, about 50,000 Da to about 130,000 Da or about 10,000 Da to about 100,000 Da. In some embodiments, the polymer has a molecular weight of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, or about 170 kilodaltons (kDa). In some embodiments, the polymer has a molecular weight greater than 28 kDa. In some embodiments, the polymer has a molecular weight of about 28 kDa to about 175 kDa. In some embodiments, the polymer has a molecular weight from about 28 kDa to about 35 kDa or about 50 kDa.

[0060] The hydrophobic unit and / or hydrophilic unit of the polymer may have pendant functional groups. The "pendant functional group" can be a functional group directly bonded to the polymer backbone or a functional group bonded to a residue bonded to the polymer backbone. The pendant functional group can be part of a hydrophobic unit and / or monomer and / or hydrophilic unit and / or monomer during polymerization, or can be added to a hydrophobic unit and / or hydrophilic unit after polymerization. In some embodiments, the pendant functional group can be bonded to a hydrophobic unit and / or hydrophilic unit after polymerization (e.g., post-polymerization functionalization). In some embodiments, the pendant functional group contains a charged group. In some embodiments, the pendant functional group is a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group (e.g., an active ester, e.g., a pentafluorophenyl ester, a succinimide ester, a 2,4-dinitrophenyl ester, etc.), an azide group, a pentafluorophenyl group, a succinimide group, a fluorophenyl group, a maleimide group, an isocyanate group or an isothiocyanate group. In some embodiments, the pendant functional group is a terminal cation (e.g., ammonium), anion (e.g., sulfonate, phosphate, carboxylate) or amphoteric (e.g., choline or a choline-like group (e.g., a derivative of choline)) group and optionally a hydrophilic group having a poly(ethylene glycol) residue and / or unit. In some embodiments, the hydrophilic group is bonded to a poly(ethylene glycol) residue and / or unit and optionally to the terminal portion of the poly(ethylene glycol) residue and / or unit.

[0061] In some embodiments, the hydrophobic unit has a pendant functional group containing alkyl (e.g., dodecyl), and / or the hydrophilic unit contains a pendant functional group containing glycol (e.g., poly(ethylene glycol)), sulfone and / or sulfonate. In some embodiments, the hydrophobic unit is prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer, and / or the hydrophilic unit is prepared from a glycol acrylate (e.g., PEG acrylate) monomer. In some embodiments, the compound of the present invention comprises at least one hydrophobic unit prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer and at least two different hydrophilic units, the hydrophilic units comprising a first hydrophilic unit prepared from a glycol acrylate (e.g., PEG acrylate) monomer and a second hydrophilic unit prepared from a sulfonic acid acrylamide monomer (e.g., 2-acrylamido-2-methylpropane sulfonic acid) and / or a sulfonate acrylate monomer.

[0062] In some embodiments, one or more hydrophobic units and / or one or more hydrophilic units may contain a charge (e.g., a positive or negative charge) and / or a charged group (e.g., a cationic or anionic group), and the charge may suppress non-specific binding to the compound or a part thereof (e.g., a part of the polymer).

[0063] In some embodiments, a hydrophobic monomer (which can be used to provide the hydrophobic unit of the polymers described herein) may have a structure represented by the following formula I.

[0064]

Chemical formula

[0065] where R is a hydrogen atom or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R1 is absent or is -O-, -NH- or -CH2-, R’ is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl or C3-C20 cycloalkyl, and R 2 is a hydrogen atom or is a halogen atom, hydroxyl, carboxyl, amino, formyl or ester (e.g., succinimidyl ester, 2,4-dinitrophenyl ester, pentafluorophenyl ester, fluorophenyl ester, etc.) group. In some embodiments, R in the compound of formula I 2 is a hydroxyl group, carboxyl group, amino group, formyl group or ester group. In some embodiments, R in the compound of formula I 2is a hydrogen atom. In some embodiments, R’ in the compound of Formula I is C2-C4 alkyl, C2-C6 alkyl, C4-C20 alkyl, C6-C20 alkyl, C8-C16 alkyl, C8-C18 alkyl, C10-C14 alkyl or C10-C12 alkyl. In some embodiments, R’ in the compound of Formula I is C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19 or C20 alkyl, alkenyl or alkynyl. In some embodiments, R’ in the compound of Formula I is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19 or C20 alkyl. In some embodiments, R’ in the compound of Formula I is C3-C5 cycloalkyl, C3-C6 cycloalkyl, C4-C20 cycloalkyl, C6-C20 cycloalkyl, C8-C16 cycloalkyl, C8-C18 cycloalkyl, C10-C14 cycloalkyl or C10-C12 cycloalkyl. In some embodiments, R’ in the compound of Formula I is C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19 or C20 cycloalkyl. One of ordinary skill in the art will recognize that in some embodiments, the monomer can be, for example, acrylate (e.g., when R 1 is an oxygen atom), acrylamide (e.g., when R 1 is NH), or vinyl ketone (e.g., when R 1 is CH2), but will readily recognize that other compounds are possible.

[0066] In some embodiments, a hydrophilic monomer (which can be used to provide the hydrophilic units of the polymers described herein) can have a structure represented by Formula II below.

[0067]

Chemical formula

[0068] Here, R is a hydrogen atom or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl), R 1 is absent or is -O-, -NH-, or -CH2-, R 3 is -(CH2CH2R 5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl-O-, and -C1-C6 alkyl-SO3- or a salt thereof, where R 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000, and R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group (e.g., dihydroxyphosphoryl), a sulfono group (e.g., hydroxysulfonyl), a phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group (e.g., pentafluorophenyl ester, succinimidyl ester, fluorophenyl ester, or 2,4-dinitrophenyl ester).

[0069] In some embodiments, R in the compound of formula II 4 is optionally a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group when R 3 is -(CH2CH2R 5 ) n -, -C1-C6 alkyl, or -C1-C6 alkyl-O-. In some embodiments, R in the compound of formula II 3 is -C1-C6 alkyl-O- or -(CH2CH2R 5 ) n -, provided that when R 5 is -O-, R 4can be a hydrogen atom, alkyl (e.g., methyl group or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine or phosphoryl group. In some embodiments, R in the compound of formula II 3 when being -C1-C6 alkyl, R 4 can be a hydroxyl group, carboxyl group, amino group, ammonio group, formyl group, ester group, phosphono group or sulfono group. In some embodiments, R in the compound of formula II 3 when being -C1-C6 alkyl-SO3- or a salt thereof, R 4 is a hydrogen atom or does not exist. In some embodiments, R in the compound of formula II 3 when being a salt of -C1-C6 alkyl-SO3- (e.g., sodium salt), and R 4 does not exist. In some embodiments, R in the compound of formula II 3 when being -(CH2CH2R 5 ) n -.

[0070] In some embodiments, the hydrophobic unit can have a structure represented by the following formula III.

[0071]

Chemical formula

[0072] Here,[[]] R is a hydrogen atom or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 does not exist or is -O-, -NH- or -CH2-, R' is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl or C3-C20 cycloalkyl, R 2is a hydrogen atom or a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester or 2,4-dinitrophenyl ester), an azide group, a maleimide group, an isocyanate group or an isothiocyanate group, and p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000 or 100,000.

[0073] In some embodiments, R in the compound of formula III 2 is a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group. In some embodiments, R in the compound of formula III 2 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group or a maleimide group, which can optionally be added and / or provided by pre-polymerization and / or post-polymerization functionalization. In some embodiments, R in the compound of formula III 2is a hydrogen atom. In some embodiments, R’ in the compound of Formula III is C2-C4 alkyl, C2-C6 alkyl, C4-C20 alkyl, C6-C20 alkyl, C8-C16 alkyl, C8-C18 alkyl, C10-C14 alkyl or C10-C12 alkyl. In some embodiments, R’ in the compound of Formula III is C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19 or C20 alkyl, alkenyl or alkynyl. In some embodiments, R’ in the compound of Formula III is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19 or C20 alkyl. In some embodiments, R’ in the compound of Formula III is C3-C5 cycloalkyl, C3-C6 cycloalkyl, C4-C20 cycloalkyl, C6-C20 cycloalkyl, C8-C16 cycloalkyl, C8-C18 cycloalkyl, C10-C14 cycloalkyl or C10-C12 cycloalkyl. In some embodiments, R’ in the compound of Formula III is C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19 or C20 cycloalkyl.

[0074] In some embodiments, the hydrophilic unit may have a structure represented by the following Formula IV.

[0075]

Chemical Formula

[0076] Here, R is a hydrogen atom or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 is absent or is -O-, -NH- or -CH2-, R 3 is -(CH2CH2R 5) n -, -C1-C6 alkyl, -C1-C6 alkoxy, and -C1-C6 alkyl-SO3- or salts thereof, wherein R 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000, R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group (e.g., dihydroxyphosphoryl), a sulfono group (e.g., hydroxysulfonyl), a phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), and p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000.

[0077] In some embodiments, R in the compound of formula IV 4 is optionally R 3 is -(CH2CH2R 5 ) n -, -C1-C6 alkyl, or -C1-C6 alkoxy, is a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group. In some embodiments, R in the compound of formula IV 3 is -C1-C6 alkoxy or -(CH2CH2R 5 ) n -, and when R 5 is -O-, R 4 can be a hydrogen atom, an alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), or phosphoryl group. In some embodiments, R in the compound of formula IV3 is -C1 to C6 alkyl or -(CH2CH2R 5 ) n -, and when R 5 is -CH2-, R 4 can be a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group or a sulfono group. In some embodiments, R 4 in the compound of formula IV is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group. In some embodiments, R 4 in the compound of formula IV is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group or a maleimide group, which can optionally be added and / or provided by pre-polymerization and / or post-polymerization functionalization. In some embodiments, when R 3 in the compound of formula IV is -C1 to C6 alkyl-SO3- or a salt thereof, R 4 is a hydrogen atom or does not exist. In some embodiments, R 3 in the compound of formula IV is a salt of -C1 to C6 alkyl-SO3- (for example, sodium salt), and R 4 does not exist. In some embodiments, R 3 in the compound of formula IV is -(CH2CH2R 5 ) n -.

[0078] In some embodiments, the compounds of the present invention can include and / or be telechelic polymers, which are polymers or prepolymers that can enter into further polymerization or other reactions via one or more of their reactive end groups. In some embodiments, the compounds of the present invention can include and / or be heterotelechelic polymers, which are the polymers or prepolymers that can enter into further polymerization or other reactions via the reactive end groups at each end of the polymer or prepolymer, and the two reactive end groups are not identical to each other. In some embodiments, the compounds of the present invention can include and / or be homotelechelic polymers, which are the polymers or prepolymers that can enter into further polymerization or other reactions via the reactive end groups at each end of the polymer or prepolymer, and the two reactive end groups are identical to each other. In some embodiments, the compounds of the present invention can include and / or be semi-telechelic polymers, which are the polymers or prepolymers that can enter into further polymerization or other reactions via the reactive end groups at each end of the polymer or prepolymer.

[0079] A bioconjugate group may optionally be present in the compounds of the present invention. The terms "bioconjugatable group", "bioconjugatable site", or "bioconjugate group" and their grammatical variations refer to residues and / or functional groups that bind to or are bound to a biomolecule (e.g., protein, peptide, DNA, RNA, etc.). Thus, the terms "bioconjugatable group", "bioconjugatable site", or "bioconjugate group" and their grammatical variations do not include biomolecules. However, in some embodiments, a bioconjugate group is used to bind to a biomolecule, or a bioconjugate group or a derivative thereof is bound to a biomolecule (e.g., protein, peptide, DNA, RNA, etc.). Exemplary bioconjugatable groups include, but are not limited to, amines (including amine derivatives), such as isocyanates, isothiocyanates, iodoacetamides, azides, diazonium salts, etc.; acids or acid derivatives, such as N-hydroxysuccinimide esters (more generally, activated esters derived from carboxylic acids, such as p-nitrophenyl esters), acid hydrazides, etc.; and other linking groups, such as aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiol groups, maleimides, aziridines, acryloyls, halogen groups, biotin, 2-iminobiotin, etc. Linking groups, such as those described above, are known and are described in U.S. Patent Nos. 6,728,129, 6,657,884, 6,212,093, and 6,208,553. For example, a compound of the present invention may have a bioconjugate group containing a carboxylic acid, and the carboxylic acid may be used for bioconjugation to a biomolecule (e.g., via carbodiimide activation and coupling with an amino-substituted biomolecule).

[0080] In some embodiments, the biomolecule can include proteins (e.g., antibodies and / or carrier proteins), peptides, DNA, RNA, etc., and / or can be proteins (e.g., antibodies and / or carrier proteins), peptides, DNA, RNA, etc. In some embodiments, the biomolecule can optionally include a residue (e.g., a polymer) that can have one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8 or more) binding sites for the compounds of the present invention. In some embodiments, the biomolecule can be a member of a specific binding pair. The terms “specific binding pair” and “ligand-receptor binding pair” are used interchangeably herein and refer to two different molecules, where one of the molecules has a region on its surface or within a cavity that specifically attracts or binds to a specific spatial or polar organization of the other molecule such that both molecules have an affinity for each other. The members of a specific binding pair can be referred to as a ligand and a receptor (antiligand). The term “ligand and receptor” is intended to include the entire ligand or receptor, or a portion of them sufficient for binding to occur between the ligand and the receptor. Ligand-receptor binding pairs include, but are not limited to: hormones and hormone receptors, such as epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor-α and tumor necrosis factor receptor, and interferon and interferon receptor; avidin and biotin or antibiotin; antibody-antigen pairs; enzymes and substrates; drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid strands; a nucleic acid strand and a complementary oligonucleotide; interleukins and interleukin receptors; stimulatory factors and their receptors, such as granulocyte-macrophage colony stimulating factor and GMCSF receptor and macrophage colony stimulating factor (MCSF) and MCSF receptor.

[0081] The compounds of the present invention may include a dye (e.g., a tetrapyrrole) covalently attached to a portion of the polymers described herein. In some embodiments, the dye may be covalently attached to the terminal portion of the polymer. When present, the bioconjugate group may also be covalently attached to a portion of the polymer, such as the terminal portion of the polymer. In some embodiments, the bioconjugate group is covalently attached to the first terminal portion (e.g., the first end) of the polymer, and the dye is covalently attached to the terminal portion on the opposite side of the polymer (e.g., the opposite end).

[0082] The compounds of the present invention may include a dye (e.g., a tetrapyrrole) covalently attached to a portion of the polymer, and the bioconjugate group may be covalently attached to a portion of the dye. In some embodiments, the bioconjugate group is covalently attached to the first portion (e.g., the first end) of the dye, and the polymer is covalently attached to the second portion (e.g., the opposite end) of the dye.

[0083] In some embodiments, the compounds of the present invention or a portion thereof have a non-rigid backbone (e.g., a non-rigid polymer backbone) and / or have conformational flexibility. The conformational flexibility of the molecular chain can be described and quantified by the "persistence length" of the compound or a portion thereof (e.g., the polymer portion). In some embodiments, the persistence length of the compounds of the present invention can be on the order of the length of a given carbon-carbon bond.

[0084] The compounds of the present invention can be self-folding, for example, self-folding in water and / or an aqueous solution. As used herein, "self-folding" refers to a compound that transfers from a partially or fully extended or unfolded structure to a structure in which at least a part of the extended or unfolded structure is folded in response to contact with a solution (e.g., an aqueous solution) or the compound, where the folding occurs spontaneously (i.e., without external control or force) in response to contact with the solution, so the folding is innate. In some embodiments, it self-folds in response to contact with water and / or an aqueous solution. The compounds of the present invention can optionally self-fold into a unimolecular micelle structure in response to contact with water and / or an aqueous solution. The aqueous solution in which the compounds of the present invention are folded can be a buffer, such as a phosphate buffer (e.g., phosphate buffered saline). In some embodiments, the aqueous solution (e.g., aqueous buffer) in which the compounds of the present invention are folded can have a low ionic strength; for example, the aqueous solution can have a mu value of about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 160 mM to about 180 mM, or about 160 mM to about 170 mM. In some embodiments, the aqueous solution in which the compounds of the present invention are folded can have a mu value of less than about 100 mM. In some embodiments, the aqueous solution in which the compounds of the present invention are folded can contain 1 M of NaCl. In some embodiments, the aqueous solution in which the compounds of the present invention are folded can contain less than 1 M of NaCl, such as less than about 0.75 M, less than about 0.5 M, or less than about 0.25 M of NaCl. In some embodiments, the aqueous solution in which the compounds of the present invention are folded contains 10 mM of NaH2PO4 and 150 mM of NaCl and has a pH of about 7.35.

[0085] In some embodiments, the compounds of the present invention can be in particulate form. The compounds of the present invention can form particles, for example, in response to contact with a solution (e.g., an aqueous solution). In some embodiments, a single (i.e., one) compound can form a particle. Thus, the compound and the particle are present in an approximately 1:1 ratio (i.e., there is one compound per particle).

[0086] The compounds of the present invention can include a part of one or more hydrophobic units in the core or internal region of the particle and / or a part of one or more hydrophilic units in the periphery or external region (e.g., shell) of the particle. In some embodiments, the particle has a micelle structure (e.g., a unimolecular micelle structure). The compounds of the present invention can include a dye, which can be bound to the polymer of the present invention, and when the compound is in a folded structure and / or particulate form (e.g., a unimolecular micelle structure), the dye can be encapsulated by a part of the compound (e.g., a part of the polymer). In some embodiments, the dye or a part thereof and one or more hydrophobic units can be present in the core or internal region of the particle, and one or more hydrophilic units can surround the dye and / or one or more hydrophobic units.

[0087] In some embodiments, the hydrophobic units present in the polymers of the present invention can be one or more hydrophobic units of Formula III. In some embodiments, the one or more hydrophobic units have an alkyl (e.g., dodecyl) pendant functional group and / or are formed from a compound of Formula I and / or an alkyl acrylate (e.g., dodecyl acrylate) monomer. In some embodiments, the hydrophilic units present in the polymers of the present invention can be one or more hydrophilic units of Formula IV and / or can be formed from a compound of Formula II. In some embodiments, the one or more hydrophilic units contain a nonionic (i.e., neutral / uncharged) pendant functional group (e.g., PEG) and / or are formed from a nonionic monomer (e.g., PEG acrylate (PEGA)). In some embodiments, the one or more hydrophilic units contain an ionic (e.g., anionic, charged) pendant functional group (e.g., sulfonic acid and / or sulfonate) and / or are formed from an ionic monomer (e.g., sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). In some embodiments, the hydrophilic units are formed from at least two different monomers, such as a nonionic (i.e., neutral / uncharged) hydrophilic monomer (e.g., PEG acrylate (PEGA)) and an ionic (e.g., anionic, charged) hydrophilic monomer (e.g., sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)), etc. One of ordinary skill in the art will understand that monomers containing acids, such as sulfonic acid, etc., can be present in the acid form and / or in their ionic form. In some embodiments, the monomer containing an acid is predominantly in its ionic form (i.e., >50%). In some embodiments, the ionic hydrophilic monomer is an acid in the deprotonated form (e.g., deprotonated sulfonic acid acrylate) and / or in the salt form, e.g., sodium sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid as the sodium salt).

