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

Abstract

To provide polymeric fluorophores including a dye, a polymer segment, and optionally a bioconjugate group.SOLUTION: Provided herein are polymeric fluorophores that include a dye, a polymer, and optionally a bioconjugate group. A polymeric fluorophore may have a structure represented by: A-B-C or C-A-B, where: A is a dye; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units; and C is optional, where C, when present, comprises a bioconjugate group. Also provided herein are compositions comprising the polymeric fluorophores and methods of preparing and using the same.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Application Information This application claims priority to U.S. Provisional Patent Application No. 62 / 609,494, filed December 22, 2017, and U.S. Provisional Patent Application No. 62 / 714,541, filed August 3, 2018, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Government support statement This invention was made with government support under Grant No. DE-SC0001035 awarded by the Department of Energy. The government has certain rights in this invention.

[0003] FIELD OF THE INVENTION The present invention relates generally to polymeric fluorophores that include a dye, a polymer segment, and optionally a bioconjugate group. The present invention also relates to compositions that include the polymeric fluorophores and methods for their preparation and use. [Background technology]

[0004] Although many applications of fluorophores are carried out in aqueous solutions, most organic fluorophores are hydrophobic or only slightly polar. Although numerous methods exist for encapsulating fluorophores, none meet the criteria of synthetic simplicity, absence of fluorophore-fluorophore quenching, and the presence of a single bioconjugable group. Summary of the Invention [Means for solving the problem]

[0005] A first aspect of the present invention is A, B, C, or CAB [In the formula, A is a dye (e.g., a fluorophore), optionally the dye having a molecular weight in the range of about 150 Daltons (Da) to about 3,000 Da; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, optionally wherein the polymer has a molecular weight ranging from about 1,000 Da, 5,000 Da, or 10,000 Da to about 175,000 Da; Optional C comprises a bioconjugate group. The present invention is directed to a compound having a structure represented by:

[0006] Another aspect of the present invention is directed to a composition comprising a compound of the present invention and optionally water. A, B, C, or CAB [In the formula, A is a dye (e.g., a fluorophore), B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units; Optional C comprises a bioconjugate group. A particle (e.g., a particle including a core and a shell) comprising a compound having a structure represented by Water and The present invention is directed to a composition comprising:

[0007] A further aspect of the present invention is directed to a method of preparing a compound comprising polymerizing a hydrophobic monomer and a hydrophilic monomer to provide a copolymer, attaching a dye to a first portion (e.g., one terminus or terminal portion) of the copolymer to provide the compound, and optionally attaching a bioconjugate group to a second portion (e.g., the other terminus or terminal portion) of the copolymer, and / or optionally cross-linking the compound. Polymerizing the hydrophobic monomer and the hydrophilic monomer may comprise polymerizing via living radical polymerization in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally a co-catalyst to provide the copolymer.

[0008] Another aspect of the present invention is directed to compounds prepared according to the methods of the present invention.

[0009] Also provided according to embodiments of the present invention are uses of compounds of the present invention and / or uses of compositions of the present invention, for example, use in flow cytometry.

[0010] A further aspect of the present invention is directed to a method of detecting cells and / or particles using flow cytometry, comprising labeling cells and / or particles with a compound of the present invention and detecting said compounds by flow cytometry, thereby detecting said cells and / or particles.

[0011] Another aspect of the present invention is directed to a method of detecting tissues and / or pathogens (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 allowing the compound to associate with the tissues and / or pathogens, and detecting the compound in the subject, thereby detecting the tissues and / or pathogens.

[0012] A further aspect of the present invention is directed to biomolecules (eg, cells, antibodies, etc.) comprising one or more (eg, 1, 2, 3, 4, 5, 6, or more) compounds of the present invention.

[0013] It should be noted that, although not specifically described therein, aspects of the invention described with respect to one embodiment may be incorporated into a different embodiment. That is, features of all embodiments and / or any embodiment may be combined in any manner and / or combination. Applicant reserves the right to modify any originally filed claims and / or file any new claims as appropriate, including the right to amend any originally filed claims to depend from and / or incorporate any feature of any other claim or claims, even if not originally claimed in that manner. These and other objects and / or aspects of the invention are described in detail in the specification set forth below. Further features, advantages, and details of the invention will be apparent to those skilled in the art from a reading of the following figures and detailed description. Such description is merely exemplary of the invention. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram illustrating an exemplary polymeric fluorophore according to an embodiment of the present invention. [Figure 2] SEC elution traces of copolymer 7 (solid line) and chlorine-loaded copolymer F2 (dashed line). Samples were eluted with THF and detected using a refractive index detector. [Figure 3]Figure 1 shows three different absorbance spectra. Panel (A) shows the absorbance spectrum of D1 in CHCl (solid line), and the absorbance (dashed line) and emission (dotted line) spectra of F1 in water at μM concentrations. Panel (B) shows the absorbance spectrum of D2 in CHCl (solid line), and the absorbance (dashed line) and emission (dotted line) spectra of F2 in water at μM concentrations. Panel (C) shows the absorbance spectrum of D3 in toluene (solid line), and the absorbance (dashed line) and emission (dotted line) spectra of F3 in water at μM concentrations. All spectra were measured at room temperature. [Figure 4] Figure 1 shows dynamic light scattering (DLS) size data for F-2 at 10 mg / mL (A), 5 mg / mL (B), and 1.0 mg / mL (C). [Figure 5] FIG. 1 shows the absorbance spectra of F-2 in 1.0 M NaCl solution (top) and water (bottom). [Figure 6] FIG. 1 shows the emission spectra of F-2 in 1.0 M NaCl solution (top) and water (bottom). DETAILED DESCRIPTION OF THE INVENTION

[0015] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. However, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0016] The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the content clearly dictates otherwise.

[0017] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Furthermore, terms such as those defined in commonly used dictionaries should be interpreted to have 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 defined otherwise herein. The terms used in describing the invention herein are for the purpose of describing particular embodiments only and are not intended to limit the invention. All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety. In the event of a conflict of terminology, the present specification controls.

[0018] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and the lack of a combination when interpreted in the alternative ("or").

[0019] Unless the context indicates otherwise, it is specifically contemplated that the various features of the invention described herein can be used in any combination. Furthermore, it is contemplated that some embodiments of the invention can exclude or reject any feature or combination of features described herein. By way of example, if the specification describes a complex as comprising components A, B, and C, it is specifically contemplated that any one of A, B, or C, or any combination thereof, can be excluded and rejected.

[0020] As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) is to be construed to include the recited materials or steps "and which do not materially affect the basic and novel characteristic(s)" of the claimed invention. See re Herz, 537 F.2d 549, 551-52, 190, USPQ 461, 463 (CCPA 1976) (emphasis in original). See also MPEP § 2111.03. Thus, as used herein, the term "consisting essentially of" should not be construed as equivalent to "comprising."

[0021] It should also be understood that the terms "example," "exemplary," and grammatical variations thereof, as used herein, are intended to refer to non-limiting example and / or variant embodiments described herein, and are not intended to indicate a preference for one or more embodiments described herein over one or more other embodiments.

[0022] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is meant to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, as well as the specified value. For example, "about X," where X is a measurable value, means including X and variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges provided herein for measurable values can include any other ranges and / or individual values therein.

[0023] As used herein, a "derivative" in reference to a chemical molecule refers to a chemical molecule in which one or more atoms (e.g., hydrogen), functional groups, and / or bonds have been modified (e.g., removed, replaced, etc.) compared to the parent molecular entity. For example, a derivative of a dye can refer to a parent dye compound in which one or more atoms (e.g., hydrogen) and / or functional groups have been modified (e.g., removed) to facilitate covalent bonding with another group or moiety (e.g., to facilitate covalent bonding with a polymer). In some embodiments, a derivative can include functional groups (e.g., substituents and / or auxochromes) that alter the absorbance spectrum of the parent molecular entity.

[0024] As used herein, "alkyl," alone or as part of another group, refers to a straight or branched chain hydrocarbon containing 1 to 20 carbon atoms, and may be referred to as a C1-C20 alkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, and the like. As used herein, "lower alkyl" is a subset of alkyl and, in some embodiments, refers to a straight or branched chain hydrocarbon group containing 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the like. Unless otherwise specified, the term "alkyl" or "lower alkyl" is intended to include both substituted and unsubstituted alkyl or lower alkyl, and these groups include but are not limited to halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating polyalkoxy such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocycloalkyloxy, mercapto, alkyl-S(O) m , haloalkyl-S(O) m , alkenyl-S(O) m , alkynyl-S(O) m , cycloalkyl-S(O) m , cycloalkylalkyl-S(O) m , aryl-S(O) m , arylalkyl-S(O) m , heterocyclo-S(O) m , heterocycloalkyl-S(O) m, amino, carboxy, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted amino, acylamino, acyloxy, ester, amido, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro, or cyano, where m=0, 1, 2, or 3.

[0025] As used herein, alone or as part of another group, "alkenyl" refers to a straight or branched chain hydrocarbon containing 1 to 20 carbon atoms (or 1 to 4 carbon atoms for lower alkenyl), which may contain 1 to 8 double bonds in the linear chain, and may be referred to as a C1-C20 alkenyl. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, and the like. Unless otherwise specified, the term "alkenyl" or "lower alkenyl" is intended to include both substituted and unsubstituted alkenyl or lower alkenyl, which groups may be substituted with groups described above in connection with alkyl and lower alkyl.

