Polymeric chromophores, compositions including the same, and methods of making and using the same

Abstract

Described herein are polymeric chromophores comprising a dye, a polymer, and optionally a bioconjugation group. The polymeric chromophores can 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 units and one or more hydrophilic units; and optionally C, where C, when present, comprises a bioconjugation group. Also described herein are compositions comprising the polymeric chromophores and methods of making and using the same.

Description

[0001] Government funding statement

[0002] This invention was carried out with government support under authorization number DE-SC0001035 granted by the Energy Department. The government holds certain rights to this invention. Technical Field

[0003] This invention generally relates to polymeric chromophores comprising dyes, polymeric segments, and optional bioconjugating groups. This invention also relates to compositions comprising polymeric chromophores and methods for their preparation and use. Background Technology

[0004] Many applications of chromophores occur in aqueous solutions; however, most organic chromophores are hydrophobic or only moderately polar. There is currently no method for encapsulating chromophores in large quantities that meets the criteria of synthetic simplicity, absence of a fluorophore—fluorophore quenching—and the presence of a single biocompatible conjugate. Summary of the Invention

[0005] The first aspect of the present invention relates to compounds having a structure represented by the following:

[0006]

[0007] in

[0008] A is a dye (e.g., a fluorophore), optionally wherein the dye has a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da;

[0009] B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, optionally wherein said polymer has a molecular weight in the range of about 1,000 Da, 5,000 Da, or 10,000 Da to about 175,000 Da; and

[0010] Optionally, C, wherein C contains a biological conjugation group.

[0011] Another aspect of the invention relates to compositions comprising the compounds of the invention and optionally water. Furthermore, aspects of the invention relate to compositions comprising: particles (e.g., particles comprising a core and a shell), wherein said particles comprise a compound having a structure represented by:

[0012]

[0013] in

[0014] A is a dye (e.g., a fluorophore);

[0015] B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units; and

[0016] Optionally, C, wherein C comprises a bioconjugated group; and

[0017] water.

[0018] Another aspect of the invention relates to a method for 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., a terminal or end portion) of the copolymer to provide the compound; optionally attaching a bioconjugating group to a second portion (e.g., another terminal or end portion) of the copolymer; and / or optionally crosslinking the compound. The polymerization of the hydrophobic and hydrophilic monomers may include polymerization 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.

[0019] Another aspect of the present invention relates to compounds prepared according to the method of the present invention.

[0020] According to embodiments of the invention, uses of the compounds of the invention and / or uses of the compositions of the invention are also provided, for example, for use in flow cytometry.

[0021] Another aspect of the present invention relates to a method for detecting cells and / or particles using flow cytometry, the method comprising labeling cells and / or particles with compounds of the present invention; and detecting the compounds by flow cytometry, thereby detecting the cells and / or particles.

[0022] Another aspect of the present invention relates to a method for detecting tissues and / or reagents (e.g., cells, infectious agents, etc.) in a subject, the method comprising: administering to the subject a compound of the present invention or a composition of the present invention, optionally wherein the compound is associated with the tissues and / or reagents; and detecting the compound in the subject, thereby detecting the tissues and / or reagents.

[0023] Another aspect of the invention relates to biomolecules (e.g., cells, antibodies, etc.) comprising one or more (e.g., 1, 2, 3, 4, 5, 6 or more) of the compounds of the invention.

[0024] It should be noted that aspects of the invention described with respect to one embodiment may be incorporated into different embodiments, even if not specifically described therein. That is, features of all and / or any embodiment can be combined in any manner and / or combination. Therefore, the applicant reserves the right to amend any originally filed claim and / or file any new claim, including the right to modify any originally filed claim to be subordinate to and / or incorporated into any feature of any other claim or claim (even if the original claim is not filed in this manner). These and other objects and / or aspects of the invention are explained in detail in the description set forth below. Other features, advantages, and details of the invention will be apparent to those skilled in the art from the following detailed description of the accompanying drawings and preferred embodiments; such description is merely illustrative. Attached Figure Description

[0025] Figure 1 A schematic diagram showing an exemplary polymer chromophore according to an embodiment of the present invention.

[0026] Figure 2 The SEC elution traces are for copolymer 7 (solid line) and copolymer F2 (dashed line) loaded with dihydroporphyrin. The sample was eluted with THF and detected with a refractive index detector.

[0027] Figure 3 Three different absorption spectra are shown. Figure (A) shows the absorption spectrum (solid line) of D1 in CH2Cl2, and the absorption spectrum (dashed line) and emission spectrum (dots) of F1 in water, at a concentration of μM. Figure (B) shows the absorption spectrum (solid line) of D2 in CH2Cl2, and the absorption spectrum (dashed line) and emission spectrum (dots) of F2 in water, at a concentration of μM. Figure (C) shows the absorption spectrum (solid line) of D3 in toluene, and the absorption spectrum (dashed line) and emission spectrum (dots) of F3 in water, at a concentration of μM. All spectra were measured at room temperature.

[0028] Figure 4 Dynamic light scattering (DLS) size data of F-2 at 10 mg / mL (A), 5 mg / mL (B), and 1.0 mg / mL (C).

[0029] Figure 5 The absorption spectrum of F-2 in 1.0 M NaCl solution is shown (top) and the absorption spectrum of F-2 in water is shown (bottom).

[0030] Figure 6 The emission spectrum of F-2 in 1.0 M NaCl solution (top) and the emission spectrum of F-2 in water (bottom) are shown.

[0031] Figure 7 DLS data for two batches of F-Ph at different concentrations in 1.0M NaCl aqueous solution are shown.

[0032] Figure 8 The absorption (left) and emission (right) spectra of Pod-Rhodamine in water are shown in the presence of various cations.

[0033] Figure 9 The fluorescence titration spectra of Au (III) (top) and Hg (II) (bottom) are shown.

[0034] Figure 10 DLS data for F-Ph in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH = 7.35) at room temperature are shown.

[0035] Figure 11 The DLS data of F-Ph in NaCl solutions of different concentrations are shown.

[0036] Figure 12 The graph shows the percentage of monomers (via DLS) in different concentrations of NaCl solution and PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH = 7.35) at room temperature.

[0037] Figure 13 The DLS data of P-S5-CD1 (28 kDa) in NaCl solutions of different concentrations at room temperature are shown.

[0038] Figure 14 DLS data for P-S5-CD1 (28 kDa) in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature are shown.

[0039] Figure 15 A graph showing the percentage of monomers (via DLS) in different concentrations of NaCl solution and PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature.

[0040] Figure 16 DLS data for F-Ph and F-PMI in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature are shown.

[0041] Figure 17DLS data for P-S5-CD1 (28 kDa) and P-S5-CD1 (28 kDa)-PMI in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature are shown.

[0042] Figure 18 DLS data for P-S5-CD1-PMI conjugates with different molecular weights are shown in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature.

[0043] Figure 19 DLS data are shown for three polymer-PMI conjugates that have nearly identical molecular weights but different side group ratios in PBS buffer (10 mM NaH2PO4, 150 mM NaCl, pH 7.35) at room temperature. Detailed Implementation

[0044] The invention will now be described more fully below with reference to the accompanying drawings, in which embodiments of the invention are illustrated. However, the 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.

[0045] The terminology used herein in the specification of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. As used in the specification and appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0046] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be further understood that terms, as defined in commonly used dictionaries, should be interpreted as having a meaning consistent with their meaning in the context of this application and related fields, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of any inconsistency in terminology, this specification shall prevail.

[0047] As used herein, “and / or” means and covers any and all possible combinations of one or more of the relevant listed items, and when interpreted in the alternative (“or”) sense, combinations that are missing.

[0048] Unless the context otherwise indicates, it is specifically meant that the various features of the invention described herein can be used in any combination. Furthermore, the invention is contemplated that, in some embodiments of the invention, any feature or combination of features described herein may be excluded or omitted. For example, if the specification states that a complex comprises components A, B, and C, it expressly means that any one or a combination of A, B, or C may be omitted and discarded.

[0049] As used herein, the transitional phrase “consistently of…” (and its grammatical variations) is interpreted to cover “those materials or steps listed in the claimed invention that do not substantially affect one or more of the essential and novel features of the claimed invention.” See also, regarding Herz , 537 F.2d 549, 551-52, 190 USPQ461,463 (CCPA 1976) (emphasized in the original); see also MPEP § 2111.03. Therefore, the term “substantially constitutes” as used herein should not be construed as equivalent to “contains”.

[0050] It should also be understood that, as used herein, the terms “instance,” “exemplary,” and their grammatical variations are intended to refer to non-limiting instances and / or variant implementations discussed herein, and not to indicate preference for one or more implementations discussed herein compared to one or more other implementations.

[0051] As used herein, the term "about" when referring to a measurable value (such as amount or concentration) is intended to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specific value, as well as the specific value itself. For example, "about X," where X is a measurable value, is intended to include X as well as variations of X of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. The ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​therein.

[0052] The term "derivative," when used herein with respect to a chemical molecule, refers to a chemical molecule having one or more modified (e.g., removed, substituted, etc.) atoms (e.g., hydrogen), functional groups, and / or bonds compared to the parent molecule. For example, a derivative of a dye can refer to a parent dye compound having one or more modified (e.g., removed) atoms (e.g., hydrogen) and / or functional groups to facilitate covalent bonding with another group or partially (e.g., facilitating covalent bonding with a polymer). In some embodiments, the derivative may include functional groups (e.g., substituents and / or auxochromes) that alter the absorption spectrum of the parent molecule.

[0053] As used herein, “alkyl” alone or as part of another group refers to a straight-chain or branched hydrocarbon containing 1 to 20 carbon atoms, which may be referred to as a C1-C20 alkyl group. Representative examples of alkyl groups 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, etc. As used herein, “lower alkyl” is a subset of alkyl groups, and in some embodiments, refers to a straight-chain or branched hydrocarbon group containing 1 to 4 carbon atoms. Representative examples of lower alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. Unless otherwise indicated, the term "alkyl" or "lower alkyl" is intended to include both substituted alkyl or lower alkyl and unsubstituted alkyl or lower alkyl groups, and these groups may be selected from halogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocyclic, heterocyclic, hydroxyl, alkoxy (thus producing polyalkoxy, such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkoxy, aryloxy, arylalkoxy, heterocyclic alkoxy, heterocyclic alkoxy, 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 Aryl-S(O) m Heterocyclic group -S(O) m Heterocyclic alkyl-S(O) m Amino, carboxyl, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, aralkylamino, heterocyclic amino, heterocyclic alkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, wherein m = 0, 1, 2 or 3.

[0054] As used herein, "alkenyl" alone or as part of another group refers to a straight-chain or branched hydrocarbon containing 1 to 20 carbon atoms (or 1 to 4 carbon atoms in lower alkenyl groups), which may contain 1 to 8 double bonds in the positive chain and may be referred to as a C1-C20 alkenyl. Representative examples of alkenyl groups include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, etc. Unless otherwise indicated, the term "alkenyl" or "lower alkenyl" is intended to include both substituted alkenyl or lower alkenyl groups and unsubstituted alkenyl or lower alkenyl groups, and these groups may be substituted by groups as described in conjunction with alkyl and lower alkyl groups above.

[0055] As used herein, "alkynyl" alone or as part of another group refers to a straight-chain or branched hydrocarbon containing 1 to 20 carbon atoms (or 1 to 4 carbon atoms in lower alkynyl groups), comprising one triple bond in the positive chain, and may be referred to as a C1-C20 alkynyl. Representative examples of alkynyl groups include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, etc. Unless otherwise indicated, the term "alkynyl" or "lower alkynyl" is intended to include both substituted alkynyl or lower alkynyl groups and unsubstituted alkynyl or lower alkynyl groups, and these groups may be substituted with the same groups described in conjunction with alkyl and lower alkyl groups above.

[0056] As used in this article, “halogen” refers to any suitable halogen, including -F, -Cl, -Br and -I.

[0057] As used in this article, "thiol group" refers to the -SH group.

[0058] As used in this article, "azido group" refers to the -N3 group.

[0059] As used in this article, "cyano" refers to the -CN group.

[0060] As used in this article, "hydroxyl group" refers to the -OH group.

[0061] As used in this article, "nitro" refers to the -NO2 group.

[0062] As used herein, "alkoxy" alone or as part of another group means an alkyl or lower alkyl group as defined herein (and therefore includes substituted versions such as polyalkoxy) attached to a parent molecule moiety by an oxygen group -O-. Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentoxy, hexoxy, etc.

[0063] As used herein alone or as part of another group, “acyl” means a -C(O)R group, where R is any suitable substituent, such as aryl, alkyl, alkenyl, ynyl, cycloalkyl, or other suitable substituent as described herein.

[0064] As used herein, "halogenated alkyl" alone or as part of another group means at least one halogen as defined herein, which is attached to a parent molecule moiety by an alkyl group as defined herein. Representative examples of halogenated alkyl groups include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, etc.

[0065] As used herein, "alkathio" alone or as part of another group refers to an alkyl group as defined herein, which is attached to a parent molecule via a thio group moiety as defined herein. Representative examples of alkathio groups include, but are not limited to, methylthio, ethylthio, tert-butylthio, hexylthio, etc.

[0066] As used herein, "cycloalkyl" alone or as part of another group refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 1 to 20 carbon atoms (optionally, as described below, the carbon atoms in the heterocyclic group are substituted). A cycloalkyl group may include 0, 1, 2, or more double or triple bonds. Representative examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclododecyl. These rings may optionally be substituted with other substituents described herein, such as halogens or lower alkyl groups. The term "cycloalkyl" is general and, unless otherwise stated, is intended to include heterocyclic groups as described below.

[0067] The term "heterocyclic group" or "heterocycle" as used alone or as part of another group refers to aliphatic (e.g., fully or partially saturated heterocycles) or aromatic (e.g., heteroaryl) monocyclic or bicyclic ring systems. Examples of monocyclic ring systems are any 5- or 6-membered rings containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. 5-membered rings contain 0-2 double bonds, and 6-membered rings contain 0-3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azahexacyclobutane, azahexacycloheptatriene, aziridine, diazacycloheptatriene, 1,3-dioxolane, dioxane, dithiazide, furan, imidazole, imidazoline, imidazoline, isothiazazole, isothiazolin, isothiazolin, isoxazole, isoxazoline, isoxazoline, morpholine, oxadiazole, oxadiazolin, oxadiazolin, oxazole, oxazoline, oxazolin, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroleline, pyrrole, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazolium, thiadiazole, thiadiazolin, thiadiazolin, thiazolidine, thiazoline, thiazoline, thiazoline, thiophene, thiomorpholine, thiomorpholine sulfone, thiaran, triazine, triazole, trithiazide, etc. Examples of bicyclic ring systems are any of the aforementioned monocyclic ring systems fused to an aryl group, a cycloalkyl group, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include, but are not limited to, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiaran, benzodioxine, 1,3-benzodioxane, cyclopentene, cyclophosphine, indazole, indole, dihydroindole, indene, naphthidine, isobenzofuran, isobenzothiaphene, isoindole, isodihydroindole, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinazine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiaranopyridine, etc. These rings include their quaternized derivatives and may optionally be selected from halogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclic, heterocyclic, hydroxyl, alkoxy, alkenyloxy, alkynoxy, haloalkoxy, cycloalkoxy, cycloalkylalkoxy, aryloxy, arylalkoxy, heterocyclic alkoxy, thiol, 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 Heterocyclic group -S(O) m Heterocyclic alkyl-S(O) mThe group is substituted with amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocyclicamino, heterocyclicalkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano groups, where m = 0, 1, 2 or 3.

[0068] As used herein, "aryl" alone or as part of another group refers to a monocyclic carbocyclic system or a bicyclic carbocyclic fused-ring system having one or more aromatic rings. Representative examples of aryl groups include, but are not limited to, azyl, dihydroindenyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, etc. Unless otherwise indicated, the term "aryl" is intended to include both substituted and unsubstituted aryl groups, and these groups may be substituted with the same groups as those described in conjunction with alkyl and lower alkyl groups above.

[0069] As used herein, "aralkyl" alone or as part of another group means an aryl group as defined herein that is attached to a parent molecule moiety by an alkyl group as defined herein. Representative examples of aralkyl groups include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, etc.

[0070] As used in this article, "amino" refers to the group -NH2.

[0071] As used in this article, “alkylamino” alone or as part of another group refers to the group -NHR, where R is alkyl.

[0072] As used in this article, “ester” alone or as part of another group refers to the -C(O)OR group, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0073] As used in this article, "formyl group" refers to the -C(O)H group.