[0088] In some embodiments, when two or more different hydrophilic units are present in the polymers of the present invention, the ratio of the two or more different hydrophilic units can vary, for example, from about 10:1 to about 1:10. For example, in some embodiments, the polymer has a ratio of about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10 (non-ionic unit: ionic unit) of a non-ionic (i.e., neutral / uncharged) hydrophilic unit (e.g., formed from pegylated methyl acrylate (PEGA)) and an ionic (e.g., anionic, charged) hydrophilic unit (e.g., formed from sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). In some embodiments, the ratio of one or more hydrophilic units to one or more hydrophobic units present in the backbone of the polymers of the present invention can vary. In some embodiments, the ratio of one or more hydrophilic units to one or more hydrophobic units present in the backbone of the polymer is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10 (hydrophobic unit: hydrophilic unit).

[0089] In some embodiments, the polymers of the present invention include hydrophobic units in an amount of about 1% to about 40% based on the total molar amount of monomers used to prepare the polymers and hydrophilic units in an amount of about 60% to about 99% based on the total molar amount of monomers used to prepare the polymers. In some embodiments, the polymers of the present invention include hydrophobic units in an amount from about 1%, about 5%, about 10%, about 15% or about 20% to about 25%, about 30%, about 35% or about 40% based on the total molar amount of monomers used to prepare the polymers and hydrophilic units in an amount from about 60%, about 65%, about 70%, about 75% or about 80% to about 85%, about 90%, about 95% or about 99% based on the total molar amount of monomers used to prepare the polymers. In some embodiments, the polymers include hydrophobic units in an amount of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40% based on the total molar amount of monomers used to prepare the polymers. In some embodiments, the polymers include less than about 30% (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10% or less than about 5%) of hydrophobic units based on the total molar amount of monomers used to prepare the polymers. In some embodiments, the polymers include hydrophilic units in an amount of about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98 or about 99% based on the total molar amount of monomers used to prepare the polymers.In some embodiments, the polymer comprises more than about 70% (e.g., more than about 75%, more than about 80%, more than about 85%, more than about 90% or more than about 95%) hydrophilic units relative to the total molar amount of monomers used to prepare the polymer.

[0090] The polymers of the present invention can have a weight fraction of hydrophobic units ranging from about 1%, about 5%, about 10%, about 15% or 20% to about 25%, about 30%, about 35% or about 40% based on the total weight of the polymer. In some embodiments, the polymer can have a weight fraction of hydrophobic units of about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39% or about 40% based on the total weight of the polymer. In some embodiments, the polymer can have a weight fraction of hydrophobic units of less than about 30% (e.g., less than about 25%, less than about 20%, less than about 15%, less than about 10% or less than 5%) based on the total weight of the polymer.

[0091] The polymer of the present invention can have a weight fraction of hydrophilic units ranging from about 60%, about 65%, about 70%, about 75% or about 80% to about 85%, about 90%, about 95% or about 99% based on the total weight of the polymer. In some embodiments, the polymer of the present invention can have a weight fraction of hydrophilic units of about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98 or about 99% based on the total weight of the polymer. In some embodiments, the polymer can have a weight fraction of hydrophilic units greater than about 70% (e.g., greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90% or greater than about 95%) based on the total weight of the polymer.

[0092] In some embodiments, the amount of unimolecular micelle structures formed in response to contact with a solution is about 50% to about 100%, about 75% to about 100%, about 85% to about 100%, or about 95% to about 100% when optionally measured using a sizing method (e.g., dynamic light scattering (DLS) spectroscopy). In some embodiments, the amount of unimolecular micelle structures formed in response to contact with a solution is about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100% when optionally measured using a sizing method (e.g., DLS spectroscopy). The solution in which the unimers are present can be an aqueous solution as described herein, e.g., an aqueous buffer. In some embodiments, the aqueous solution in which the unimers are present can be a phosphate buffer (e.g., phosphate buffered saline). In some embodiments, the aqueous solution (e.g., aqueous buffer) in which the unimers are present has a low ionic strength (e.g., can have a μ value of about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 160 mM to about 180 mM, or about 160 mM to about 170 mM). In some embodiments, the aqueous solution in which the unimers are present contains 10 mM of NaH2PO4 and 150 mM of NaCl and has a pH of about 7.35.

[0093] In some embodiments, dilution of a solution containing a compound of the invention in the form of a unimolecular micelle structure results in no loss or less than about 20% loss of the unimolecular micelle structure present in the solution as compared to the amount of the unimolecular micelle structure present in the solution prior to dilution. In some embodiments, the amount of the unimolecular micelle structure present in the solution does not change in response to dilution or changes by less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% or less than about 0.1% as compared to the amount of the unimolecular micelle structure present in the solution prior to dilution.

[0094] In some embodiments, a solution comprising a compound of the invention in the form of a unimolecular micelle structure contains less than about 50% aggregates (e.g., less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than about 32%, less than about 31%, less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% or less than about 0.1%). Thus, at least 50% or more of the compound can be non-aggregated and in the form of a unimolecular micelle structure. In some embodiments, dilution of a solution comprising a compound of the invention in the form of a unimolecular micelle structure produces no additional aggregates or minimal additional aggregate formation compared to the amount of aggregates present in the solution prior to dilution. In some embodiments, the amount of aggregates present in a solution comprising a compound of the invention does not change upon dilution or changes by less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% or less than about 0.1% compared to the amount of aggregates present in the solution prior to dilution.In some embodiments, the diluted solution comprises less than about 50% aggregates (e.g., less than about 49%, less than about 48%, less than about 47%, less than about 46%, less than about 45%, less than about 44%, less than about 43%, less than about 42%, less than about 41%, less than about 40%, less than about 39%, less than about 38%, less than about 37%, less than about 36%, less than about 35%, less than about 34%, less than about 33%, less than about 32%, less than about 31%, less than about 30%, less than about 29%, less than about 28%, less than about 27%, less than about 26%, less than about 25%, less than about 24%, less than about 23%, less than about 22%, less than about 21%, less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1% or less than about 0.1%).

[0095] The compounds of the present invention can have a diameter in the range of about 1 nm to about 50 nm or about 3 nm to about 30 nm (e.g., when folded like a unimolecular micelle structure) in water and / or an aqueous solution. In some embodiments, the compound can have a diameter of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49 or about 50 nm (e.g., when folded like a unimolecular micelle structure) in water and / or an aqueous solution. In some embodiments, the compounds of the present invention can have a particulate form (i.e., at least partially folded structure).

[0096] In some embodiments, the compounds of the invention are crosslinked, where optionally, when the compound is in a folded structure, the compound is crosslinked. In some embodiments, the compounds of the invention can be in solution (e.g., an aqueous solution) and / or can be crosslinked with a crosslinking agent. Crosslinking the compounds of the invention can involve linking together two or more residues and / or functional groups (e.g., pendant functional groups) of one or more hydrophobic units and / or one or more hydrophilic units. Crosslinking can provide a compound in a folded structure that cannot be unfolded without breaking one or more bonds formed by the crosslinking. The degree or amount of crosslinking can be controlled, modified, and / or adjusted, for example, by the amount of crosslinking agent reacted with the compound. In some embodiments, the step of crosslinking the compound can involve reactions and / or reactive entities (e.g., functional groups) listed in Table 1.

[0097] [Table 1]

[0098] In the compounds of the invention, the fluorescence quantum yield of the dye when the compound is present in water and / or an aqueous solution can be reduced by about 10% or less (e.g., about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, about 2% or less, about 1% or less) compared to the fluorescence quantum yield of the dye when the compound is present in a hydrophobic solvent (e.g., in toluene). In response to the bioconjugation reaction of the compounds of the invention with a biomolecule (e.g., a protein), the fluorescence quantum yield of the dye can be the same as or substantially the same (e.g., within ±20%) as the fluorescence quantum yield of the dye in water and / or a hydrophobic solvent. In some embodiments, when the fluorescence quantum yield of the dye is 1.00 (theoretical maximum), a decrease of less than one-tenth (e.g., about one-tenth or less, about one-ninth or less, about one-eighth or less, about one-seventh or less, about one-sixth or less, about one-fifth or less, about one-fourth or less, about one-third or less, about one-half or less) is acceptable.

[0099] In some embodiments, the compounds of the present invention are water-soluble. The compound may have a solubility in water in the range of about 1 mg / mL to about 10 mg / mL at room temperature. In some embodiments, the compound has a solubility in water of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL or about 10 mg / mL at room temperature.

[0100] In some embodiments, the compounds and / or particles of the present invention are resistant to dilution. As used herein, "resistant to dilution" refers to compounds and / or particles that retain their structure and / or properties. In some embodiments, being resistant to dilution refers to compounds and / or particles that retain a folded structure (e.g., a unimolecular micelle structure), which can be determined by measuring the diameter of the particles before and after dilution, and the diameter after dilution can remain within ±50% or less, ±40% or less, ±30% or less, ±20% or less, ±10% or less of the diameter before dilution. In some embodiments, being resistant to dilution refers to compounds and / or particles that retain the fluorescence quantum yield of the dye after dilution within the range of ±50% or less, ±40% or less, ±30% or less, ±20% or less, ±10% or less of the fluorescence quantum yield of the dye before dilution. In some embodiments, the compounds and / or particles of the present invention remain in a folded structure when diluted up to 25-fold, 50-fold, 75-fold or 100-fold or when diluted to a concentration below micromolar.

[0101] According to some embodiments of the present invention, there is provided a method for preparing a compound and / or composition of the present invention. In some embodiments, the method for preparing a compound of the present invention comprises polymerizing a hydrophobic monomer and a hydrophilic monomer to provide a copolymer, attaching a dye to a first portion (e.g., a terminal or end portion) of the copolymer, and optionally attaching a bioconjugate group (e.g., a group capable of a bioconjugation reaction) to a second portion (e.g., another terminal or end portion) of the copolymer, thereby providing the compound. The hydrophobic monomer and the hydrophilic monomer can be polymerized using any method known to those skilled in the art, such as, but not limited to, a condensation reaction (e.g., reaction with a diol and a diacid) and / or living radical polymerization (e.g., atom-transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT)). In some embodiments, polymerizing the hydrophobic monomer and the hydrophilic monomer is performed by a method that provides a copolymer having one or both reactive groups of the copolymer (i.e., one or both terminal groups of the copolymer can enter into further polymerization or reaction), and the two terminal groups may be the same or different. In some embodiments, polymerizing the hydrophobic monomer and the hydrophilic monomer is via living radical polymerization (e.g., ATRP) in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally, a co-catalyst, to provide a copolymer. In some embodiments, polymerizing the hydrophobic monomer and the hydrophilic monomer is via living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT agent (e.g., a thiocarbonylthio compound).

[0102] In some embodiments, attaching the dye to the first portion of the copolymer can include reacting a monomer containing the dye with a hydrophobic monomer and / or hydrophobic unit and / or hydrophilic monomer and / or hydrophilic unit. Thus, in some embodiments, the step of attaching the dye to the copolymer can occur during or after the polymerization step. In some embodiments, the method includes reacting a monomer containing the dye with one or more (e.g., two or three) hydrophobic monomers and / or hydrophobic units and / or one or more (e.g., two or three) hydrophilic monomers and / or hydrophilic units during the step of polymerizing the hydrophobic monomer and the hydrophilic monomer. In some embodiments, the polymerization of one or more hydrophobic monomers and one or more hydrophilic monomers occurs via living radical polymerization (e.g., ATRP) in the presence of an initiator, and the initiator contains the dye. In some embodiments, the polymerization of one or more hydrophobic monomers and one or more hydrophilic monomers occurs via living radical polymerization (e.g., RAFT) in the presence of a radical initiator and a RAFT agent, where optionally the RAFT agent contains the dye.

[0103] Exemplary end functional groups on the copolymer, when the copolymer is available for immediate dye attachment or bioconjugation reactions, include, but are not limited to, the end functional groups listed in Table 2. These end functional groups are not pendant functional groups but can be present at either end of the copolymer.

[0104] [Table 2]

[0105] Some functional groups can be unstable under certain polymerization conditions. Thus, in some embodiments, the functional groups can be introduced in a protected form. As a result, these functional groups can be available for dye attachment or bioconjugation reactions upon deprotection. Exemplary protected forms of certain functional groups include, but are not limited to, those listed in Table 3.

[0106]

Table 3

[0107] In some embodiments, a portion of the copolymer (e.g., the end or terminus) may contain a halogen group (e.g., Cl, Br, I). The halide portion of the copolymer can be derivatized with a nucleophile or end-capping reagent to generate a functional group for dye attachment or bioconjugation reactions. In some embodiments, a portion of the copolymer (e.g., the end or terminus) may have a thiol group, which can be derivatized with a reagent containing a thiol-reactive group to generate a functional group for dye attachment or bioconjugation reactions. Examples of thiol-reactive groups include, but are not limited to, halides (e.g., bromine atom, chlorine atom, iodine atom), alkynes, aldehydes, vinyl ketones, and / or maleimide functional groups. All of the functional groups listed in Tables 2 and 3 are compatible with these strategies, and additional exemplary functional groups include, but are not limited to, those listed in Table 4.

[0108]

Table 4

[0109] (Optionally, via ATRP or RAFT) polymerizing the hydrophobic monomer and the hydrophilic monomer can include polymerizing the hydrophobic monomer and the hydrophilic monomer at a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10 (1 or more hydrophobic monomers: 1 or more hydrophilic monomers). In some embodiments, the ratio can be from about 1:1 to about 1:3 or about 1:6. In some embodiments, the hydrophobic monomer is an alkyl acrylate (e.g., dodecyl acrylate), and / or the hydrophilic monomer is a glycol acrylate (e.g., PEG acrylate). In some embodiments, 1 or more hydrophobic monomers are polymerized (optionally, via RAFT or ATRP) with 2 or more different hydrophilic monomers at a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10 (1 or more hydrophobic monomers: 1 or more hydrophilic monomers). For example, in some embodiments, the first hydrophilic monomer can be ionic (e.g., a sulfonic acid acrylate monomer (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or a sulfonate monomer), and the second hydrophilic monomer can be non-ionic (e.g., a glycol acrylate (e.g., a pegylated methyl acrylate)). The ratio of the first hydrophilic monomer to the second hydrophilic monomer can vary (e.g., the ratio of the first hydrophilic monomer: the second hydrophilic monomer can be about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5 or about 1:6).

[0110] Exemplary catalysts that can be used in the method of the present invention include, but are not limited to, ruthenium complexes, iron complexes, copper complexes, nickel complexes, palladium complexes, rhodium complexes and rhenium complexes. Exemplary ruthenium complexes include, but are not limited to, dichlorotris(triphenylphosphine)ruthenium(II) [RuCl2(PPh3)3], pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride [RuCp*Cl(PPh3)2], chloro(cyclopentadienyl)bis(triphenylphosphine)ruthenium [RuCpCl(PPh3)2], dihydridotetrakis(triphenylphosphine)ruthenium(II) [RuH2(PPh3)4] and dichloro(p-cymene)ruthenium(II) dimer. Exemplary iron complexes include, but are not limited to, dichlorobis(triphenylphosphine)iron(II) [FeCl2(PPh3)2], bromo(cyclopentadienyl)dicarbonyliron(II) [FeCpBr(CO)2] and cyclopentadienyliron dicarbonyl dimer. In some embodiments, copper complexes generated in-situ with copper salts and ligands can be used, and exemplary copper salts include, but are not limited to, copper chloride, copper bromide, copper triflate, copper hexafluorophosphate and copper acetate. Exemplary nitrogen-based ligands include, but are not limited to, 2,2'-bipyridine and its derivatives, 1,10-phenanthroline and its derivatives, sparteine and other diamines, and terpyridine and its derivatives. Exemplary nickel complexes include, but are not limited to, dibromobis(triphenylphosphine)nickel(II) [NiBr2(PPh3)2] and tetrakis(triphenylphosphine)nickel [Ni(PPh3)4]. An exemplary palladium complex is tetrakis(triphenylphosphine)palladium [Pd(PPh3)4]. An exemplary rhodium complex is tris(triphenylphosphine)rhodium bromide. An exemplary rhenium complex is dioxobis(triphenylphosphine)rhenium iodide. In some embodiments, the catalyst is pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride.

[0111] The cocatalyst may optionally be present in the method of the present invention, for example, in the step of polymerizing the hydrophobic monomer and the hydrophilic monomer. In some embodiments, a cocatalyst may be present and may be 4-(dimethylamino)-1-butanol.

[0112] In some embodiments, the method of the present invention includes hydrolyzing the copolymer, optionally in the presence of trifluoroacetic acid and water, to provide a formyl group at a first portion (e.g., a first end) of the copolymer. The method may include reacting the dye with the formyl group of the copolymer to form a hydrazone bond between the dye and the copolymer, optionally via aldehyde-hydrazide chemistry, thereby binding the dye to the first portion of the copolymer. In some embodiments, the biomolecule may be bound by reacting the formyl group with an amine group in a bioconjugate group via reductive amination.

[0113] In some embodiments, the method of the present invention includes reacting the copolymer with mercaptoacetic acid and triethylamine to provide a carboxymethylthioether group at a second portion (e.g., a second end) of the copolymer. The carboxymethylthioether group may be derivatized to provide an N-hydroxysuccinimide ester at the second portion of the copolymer. A biomolecule (e.g., avidin) may be bound to the N-hydroxysuccinimide ester at the second portion of the copolymer.

[0114] In some embodiments, the method of the present invention includes reacting the copolymer with sodium azide to provide an azide group and, optionally, binding a dye to the azide group via copper-catalyzed azide-alkyne chemistry.

[0115] In some embodiments, the method of the present invention includes RAFT polymerization. In some embodiments, the RAFT polymerization occurs in the presence of a radical initiator (e.g., AIBN) and a RAFT agent, such as a thiocarbonylthio compound, etc. Additional examples of RAFT agents include, but are not limited to, dithioesters, dithiocarbamates, trithiocarbonates, dithiobenzoates, and / or xanthates.

[0116] In some embodiments, the method of the present invention includes cleaving the thiocarbonylthio functionality present at the terminal ends of the copolymer obtained using RAFT polymerization. Such cleavage can occur using any of the general methods known in the art. For example, in some embodiments, the thiocarbonylthio functionality is cleaved via aminolysis, for example, in the presence of ethanolamine, to give a free thiol. In some embodiments, the free thiol can be coupled to a dye containing maleimide functionality, thereby coupling the dye to the first portion (e.g., the terminal end) of the copolymer. In some embodiments, a biomolecule can be coupled to the free thiol group of the first portion (e.g., the terminal end). In some embodiments, a biomolecule can be coupled to the opposite terminal end of the polymer.

[0117] According to some embodiments, the compounds and / or compositions of the present invention can be used in flow cytometry. Flow cytometry is known and is described, for example, in U.S. Patent Nos. 5,167; 5,915,925; 6,248,590; 6,589,792; and 6,890,487. In some embodiments, the particles to be detected, such as cells, are labeled with a luminescent compound, such as a compound of the present invention, for detection. The labeling can be carried out by any suitable technique, such as by binding the luminescent compound (e.g., a compound of the present invention) to the particle or cell, for example, via an antibody that specifically binds to the particle or cell, by uptake or internalization of the luminescent compound into the cell or particle, by non-specific absorption of the luminescent compound into the cell or particle, etc. The compounds described herein can be useful as such luminescent compounds in flow cytometry, and the flow cytometry technique (fluorescent activated cell sorting, i.e., FACS) can be carried out according to known techniques or modifications thereof that are apparent to those skilled in the art based on the present disclosure.