[0026] As used herein, "alkynyl" alone or as part of another group refers to a straight or branched chain hydrocarbon containing 1 to 20 carbon atoms (or 1 to 4 carbon atoms for lower alkynyl) containing one triple bond in the linear chain, and can be referred to as a C1-C20 alkynyl. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. Unless otherwise specified, the term "alkynyl" or "lower alkynyl" is intended to include both substituted and unsubstituted alkynyl or lower alkynyl, which groups may be substituted with the same groups as described above in connection with alkyl and lower alkyl.

[0027] As used herein, "halo" refers to any suitable halogen, including -F, -Cl, -Br, and -I.

[0028] As used herein, "mercapto" refers to the group --SH.

[0029] As used herein, "azido" refers to the group --N3.

[0030] As used herein, "cyano" refers to the group --CN.

[0031] As used herein, "hydroxyl" refers to an --OH group.

[0032] As used herein, "nitro" refers to the group --NO.sub.2.

[0033] "Alkoxy," as used herein alone or as part of another group, refers to an alkyl or lower alkyl group, as defined herein, appended to the parent molecular moiety through an oxy group -O- (and thus includes substituted versions such as polyalkoxy). Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentyloxy, hexyloxy, and the like.

[0034] As used herein, "acyl" alone or as part of another group refers to the group -C(O)R, where R is any suitable substituent, such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl, or other suitable substituent described herein.

[0035] "Haloalkyl," as used herein alone or as part of another group, refers to at least one halogen, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of haloalkyl include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, and the like.

[0036] "Alkylthio," as used herein alone or as part of another group, refers to an alkyl group, as defined herein, appended to the parent molecular moiety through a thio moiety, as defined herein. Representative examples of alkylthio include, but are not limited to, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

[0037] As used herein, "aryl," alone or as part of another group, refers to a monocyclic carbocyclic ring system or a bicyclic carbocyclic fused ring system having one or more aromatic rings. Representative examples of aryl include, but are not limited to, azulenyl, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, and the like. Unless otherwise specified, the term "aryl" is intended to include both substituted and unsubstituted aryl, which may be substituted with the same groups as those described above in connection with alkyl and lower alkyl.

[0038] "Arylalkyl," as used herein alone or as part of another group, refers to an aryl group, as defined herein, appended to the parent molecular moiety through an alkyl group, as defined herein. Representative examples of arylalkyl include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, and the like.

[0039] As used herein, "amino" refers to the group --NH.sub.2.

[0040] The term "alkylamino" as used herein alone or as part of another group refers to the group --NHR, where R is an alkyl group.

[0041] As used herein, "ester" alone or as part of another group refers to a -C(O)OR group, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0042] As used herein, "formyl" refers to the group --C(O)H.

[0043] As used herein, "carboxylic acid" refers to the group --C(O)OH.

[0044] As used herein, "sulfoxyl" refers to a compound of formula -S(O)R, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

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

[0046] As used herein, "sulfonate" refers to a salt of sulfonic acid (e.g., the sodium (Na) salt) and / or a compound of formula -S(O)(O)OR, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0047] As used herein, "sulfonic acid" refers to a compound of formula -S(O)(O)OH.

[0048] As used herein, "amide" alone or as part of another group refers to -C(O)NR a R b The group [wherein, R a and R bis any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0049] As used herein, "sulfonamide" alone or as part of another group refers to -S(O)NR a R b The group [wherein, R a and R b is any suitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.

[0050] The compounds of the present invention include polymeric fluorophores. The compounds of the present invention include a single (i.e., one) polymer attached to a single (i.e., one) dye, and optionally a single (i.e., one) bioconjugate group that may have a single binding site for a biomolecule. Exemplary compounds are shown in Figure 1. In some embodiments, one polymer is attached to both the dye and the bioconjugate group (if present). In some embodiments, one dye is attached to both the polymer and the bioconjugate group (if present). In some embodiments, the compositions of the present invention include compounds of the present invention in solution, such as, for example, water, an aqueous solution, and / or a hydrophobic solvent.

[0051] While a compound of the invention may be attached to a single biomolecule via a bioconjugate group, the biomolecule may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) compounds of the invention. Thus, in some embodiments, a biomolecule and / or portion thereof comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) compounds of the invention.

[0052] In some embodiments, the compounds of the present invention are A, B, C, or CAB [In the formula, A is a dye (e.g., a fluorophore), B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units; and optional C, if present, comprises a bioconjugate group. It has a structure represented by:

[0053] The terms "dye" and "fluorophore" are used interchangeably herein to refer to molecular entities that emit fluorescence. A dye or fluorophore can also be referred to as a chromophore having particular spectroscopic features and / or properties. In some embodiments, the dye has a molecular weight ranging from about 150 Daltons (Da) to about 3,000 Da, from about 400 Da to about 1,100 Da, or from about 300 Da to about 1,000 Da. In some embodiments, the dye has a molecular weight of about 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 Da. Exemplary dyes include, but are not limited to, tetrapyrroles; rylenes such as perylene, terrylene, and quattarylene; fluoresceins such as TET (tetramethylfluorescein), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyfluorescein (HEX), and 5-carboxyfluorescein (5-FAM); phycoerythrin; resorufin dyes; coumarin dyes; rhodamine dyes such as 6-carboxy-X-rhodamine (ROX), Texas Red, and N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); cyanine dyes; phthalocyanines; boron-dipyrromethene (BODIPY) dyes; quinolines; pyrenes; acridines; stilbenes; and derivatives thereof. In some embodiments, the dye is a tetrapyrrole, which includes porphyrins, chlorins, and bacteriochlorins, and derivatives thereof.Exemplary tetrapyrroles include, but are not limited to, those described in 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, 6,60 No. 3,070, No. 6,849,730, No. 7,005,237, No. 6,916,982, No. 6,944,047, No. 7,884,280 , No. 7,332,599, No. 7,148,361, No. 7,022,862, No. 6,924,375, No. 7,501,507, No. 7,323 ,561, No. 7,153,975, No. 7,317,108, No. 7,501,508, No. 7,378,520, No. 7,534,807, No. 7,919,770, No. 7,799,910, No. 7,582,751, No. 8,097,609, No. 8,187,824, No. 8,207, 329, 7,633,007, 7,745,618, 7,994,312, 8,278,340, 9,303,165, and 9,365,722, and International Application Nos. PCT / US17 / 47266 and PCT / US17 / 63251. In some embodiments, the dye is hydrophobic. In some embodiments, the dye may be attached and / or bound to a monomer polymerized with one or more different monomers (e.g., polymerized with a hydrophobic monomer and / or a hydrophilic monomer). In some embodiments, the dye is a luminophore (i.e., a substance and / or compound capable of emitting light, without specifying the nature of the emission (e.g., singlet, triplet, and / or other state)). Exemplary luminophores include, but are not limited to, phosphorescent and / or fluorophore, which result in phosphorescence and / or fluorescence, respectively.

[0054] Polymers of the compounds of the invention can include one or more (e.g., 1, 5, 10, 50, 100, or more) hydrophobic units and one or more (e.g., 1, 5, 10, 50, 100, or more) hydrophilic units. The polymers can be prepared from one or more (e.g., 1, 5, 10, 50, 100, or more) hydrophobic monomers and one or more (e.g., 1, 5, 10, 50, 100, or more) hydrophilic monomers using any type of polymerization to provide a polymer comprising one or more hydrophobic units and one or more hydrophilic units. In some embodiments, the polymers can be prepared from two or more (e.g., 2, 3, 4, 5, or more) hydrophobic monomers that are different from each other and / or two or more (e.g., 2, 3, 4, 5, or more) hydrophilic monomers that are different from each other. For example, in some embodiments, the polymeric compounds of the present invention may be prepared from at least one hydrophobic monomer, at least one of the first hydrophilic monomers, and at least one of the second hydrophilic monomers, where the first hydrophilic monomer and the second hydrophilic monomer are different from each other.

[0055] As used herein, a "hydrophilic monomer" refers to a monomer that includes a hydrophilic (e.g., ionic and / or polar) functional group (e.g., a hydrophilic pendant functional group), optionally located at a moiety and / or terminal end of the monomer. As will be appreciated by those skilled in the art, a hydrophilic monomer can be referred to as a hydrophilic monomer even if a portion of the monomer is hydrophobic, such as a portion that forms a polymer backbone when polymerized with other monomers and / or functional group moieties (e.g., a hydrocarbon chain). As used herein, a "hydrophilic unit" refers to a section or unit of a polymer prepared from a corresponding hydrophilic monomer. As used herein, a "hydrophobic monomer" refers to a monomer that includes a hydrophobic functional group (e.g., a hydrophobic pendant functional group), optionally located at a moiety and / or terminal end of the monomer. In some embodiments, the hydrophobic functional group is a hydrocarbon moiety (e.g., alkyl). As used herein, "hydrophobic unit" refers to a section or unit of a polymer prepared from the corresponding hydrophobic monomer.

[0056] 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. One or more hydrophobic units and 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. One or more hydrophobic units and one or more hydrophilic units may be present in the polymer in a ratio (hydrophobic units:hydrophilic units) of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10. The length of the polymer may be varied and / or controlled. In some embodiments, the polymer has a molecular weight ranging from about 1,000 Da to about 175,000 Da, from about 5,000 Da to about 175,000 Da, from about 10,000 Da to about 175,000 Da, from about 100,000 Da to about 150,000 Da, from about 50,000 Da to about 130,000 Da, or from 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).