[0074] As used in this article, "carboxylic acid" refers to the -C(O)OH group.

[0075] As used herein, “sulfonoxy” refers to compounds of the formula -S(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0076] 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.

[0077] As used herein, “sulfonate” refers to a salt of sulfonic acid (e.g., a 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.

[0078] As used in this article, "sulfonic acid" refers to compounds of the formula -S(O)(O)OH.

[0079] As used in this article, "amide" alone or as part of another group refers to -C(O)NR a R b Group, wherein R a and R b It can be any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0080] As used in this article, "sulfonamide" alone or as part of another group refers to -S(O)2NR. a R b Group, wherein R a and R b It can be any suitable substituent, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.

[0081] The compounds of the present invention comprise polymeric fluorophores. The compounds of the present invention comprise a single polymer linked to a single dye and optionally contain a single bioconjugating group, which may have a single binding site of a biomolecule. Exemplary compounds are shown in... Figure 1 In some embodiments, a polymer is linked to both a dye and a bioconjugating group (when present). In some embodiments, a dye is linked to both a polymer and a bioconjugating group (when present). In some embodiments, the compositions of the present invention comprise the compounds of the present invention in a solution (such as water, aqueous solutions, and / or hydrophobic solvents).

[0082] While the compounds of the present invention can be linked to a single biomolecule via a bioconjugation group, the biomolecule may contain one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the compounds of the present invention. Therefore, in some embodiments, the biomolecule and / or a portion thereof contains one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the compounds of the present invention.

[0083] In some embodiments, the compounds of the present invention have a structure represented by the following:

[0084]

[0085] in

[0086] A is a dye (e.g., a fluorophore);

[0087] B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units; and

[0088] Optionally, C, wherein C contains a biological conjugate group when present.

[0089] The terms “dye” and “chromophore” are used interchangeably herein to refer to luminescent (e.g., fluorescent and / or phosphorescent molecular entities) and / or non-fluorescent molecular entities (e.g., non-fluorescent and / or non-phosphorescent molecular entities). As used herein, the term “non-fluorescent molecular entity” means a molecular entity that does not have fluorescence or has negligible fluorescence. In some embodiments, non-fluorescent molecular entities do not form excited states with any significant lifetime and / or relax rapidly and substantially quantitatively to the ground state. In some embodiments, non-fluorescent molecular entities have excited-state lifetimes of less than about 100, 75, 50, 25, 10, 5, 1, 0.5, or 0.1 picoseconds. In some embodiments, non-fluorescent molecular entities have quantum yields of internal transformations greater than about 0.8, 0.85, 0.9, 0.95, 0.99, 0.999, 0.9999, or 0.99999, where a quantum yield of 1.0 corresponds to 100%. In some embodiments, the non-fluorescent molecular entities have fluorescence quantum yields of less than about 0.2, 0.15, 0.1, 0.05, 0.01, 0.001, 0.0001, or 0.00001, where a quantum yield of 1.0 corresponds to 100%. Fluorescence quantum yield is known to originate from a competing process of radiative decay relative to the sum of all processes that reduce the excited-state manifold. Such compounds are often referred to as “non-fluorescent”, although sensitive detection techniques can typically detect trace amounts of residual fluorescence, as expected with such low fluorescence quantum yields. Small amounts of fluorescence may not adversely affect some applications, such as photoacoustic imaging methods, although the maximum possible conversion of light input to thermal output is desired. Therefore, the term “non-fluorescent” is used herein to refer to molecular entities with no fluorescence or negligible fluorescence. In some embodiments, the compounds of the present invention comprise a dye, and the dye is a non-fluorescent molecular entity (e.g., a non-fluorescent and / or non-phosphorescent molecular entity). In some embodiments, the compounds of the present invention comprise a dye, and the dye is a luminescent (e.g., a fluorescent and / or phosphorescent molecular entity). In this article, "fluorescent molecular entity" and "fluorophore" are used interchangeably and refer to molecular entities that emit fluorescence.

[0090] The dyes of the present invention may have certain spectral characteristics and / or properties, such as those suitable for use in the methods of the present invention. In some embodiments, the dyes have a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da, about 400 Da to about 1,100 Da, or about 300 Da to about 1,000 Da. In some embodiments, the dye has about 150 Da, 200 Da, 300 Da, 400 Da, 500 Da, 600 Da, 700 Da, 800 Da, 900 Da, 1000 Da, 1200 Da, 1300 Da, 1400 Da, 1500 Da, 1600 Da, 1700 Da, 1800 Da, 1900 Da, 2000 Da Molecular weight of Da, 2200 Da, 2300 Da, 2400 Da, 2500 Da, 2600 Da, 2700 Da, 2800 Da, 2900 Da or 3000 Da. Exemplary dyes include, but are not limited to, tetrapyrrole; naphthalene-based dyes, such as dinaphthalene, trinaphthalene, and tetranaphthalene; fluoresceins, such as TET (tetramethylfluorescein), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyfluorescein (HEX), and 5-carboxyfluorescein (5-FAM); phycoerythrin; halogen dyes; coumarin dyes; rhodamine dyes, such as 6-carboxy-X-rhodamine (ROX), Texas Red, and N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); anthocyanin dyes; phthalocyanine; boron-dipyrrole methylene (BODIPY) dyes; quinoline; pyrene; acridine; stilbene; and their derivatives. In some embodiments, the dye is tetrapyrrole, which includes porphyrin, dihydroporphyrin, and chlorophyll and their derivatives.Exemplary tetrapyrroles include, but are not limited to, 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; and 7,323,560. 1; 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 those described in International Applications PCT / US17 / 47266 and PCT / US17 / 63251. In some embodiments, the dye is hydrophobic. In some embodiments, the dye may be linked to and / or incorporated to a monomer that polymerizes with one or more different monomers (e.g., with hydrophobic and / or hydrophilic monomers). In some embodiments, the dye is a luminescent material (i.e., a material and / or compound that can emit light and whose initial state (e.g., singlet, triplet, and / or another state) is not specified). Exemplary luminescent materials include, but are not limited to, phosphors and / or fluorophores that respectively provide phosphorescence and / or fluorescence.

[0091] In some embodiments, the compounds of the present invention comprise a recognition motif. In some embodiments, the dyes of the present invention may comprise a recognition motif and / or the connectors linking the dyes of the present invention to the polymers of the present invention may comprise a recognition motif. The recognition motif may be attached to the dye and / or the connector. As used herein, “recognition motif” refers to a molecular entity that can bind to a binding entity, and such binding alters the absorption spectrum of the dye and / or activates the fluorescence of the dye. Recognition motifs and binding entities known to those skilled in the art can be used in the compounds of the present invention. Exemplary recognition motifs include, but are not limited to, crown ethers, cavitation ligands, pincers, and / or chelating motifs. Exemplary binding entities are metal ions (e.g., Hg, Cr, Li, etc.). The mechanism by which the absorption spectrum of the dye is altered and / or the fluorescence of the dye is activated can be achieved by a variety of means, such as: (i) metal ion binding promotes ring-opening of the ring that produces the conjugated chromophore; or (ii) metal ion binding to an electron-rich group that causes fluorescence quenching when it is not bound, thereby binding causing quenching shutdown. In some embodiments, the compounds of the present invention are used as and / or function as colorimetric sensors and / or fluorescence sensors. In some embodiments, the compounds of the present invention provide and / or enable metal ion sensing in water, optionally without the addition and / or in the absence of organic solvents. In some embodiments, the compounds of the present invention are used in sensing applications and / or sensors. For example, in some embodiments, the compounds of the present invention are present (e.g., embedded in) a sensor and / or on a sensor. The sensor may be an in vivo sensor and / or for in vivo sensing applications and / or may be an environmental sensor and / or for environmental sensing applications. The recognition motif may be at least partially solvent-accessible and / or available to allow binding to the binding entity. In some embodiments, the compounds of the present invention include a recognition motif and may be used in aqueous solutions, optionally for sensing applications and / or for photoacoustic imaging methods. In some embodiments, when the compounds of the present invention are used in photoacoustic imaging methods and the compounds contain a recognition motif, the recognition motif may cause a shift in the absorption spectrum of the dye upon binding to the binding entity.

[0092] The polymers of the compounds of the present invention may comprise 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 polymers may be prepared by any type of polymerization using 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 to provide a polymer comprising one or more hydrophobic units and one or more hydrophilic units. In some embodiments, the polymer may 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 polymers of the compounds of the present invention may be prepared from at least one hydrophobic monomer, at least one first hydrophilic monomer, and at least one second hydrophilic monomer, wherein the first hydrophilic monomer and the second hydrophilic monomer are different from each other.

[0093] As used herein, “hydrophilic monomer” refers to a monomer containing hydrophilic (e.g., ionic and / or polar) functional groups (e.g., hydrophilic side-group functional groups), optionally wherein the hydrophilic functional group is located at the terminal portion of a portion and / or a terminal portion of the monomer. As those skilled in the art will understand, a portion of a hydrophilic monomer may be hydrophobic, such as a portion forming the polymer backbone when polymerized with other monomers and / or a portion containing functional groups including ionic moieties (e.g., hydrocarbon chains), but if it contains hydrophilic functional groups, it is still referred to as a hydrophilic monomer. As used herein, “hydrophilic unit” refers to a portion or unit of a polymer prepared from a corresponding hydrophilic monomer. As used herein, “hydrophobic monomer” refers to a monomer containing hydrophobic functional groups (e.g., hydrophobic side-group functional groups), optionally wherein the hydrophobic functional group is located at the terminal portion of a portion and / or a terminal portion of the monomer. In some embodiments, the hydrophobic functional group is a hydrocarbon moieties (e.g., alkyl groups). As used herein, “hydrophobic unit” refers to a portion or unit of a polymer prepared from a corresponding hydrophobic monomer.

[0094] In some embodiments, the polymer of the compound of the present invention may also be referred to as a polymer segment of the compound of the present 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 may be an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer. The one or more hydrophobic units and the one or more hydrophilic units may be present in the polymer at a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (hydrophobic unit: hydrophilic unit). In some embodiments, the ratio of hydrophobic units to hydrophilic units is about 1:4 to about 1:6. The length of the polymer may 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 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 of about 28 kDa to about 35 or 50 kDa.

[0095] The hydrophobic and / or hydrophilic units of the polymer may contain side functional groups. A "side functional group" can be a functional group directly attached to the polymer backbone or directly attached to a portion of the polymer backbone. The side functional group may be part of the hydrophobic unit and / or monomer and / or hydrophilic unit and / or monomer during polymerization, or may be added to the hydrophobic and / or hydrophilic unit after polymerization. In some embodiments, the side functional group may be added to the hydrophobic and / or hydrophilic unit after polymerization (e.g., post-polymerization functionalization). In some embodiments, the side functional group contains a charged group. In some embodiments, the side functional group is a halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., active esters such as pentafluorophenyl ester, succinimidyl ester, 2,4-dinitrophenyl ester, etc.), azide, pentafluorophenyl, succinimidyl, fluorophenyl, maleimide, isocyanate, or isothiocyanate. In some embodiments, the side functional group is a hydrophilic group comprising a terminal cation (e.g., ammonium), anion (e.g., sulfonate, phosphate, carboxylate), or zwitterionic group (e.g., choline or choline-like group (e.g., choline derivative)) group and optionally a poly(ethylene glycol) moiety and / or unit. In some embodiments, the hydrophilic group is attached to the poly(ethylene glycol) moiety and / or unit, optionally to the terminal portion of the poly(ethylene glycol) moiety and / or unit.

[0096] In some embodiments, the hydrophobic unit comprises a side functional group containing an alkyl group (e.g., dodecyl) and / or the hydrophilic unit comprises a side functional group containing a glycol (e.g., polyethylene glycol), a sulfonic acid, and / or a 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 compounds of the present invention comprise at least one hydrophobic unit prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer and at least two different hydrophilic units, said hydrophilic unit comprising a first hydrophilic unit prepared from a glycol acrylate (e.g., PEG acrylate) monomer and a second hydrophilic unit prepared from a sulfonate acrylamide monomer (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or a sulfonate acrylate monomer.

[0097] In some embodiments, one or more hydrophobic units and / or one or more hydrophilic units may contain charged (e.g., positive or negative) and / or charged groups (e.g., cationic or anionic groups), and said charge may suppress non-specific binding to the compound or a portion thereof (e.g., a portion of a polymer).

[0098] In some embodiments, the hydrophobic monomer (which can be used to provide the hydrophobic unit of the polymer as described herein) can have a structure represented by Formula I:

[0099] ,

[0100] in:

[0101] R is hydrogen or a C1-C8 alkyl group (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl group);

[0102] R 1 It either does not exist or is -O-, -NH-, or -CH2-;

[0103] R' is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 ynyl, or C3-C20 cycloalkyl; and

[0104] R 2 It is hydrogen or a halogen, hydroxyl, carboxyl, amino, formyl, or ester group (e.g., succinimide ester, 2,4-dinitrophenyl ester, pentafluorophenyl ester, fluorophenyl ester, etc.). In some embodiments, R in the compound of formula I 2 It is a hydroxyl, carboxyl, amino, formyl, or ester group. In some embodiments, R in the compound of formula I... 2It is hydrogen. 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, C19, or C20 alkyl, alkenyl, or alkynyl. In some embodiments, R' in the compound of Formula I is a 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 a 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 a C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 cycloalkyl. As will be readily recognized by those skilled in the art, in some embodiments, the monomer may be, for example, an acrylate (e.g., when R...). 1 When it is oxygen), acrylamide (for example, when R 1 (when it is NH) or vinyl ketone (e.g., when R) 1 (When it is CH2), but other compounds are also possible.

[0105] In some embodiments, the hydrophilic monomer (which can be used to provide the hydrophilic unit of the polymer as described herein) can have a structure represented by Formula II:

[0106] ,

[0107] in:

[0108] R is hydrogen or a C1-C8 alkyl group (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl group);

[0109] R 1 It does not exist or is either -O-, -NH-, or -CH2-;

[0110] R 3 Groups selected from the following: -(CH2CH2R) 5 ) n-, -C1-C6 alkyl, -C1-C6 alkyl-O- and -C1-C6 alkyl-SO3- or their salts, wherein R 5 It 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

[0111] R 4 It is absent or is hydrogen, alkyl, phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), phosphoryl, halogen, hydroxy, carboxyl, amino, ammonium, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester).

[0112] In some embodiments, R in the compound of formula II 4 It is a hydroxyl, carboxyl, amino, formyl, or ester group, optionally when R 3 Yes -(CH2CH2R) 5 ) n -, -C1-C6 alkyl or -C1-C6 alkyl-O-. In some embodiments, when R in the compound of formula II... 3 It is a -C1-C6 alkyl-O- or -(CH2CH2R) 5 ) n - when, where R 5 It's -O-, R 4 It can be hydrogen, alkyl (e.g., methyl or ethyl), phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine, or phosphoryl. In some embodiments, when R in the compound of formula II... 3 When it is a -C1-C6 alkyl group, R 4 It can be hydroxyl, carboxyl, amino, ammonium, formyl, ester, phosphonyl, or sulfonyl. In some embodiments, when R in the compound of formula II... 3 When it is a -C1-C6 alkyl-SO3- or a salt thereof, R 4 It is hydrogen or absent. In some embodiments, R in the compound of formula II 3 It is a salt of -C1-C6 alkyl-SO3- (e.g., sodium salt), and R 4 It does not exist. In some embodiments, R in the compound of formula II 3 Yes -(CH2CH2R) 5 ) n -

[0113] In some implementations, the hydrophobic unit may have a structure represented by Equation III:

[0114] ,

[0115] in:

[0116] R is hydrogen or a C1-C8 alkyl group (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl group);

[0117] R 1 It either does not exist or is -O-, -NH-, or -CH2-;

[0118] R' is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, or C3-C20 cycloalkyl;

[0119] R 2 It is a hydrogen or halogen group, hydroxyl group, carboxyl group, amino group, formyl group, vinyl group, epoxy group, mercapto group, ester group (e.g., pentafluorophenyl ester, succinimidyl ester, fluorophenyl ester or 2,4-dinitrophenyl ester), azide group, maleimide group, isocyanate group or isothiocyanate group; and

[0120] p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000.