[0118] In some embodiments, a method of detecting cells and / or particles using flow cytometry is provided, the method comprising labeling the cells and / or particles with a compound of the present invention and detecting the compound by flow cytometry, thereby detecting the cells and / or particles.

[0119] In some embodiments, a method of detecting tissue and / or an agent (e.g., a cell, an infectious agent, etc.) in a subject is provided, the method comprising administering to the subject a compound and / or composition of the present invention, wherein optionally the compound associates with the tissue and / or agent, and detecting the compound in the subject, thereby detecting the tissue and / or agent.

[0120] In some embodiments, methods of using the compounds of the present invention in photodynamic therapy (PDT) and / or photodynamic inactivation (PDI) are provided. Photodynamic therapy (PDT) is a form of phototherapy that involves light and a photosensitizing chemical used in combination with molecular oxygen to induce cell death (phototoxicity). PDT can be used to kill microbial cells, including bacteria, fungi, and viruses. PDT can also be used to treat cancer. When light energy is administered by photodynamic therapy (PDT) to destroy a tumor, various forms of energy are within the scope of the present invention as would be understood by one of ordinary skill in the art. Such forms of energy include, but are not limited to, heat, sound waves, ultrasound, chemicals, light, microwaves, ionization (e.g., X-rays and gamma rays), mechanical, and / or electrical. For example, factors that are sonodynamically induced or activated include, but are not limited to, gallium-porphyrin complexes (see Yumita et al., Cancer Letters 112:79-86 (1997)), other porphyrin complexes such as protoporphyrin and hematoporphyrin (see Umemura et al., Ultrasonics Sonochemistry 3:S187-S191 (1996)); other anti-cancer agents such as daunorubicin and adriamycin used in the presence of sonotherapy (see Yumita et al., Japan J. Hyperthermic Oncology 3(2):175-182 (1987)).

[0121] Examples of treatment areas for PDT and / or PDI include, but are not limited to, the following.

[0122] (i) Management of opportunistic infections. The compounds, compositions and / or methods of the present invention may be useful for the PDT of opportunistic infections, particularly of soft tissues. For the antibacterial treatment of infections (via PDT), particularly wound infections, the infectious organisms may include, as non-limiting examples, Staphylococcus aureus, Pseudomonas aeruginosa and / or Escherichia coli. In nosocomial infections, Pseudomonas aeruginosa is a factor in 8% of surgical wound infections and 10% of bloodstream infections. In some embodiments, the subject is an immunocompromised subject, e.g., a subject suffering from AIDS and / or a subject undergoing treatment with immunosuppressive agents.

[0123] (ii) Treatment of burns. Infections by Staphylococcus aureus and Gram-positive bacteria are generally particularly prominent in burns (Lambrechts, 2005). The multidrug resistance of Staphylococcus aureus presents a significant medical problem. In this regard, the compounds, compositions and / or methods of the present invention may be useful for the treatment of opportunistic infections in burns.

[0124] (iii) Septicemia. The compounds, compositions and / or methods of the present invention may be useful for the PDT treatment of subjects suffering from opportunistic infections of Vibrio vulnificus. The Gram-negative bacterium Vibrio vulnificus causes primary septicemia, wound infections and / or gastrointestinal diseases in humans.

[0125] (iv) Ulcers. The compounds, compositions and / or methods of the present invention may be useful for the PDT treatment of bacteria that cause ulcers (Helicobacter pylori). Clinically, the treatment may be performed in any suitable manner, e.g., by inserting a light fiber cable (similar to an endoscope but equipped for the delivery of red or near-infrared light) into the stomach and / or the diseased area.

[0126] (v) Periodontal disease. The compounds, compositions and / or methods of the present invention may be useful in PDT for the treatment of periodontal disease, including gingivitis. Periodontal disease is caused by the abnormal growth of bacteria, such as the gram-negative anaerobic bacterium Porphyromonas gingivalis. As with many PDT treatments, a targeting or solubilizing entity combined with a photoactive species is essential for proper delivery of the photoactive species to the desired cells. Oral pathogens targeted include, but are not limited to, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp., Polymorphum, Actinomyces viscosus and streptococci. For such uses, the compounds and / or compositions of the present invention may be administered topically (e.g., as a mouse wash or mouse rinse) and then photo-administered with an external device, intraoral appliance, or combination thereof.

[0127] (vi) Atherosclerosis. The compounds, compositions and / or methods of the present invention may be useful in PDT for treating vulnerable atherosclerotic plaque. Without wishing to be bound by a particular theory, invading inflammatory macrophages secrete metalloproteinases that degrade the thin layer of collagen in coronary arteries, resulting in thrombosis, which is often fatal (Demidova and amblin, 2004). Bacteriochlorins that target such inflammatory macrophages may be useful for PDT of unstable plaques.

[0128] (vii) Cosmetic and dermatological uses. The compounds, compositions and / or methods of the present invention may be useful in PDT for treating a wide range of cosmetic dermatological problems, such as hair removal, treatment of psoriasis and / or removal of skin discoloration. Ruby lasers are currently used for hair removal. In many laser treatments, melanin is the photosensitized chromophore. Such treatments work moderately well for fair-skinned people with dark hair. The compounds, compositions and / or methods of the present invention can be used as near-infrared photosensitizers for hair removal, which makes it possible to target chromophores with more specific and / or sharp absorption bands.

[0129] (viii) Acne. The compounds, compositions and / or methods of the present invention may be useful in PDT for treating acne. Acne vulgaris is caused by Propionibacterium acnes, which infects the sebaceous glands. Approximately 80% of young people are affected. Here too, the increasing resistance of bacteria to antibiotic treatment has led to a sharp increase in acne that is difficult to treat. Current PDT treatments for acne typically rely on the addition of aminolevulinic acid, which is converted to free base porphyrin in hair follicles or sebaceous glands. The compounds and / or compositions of the present invention can be administered to a subject locally or parenterally (e.g., by subcutaneous injection), depending on the particular condition.

[0130] (ix) Infectious diseases. The compounds, compositions and / or methods of the present invention may be useful in PDT for treating infectious diseases. For example, cutaneous leishmaniasis and subcutaneous leishmaniasis, which are widespread in the Mediterranean and Middle East regions, are currently treated with arsenic-containing compounds. PDT has recently been used in at least one case to produce reasonable effects in human subjects. The use of the compounds and / or compositions of the present invention may be similarly useful and potentially offer advantages such as ease of synthesis and better spectral absorption characteristics.

[0131] (x) Tissue sealants. The compounds, compositions and / or methods of the present invention may be useful in PDT as tissue sealants in a subject in need thereof. Photoactivated tissue sealants are attractive for sealing wounds, binding tissues and / or closing defects in tissues. There are many applications where sutures and / or staples are undesirable, and the use of such mechanical sealing methods often results in infection and / or scarring.

[0132] (xi) Neoplastic diseases. The compounds, compositions and / or methods of the present invention may be useful in PDT for treating neoplastic diseases and / or cancers, such as those including skin cancer, lung cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, ovarian cancer, basal cell carcinoma, leukemia, lymphoma, squamous cell carcinoma, melanoma, plaque-stage cutaneous T-cell lymphoma and / or Kaposi's sarcoma.

[0133] During photomechanical therapy, the compounds of the present invention are administered to a subject in need thereof (e.g., a subject having any of the diseases described above). The administered compound can bind to diseased tissue present within the subject, and exposure of the subject to a light source emitting light of an appropriate wavelength and intensity can activate the compound within the diseased tissue (e.g., release reactive oxygen species (ROS)), thereby treating the diseased tissue, optionally without affecting healthy tissue. For example, in some embodiments, the diseased tissue is hyperproliferative tissue (e.g., a tumor).

[0134] In some embodiments, methods of using the compounds of the present invention in photoacoustic imaging are provided. According to some embodiments, the method of the present invention includes a method of performing photoacoustic imaging. Photoacoustic imaging (PAI) is attractive in that it does not rely on optical luminescence for detection (Haisch, C., Quantitative analysis in medicine using photoacoustic tomography. Anal. Bioanal. Chem. 2009, 393, 473 - 479; Cox, B.; Laufer, J.G.; Arridge, S.R.; Beard, P.C. Quantitative spectroscopic photoacoustic imaging: a review. J. Biomed. Opt. 2012, 17, 061202). Optical luminescence can be affected by light scattering. In PAI, after laser irradiation (e.g., optionally performed using non - ionizing laser pulses), thermoelastic expansion and ultrasonic pressure waves follow. Detection of the ultrasonic pressure waves can be achieved via conventional ultrasonic detectors. Essentially, ultrasound imaging can be performed with a laser input. Notably, in contrast to X - ray imaging methods, PAI does not rely on ionizing radiation.

[0135] The method of the present invention may include administering a compound and / or composition of the present invention to a subject, where optionally, the compound associates with tissues and / or cells in the subject; irradiating at least a part or portion of the subject with a laser, where optionally, the part or portion of the subject contains the compound of the present invention; and imaging at least the part or portion of the subject, where optionally, the imaging includes ultrasound imaging.

[0136] PAI can be performed without utilizing any exogenous contrast agents or chemical probes. In such cases, the distinct absorption of endogenous chromophores in natural tissues produces a distinct signal. Absorption by hemoglobin, for example, facilitates the depiction of the presence of blood vessels. However, the molar extinction coefficient of hemoglobin is low and may be insufficient for distinct depiction of deep tissues. In such cases, the use of contrast agents is very attractive. In some embodiments, the compounds of the present invention are used as contrast agents in PAI and / or contain dyes that can be used as contrast agents in PAI.

[0137] Various substances are being investigated for use as contrast agents in PAI. Exemplary dyes for use in PAI include, but are not limited to, gold nanomaterials, carbon nanotubes, porphyrins in liposomes, semiconductor polymers, and naphthalocyanines (Chitgupi, U.; Lovell, J.F. Naphthalocyanines as contrast agents for photoacoustic and multimodal imaging. Biomed. Eng. Lett. 2018, 8, 215 - 221; de la Zerda, A., et al., Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics. Contrast Media Mol. Imaging 2011, 6, 346 - 369). In some embodiments, the dyes present in the compounds of the invention and / or the compounds of the invention have the following photophysical properties, which are that following absorption of light, the dye / compound relaxes immediately and quantitatively to the ground state without emission of light or formation of any significant - lifetime metastable state. In other words, the yield of internal conversion (i.e., non - radiative decay) must be quantitative, and ideally, the rate of internal conversion is very fast and the lifetime of the excited state must be less than 1 picosecond. This description essentially represents the “optical - to - acoustic conversion efficiency” from the perspective of molecular photophysics (Cheng, K.; Cheng, Z. Near infrared receptor - targeted nanoprobes for early diagnosis of cancers. Curr. Med. Chem. 2012, 19, 4767 - 4785). The attraction of such rapid and quantitative internal conversion is to convert all of the absorbed light into heat, i.e., the thermal expansion that generates ultrasound.A research group refers to such contrast agents as "sonochromes" (Duffy, M.J., et al., Towards optimized naphthalocyanines as sonochromes for photoacoustic imaging in vivo. Photoacoustics 2018, 9, 49 - 61), and distinguishes them from the more commonly known lumichromes or fluorochromes or chromophores, where all of these mean emission following absorption of incident light. In some embodiments, the compounds of the present invention are sonochromes and / or contain sonochromes.

[0138] In some embodiments, the dyes present in the compounds of the present invention and / or the compounds of the present invention absorb light in the red or in the near - infrared region (NIR). For example, in some embodiments, the compounds of the present invention can be used to image deep tissues, where this region presents an optical window that allows light transmission, so absorption in the red or near - infrared region (NIR) is desired. At shorter wavelengths, absorption by endogenous chromophores (e.g., hemoglobin, melanin) can occur. At longer wavelengths, light scattering by the overtone vibration bands of water can be observed. In some embodiments, the dyes present in the compounds of the present invention and / or the compounds of the present invention absorb red or NIR, and the molar extinction coefficient is as large as possible, producing excellent sensitivity, e.g., 1,000 M -1 cm -1 , 10,000 M -1 cm -1 , 100,000 M -1 cm -1 or values of molar extinction coefficients above, and in some embodiments, chlorin exhibits a Q -1 cm -1 ~ about 100,000 M -1 cm -1 band molar extinction coefficient in the range of. In some embodiments, bacteriochlorin exhibits a band molar extinction coefficient of about 50,000 M y cm -1 cm-1 ~ about 200,000 M -1 cm -1 Q in the range of y shows the band molar extinction coefficient.

[0139] In some embodiments, the methods of the invention provide multi-wavelength multiplexing. Multi-wavelength multiplexing can be achieved by using two or more absorbers as PAI contrast agents, all of which exhibit quantitative (or near quantitative) internal conversion, where two or more absorbers are two or more different compounds of the invention. Two or more different compounds of the invention can have mostly non-overlapping absorption bands. Multiplexing can be achieved by sweeping an incident light source (e.g., a laser) across the NIR and red spectral regions, along with detection of the resulting ultrasound in response to the sequential absorption of each spectrally distinct contrast agent. Alternatively, a set of multiple lasers can be used, each with a dedicated laser for a different PAI contrast agent.

[0140] In some embodiments, the dye present in the compounds of the invention comprises chlorin or bacteriochlorin, where optionally the compound is used in the methods of the invention for PAI. Chlorin and / or bacteriochlorin can be ideal for photoacoustic imaging, based on their strong and sharp long wavelength (Q y ) absorption bands. Chlorin and / or bacteriochlorin can be modified to produce a high yield of internal conversion and / or packaged in a manner to achieve solubilization in an aqueous medium.

[0141] For example, tetrapyrrole macrocyclic molecules that are fluorescent in their free base form can be made non-fluorescent by metallation with an appropriate metal. Tetrapyrroles include porphyrins and hydroporphyrins, the latter of which include chlorins and bacteriochlorins. Based on extensive research on the preparation and study of metallotetrapyrroles over nearly a century, there exists a true "periodic table of metallotetrapyrroles". Metals that give non-emissive tetrapyrrole chelates are well-known (see, for example, Gouterman, M. Optical spectra and electronic structure. In The Porphyrins; Dolphin, D. (Ed.), Vol. III, Academic Press: New York, 1978, pp1-165). Examples of metals that can give non-emissive tetrapyrrole chelates (valences not shown for clarity) include, but are not limited to, Fe, Co, Ni, Cu, Zr, Ru, and the lanthanides. In some embodiments, the dye present in the compounds of the present invention is a tetrapyrrole macrocyclic molecule containing iron. Iron is particularly attractive given the presence of iron as a natural component in human metabolism, the extensive research focused on iron tetrapyrroles (given the fact that heme is an iron chelate of protoporphyrin IX), and the very short excited state lifetimes of iron porphyrins. In some embodiments, the compounds of the present invention contain iron chlorin or iron bacteriochlorin. In some embodiments, the method of the present invention comprises administering a compound of the present invention containing iron chlorin or iron bacteriochlorin as a PAI contrast agent to a subject and performing photoacoustic imaging. In some embodiments, the dye present in the compounds of the present invention is a tetrapyrrole macrocyclic molecule containing copper (e.g., Cu(II)). In some embodiments, the dye present in the compounds of the present invention contains copper (e.g., Cu(II)) and is optionally used for photoacoustic imaging. In some embodiments, the dye present in the compounds of the present invention contains iron (e.g., Fe(II)) and can optionally be used for oxygen sensing.

[0142] In some embodiments, the compounds of the invention include sterically hindered and / or Fe(II) tetrapyrroles that do not form mu-oxo oxo dimers of Fe(III) tetrapyrroles. In some embodiments, the compounds of the invention include Fe(III) tetrapyrroles. It should be noted that Fe(II) tetrapyrroles can coordinate to molecular oxygen and, in the absence of steric hindrance, can undergo a chemical reaction to yield mu-oxo dimers of Fe(III) tetrapyrroles. In contrast, Fe(III) tetrapyrroles do not coordinate to molecular oxygen and do not give rise to mu-oxo dimer formation. Fe(III) tetrapyrroles are the preferred oxidation state of iron tetrapyrroles in response to formation under aerobic conditions. A variety of well-established methods over the years are available for the formation of Fe(III) tetrapyrroles and for the conversion of Fe(II) tetrapyrroles to Fe(III) tetrapyrroles.

[0143] The free base tetrapyrrole can give a certain amount of fluorescence (e.g., a maximum of about 10% quantum yield), a certain amount of triplet state formation (e.g., a maximum of about 70% quantum yield), and the remainder is internal conversion (e.g., a maximum of about 20% quantum yield). As described above, a convenient way to achieve a quantum yield of about 100% for internal conversion (i.e., non-radiative decay) is to metallate the tetrapyrrole with a metal that relaxes the excited state to the ground state rapidly and essentially quantitatively by one or more mechanisms. An alternative approach to promoting internal conversion versus radiative decay (i.e., fluorescence) and intersystem crossing (i.e., triplet state formation) is to attach appropriate substituents to the tetrapyrrole. Typical substituents include substituents that give rise to spin-orbit coupling, such as heavier halogen atoms, e.g., heavier halogen atoms including bromine atoms, iodine atoms, and astatine. Thus, in some embodiments, the introduction of one or more halogen atoms in the dyes and / or compounds of the invention can be used alone or in combination with a metal that gives limited emission by itself, thereby providing rapid and essentially quantitative relaxation to the ground state. Such metals include many of the metals in the periodic table. Methods for metallating tetrapyrroles are well known (Buchler, J.W. Static coordination chemistry of metalloporphyrins. In Porphyrins and Metalloporphyrins; Smith, K.M. (Ed.), 1975, Elsevier Scientific Publishing Co.: Amsterdam, pp157 - 231; Sanders, J.K.M., et al., Axial coordination chemistry of metalloporphyrins. In The Porphyrin Handbook; Kadish, K.M.; Smith, K.M.; Guilard, R. (Eds.), Vol. 3, 2000, Academic Press: San Diego, pp1 - 48). Since it is well known that heavy atoms attached to arenes result in rapid relaxation of the excited state, arenes substituted with a wide variety of heavy atoms are excellent candidates for the use of PAI according to the method of the invention.In some embodiments, the compounds of the invention include a tetrapyrrole (e.g., a tetrapyrrole having a heavy atom substituent at the periphery of the macrocyclic compound and / or a metal chelated at the center that does not give emission). Such tetrapyrroles (e.g., chlorin or bacteriochlorin) can provide many possible narrowband absorptions across the red and NIR spectral regions.

[0144] While various mechanisms are described by which the excited state of the compound can return rapidly and essentially quantitatively to the ground state, the invention is not limited thereto and other mechanisms known in the art can be used. For example, such mechanisms can arise from: (1) a high rate of internal conversion relative to the rates of radiative decay and intersystem crossing; (2) a high rate of intersystem crossing relative to the high rates of radiative decay and internal conversion, followed by an immediate and non-radiative decay from the excited multiplet state to the ground state; and / or, (3) a high rate of charge transfer relative to all other rates for the depopulation of the excited state, followed by charge recombination that quantitatively yields the ground state. Another example is to sterically distort the macrocyclic compound from its essential planarity. Other mechanisms are known to those skilled in the art. Regardless of the mechanism, established methods known to those skilled in the art can be used to create tetrapyrroles that exhibit an excited state with a very short lifetime and an essentially quantitative relaxation to the ground state. The rapid and nearly quantitative relaxation to the ground state can provide what is herein called a "non-emissive" molecular entity that can be used in PAI. Another example is to sterically distort the macrocyclic compound from its essential planarity. Other mechanisms are known to those skilled in the art. Regardless of the mechanism, established methods known to those skilled in the art are used to create tetrapyrroles that exhibit an excited state with a very short lifetime and an essentially quantitative relaxation to the ground state. The rapid and nearly quantitative relaxation to the ground state can provide what is herein called a "non-emissive" molecular entity that can be used in PAI.