[0057] The hydrophobic and / or hydrophilic units of the polymer can include pendant functional groups. A "pendant functional group" can be a functional group directly attached to the polymer backbone or to a moiety attached to the polymer backbone. The pendant functional group can be part of the hydrophobic and / or hydrophilic units and / or monomers during polymerization, or can be added to the hydrophobic and / or hydrophilic units after polymerization. In some embodiments, the pendant functional group can be added to the hydrophobic and / or hydrophilic units after polymerization (e.g., post-polymerization functionalization). In some embodiments, the pendant functional group comprises a charged group. In some embodiments, the pendant functional group is a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., active esters such as pentafluorophenyl ester, succinimide ester, 2,4-dinitrophenyl ester), azido, pentafluorophenyl, succinimide, fluorophenyl, maleimide, isocyanato, or isothiocyanato group. In some embodiments, the pendant functional groups are hydrophilic groups comprising terminal cationic (e.g., ammonium), anionic (e.g., sulfonate, phosphate, carboxylate), or zwitterionic (e.g., choline or choline-like groups (e.g., derivatives of choline)) groups, and optionally poly(ethylene glycol) moieties and / or units. In some embodiments, the hydrophilic groups are attached to the poly(ethylene glycol) moieties and / or units, and optionally to the terminal portions of the poly(ethylene glycol) moieties and / or units.

[0058] In some embodiments, the hydrophobic units comprise pendant functional groups comprising alkyl (e.g., dodecylmethyl), and / or the hydrophilic units comprise pendant functional groups comprising glycol (e.g., poly(ethylene glycol)), sulfonic acid, and / or sulfonate. In some embodiments, the hydrophobic units are prepared from alkyl acrylate (e.g., dodecylmethyl acrylate) monomers, and / or the hydrophilic units are prepared from glycol acrylate (e.g., PEGylated methyl acrylate) monomers. In some embodiments, the compounds of the present invention comprise at least one hydrophobic unit prepared from an alkyl acrylate (e.g., dodecylmethyl acrylate) monomer and at least two different hydrophilic units, including a first hydrophilic unit prepared from a glycol acrylate (e.g., PEGylated methyl acrylate) monomer and a second hydrophilic unit prepared from a sulfonic acid acrylate monomer (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or a sulfonate acrylate monomer.

[0059] In some embodiments, one or more of the hydrophobic units and / or one or more of the hydrophilic units may comprise a charge (e.g., a positive or negative charge) and / or a charged group (e.g., a cationic or anionic group), which may inhibit non-specific binding with a compound or portion thereof (e.g., a portion of a polymer).

[0060] In some embodiments, a hydrophobic monomer, which may be used to provide the hydrophobic units of the polymers described herein, may have a structure represented by Formula I:

[0061] [ka]

[0062] [In the formula, R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl); R 1is absent or is -O-, -NH-, or -CH2-, A is a C1-C20 alkyl, a C2-C20 alkenyl, or a C2-C20 alkynyl; R 2 is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, or ester (e.g., succinimide ester, 2,4-dinitrophenyl ester, pentafluorophenyl ester, fluorophenyl ester, etc.) group. In some embodiments, R in the compound of Formula I 2 is a hydroxyl, carboxyl, amino, formyl, or ester group. In some embodiments, R in the compound of Formula I 2 is hydrogen. In some embodiments, A in compounds 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, A in compounds of Formula I is C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl, alkenyl, or alkynyl. In some embodiments, A in the compound of formula I is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl.

[0063] In some embodiments, a hydrophilic monomer, which may be used to provide the polymeric hydrophilic units described herein, may have a structure represented by Formula II:

[0064] [ka]

[0065] [In the formula, R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl); R 1 is absent or is —O—, —NH—, or —CH—; R 3 is -(CH2CH2R 5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl-O-, and -C1-C6 alkyl-SO3-, or a salt thereof, 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; R 4 is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group.

[0066] In some embodiments, optionally R 3 -(CH2CH2R 5 ) n -, -C1-C6 alkyl, or -C1-C6 alkyl-O-, R in the compound of formula II 4 is a hydroxyl, carboxyl, amino, formyl, or ester group. In some embodiments, R in the compound of Formula II 3 is -C1-C6 alkyl-O- or -(CH2CH2R 5 ) n - and R 5 is -O-, then R 4 can be hydrogen, alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine, or phosphoryl group. In some embodiments, R in the compound of Formula II 3is -C1-C6 alkyl, R 4 can be a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group. In some embodiments, R in the compound of Formula II 3 is -C1-C6 alkyl-SO3- or a salt thereof, R 4 is hydrogen or absent. In some embodiments, R in the compound of formula II 3 is a salt of -C1-C6 alkyl-SO3- (e.g., sodium salt), and R 4 is absent. In some embodiments, R in the compound of Formula II 3 Ha-(CH2CH2R 5 ) n -It is.

[0067] In some embodiments, the hydrophobic unit can have a structure represented by Formula III:

[0068] [ka]

[0069] [In the formula, R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl); R 1 is absent or is -O-, -NH-, or -CH2-, A is a C1-C20 alkyl, a C2-C20 alkenyl, or a C2-C20 alkynyl; R 2 is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azido, maleimide, isocyanato, or isothiocyanato group; p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000].

[0070] In some embodiments, R in the compound of Formula III 2 is a hydroxyl, carboxyl, amino, formyl, or ester group. In some embodiments, R in the compound of Formula III 2 is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimide group, which may optionally be added and / or provided after polymerization and / or by post-polymerization functionalization. In some embodiments, R in the compound of Formula III 2 is hydrogen. In some embodiments, A 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, A in the compound of Formula III is C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl, alkenyl, or alkynyl. In some embodiments, A in the compound of formula III is C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl.

[0071] In some embodiments, the hydrophilic unit can have a structure represented by formula IV:

[0072] [ka]

[0073] [In the formula, R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl); R 1 is absent or is —O—, —NH—, or —CH—; R 3 is -(CH2CH2R5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl-O-, and -C1-C6 alkyl-SO3-, or a salt thereof, 5 is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; R 4 is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group; p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000].

[0074] In some embodiments, optionally R 3 -(CH2CH2R 5 ) n -, -C1-C6 alkyl, or -C1-C6 alkyl-O-, R in the compound of formula IV 4 is a hydroxyl, carboxyl, amino, formyl, or ester group. In some embodiments, R in the compound of formula IV 3 is -C1-C6 alkyl-O- or -(CH2CH2R 5 ) n - and R 5 is -O-, then R 4 can be hydrogen, alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), or phosphoryl group. In some embodiments, R in the compound of formula IV3 is -C1-C6 alkyl or -(CH2CH2R 5 ) n - and R 5 is -CH2-, R 4 can be a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group. In some embodiments, R in the compound of formula IV 4 is hydrogen, alkyl, phosphono, sulfono, phosphatidylcholine, phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or an ester group. 4 is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimide group, which may optionally be added and / or provided after polymerization and / or by post-polymerization functionalization. In some embodiments, R in the compound of formula IV 3 is -C1-C6 alkyl-SO3- or a salt thereof, R 4 is hydrogen or absent. In some embodiments, R in the compound of formula IV 3 is a salt of -C1-C6 alkyl-SO3- (e.g., sodium salt), and R 4 is absent. In some embodiments, R in the compound of Formula II 3 Ha-(CH2CH2R 5 ) n -It is.

[0075] In some embodiments, the compounds of the present invention may include and / or be telechelic polymers, which are polymers or prepolymers that can participate in further polymerization or other reactions through one or more of their reactive end groups. In some embodiments, the compounds of the present invention may include and / or be heterotelechelic polymers, which are polymers or prepolymers that can participate in further polymerization or other reactions through reactive end groups at each end of the polymer or prepolymer (where the two reactive end groups are not identical). In some embodiments, the compounds of the present invention may include and / or be homotelechelic polymers, which are polymers or prepolymers that can participate in further polymerization or other reactions through reactive end groups at each end of the polymer or prepolymer (where the two reactive end groups are identical). In some embodiments, the compounds of the present invention may include and / or be semitelechelic polymers, which are polymers or prepolymers that can participate in further polymerization or other reactions through a reactive end group at one end of the polymer or prepolymer.

[0076] Bioconjugate groups may optionally be present in the compounds of the present invention. A "bioconjugable group," "bioconjugable moiety," or "bioconjugate group," and grammatical variations thereof, refer to a moiety and / or functional group that can be used to bind to or is attached to a biomolecule (e.g., a protein, peptide, DNA, RNA, etc.). Thus, a "bioconjugable group," "bioconjugable moiety," or "bioconjugate group," and grammatical variations thereof, do not include a biomolecule. However, in some embodiments, a bioconjugate group is used to bind to a biomolecule, or a bioconjugate group or a derivative thereof is attached to a biomolecule (e.g., a protein, peptide, DNA, RNA, etc.). Exemplary bioconjugable groups include, but are not limited to, amines (including amine derivatives) such as isocyanates, isothiocyanates, iodoacetamides, azides, diazonium salts, etc.; acids or acid derivatives such as N-hydroxysuccinimide esters (more commonly activated esters derived from carboxylic acids, e.g., p-nitrophenyl esters), acid hydrazides, etc.; and other linking groups such as aldehydes, sulfonyl chlorides, sulfonylhydrazides, epoxides, hydroxyl groups, thiol groups, maleimides, aziridines, acryloyl, halo groups, biotin, 2-iminobiotin, etc. Linking groups such as the foregoing are known and are described in U.S. Patent Nos. 6,728,129, 6,657,884, 6,212,093, and 6,208,553. For example, compounds of the invention may include a bioconjugate group that includes a carboxylic acid, which may be used for bioconjugation with a biomolecule (e.g., via carbodiimide activation and coupling of an amino substituent with a biomolecule).