[0121] In some embodiments, R in the compound of formula III 2 It is a hydroxyl, carboxyl, amino, formyl, or ester group. In some embodiments, R in the compound of formula III... 2 It is a vinyl, epoxy, mercapto, azide, isocyanate, isothiocyanate, or maleimide group, which may optionally be added and / or provided before and / or through post-polymerization functionalization. In some embodiments, R in the compound of formula III 2It is hydrogen. 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, C19, or C20 alkyl, alkenyl, or alkynyl. In some embodiments, R' in the compound of formula III is a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl. In some embodiments, R' in the compound of formula III is a 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 a C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19 or C20 cycloalkyl group.

[0122] In some implementations, the hydrophilic unit may have a structure represented by formula IV:

[0123] ,

[0124] in:

[0125] R is hydrogen or a C1-C8 alkyl group (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl group);

[0126] R 1 It does not exist or is either -O-, -NH-, or -CH2-;

[0127] R 3 Groups selected from the following: -(CH2CH2R) 5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl-O- and -C1-C6 alkyl-SO3- or their salts, wherein R 5 It 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;

[0128] R 4The following groups are absent or have hydrogen, alkyl, phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), phosphoryl, halogen, hydroxy, carboxyl, amino, ammonium, formyl, or ester (e.g., pentafluorophenyl ester, succinimidyl ester, fluorophenyl ester, or 2,4-dinitrophenyl ester); and

[0129] p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000.

[0130] In some embodiments, R in the compound of formula IV 4 It is a hydroxyl, carboxyl, amino, formyl, or ester group, optionally when R 3 Yes -(CH2CH2R) 5 ) n -, -C1-C6 alkyl or -C1-C6 alkyl-O-. In some embodiments, when R in the compound of formula IV... 3 It is a -C1-C6 alkyl-O- or -(CH2CH2R) 5 ) n - when, where R 5 It's -O-, R 4 It can be hydrogen, alkyl (e.g., methyl or ethyl), phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), or phosphoryl. In some embodiments, when R in the compound of formula IV... 3 It is a -C1-C6 alkyl group or -(CH2CH2R) 5 ) n - when, where R 5 It is -CH2-, R 4 It can be hydroxyl, carboxyl, amino, ammonium, formyl, ester, phosphonyl, or sulfonyl. In some embodiments, R in the compound of formula IV... 4 It is hydrogen, alkyl, phosphonoyl, sulfonyl, phosphatidylcholine, phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester group. In some embodiments, R in the compound of formula IV 4 It is a vinyl, epoxy, mercapto, azide, isocyanate, isothiocyanate, or maleimide group, which may optionally be added and / or provided before and / or through post-polymerization functionalization. In some embodiments, when R in the compound of formula IV... 3 When it is a -C1-C6 alkyl-SO3- or a salt thereof, R 4 It is hydrogen or absent. In some embodiments, R in the compound of formula IV 3It is a salt of -C1-C6 alkyl-SO3- (e.g., sodium salt), and R 4 It does not exist. In some embodiments, R in the compound of formula IV 3 Yes -(CH2CH2R) 5 ) n -

[0131] In some embodiments, the compounds of the present invention may comprise and / or a telechelicer polymer, which is a polymer or prepolymer capable of participating in further polymerization or other reactions via one or more of its reactive end groups. In some embodiments, the compounds of the present invention may comprise and / or a heterotelechelicer polymer, which is a polymer or prepolymer capable of participating in further polymerization or other reactions via reactive end groups at each end of the polymer or prepolymer, and the two reactive end groups are different from each other. In some embodiments, the compounds of the present invention may comprise and / or a homotelechelicer polymer, which is a polymer or prepolymer capable of participating in further polymerization or other reactions via reactive end groups at each end of the polymer or prepolymer, and the two reactive end groups are different from each other. In some embodiments, the compounds of the present invention may comprise and / or a semitelechelicer polymer, which is a polymer or prepolymer capable of participating in further polymerization or other reactions via a reactive end group at one end of the polymer or prepolymer.

[0132] Bioconjugable groups may optionally be present in the compounds of the present invention. The terms "bioconjugable group," "bioconjugable site," or "bioconjugable group" and their grammatical variations refer to portions and / or functional groups that can bind or are used to bind biomolecules (e.g., proteins, peptides, DNA, RNA, etc.). Therefore, "bioconjugable group," "bioconjugable site," or "bioconjugable group" and their grammatical variations do not contain biomolecules. However, in some embodiments, the bioconjugable group is used to bind biomolecules, or the bioconjugable group or its derivatives bind biomolecules (e.g., proteins, peptides, DNA, RNA, etc.). Exemplary bioconjugable groups include, but are not limited to, amines (including amine derivatives), such as isocyanates, isothiocyanates, iodoacetamide, azides, diazonium salts, etc.; acids or acid derivatives, such as N-hydroxysuccinimide esters (more generally, active esters derived from carboxylic acids, such as p-nitrophenyl esters), hydrazides, etc.; and other linking groups, such as aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiols, maleimides, aziridines, acryloyl groups, halogen groups, biotin, 2-iminobiotin, etc.; linking groups (such as those described above) are known and described in U.S. Patent Nos. 6,728,129; 6,657,884; 6,212,093; and 6,208,553. For example, the compounds of the present invention may contain a bioconjugable group comprising a carboxylic acid, and the carboxylic acid may be used for bioconjugation to biomolecules (e.g., by activation with carbodiimide and coupling with amino-substituted biomolecules).

[0133] In some embodiments, the biomolecule may comprise and / or proteins (e.g., antibodies and / or carrier proteins), peptides, DNA, RNA, etc. In some embodiments, the biomolecule may comprise portions (e.g., polymers) that optionally include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) binding sites of the compounds of the present invention. In some embodiments, the biomolecule may 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 distinct molecules, one of which has a specific spatial or polar region on its surface or within a cavity of the molecule that specifically attracts or binds to the other molecule, resulting in an affinity between the two molecules. Members of a specific binding pair may be referred to as the ligand and receptor (anti-ligand). The terms ligand and receptor are intended to encompass the entire ligand or receptor or portions thereof sufficient to cause binding between the ligand and receptor. Examples of 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 avidin; antibody and antigen pairs; enzymes and substrates; drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid chains; nucleic acid chains and complementary oligonucleotides; interleukins and interleukin receptors; and stimulatory factors and their receptors, such as granulocyte-macrophage colony-stimulating factor (GMCSF) and GMCSF receptor, and macrophage colony-stimulating factor (MCSF) and MCSF receptor.

[0134] The compounds of the present invention may comprise a dye (e.g., tetrapyrrole) covalently linked to a portion of a polymer as described herein. In some embodiments, the dye may be covalently linked to an end portion of the polymer. When present, a bioconjugating group may also be covalently linked to a portion of the polymer, such as an end portion of the polymer. In some embodiments, the bioconjugating group is covalently linked to a first end portion (e.g., a first end) of the polymer, and the dye is covalently linked to an opposite end portion (e.g., an opposite end) of the polymer.

[0135] The compounds of the present invention may comprise a dye (e.g., tetrapyrrole) covalently attached to a portion of a polymer, and a bioconjugating group may be covalently attached to a portion of the dye. In some embodiments, the bioconjugating group is covalently attached to a first portion (e.g., a first end) of the dye, and the polymer is covalently attached to a second portion (e.g., a opposite end) of the dye.

[0136] In some embodiments, the compounds of the present invention, or portions thereof, have a non-rigid backbone (e.g., a non-rigid polymer backbone) and / or exhibit conformational flexibility. The conformational flexibility of the molecular chain can be described and quantified by the “relevant length” of the compound or a portion thereof (e.g., a polymer portion). In some embodiments, the relevant length of the compounds of the present invention can be approximately the length of a given carbon-carbon bond.

[0137] The compounds of the present invention may be self-folding, for example, self-folding in water and / or aqueous solutions. As used herein, “self-folding” means a compound transforming from a partially or fully extended or folded structure to a structure in which at least a portion of the extended or folded structure becomes folded upon contact with a solution (e.g., an aqueous solution) or the compound, and the folding is inherent because it occurs spontaneously upon contact with the solution (i.e., without external control or force). In some embodiments, the compounds of the present invention self-fold upon contact with water and / or aqueous solutions. The compounds of the present invention may optionally self-fold into a monomeric micelle structure upon contact with water and / or aqueous solutions. The aqueous solution in which the compounds of the present invention fold can be a buffer solution, such as a phosphate buffer (e.g., phosphate-buffered saline). In some embodiments, the aqueous solution (e.g., an aqueous buffer) in which the compound of the present invention folds may have a low ionic strength; for example, the μ value of the aqueous solution may be 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 compound of the present invention folds may have a μ value of less than about 100 mM. In some embodiments, the aqueous solution in which the compound of the present invention folds may contain 1 M NaCl. In some embodiments, the aqueous solution in which the compound of the present invention folds may contain less than 1 M NaCl, for example less than about 0.75 M, 0.5 M, or 0.25 M NaCl. In some embodiments, the aqueous solution in which the compound of the present invention folds contains 10 mM NaH2PO4 and 150 mM NaCl, and the pH is about 7.35.

[0138] In some embodiments, the compounds of the present invention may be in particulate form. The compounds of the present invention may form particles, for example, upon contact with a solution (e.g., an aqueous solution). In some embodiments, a single (i.e., one) compound may form particles. Thus, the compound and particles are present in a ratio of approximately 1:1 (i.e., one compound per particle).

[0139] The compounds of the present invention may contain a portion of one or more hydrophobic units in the core or internal region of the particle and / or a portion of one or more hydrophilic units in the peripheral or external region (e.g., shell) of the particle. In some embodiments, the particles have a micellar structure (e.g., a monomeric micellar structure). The compounds of the present invention may contain a dye that may be attached to the polymer of the present invention, and when the compound is in a folded structure and / or particulate form (e.g., a monomeric micellar structure), the dye may be encapsulated by a portion of the compound (e.g., a portion of the polymer). In some embodiments, the dye or a portion thereof and one or more hydrophobic units may be present in the core or internal region of the particle, and one or more hydrophilic units may surround the dye and / or one or more hydrophobic units.

[0140] In some embodiments, the hydrophobic units present in the polymers of the present invention may be one or more of the hydrophobic units of Formula III. In some embodiments, one or more of the hydrophobic units comprise alkyl (e.g., dodecyl) side functional groups and / or are formed from compounds of Formula I and / or alkyl acrylate (e.g., dodecyl acrylate) monomers. In some embodiments, the hydrophilic units present in the polymers of the present invention may be one or more of the hydrophilic units of Formula IV and / or may be formed from compounds of Formula II. In some embodiments, one or more of the hydrophilic units comprise nonionic (i.e., neutral / uncharged) side functional groups (e.g., PEG) and / or are formed from nonionic monomers (e.g., PEG acrylate (PEGA)). In some embodiments, one or more of the hydrophilic units comprise ionic (e.g., anionic, charged) side functional groups (e.g., sulfonic acids and / or sulfonates) and / or are formed from ionic monomers (e.g., sulfonate acrylates (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). In some embodiments, the hydrophilic unit is formed from at least two different monomers, such as nonionic (i.e., neutral / uncharged) hydrophilic monomers (e.g., PEG acrylate (PEGA)) and ionic (e.g., anionic, charged) hydrophilic monomers (e.g., sulfonate acrylates (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). As those skilled in the art will understand, monomers containing acids (such as sulfonic acids) may be present in acidic form and / or in their ionic form. In some embodiments, the acid-containing monomer is predominantly (i.e., greater than 50%) in its ionic form. In some embodiments, the ionic hydrophilic monomer is an acid in a deprotonated form (e.g., deprotonated sulfonate acrylate) and / or in a salt form (e.g., sodium sulfonate acrylate, such as 2-acrylamido-2-methylpropanesulfonic acid as a sodium salt).

[0141] In some embodiments, when two or more different hydrophilic units are present in the polymer of the present invention, the ratio of the two or more different hydrophilic units can be varied, for example, from about 10:1 to about 1:10. For example, in some embodiments, the polymer comprises nonionic (i.e., neutral / uncharged) hydrophilic units (e.g., formed from polyethylene glycol-modified methyl acrylate (PEGA)) and ionic (e.g., anionic, charged) hydrophilic units (e.g., formed from sulfonated acrylates (e.g., 2-acrylamido-2-methylpropanesulfonic acid)) in a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (nonionic unit: ionic unit). In some embodiments, the ratio of one or more hydrophilic units to one or more hydrophobic units present in the main chain of the polymer of the present invention can be varied. In some embodiments, the ratio of one or more hydrophilic units to one or more hydrophobic units in the polymer backbone is about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 (hydrophobic unit: hydrophilic unit).

[0142] In some embodiments, the polymer of the present invention comprises about 1% to about 40% hydrophobic units and about 60% to about 99% hydrophilic units based on the total molar amount of the monomers used to prepare the polymer. In some embodiments, the polymer of the present invention comprises about 1%, 5%, 10%, 15%, or 20% to about 25%, 30%, 35%, or 40% hydrophobic units and about 60%, 65%, 70%, 75%, or 80% to about 85%, 90%, 95%, or 99% hydrophilic units based on the total molar amount of the monomers used to prepare the polymer. In some embodiments, the polymer comprises about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% hydrophobic units based on the total molar amount of monomers used to prepare the polymer. In some embodiments, the polymer comprises less than about 30% (e.g., less than about 25%, 20%, 15%, 10%, or 5%) of hydrophobic units based on the total molar amount of monomers used to prepare the polymer. In some embodiments, the polymer comprises about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% hydrophilic units based on the total molar amount of the monomers used to prepare the polymer. In some embodiments, the polymer comprises more than about 70% (e.g., more than about 75%, 80%, 85%, 90%, or 95%) hydrophilic units based on the total molar amount of the monomers used to prepare the polymer.

[0143] The polymers of the present invention may have a weight fraction of hydrophobic units of about 1%, 5%, 10%, 15%, or 20% to about 25%, 30%, 35%, or 40% based on the total weight of the polymer. In some embodiments, the polymer may have a weight fraction of hydrophobic units of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% based on the total weight of the polymer. In some embodiments, the polymer may have a weight fraction of hydrophobic units of less than about 30% (e.g., less than about 25%, 20%, 15%, 10%, or 5%) based on the total weight of the polymer.

[0144] The polymers of the present invention may have a weight fraction of hydrophilic units of about 60%, 65%, 70%, 75%, or 80% to about 85%, 90%, 95%, or 99% based on the total weight of the polymer. In some embodiments, the polymers of the present invention may have a weight fraction of hydrophilic units of about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% based on the total weight of the polymer. In some embodiments, the polymer may have a weight fraction of hydrophilic units greater than about 70% (e.g., greater than about 75%, 80%, 85%, 90%, or 95%) based on the total weight of the polymer.

[0145] In some embodiments, the amount of monomeric micelle structures formed upon contact with the solution is about 50% to about 100%, about 75% to about 100%, about 85% to about 100%, or about 95% to about 100%, optionally measured using a dimensional method (e.g., dynamic light scattering (DLS) spectroscopy). In some embodiments, the amount of monomeric micelle structures formed upon contact with the solution is about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, optionally measured using a dimensional method (e.g., dynamic light scattering (DLS) spectroscopy). The solution in which the monomers are present can be an aqueous solution as described herein, such as an aqueous buffer solution. In some embodiments, the aqueous solution in which the monomers are present is a phosphate buffer solution (e.g., phosphate-buffered saline). In some embodiments, the aqueous solution containing the monomer (e.g., an aqueous buffer solution) has a low ionic strength (e.g., 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 containing the monomer comprises 10 mM NaH2PO4 and 150 mM NaCl, with a pH of about 7.35.

[0146] In some embodiments, diluting a solution containing the compound of the invention in the form of a monomeric micelle structure does not result in a loss of the monomeric micelle structure present in the solution, or the loss is less than about 20%, compared to the amount of monomeric micelle structure present in the solution before dilution. In some embodiments, the amount of monomeric micelle structure present in the solution does not change upon dilution, or changes by less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1%, compared to the amount of monomeric micelle structure present in the solution before dilution.

[0147] In some embodiments, solutions containing the compounds of the invention in the form of monomeric micelles contain less than about 50% (e.g., less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1%) of aggregates. Therefore, at least 50% or more of the compound does not aggregate and can be in the form of monomeric micelles. In some embodiments, diluting a solution containing the compounds of the invention in the form of monomeric micelles results in little or no additional aggregate formation compared to the amount of aggregates present in the solution before dilution. In some embodiments, the amount of aggregates present in a solution containing the compound of the present invention does not change upon dilution, or changes by less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1%, compared to the amount of aggregates present in the solution before dilution. In some embodiments, the diluted solution contains less than about 50% (e.g., less than about 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.1%) of aggregates.