[0145] The compounds of the present invention may optionally package a metallotetrapyrrole for use in PAIs. The metallotetrapyrrole may have a bioconjugable group that can be used to attach the metallotetrapyrrole to the polymers described herein to prepare the compounds of the present invention. Thus, the compounds of the present invention may include a single metallotetrapyrrole. In some embodiments, the compounds of the present invention may optionally maintain the inherent spectral characteristics of the dye (e.g., absorption spectrum, fluorescence spectrum, fluorescence quantum yield, etc.) without modification due to interactions with external entities, such as other dyes and / or biological materials (e.g., cellular components, proteins, etc.), by packaging the dye within a portion of the compound (e.g., within a polymer portion). The inclusion of a single dye (e.g., Fe(III) tetrapyrrole) within the compounds of the present invention may preserve the inherent absorption spectrum of the dye.

[0146] According to some embodiments, the dye present in the compound of the present invention may be a non-luminescent molecular entity (e.g., a non-fluorescent and / or non-phosphorescent molecular entity), where optionally the compound is used in a PAI. The dye may have a rapid optical to acoustic conversion. In some embodiments, the dye is a non-luminescent molecular entity and has a short excited state lifetime, where optionally the excited state lifetime is in the sub-picosecond range. Upon irradiation, the excited state may quickly return to the ground state and release heat. The heat generates a "sound wave" that can be detected by a microphone. The structure of the compound of the present invention may protect the dye from the physiological environment and / or be suitable for use in a method of performing a PAI.

[0147] In some embodiments, the compounds of the present invention provide a means for packaging hydrophobic chromophores, which can allow high solubility in water to be achieved, and / or a means for preventing the chromophores from aggregating, which can alter the appearance of the absorption band, including wavelength position, molar extinction coefficient, and band width.

[0148] The present invention will be described in more detail in the following non-limiting examples.

[0149] Example

[0150] Example 1 - Single Polymer Capsule Encapsulation of a Single Hydrophobic Chromophore Studies of random copolymers with pendant - pegylated chromophores and polymerized micelles containing hydrophobic fluorophores were conducted. Finally, a design involving a heterotelechelic, amphiphilic, random copolymer derived via living radical polymerization from two acrylate monomers (a hydrophilic (pendant PEG - 6) monomer and a hydrophobic (dodecyl) monomer in a 3:1 ratio) (RuCp*Cl(PPh3)2, 4 - (dimethylamino)-1 - butanol and an acetal - substituted initiator, in ethanol, at 40 °C, via) was identified. Hydrolysis of the acetal and subsequent reaction with hydrophobic chlorin - hydrazide gave a polymer with a single chlorin - hydrazone (i.e., a foldamer or single - chain nanoparticle, abbreviated as scNp). Testing of the chlorin - polymer in aqueous solution revealed sharp absorption / fluorescence bands and an undiminished fluorescence quantum yield compared to chlorin in toluene. This approach separates the chromophore selection and aqueous solubilization strategies into individual realms, and the latter implementation is currently very straightforward.

[0151] F1 to F3, which are amphiphilic copolymers labeled with three hydrophobic dyes with self-folding properties, were synthesized and spectroscopically characterized. The conformational features of the hydrophobic dyes and the polymer backbone are shown in Scheme 1 below. The amphiphilic copolymer is composed of a hydrophilic segment (PEG segment) and a hydrophobic segment (dodecyl segment) in a ratio of 3:1, and the molecular weight is about 120 kDa. As a random block copolymer, the copolymer in water self-folds to form a hydrophobic core, encapsulating the hydrophobic dye in it, thereby protecting the dye from aggregation. Three hydrophobic dyes (i.e., BODIPY, chlorin, and phthalocyanine, which differ in molecular size and absorption wavelength (540, 640, and 700 nm, respectively)) were loaded onto the same polymer backbone and subjected to spectroscopic measurements. Without wishing to be bound by any particular theory, the distinct fluorescence properties resulting from the dye-loaded copolymer in water suggest that the effectiveness of dye encapsulation can depend on the molecular size of the dye and the length of the polymer backbone.

[0152]

Chemical Structure

[0153] Synthesis of hydrophobic fluorophores. Generally, the dye-hydrazide used here for dye conjugation is prepared from the corresponding carboxylic acid ester via amide formation. Treatment of the activated carboxyl species BODIPY-NHS ester 1 with hydrazine hydrate gave the desired BODIPY-hydrazide D1 in 40% yield (Scheme 2 below).

[0154]

Chemical Structure

[0155] Iodochlorin 2 was quantitatively converted to methyl ester 3 via carbonyl insertion in the presence of Pd(PPh3)4, methanol, and carbon monoxide (Scheme 3 below). Next, methyl ester 3 was treated with hydrazine hydrate under reflux conditions to produce the desired chlorin-hydrazide D2 in 83% yield. It was noted that the reaction needed to be carried out at a concentration of less than 50 mM because more concentrated solutions resulted in the reduction of D2 to the corresponding bacteriochlorin.

[0156]

Chemical formula

[0157] The preparation of phthalocyanine-hydrazide D3 required more effort due to the solubility limitations of the macrocyclic compound. Ethynylphthalocyanine 4 was coupled with methyl 3-(4-bromophenyl)propionate in the presence of Pd(OAc)2 / P(o-tol)3 to give methyl ester 5 in 13% yield (Scheme 4 below). Again, the low solubility of the macrocyclic compound in the reaction system accounted for the low yield of the Sonogashira coupling reaction. Next, methyl ester 5 was treated with hydrazine in a mixture of toluene and methanol to produce the desired hydrazide D3.

[0158]

Chemical formula

[0159] Synthesis of the copolymer. The living radical polymerization of the monomers PEGA and LA was carried out with the initiator 6, reported in the presence of RuCp*Cl(PPh3)2 and 4-dimethylaminobutanol, at a ratio of 3 to 1 (Scheme 5 below). The resulting copolymer 7 is heterotelechelic with an acetal at one end and a bromine atom at the other end. The two functional groups were each derivatized for the introduction of a handle capable of dye binding and bioconjugation reactions. The bromine atom in 7 was replaced with mercaptoacetic acid to give a carboxyl group at the end of the copolymer open to bioconjugation reactions. Hydrolysis of the acetal end under acidic conditions resulted in the formyl copolymer 8. This copolymer 8 functioned as a platform for dye conjugation and generated the copolymers F1 - F3 loaded with the desired dyes via treatment with hydrazides D1 - D3, respectively.

[0160]

Chemical Structure

[0161] SEC analysis. Taking F2 as an example, analytical SEC was used to monitor the process of the dye conjugation reaction. The SEC trace shown in Figure 2 indicates that the size increases in response to the binding of chlorin onto the copolymer. Also, the molecular weight of the copolymer 7 was estimated to be 1.2×10 5 g / mol based on SEC analysis.

[0162] Measurement of absorption and emission spectra. Next, copolymers F1 - F3 loaded with the dyes of interest were subjected to an investigation of their spectroscopic properties in both organic solvents and aqueous solutions. The spectra are shown in Figure 3. For both F1 (loaded with BODIPY, Figure 3, panel A) and F2 (loaded with chlorin, Figure 3, panel B), the absorption spectra of the samples in aqueous solution at μM concentrations are comparable to the absorption spectra of the organic solvents. The binding to the 120 kDa amphiphilic copolymer dramatically enhances the water solubility of BODIPY D1 and chlorin D2 without strongly perturbing the spectroscopic properties. The emission bands for F1 and F2 in water remain the same as those measured in organic solvents, indicating minimal dye - dye interactions for the aqueous solutions of F1 and F2 at μM concentrations. Nevertheless, copolymer F3 bearing phthalocyanine, with a maximum molecular size of the dye, shows an absorption spectrum in water that is completely different from that in toluene (Figure 3, panel C) and has completely quenched fluorescence. This negative result could be due to an inappropriate size of the copolymer backbone. Larger polymers may be required to encapsulate large hydrophobic chromophores such as phthalocyanine D3.

[0163] Fluorescence quantum yield. The values of the fluorescence quantum yield were also measured for F1 - F3 in water at room temperature. The data are summarized in Table 5 together with other spectroscopic data. Taking the chlorin - conjugated copolymer F2 as an example, the dye - polymer conjugate shows a fluorescence quantum yield of 0.18 at μM concentration (item 6), which is similar to the value from the CH2Cl2 solution of the dye D2 alone (0.19, item 4). Similar results were observed for the BODIPY - labeled copolymer F1 (Φf = 0.058, item 3) and the BODIPY dye D1 (0.065, item 1). These comparisons indicate that there is no dye - dye quenching due to aggregation for F1 and F2 in μM aqueous solutions. The results show amphiphilic copolymers as a successful platform for encapsulating hydrophobic chromophores in water when the polymer chain length is appropriate. However, as described above, the copolymer labeled with phthalocyanine has completely quenched fluorescence. Longer polymer chains may be more effective for encapsulating larger chromophores such as D3. Also, smaller phthalocyanine skeletons (e.g., having methyl instead of heptyl as a peripheral group) can be successfully encapsulated with copolymers of the current length.

[0164]

Table 5

[0165] Experimental section

[0166] General methods. All commercially obtained chemical substances were used as received, unless otherwise specified. Reagent-grade solvents (CH2Cl2, THF, methanol) and HPLC-grade water were used as received. NMR data were measured in CDCl3 solution, unless otherwise specified. Compounds 1, 2, and 4 that are not commercially available were prepared according to the literature procedures. Analytical SEC experiments were performed using a Plgel 10000 Å SEC column, eluted at 35 °C at a flow rate of 1 mL / min using ACS-grade THF (stabilized with 400 ppm BHT). Samples were detected using an Agilent 1260 Infinity refractive index detector. Absorption spectra were measured at room temperature using an Agilent 8453 and a Shimadzu UV1800 instrument with diluted (μM) solutions of the compounds and solvent blanks in UV-transparent (e.g., quartz) cuvettes.

[0167] 2-[6-(N-Aminocarbamoyl)hex-1-yn-1-yl]-8-mesityl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (D1). A solution of 1 (9.0 mg) in THF (500 μL) was treated with hydrazine hydrate (5.3 μL) at room temperature for 30 min. The solution was then concentrated and subjected to chromatography (silica gel, CH3OH / acetic acid = 9:1) to give a red solid (3.0 mg, 39%): 1 H NMR (DMSO-d6, 300 MHz) δ 8.85 (br, 1H), 7.99 (s, 1H), 7.94 (s, 1H), 6.95 (s, 2H), 6.76 (d, J = 4.2 Hz, 1H), 6.72 (s, 1H), 6.53 (d, J = 4.2 Hz, 1H), 2.45~2.47 (m, 2H), 2.36 (s, 3H), 2.17~2.20 (m, 2H), 2.09 (s, 6H), 1.58~1.42 (m, 4H); MALDI-MS observed 449.1 [(M + H) + , 429.2 [(M - F) + , calculated 448.2 (M = C 25 H 27 BF2N4O).

[0168] 10-Mesityl-5-(4-methoxycarbonyl)phenyl-18,18-dimethylcorrole (3). Toluene and methanol were degassed by bubbling with argon for 1 h. (20 mg, 0.030 mmol, 1.0 equiv) and Pd(PPh3)4 (3.5 mg, 3.0 μmol, 0.10 equiv) were placed in a conical vial equipped with a rubber septum and then evacuated under high vacuum. Next, the vial was refilled with argon. This evacuation-purge process was repeated three times. Degassed toluene (0.50 mL) and methanol (0.50 mL) and triethylamine (21 μL, 0.15 mmol, 5.0 equiv) were added to the vial under argon. The solution was degassed again by three freeze-pump-thaw cycles. The vial was evacuated under high vacuum at 77 K and then refilled with carbon monoxide. A balloon filled with CO was also connected to the vial to provide an excess pressure. The solution was stirred at 65 °C for 23 h, concentrated, and subjected to chromatography (silica gel, hexane / CH2Cl2 = 1:1) to give a green solid (18 mg, 100%): TLC (silica, hexane / CH2Cl2 = 1:1) R f = 0.28; 1 H NMR (300 MHz) δ 8.92 (s, 1H), 8.87 (s, 1H), 8.82 (d, J = 4.8 Hz, 1H), 8.73 (d, J = 4.7 Hz, 1H), 8.69 (d, J = 4.7 Hz, 1H), 8.61 (d, J = 4.7 Hz, 1H), 8.38 (d, J = 8.1 Hz, 2H), 8.37 (s, 1H), 8.36 (s, 1H), 8.22 (d, J = 8.3 Hz, 2H), 7.22 (s, 2H), 4.57 (s, 2H), 4.08 (s, 3H), 2.58 (s, 3H), 2.03 (s, 6H), 1.84 (s, 6H), -1.87 (br, s, 2H); 1313C NMR (100 MHz) δ 175.2, 167.6, 163.6, 152.4, 151.5, 147.2, 140.9, 140.4, 139.2, 138.3, 137.7, 134.7, 134.4, 134.1, 132.1, 131.1, 129.5, 128.1, 128.0, 127.8, 123.7, 123.6, 120.59, 120.57, 96.81, 94.99, 52.49, 51.86, 46.63, 31.31, 21.57, 21.45; ESI-MS observed value 592.2851 [(M+H] + , calculated value 592.2838 (M = C 39 H 36 N4O2); Literature value (CH2Cl2) 415, 509, 533, 590, 641 nm.

[0169] 5-[4-(N - Aminocarbamoyl)phenyl]-10 - mesityl - 18,18 - dimethylchlorin (D2). A solution of chlorin 3 (44 mg, 75 μmol, 1.0 equiv) in THF (1.0 mL) was treated with methanol (1.0 mL) and hydrazine hydrate (0.21 mL, 3.8 mmol, 50 equiv) at 50 °C for 24 h. [Note: If the concentration is above 50 mM, reduction of the corresponding bacteriochlorin - hydrazine of chlorin - hydrazine will occur. The bacteriochlorin can be oxidized back to the desired chlorin by treating with DDQ (1.0 equiv) in CH2Cl2 at room temperature for 30 min.] Next, the solution was diluted with ethyl acetate, washed with water, dried over sodium sulfate, concentrated, and subjected to chromatography (silica gel, hexane / EtOAc = 1:2 to CH2Cl2 / CH3OH = 9:1) to give a green solid (37 mg, 84%): 11H NMR (400 MHz) δ 8.96 (s, 1H), 8.88 (s, 1H), 8.76 (d, J = 4.5 Hz, 1H), 8.75 (d, J = 4.5 Hz, 1H), 8.62 (d, J = 4.7 Hz, 1H), 8.56 (d, J = 4.7 Hz, 1H), 8.44 (d, J = 8.1 Hz, 2H), 8.39 (s, 1H), 8.38 (s, 1H), 8.30 (d, J = 8.0 Hz, 2H), 7.68 - 7.64 (m, 2H), 5.02 (br, 2H), 4.62 (s, 2H), 2.60 (s, 3H), 2.06 (s, 6H), 1.85 (s, 6H), -1.85 (br, s, 2H); 13 13C NMR (100 MHz) δ 165.7, 164.8, 163.5, 153.44, 153.38, 144.3, 140.8, 139.0, 138.0, 134.3, 132.1, 132.0, 128.9, 128.6, 128.5, 127.7, 126.8, 123.7, 88.75, 82.21, 53.77, 42.04, 31.14, 30.29, 29.65, 21.27, 18.40, 17.37, 12.06; MALDI-MS observed value 593.1 [(M + H) + , calculated value 592.3 (M = C 38 H 36 N6O).

[0170] 2-[4-(2 - Methoxy - 2 - oxoethyl)phenyl]ethynyl - 9,10,16,17,23,24 - hexakisheptylphthalocyanine (5). According to the standard Sonogashira coupling reaction procedure, a solution of 4 (20 mg, 18 μmol), methyl 3-(4 - bromophenyl)propionate (4.8 mg, 20 μmol), Pd(OAc)2 (1.1 mg, 13 μmol) and P(o - tol)3 (5.5 mg, 18 μmol) in degassed toluene (6.0 mL) was degassed by three freeze - pump - thaw cycles. The mixture was stirred at 60 °C for 18 h. The resulting reaction mixture was concentrated and subjected to column chromatography by three column strategies [(1) silica, CH2Cl2, (2) SEC, toluene, (3) silica, CH2Cl2] to give a green solid (3.0 mg, 13%). MALDI - MS: observed value 1289.4 [(M + H) +, Calculated value 1288.9 (M = C 86 H 112 N8O2).

[0171] 2-[4-(N-Aminocarbamoyl)methylphenylethynyl]-9,10,16,17,23,24-hexaheptylphthalocyanine (D3). A solution of 5 (3.0 mg, 2.3 μmol) in toluene (140 μL) was treated with 6.5 μL of hydrazine hydrate (55 wt%) and methanol (10 μL). The resulting mixture was stirred at 50 °C for 16 h, after which ethyl acetate and water were added to the mixture. The organic extract was washed with brine, dried (Na2SO4), and concentrated to give a green solid, which was used directly in the next step of the synthesis.

[0172] Example 2 A general approach for polymer preparation and derivatization according to some embodiments of the present invention is shown in Scheme 6 below.

[0173]

Chemical formula

[0174] In the approach shown in Scheme 6, the initiator is Q-X, where X is a halo halogen atom (e.g., Cl, Br, I) or a sulfonate (e.g., triflate), and Q can have a dye or a functional group and remains intact throughout the polymerization process.

[0175] In the case of further derivatization, the rich group required for dye binding is incorporated (in the Q unit) prior to polymerization and can be used directly. Alternatively, derivatization of Q in the synthetic polymer I after polymerization can give a modified Q (denoted as Q') in the polymer II for dye binding.

[0176] The definition of the bond to the biomolecule (ω-terminus of the polymer) is in some cases by the direct use of the X substituent in Polymer I. Alternatively, the X group can be substituted to give the functional group W in Polymer II for the binding of the biomolecule. Examples of W include azide, isocyanate, isothiocyanate, active ester (e.g., pentafluorophenyl ester, succinimide ester, 2,4-dinitrophenyl ester), maleimide, vinyl, mercapto, amino, and carboxylic acid. Derivatization at the ω-terminus in Polymer I is achieved by a single step or multiple steps (e.g., nucleophilic substitution and / or deprotection) to give the desired functional group W in Polymer II. For the prepolymerization method, the functional group is first attached within the initiator (Q unit of Q-X, Scheme 6) and remains as such throughout the polymerization process.