[0077] In some embodiments, a biomolecule may include and / or be a protein (e.g., an antibody and / or a carrier protein), a peptide, DNA, RNA, etc. In some embodiments, a biomolecule may include a moiety (e.g., a polymer) that may optionally include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) binding sites for a compound of the invention. In some embodiments, a 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 different molecules, one of which has an area on its surface or in a cavity that specifically attracts or binds to a particular spatial or polar structure of the other molecule, causing the two molecules to have affinity for each other. Members of a specific binding pair may be referred to as a ligand and receptor (antiligand). The terms ligand and receptor are intended to encompass the entire ligand or receptor, or a portion thereof sufficient for binding between the ligand and receptor to occur. Examples of ligand-receptor binding pairs include, but are not limited to, hormones and hormone receptors, e.g., epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor-α and tumor necrosis factor receptor, and interferon and interferon receptor; avidin and biotin or antibiotin; antibody and antigen pairs; enzymes and substrates; drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid strands; a nucleic acid strand and a complementary oligonucleotide; an interleukin and an interleukin receptor; 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.

[0078] The compounds of the present invention may include a dye (e.g., a tetrapyrrole) covalently attached to a portion of a polymer described herein. In some embodiments, the dye may be covalently attached to a terminal portion of the polymer. If present, a bioconjugate group may also be covalently attached to a portion of the polymer, such as a terminal portion of the polymer. In some embodiments, the bioconjugate group is covalently attached to a first terminal portion (e.g., the first end) of the polymer and the dye is covalently attached to an opposite terminal portion (e.g., the opposite end) of the polymer.

[0079] The compounds of the invention may include a dye (e.g., a tetrapyrrole) covalently attached to a polymer moiety, and a bioconjugate group may be covalently attached to the dye moiety. In some embodiments, the bioconjugate group is covalently attached to a first moiety (e.g., a first end) of the dye, and the polymer is covalently attached to a second moiety (e.g., an opposite end) of the dye.

[0080] In some embodiments, compounds of the present invention, or portions thereof, have a non-rigid backbone (e.g., a non-rigid polymer backbone) and / or have conformational flexibility. The conformational flexibility of a molecular chain can be described and quantified by the "persistence length" of the compound or portion thereof (e.g., a polymer portion). In some embodiments, the persistence length of a compound of the present invention can be on the order of magnitude of the length of a given carbon-carbon bond.

[0081] 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" refers to a compound that transitions from a partially or completely extended or unfolded structure to a structure in which at least a portion of the extended or unfolded structure is folded upon contact with a solution (e.g., aqueous solution) or compound, and the folding is intrinsic because it occurs spontaneously (i.e., without external control or force) upon contact with the solution. 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 unimeric micellar structure upon contact with water and / or aqueous solutions.

[0082] In some embodiments, the compound of the present invention can be in the form of particles.For example, the compound of the present invention can form particles when it is contacted with a solution (for example, an aqueous solution).In some embodiments, a single (i.e., one) compound can form particles.Therefore, the compound and the particle are present in a ratio of about 1:1 (i.e., one compound is present per particle).

[0083] The compounds of the present invention may comprise one or more hydrophobic unit moieties in the core or interior region of the particle, and / or one or more hydrophilic unit moieties in the peripheral or external region (e.g., shell) of the particle. In some embodiments, the particles have a micellar structure (e.g., a unimeric micellar structure). The compounds of the present invention may comprise a dye that can be attached to a polymer of the present invention, and the dye may be encapsulated by a moiety of the compound (e.g., a polymer moiety) when the compound is in a folded structure and / or particle form (e.g., a unimeric micellar structure). In some embodiments, the dye or a moiety and one or more hydrophobic units may be present in the core or interior region of the particle, and one or more hydrophilic units may surround the dye and / or one or more hydrophobic units.

[0084] 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 include an alkyl (e.g., dodecylmethyl) pendant functional group and / or are formed from a compound of Formula I and / or an alkyl acrylate (e.g., dodecylmethyl acrylate) monomer. 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 a compound of Formula II. In some embodiments, one or more of the hydrophilic units include a non-ionic (i.e., neutral / uncharged) pendant functional group (e.g., PEG) and / or are formed from a non-ionic monomer (e.g., pegylated methyl acrylate (PEGA)). In some embodiments, one or more of the hydrophilic units include an ionic (e.g., anionic, charged) pendant functional group (e.g., sulfonic acid and / or sulfonate) and / or are formed from an ionic monomer (e.g., sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). In some embodiments, the hydrophilic units are formed from at least two different monomers, such as, for example, a nonionic (i.e., neutral / uncharged) hydrophilic monomer (e.g., pegylated methyl acrylate (PEGA)) and an ionic (e.g., anionic, charged) hydrophilic monomer (e.g., sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). As will be appreciated by those skilled in the art, an acid-containing monomer, such as, for example, a sulfonic acid, can exist in the acid form and / or its 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 its deprotonated form (e.g., a deprotonated sulfonic acid acrylate) and / or in its salt form, such as sodium sulfonate acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid as the sodium salt).

[0085] In some embodiments, when two or more different hydrophilic units are present in a polymer of the invention, the ratio of the two or more different hydrophilic units can vary, for example, from about 10:1 to about 1:10. For example, in some embodiments, the polymer comprises nonionic (i.e., neutral / uncharged) hydrophilic units (e.g., formed from pegylated methyl acrylate (PEGA)) and ionic (e.g., anionic, charged) hydrophilic units (e.g., formed from sulfonic acid acrylate (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 units:ionic units). In some embodiments, the ratio of hydrophilic units to hydrophobic units present in the backbone of a polymer of the invention can vary. In some embodiments, the ratio of hydrophilic to hydrophobic units present in the backbone of the polymer is about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (hydrophobic units:hydrophilic units).

[0086] In some embodiments, the polymers of the present invention comprise from about 1% to about 40% hydrophobic units, based on the total molar amount of monomers used to prepare the polymer, and from about 60% to about 99% hydrophilic units, based on the total molar amount of monomers used to prepare the polymer. In some embodiments, the polymers of the present invention comprise from about 1%, 5%, 10%, 15%, or 20% to about 25%, 30%, 35%, or 40% hydrophobic units, based on the total molar amount of monomers used to prepare the polymer, and from about 60%, 65%, 70%, 75%, or 80% to about 85%, 90%, 95%, or 99% hydrophilic units, based on the total molar amount of 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%) 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 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 monomers used to prepare the polymer.

[0087] 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 polymers 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 can have a weight fraction of hydrophobic units of less than about 30% (eg, less than about 25%, 20%, 15%, 10%, or 5%) based on the total weight of the polymer.

[0088] 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 can have a weight fraction of hydrophilic units greater than about 70% (eg, greater than about 75%, 80%, 85%, 90%, or 95%) based on the total weight of the polymer.

[0089] In some embodiments, the amount of unimeric micellar structures formed upon contact with the solution is between about 50% and about 100%, between about 75% and about 100%, between about 85% and about 100%, or between about 95% and about 100%, optionally as measured using a sizing method (e.g., dynamic light scattering (DLS)). In some embodiments, the amount of unimeric micellar 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 as measured using a sizing method (e.g., dynamic light scattering (DLS)).

[0090] In some embodiments, dilution of a solution containing a compound of the invention in the form of a unimeric micellar structure results in no loss or a loss of less than about 20% of the unimeric micellar structure present in the solution compared to the amount of unimeric micellar structure present in the solution prior to dilution. In some embodiments, the amount of unimeric micellar 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 unimeric micellar structure present in the solution prior to dilution.

[0091] In some embodiments, a solution containing a compound of the invention in the form of a unimeric micellar structure contains less than about 50% aggregates (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%). Thus, at least 50% or more of the compound is not aggregated and may be in the form of a unimeric micellar structure. In some embodiments, dilution of a solution comprising a compound of the invention in the form of a unimeric micellar structure results in no or only minimal additional aggregate formation compared to the amount of aggregates present in the solution prior to dilution, hi some embodiments, the amount of aggregates present in a solution comprising a compound of the invention does not change 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% upon dilution compared to the amount of aggregates present in the solution prior to dilution. In some embodiments, the diluted solution contains less than about 50% aggregates (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%).

[0092] The compounds of the present invention may have a diameter in the range of about 1 nm to about 50 nm or about 3 nm to about 30 nm in water and / or aqueous solutions (e.g., when folded into a unimeric micelle structure, etc.). In some embodiments, the compounds may have a diameter 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 in water and / or aqueous solutions (e.g., when folded into a unimeric micelle structure, etc.). In some embodiments, the compounds of the present invention may be in the form of particles (i.e., at least partially folded).

[0093] In some embodiments, the compounds of the present invention are cross-linked, optionally when the compound is in a folded conformation. In some embodiments, the compounds of the present invention may be in solution (e.g., aqueous solution) and / or may be cross-linked using a cross-linking agent. Cross-linking the compounds of the present invention may involve linking together two or more moieties and / or functional groups (e.g., pendant functional groups) of hydrophobic and / or hydrophilic units. Cross-linking may provide the compound with a folded conformation that cannot unfold without cleaving one or more of the links formed by the cross-linking. The degree or amount of cross-linking may be controlled, modified, and / or adjusted, for example, by the amount of cross-linking agent reacted with the compound. In some embodiments, cross-linking the compound may include the reactions and / or reactive entities (e.g., functional groups) listed in Table 1.

[0094] [Table 1]

[0095] In compounds of the present invention, the fluorescence quantum yield of the dye when the compound is present in water and / or aqueous solution may be reduced by about 10% or less (e.g., 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) compared to the fluorescence quantum yield of the dye when the compound is present in a hydrophobic solvent (e.g., toluene). Upon bioconjugation of a compound of the present invention with a biomolecule (e.g., a protein), the fluorescence quantum yield of the dye may be the same or substantially the same (e.g., within ±20%) as the fluorescence quantum yield of the dye in water and / or hydrophobic solvent. In some embodiments, when 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) may be tolerated.