[0148] The compounds of the present invention may have a diameter in water and / or aqueous solutions ranging from about 1 nm to about 50 nm or from about 3 nm to about 30 nm (e.g., when folded, as in a monomeric micelle structure). In some embodiments, the compounds may have a diameter in water and / or aqueous solutions of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nm (e.g., when folded, as in a monomeric micelle structure). In some embodiments, the compounds of the present invention may be in particulate form (i.e., at least partially folded).

[0149] In some embodiments, the compounds of the present invention are crosslinked, optionally wherein the compounds are crosslinked when they have a folded structure. In some embodiments, the compounds of the present invention may be crosslinked in a solution (e.g., an aqueous solution) and / or may be crosslinked with a crosslinking agent. Crosslinking the compounds of the present invention may include linking two or more portions and / or functional groups (e.g., side functional groups) of one or more hydrophobic units and / or one or more hydrophilic units together. Crosslinking can provide compounds with a folded structure that cannot unfold without breaking one or more bonds formed by crosslinking. The degree or amount of crosslinking may be controlled, modified, and / or tuned, for example, by the amount of crosslinking agent reacting with the compound. In some embodiments, the step of crosslinking the compounds may include reactions and / or reactive entities (e.g., functional groups) as listed in Table 1.

[0150] Table 1: Exemplary crosslinking reactions and functional groups.

[0151] reaction Functional groups polymerization Olefins polymerization acrylate Thiol-ene reaction Thiol group + olefin azide-acetylene reaction Azide group + alkyne Thiol-maleimide reaction Thiol group + maleimide Hydroxyl + glutaraldehyde hydroxyl + aldehyde amine + glutaraldehyde amino + aldehyde Disulfide formation Thiol + Thiol amide formation amine + carboxylic acid ester formation Hydroxyl + carboxylic acid Acetylurea formation Carbodiimide + Carboxylic Acid hydrazone formation Acylhydrazine + aldehyde

[0152] In the compounds of the present invention, the fluorescence quantum yield of the dye may be reduced by about 10% or less (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less) when the compound is present in water and / or aqueous solutions, compared to the fluorescence quantum yield of the dye when the compound is present in a hydrophobic solvent (e.g., toluene). When the compounds of the present invention are bioconjugated to biomolecules (e.g., proteins), the fluorescence quantum yield of the dye may be the same as or substantially the same as the fluorescence quantum yield of the dye in water and / or hydrophobic solvents (e.g., within ±20%). In some embodiments, if the fluorescence quantum yield of the dye is 1.00 (theoretical maximum), a reduction of 10-fold or less (e.g., about 10, 9, 8, 7, 6, 5, 4, 3, 2-fold or less) is acceptable.

[0153] In some embodiments, the compounds of the present invention are water-soluble. The solubility of the compounds in water at room temperature can be in the range of about 1 mg / mL to about 10 mg / mL. In some embodiments, the solubility of the compounds in water at room temperature is about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / mL.

[0154] In some embodiments, the compounds and / or particles of the present invention are dilution-resistant. As used herein, “dilution-resistant” means that the compounds and / or particles retain their structure and / or properties. In some embodiments, dilution-resistant means that the compounds and / or particles retain a folded structure (e.g., a monomeric micelle structure), which can be determined by measuring the diameter of the particles before and after dilution, and the diameter after dilution can be maintained within ±50%, 40%, 30%, 20%, 10% or less of the diameter before dilution. In some embodiments, dilution-resistant means that the fluorescence quantum yield of the dye after dilution is within ±50%, 40%, 30%, 20%, 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 retain a folded structure when diluted to up to 25x, 50x, 75x or 100x or when diluted to submicromolar concentrations.

[0155] Methods for preparing the compounds and / or compositions of the present invention are provided according to some embodiments of the invention. In some embodiments, the method for preparing the compounds 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 bioconjugating group (e.g., a biocompatible group) 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, by condensation reactions (e.g., reactions with diols and diacids) and / or living radical polymerization (e.g., atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT)). In some embodiments, the hydrophobic monomer and the hydrophilic monomer are polymerized using a method for providing copolymers, wherein one or both end groups of the copolymer are reactive (i.e., one or both end groups of the copolymer are capable of participating in further polymerization or reaction), and the two end groups may be the same or different. In some embodiments, hydrophobic and hydrophilic monomers are polymerized 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 copolymers. In some embodiments, hydrophobic and hydrophilic monomers are polymerized via living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT reagent (e.g., a thiocarbonyl sulfide compound).

[0156] In some embodiments, attaching a dye to a first portion of the copolymer may include reacting a dye-containing monomer with hydrophobic monomers and / or units and / or hydrophilic monomers and / or units. Therefore, in some embodiments, the step of attaching the dye to the copolymer may occur during or after the polymerization step. In some embodiments, the method includes reacting a dye-containing monomer with one or more (e.g., two or three) hydrophobic monomers and / or units and / or one or more (e.g., two or three) hydrophilic monomers and / or units during the step of polymerizing the hydrophobic and hydrophilic monomers. 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 said initiator contains the dye. In some embodiments, the polymerization of one or more hydrophobic monomers and / or one or more hydrophilic monomers occurs via living radical polymerization (e.g., RAFT) in the presence of a radical initiator and a RAFT reagent, optionally wherein said RAFT reagent contains the dye.

[0157] When copolymers are used for direct dye linking or bioconjugation, exemplary terminal functional groups that the copolymer may contain include, but are not limited to, those described in Table 2. These terminal functional groups are not side group functional groups, but may be present at either end of the copolymer.

[0158] Table 2: Exemplary terminal functional groups (FG) on copolymers and dyes or biomolecules, along with exemplary bonds and chemistry.

[0159]

[0160] Some functional groups may be unstable under certain polymerization conditions. Therefore, in some embodiments, functional groups may be introduced in a protected form. As a result, these functional groups can be used for dye linking or bioconjugation upon deprotection. Exemplary forms of protection for certain functional groups include, but are not limited to, those listed in Table 3.

[0161] Table 3: Exemplary forms of protection for certain functional groups.

[0162]

[0163] In some embodiments, a portion of the copolymer (e.g., the terminal or end portion) may contain a halogen group (e.g., Cl, Br, I). The halide portion of the copolymer may be derivatized with a nucleophile or end-capping agent to generate a functional group for dye linking or bioconjugation. In some embodiments, a portion of the copolymer (e.g., the terminal portion) may contain a thiol group, which may be derivatized with a reagent containing a thiol reactive group to generate a functional group for dye linking or bioconjugation. Examples of thiol reactive groups include, but are not limited to, halides (e.g., bromine, chlorine, iodine), alkynes, aldehydes, vinyl ketones, and / or maleimide functional groups. All 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.

[0164] Table 4: Exemplary terminal functional groups (FG) on the derived copolymers and on the dyes or biomolecules, along with exemplary bonds and chemistry.

[0165]

[0166] Polymerizing hydrophobic and hydrophilic monomers (optionally via ATRP or RAFT) can include polymerizing the hydrophobic and hydrophilic monomers at a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (one or more hydrophobic monomers: one or more hydrophilic monomers). In some embodiments, the ratio may be 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, one or more hydrophobic monomers are polymerized with two or more different hydrophilic monomers (optionally via RAFT or ATRP) at a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (one or more hydrophobic monomers: one or more hydrophilic monomers). For example, in some embodiments, the first hydrophilic monomer may be ionic (e.g., sulfonate acrylate monomers (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or sulfonate monomers), and the second hydrophilic monomer may be nonionic (e.g., glycol acrylates (e.g., polyethylene glycolated methyl acrylate)). The ratio of the first hydrophilic monomer to the second hydrophilic monomer may vary (e.g., the ratio of the first hydrophilic monomer to the second hydrophilic monomer may be about 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, or 1:6).

[0167] Exemplary catalysts that can be used in the methods 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) [RuCp*Cl(PPh3)2], chloro(cyclopentadienyl)bis(triphenylphosphine)ruthenium[RuCpCl(PPh3)2], dihydrotetrahydrotetra(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 cyclopentadienyldicarbonyliron dimer. In some embodiments, copper complexes generated in situ using copper salts and ligands can be used, and exemplary copper salts include, but are not limited to, cuprous chloride, cuprous bromide, cuprous trifluoromethanesulfonate, cuprous hexafluorophosphate, and cuprous acetate. Exemplary nitrogen-based ligands include, but are not limited to, 2,2'-bipyridine and its derivatives, 1,10-phenanthroline and its derivatives, stigmine 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 tetra(triphenylphosphine)nickel [Ni(PPh3)4]. An exemplary palladium complex is tetra(triphenylphosphine)palladium [Pd(PPh3)4]. An exemplary rhodium complex is tri(triphenylphosphine)rhodium bromide. An exemplary rhenium complex is dioxobis(triphenylphosphine)rhenium iodide. In some embodiments, the catalyst is pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride.

[0168] A co-catalyst 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 co-catalyst may be present, and it may be 4-(dimethylamino)-1-butanol.

[0169] In some embodiments, the method of the present invention includes optionally hydrolyzing the copolymer 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 a dye with the formyl group of the copolymer to optionally form an hydrazone bond between the dye and the copolymer via aldehyde-hydrazide chemistry, thereby attaching the dye to the first portion of the copolymer. In some embodiments, biomolecules may be linked by reacting the formyl group with an amino group on a bioconjugating group via reductive amination.

[0170] In some embodiments, the method of the present invention includes reacting the copolymer with mercaptoacetic acid and triethylamine to provide a carboxymethyl sulfide group at a second portion (e.g., a second end) of the copolymer. The carboxymethyl sulfide group can be derived to provide the second portion of the copolymer. N 2-Hydroxysuccinimide ester. Biomolecules (e.g., avidin) can be attached to the second part of the copolymer. N -Hydroxysuccinimide ester.

[0171] In some embodiments, the method of the present invention includes reacting the copolymer with sodium azide to provide an azide group, and optionally attaching the dye to the azide group via copper-catalyzed azide-acetylene chemistry.

[0172] In some embodiments, the method of the present invention includes RAFT polymerization. In some embodiments, RAFT polymerization is carried out in the presence of a free radical initiator (e.g., AIBN) and a RAFT reagent (such as a thiocarbonyl sulfide compound). Other examples of RAFT reagents, but not limited to, are dithioesters, dithiocarbamates, trithiocarbonates, dithiobenzoates, and / or xanthates.

[0173] In some embodiments, the method of the present invention includes cleaving the thiocarbonyl thiofunctionality present at the ends of the copolymer obtained using RAFT polymerization. Such cleavage can be performed using any general method known in the art. For example, in some embodiments, the thiocarbonyl thiofunctionality is cleaved by ammonolysis, for example, in the presence of ethanolamine, to provide free thiols. In some embodiments, the free thiols can be coupled with a dye containing maleimide functionality, thereby attaching the dye to a first portion (e.g., the ends) of the copolymer. In some embodiments, biomolecules can be attached to the free thiol groups of the first portion (e.g., the ends). In some embodiments, biomolecules can be attached to opposite ends of the polymer.

[0174] According to some embodiments, the compounds and / or compositions of the present invention can be used in flow cytometry. Flow cytometry is known and 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 (e.g., cells) to be detected are labeled with a luminescent compound (such as the compounds of the present invention) for detection. Labeling can be performed by any suitable technique, such as binding the luminescent compound (e.g., the compounds of the present invention) to the particles or cells, for example by using antibodies that specifically bind to the particles or cells, by taking up or internalizing the luminescent compound into the cells or particles, by non-specifically adsorbing the luminescent compound onto the cells or particles, etc. The compounds described herein can be used as such luminescent compounds in flow cytometry techniques (including fluorescence activated cell sorting or FACS) that can be performed according to known techniques or variations thereof, which will be apparent to those skilled in the art based on this disclosure.

[0175] In some embodiments, a method for detecting cells and / or particles using flow cytometry is provided, the method comprising labeling cells and / or particles with the compounds of the present invention, and detecting the compounds by flow cytometry, thereby detecting the cells and / or particles.

[0176] In some embodiments, a method is provided for detecting tissues and / or reagents (e.g., cells, infectious agents, etc.) in a subject, the method comprising: administering the subject a compound and / or composition of the present invention, optionally wherein the compound is associated with the tissue and / or reagent; and detecting the compound in the subject, thereby detecting the tissue and / or reagent.

[0177] 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 involving light and photosensitive chemicals (e.g., the compounds of the present invention), which are 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 applied in photodynamic therapy (PDT) to destroy tumors, various forms of energy are within the scope of the present invention, as understood by those skilled in the art. Such forms of energy include, but are not limited to, heat, sound waves, ultrasound, chemical, light, microwaves, ionizing (e.g., X-rays and gamma rays), mechanical, and / or electrical energy. For example, sonokinetic inducing or activating agents 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 cancer drugs used in the presence of ultrasound therapy, such as daunorubicin and doxorubicin (see Yumita et al., Japan J. Hyperthermic Oncology 3(2):175-182 (1987)).

[0178] Examples of processing areas for PDT and / or PDI include, but are not limited to, the following:

[0179] (i) Management of opportunistic infections. The compounds, compositions, and / or methods of the present invention can be used for prophylactic treatment (PDT) of opportunistic infections, particularly soft tissue PDT. For antimicrobial treatment (via PDT) of infections (particularly wound infections), the infecting organism may include (as non-limiting examples) Staphylococcus aureus, Pseudomonas aeruginosa, and / or Escherichia coli. In hospital-acquired infections, Pseudomonas aeruginosa causes 8% of surgical wound infections and 10% of bloodstream infections. In some embodiments, the subject is an immunocompromised subject, such as those with AIDS and / or those undergoing immunosuppressive treatment.

[0180] (ii) Treatment of burns. Infections with Staphylococcus aureus and Gram-positive bacteria are particularly prevalent in burns (Lambrechts, 2005). Multidrug resistance of Staphylococcus aureus presents a significant medical challenge. In this regard, the compounds, compositions, and / or methods of the present invention can be used to treat opportunistic infections in burns.

[0181] (iii) septicemia.The compounds, compositions, and / or methods of this invention can be used in PDT treatment of subjects suffering from opportunistic infections of Vibrio vulnificus. Vibrio vulnificus, a Gram-negative bacterium, causes primary sepsis, wound infections, and / or gastrointestinal diseases in humans.

[0182] (iv) ulcer. The compounds, compositions, and / or methods of the present invention can be used for PDT treatment of ulcer-causing bacteria (Helicobacter pylori). In clinical practice, the treatment can be carried out in any suitable manner, for example by inserting an optical fiber (similar to an endoscope but provided for delivering red or near-infrared light) into the stomach and / or the affected area.

[0183] (v) Periodontal disease. The compounds, compositions, and / or methods of this invention can be used in photodynamic therapy (PDT) to treat periodontal disease (including gingivitis). Periodontal disease is caused by the overgrowth of bacteria such as the Gram-negative anaerobic bacterium *Porphyromonas gingivalis*. As with many PDT treatments, targeting or solubilizing the organism with a photoactive substance is necessary for the proper delivery of the photoactive substance to the desired cells. Oral pathogens of interest for targeting include, but are not limited to, *Porphyromonas gingivalis*, *Actinomyces surae*, etc. Actinobacillus actinomycetemcomitans Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, and Fusobacterium polymorphum subspecies ( Fusobacterium nucleatum subsp. Polymorphum ( ), adhesive actinomycetes and streptococci. For such applications, the compounds and / or compositions of the present invention can be administered topically (e.g., as a mouthwash or gargle), followed by light delivery using an external device, an in-mouth appliance, or a combination thereof.

[0184] (vi) Atherosclerosis. The compounds, compositions, and / or methods of this invention can be used in photodynamic therapy (PDT) to treat vulnerable atherosclerotic plaques. Without wishing to be bound by any particular theory, it is believed that invading inflammatory macrophages secrete metalloproteinases that degrade the collagen layer in the coronary arteries, leading to thrombosis, which is often fatal (Demidova and Hamblin, 2004). Chlorophyll targeting such inflammatory macrophages can be used in PDT for vulnerable plaques.

[0185] (vii) Cosmetic and dermatological applications.The compounds, compositions, and / or methods of this invention can be used in phototherapy (PDT) to treat various cosmetic dermatological problems such as hair removal, psoriasis treatment, and / or removal of skin discoloration. Currently, ruby ​​lasers are used for hair removal; in many laser treatments, melanin is the photosensitive chromophore. Such treatments are quite effective for people with dark hair and fair skin. The compounds, compositions, and / or methods of this invention can be used as near-infrared photosensitizers for hair removal, capable of targeting chromophores with more specific and / or sharp absorption bands.