[0177] Some examples of Q and Q-X are shown in Scheme 7. As shown in Scheme 7, Q can include a hydroxy group 1,2 , a carboxy group 3 , an amino group 4 , a formyl group 4 , a vinyl group 5,6 , an epoxy group 7 , an anhydride 8 , a haloaryl group 7 , an ester group 3 or an oxazoline group 8 . A vinyl group or an allyl group can be attached via the initiator and can remain as such during polymerization without causing additional problems during crosslinking 1,5,6 . This can be achieved mainly by selecting an appropriate ligand in the presence of a copper(I) catalyst. However, some functional groups commonly used in dye binding (e.g., azide group) or bioconjugation reactions cannot be introduced by the prepolymerization method (shown in Table 6).

[0178]

Chemical Structure

[0179] JPEG2025090738000018.jpg97170

[0180] It should be noted that the examples discussed in this specification describe the binding of the dye to the α-terminus of the polymer and the binding of the biomolecule to the ω-terminus of the polymer. However, the use of the two termini can be reversed if necessary, in which case the biomolecule is bound to the α-terminus of the polymer and the dye is bound to the ω-terminus of the polymer.

[0181] References JPEG2025090738000019.jpg147170

[0182] Example 3 - Exemplary Reactions An exemplary reaction for preparing a compound of the present invention including crosslinking is provided in Scheme 8 below.

[0183]

Chem.

[0184] An exemplary reaction for preparing a compound of the present invention including sulfonation and crosslinking is provided in Scheme 9 below.

[0185]

Chem.

[0186] Example 4 Exemplary approaches for polymer preparation and derivatization according to some embodiments of the present invention are shown in Scheme 10 below.

[0187]

Chem.

[0188] In the approach shown in Scheme 10, Z in the RAFT agent is aryl, alkyl or thioalkyl, and Q can have a functional group that remains as such during the polymerization process.

[0189] In the case of further derivatization, the functional groups required for attachment to a dye or biomolecule are incorporated (in the Q unit) prior to polymerization and can be used directly. Such functional groups are first introduced into the Q unit of the RAFT agent and can remain as such throughout the polymerization process. Alternatively, after polymerization, derivatization of Q in the synthetic polymer can provide a modified Q for attachment of a dye or biomolecule.

[0190] Some examples of Z and Q in the RAFT agent are shown in Chart 1 below. Examples of Z in the RAFT agent include, but are not limited to, phenyl (optionally substituted) and / or thioalkyl groups (including branched and / or unbranched C1-C25 thioalkyl groups).

[0191] Examples of Q in the RAFT agent include, but are not limited to, carboxylate, azide, hydroxy, N-succinimidyl, vinyl, phthalimide and / or biotinyl.

[0192]

Chemical Structure

[0193] Before binding a dye or a biomolecule at the terminal end of a polymer containing a thiocarbonylthio group, the thiol group can be liberated by cleaving the thiocarbonylthio group using methods known in the art. The liberated thiol group can either directly bind to the dye or biomolecule or can be further modified with a factor L-W to provide a capped thiol (e.g., thioether) having an appropriate functional group W for coupling with the dye or biomolecule. The factor L-W contains a thiol-reactive group L, which reacts with the liberated thiol group and also functions as a linker L' between the thiol and the functional group W in the capped product.

[0194] Some examples of L and W in L-W are shown in Chart 2 below. Examples of the L group in the L-W factor include, but are not limited to, substituted halides (e.g., substituted benzyl bromide and / or α-acid), substituted alkynes (e.g., substituted benzyl alkyne), substituted vinyl esters (e.g., α-vinyl ester) and / or substituted succinimides (e.g., ethylamine succinimide, ethanol succinimide).

[0195] Examples of the functional group W include, but are not limited to, carboxylic acids (e.g., -COOH, -CH2CH2COOH), amines (e.g., -NH2, -CH2CH2NH2, optionally having a protecting group: NHBoc, -CH2CH2NHBoc), aldehydes, alcohols (e.g., -CH2CH2OH) and / or alkylated alcohols (e.g., -OCH2CH2OH, -OCH2CH2NHBoc, -OCH2CH2N3, -OCH2C ≡ CH, -OCH2CH=CH2).

[0196] Derivatization of the liberated thiol group can be achieved via a single step or multiple steps (e.g., nucleophilic substitution and / or deprotection) to give the desired functional group W.

[0197]

Chemical formula

[0198] Further examples of RAFT polymerization are shown in Scheme 11. The hydrophobic monomer dodecyl acrylate (LA) is polymerized in the presence of the hydrophilic monomer 2-acrylamido-2-methylpropanesulfonic acid as the sodium salt (AMPS) and PEG acrylate (PEGA) to produce a polymer. In some embodiments, one or more functional groups are present (e.g., pre-introduced) in the RAFT agent prior to polymerization. Examples of such one or more functional groups are shown in Scheme 11. After polymerization, the one or more pre-introduced functional groups are located at one terminal end of the polymer and can be used for coupling to a biomolecule or a dye.

[0199]

Chemical formula

[0200] References JPEG2025090738000029.jpg219170

[0201] Example 5 Synthesis of Amphiphilic Random Copolymers via Reversible Addition-Fragmentation chain Transfer (RAFT) A model study of the synthesis of sulfonated amphiphilic random copolymers is shown in Scheme 12 below. Three monomers were used, one of which was hydrophobic (dodecyl acrylate (LA)), and two of which were hydrophilic as the sodium salts (AMPS) (2-acrylamido-2-methylpropanesulfonic acid and PEG acrylate (PEGA)). AMPS can be prepared by basifying commercially available 2-acrylamido-2-methylpropanesulfonic acid with sodium hydroxide and / or basifying the sodium salt of commercially available 2-acrylamido-2-methylpropanesulfonic acid having a small amount of free acid present as a minor contaminant in the commercially available AMPS material. As was available in the lab, RAFT chain transfer agent 1 was used. Polymerizations with various monomer ratios were carried out in DMF (80 °C) containing AIBN as the radical initiator and mesitylene as the internal standard. After polymerization, the crude product was poured into a large excess of ethyl ether to precipitate the polymer. The precipitate was then dialyzed against water to give the purified polymer.

[0202] [Chemical Formula]

[0203] Dynamic light scattering (DLS) size analysis of amphiphilic polymers. Each polymer was dissolved in a 1.0 M aqueous NaCl solution and passed through a 200 μm membrane filter. The filtrate was examined by DLS to determine the size of the nanoparticles. The DLS size data of various polymers are summarized in Table 7. According to the data, for polymers without PEG groups, the best results were obtained using sulfonate groups and lauryl groups in a ratio of 6:1, which gave 65% unimers in aqueous solution. As the PEG group was introduced, the percentage of unimers increased when the ratio of the PEG group to the sulfonic acid group decreased from 1:1 to 1:5. At a ratio of AMPS:PEGA:LA = 5:1:1, the unimer appeared to be the major species in the aqueous solution.

[0204]

Table 7

[0205] Synthesis of polymer-chromophore conjugates via RAFT polymerization. The living radical polymerization of the monomers PEGA, LA, and AMPS was carried out in the presence of 2,2'-azobis(2-methylpropionitrile) (AIBN), a radical initiator, using 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid 2, a RAFT agent, in a ratio of 1:1:5 (i.e., hydrophilic / hydrophobic ratio = 6:1) (Scheme 13). The resulting polymer 3 is heterotelechelic, containing a carboxyl group at one end and a thiocarbonylthio group at the other end. Aminolysis of polymer 3 with ethanolamine cleaved the thiocarbonyl group and revealed a free thiol group. In situ coupling using bacteriochlorin D1 substituted with the hydrophobic maleimide of the latter (free thiol group) gave the desired polymer-chromophore conjugate F-2.

[0206]

Chem.

[0207] Dynamic light scattering (DLS) size analysis of the polymer-chromophore conjugate. The polymer-chromophore sample was dissolved in a 1.0 M aqueous NaCl solution and passed through a 200 μm membrane filter. The filtered solution was examined by DLS to determine the size of the nanoparticles. The DLS size data for the various polymers are summarized in Table 8. The polymer-chromophore sample F-2 showed a unimolecular type at various concentrations (Figure 4).

[0208]

Table 8

[0209] Measurement of the absorption and emission spectra and fluorescence quantum yield of F-2. The absorption and emission spectra of the target polymer-chromophore conjugate F-2 were measured at room temperature in both water and aqueous buffer (Figures 5 and 6). The spectroscopic data and fluorescence quantum yield data are summarized in Table 9 below.

[0210] The absorption and emission spectra of F-2 in aqueous solution are comparable to those of D1 in toluene, and the broadening and decrease of the Qy absorbance are minimized. The fluorescence yield of F-2 in the aqueous medium is 93% (in buffer) and 80% (in water) relative to the fluorescence yield of D1 in toluene. These data are consistent with a slight aggregation of the chromophores in the aqueous medium. The single chromophores are encapsulated in the amphiphilic polymer and maintain their intrinsic fluorescence upon immersion in the aqueous environment.

[0211]

Table 9

[0212] Example 6 Developing a novel method of molecular fluorescence or luminescence for chemical sensing using an organic chromophore as a recognition unit is of particular interest, especially in chemistry, biology, environmental science, clinical and medical fields (References 1 - 3). Detection is based on (1) a shift in the absorption or emission wavelength of the fluorophore, or (2) a change in the intensity of absorption or emission. Structural features that control the change in the wavelength or intensity of absorption or fluorescence include, but are not limited to, the twist of double bonds, changes in the conjugation pattern, "heavy" atoms, weak bonds, and opportunities for photoinduced electron transfer (PET) or electronic energy transfer (EET) (References 4 - 10). The advantages of such detection via optical signals are as follows: high sensitivity; "on - off" switchability; qualitative or quantitative analysis; detection by the naked eye, etc. (References 11 - 15).

[0213] Heavy metal ions have harmful effects on the environment and human health, and thus there is great concern among chemists, biologists, environmental scientists, and medical doctors (Reference 16). The demand for highly sensitive and selective fluorogenic sensors targeting toxic heavy metal ions is continuously increasing because the issue is important. In 1997, Czarnik and his colleagues reported the spiro lactam ring-opening that induces the fluorescence of rhodamine-B hydrazide for the detection of Cu(II) in aqueous solution (Reference 17). As shown in Scheme 14, the non-fluorescent rhodamine-hydrazide gives a conjugated and fluorescent rhodamine structure through ring-opening by hydrolysis catalyzed by metal cations. The opening of the ring depends on the nature of the cation. The cations tested in this study included Ag(I), Al(III), Ca(II), Cd(II), Co(II), Cr(III), Cu(II), Eu(III), Fe(III), Ga(III), Gd(III), Hg(II), In(III), K(I), Li(I), Mg(II), Mn(II), Na(I), Ni(II), Pb(II), Rb(I), Sn(IV), Sr(II), U(IV), Yb(III), Zn(II), Cu(II), and Hg(II). Among these, only Cu(II) and Hg(II) brought about significant changes in the absorption or fluorescence spectrum. The selective detection of Cu(II) was sensitive and quantitative for Cu(II) at a concentration of 10 -7 M and was highly sensitive and quantitative for Cu(II).

[0214] [Chemical formula]

[0215] Several rhodamine-hydrazide analogs have been synthesized and analyzed for the detection of metal ions such as Pb(II) (reference 18), Cd(II), Fe(III), Hg(II) (reference 19), and Sn(II) (reference 20). However, this application in aqueous solutions requires the incorporation of organic solvents such as acetonitrile and methanol due to the hydrophobic nature of rhodamine-hydrazide. In this regard, in order to study metal ion sensing in pure water without the addition of organic solvents, the inventors designed and synthesized pod-rhodamine.

[0216] Pod-rhodamine was synthesized by first preparing an amphiphilic random copolymer. The synthesis of the targeted sulfonated amphiphilic random copolymer is shown in Scheme 15.

[0217]

Chemical formula

[0218] The polymerization was carried out as described herein to give F-Ph, where the ratio of m:n:p is 1.0:1.0:5.0 based on the reaction stoichiometry and by both 1H NMR spectroscopy of the synthesized polymer. The size of the targeted amphiphilic random copolymer F-Ph was also measured using dynamic light scattering (DLS) spectroscopy in aqueous solutions at various concentrations of the polymer (Figure 7). The data indicate that the polymer exhibits exclusively unimolecular behavior in aqueous solution, with the size distribution reaching a peak at 10 nm and no detectable aggregation. 1 The dithioester of F-Ph was removed by reaction with hydrazine hydrate in DMF to give polymer F-SH containing free thiol end groups. The thiol groups of F-SH were further derivatized to F-CHO with formyl groups by reacting with p-bromomethylbenzaldehyde in DMF.

[0219] 1 ​The inspection of F-CHO by 1H NMR spectroscopy (in D2O) gave m, n, and p values of 22, 21, and 104, respectively. The m, n, and p values are obtained based on a single carboxaldehyde proton. Next, the data is consistent with the ratio of m:n:p of 1.0:1.0:5.0 predicted from the first monomer stoichiometry. 1 It should be noted that the calculated molecular weight of F-CHO given by the m, n, and p values from 1H NMR measurement is 39.6 kDa, which is compared with the estimated molecular weight of F-Ph of 41.4 kDa estimated from HPLC analysis (the two polymers have molecular weights that differ by only 2 Da in mass). This comparison is excellent for both HPLC measurement and NMR measurement.

[0220] Pod-rhodamine was prepared by the reaction of F-CHO with rhodamine-hydrazide I in N,N-dimethylformamide at 40 °C for 15 h. Subsequently, removal of the unreacted dye by dialysis gave the desired pod-rhodamine in 91% yield.

[0221] [Chemical formula]

[0222] Pod-rhodamine was subjected to test absorption and emission in water in the presence of various metal ions. In a vial, 1.0 mg of pod-rhodamine was treated with a solution of a metal salt in water (1.0 mL, 2 mM, 100 molar equivalents of pod-rhodamine). The final concentration of pod-rhodamine was 20 μM. The resulting solution was stirred at room temperature for 1 h, after which the solution was measured by absorption and fluorescence spectroscopy. In this study, the cations tested were as follows: Au(III), Al(III), Ce(III), Cd(II), Co(II), Cr(II), Cu(II), Fe(III), Ga(III), Hg(II), In(III), Mg(II), Mn(II), Ni(II), Pb(II), Yb(III), and Zn(II).

[0223] The absorption and emission spectra of various solutions are shown in Figure 8. For absorption analysis, Au(III), Cr(II), Cu(II), Fe(III), Hg(II) and In(III) showed changes in absorption. For fluorescence analysis, Au(III), Ga(III), Hg(II) and In(III) showed increased fluorescence intensity compared to the blank control. The loss of fluorescence in the Cu(II) and Fe(III) samples may be due to the heavy atom effect. Photographs of various reaction solutions were obtained with or without illumination. For Cr(II), a precipitate was observed during the reaction, so the absorption with Cr(II) was measured using the supernatant.

[0224] Fluorescence titrations were performed with Au(III) and Hg(II) using 10 μM pod-rhodamine and 0 - 1.0 μM cation (excited at 510 nm). Figure 9 shows the titration fluorescence spectra.

[0225] In summary, the important point of this work is that the rhodamine sensor remains active upon conjugation with the heterotelechelic polymer and can be used in pure water for the purpose of ion sensing. In contrast, the literature data indicate that the use of only the rhodamine sensor requires the use of a mixture of organic and aqueous media. Without wishing to be bound by a particular theory, this suggests that the polymer provides an organic solubilizing function for the conjugated rhodamine sensor.

[0226] References: JPEG2025090738000038.jpg204170

[0227] Example 7 Embodiments of the present invention relate to heterotelechelic polymers having a single bioconjugatable group and a single chromophore for use in aqueous solutions, which is counterintuitive. The counterintuitive nature stems from the widespread belief in the field, as shown in numerous papers over about 50 years, that in order to achieve an appropriate signal (e.g., brightness), the polymer or other structure needs to load as many chromophores as possible. One aspect of the inventors' strategy is to site-isolate the desired chromophore as a single cargo item at one end of each heterotelechelic polymer, achieve a high degree of unimolecular self-assembly in an aqueous solution, and utilize the other end of the polymer for bioconjugation reactions.

[0228] One approach for solubilizing hydrophobic fluorophores in aqueous solutions involves attachment to amphiphilic polymers, which self-assemble with the hydrophobic fluorophore immobilized within the hydrophobic interior. The single-polymer–single-cargo strategy was previously reported by the inventors' group 9 wherein amphiphilic polymers containing reactive end groups and pendant hydrophobic and hydrophilic groups were used to package hydrophobic fluorophores in aqueous solutions. One polymer that was specifically studied contained acrylate and acrylamide units in a backbone having pendant hydrophobic (dodecyl) groups, nonionic hydrophilic (PEG9) groups, and ionic hydrophilic (sulfonate-terminated) groups. The polymer was heterotelechelic with benzothioate and carboxylic acid end groups. The polymer, designated F-Ph, exhibited a molecular weight of approximately 40 kDa and contained lauryl, PEG9, and sulfonate as pendant groups in a ratio of 1:1:5. The construct dissolved freely in aqueous media. Studies showed that the polymer F-Ph formed unimers quantitatively in 1 M NaCl solution at room temperature, as evaluated by dynamic light scattering (DLS) spectroscopy. Attachment of the hydrophobic fluorophore (extracted from approximately 8 classes of fluorophores) was carried out at the end of the F-Ph polymer (i.e., one fluorophore per polymer). The resulting polymer–fluorophore retained the intrinsic brightness observed for the fluorophore alone in organic solvents.

[0229] Most antibodies or cells cannot survive in an aqueous solution containing such a high concentration of NaCl. The present inventors recently decided to examine F-Ph and fluorophore-containing derivatives of F-Ph in aqueous media of the type used in biology. In PBS buffer commonly used in biology, F-Ph contained 68% unimers (peak at about 13 nm) and 32% aggregated particles with dimensions greater than 100 nm (Figure 10). The PBS buffer contains 0.15 M NaCl and 0.01 M phosphate. These results suggest the importance of ionic strength in the self-assembly of amphiphilic polymers. h A high percentage of unimers (relative to aggregates) at low ionic strength is considered important for many biological applications, so the present inventors initiated studies to investigate many factors thought to affect the assembly process of unimers. The most interesting application for amphiphilic polymers is flow cytometry. The study required examination of F-Ph as a function of ionic strength, as well as preparation and examination of new polymers. The new polymers differed in the nature of the pendant groups, the ratio of pendant groups, molecular weight, and the presence or absence of a hydrophobic fluorophore. The laser in the DLS apparatus irradiates the sample at 632 nm, which eliminates the possibility of using chromophores that absorb in this region. The fluorophore selected for the studies described herein is perylene-monimide. The perylene-monimide (which does not absorb at 632 nm) is a large hydrophobic arene and is thus considered a viable surrogate for various tetrapyrrole macrocyclic molecules (e.g., chlorin and bacteriochlorin).

[0230]

[0231] ​1. Synthesis of polymers. A new series of amphiphilic polymers containing cyclododecyl groups and sulfonate pendant groups were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerization. The cyclododecyl groups were introduced via cyclododecyl acrylate. The sulfonate was introduced at the end of N-alkyl substituted acrylamide. Since cyclododecyl (CD) has previously been shown to have a much stronger tendency for intramolecular self-association than the lauryl group 2 , the lauryl in F-Ph was replaced by cyclododecyl. And PEG9 was also removed. Thus, the resulting polymers contained CD and sulfonate pendant groups, did not contain PEG groups, and retained benzothioate and carboxylic acid termini. For the studies described below, polymers were prepared that included different ratios of the two types of pendant groups and / or variations in the overall molecular weight. Treatment of such polymers with ethanolamine gave cleavage of the thiobenzoyl group and a free thiol at the terminus. The resulting free thiol reacted with perylene-monoimide maleimide (PMI-mal) to give the corresponding polymer fluorophore conjugate (Scheme 17).