[0096] In some embodiments, the compounds of the present invention are water-soluble. The compounds may have a solubility in water ranging from about 1 mg / mL to about 10 mg / mL at room temperature. In some embodiments, the compounds have a solubility in water of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / mL at room temperature.

[0097] In some embodiments, the compounds and / or particles of the present invention are resistant to dilution. As used herein, "resistant to dilution" refers to the compounds and / or particles retaining their structure and / or properties. In some embodiments, resistant to dilution refers to the compounds and / or particles retaining a folded structure (e.g., a unimeric micellar structure), which may be determined by measuring the particle diameter before and after dilution, where the diameter after dilution may remain within ±50%, 40%, 30%, 20%, 10% or less of the diameter before dilution. In some embodiments, resistant to dilution refers to the compounds and / or particles retaining a fluorescence quantum yield of the dye after dilution 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 up to 25x, 50x, 75x, or 100x, or when diluted to a concentration of micromolar or less.

[0098] According to some embodiments of the present invention, methods for preparing the compounds and / or compositions of the present invention are provided. In some embodiments, the method for preparing the compounds of the present invention includes polymerizing a hydrophobic monomer and a hydrophilic monomer to provide a copolymer, attaching a dye to a first portion of the copolymer (e.g., at one end or terminal portion), and optionally attaching a bioconjugate group (e.g., a bioconjugable group) to a second portion of the copolymer (e.g., at the other end or terminal portion), thereby providing the compound. The hydrophobic and hydrophilic monomers may be polymerized using any method known to those skilled in the art, including, but not limited to, via condensation reactions (e.g., reactions using a diol and a diacid) and / or living radical polymerization (e.g., atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT)). In some embodiments, polymerizing the hydrophobic and hydrophilic monomers is carried out using a method that provides a copolymer in which one or both end groups of the copolymer are reactive (i.e., one or both of the end groups of the copolymer can participate in further polymerization or reaction), where the two end groups can be the same or different. In some embodiments, polymerizing the hydrophobic and hydrophilic monomers is via living radical polymerization (e.g., ATRP) in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally a co-catalyst to provide the copolymer. In some embodiments, polymerizing the hydrophobic and hydrophilic monomers is via living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT agent (e.g., a thiocarbonylthio compound).

[0099] In some embodiments, attaching the dye to the first portion of the copolymer can include reacting a monomer comprising the dye with a hydrophobic monomer and / or unit and / or a hydrophilic monomer and / or unit. Thus, in some embodiments, attaching the dye to the copolymer can occur during or after the polymerization step. In some embodiments, the method includes reacting a monomer comprising the dye 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, polymerization of the 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, where the initiator comprises a dye. In some embodiments, 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 agent, optionally the RAFT agent comprising a dye.

[0100] Exemplary terminal functional groups of the copolymer, where the copolymer is available for immediate dye attachment or bioconjugation, may include, but are not limited to, those listed in Table 2. These terminal functional groups are not pendant functional groups, but may be present at either end of the copolymer.

[0101] [Table 2]

[0102] Some functional groups may be unstable under certain polymerization conditions. Therefore, in some embodiments, functional groups may be introduced in protected form. These functional groups may then become available for dye attachment or bioconjugation upon deprotection. Exemplary protected forms of certain functional groups include, but are not limited to, those listed in Table 3.

[0103] [Table 3]

[0104] In some embodiments, a portion of the copolymer (e.g., terminal or end portion) may contain a halo group (e.g., Cl, Br, I). The halide portion of the copolymer may be derivatized with a nucleophile or end-capping reagent to generate functional groups for dye attachment or bioconjugation. In some embodiments, a portion of the copolymer (e.g., terminal end portion) may contain a thiol group, which may be derivatized with a reagent containing a thiol-reactive group to generate functional groups for dye attachment or bioconjugation. Examples of thiol-reactive groups include, but are not limited to, halide (e.g., bromo, chloro, iodo), alkyne, aldehyde, vinyl ketone, and / or maleimide functional groups. All functional groups listed in Tables 2 and 3 are compatible with these strategies, and further exemplary functional groups include, but are not limited to, those listed in Table 4.

[0105] [Table 4]

[0106] Polymerizing the hydrophobic and hydrophilic monomers (optionally via ATRP or RAFT) may include polymerizing the hydrophobic and hydrophilic monomers in 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 monomer:hydrophilic monomer). In some embodiments, the ratio may be from about 1:1 to about 1:3 or about 1:6. In some embodiments, the hydrophobic monomer is an alkyl acrylate (e.g., dodecyl methyl acrylate) and / or the hydrophilic monomer is a glycol acrylate (e.g., PEGylated methyl acrylate). In some embodiments, one or more hydrophobic monomers are polymerized with two or more different hydrophilic monomers (optionally via RAFT or ATRP) in 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 monomer:hydrophilic monomer). For example, in some embodiments, a first hydrophilic monomer can be ionic (e.g., a sulfonic acid acrylate monomer (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or a sulfonate monomer), and a second hydrophilic monomer can be non-ionic (e.g., a glycol acrylate (e.g., PEGylated methyl acrylate)). The ratio of the first hydrophilic monomer to the second hydrophilic monomer can vary (e.g., the ratio of first hydrophilic monomer:second hydrophilic monomer can 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.

[0107] Exemplary catalysts that may 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) chloride [RuCp *Cl(PPh3)2], chloro(cyclopentadienyl)bis(triphenylphosphine)ruthenium (II) [RuCpCl(PPh3)2], dihydridotetrakis(triphenylphosphine)ruthenium(II) [RuH2(PPh3)4], and dichloro(p-cymene)ruthenium(II) dimer. Exemplary iron complexes include, but are not limited to, dichlorobis(triphenylphosphine)iron(II) [FeCl2(PPh3)2], bromo(cyclopentadienyl)dicarbonyliron(II) [FeCpBr(CO)2], and cyclopentadienyliron dicarbonyl dimer. In some embodiments, copper complexes prepared in situ using copper salts and ligands may be used; exemplary copper salts include, but are not limited to, cuprous chloride, cuprous bromide, copper(I) triflate, copper(I) 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, sparteine and other diamines, and terpyridine and its derivatives. Exemplary nickel complexes include, but are not limited to, dibromobis(triphenylphosphine)nickel(II) [NiBr2(PPh3)2] and tetrakis(triphenylphosphine)nickel [Ni(PPh3)4]. An exemplary palladium complex is tetrakis(triphenylphosphine)palladium [Pd(PPh3)4]. An exemplary rhodium complex is tris(triphenylphosphine)rhodium bromide. An exemplary rhenium complex is dioxobis(triphenylphosphine)rhenium iodide. In some embodiments, the catalyst is pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride.

[0108] A co-catalyst may optionally be present in the methods of the present invention, such as in the step of polymerizing the hydrophobic and hydrophilic monomers. In some embodiments, a co-catalyst may be present and may be 4-(dimethylamino)-1-butanol.

[0109] In some embodiments, the methods of the present invention include hydrolyzing the copolymer, optionally in the presence of trifluoroacetic acid and water, to provide a formyl group at the first portion (e.g., first end) of the copolymer. The methods may include reacting a dye with the formyl group of the copolymer, optionally forming a 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, a biomolecule may be attached by reacting the formyl group with an amine group on a bioconjugate group via reductive amination.

[0110] In some embodiments, the method includes reacting the copolymer with mercaptoacetic acid and triethylamine to provide a carboxymethyl thioether group at a second portion (e.g., a second terminus) of the copolymer. The carboxymethyl thioether group can be derivatized to provide an N-hydroxysuccinimide ester at the second portion of the copolymer. A biomolecule (e.g., avidin) can be attached to the N-hydroxysuccinimide ester at the second portion of the copolymer.

[0111] In some embodiments, the method of the present invention comprises reacting the copolymer with sodium azide to provide an azide group, and optionally attaching a dye to the azide group via copper-catalyzed azide-alkyne chemistry.

[0112] In some embodiments, the methods of the present invention involve RAFT polymerization. In some embodiments, the RAFT polymerization occurs in the presence of a radical initiator (e.g., AIBN) and a RAFT agent, such as a thiocarbonylthio compound. Further examples of RAFT agents include, but are not limited to, dithioesters, dithiocarbamates, trithiocarbonates, dithiobenzoates, and / or xanthates.

[0113] In some embodiments, the methods of the present invention include cleaving the thiocarbonylthio functionality present at the terminal end of a copolymer obtained using RAFT polymerization. Such cleavage can occur using any common method known in the art. For example, in some embodiments, the thiocarbonylthio functionality is cleaved via aminolysis, e.g., in the presence of ethanolamine, to provide a free thiol. In some embodiments, the free thiol can be coupled to a dye containing a maleimide functionality, thereby attaching the dye to the first portion (e.g., the terminal end) of the copolymer. In some embodiments, a biomolecule can be attached to the free thiol group of the first portion (e.g., the terminal end). In some embodiments, a biomolecule can be attached to the opposite terminal end of the polymer.

[0114] In some embodiments, the compounds and / or compositions of the present invention can be used in flow cytometry. Flow cytometry is known and is described, for example, in U.S. Patent Nos. 5,167, 5,915,925, 6,248,590, 6,589,792, and 6,890,487. In some embodiments, for detection, particles to be detected, such as cells, are labeled with a luminescent compound, such as a compound of the present invention. Labeling can be performed by any suitable technique, such as by binding a luminescent compound (e.g., a compound of the present invention) to a particle or cell, for example, via an antibody that specifically binds to the particle or cell, by uptake or internalization of the luminescent compound into the cell or particle, or by nonspecific adsorption of the luminescent compound to the cell or particle. The compounds described herein can be useful as such luminescent compounds in flow cytometry. Flow cytometry techniques (including fluorescence activated cell sorting or FACS) may be performed according to known techniques or variations thereof that will be apparent to those skilled in the art based on the present disclosure.