[0186] (viii) Acne. The compounds, compositions, and / or methods of the present invention can be used in photodynamic therapy (PDT) to treat acne. Acne vulgaris is caused by Propionibacterium acnes, which infects the sebaceous glands; approximately 80% of young people are affected. Here, the increasing resistance of bacteria to antibiotic treatment again leads to outbreaks of difficult-to-treat acne. Current PDT treatments for acne typically rely on the addition of aminolevulinic acid, which is converted into free basal porphyrin in the hair follicle or sebaceous gland. The compounds and / or compositions of the present invention can be administered to subjects topically or parenterally (e.g., by subcutaneous injection), depending on the specific condition.

[0187] (ix) Infectious diseases. The compounds, compositions, and / or methods of the present invention can be used in photodynamic therapy (PDT) to treat infectious diseases. For example, cutaneous leishmaniasis and subcutaneous leishmaniasis, which are prevalent in the Mediterranean and Middle East, are currently treated with arsenic-containing compounds. PDT has recently been used, in at least one instance, to rationally act on human subjects. The use of the compounds and / or compositions of the present invention is equally useful and potentially offers advantages such as ease of synthesis and better spectral absorption properties.

[0188] (x) Tissue sealing agent. The compounds, compositions, and / or methods of the present invention can be used in photodynamic therapy (PDT) as tissue sealants in subjects who require them. Photoactivated tissue sealants are attractive for closing wounds, adhesive tissues, and / or closing defects in tissues. There are many applications where the use of sutures and / or staples is undesirable, and the use of such mechanical closure methods often results in infection and / or scarring.

[0189] (xi) Excessive sexual characteristics. The compounds, compositions, and / or methods of the present invention can be used in PDT to treat proliferative diseases and / or cancers, including skin cancer, lung cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, ovarian cancer, basal cell carcinoma, leukemia, lymphoma, squamous cell carcinoma, melanoma, macular stage cutaneous T-cell lymphoma, and / or Kaposi's sarcoma.

[0190] During photodynamic therapy, the compounds of the present invention are administered to a subject who requires them (e.g., a subject suffering from any of the aforementioned diseases). The administered compound can be associated with diseased tissue present within the subject, and exposure of the subject to a suitable light source emitting appropriate wavelength and intensity can activate the compound (e.g., release reactive oxygen species (ROS)) into the diseased tissue, 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).

[0191] In some embodiments, methods of using the compounds of the present invention in photoacoustic imaging are provided. According to some embodiments, the methods of the present invention include a method of performing photoacoustic imaging. Photoacoustic imaging (PAI) is attractive in that it is independent of light emission used for detection (Haisch, C., Quantitative analysis in medicine using photoacoustic tomography. Anal. Bioanal. Chem. 2009, 393, 473-479; Cox, B.; Laufer, JG; Arridge, SR; Beard, PC Quantitative spectroscopic photoacoustic imaging: a review. J. Biomed. Opt. 2012, 17, 061202). Light emission can be affected by light scattering. In PAI, laser irradiation (e.g., optionally with a non-ionizing laser pulse) is followed by thermoelastic expansion and ultrasonic pressure waves. Detection of the ultrasonic pressure waves can be achieved using conventional ultrasonic detectors. Essentially, ultrasonic imaging can be performed using a laser input. Notably, unlike X-ray imaging methods, PAI is independent of ionizing radiation.

[0192] The method of the present invention may include administering a subject a compound and / or composition of the present invention, optionally wherein the compound binds to tissues and / or cells in the subject; irradiating at least a portion or part of the subject with a laser, optionally wherein the portion or part of the subject contains a compound of the present invention; and imaging at least a portion or part of the subject, optionally wherein the imaging includes ultrasound imaging.

[0193] PAI can be performed without the use of any exogenous contrast agents or chemical probes. In this case, different absorptions of endogenous chromophores in natural tissues produce different signals. For example, the absorption of hemoglobin facilitates the depiction of the presence of blood vessels. However, hemoglobin has a low molar absorption coefficient and may not be sufficient for clear depiction in deep tissues. In such cases, the use of a contrast agent 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.

[0194] Various substances have been tested for use as contrast agents in photoacoustic imaging (PAI). Examples of dyes used in PAI include, but are not limited to, gold nanomaterials, carbon nanotubes, porphyrins in liposomes, semiconductor polymers, and naphthalocyanines (Chitgupi, U.; Lovell, JF 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 compounds of the present invention and / or the dyes present in the compounds of the present invention have the photophysical property that, upon absorption of light, the dye / compound immediately and quantitatively relaxes to the ground state without emitting light or forming any metastable state with a significant lifetime. In other words, the yield of internal conversion (i.e., without radiative decay) should be quantifiable, and ideally, the rate of internal conversion should be exceptionally fast, with an excited-state lifetime of less than 1 picosecond. This specification primarily addresses the molecular photophysics aspect of "photoacoustic conversion efficiency" (Cheng, K.; Cheng, Z. Near infrared receptor-targeted nanoprobes for early diagnosis of cancers. Curr. Med. Chem. 2012, 19, 4767-4785). The appeal of this rapid and quantifiable internal conversion is the conversion of all absorbed light into heat, i.e., thermal expansion that produces ultrasound. One research group termed such contrast agents "sonochromes" (Duffy, MJ, et al., Towards optimized naphthalocyanines as sonochromes for photoacoustic imaging in vivo. Photoacoustics 2018, 9, 49-61) to distinguish them from the more generally known luminescent dyes or fluorescent dyes or luminescent bodies, all of which imply the emission of light after absorbing incident light. In some embodiments, the compounds of the present invention are and / or contain sonochrome.

[0195] In some embodiments, the compounds of the present invention and / or the dyes present in the compounds of the present invention absorb red or near-infrared (NIR) light. For example, in some embodiments, the compounds of the present invention can be used for imaging deep tissues, where absorption in the red or near-infrared (NIR) region is desirable because this region provides an optical window that allows light to pass through. At shorter wavelengths, absorption by endogenous chromophores (e.g., hemoglobin, melanin) can occur; at longer wavelengths, scattering of light by the overtone bands of water can be observed. In some embodiments, the compounds of the present invention and / or the dyes present in the compounds of the present invention absorb red or NIR light, and the molar absorptivity is as large as possible to produce high sensitivity, for example, a molar absorptivity value of 1,000 MΩ. -1 cm -1 10,000M -1 cm -1 100,000M -1 cm -1 Or even larger. In some embodiments, dihydroporphyrin exhibits approximately 10,000 M. -1 cm -1 To approximately 100,000M -1 cm -1 The Qy band molar absorption coefficient ranges, and in some embodiments, chlorophyll exhibits approximately 50,000 M. -1 cm -1 Approximately 200,000M -1 cm -1 The molar absorption coefficient of the Qy band in the range.

[0196] In some embodiments, the method of the present invention provides multi-wavelength multiplexing. Multi-wavelength multiplexing can be achieved by using two or more absorbers as PAI contrast agents, all exhibiting quantitative (or near-quantitative) internal switching, wherein the two or more absorbers are two or more different compounds of the present invention. The two or more different compounds of the present invention can have substantially 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, detecting the generated ultrasound as each spectrally different contrast agent is absorbed sequentially. Alternatively, a set of multiple lasers can be used, each dedicated to a different PAI contrast agent.

[0197] In some embodiments, the dye present in the compounds of the present invention comprises dihydroporphyrin or chlorophyll, optionally wherein the compound is used in the method of the present invention for photoacoustic imaging (PAI). Given the strong and sharp long-wavelength (Qy) absorption bands, dihydroporphyrin and / or chlorophyll may be ideal for photoacoustic imaging. Dihydroporphyrin and / or chlorophyll can be modified to produce high yields of internal conversion and / or packaged in a manner that allows for dissolution in aqueous media.

[0198] For example, tetrapyrrole macrocycles that are fluorescent in their free base form can become non-fluorescent by metallization with suitable metals. Tetrapyrroles include porphyrins and hydride porphyrins; the latter include dihydroporphyrin and chlorophyll. A true “periodic table of metal tetrapyrroles” exists, and extensive work has been done on the preparation and study of metal tetrapyrroles over the past century. Metals that provide non-fluorescent 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, pp 1–165). Examples of metals that can provide non-fluorescent tetrapyrrole chelates (valences not shown for clarity) include, but are not limited to, Fe, Co, Ni, Cu, Zr, Ru, and lanthanides. In some embodiments, the dye present in the compounds of the present invention is a tetrapyrrole macrocycle containing iron. Given the presence of iron as a naturally occurring component of human metabolism, the extensive research already conducted on tetrapyrrole iron (considering the fact that heme is an iron chelate of protoporphyrin IX), and the particularly short excited-state lifetime of iron porphyrins, iron may be particularly attractive. In some embodiments, the compounds of the present invention comprise ferric dihydroporphyrin or ferric chlorophyll. In some embodiments, the method of the present invention comprises administering a subject a compound of the present invention containing ferric dihydroporphyrin or ferric chlorophyll as a PAI contrast agent, and performing photoacoustic imaging. In some embodiments, the dye present in the compounds of the present invention is a tetrapyrrole macrocycle comprising copper (e.g., Cu(II)). In some embodiments, the dye present in the compounds of the present invention comprises 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 comprises iron (e.g., Fe(II)) and is optionally used for oxygen sensing.

[0199] In some embodiments, the compounds of the present invention comprise sterically hindered and / or non-forming μ-oxodimers of Fe(III)tetrapyrrole. In some embodiments, the compounds of the present invention comprise Fe(III)tetrapyrrole. It is reasonable to mention that Fe(II)tetrapyrrole can coordinate with molecular oxygen and, if sterically hindered, can induce a chemical reaction leading to μ-oxodimers of Fe(III)tetrapyrrole. Conversely, Fe(III)tetrapyrrole does not coordinate with molecular oxygen and does not undergo μ-oxodimer formation. Fe(III)tetrapyrrole is the preferred oxidation state of iron tetrapyrrole when formed under aerobic conditions. Various long-established methods can be used to form Fe(III)tetrapyrrole and to convert Fe(II)tetrapyrrole to the corresponding Fe(III)tetrapyrrole.

[0200] Free base tetrapyrrole can provide a certain amount of fluorescence (e.g., quantum yield up to ~10%), a certain amount of triplet formation (e.g., quantum yield up to ~70%), and the remainder of internal transformation (e.g., quantum yield up to ~20%). As mentioned above, a convenient method for achieving a quantum yield of ~100% for internal transformation (i.e., no radiative decay) is to metallize tetrapyrrole with a metal through one or more mechanisms, causing the excited state to relax rapidly and substantially quantitatively to the ground state. An alternative method to promote internal transformation for radiative decay (i.e., fluorescence) and intersystem crossing (i.e., triplet formation) is to attach suitable substituents to tetrapyrrole. Typical substituents are those that cause spin-orbit coupling, such as heavier halogens, including bromine, iodine, and astatine. Thus, in some embodiments, the introduction of one or more halogens into the dyes and / or compounds of the present invention can be used alone or in conjunction with a metal that itself provides limited luminescence, thereby providing rapid and substantially quantitative relaxation to the ground state. These metals include many metals in the periodic table. The method of tetrapyrrole metallization is well known (Buchler, JW Static coordination chemistry of metalloporphyrins. In Porphyrins and Metalloporphyrins; Smith, KM (Ed.), 1975, Elsevier Scientific Publishing Co.: Amsterdam, pp 157–231; Sanders, JKM, et al., Axial coordination chemistry of metalloporphyrins. In The Porphyrin Handbook; Kadish, KM; Smith, KM; Guilard, R. (Eds.), Vol. 3, 2000, Academic Press: San Diego, pp 1–48). Because it is well known that heavy atoms attached to aromatics induce rapid relaxation of excited states, various heavily substituted aromatics are excellent candidates for PAI according to the method of the present invention. In some embodiments, the compounds of the present invention comprise tetrapyrrole (e.g., tetrapyrrole with heavy atom substituents around the macrocycle and / or a metal providing a non-luminescent central chelate). This tetrapyrrole (e.g., dihydroporphyrin or chlorophyll) can provide many possible narrow-band absorptions across the red and NIR spectral regions.

[0201] While various mechanisms by which excited states of compounds can rapidly and substantially quantitatively revert to the ground state have been described, the present invention is not limited thereto, and other mechanisms known in the art may be used. For example, such mechanisms may arise from (1) a high internal conversion rate relative to the rate of radiative decay and intersystemic crossover; (2) a high rate of intersystemic crossover relative to the rate of radiative decay and internal conversion, followed by immediate and nonradiative decay from the excited multistate to the ground state; and / or (3) a high charge transfer rate relative to all other rates of reduction in the number of excited-state particles, followed by charge recombination quantitatively leading to the ground state. Another example is the deformation of macrocyclic structures away from their substantially planar nature. 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 produce tetrapyrroles, which exhibit excited states with extremely short lifetimes and substantially quantitative relaxation to the ground state. Rapid and near-quantitative relaxation to the ground state can provide substances referred to herein as “non-fluorescent” molecular entities, which can be used for PAIs.

[0202] The compounds of the present invention can package a metal tetrapyrrole, optionally for use in PAI. The metal tetrapyrrole may have biocombinable groups that can be used to attach the metal tetrapyrrole to the polymer described herein to provide the compounds of the present invention. Therefore, the compounds of the present invention may comprise a single metal tetrapyrrole. In some embodiments, the compounds of the present invention can maintain the inherent spectral characteristics (e.g., absorption spectrum, fluorescence spectrum, fluorescence quantum yield, etc.) of the dye by packaging the dye within a portion of the compound (e.g., within a polymer portion), optionally without alteration due to interaction with external entities (e.g., other dyes and / or biological material (e.g., cellular components, proteins, etc.)). Including a single dye (e.g., Fe(III) tetrapyrrole) in the compounds of the present invention can maintain the inherent absorption spectrum of the dye.

[0203] According to some embodiments, the dye present in the compounds of the present invention can be a non-fluorescent molecular entity (e.g., a non-fluorescent and / or non-phosphorescent molecular entity), optionally wherein the compound is used for PAI. The dye can have a rapid optical-to-acoustic transition. In some embodiments, the dye is a non-fluorescent molecular entity and has a short excited-state lifetime, optionally wherein the excited-state lifetime is in the sub-picosecond range. Upon irradiation, the excited state can immediately return to the ground state, releasing heat. The heat generates "sound waves," which can be detected by a microphone. The structure of the compounds of the present invention can protect the dye from physiological environmental influences and / or be suitable for methods of performing PAI.

[0204] In some embodiments, the compounds of the present invention provide means for packaging hydrophobic chromophores that may allow for high solubility in water, and / or means for preventing chromophore aggregation, since aggregation can alter the appearance of the absorption band, including wavelength position, molar absorption coefficient, and band width.

[0205] The invention is explained in more detail in the following non-limiting embodiments. Example

[0206] Example 1 - Single polymer encapsulation of a single hydrophobic chromophore

[0207] Random copolymers with side-group PEGylated chromophores and polymeric micelles containing hydrophobic fluorophores were investigated. Ultimately, a design requiring hetero-truncate amphiphilic random copolymers was determined, which were derived by living radical polymerization of two acrylate monomers—a hydrophilic (side-group PEG-6) monomer and a hydrophobic (dodecyl) monomer—in ethanol at a 3:1 ratio (using an initiator substituted with RuCp*Cl(PPh3)2, 4-(dimethylamino)-1-butanol, and an acetal at 40 °C. The acetal was hydrolyzed and subsequently reacted with a hydrophobic dihydroporphyrin-hydrazide to provide a polymer with a single dihydroporphyrin-hydrazone (i.e., folded bodies or single-chain nanoparticles, abbreviated as scNp). The dihydroporphyrin polymers in aqueous solution exhibited sharp absorption / fluorescence bands and no reduction in fluorescence quantum yield compared to dihydroporphyrin in toluene. This method separates chromophore selection and water-soluble strategies into different domains, the latter of which is now quite straightforward to implement.

[0208] Three hydrophobic dye-labeled amphiphilic copolymers F1-F3 with self-folding properties were synthesized and spectroscopically characterized. The structural features of the hydrophobic dyes and polymer backbones are shown in Scheme 1. The amphiphilic copolymers consist of hydrophilic segments (PEG segments) and hydrophobic segments (dodecyl segments) in a 3:1 ratio, with a molecular weight of approximately 120 kDa. As random block copolymers, these copolymers can self-fold in water to generate hydrophobic centers, encapsulating the hydrophobic dyes and thereby protecting them from aggregation. Three hydrophobic dyes, namely BODIPY, dihydroporphyrin, and phthalocyanine, with different molecular sizes and absorption wavelengths (540 nm, 640 nm, and 700 nm, respectively), were loaded onto the same polymer backbone, and spectroscopic measurements were performed. While not wishing to be bound by any particular theory, the unique fluorescence properties obtained in water of the dye-loaded copolymers suggest that the effectiveness of dye encapsulation can depend on the molecular size of the dyes and the length of the copolymer backbone.