[0232] [Chemical formula]

[0233] Depending on the use of various initial ratios of cyclododecyl acrylate (CDA, a hydrophobic monomer), sulfonate-terminated acrylamide (AMPS, a hydrophilic monomer / ionic monomer), and a chain-transfer reagent (CTR), seven P-S-CD polymers were prepared. Subsequent derivatization gave the corresponding polymer-PMI conjugates (Table 10). The molecular weight of each P-S-CD polymer was estimated from HPLC analysis. Here, the present inventors examined the percentage of unimers observed in response to changes in the following parameters: (1) the ionic strength of the solution, (2) the ratio of the two types of pendant groups, (3) the molecular weight of the polymer alone (lacking the fluorophore), and (4) the presence of the fluorophore - in other words, the effect of the presence and absence of the fluorophore attached to the polymer.

[0234]

Table 10

[0235] 2. Study of the effects in unimer assembly. A. Ionic strength. DLS studies of the previous F-Ph and the new polymer P-S5-CD1 (28 kDa) (Item 5, Table 10) were carried out in the following six media: pure water, PBS buffer, and aqueous solutions with various concentrations of NaCl (0.25 M, 0.50 M, 0.75 M, and 1.0 M). The raw DLS data are shown in Figure 11. In pure water, F-Ph showed three different sizes according to DLS examination. The peak at about 1 nm could be due to the unfolded polymer, but a part of the polymer could aggregate to give a size distribution reaching a peak at about 400 nm. The ionic strength of the PBS buffer also did not result in the complete unimer behavior of F-Ph (see Figure 10). However, in the 0.25 M NaCl solution and solutions with higher ionic strength, F-Ph provided unimers quantitatively (Figure 11). The DLS data of Figure 11 regarding the percentage of unimers were plotted graphically in Figure 12.

[0236] Next, the present inventors turned their attention to a similar study of the new polymer P-S5-CD1(28 kDa). The raw DLS results in pure water and in aqueous solutions of various NaCl concentrations are shown in FIG. 13, while the results in PBS buffer are shown in FIG. 14. These results are plotted in a graph in FIG. 15. The graph shows that the polymer P-S5-CD1(28 kDa) self-organizes to produce unimers in a quantitative manner in an aqueous medium containing 0.50 M or more of NaCl. Apparently, the polymer tends to aggregate into particles of uniform size in an aqueous medium of high ionic strength.

[0237] 2.B. Effect of pendants and presence of hydrophobic fluorophores. Samples of F-Ph or F-PMI were dissolved in PBS buffer and examined by DLS spectroscopy. The percentage of unimers for the F-PMI conjugate was higher than the percentage of F-Ph in PBS buffer (FIG. 16). Thus, the presence of the hydrophobic fluorophore cargo appears to drive the assembly of the polymer in PBS buffer.

[0238] A similar comparison was made for P-S5-CD1(28 kDa) and P-S5-CD1(28 kDa)-PMI (FIG. 17). The results show that P-S5-CD1(28 kDa)-PMI was quantitatively assembled into unimer particles in PBS buffer, whereas the polymer lacking the PMI unit was not quantitatively assembled into unimers. Again, the presence of the hydrophobic cargo appears to drive the assembly of the overall structure into the unimer architecture.

[0239] 2.C. Molecular weight. DLS of a series of cyclododecyl / sulfonate polymer-PMI conjugates containing the same ratio of pendants (5:1) but having different degrees of polymerization was measured. The molecular weight directly reflecting the degree of polymerization when a constant ratio of the reacting monomers was given was in the range of 10 - 35 kDa. The raw DLS data for the polymer-PMI conjugates in PBS buffer are shown in Figure 18. For P-S5-CD1(28 kDa)-PMI and P-S5-CD1(35 kDa)-PMI, the percentage of unimers was 100%, but the constructs derived from the low molecular weight polymers, namely P-S5-CD1(10 kDa)-PMI and P-S5-CD1(18 kDa)-PMI, were not 100%. Based on this dataset, the 28 kDa polymer is of sufficient size to provide 100% unimer formation for constructs containing PMI (however, as described above, PMI is not completely absent).

[0240] 2.D. Ratio of pendants. The effect of the ratio of pendants was also studied. Three polymer-PMI conjugates were prepared, where the polymers had approximately the same molecular weight (about 28 kDa) but showed different ratios of pendants. The ratios of the pendant groups were 4:1, 5:1, and 6:1 for sulfonate to cyclododecyl. The raw DLS data for each construct in PBS are shown in Figure 19. Each construct was quantitatively assembled into unimer particles and had similar size and polydispersity index (PDI) in each case. Therefore, a slight change in the ratio of the pendant groups does not result in aggregation in PBS buffer, at least for this study of polymers of about 28 kDa.

[0241] The results of the above experiments are summarized in Table 11 below. For the seven P-S-CD polymers prepared by the present inventors, all the polymers (lacking the hydrophobic fluorophore) formed unimers quantitatively in 1 M NaCl solution but did not form in PBS buffer. However, the attachment to the hydrophobic fluorophore enabled polymers with a molecular weight sufficient to aggregate unimolecularly, regardless of the ratio of the pendants.

[0242]

Table 11

[0243] 3. Experimental section General method for preparing polymers. A solution flask of CDA (715 mg, 3.0 mmol), AMPS (12 mmol - 18 mmol), chain transfer reagent (0.06 mmol - 0.27 mmol) and mesitylene (360 mg, 3.0 mmol) in DMF (27 mL) in a Schlenk tube was degassed by three freeze-pump-thaw cycles. Next, AIBN (4.9 mg, 0.030 mmol) was added to the flask. The resulting mixture was stirred at 80 °C for 24 h. Mesitylene was 1 included as an internal standard for evaluating the conversion of each monomer via 1H NMR spectroscopy. The mixture was cooled to room temperature and then poured into 200 mL of diethyl ether. The precipitate was washed three times with diethyl ether. Next, the crude polymer was dissolved in deionized water and placed inside a dialysis membrane tube equipped with two closures through which compounds with a molecular weight less than 3.5 kDa could pass. The solution was dialyzed against deionized water and the reservoir volume was exchanged with fresh deionized water four times over a period of about 24 h. The dialyzed solution was lyophilized under high vacuum to give a pale pink solid (1.2 - 1.4 g).

[0244] General method for preparing polymer-PMI conjugate. Taking F-S5-CD1(28kDa)-PMI as a representative example, a solution of PMI-maleimide (1.0 mg, 0.97 μmol) and F-S5-CD1(28kDa) (18.1 mg, 0.65 μmol) in DMF (1 mL) was treated with ethanolamine (1 drop). The mixture was stirred at 35 °C for 16 hours. Next, the mixture was transferred into a dialysis membrane tube equipped with two closures. Then, the solution was dialyzed in DMF to remove the excess fluorophore. The dialysis reservoir volume was exchanged with fresh DMF four times over about 24 hours. The resulting solution was dried under high vacuum at 30 °C. The resulting solid was dissolved in deionized water and then lyophilized to give a pink solid (17 mg).

[0245] 4. Discussion A set of amphiphilic polymers was prepared with various ratios of pendant groups and various degrees of polymerization to investigate factors related to the extent of unimers formation in PBS buffer. PBS buffer is a widely accepted medium for biological assays and many clinical studies. Substitution of lauryl and PEG groups (previously used 9) with cyclododecyl groups was readily achieved. Comparison of polymers containing lauryl and PEG with those containing cyclododecyl showed that the latter promoted intramolecular aggregation. One of the surprising results was that the presence of a single hydrophobic fluorophore induced self-folding of the amphiphilic polymer in aqueous solution. A polymer with a high molecular weight (about 28 kDa) was sufficient to completely package the hydrophobic fluorophore in PBS buffer; that is, unimers were quantitatively formed. The ratio of sulfonate groups to cyclododecyl pendant groups could be varied in the range of 4 - 6:1 as long as the molecular weight was above 28 kDa, and accordingly unimers were quantitatively formed in PBS buffer. Without wishing to be bound by any particular theory, the interaction of three factors (molecular weight, ratio of pendant groups, and presence of hydrophobic cargo) is thought to affect the aggregation of the polymer, which was clearly evident in PBS buffer but perhaps unclear under high ionic strength conditions of 1 M NaCl, where the medium has been widely used by others and by the inventors (although not nearly universally) in studies of other foldamer formation. In summary, the hetero-telechelic P-S-CD polymers described herein provide a simple and accessible platform for use with hydrophobic fluorophores in potential applications in biochemistry.