[0115] In some embodiments, there is provided a method of detecting cells and / or particles using flow cytometry, the method comprising labeling the cells and / or particles with a compound of the invention and detecting the compound by flow cytometry, thereby detecting the cells and / or particles.

[0116] In some embodiments, methods are provided for detecting tissues and / or pathogens (e.g., cells, infectious agents, etc.) in a subject, the methods comprising administering to the subject a compound and / or composition of the invention, optionally allowing the compound to associate with the tissue and / or pathogen, and detecting the compound in the subject, thereby detecting the tissue and / or pathogen.

[0117] In some embodiments, methods are provided for using the compounds of the present invention in photodynamic therapy (PDT) and / or photodynamic inactivation (PDI). Photodynamic therapy (PDT) is a form of phototherapy that involves light and a photosensitizing chemical (e.g., a compound of the present invention) used in conjunction with molecular oxygen to induce cell death (phototoxicity). PDT can be used to kill microbial cells, including bacteria, fungi, and viruses. PDT can also be used to treat cancer. When light energy is administered in photodynamic therapy (PDT) to destroy tumors, various forms of energy are within the scope of the present invention, as will be understood by those skilled in the art. Such forms of energy include, but are not limited to, heat, acoustic, ultrasound, chemical, light, microwave, ionization (such as X-rays and gamma rays), mechanical, and / or electrical. For example, sonodynamically induced or activated drugs 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)), and other cancer drugs such as daunorubicin and adriamycin used in the presence of ultrasound therapy (see Yumita et al., Japan J. Hyperthermic Oncology, 3(2):175-182 (1987)).

[0118] Examples of treatment areas include, but are not limited to:

[0119] (i) Treatment of opportunistic infections. The compounds, compositions, and / or methods of the present invention may be useful for opportunistic infections, particularly PDT of soft tissues. For antimicrobial treatment (via PDT) of infections, particularly wound infections, infectious organisms may include (by way of non-limiting examples) Staphylococcus aureus, Pseudomonas aeruginosa, and / or Escherichia coli. In hospital-acquired infections, Pseudomonas aeruginosa is responsible for 8% of surgical wound infections and 10% of bloodstream infections. In some embodiments, the subject is an immunocompromised subject, such as, for example, a person with AIDS and / or a person receiving treatment with an immunosuppressant.

[0120] (ii) Treatment of burns. Infections caused by Staphylococcus aureus and gram-positive bacteria in general are particularly prevalent in burns (Lambrechts, 2005). Multidrug resistance of Staphylococcus aureus presents significant medical challenges. In this regard, the compounds, compositions, and / or methods of the present invention may be useful for treating opportunistic infections in burns.

[0121] (iii) Sepsis. The compounds, compositions, and / or methods of the present invention may be useful for PDT treatment of subjects suffering from opportunistic infections caused by Vibrio vulnificus. V. vulnificus is a Gram-negative bacterium that causes primary sepsis, wound infections, and / or gastrointestinal disease in humans.

[0122] (iv) Ulcers. The compounds, compositions, and / or methods of the present invention may be useful for PDT treatment of ulcer-causing bacteria (Helicobacter pylori). In the clinic, treatment may be achieved in any suitable manner, such as by inserting a fiber optic cable (similar to an endoscope, but providing red or near-infrared light delivery) into the stomach and / or affected area.

[0123] (v) Periodontal Disease. The compounds, compositions, and / or methods of the present invention may be useful in PDT to treat periodontal disease, including gingivitis. Periodontal disease is caused by bacterial overgrowth, such as the Gram-negative anaerobe Porphyromonas gingivalis. As with many PDT treatments, targeting or solubilizing the entity in conjunction with the photoactive species is essential for proper delivery of the photoactive species to the desired cells. Oral pathogens of interest for targeting include, but are not limited to, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythis, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. polymorphum, Actinomyces viscosus, and Streptococcus. For such applications, the compounds and / or compositions of the present invention may be applied topically (e.g., as a mouthwash or rinse) and then light may be administered using an external device, an intraoral appliance, or a combination thereof.

[0124] (vi) Atherosclerosis. The compounds, compositions, and / or methods of the present invention may be useful in 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 thin layer of collagen in coronary arteries, often leading to fatal thrombosis (Demidova and Hamblin, 2004). Bacteriochlorins that target such inflammatory macrophages may be useful in PDT of vulnerable plaques.

[0125] (vii) Cosmetic and dermatological applications. The compounds, compositions, and / or methods of the present invention may be useful in PDT to treat a wide range of cosmetic and dermatological problems, such as hair removal, treatment of psoriasis, and / or removal of skin discoloration. Ruby lasers are currently used for hair removal. In many laser treatments, melanin is the photosensitizing chromophore. Such treatments work better in individuals with darker hair and lighter skin. The compounds, compositions, and / or methods of the present invention may be used as near-infrared sensitizers for hair removal, allowing for more specific and / or sharper absorption band targeting of the chromophore.

[0126] (viii) Acne. The compounds, compositions, and / or methods of the present invention can be useful in PDT to treat acne. Acne vulgaris is caused by Propionibacterium acnes, which infects the sebaceous glands and affects approximately 80% of young people. Again, the increasing resistance of bacteria to antibiotic treatment has led to a surge in difficult-to-treat acne. Current PDT treatments for acne typically rely on the addition of aminolevulinic acid, which is converted to free-base porphyrin in hair follicles or sebaceous glands. The compounds and / or compositions of the present invention can be administered to a subject topically or parenterally (e.g., by subcutaneous injection), depending on the specific condition.

[0127] (ix) Infectious diseases. The compounds, compositions, and / or methods of the present invention may be useful in PDT for treating infectious diseases. For example, cutaneous leishmaniasis and subcutaneous leishmaniasis, which are widespread in the Mediterranean and Middle Eastern regions, are currently treated with arsenic-containing compounds. PDT has recently been used with reasonable effectiveness, at least in some cases in human subjects. The use of the compounds and / or compositions of the present invention is similarly useful and potentially offers advantages such as ease of synthesis and better spectral absorption properties.

[0128] (x) Tissue sealant. The compounds, compositions, and / or methods of the present invention can be useful in PDT as tissue sealants in subjects in need thereof. Light-activated tissue sealants are attractive for sealing wounds, joining tissues, and / or closing defects in tissues. There are many applications where sutures and / or staples are undesirable, and the use of such mechanical sealing methods often leads to infection and / or scarring.

[0129] (xi) Neoplastic diseases. The compounds, compositions, and / or methods of the present invention may be useful in PDT to treat neoplastic 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, cutaneous T-cell lymphoma in the plaque stage, and / or Kaposi's sarcoma.

[0130] During photodynamic therapy, a compound of the present invention is administered to a subject in need thereof (e.g., a subject suffering from any of the above-mentioned diseases). The administered compound may bind to diseased tissue present within the subject, and exposing the subject to a light source emitting appropriate light having a suitable wavelength and intensity activates the compound (e.g., releases reactive oxygen species (ROS)) within the diseased tissue, thereby treating the diseased tissue, optionally without affecting healthy tissue. For example, in some embodiments, the diseased tissue is a hyperproliferative tissue (e.g., a tumor).

[0131] The present invention is described in further detail in the following non-limiting examples. [Example]

[0132] Single-polymer encapsulation of a single hydrophobic fluorophore Studies were carried out on random copolymers bearing pendant PEGylated fluorophores and polymerized micelles containing hydrophobic fluorophores. Finally, the polymerized micelles (RuCp *We identified a design requiring heterotelechelic, amphiphilic, random copolymers derived from two acrylate monomers—a hydrophilic (pendant PEG-6) monomer and a hydrophobic (dodecyl) monomer—in a 3:1 ratio via Cl(PPh3)2, 4-(dimethylamino)-1-butanol, and an acetal-substituted initiator (in ethanol at 40 °C). Hydrolysis of the acetal followed by reaction with a hydrophobic chlorin-hydrazide afforded polymers bearing a single chlorin-hydrazone (i.e., foldamers or single-chain nanoparticles, abbreviated as scNp). Examination of the chlorin polymers in aqueous solution revealed sharp absorption / emission bands and undampened fluorescence quantum yields compared to chlorins in toluene. This approach separates fluorophore selection and water-solubilization strategies into distinct domains, the latter of which is now significantly easier to implement.

[0133] 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 are composed of a 3:1 ratio of hydrophilic (PEG) and hydrophobic (dodecyl) segments and have a molecular weight of approximately 120 kDa. As random block copolymers, the copolymers in water undergo self-folding to create a hydrophobic center, encapsulating the hydrophobic dye and thereby protecting it from aggregation. Three hydrophobic dyes, namely, BODIPY, chlorin, and phthalocyanine, with different molecular sizes and absorption wavelengths (540, 640, 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 resulting distinctly different fluorescence properties of the dye-loaded copolymers in water suggest that the effectiveness of dye encapsulation may depend on the molecular size of the dye and the length of the copolymer backbone.

[0134] [ka]

[0135] Synthesis of Hydrophobic Fluorophores. Generally, the dye-hydrazides used here for dye attachment were prepared from the corresponding carboxylic acid esters via amide formation. Treatment of the activated carboxylic species, BODIPY-NHS ester 1, with hydrazine hydrate afforded the desired BODIPY-hydrazide D1 in 40% yield (Scheme 2).