[0209]

[0210] Option 1. Copolymers F1-F3 of the target amphiphilic loaded dye.

[0211] Synthesis of hydrophobic fluorophores. Typically, the dye hydrazides used here for dye linkage are prepared from the corresponding carboxylic acid esters via amide formation. Treatment of BODIPY-NHS ester 1, which serves as the activating carboxyl group, with hydrazine hydrate yielded the desired BODIPY-hydrazide D1 in 40% yield (Scheme 2).

[0212]

[0213] Scheme 2. Synthesis of BODIPY-hydrazide D1.

[0214] Iodine dihydroporphyrin 2 was quantitatively converted to methyl ester 3 via carbonyl insertion in the presence of Pd(PPh3)4, methanol, and carbon monoxide (Scheme 3). Methyl ester 3 was then treated with hydrazine hydrate under reflux to produce the desired dihydroporphyrin-hydrazide D2 in 83% yield. It is noted that the reaction needs to be carried out at concentrations below 50 mM, as higher concentrations lead to the reduction of D2 to the corresponding chlorophyll.

[0215]

[0216] Scheme 3. Synthesis of dihydroporphyrin-hydrazide D2.

[0217] Due to the solubility limitations of macrocyclic compounds, the preparation of phthalocyanine-hydrazide D3 requires further effort. In Pd(OAc)2 / P( o In the presence of α-(tol)3, ethynylphthalocyanine 4 is coupled with methyl 3-(4-bromophenyl)propionate to provide methyl ester 5 in 13% yield (Scheme 4). Furthermore, the low solubility of the macrocycle in the reaction system is the reason for the low yield of the Sonogashira coupling reaction. Methyl ester 5 is then treated with hydrazine in a mixture of toluene and methanol to produce the desired hydrazide D3.

[0218]

[0219] Scheme 4. Synthesis of phthalocyanine-hydrazide D3.

[0220] Synthesis of copolymers. Living radical polymerization of monomers PEGA and LA was carried out in the presence of RuCp*Cl(PPh3)2 and 4-dimethylaminobutanol using a reported initiator 6 at a ratio of 3:1 (Scheme 5). The resulting copolymer 7 was hetero-clawed, with one end being an acetal and the other a bromide. These two functional groups were derived for dye linking and mounting bioconjugable handles, respectively. The bromide in 7 was replaced with thioglycolic acid, providing a carboxyl group at the copolymer's end to open for bioconjugation. Hydrolysis of the acetal end under acidic conditions yielded a formyl copolymer 8. This copolymer 8 was used as a platform for dye conjugation, and treatment with hydrazides D1-D3, respectively, yielded copolymers F1-F3 for the target dye-loaded copolymers.

[0221]

[0222] Scheme 5. Preparation of amphiphilic copolymers F1-F3 loaded with dyes.

[0223] SEC analysis. Taking F2 as an example, SEC analysis is used to monitor the dye-linking reaction process. (Shown...) Figure 2 The SEC traces indicate size increase when dihydroporphyrin is bonded to the copolymer. Similarly, the molecular weight of copolymer 7 is estimated to be 1.2 × 10⁻⁶ based on SEC analysis. 5 g / mol.

[0224] Absorption and emission spectra were measured. The spectroscopic properties of the target dye-loaded copolymers F1-F3 were then studied in both organic solvents and aqueous solutions. The spectra are shown below. Figure 3 In the middle. For F1 (load BODIPY, Figure 3 (Figure A) and F2 (supported dihydroporphyrin, Figure 3 (Figure B) Both, the absorption spectra of the samples in aqueous solution at μM concentration are comparable to their absorption spectra in organic solutions. The linkage with the 120-kDa amphiphilic copolymer strongly enhances the water solubility of BODIPY D1 and dihydroporphyrin D2 without significantly interfering with the spectroscopic properties. The emission bands of F1 and F2 in water remain the same as those measured in organic solutions, indicating minimal dye-dye interactions involved in F1 and F2 in aqueous solution at μM concentration. However, as the dye molecule size is at its maximum, the phthalocyanine-loaded copolymer F3 provides an absorption spectrum in water that is completely different from its absorption spectrum in toluene. Figure 3 (Figure C) shows completely quenched fluorescence. This negative result may be due to inappropriate copolymer backbone size. Larger polymers, such as phthalocyanine D3, may be needed to encapsulate large hydrophobic chromophores.

[0225] Fluorescence quantum yield. Fluorescence quantum yield values ​​for F1–F3 in water were also measured at room temperature. The data are summarized in Table 5 along with other spectroscopic data. Taking the dihydroporphyrin-linked copolymer F2 as an example, the dye-copolymer conjugate exhibited a fluorescence quantum yield of 0.18 at a concentration of μM in water (item 6), similar to the value in CH2Cl2 solution of dye D2 alone (0.19, item 4). For the BODIPY-labeled copolymer F1 ( Similar results were observed for F1 (0.058, entry 3) and BODIPY dye D1 (0.065, entry 1). These comparisons indicate the absence of dye-dye quenching caused by the aggregation of F1 and F2 in μM aqueous solution. The results demonstrate that amphiphilic copolymers serve as a successful platform for encapsulating hydrophobic chromophores in water when the polymer chain length is appropriate. However, as mentioned above, phthalocyanine-labeled copolymers exhibit completely quenched fluorescence. The longer the polymer chain, the more effective it may be for encapsulating larger chromophores (such as D3). Furthermore, smaller phthalocyanine backbones (e.g., with methyl groups replacing heptyl groups as peripheral groups) can be successfully encapsulated with copolymers of the current length.

[0226] Table 5. Spectroscopic properties of copolymers F1-F3 and chromophores D1-D3.

[0227]

[0228] Experimental Section

[0229] General methods. Unless otherwise specified, all commercially available chemicals were used as is. Reagent-grade solvents (CH₂Cl₂, THF, methanol) and HPLC-grade water were used as is. Unless otherwise specified, NMR data were measured in CDCl₃ solution. Non-commercial compounds 1, 2, and 4 were prepared according to literature procedures. Analytical SEC experiments were performed using a PLgel 10000 Å SEC column, eluted with ACS-grade THF (stabilized with 400 ppm BHT) at 35 °C at a flow rate of 1 mL / min. Samples were detected using an Agilent 1260 infinite refractive index detector. Absorption spectra of the compounds were measured at room temperature using dilute (μmol) solutions of the compounds in UV-transparent (e.g., quartz) cuvettes with solvent blanks on Agilent 8453 and Shimadzu UV1800 instruments.

[0230] 2-[6-( N [-aminocarbamoyl]hex-1-yn-1-yl]-8-trimethyl-4,4-difluoro-4-boron-3a,4a-diaza- s -Dicyclopentadienzobenzene (D1). A solution of 1 (9.0 mg) in THF (500 μL) was treated with hydrazine hydrate (5.3 μL) for 30 min at room temperature. The solution was then concentrated and subjected to chromatographic separation (silica gel, CH3OH / acetic acid = 9:1), yielding a red solid (3.0 mg, 39%): 1H NMR (DMSO- d 6, 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, observation: 449.1 [(M+H)] + ], 429.2[(MF) + ], Calculated value: 448.2 (M = C 25 H 27 BF2N4O).

[0231] 10-Trimethyl-5-(4-methoxycarbonyl)phenyl-18,18-dimethyldihydroporphyrin (3). Toluene and methanol were degassed by bubbling with argon for 1 hour. Iodine dihydroporphyrin 2 (20 mg, 0.030 mmol, 1.0 equivalent) and Pd(PPh3)4 (3.5 mg, 3.0 μmol, 0.10 equivalent) were added to an Erlenmeyer flask with a rubber septum and then evacuated under high vacuum. The flask was then refilled with argon. This evacuation-purging process was repeated three times. Degassed toluene (0.50 mL) and methanol (0.50 mL) and triethylamine (21 μL, 0.15 mmol, 5.0 equivalent) were added to the flask under argon. The solution was degassed again by three freeze-purge-thaw cycles. The flask was evacuated under high vacuum at 77 K and then refilled with carbon monoxide. A CO-filled balloon was also attached to the vial to provide additional pressure. The solution was stirred at 65°C for 23 hours, concentrated, and separated chromatographically (silica gel, hexane / CH2Cl2 = 1:1) to provide a green solid (18 mg, 100%): TLC (silica gel, 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); 13 C 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, Observation value: 592.2851 [(M+H)] + ], Calculated value: 592.2838 (M = C 39 H 36 N4O2); Laboratory (CH2Cl2) 415, 509, 533, 590, 641 nm.

[0232] 5-[4-( N [-aminocarbamoyl]phenyl-10-trimethylmethyl-18,18-dimethyldihydroporphyrin (D2). A solution of dihydroporphyrin 3 (44 mg, 75 μmol, 1.0 equivalent) in THF (1.0 mL) was treated with methanol (1.0 mL) and hydrazine hydrate (0.21 mL, 3.8 mmol, 50 equivalent) at 50 °C for 24 h. [Note: If the concentration is greater than 50 mM, the dihydroporphyrin-hydrazine will be reduced to the corresponding chlorophyll-hydrazine. Chlorophyll can be oxidized back to the desired dihydroporphyrin by treating DDQ (1.0 equivalent) in CH2Cl2 for 30 min at room temperature.] The solution was then diluted with ethyl acetate, washed with water, dried over sodium sulfate, concentrated, and chromatographically separated (silica gel, hexane / EtOAc = 1:2 to CH2Cl2 / CH3OH = 9:1) to provide a green solid (37 mg, 84%). 1 H NMR (400 MHz) δ 8.96 (s, 1H), 8.88 (s, 1H), 8.76 (d, J= 4.5Hz, 1H), 8.75 (d, J = 4.5 Hz, 1H), 8.62 (d, J = 4.7 Hz, 1H), 8.56 (d, J = 4.7Hz, 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 C 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, observation: 593.1 [(M+H)] + ], Calculated value: 592.3 (M = C 38 H 36 N6O).

[0233] 2-[4-(2-methoxy-2-oxoethyl)phenyl]ethynyl-9,10,16,17,23,24-hexaheptaphyllocyanine (5). Following the standard Sonogashira coupling procedure, 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( oThe solution of 3(5.5 mg, 18 μmol) in degassed toluene (6.0 mL) was degassed. The mixture was stirred at 60 °C for 18 hours. The resulting reaction mixture was concentrated and separated by column chromatography using a three-column strategy [(1) silica, CH2Cl2, (2) SEC, toluene, (3) silica, CH2Cl2], yielding 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).

[0234] 2-[4-( N [-aminocarbamoyl]methylphenylethynyl]-9,10,16,17,23,24-hexaheptaphyllocyanine (D3). A solution of 5 (3.0 mg, 2.3 μmol) in toluene (140 μL) was treated with 6.5 μL hydrazine hydrate (55 wt%) and methanol (10 μL). The resulting mixture was stirred at 50 °C for 16 h, followed by the addition of ethyl acetate and water. The organic extract was washed with brine, dried (Na2SO4), and concentrated to provide a green solid, which was used directly in the next synthetic step.

[0235] Example 2

[0236] According to some embodiments of the present invention, general methods for polymer preparation and derivation are shown in Scheme 6 below.

[0237]

[0238] Option 6.

[0239] In the method shown in Scheme 6, the initiator is QX, where X can be a halogen (e.g., Cl, Br, I) or a sulfonate (e.g., trifluoromethanesulfonate), and Q can carry a dye or can carry functional groups and remain intact during polymerization.

[0240] In further derivation, the functional groups required for dye linking can be incorporated (in the Q unit) and used directly before polymerization. Alternatively, after polymerization, the derivatization of Q in polymer I can provide a modified Q (denoted as Q') for dye linking in polymer II.

[0241] In one case, the X-substituent in polymer I provides a link to the biomolecule (the ω-end of the polymer) by direct use. Alternatively, the X-group can be substituted to give a functional group W in polymer II for linking the biomolecule. Examples of W include azide, isocyanate, isothiocyanate, active esters (e.g., pentafluorophenyl ester, succinimidyl ester, 2,4-dinitrophenyl ester), maleimide, vinyl, mercapto, amino, and carboxylic acids. Derivatization of the ω-end of polymer I can be achieved in one or more steps (e.g., nucleophilic substitution and / or deprotection) to give the desired functional group W in polymer II. For prepolymerization methods, the functional group is first attached to the Q unit of the initiator (Scheme 6) and remains intact during polymerization.

[0242] Some examples of Q and QX are shown in Scheme 7. As shown in Scheme 7, Q may include hydroxyl groups, 1,2 carboxyl, 3 amino, 4 Formyl, 4 vinyl, 5,6 Epoxy groups, 7 acid anhydride, 8 Halogenated aryl, 7 ester group 3 or oxazolinyl 8 Vinyl or allyl groups can be installed using an initiator and remain intact during polymerization without causing additional problems during crosslinking. 1,5,6 This can be achieved by selecting appropriate ligands, primarily in the presence of a copper (I) catalyst. However, some functional groups commonly used for dye linkage (e.g., azide groups) or for bioconjugation cannot be installed via prepolymerization methods (shown in Table 6).

[0243]

[0244] Option 7. Functional groups compatible with pre-polymerized assembly.

[0245] Table 6. Commonly used functional groups that need to be installed after polymerization.

[0246]

[0247] Note that the embodiments discussed herein describe the attachment of the dye to the α-end of the polymer and the attachment of the biomolecule to the ω-end of the polymer. However, the use of these two ends can be reversed as needed, so that the biomolecule is attached to the α-end of the polymer and the dye is attached to the ω-end of the polymer.

[0248] References

[0249] (1) Kamigaito, M.; Ando, T.; Sawamoto, M. Metal-Catalyzed LivingRadical Polymerization. Chem.Rev. 2001, 101 , 3689-3745.

[0250] (2) Haddleton, D. M.; Waterson, C.; Derrick, P. J.; Jasieczek, C. B.;Shooter, A. J. Monohydroxy Terminally Functionalized Poly(methylmethacrylate) from Atom Transfer Radical Polymerisation. Chem.Commun. 1997,683-684.

[0251] (3) Zhang, X.; Matyjaszewski, K. Synthesis of Functional Polystyrenesby Atom Transfer Radical Polymerization Using Protected and UnprotectedCarboxylic Acid Initiators. Macromolecules 1999, 32 , 7349-7353.

[0252] (4) Haddleton, D. M.; Waterson, C. Phenolic Ester-Based Initiatorsfor Transition Metal Mediated Living Polymerization. Macromolecules 1999, 32 ,8732-8739.

[0253] (5) Zeng, F.; Shen, Y.; Zhu, S.; Pelton, R. Synthesis andCharaterization of Comb-Branched Polyeletrolytes.1. Preparation of Cationicmacromonomer of 2-(Dimethylamino)ethyl Methacrylate by Atom Transfer RadicalPolymerization. Macromolecules 2000, 33 , 1628-1635.

[0254] (6) Shen, Y.; Zhu, S.; Zeng, F.; Pelton, R. Synthesis of MethacrylateMacromonomers Using Silica Gel Supported Atom Transfer Radical Polymerization. Macromol.Chem.Phys. 2000, 201 , 1387-1394.

[0255] (7) Zhang X.; Xia, J.; Matyjaszewski K. End-Functional Poly(tert-butyl acrylate) Star Polymers by Controlled RadicalPolymerization. Macromolecules 2000, 33 , 2340-2345.

[0256] (8) Malz, H.; Komber, H.; Voigt, D.; Hopfe, I., Pionteck, J.Synthesis of Functional Polymers by Atom Transfer Radical Polymerization. Mac romol.Chem.Phys. 1999, 200 , 642-651.

[0257] Example 3. Example Response

[0258] Exemplary reactions for preparing the compounds of the present invention, including cross-linking, are provided in Scheme 8.

[0259]

[0260]

[0261] Option 8: An exemplary reaction having a crosslinking step.

[0262] Exemplary reactions for preparing the compounds of the present invention, including sulfonation and crosslinking, are provided in Scheme 9.

[0263]

[0264]

[0265]

[0266] Option 9: An exemplary reaction having sulfonation and crosslinking steps.