[0246] 5. References JPEG2025090738000042.jpg221170

[0247] The foregoing are examples of the present invention and should not be construed as limiting thereof. The present invention is defined by the following claims, and equivalents of the claims are included therein. All publications, patent applications, patents, patent publications, and other references cited herein are hereby incorporated by reference in their entirety for the teachings relevant to the sentences and / or paragraphs in which the references are presented. The first aspect of the present invention is as follows. [Item A1] A compound having a structure represented by the following A - B - C, or C - A - B Here, A is a dye (e.g., a fluorophore (e.g., a fluorescent dye) or a non - fluorescent molecular entity), where optionally, the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da. B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, where optionally, the polymer has a molecular weight in the range from about 1,000 Da, 5,000 Da or 10,000 Da to about 175,000 Da, and C is optional, where C contains a bioconjugate group, and Here, the compound further comprises a recognition motif, where optionally, the recognition motif is bound to the dye. [Item A2] The compound according to Item A1, wherein the dye (e.g., a tetrapyrrole macrocyclic molecule) is covalently bound to a part (e.g., a terminal) of the polymer. [Item A3] The compound according to Item A1 or 2, wherein the one or more hydrophobic units and the one or more hydrophilic units are randomly distributed in the polymer. [Item A4] The one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10, where optionally, the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:6 (hydrophobic unit: hydrophilic unit), the compound according to any one of items A1 to 3. [Item A5] The compound according to any one of items A1 to 4, wherein the compound has conformational flexibility. [Item A6] The compound according to any one of items A1 to 5, wherein the compound self-folds in an aqueous solution (for example, an aqueous solution containing 10 mM NaH2PO4 and 150 mM NaCl and having a pH of 7.35), and optionally self-folds into a unimolecular micelle structure. [Item A7] The compound according to any one of items A1 to 6, wherein the polymer is an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer. [Item A8] The compound according to any one of items A1 to 7, wherein the compound is cross-linked, where optionally, when the compound has a folded structure, the compound is cross-linked. [Item A9] The compound according to any one of items A1 to 8, wherein the compound folds to provide particles, where optionally, the particles have a diameter in the range from about 1 nm or about 3 nm to about 30 nm or about 40 nm. [Item A10] The compound according to any one of items A1 to 9, wherein at least a portion of the one or more hydrophobic units is present in the core of the particles, and / or at least a portion of the one or more hydrophilic units is present at the periphery (for example, the shell) of the particles. [Item A11] The compound according to any one of items A1 to 10, wherein when the compound is in a folded structure, the dye is encapsulated by a part of the compound (for example, a part of the polymer). [Item A12] The compound according to any one of items A1 to 11, wherein the polymer is a telechelic polymer or a heterotelechelic polymer. [Item A13] The compound according to any one of items A1 to 12, wherein the dye (for example, a tetrapyrrole macrocyclic molecule) is hydrophobic. [Item A14] The compound according to any one of items A1 to 13, wherein the compound is water-soluble, and optionally, the compound has solubility in water in the range of about 1 mg / mL to about 10 mg / mL at room temperature. [Item A15] At least one of the one or more hydrophobic units and / or the one or more hydrophilic units has a pendant functional group, and optionally, the pendant functional group is a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group (for example, a pentafluorophenyl ester, a succinimide ester or a fluorophenyl ester), an azide group, a maleimide group, an isocyanate group, an isothiocyanate group, a phosphono group, a sulfono group, an ammonio group or a phosphatidylcholine group, and / or the pendant functional group is a hydrophilic group having a terminal cation (for example, ammonium), an anion (for example, sulfonate, phosphate, carboxylate or phosphonate) or an amphoteric group (for example, a choline-like) group, and optionally, a poly(ethylene glycol) residue. The compound according to any one of items A1 to 14. [Item A16] The compound according to any one of items A1 to 15, wherein at least one of the one or more hydrophobic units contains an alkyl pendant group (for example, dodecyl), and / or at least one of the one or more hydrophilic units contains a glycol pendant group (for example, poly(ethylene glycol)). [Item A17] The compound according to any one of items A1 to 16, wherein the polymer is bonded to one dye and optionally to one bioconjugate group. [Item A18] The compound according to any one of items A1 to 17, wherein the hydrophobic unit has a structure represented by the following formula III [Chemical formula] Herein R is a hydrogen atom or C1-C8 alkyl (for example, C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 is absent or is -O-, -NH- or -CH2-, R' is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl or C3-C20 cycloalkyl, R 2 is a hydrogen atom or a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group (for example, pentafluorophenyl ester, succinimide ester, fluorophenyl ester or 2,4-dinitrophenyl ester), an azide group, a maleimide group, an isocyanate group or an isothiocyanate group, and p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000 or 100,000. [Item A19] R in the hydrophobic unit 2 is a hydrogen atom or a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group, the compound according to claim 18. [Item A20] R in the hydrophobic unit 2 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group or a maleimide group, the compound according to item A18. [Item A21] The compound according to any one of items A1 to 20, wherein the hydrophilic unit has a structure represented by the following formula IV [Chemical formula] Herein R is a hydrogen atom or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 is absent or is -O-, -NH- or -CH2-, R 3 is -(CH2CH2R 5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl -O- and -C1-C6 alkyl -SO3- or a salt thereof, selected from the group consisting of, wherein R 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000, R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group (e.g., dihydroxyphosphoryl), a sulfono group (e.g., hydroxysulfonyl), a phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester or 2,4-dinitrophenyl ester), and p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000 or 100,000. [Item A22] The compound according to item A21, wherein R in the hydrophilic unit 4 is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group. [Item A23] The compound according to item A21, wherein R in the hydrophilic unit 4 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group or a maleimide group. [Item A24] R 3 is -C1-C6 alkyl -O- or -(CH2CH2R 5 ) n -, where R 5 is -O-, and R in the hydrophilic unit 4 is a hydrogen atom, an alkyl group (e.g., a methyl group or an ethyl group), a phosphono group (e.g., dihydroxyphosphoryl), a sulfono group (e.g., hydroxysulfonyl), a phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl) or a phosphoryl group, the compound according to item A21 or 22. [Item A25] R 3 is C1-C6 alkyl or -(CH2CH2R 5 ) n -, where R 5 is -CH2-, and R in the hydrophilic unit 4 is a hydroxyl, carboxyl group, amino group, ammonio group, formyl group, ester group, phosphono group or sulfono group, the compound according to item A21 or 22. [Item A26] R 3 is -C1-C6 alkyl -SO3- or a salt thereof, the compound according to item A21 or 22. [Item A27] The recognition motif is selected from the group consisting of crown ethers, cryptands, pincers, chelating motifs and any combination thereof, the compound according to any one of items A1-26. [Item A28] The compound contains an Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole (e.g., porphyrin), the compound according to any one of items A1-27. [Item A29] The compound is optionally a sensor (e.g., a colorimetric sensor, a fluorescence sensor, an in vivo sensor, an oxygen sensor, an environmental sensor, etc.) without the addition and / or presence of an organic solvent and / or functions as such a sensor, the compound according to any one of items A1-28. [Item A30] A compound having a structure represented by the following A - B - C, or C - A - B Herein,[ A is a dye (e.g., a fluorophore (e.g., a fluorescent emitter) or a non - emitting molecular entity), where optionally, the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, where optionally, the polymer has a molecular weight in the range from about 1,000 Da, 5,000 Da or 10,000 Da to about 175,000 Da, and C is optional, where C contains a bioconjugate group, and wherein the compound contains at least one unit having a structure represented by the following formula III,[ [Chemical formula] Herein,[ R is a hydrogen atom or C1 - C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 is absent or is - O -, - NH - or - CH2 -, R’ is C3 - C20 cycloalkyl, R 2 is a hydrogen atom or a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester or 2,4 - dinitrophenyl ester), an azide group, a maleimide group, an isocyanate group or an isothiocyanate group, and p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000 or 100,000. [Item A31] The compound according to item A30, wherein the pigment (for example, a tetrapyrrole macrocyclic molecule) is covalently bonded to a part (for example, the end) of the polymer. [Item A32] The compound according to item A30 or 31, wherein the one or more hydrophobic units and the one or more hydrophilic units are randomly distributed in the polymer. [Item A33] The compound according to any one of items A30 to A32, wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10, and optionally, the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:6 (hydrophobic unit: hydrophilic unit). [Item A34] The compound according to any one of items A30 to A33, wherein the compound has conformational flexibility. [Item A35] The compound according to any one of items A30 to A34, wherein the compound self-folds in an aqueous solution (for example, an aqueous solution containing 10 mM NaH2PO4 and 150 mM NaCl and having a pH of 7.35), and optionally, self-folds into a unimolecular micelle structure. [Item A36] The compound according to any one of items A30 to A35, wherein the polymer is an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer. [Item A37] The compound according to any one of items A30 to A36, wherein the compound is cross-linked, and optionally, when the compound has a folded structure, the compound is cross-linked. [Item A38] The compound according to any one of items A30 to A37, wherein the compound is folded to provide particles, and optionally, the particles have a diameter in the range from about 1 nm or about 3 nm to about 30 nm or about 40 nm. [Item A39] The compound according to any one of items A30 to 38, wherein at least a part of the one or more hydrophobic units is present in the core of the particle, and / or at least a part of the one or more hydrophilic units is present in the periphery (e.g., shell) of the particle. [Item A40] The compound according to any one of items A30 to 39, wherein when the compound is in a folded structure, the dye is encapsulated by a part of the compound (e.g., a part of the polymer). [Item A41] The compound according to any one of items A30 to 40, wherein the polymer is a telechelic polymer or a heterotelechelic polymer. [Item A42] The compound according to any one of items A30 to 41, wherein the dye (e.g., a tetrapyrrole macrocyclic molecule) is hydrophobic. [Item A43] The compound according to any one of items A30 to 42, wherein the compound is water-soluble, and optionally, the compound has solubility in water in the range of about 1 mg / mL to about 10 mg / mL at room temperature. [Item A44] The compound according to any one of items A30 to 43, wherein at least one of the one or more hydrophobic units and / or the one or more hydrophilic units has a pendant functional group, and optionally, the pendant functional group is a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group (e.g., a pentafluorophenyl ester, a succinimide ester or a fluorophenyl ester), an azide group, a maleimide group, an isocyanate group, an isothiocyanate group, a phosphono group, a sulfono group, an ammonio group or a phosphatidylcholine group, and / or the pendant functional group is a hydrophilic group having a terminal cation (e.g., ammonium), an anion (e.g., sulfonate, phosphate, carboxylate or phosphonate) or an amphoteric group (e.g., a choline-like) group, and optionally, a poly(ethylene glycol) residue. [Item A45] The compound according to any one of items A30 to 44, wherein at least one of the above-mentioned hydrophobic units contains an alkyl pendant group (for example, dodecyl), and / or at least one of the above-mentioned hydrophilic units contains a glycol pendant group (for example, poly(ethylene glycol)). [Item A46] The compound according to any one of items A30 to 45, wherein the polymer is bonded to one dye and optionally to one bioconjugate group. [Item A47] The compound according to any one of items A30 to 46, wherein R’ is C8 - C12 cycloalkyl. [Item A48] R in the hydrophobic unit 2 is a hydrogen atom or a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group, the compound according to any one of items A30 to 47. [Item A49] R in the hydrophobic unit 2 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group or a maleimide group, the compound according to any one of items A30 to 48. [Item A50] The compound according to any one of items A30 to 49, wherein the hydrophilic unit has a structure represented by the following formula IV [Chemical formula] Here R is a hydrogen atom or C1 - C8 alkyl (for example, C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 is absent or is -O-, -NH- or -CH2-, R 3 is -(CH2CH2R 5 ) n -, -C1 - C6 alkyl, -C1 - C6 alkyl - O- and -C1 - C6 alkyl - SO3- or a salt thereof, where R 5is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000, R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group (e.g., dihydroxyphosphoryl), a sulfono group (e.g., hydroxysulfonyl), a phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester or 2,4-dinitrophenyl ester), and p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000 or 100,000. [Item A51] R in the hydrophilic unit 4 is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group, the compound according to Item A50. [Item A52] R in the hydrophilic unit 4 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group or a maleimide group, the compound according to Item A50. [Item A53] R 3 is -C1-C6 alkyl -O- or -(CH2CH2R 5 ) n -, where R 5 is -O-, and R in the hydrophilic unit 4The compound according to item A50 or 51, wherein the group is a hydrogen atom, an alkyl group (e.g., a methyl group or an ethyl group), a phosphono group (e.g., dihydroxyphosphoryl), a sulfono group (e.g., hydroxysulfonyl), a phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl) or a phosphoryl group. [Item A54] R 3 is C1-C6 alkyl or -(CH2CH2R 5 ) n -, where R 5 is -CH2-, and R 4 in the hydrophilic unit is a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group or a sulfono group; the compound according to item A50 or 51. [Item A55] R 3 is -C1-C6 alkyl-SO3- or a salt thereof; the compound according to item A50 or 51. [Item A56] Further comprising a recognition motif, optionally, the recognition motif being bound to a dye; the compound according to items A30-55. [Item A57] The recognition motif is selected from the group consisting of a crown ether, a cryptand, a pincer, a chelating motif and any combination thereof, optionally, the compound comprising an Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole (e.g., porphyrin); the compound according to item A56. [Item A58] The compound is optionally a sensor (e.g., a chromogenic sensor, a fluorescent sensor, an in vivo sensor, an oxygen sensor, an environmental sensor, etc.) without the addition and / or presence of an organic solvent and / or functions as the sensor; the compound according to any one of items A30-57. [Item A59] A composition comprising the compound according to any one of items A1-58. [Item A60] The composition according to item A59, wherein the compound forms particles. [Item A61] The composition according to item A59 or 60, wherein the compound and the particles are present in the composition in a ratio of about 1:1 (for example, one compound per particle). [Item A62] The composition according to any one of items A59 to 61, wherein at least a part of the one or more hydrophobic units is present in the core of the particles. [Item A63] The composition according to any one of items A59 to 62, wherein at least a part of the one or more hydrophilic units is present in the shell (for example, the periphery) of the particles. [Item A64] The composition according to any one of items A59 to 63, wherein the particles are resistant to dilution, and optionally, when the composition is diluted up to 100-fold or to a concentration below micromolar, the particles remain in a folded structure. [Item A65] The compound according to any one of items A59 to 64, wherein the dye is present in the core of the particles and / or encapsulated by at least a part of the polymer. [Item A66] The compound according to any one of items A59 to 65, wherein the composition does not have an organic solvent. [Item A67] A method for preparing a compound according to any one of items A1 to 58, comprising: Polymerizing a hydrophobic monomer and a hydrophilic monomer to prepare a copolymer containing hydrophobic units and hydrophilic units; Binding a dye (for example, a fluorophore (for example, a fluorescent fluorophore) or a non-fluorescent molecular entity) to a first part (for example, the end or the terminal part) of the copolymer, thereby preparing the compound, wherein the dye optionally contains a recognition motif; and Optionally, binding a bioconjugate group to a second part (for example, another end or terminal part) of the copolymer; and / or Optionally, crosslinking the compound. The method as described above, including... [Item A68] The polymerization of the hydrophobic monomer and the hydrophilic monomer includes preparing a copolymer by polymerizing the hydrophobic monomer and the hydrophilic monomer via living radical polymerization (e.g., ATRP) in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally a cocatalyst, as described in Item A67. [Item A69] The polymerization of the hydrophobic monomer and the hydrophilic monomer includes preparing a copolymer by polymerizing the hydrophobic monomer and the hydrophilic monomer via living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT agent (e.g., a thiocarbonylthio compound), as described in Item A67. [Item A70] The polymerization of the hydrophobic monomer and the hydrophilic monomer includes polymerizing the hydrophobic monomer and the hydrophilic monomer at a ratio of about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10, as described in any one of Items A67 - 69. [Item A71] The method as described in any one of Items A67 - 70, wherein the hydrophobic monomer has a structure represented by the following Formula I [Chemical formula] Here, R is a hydrogen atom or C1 - C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl), R 1 either does not exist or is -O-, -NH-, or -CH2-, R’ is C1 - C20 alkyl, C2 - C20 alkenyl, C2 - C20 alkynyl, C3 - C20 cycloalkyl, and R 2is a hydrogen atom or a halogen atom, a hydroxyl group, a carboxyl group, an amino group, formyl or an ester group (e.g., succinimide ester, 2,4-dinitrophenyl ester, pentafluorophenyl ester, fluorophenyl ester, etc.). [Item A72] R in the hydrophobic monomer 2 is a hydrogen atom, or a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group, the method according to Item A71. [Item A73] The hydrophilic monomer has a structure represented by the following formula II, the method according to any one of Items A67 to 72 [Chemical formula] Here,[[]]END]] R is a hydrogen atom or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl), R 1 does not exist or is -O-, -NH- or -CH2-, R 3 is -(CH2CH2R 5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl-O- and -C1-C6 alkyl-SO3- or a salt thereof, where R 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000, and R 4is absent or is a hydrogen atom, alkyl, phosphono group (e.g., dihydroxyphosphoryl), sulfono group (e.g., hydroxysulfonyl), phosphatidylcholine group (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl group, halogen atom, hydroxyl group, carboxyl group, amino group, ammonio group, formyl group or ester group (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester or 2,4-dinitrophenyl ester). [Item A74] R in the hydrophilic unit 4 is a hydroxyl group, carboxyl group, amino group, formyl group or ester group, and the method according to Item A73. [Item A75] R 3 is -C1-C6 alkyl-O-, and R in the hydrophilic monomer 4 is a hydrogen atom, alkyl group (e.g., methyl group or ethyl group), phosphono group (e.g., dihydroxyphosphoryl), sulfono group (e.g., hydroxysulfonyl), phosphatidylcholine group or phosphoryl group, and the method according to Item A73. [Item A76] R 3 is -C1-C6 alkyl or -(CH2CH2R 5 ) n -, R 5 is -O-, and R in the hydrophilic monomer 4 is a hydroxyl group, carboxyl group, amino group, ammonio group, formyl group, ester group, phosphono group or sulfono group, and the method according to Item A73. [Item A77] The method according to any one of Items A67-76, further comprising binding a recognition motif to the compound, optionally to a part of the dye or copolymer. [Item A78] A method of using the compound according to any one of Items A1-58 or the composition according to any one of Items A59-66 in flow cytometry. [Item A79] A method for detecting cells and / or particles using flow cytometry, comprising labeling the cells and / or particles with a compound according to any one of items A1 to 58 or a compound prepared according to the method according to any one of items A67 to 77, and detecting the compound by flow cytometry, thereby detecting the cells and / or particles. [Item A80] A method for detecting tissues and / or factors (e.g., cells, infectious agents, etc.) in a subject, comprising administering to the subject a compound according to any one of items A1 to 58, a composition according to any one of items A59 to 66, or a compound prepared according to the method according to any one of items A67 to 77, where optionally the compound associates with the tissue and / or factor, and detecting the compound within the subject, thereby detecting the tissue and / or factor. [Item A81] A method for treating a subject in need of treatment of cells and / or tissues (e.g., diseased cells and / or tissues), comprising administering to the subject a compound according to any one of items A1 to 58, a composition according to any one of items A59 to 66, or a compound prepared according to the method according to any one of items A67 to 77, where optionally the compound associates with the cells and / or tissue, and irradiating the subject or a part of the subject (e.g., the site where the cells and / or tissue are present) with light of a wavelength and intensity sufficient to treat the cells and / or tissue, where optionally the light activates the compound or a part of the compound, comprising the above. [Item A82] The method according to item A81, wherein the cells and / or tissue are hyperproliferative tissue (e.g., a tumor). [Item A83] A photodynamic therapy method for treating a subject in need of treatment of hyperproliferative tissue, comprising Administering to a subject a compound according to any one of items A1 to 58, a composition according to any one of items A59 to 66, or a compound prepared according to the method according to any one of items A67 to 77, where optionally, the compound associates with the hyperproliferative tissue, and irradiating the hyperproliferative tissue with light of a wavelength and intensity sufficient to activate the compound or a portion of the compound, thereby treating the hyperproliferative tissue The method comprising the above. [Item A84] A biomolecule comprising a compound according to any one of items A1 to 58 or a compound prepared according to the method according to any one of items A67 to 77. [Item A85] The biomolecule according to item A84, comprising two or more compounds according to any one of items A1 to 58 or two or more compounds prepared according to the method according to any one of items A67 to 77. [Item A86] In a photodynamic therapy method, a method of using a compound according to any one of items A1 to 58 or a composition according to any one of items A59 to 66. [Item A87] A method of using a compound in a photoacoustic imaging method, wherein the compound has a structure represented by the following, said method A - B - C, or C - A - B where A is a dye (e.g., a fluorophore (e.g., a fluorescent emitter) or a non - emissive molecular entity), where optionally, the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, where optionally, the polymer has a molecular weight in the range from about 1,000 Da, 5,000 Da or 10,000 Da to about 175,000 Da, and C is optional, where C comprises a bioconjugate group, where optionally, the compound is a compound according to any one of items A1 to 58 or a composition according to any one of items A59 to 66. [Item A88] A method for imaging tissues and / or factors (e.g., cells, infectious agents, etc.) in a subject, comprising: administering to the subject a compound having a structure represented by: A-B-C, or C-A-B wherein: A is a dye (e.g., a fluorophore (e.g., a fluorescent fluorophore) or a non-fluorescent molecular entity), optionally having a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, optionally having a molecular weight in the range from about 1,000 Da, 5,000 Da or 10,000 Da to about 175,000 Da; and C is optional and comprises a bioconjugate group, optionally such that the compound associates with the tissue and / or factor; and detecting the compound within the subject, thereby imaging the tissue and / or factor. Including: Optionally, the compound is a compound according to any one of Items A1 - 58 or a composition according to any one of Items A59 - 66. The method. [Item A89] Detecting the compound within the subject includes irradiating the subject or a part of the subject (e.g., the site where the compound is present and / or the site to be imaged) with light having a wavelength and intensity sufficient to generate ultrasound (e.g., an ultrasonic pressure wave), optionally using a laser and / or exposing the subject to one or more non-ionizing laser pulses, according to the method of Item A88. [Item A90] Detecting the compound within the subject optionally includes detecting ultrasound using an ultrasound detector, according to the method of Item A88 or 89. [Item A91] The method according to any one of items A88 to 90, wherein the method for imaging tissues and / or factors in the subject includes photoacoustic imaging of the tissues and / or factors. [Item A92] A method of using a compound as a sensor (e.g., a chromogenic sensor, a fluorescent sensor, an in-vivo sensor, an oxygen sensor, etc.) optionally without the addition and / or presence of an organic solvent, wherein the compound has a structure represented by the following: the method A - B - C, or C - A - B Here, A is a dye (e.g., a fluorophore (e.g., a fluorescent fluorophore) or a non-fluorescent molecular entity), where optionally, the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da. B is a polymer containing one or more hydrophobic units and one or more hydrophilic units, where optionally, the polymer has a molecular weight in the range from about 1,000 Da, 5,000 Da or 10,000 Da to about 175,000 Da, and C is optional, where C contains a bioconjugate group, and Here optionally, the compound is a compound according to any one of items A1 to 58 or a composition according to any one of items A59 to 66. [Item A93] A method for sensing metal ions, preparing a compound having a structure represented by the following A - B - C, or C - A - B Here, A is a dye (e.g., a fluorophore (e.g., a fluorescent fluorophore) or a non-fluorescent molecular entity), where optionally, the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da; B is a polymer containing one or more hydrophobic units and one or more hydrophilic units, where optionally, the polymer has a molecular weight in the range from about 1,000 Da, 5,000 Da or 10,000 Da to about 175,000 Da, and C is optional, where C contains a bioconjugate group, and, contacting the metal ion with the compound, thereby sensing the metal ion comprising wherein optionally, the compound is a compound according to any one of items A1 to 58 or a composition according to any one of items A59 to 66 the method [Item A94] The method according to item A93, wherein the compound and / or metal ion are present in water and optionally do not have an organic solvent [Item A95] A method of using a compound as an oxygen sensor and / or a substance that binds oxygen, wherein the compound has a structure represented by the following A - B - C, or C - A - B wherein A is a dye (e.g., a fluorophore (e.g., a fluorescing group) or a non - fluorescent molecular entity), wherein optionally, the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, wherein optionally, the polymer has a molecular weight in the range from about 1,000 Da, 5,000 Da or 10,000 Da to about 175,000 Da, and C is optional, wherein C contains a bioconjugate group wherein optionally, the compound is a compound according to any one of items A1 to 58 or a composition according to any one of items A59 to 66 [Item A96] The method according to item A92 or 95 or 93 or 94, wherein the compound comprises an Fe(II) - chelated tetrapyrrole (e.g., porphyrin) or a Cu(II) - chelated tetrapyrrole (e.g., porphyrin) The second aspect of the present invention is as follows [Item B1] A compound having a structure represented by the following A - B - C, or C - A - B Here, A is a dye; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C contains a bioconjugate group, and here, the compound contains at least one unit having a structure represented by the following formula III, [Chemical formula] Here, R is a hydrogen atom or C1-C8 alkyl, R 1 is absent or is -O-, -NH- or -CH2-, R' is C3-C20 cycloalkyl, R 2 is a hydrogen atom or a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group, an azide group, a maleimide group, an isocyanate group or an isothiocyanate group, and p is an integer from 1 to 100,000. [Item B2] The compound according to item B1, wherein the dye is a lumophore or a non-luminescent molecular entity. [Item B3] The compound according to item B1 or 2, wherein the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da. [Item B4] The compound according to any one of items B1 to 3, wherein the polymer has a molecular weight in the range of about 1,000 Da to about 175,000 Da. [Item B5] R 2 is an ester selected from pentafluorophenyl ester, succinimide ester, fluorophenyl ester and 2,4-dinitrophenyl ester, and the compound according to any one of items B1 to 4. [Item B6] The compound according to any one of items B1 to 5, wherein the dye is covalently bonded to a part of the polymer. [Item B7] The compound according to any one of items B1 to 6, wherein the one or more hydrophobic units and the one or more hydrophilic units are randomly distributed in the polymer. [Item B8] The compound according to any one of items B1 to 7, wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:1 to about 1:10. [Item B9] The compound according to any one of items B1 to 8, wherein the compound has conformational flexibility. [Item B10] The compound according to any one of items B1 to 9, wherein the compound self-folds in an aqueous solution. [Item B11] The compound according to any one of items B1 to 10, wherein the polymer is an amphiphilic random copolymer. [Item B12] The compound according to any one of items B1 to 11, wherein the compound is crosslinked. [Item B13] The compound according to any one of items B1 to 12, wherein the compound is folded to provide particles. [Item B14] The compound according to any one of items B1 to 13, wherein at least a part of the one or more hydrophobic units is present in the core of the particles and / or at least a part of the one or more hydrophilic units is present on the periphery of the particles. [Item B15] The compound according to any one of items B1 to 14, wherein when the compound is in a folded structure, the dye is encapsulated by a part of the compound. [Item B16] The compound according to any one of items B1 to 15, wherein the polymer is a telechelic polymer or a heterotelechelic polymer. [Item B17] The compound according to any one of items B1 to 16, wherein the dye is hydrophobic. [Item B18] The compound according to any one of Items B1 to 17, wherein the compound is water-soluble. [Item B19] The compound according to any one of Items B1 to 18, wherein at least one of the one or more hydrophobic units and / or the one or more hydrophilic units has a pendant functional group. [Item B20] The compound according to any one of Items B1 to 19, wherein at least one of the one or more hydrophobic units contains an alkyl pendant group and / or at least one of the one or more hydrophilic units contains a glycol pendant group. [Item B21] The compound according to any one of Items B1 to 20, wherein the polymer is bound to one dye and may be bound to one bioconjugate group. [Item B22] The compound according to any one of Items B1 to 21, wherein R’ is C8 - C12 cycloalkyl. [Item B23] R in the hydrophobic unit 2 is a hydrogen atom or a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group, and the compound according to any one of Items B1 to 22. [Item B24] R in the hydrophobic unit 2 is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group or a maleimide group, and the compound according to any one of Items B1 to 23. [Item B25] The compound according to any one of Items B1 to 24, wherein the hydrophilic unit has a structure represented by the following Formula IV [Chemical formula] Here, R is a hydrogen atom or C1 - C8 alkyl, R 1 is absent or is -O-, -NH- or -CH2-, R 3 is selected from the group consisting of -(CH2CH2R 5 ), -C1-C6 alkyl, -C1-C6 alkyl-O-, and -C1-C6 alkyl-SO3- or salts thereof, wherein R n is -O- or -CH2-, and n is an integer from 1 to 10,000, 5 R R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group, and p is an integer from 1 to 100,000. [Item B26] The compound according to Item B25, wherein R 4 in the hydrophilic unit is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group. [Item B27] The compound according to Item B25, wherein R 4 in the hydrophilic unit is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group or a maleimide group. [Item B28] R 3 is -C1-C6 alkyl-O- or -(CH2CH2R 5 ), n wherein R 5 is -O-, and the compound according to Item B25 or 26, wherein R 4 in the hydrophilic unit is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group or a phosphoryl group. [Item B29] R 3 is C1-C6 alkyl or -(CH2CH2R 5 ), n wherein R 5is -CH2-, and R in the hydrophilic unit 4 is hydroxyl, carboxyl group, amino group, ammonium group, formyl group, ester group, phosphono group or sulfono group, the compound according to item B25 or 26. [Item B30] R 3 is -C1-C6 alkyl -SO3- or a salt thereof, the compound according to item B25 or 26. [Item B31] The compound is a sensor and / or functions as the sensor, the compound according to any one of items B1 to 30. [Item B32] A composition comprising the compound according to any one of items B1 to 31. [Item B33] The compound forms particles, the composition according to item B32. [Item B34] The compound and the particles are present in a ratio of about 1:1 in the composition, the composition according to item B32 or 33. [Item B35] At least a part of the one or more hydrophobic units is present in the core of the particles, the composition according to any one of items B32 to 34. [Item B36] At least a part of the one or more hydrophilic units is present on the shell of the particles, the composition according to any one of items B32 to 35. [Item B37] The particles are resistant to dilution, the composition according to any one of items B32 to 36. [Item B38] The pigment is present in the core of the particles and / or encapsulated by at least a part of the polymer, the compound according to any one of items B32 to 37. [Item B39] The composition has no organic solvent, the compound according to any one of items B32 to 38. [Item B40] A method for preparing the compound defined in any one of items B1 to 31, wherein Prepare a copolymer containing a hydrophobic unit and a hydrophilic unit by polymerizing a hydrophobic monomer and a hydrophilic monomer. Bind a dye to the first part of the copolymer, thereby preparing the compound. The method as described above. [Item B41] The method according to item B40, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer includes polymerizing the hydrophobic monomer and the hydrophilic monomer via living radical polymerization in the presence of an initiator and a catalyst to prepare a copolymer. [Item B42] The method according to item B40, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer includes polymerizing the hydrophobic monomer and the hydrophilic monomer via living radical polymerization in the presence of an initiator and a RAFT agent to prepare a copolymer. [Item B43] The method according to any one of items B40 to B42, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer includes polymerizing the hydrophobic monomer and the hydrophilic monomer at a ratio of about 1:1 to about 1:10. [Item B44] The method according to any one of items B40 to B43, wherein the hydrophobic monomer has a structure represented by the following formula I [Chemical formula] Here, R is a hydrogen atom or C1-C8 alkyl, R 1 either does not exist or is -O-, -NH- or -CH2-, R' is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, and R 2 is a hydrogen atom or a halogen atom, a hydroxyl group, a carboxyl group, an amino group, formyl or an ester group. [Item B45] R in the hydrophobic monomer 2is a hydrogen atom or is a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group, item B 44 The method according to [Item B46] The method according to any one of items B40 to 45, wherein the hydrophilic monomer has a structure represented by the following formula II [Chemical formula] Here R is a hydrogen atom or C1-C8 alkyl R 1 is absent or is -O-, -NH- or -CH2- R 3 is -(CH2CH2R 5 ) n is selected from the group consisting of -, -C1-C6 alkyl, -C1-C6 alkyl-O- and -C1-C6 alkyl-SO3- or salts thereof, where R 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000, and R 4 is absent or is a hydrogen atom, alkyl, phosphono group, sulfono group, phosphatidylcholine group, phosphoryl group, halogen atom, hydroxyl group, carboxyl group, amino group, ammonium group, formyl group or ester group. [Item B47] R in the hydrophilic unit 4 is a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group, the method according to item B46. [Item B48] R 3 is -C1-C6 alkyl-O-, and R in the hydrophilic monomer 4 is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group or a phosphoryl group, the method according to item B46. [Item B49] R 3 is -C1-C6 alkyl or -(CH2CH2R5 ) n - wherein R 5 is -O- and R in the hydrophilic monomer 4 is a hydroxyl group, carboxyl group, amino group, ammonium group, formyl group, ester group, phosphono group or sulfono group, the method according to item B46. [Item B50] A method of using the compound according to any one of items B1 to B31 or the composition according to any one of items B32 to B39 in flow cytometry. [Item B51] A method for detecting cells and / or particles using flow cytometry, comprising labeling the cells and / or particles with the compound according to any one of items B1 to B31 or the compound prepared according to the method according to any one of items B32 to B39, and detecting the compound by flow cytometry, thereby detecting the cells and / or particles. [Item B52] An agent used for detecting a tissue and / or a factor in a subject, the agent comprising the compound according to any one of items B1 to B31, the composition according to any one of items B32 to B39 or the compound prepared according to the method according to any one of items B40 to B49, wherein the agent is administered to the subject and the compound in the subject is detected, thereby detecting the tissue and / or the factor. [Item B53] An agent used for treating a subject in need of treatment of cells and / or tissues, the agent comprising the compound according to any one of items B1 to B31, the composition according to any one of items B32 to B39 or the compound prepared according to the method according to any one of items B40 to B49, wherein the agent is administered to the subject and the subject or a part of the subject is irradiated with light of sufficient wavelength and intensity to treat the cells and / or tissues. [Item B54] The agent according to item B53, wherein the cells and / or tissues are hyperproliferative tissues. [Item B55] An agent used for photodynamic therapy for treating a subject in need of treatment of hyperproliferative tissue, said agent comprising a compound according to any one of items B1 to B31, a composition according to any one of items B32 to B39 or a compound prepared according to a method according to any one of items B40 to B49, wherein said agent is administered to said subject and said hyperproliferative tissue is irradiated with light having a wavelength and intensity sufficient to activate said compound or a part of said compound, whereby said hyperproliferative tissue is treated, said agent. [Item B56] A biomolecule comprising a compound according to any one of items B1 to B31 or a compound prepared according to a method according to any one of items B40 to B49. [Item B57] The biomolecule according to item B56, comprising two or more compounds according to any one of items B1 to B31 or two or more compounds prepared according to a method according to any one of items B40 to B49. [Item B58] An agent used for photodynamic therapy, said agent comprising a compound according to any one of items B1 to B31 or a composition according to any one of items B32 to B39. [Item B59] A method of using a compound in a photoacoustic imaging method, said compound having a structure represented by the following: A - B - C, or C - A - B wherein, A is a dye; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C comprises a bioconjugate group. [Item B60] An agent used for imaging tissue and / or factors in a subject, said agent comprising a compound having a structure represented by the following: A - B - C, or C - A - B wherein, A is a dye, B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, and C is optional, wherein C comprises a bioconjugate group, and the compound is administered to a subject, and the compound within the subject is detected, whereby the tissue and / or factor is imaged the agent. [Item B61] The agent according to item B60, wherein the compound within the subject is detected by irradiating the subject or a part of the subject with light having a wavelength and intensity sufficient to generate ultrasound. [Item B62] The agent according to item B60 or 61, wherein the compound within the subject is detected by detecting ultrasound. [Item B63] The agent according to any one of items B60 to 62, wherein imaging of the tissue and / or factor in the subject is performed by photoacoustic imaging of the tissue and / or factor. [Item B64] A method of using a compound as a sensor, wherein the compound has a structure represented by the following A-B-C, or C-A-B wherein A is a dye, B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, and C is optional, wherein C comprises a bioconjugate group. [Item B65] A method of sensing a metal ion, comprising providing a compound having a structure represented by the following A-B-C, or C-A-B wherein A is a dye; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, and C is optional, wherein C comprises a bioconjugate group, and Contacting the metal ion with the compound, thereby sensing the metal ion comprising the method. [Item B66] The method according to Item B65, wherein the compound and / or the metal ion are present in water. [Item B67] A method of using a compound as an oxygen sensor and / or a substance that binds oxygen, the method wherein the compound has a structure represented by the following A - B - C, or C - A - B wherein A is a dye, B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C contains a bioconjugate group. [Item B68] The method according to Item B64 or 67, or the method according to Item B65 or 66, wherein the compound comprises an Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole.