[0136] [ka]

[0137] In the presence of Pd(PPh), methanol, and carbon monoxide, iodochlorin 2 was quantitatively converted to methyl ester 3 via carbonyl insertion (Scheme 3). Subsequent treatment of methyl ester 3 with hydrazine hydrate under reflux conditions afforded the desired chlorin-hydrazide D2 in 83% yield. It was found necessary to perform the reaction at concentrations below 50 mM, as more concentrated solutions resulted in the reduction of D2 to the corresponding bacteriochlorin.

[0138] [ka]

[0139] The preparation of phthalocyanine-hydrazide D3 was more laborious due to the limited solubility of the macrocycle. Ethynylphthalocyanine 4 was coupled with methyl 3-(4-bromophenyl)propanoate in the presence of Pd(OAc) / P(o-tol) to give the methyl ester 5 in 13% yield (Scheme 4). Again, the low solubility of the macrocycle in the reaction system is responsible for the low yield of the Sonogashira coupling reaction. Subsequent treatment of methyl ester 5 with hydrazine in a mixture of toluene and methanol afforded the desired hydrazide D3.

[0140] [ka]

[0141] Synthesis of copolymers. Living radical polymerization of the monomers PEGA and LA was carried out in a 3:1 ratio using the reported initiator 6 with RuCp * This was carried out in the presence of Cl(PPh3)2 and 4-dimethylaminobutanol (Scheme 5). The resulting copolymer 7 was heterotelechelic, possessing an acetal group at one end and a bromine group at the other end. The two functional groups were derivatized for further dye attachment and bioconjugation-enabled handles, respectively. The bromine group in 7 was replaced with mercaptoacetic acid, resulting in a carboxyl group at the copolymer end, amenable to bioconjugation. Hydrolysis of the acetal end under acidic conditions afforded formyl copolymer 8. This copolymer 8 served as a platform for dye conjugation, affording target dye-loaded copolymers F1–F3 via treatment with hydrazides D1–D3, respectively.

[0142] [ka]

[0143] SEC analysis. Taking F2 as an example, analytical SEC was used to monitor the process of the dye attachment reaction. The SEC trace shown in Figure 2 shows the size increase when chlorin was attached onto the copolymer. Also, the molecular weight of copolymer 7 was estimated to be 1.2 × 10 based on SEC analysis. 5 It was estimated to be g / mol.

[0144] Measurement of absorbance and emission spectra. The target dye-loaded copolymers F1–F3 were then subjected to investigation of their spectroscopic properties in both organic and aqueous solutions. The spectra are shown in Figure 3. For both F1 (BODIPY-loaded, Figure 3, Panel A) and F2 (chlorin-loaded, Figure 3, Panel B), the absorbance spectra of the samples in aqueous solutions at μM concentrations were comparable to those in organic solutions. Attachment to the 120 kDa amphiphilic copolymer dramatically enhances the water solubility of BODIPY D1 and chlorin D2 without disrupting their spectroscopic properties. The emission bands of F1 and F2 in water remained the same as those measured in organic solution, indicating minimal dye-dye interactions are involved in the aqueous solutions of F1 and F2 at μM concentrations. Nevertheless, for the phthalocyanine-loaded copolymer F3, which has the largest dye molecular size, a completely different absorbance spectrum was obtained in water from that in toluene (Figure 3, Panel C), indicating completely quenched fluorescence. This negative result may be due to inappropriate copolymer backbone size. Larger sized polymers may be required to encapsulate large hydrophobic fluorophores such as phthalocyanine D3.

[0145] Fluorescence quantum yield. Fluorescence quantum yields were also measured for F1-F3 in water at room temperature. The data are summarized in Table 5 along with other spectroscopic data. Taking the chlorin-attached copolymer F2 as an example, the dye-copolymer conjugate exhibited a fluorescence quantum yield of 0.18 in water at μM concentrations (entry 6), which is similar to the value for dye D2 alone in CHCl (0.19, entry 4). Similar results were obtained for BODIPY-labeled copolymer F1 (Φ f= 0.058, entry 3) and BODIPY dye D1 (0.065, entry 1). These comparisons demonstrate the absence of dye-dye quenching resulting from aggregation of F1 and F2 in μM aqueous solutions. This result demonstrates amphiphilic copolymers as a successful platform for encapsulation of hydrophobic fluorophores in water, provided the polymer chain length is appropriate. However, as noted above, the phthalocyanine-labeled copolymers have completely quenched fluorescence. Longer polymer chains may be more effective for encapsulating larger fluorophores, such as D3. Also, smaller phthalocyanine skeletons (e.g., those with methyl instead of heptyl as peripheral groups) may be successfully encapsulated using copolymers of the current length.

[0146] [Table 5]

[0147] Experimental Section General Methods. All commercially purchased chemicals were used as received unless otherwise noted. Reagent-grade solvents (CHCl, THF, methanol) and HPLC-grade water were used as received. NMR data were measured in CDCl solutions unless otherwise noted. Non-commercially available compounds 1, 2, and 4 were prepared according to literature procedures. Analytical SEC experiments were performed on a PLgel 10000Å SEC column and 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 infinity refractive index detector. Absorbance spectra were measured at room temperature on Agilent 8453 and Shimadzu UV1800 instruments using dilute solutions (μmolar) of compound in UV-transparent (e.g., quartz) cuvettes versus solvent blanks.

[0148] 2-[6-(N-aminocarbamoyl)hex-1-yn-1-yl]-8-mesityl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (D1). A solution of 1 (9.0 mg) in THF (500 μL) was treated with hydrazine hydrate (5.3 μL) at room temperature for 30 min. The solution was then concentrated and chromatographed (silica gel, CHOH / acetic acid 9:1) to give a red solid (3.0 mg, 39%): H NMR (DMSO-d, 300 MHz) δ 8.85 (br, 1H), 7.99 (s, 1H), 7.94 (s, 1H), 6.95 (s, 2H), 6.76 (d, J = 4.2 Hz, 1H), 6.72 (s, 1H), 6.53 (d, J = 4.2 Hz, 1H), 2.45-2.47 (m, 2H), 2.36 (s, 3H), 2.17-2.20 (m, 2H), 2.09 (s, 6H), 1.58-1.42 (m, 4H); MALDI-MS observed 449.1 [(M+H) + ], 429.2[(MF) + ], calculated value 448.2 (M = C 25 H 27 BF2N4O).

[0149] 10-Mesityl-5-(4-methoxycarbonyl)phenyl-18,18-dimethylchlorin (3). Toluene and methanol were degassed by bubbling argon through them for 1 h. Iodochlorin 2 (20 mg, 0.030 mmol, 1.0 equiv) and Pd(PPh3)4 (3.5 mg, 3.0 μmol, 0.10 equiv) were placed in a conical vial equipped with a rubber septum and then evacuated under high vacuum. The vial was then refilled with argon. This evacuation-purge process was repeated three times. Degassed toluene (0.50 mL) and methanol (0.50 mL) were added to the vial under argon, as well as triethylamine (21 μL, 0.15 mmol, 5.0 equiv). The solution was again degassed using three freeze-purge cycles. The vial was evacuated under high vacuum at 77 K and then refilled with carbon monoxide. A CO-filled balloon was also connected to the vial to provide excess pressure. The solution was stirred at 65 °C for 23 h, concentrated, and chromatographed (silica gel, hexane / CHCl 1:1) to give a green solid (18 mg, 100%): TLC (silica, hexane / CHCl 1:1) R f =0.28; 1 H NMR (300MHz) δ8.92(s, 1H), 8.87(s, 1H), 8.82(d, J=4.8Hz, 1H), 8.73(d, J= 4.7Hz, 1H), 8.69(d, J=4.7Hz, 1H), 8.61(d, J=4.7Hz, 1H), 8.38(d, J=8.1Hz, 2H), 8.37(s, 1H), 8.36(s, 1H), 8.22(d, J=8.3Hz, 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); 13C NMR (100MHz) δ175.2, 167.6, 163.6, 152.4, 151.5, 147.2, 140.9, 140.4, 139.2, 138.3, 137.7, 134.7, 134.4, 134.1, 132.1, 131.1, 129.5, 128.1, 128.0, 127.8, 123.7, 123.6, 120.59, 120.57, 96.81, 94.99, 52.49, 51.86, 46.63, 31.31, 21.57, 21.45; ESI-MS observed value 592.2851 [(M+H] + ], calculated value 592.2838 (M = C 39 H 36 N4O2); experimental values (CH2Cl2) 415, 509, 533, 590, 641 nm.

[0150] 5-[4-(N-aminocarbamoyl)phenyl]-10-mesityl-18,18-dimethylchlorin (D2). A solution of chlorin 3 (44 mg, 75 μmol, 1.0 equiv.) in THF (1.0 mL) was treated with methanol (1.0 mL) and hydrazine hydrate (0.21 mL, 3.8 mmol, 50 equiv.) at 50°C for 24 h. [Note: Reduction of the chlorin-hydrazine to the corresponding bacteriochlorin-hydrazine occurs at concentrations greater than 50 mM. The bacteriochlorin can be oxidized back to the desired chlorin by treatment with DDQ (1.0 equiv.) in CHCl at room temperature for 30 min.] The solution was then diluted with ethyl acetate, washed with water, dried over sodium sulfate, concentrated, and chromatographed (silica gel, hexane / EtOAc 1:2 to CH2Cl2 / CH3OH 9:1) to give a green solid (37 mg, 84%): 1H NMR (400MHz) δ8.96(s, 1H), 8.88(s, 1H), 8.76(d, J=4.5Hz, 1H), 8.75(d, J=4. 5Hz, 1H), 8.62(d, J=4.7Hz, 1H), 8.56(d, J=4.7Hz, 1H), 8.44(d, J=8.1Hz, 2H) , 8.39(s, 1H), 8.38(s, 1H), 8.30(d, J=8.0Hz, 2H), 7.68-7.64(m, 2H), 5.02(b r, 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 observed 593.1 [(M+H) + ], calculated value 592.3 (M = C 38 H 36 N6O).