[0267] Example 4

[0268] Example methods for polymer preparation and derivation according to some embodiments of the present invention are shown in Scheme 10 below.

[0269]

[0270] Scheme 10. Example synthesis of RAFT heteroclinic random copolymers.

[0271] In the method shown in Scheme 10, Z in the RAFT reagent is aryl, alkyl, or thioalkyl, and Q may carry functional groups that remain intact during polymerization.

[0272] In further derivation, the functional groups required for linking to dyes or biomolecules can be incorporated (into the Q unit) and used directly before polymerization. Such functional groups can be initially installed into the Q unit of the RAFT reagent and remain intact during polymerization. Alternatively, derivatization of Q in the synthetic polymer after polymerization can provide modified Q units for dye or biomolecule linking.

[0273] Some examples of Z and Q in RAFT reagents are shown in Figure 1. Examples of Z in RAFT reagents include, but are not limited to, phenyl (optionally substituted) and / or thioalkyl (including branched and / or unbranched C1-C25 thioalkyl).

[0274] Examples of Q in RAFT reagents include, but are not limited to, carboxylic esters, azide groups, hydroxyl groups, N-succinimide groups, vinyl groups, phthalimide groups, and / or biotin groups.

[0275]

[0276] Figure 1. Examples of RAFT reagents with terminal functional groups that can be provided.

[0277] Before attaching dyes or biomolecules to the ends of polymers containing thiocarbonyl thio groups, thiol groups can be released by cleaving the thiocarbonyl thio groups using methods known in the art. The free thiol groups can be directly coupled to the dyes or biomolecules, or they can be further modified with reagent LW to provide end-capped thiols (e.g., thioethers) having suitable functional groups W for coupling with dyes or biomolecules. Reagent LW comprises a thiol reactive group L that reacts with the free thiol group and also acts as a linker L' between the thiol and the functional group W in the end-capped product.

[0278] Examples of L and W in LW are shown in Figure 2. Examples of L groups in reagent LW include, but are not limited to, substituted halides (e.g., substituted benzyl bromides and / or α-acids), substituted alkynes (e.g., substituted benzyl alkynes), substituted vinyl esters (e.g., α-vinyl esters), and / or substituted succinimides (e.g., ethylamine succinimide, ethanol succinimide).

[0279] Examples of functional group W include, but are not limited to, carboxylic acids (e.g., -COOH, -CH2CH2COOH), amino groups (e.g., -NH2, -CH2CH2NH2, optionally having protecting groups: NHBoc, -CH2CH2NHBoc), aldehydes, alcohols (e.g., -CH2CH2OH) and / or alkylated alcohols (e.g., -OCH2CH2OH, -OCH2CH2NHBoc, -OCH2CH2N3, -OCH2C). ≡ CH, -OCH2CH=CH2).

[0280] Derivatization of free thiol groups can be achieved in one or more steps (e.g., nucleophilic substitution and / or deprotection) to give the desired functional group W.

[0281]

[0282] Figure 2. Examples of thiol reactive groups with additional functional groups.

[0283] Another example of RAFT polymerization is shown in Scheme 11. The hydrophobic monomer dodecyl acrylate (LA) is polymerized with the hydrophilic monomer 2-acrylamido-2-methylpropanesulfonic acid as its sodium salt (AMPS) and PEG acrylate (PEGA) in the presence of a RAFT reagent and a free radical initiator to produce a polymer. In some embodiments, one or more functional groups are present (e.g., pre-installed) on the RAFT reagent prior to polymerization. An example of such one or more functional groups is shown in Scheme 11. After polymerization, the pre-installed one or more functional groups will be located at one end of the polymer and can be used for coupling to biomolecules or dyes.

[0284]

[0285] Option 11. Functional groups pre-installed on RAFT reagents.

[0286] References

[0287] (9) Sumerlin, BS; Donovan, MS; Mitsukami, Y.; Lowe, AB; McCormick, CLWater-Soluble Polymers. 84. Controlled Polymerization in AqueousMedia of Anionic Acrylamido Monomers via RAFT. Macromolecules 2001, 34 , 6561-6564.

[0288] (10) Gondi, SR; Vogt, AP; Sumerlin, BSVersatile Pathway to Functional Telechelics via RAFT Polymerization and ClickChemistry. Macromolecules 2007, 40 , 474-481.

[0289] (11) Chong,YK; Krstina, J.; Le, TPT; Moad, G.; Postma, A.;Rizzardo, E.; Thang, SHThiocarbonylthio Compounds [S C(Ph)S−R] in FreeRadical Polymerization with Reversible Addition-Fragmentation Chain Transfer(RAFT Polymerization).Role of the Free-Radical Leaving Group (R). Macromolecules 2003, 36 , 2256-2272.

[0290] (12) Bathfield, M.; D'Agosto, F.; Spitz, R.; Charreyre M.; Delair, T.Versatile Precursors of Functional RAFT Agents.Application to the Synthesisof Bio-Related End-Functionalized Polymers. J. Am. Chem. Soc. 2006, 128 , 2546-2547.

[0291] (13) Patton, D.L.; Advincula, R.C.A Versatile Synthetic Route toMacromonomers via Polymerization. Macromolecules 2006, 39 , 8674-8683.

[0292] (14) Moad, G.; Chong, Y.K.; Postma, A.; Rizzardo, E.; Thang,S.H. Polymer 2005, 46 , 8458-8468.

[0293] (15) Postma, A.; Davis, T.P.; Evans, R.A.; Li, G.; Moad, G.; O'Shea,M.S.Synthesis of Well-Defined Polystyrene with Primary Amine End Groupsthrough the Use of Phthalimido-Functional RAFT Agents. Macromolecules 2006,39 , 5293-5306.

[0294] (16) Hong, CY.; Pan, CY. Direct Synthesis of Biotinylated Stimuli-Responsive Polymer and Diblock Copolymer by RAFT Polymerization UsingBiotinylated Trithiocarbonate as RAFT Agent. Macromolecules 2006, 39 , 3517-3524.

[0295] Example 5. Synthesis of amphiphilic random copolymers by reversible addition-fragmentation chain transfer (RAFT) polymerization.

[0296] A model study for the synthesis of sulfonated amphiphilic random copolymers is shown in Scheme 12. Three monomers were used, one of which was hydrophobic (dodecyl acrylate (LA)) and two of which were hydrophilic (2-acrylamido-2-methylpropanesulfonic acid as sodium salt (AMPS) and PEG acrylate (PEGA)). AMPS could be prepared by alkalizing commercially available 2-acrylamido-2-methylpropanesulfonic acid with sodium hydroxide and / or alkalizing a commercially available sodium salt of 2-acrylamido-2-methylpropanesulfonic acid, which has a small amount of free acid present as a minor contaminant in commercially available AMPS materials. RAFT chain transfer agent 1 was used exactly as obtained in the laboratory. Polymerization was carried out in DMF (80°C) containing AIBN as a radical initiator and mesitylene as an internal standard at different monomer ratios. After polymerization, the crude product was poured into a large excess of diethyl ether to precipitate the polymer. The precipitate was then dialyzed against water to give purified polymer.

[0297]

[0298] Option 12. Synthesize sulfonated amphiphilic random copolymers via RAFT polymerization.

[0299] Dynamic light scattering (DLS) size analysis of amphiphilic polymers. Each polymer was dissolved in a 1.0 M NaCl aqueous solution and passed through a 200 nm membrane filter. The filtrate was examined by DLS to determine the size of the nanoparticles. DLS size data for different polymers are summarized in Table 7. Based on the data, for polymers without PEG groups, a sulfonate to lauryl ratio of 6:1 yielded the best results, providing 65% monomers in aqueous solution. When PEG groups were introduced, the percentage of monomers increased as the ratio of PEG groups to sulfonate groups decreased from 1:1 to 1:5. At AMPS:PEGA:LA = 5:1:1, monomers appeared to be the dominant substance in aqueous solution.

[0300] Table 7. DLS size data for polymers.

[0301]

[0302] Polymer-chromophore conjugates were synthesized via RAFT polymerization. Living radical polymerization of monomers PEGA, LA, and AMPS was carried out in a 1:1:5 ratio (i.e., hydrophilic / hydrophobic ratio = 6:1) using the RAFT reagent 4-cyano-4-(phenylthiocarboxythio)valerate 2 in the presence of the radical initiator 2,2'-azobis(2-methylpropionitrile) (AIBN) (Scheme 13). The resulting polymer 3 was hetero-articulate, containing a carboxyl group at one end and a thiocarbonyl thio group at the other. Ammonolysis of polymer 3 with ethanolamine cleaved the thiocarbonyl group, revealing a free thiol group. The latter, in situ coupling with hydrophobically maleimide-substituted chlorophyll D1, yielded the target polymer-chromophore conjugate F-2.

[0303]

[0304] Option 13. Synthesize polymer-chromophores via RAFT polymerization.

[0305] Dynamic light scattering (DLS) size analysis of polymer-chromophore conjugates. Polymer-chromophore samples were dissolved in 1.0 M NaCl aqueous solution and passed through a 200 nm membrane filter. The filtered solution was examined by DLS to determine the size of the nanoparticles. DLS size data for different polymers are summarized in Table 8. Polymer-chromophore sample F-2 exhibited a monomeric form within a certain concentration range (…). Figure 4 ).

[0306] Table 8. DLS size data of F-2 in aqueous solution.

[0307]

[0308] The absorption and emission spectra of F-2, as well as the fluorescence quantum yield, were measured. The absorption and emission spectra of the target polymer-chromophore conjugate F-2 were measured in both water and buffered aqueous solutions at room temperature. Figure 5 and Figure 6 ) The spectroscopic data and fluorescence quantum yield data are summarized in Table 9.

[0309] The absorption and emission spectra of F-2 in aqueous solution are comparable to those of D1 in toluene, where Q y Absorbance broadened and decreased minimally. The fluorescence yield of F-2 in aqueous media was 93% (in buffer) and 80% (in water) relative to the fluorescence yield of D1 in toluene. These data are consistent with the inconspicuous chromophore aggregation in aqueous media. Individual chromophores are encapsulated in amphiphilic polymers and retain their intrinsic fluorescence upon immersion in an aqueous environment.

[0310] Table 9. Spectroscopic data and fluorescence quantum yield of F-BC in aqueous solution.

[0311]

[0312] Example 6

[0313] Developing novel methods for molecular fluorescence or luminescence in chemical sensing using organic chromophores as recognition units is of great interest, particularly 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 absorption or emission intensity. Structural features that control changes in the wavelength or intensity of absorption or fluorescence include, but are not limited to: double bond twisting, changes in coupling patterns, “heavy” atoms, weak bonds, and opportunities for photoinduced electron transfer (PET) or electron energy transfer (EET) (References 4-10). The advantages of this detection via optical signals include: high sensitivity; “on and off” switchability; qualitative or quantitative analysis; and visual detection (References 11-15).

[0314] Heavy metal ions pose a significant threat to the environment and human health, thus attracting considerable attention from chemists, biologists, environmental scientists, and medical scientists (Reference 16). Due to the considerable challenges involved, the demand for sensitive and selective fluorophore sensors targeting toxic heavy metal ions continues to increase. In 1997, Czarnik and colleagues reported the ring-opening of a spirolactam ring, which induced fluorescence in rhodamine-β-hydrazide for the detection of Cu(II) in aqueous solution (Reference 17). As shown in Scheme 14, the non-fluorescent rhodamine-β-hydrazide undergoes hydrolytic ring-opening catalyzed by a metal cation to yield a conjugated and fluorescent rhodamine structure. The ring opening depends on the nature of the cation. The cations tested in this work 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). Only Cu(II) and Hg(II) showed significant changes in their absorption or fluorescence spectra. The selective detection of Cu(II) was highly sensitive, and quantification was performed using Cu(II) at a concentration of 10⁻⁷ M.

[0315]

[0316] Scheme 14. Hydrolysis of rhodamine hydrazide catalyzed by metal ions.

[0317] Several rhodamine-hydrazide analogs were 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, due to the hydrophobicity of rhodamine-hydrazides, this application in aqueous solutions requires the addition of organic solvents such as acetonitrile and methanol. In this regard, we designed and synthesized Pod-rhodamine to investigate metal ion sensing in pure water without the addition of organic solvents.

[0318] Pod-rhodamine was synthesized by first preparing an amphiphilic random copolymer. The target sulfonated amphiphilic random copolymer was synthesized as shown in Scheme 15.

[0319]

[0320] Scheme 15. Synthesis of F-CHO.

[0321] Polymerization was carried out as described in this paper to obtain F-Ph, where m:n:p is 1.0:1.0:5.0, both based on reaction stoichiometry and the synthesis of the polymer. 1 1H NMR spectroscopy measurements were performed. The size of the target amphiphilic random copolymer F-Ph was also measured in aqueous solutions of various polymer concentrations using dynamic light scattering (DLS) spectroscopy. Figure 7 Data show that the polymer exhibits unique monopolymer behavior in aqueous solution, with its size distribution peaking at 10 nm and no detectable aggregation.

[0322] The dithioester of F-Ph was removed by reaction with hydrazine hydrate in DMF, yielding the polymer F-SH containing free thiol end groups. The thiol groups of F-SH were further derived into formyl-terminated F-CHO by reaction with p-bromomethylbenzaldehyde in DMF. 1 F-CHO was examined by 1H NMR spectroscopy (in D2O), yielding m, n, and p values ​​of 22, 21, and 104, respectively. These m, n, and p values ​​were obtained based on a single formaldehyde proton. The data were then consistent with the expected m:n:p ratio of 1.0:1.0:5.0 from the initial monomer stoichiometry. Note that... 1 The m, n, and p values ​​measured by ¹H NMR gave a calculated molecular weight of 39.6 kDa for F-CHO, which was compared with the estimated molecular weight of 41.4 kDa for F-Ph derived from HPLC analysis (the two polymers have molecular formulas that differ in mass by only 2 Da). The comparison between HPLC and NMR determinations was satisfactory.

[0323] Pod-Rhodamine was prepared by reacting F-CHO with rhodamine-hydrazide I in N,N-dimethylformamide at 40 °C for 15 h. Unreacted dye was then removed by dialysis, yielding the target Pod-Rhodamine in 91% yield (Scheme 16).

[0324]

[0325] Scheme 16. Synthesis of Pod-Rhodamine. +

[0326] The absorption and emission of Pod-Rhodamine in water were tested in the presence of various metal ions. 1.0 mg of Pod-Rhodamine was treated in a vial with a solution of a metal salt (1.0 mL, 2 mM, 100 molar equivalents of Pod-Rhodamine) in water. The final concentration of Pod-Rhodamine was 20 μM. The resulting solution was stirred at room temperature for 1 hour, and then the absorption and emission spectra were measured. The cations tested in this study were: 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).

[0327] 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) produced increased fluorescence intensity compared to the blank control. The fluorescence loss on the Cu(II) and Fe(III) samples may be due to the heavy atom effect. Photographs of various reaction solutions with and without illumination were obtained. For Cr(II), precipitation was observed during the reaction; therefore, the absorption of Cr(II) was measured using the supernatant.

[0328] Fluorescent titration was performed with Au (III) and Hg (II) with 10 μM Pod-Rhodamine and 0–1.0 μM cations (excited at 510 nm). Figure 9 The titration fluorescence spectrum is shown.

[0329] In summary, the key point of this work is that the rhodamine sensor remains active when combined with the heteroclinic polymer and can be used for ion sensing purposes in pure water. Conversely, literature data suggests that using the rhodamine sensor alone requires a mixture of organic and aqueous media. Without wishing to be bound by any particular theory, this indicates that the polymer provides the organic solubilizing properties for the conjugated rhodamine sensor.

[0330] References

[0331]

[0332]

[0333] Example 7

[0334] Embodiments of the present invention relate to a heteroclinic polymer with a single bioconjugable group and a single chromophore for use in aqueous solutions, which is counterintuitive. The counterintuitive nature stems from the prevailing view in the art, as illustrated in numerous papers over approximately 50 years, that to obtain sufficient signal (e.g., brightness), the polymer or other construct needs to be loaded with as many chromophores as possible. One aspect of our strategy is to isolate the desired chromophore at one end of each heteroclinic polymer as a single cargo item in-situ, achieving a high degree of monomeric self-assembly in aqueous solution, and utilizing the other end of the polymer for bioconjugation.