Claims

1. A compound having a structure represented by A,B,C, or CAB Where: A is a dye; B is a polymer containing one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C comprises a bioconjugate group; and wherein the compound comprises at least one unit having a structure represented by formula III: 【Chemistry 1】 Where: R is a hydrogen atom or a C1-C8 alkyl group; R 1 is not present or is -O-, -NH- or -CH 2 - and R' is a C3-C20 cycloalkyl; R 2 is a hydrogen atom, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group, a vinyl group, an epoxy group, a mercapto group, an ester group, an azide group, a maleimide group, an isocyanate group, or an isothiocyanate group, and p is an integer from 1 to 100,000.

2. The compound of claim 1 , wherein the dye is a luminophore or a non-luminescent molecular entity.

3. 3. The compound according to claim 1 or 2, wherein the dye has a molecular weight ranging from about 150 Daltons (Da) to about 3,000 Da.

4. The compound according to any one of claims 1 to 3, wherein the polymer has a molecular weight ranging from about 1,000 Da to about 175,000 Da.

5. R 2 A compound according to any one of claims 1 to 4, wherein is an ester selected from pentafluorophenyl ester, succinimide ester, fluorophenyl ester and 2,4-dinitrophenyl ester.

6. The compound according to any one of claims 1 to 5, wherein the dye is covalently attached to a portion of the polymer.

7. The compound according to any one of claims 1 to 6, wherein the one or more hydrophobic units and the one or more hydrophilic units are randomly distributed in the polymer.

8. The compound according to any one of claims 1 to 7, wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:1 to about 1:

10.

9. The compound according to any one of claims 1 to 8, wherein the compound has conformational flexibility.

10. The compound according to any one of claims 1 to 9, wherein the compound self-folds in aqueous solution.

11. The compound according to any one of claims 1 to 10, wherein the polymer is an amphiphilic random copolymer.

12. The compound according to any one of claims 1 to 11, wherein the compound is crosslinked.

13. The compound according to any one of claims 1 to 12, wherein the compound folds to provide a particle.

14. 14. The compound according to claim 1, wherein at least a portion of said one or more hydrophobic units is present in the core of the particle and / or at least a portion of said one or more hydrophilic units is present at the periphery of the particle.

15. The compound of any one of claims 1 to 14, wherein the dye is encapsulated by a portion of the compound when the compound is in a folded conformation.

16. The compound according to any one of claims 1 to 15, wherein the polymer is a telechelic polymer or a heterotelechelic polymer.

17. The compound according to any one of claims 1 to 16, wherein the dye is hydrophobic.

18. The compound according to any one of claims 1 to 17, wherein the compound is water soluble.

19. 19. The compound according to any one of claims 1 to 18, wherein at least one of the one or more hydrophobic units and / or the one or more hydrophilic units has a pendant functional group.

20. 20. The compound of claim 1, wherein at least one of the one or more hydrophobic units comprises an alkyl pendant group and / or at least one of the one or more hydrophilic units comprises a glycol pendant group.

21. The compound according to any one of claims 1 to 20, wherein the polymer is attached to one dye and optionally to one bioconjugate group.

22. The compound of any one of claims 1 to 21, wherein R' is a C8 to C12 cycloalkyl.

23. R in the hydrophobic unit 2 The compound according to any one of claims 1 to 22, wherein is a hydrogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group.

24. R in the hydrophobic unit 2 The compound according to any one of claims 1 to 23, wherein is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group, or a maleimide group.

25. The compound according to any one of claims 1 to 24, wherein the hydrophilic unit has a structure represented by formula IV: 【Chemistry 2】 Where: R is a hydrogen atom or a C1-C8 alkyl group; R 1 is not present or is -O-, -NH- or -CH 2 - and R 3 is -(CH 2 CH 2 R 5 ) n -, -C 1 ~C 6 Alkyl, -C 1 ~C 6 Alkyl-O- and -C 1 ~C 6 Alkyl-SO 3 - or salts thereof, wherein R 5 is -O- or -CH 2 and n is an integer from 1 to 10,000. R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group, and p is an integer from 1 to 100,000.

26. R in the hydrophilic unit 4 The compound according to claim 25, wherein is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group or an ester group.

27. R in the hydrophilic unit 4 The compound according to claim 25, wherein is a vinyl group, an epoxy group, a mercapto group, an azide group, an isocyanate group, an isothiocyanate group, or a maleimide group.

28. R 3 But -C 1 ~C 6 Alkyl-O- or -(CH 2 CH 2 R 5 ) n - where R 5 is -O-, and R in the hydrophilic unit 4 The compound according to claim 25 or 26, wherein is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group or a phosphoryl group.

29. R 3 But, C 1 ~C 6 Alkyl or -(CH 2 CH 2 R 5 ) n - where R 5 Ga-CH 2 and R in the hydrophilic unit 4 27. The compound according to claim 25 or 26, wherein is a hydroxyl, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group or a sulfono group.

30. R 3 But -C 1 ~C 6 Alkyl-SO 3 - or a salt thereof.

31. The compound according to any one of claims 1 to 30, wherein the compound is and / or functions as a sensor.

32. A composition comprising a compound according to any one of claims 1 to 31.

33. The composition of claim 32 , wherein the compound forms a particle.

34. 34. The composition of claim 32 or 33, wherein the compound and the particles are present in the composition in a ratio of about 1:

1.

35. A composition according to any one of claims 32 to 34, wherein at least a portion of said one or more hydrophobic units are present in the core of said particle.

36. A composition according to any one of claims 32 to 35, wherein at least a portion of said one or more hydrophilic units is present in the shell of said particle.

37. The composition of any one of claims 32 to 36, wherein the particles are resistant to dilution.

38. A compound according to any one of claims 32 to 37, wherein the dye is present in the core of the particle and / or is encapsulated by at least a portion of the polymer.

39. The compound according to any one of claims 32 to 38, wherein the composition is free of organic solvents.

40. A method for preparing a compound as defined in any one of claims 1 to 31, comprising the steps of: polymerizing a hydrophobic monomer and a hydrophilic monomer to provide a copolymer comprising hydrophobic and hydrophilic units; attaching a dye to a first portion of said copolymer, thereby providing said compound; The method comprising:

41. 41. The method of claim 40, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer via living radical polymerization in the presence of an initiator and a catalyst to provide a copolymer.

42. 41. The method of claim 40, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer via living radical polymerization in the presence of an initiator and a RAFT agent to provide a copolymer.

43. 43. The method of any one of claims 40-42, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer in a ratio of about 1:1 to about 1:

10.

44. The method of any one of claims 40 to 43, wherein the hydrophobic monomer has a structure represented by Formula I: 【Chemistry 3】 Where: R is a hydrogen atom or a C1-C8 alkyl; R 1 is not present or is -O-, -NH- or -CH 2 - and R' is C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, and R 2 is a hydrogen atom or a halogen atom, a hydroxyl group, a carboxyl group, an amino group, a formyl group or an ester group.

45. R in the hydrophobic monomer 2 The method of claim 44, wherein is a hydrogen atom, or a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group.

46. The method of any one of claims 40 to 45, wherein the hydrophilic monomer has a structure represented by Formula II: 【Chemistry 4】 Where: R is a hydrogen atom or a C1-C8 alkyl; R 1 is not present or is -O-, -NH- or -CH 2 - and R 3 But -(CH 2 CH 2 R 5 ) n -, -C 1 ~C 6 Alkyl, -C 1 ~C 6 Alkyl-O- and -C 1 ~C 6 Alkyl-SO 3 - or salts thereof, wherein R 5 is -O- or -CH 2 and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000; and R 4 is absent or is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, a phosphoryl group, a halogen atom, a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, or an ester group.

47. R in the hydrophilic unit 4 The method of claim 46, wherein is a hydroxyl group, a carboxyl group, an amino group, a formyl group, or an ester group.

48. R 3 Ga-C 1 ~C 6 alkyl-O-, and R in the hydrophilic monomer 4 The method of claim 46, wherein is a hydrogen atom, an alkyl group, a phosphono group, a sulfono group, a phosphatidylcholine group, or a phosphoryl group.

49. R 3 Ga-C 1 ~C 6 Alkyl or -(CH 2 CH 2 R 5 ) n - and R 5 is -O-, and R in the hydrophilic monomer 4 The method of claim 46, wherein is a hydroxyl group, a carboxyl group, an amino group, an ammonio group, a formyl group, an ester group, a phosphono group, or a sulfono group.

50. A method of using a compound according to any one of claims 1 to 31 or a composition according to any one of claims 32 to 39 in flow cytometry.

51. A method for detecting cells and / or particles using flow cytometry, comprising labeling the cells and / or particles with a compound according to any one of claims 1 to 31 or a compound prepared according to the method according to any one of claims 32 to 39, and detecting said compounds by flow cytometry, thereby detecting said cells and / or particles.

52. 50. An agent for use in detecting tissues and / or factors in a subject, the agent comprising a compound according to any one of claims 1 to 31, a composition according to any one of claims 32 to 39, or a compound prepared according to a method according to any one of claims 40 to 49, wherein the agent is administered to the subject and the compound is detected in the subject, thereby detecting the tissues and / or factors.

53. 50. An agent for use in treating cells and / or tissues in a subject in need thereof, the agent comprising a compound according to any one of claims 1 to 31, a composition according to any one of claims 32 to 39, or a compound prepared according to a method according to any one of claims 40 to 49, wherein the agent is administered to the subject and the subject or a part of the subject is irradiated with light of a sufficient wavelength and intensity to treat the cells and / or tissues.

54. The method of claim 53, wherein the cells and / or tissues are hyperproliferative tissues.

55. 50. An agent for use in photodynamic therapy to treat hyperproliferative tissue in a subject in need thereof, the agent comprising a compound according to any one of claims 1 to 31, a composition according to any one of claims 32 to 39, or a compound prepared according to a method according to any one of claims 40 to 49, wherein the agent is administered to the subject and the hyperproliferative tissue is irradiated with light of a wavelength and intensity sufficient to activate the compound or a portion of the compound, thereby treating the hyperproliferative tissue.

56. A biomolecule comprising a compound according to any one of claims 1 to 31 or a compound prepared according to the method according to any one of claims 40 to 49.

57. 57. The biomolecule of claim 56, comprising two or more compounds according to any one of claims 1 to 31 or two or more compounds prepared according to the method of any one of claims 40 to 49.

58. 40. An agent for use in photodynamic therapy, said agent comprising a compound according to any one of claims 1 to 31 or a composition according to any one of claims 32 to 39.

59. 1. A method of using a compound in photoacoustic imaging, the compound having a structure represented by: A,B,C, or CAB Where: A is a dye; B is a polymer containing one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C comprises a bioconjugate group.

60. 1. An agent for use in imaging tissues and / or factors in a subject, the agent comprising a compound having a structure represented by: A,B,C, or CAB Where: A is a dye, B is a polymer containing one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C comprises a bioconjugate group; and The compound is administered to a subject and the compound is detected within the subject, thereby imaging the tissue and / or factor. The agent.

61. 61. The method of claim 60, wherein the compound in the subject is detected by illuminating the subject or a portion of the subject with light of a wavelength and intensity sufficient to generate ultrasound.

62. 62. The method of claim 60 or 61, wherein the compound in the subject is detected by detecting ultrasound.

63. The method according to any one of claims 60 to 62, wherein imaging of tissues and / or factors in the subject is performed by photoacoustic imaging of the tissues and / or factors.

64. 1. A method of using a compound as a sensor, wherein the compound has a structure represented by: A,B,C, or CAB Where: A is a dye, B is a polymer containing one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C comprises a bioconjugate group.

65. 1. A method for sensing metal ions, comprising: Providing a compound having a structure represented by: A,B,C, or CAB Where: A is a dye; B is a polymer containing one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C comprises a bioconjugate group; and contacting said metal ion with said compound, thereby sensing said metal ion; Including, The method.

66. 66. The method of claim 65, wherein the compound and / or metal ion is present in water.

67. 1. A method of using a compound as an oxygen sensor and / or oxygen binding material, the compound having a structure represented by: A,B,C, or CAB Where: A is a dye, B is a polymer containing one or more hydrophobic units and one or more hydrophilic units, and C is optional, where C comprises a bioconjugate group.

68. 67. The method of claim 64 or 67, or the method of claim 65 or 66, wherein the compound comprises an Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole.

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