[0335] One method for dissolving hydrophobic fluorophores in aqueous solutions requires linkage with an amphiphilic polymer, which self-assembles with the hydrophobic fluorophore immobilized within the hydrophobic structure. Our group previously... 9 This paper reports a single-polymer-single-cargo strategy in which hydrophobic fluorophores are packaged in aqueous solutions using amphiphilic polymers containing reactive end groups and hydrophobic and hydrophilic side groups. One polymer studied in particular contains acrylate and acrylamide units in its backbone, with side-hydrophobic (dodecyl), nonionic hydrophilic (PEG9), and ionic hydrophilic (sulfonate-terminated) groups. This polymer is a heteroclinic polymer with benzothioate and carboxylic acid end groups. This polymer, named F-Ph, exhibits ~40 kDa and contains lauryl, PEG9, and sulfonate as side groups in a 1:1:5 ratio. The construct is freely soluble in an aqueous medium. The study shows that polymer F-Ph quantitatively forms monomers in 1M NaCl solution at room temperature, as assessed by dynamic light scattering (DLS) spectroscopy. Hydrophobic fluorophores (derived from several Class 8 fluorophores) are attached to the ends of the F-Ph polymer (i.e., one fluorophore per polymer). The polymer-fluorophore constructs obtained in aqueous solution retain the intrinsic brightness observed for the fluorophore alone in organic solvents.

[0336] Most antibodies or cells cannot survive in aqueous solutions containing such high concentrations of NaCl. We recently turned to the detection of F-Ph and its fluorophore-containing derivatives in aqueous media of the type used in biology. In PBS buffer, which is commonly used in biology, F-Ph contains 68% monomers (D... h The peak is at ~13 nm) and 32% of the aggregated particles are larger than 100 nm. Figure 10 The PBS buffer contained 0.15 M NaCl and 0.01 M phosphate. The results suggest the importance of ionic strength in the self-assembly of amphiphilic polymers.

[0337] Because a high percentage of monomers (as opposed to aggregates) at low ionic strength is considered important for many biological applications, we began our investigation to explore the many factors believed to influence the monomer assembly process. Flow cytometry is the most significant application for amphiphilic polymers. The study required examining F-Ph as a function of ionic strength, as well as preparing and examining new polymers. New polymers varied in the nature of their side groups, the ratio of side groups, their molecular weight, and the presence or absence of hydrophobic fluorophores. The laser in the DLS instrument illuminated the sample at 632 nm, eliminating the possibility of using chromophores that absorb in this region. The fluorophore chosen for the study described herein was dinoflagellated phenylimide. Dinoflagellated phenylimide (which does not absorb at 632 nm) is a large hydrophobic aromatic and was therefore considered a viable alternative to various tetrapyrrole macrocycles (e.g., dihydroporphyrin and chlorophyll).

[0338] 1. Polymer Synthesis. A new series of amphiphilic polymers containing cyclododecyl and sulfonate side groups were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. The cyclododecyl group was introduced via cyclododecyl acrylate. The sulfonate was positioned at the end of an N-alkyl-substituted acrylamide. Cyclododecyl (CD) has previously been shown to exhibit a much stronger intramolecular self-binding tendency than lauryl groups. 2 Therefore, the lauryl group in F-Ph is replaced by a cyclododecyl group. PEG9 is also removed. Thus, the resulting polymer contains CD and sulfonate side groups, lacks the PEG group, and retains the thiobenzoate and carboxyl termini. For the studies described below, polymers containing two types of side groups in varying ratios and / or with varying total molecular weights were prepared. Treatment of these polymers with ethanolamine caused the cleavage of the thiobenzoyl group, producing free thiols at the termini. The resulting free thiols reacted with dinaphthalene-benzene monoimide maleimide (PMI-mal) to give the corresponding polymer-fluorophore conjugate (Scheme 17).

[0339]

[0340]

[0341] Scheme 17. Synthesize PS-CD polymers and corresponding PS-CD-PMI conjugates.

[0342] Seven PS-CD polymers were prepared using various initial ratios of cyclododecyl acrylate (CDA, hydrophobic monomer), sulfonate-terminated acrylamide (AMPS, hydrophilic / ionic monomer), and chain transfer agent (CTR). Subsequent derivatization yielded the corresponding polymer-PMI conjugates (Table 10). The molecular weight of each PS-CD polymer was deduced by HPLC analysis. Here, we examined the percentage of monomers observed when the following parameters varied: (1) the ionic strength of the solution, (2) the ratio of the two types of side groups, (3) the molecular weight of the individual polymer (lacking a fluorophore), and (4) the presence of a fluorophore—in other words, the effect of the presence of a fluorophore associated with the polymer relative to its absence.

[0343] Table 10. PS-CD polymers with different side group ratios and molecular weights.

[0344]

[0345] 2. Study on the effect on monomer assembly. A. Ionic strength. DLS studies of the previous F-Ph and the new polymer P-S5-CD1 (28 kDa) (entry 5, Table 10) were conducted in six media: pure water, PBS buffer, and aqueous solutions with different NaCl concentrations (0.25 M, 0.50 M, 0.75 M, 1.0 M). Raw DLS data are shown in... Figure 11 In pure water, F-Ph exhibits three distinct sizes during DLS examination. A peak at ~1 nm can be generated by unfolded polymers, while partial polymer aggregation causes a size distribution peak at ~400 nm. The ionic strength of the PBS buffer does not induce complete monopolymerization of F-Ph (see [link to PBS]). Figure 10 However, in 0.25 M NaCl solution and solutions with higher ionic strength, F-Ph quantitatively provided monomers ( Figure 11 ). Figure 12 The diagram is drawn in Chinese. Figure 11 The data includes DLS data on the percentage of monomers.

[0346] We then conducted similar studies on the novel polymer P-S5-CD1 (28 kDa). Crude DLS results in pure water and aqueous solutions of various NaCl concentrations are shown below. Figure 13 The crude DLS results in PBS buffer are shown in Figure 14 The results are plotted graphically. Figure 15 The figure shows that the polymer P-S5-CD1 (28 kDa) self-assembles in an aqueous medium containing ≥0.50 M NaCl to quantitatively provide monomers. Clearly, the polymer tends to aggregate into uniformly sized particles in an aqueous medium with high ionic strength.

[0347] 2. B. The role of side groups and the presence of hydrophobic fluorophores. F-Ph or F-PMI samples were dissolved in PBS buffer and detected by DLS spectroscopy. The percentage of F-PMI conjugate monomers was higher than the percentage of F-Ph monomers in PBS buffer. Figure 16 Therefore, the presence of hydrophobic fluorophores appears to drive the assembly of polymers in PBS buffer.

[0348] A similar comparison was made between P-S5-CD1(28 kDa) and P-S5-CD1(28 kDa)-PMI. Figure 17 The results showed that P-S5-CD1(28 kDa)-PMI quantitatively assembled into monomeric particles in PBS buffer, while polymers lacking PMI units did not quantitatively assemble into monomers. Similarly, the presence of hydrophobic cargoes appeared to drive the assembly of the entire construct into a monomeric structure.

[0349] 2. C. Molecular Weight. DLS was measured for a series of cyclododecyl / sulfonate polymer-PMI conjugates containing the same ratio of side groups (5:1) but with varying degrees of polymerization. Molecular weights ranged from 10–35 kDa, directly reflecting the degree of polymerization for a given constant ratio of reactants. Raw DLS data for the polymer-PMI conjugates in PBS buffer are shown below. Figure 18 The monomer percentages of P-S5-CD1(28 kDa)-PMI and P-S5-CD1(35 kDa)-PMI are 100%, while the monomer percentages of the constructs derived from lower molecular weight polymers, namely P-S5-CD1(10 kDa)-PMI and P-S5-CD1(18 kDa)-PMI, are not 100%. According to this dataset, the size of the 28 kDa polymer is sufficient to provide 100% monomer formation for the constructs containing PMI (but as mentioned above, incomplete formation occurs without PMI).

[0350] 2. D. Side group ratio. The effect of the side group ratio was also investigated. Three polymer-PMI conjugates were prepared, where the polymers exhibited nearly identical molecular weights (~28 kDa) but different side group ratios. For sulfonate versus cyclodecyl, the side group ratios were 4:1, 5:1, and 6:1. Raw DLS data for each construct in PBS are shown in [reference needed]. Figure 19 Each construct was quantitatively assembled into monomeric particles and had similar size and polydispersity index (PDI) in each case. Therefore, small variations in the side group ratio did not lead to aggregation in PBS buffer, at least not for the approximately 28 kDa polymers in this study.

[0351] The results of the above experiments are summarized in Table 11. For the seven PS-CD polymers we prepared, all polymers (lacking hydrophobic fluorophores) quantitatively formed monomers in 1M NaCl solution, but not in PBS buffer. However, the connection with the hydrophobic fluorophore enabled the polymers to have sufficient molecular weight to assemble monomericly, regardless of the ratio of side groups.

[0352] Table 11. Summary of the properties and characteristics of PS-CD polymers.

[0353]

[0354] 3. Experimental slides

[0355] A general method for preparing the polymer. A solution of CDA (715 mg, 3.0 mmol), AMPS (12 mmol–18 mmol), chain transfer agent (0.06 mmol–0.27 mmol), and mesitylene (360 mg, 3.0 mmol) in DMF (27 mL) in a Schlenk flask was degassed by three freeze-pump-thaw cycles. AIBN (4.9 mg, 0.030 mmol) was then added to the flask. The resulting mixture was stirred at 80 °C for 24 hours, including mesitylene as an internal standard, to obtain the polymer. 1 The conversion of each monomer was evaluated by 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. The crude polymer was then dissolved in DI water and placed in a dialysis membrane tube equipped with two closures, allowing compounds with a molecular weight less than 3.5 kDa to pass through. The solution was dialyzed in DI water, and the reservoir volume was replaced four times with fresh DI water over ~24 hours. The dialyzed solution was freeze-dried under high vacuum to give a light pink solid (1.2–1.4 g).

[0356] A general method for preparing polymer-PMI conjugates. Using F-S5-CD1 (28 kDa)-PMI as a representative example, a DMF solution of PMI-maleimide (1.0 mg, 0.97 μmol) and F-S5-CD1 (28 kDa) (18.1 mg, 0.65 μmol) was treated with ethanolamine (1 drop) in 1 mL. The mixture was stirred at 35 °C for 16 h, and then transferred to a dialysis membrane tube equipped with two closures. The solution was then dialyzed in DMF to remove excess fluorophores. The dialysis vessel volume was replaced four times with fresh DMF over ~24 hours, and the resulting solution was dried under high vacuum at 30 °C. The resulting solid was dissolved in DI water and then freeze-dried to give a pink solid (17 mg).

[0357] 4. Discussion

[0358] A group of amphiphilic polymers with different side group ratios and degrees of polymerization were prepared to investigate factors related to the degree of monomer formation in PBS buffer. PBS buffer is a widely accepted medium for biological assays and many clinical studies. It is easy to replace the lauryl and PEG groups with cyclododecyl groups (previously used...) 9 Polymers with lauryl and PEG groups, compared to those with cyclododecyl groups, showed that the latter promoted intramolecular assembly. A surprising result was that the presence of a single hydrophobic fluorophore induced self-folding of the amphiphilic polymer in aqueous solution. High molecular weight (~28 kDa) polymers were sufficient to completely encapsulate the hydrophobic fluorophore in PBS buffer; i.e., quantitatively forming monomers. As long as the molecular weight was greater than 28 kDa, the ratio of sulfonate to cyclododecyl side groups could vary between 4–6:1, thus quantitatively forming monomers in PBS buffer. While not wishing to be bound by any particular theory, it is believed that the interaction of three factors (molecular weight, side group ratio, and the presence of hydrophobic cargo) influences polymer assembly, which is clearly evident in PBS buffer but may be obscured under the high ionic strength conditions of 1 M NaCl, a medium that has been extensively (if not almost universally) used by others and by us in studies of fold formation. In summary, the heteroatomic PS-CD polymers described herein provide a concise and readily available platform for use with hydrophobic fluorophores in potential life science applications.

[0359] 5. References

[0360]

[0361]

[0362] The above is a description of the invention and should not be construed as limiting the invention. The invention is defined by the appended claims, and equivalents of the claims are included therein. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated herein by reference in their entirety for the purpose of teaching in relation to the sentences and / or paragraphs in which the references are presented.

Claims

1. A compound that is F1 or F2: The compound said compound comprises: dye; A polymer having a polymer backbone comprising one or more hydrophobic units and one or more hydrophilic units, wherein the polymer backbone comprises a first end and a second end, wherein the first end and the second end are opposite ends of the polymer backbone; and Biological conjugation groups; The dye is covalently attached to the first or second end of the polymer backbone.

2. A composition comprising the compound of claim 1.

3. A method for preparing the compound of claim 1, comprising: To polymerize hydrophobic and hydrophilic monomers to provide a polymer backbone comprising one or more hydrophobic units and one or more hydrophilic units; The dye is attached to the first or second end of the polymer backbone to provide the compound.

4. The method of claim 3, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer by living radical polymerization in the presence of an initiator, a catalyst and optionally a co-catalyst to provide a polymer.

5. The method of claim 4, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer by living radical polymerization in the presence of an initiator and a RAFT reagent to provide a polymer.

6. The in vitro non-therapeutic use of the compound of claim 1 or the composition of claim 2 in flow cytometry.

7. A non-therapeutic method for detecting cells and / or particles in vitro using flow cytometry, said method comprising labeling cells and / or particles with the compound of claim 1 or a compound prepared by the method of any one of claims 3-5; and The compounds are detected by flow cytometry, thereby detecting the cells and / or particles.

8. Use of the compound of claim 1, the composition of claim 2, or the compound prepared by the method according to any one of claims 3-5 in the manufacture of a medicament for detecting tissues and / or reagents in a subject, optionally wherein the compound is associated with the tissues and / or reagents.

9. Use in the manufacture of a medicament of the compound of claim 1, the composition of claim 2, or a compound prepared by the method according to any one of claims 3-5, for treating cells and / or tissues in a subject in need of the medicament, optionally wherein the compound is associated with the cells and / or tissues, and The treatment includes irradiating the subject or a portion thereof with light of a wavelength and intensity sufficient to treat the cells and / or tissue, optionally wherein the light activates the compound or a portion thereof.

10. The use according to claim 9, wherein the cell and / or tissue is a hyperproliferating tissue.

11. Use in the manufacture of a medicament of the compound of claim 1, the composition of claim 2, or a compound prepared by the method according to any one of claims 3-5, the medicament being used as a photodynamic therapy for treating hyperproliferating tissues of a subject in need of it, wherein the photodynamic therapy comprises: The subject is given the compound of claim 1, the composition of claim 2, or a compound prepared by the method according to any one of claims 3-5, optionally wherein the compound is associated with the hyperproliferating tissue, and the hyperproliferating tissue is treated by irradiating the hyperproliferating tissue with light of a wavelength and intensity sufficient to activate the compound or a portion thereof.

12. A reagent comprising the compound of claim 1 or a compound prepared by the method according to any one of claims 3-5.

13. The reagent of claim 12, wherein the biomolecule comprises two or more compounds of claim 1 or two or more compounds prepared by the method of any one of claims 3-5.

14. Use of the compound of claim 1 or the composition of claim 2 in the manufacture of a medicament for use in photodynamic therapy.

15. Use of the compound of claim 1 or the composition of claim 2 in the manufacture of a medicament for use in a photoacoustic imaging method.

16. Use of a compound or composition in the manufacture of a medicament for imaging tissues and / or reagents in a subject. The imaging wherein the imaging comprises administering the subject the compound of claim 1 or the composition of claim 2, and detecting the compound in the subject, thereby imaging the tissue and / or reagent.

17. The use of claim 16, wherein detecting the compound in the subject comprises irradiating the subject or a portion thereof with light of a wavelength and intensity sufficient to generate ultrasound, optionally wherein the irradiation is performed using a laser and / or by exposing the subject to one or more non-ionizing laser pulses.

18. The use according to claim 16 or 17, wherein detecting the compound in the subject comprises detecting ultrasound, optionally using an ultrasound detector.

19. The use according to claim 16 or 17, wherein imaging of the tissue and / or reagent in the subject comprises photoacoustic imaging of the tissue and / or reagent.

20. Use of the compound of claim 1 or the composition of claim 2 as a sensor, optionally without the addition and / or presence of organic solvents.

21. A method for sensing metal ions, the method comprising: Provide the compound of claim 1 or the composition of claim 2; as well as The metal ions are brought into contact with the compound to sense the metal ions.

22. The method of claim 21, wherein the compound and / or metal ions are present in water, optionally free of organic solvents.

23. Use of the compound of claim 1 or the composition of claim 2 as an oxygen sensor and / or oxygen-binding material.

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