[0151] 2-[4-(2-Methoxy-2-oxoethyl)phenyl]ethynyl-9,10,16,17,23,24-hexaheptylphthalocyanine (5). Following the standard Sonogashira coupling procedure, a solution of 4 (20 mg, 18 μmol), methyl 3-(4-bromophenyl)propanoate (4.8 mg, 20 μmol), Pd(OAc) (1.1 mg, 13 μmol), and P(o-tol) (5.5 mg, 18 μmol) in degassed toluene (6.0 mL) was degassed by three freeze-degass cycles. The mixture was stirred at 60 °C for 18 h. The resulting reaction mixture was concentrated and subjected to column chromatography using a three-column strategy [(1) silica, CHCl, (2) SEC, toluene, (3) silica, CHCl] to give a green solid (3.0 mg, 13%). MALDI-MS: Observed value 1289.4 [(M+H) + ], calculated value 1288.9 (M = C 86 H112 N8O2).

[0152] 2-[4-(N-aminocarbamoyl)methylphenylethynyl]-9,10,16,17,23,24-hexaheptylphthalocyanine (D3). A solution of 5 (3.0 mg, 2.3 μmol) in toluene (140 μL) was treated with 6.5 μL of hydrazine hydrate (55 wt%) and methanol (10 μL). The resulting mixture was stirred at 50 °C for 16 h, at which point ethyl acetate and water were added to the mixture. The organic extract was washed with brine, dried (NaSO), and concentrated to give a green solid, which was used directly in the next step of the synthesis. [Example]

[0153] A general method for the preparation and derivatization of polymers according to some embodiments of the present invention is shown below in Scheme 6.

[0154] [ka]

[0155] In the approach shown in Scheme 6, the initiator is QX, where X can be a halo (e.g., Cl, Br, I) or a sulfonate (e.g., triflate), and Q can carry a dye or a functional group that remains intact throughout the polymerization process.

[0156] For further derivatization, the functional groups required for dye attachment can be incorporated (into the Q unit) prior to polymerization and used directly. Alternatively, after polymerization, derivatization of Q in the synthetic polymer I can provide a modified Q (denoted Q') in polymer I for dye attachment.

[0157] Provision of attachment to a biomolecule (at the ω-terminus of the polymer) occurs, in one instance, through the direct use of an X substituent in polymer I. Alternatively, the X group can be substituted to provide a functional group W in polymer II for attachment of a biomolecule. Examples of W include azide, isocyanato, isothiocyanato, active esters (e.g., pentafluorophenyl ester, succinimide ester, 2,4-dinitrophenyl ester), maleimide, vinyl, mercapto, amino, and carboxylic acid. Derivatization at the ω-terminus in polymer I can be accomplished by a single step or multiple steps (e.g., nucleophilic substitution and / or deprotection) to provide the desired functional group W in polymer II. In prepolymerization methods, the functional group is first installed in the initiator (Q unit of QX, Scheme 6) and remains intact throughout the polymerization process.

[0158] Some examples of Q and QX are shown in Scheme 7. As shown in Scheme 7, Q can include hydroxy groups. 1、2 , carboxy 3 ,amino 4 , formyl 4 , vinyl 5、6 , epoxy 7 , anhydride 8 , haloaryl 7 ,ester 3 , or oxazoline 8 Vinyl or allyl groups can be installed via the initiator and remain intact during polymerization without causing additional problems during cross-linking. 1、5、6 This can be achieved by selecting appropriate ligands, primarily in the presence of copper(I) catalysts. However, some functional groups commonly used for dye attachment (e.g., azide groups) or bioconjugation cannot be installed by prepolymerization methods (shown in Table 6).

[0159] [ka]

[0160] [Table 6]

[0161] It should be noted that the examples described herein describe the attachment of a dye to the α-terminus of the polymer and a biomolecule to the ω-terminus of the polymer, however, the use of the two termini can be reversed if desired, with the biomolecule attached to the α-terminus of the polymer and the dye attached to the ω-terminus of the polymer.

[0162] References JPEG2025118679000018.jpg141166 [Example]

[0163] Reaction examples An exemplary reaction for preparing compounds of the invention, including cross-linking, is provided in Scheme 8.

[0164] [ka] JPEG2025118679000020.jpg113166

[0165] Exemplary reactions for preparing compounds of the invention, including sulfonation and cross-linking, are provided in Scheme 9.

[0166] [ka] JPEG2025118679000022.jpg172166JPEG2025118679000023.jpg225166 [Example]

[0167] An example of a method for preparing and derivatizing polymers according to some embodiments of the present invention is shown below in Scheme 10.

[0168] [ka]

[0169] In the approach shown in Scheme 10, Z in the RAFT agent can be aryl, alkyl, or thioalkyl, and Q can carry a functional group that remains intact throughout the polymerization process.

[0170] For further derivatization, functional groups required for attachment to dyes or biomolecules can be incorporated (into the Q units) prior to polymerization and used directly. Such functional groups can be initially installed within the Q units of the RAFT agent and remain intact throughout the polymerization process. Alternatively, derivatization of Q in the synthetic polymer after polymerization can yield modified Q for attachment of dyes or biomolecules.

[0171] Some examples of Z and Q in RAFT agents are shown in Chart 1. Examples of Z in RAFT agents include, but are not limited to, phenyl (optionally substituted) and / or thioalkyl groups (including branched and / or unbranched C1-C25 thioalkyl groups).

[0172] Examples of Q in a RAFT agent include, but are not limited to, carboxylate, azide, hydroxy, N-succinimidyl, vinyl, phthalimide, and / or biotinyl.

[0173] [ka]

[0174] Prior to attaching a dye or biomolecule to the terminal end of a polymer containing a thiocarbonylthio group, the thiol group can be liberated by cleavage of the thiocarbonylthio group using methods known in the art. The free thiol group can be directly coupled to a dye or biomolecule or can be further modified with an agent LW to provide a capped thiol (e.g., a thioether) with an appropriate functional group W for coupling to a dye or biomolecule. The agent LW contains a thiol-reactive group L, which reacts with the free thiol group and also serves as a linker L' between the thiol and functional group W in the capped product.

[0175] Some examples of L and W in LW are shown in Chart 2. Examples of L groups in LW agents 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).

[0176] Examples of functional groups W include, but are not limited to, carboxylic acids (e.g., -COOH, -CH2CH2COOH), aminos (e.g., -NH2, -CH2CH2NH2, optionally with a protecting group, i.e., NHBoc, -CH2CH2NHBoc), aldehydes, alcohols (e.g., -CH2CH2OH), and / or alkylated alcohols (e.g., -OCH2CH2OH, -OCH2CH2NHBoc, -OCH2CH2N3, -OC≡CH, -OCH2CH=CH2).

[0177] Derivatization of the free thiol group can be accomplished in a single step or in multiple steps (eg, nucleophilic substitution and / or deprotection) to provide the desired functional group W.

[0178] [ka]

[0179] A further example of RAFT polymerization is shown in Scheme 11. The hydrophobic monomer dodecylmethylacrylate (LA) is polymerized with the hydrophilic monomers 2-acrylamido-2-methylpropanesulfonic acid (AMPS) as the sodium salt and PEGylated methylacrylate (PEGA) in the presence of a RAFT agent and a radical initiator to produce a polymer. In some embodiments, one or more functional groups are present (e.g., pre-installed) on the RAFT agent prior to polymerization. Examples of such functional groups are shown in Scheme 11. After polymerization, the pre-installed functional group is located at one terminal end of the polymer and can be used for coupling to biomolecules or dyes.

[0180] [ka]

[0181] References JPEG2025118679000028.jpg152166 [Example]

[0182] Synthesis of amphiphilic random copolymers via reversible addition-fragmentation chain transfer (RAFT) polymerization. A model study of the synthesis of sulfonated amphiphilic random copolymers is shown in Scheme 12. Three monomers were used, one hydrophobic (dodecylmethyl acrylate (LA)) and two hydrophilic (2-acrylamido-2-methylpropanesulfonic acid (AMPS) as the sodium salt and PEGylated methyl acrylate (PEGA)). AMPS can be prepared by basifying commercially available 2-acrylamido-2-methylpropanesulfonic acid with sodium hydroxide and / or by basifying the commercially available sodium salt of 2-acrylamido-2-methylpropanesulfonic acid, which has a small amount of free acid present as a trace contaminant in the commercially available AMPS material. RAFT chain transfer agent 1 was used as available in the laboratory. Polymerizations using varying monomer ratios were carried out in DMF (80 °C) containing AIBN as the radical initiator and mesitylene as the internal standard. After polymerization, the crude product was poured into a large excess of ethyl ether to precipitate the polymer. The precipitate was then dialyzed against water to obtain the purified polymer.

[0183] [ka]

[0184] Dynamic light scattering (DLS) size analysis of amphiphilic polymers. Each polymer was dissolved in 1.0 M aqueous NaCl and passed through a 200 nm membrane filter. The filtrate was examined by DLS to determine the nanoparticle size. DLS size data for various polymers are summarized in Table 7. The data indicated that for polymers without PEG groups, a 6:1 ratio of sulfonic acid groups to lauryl groups produced the best results, yielding 65% unimers in aqueous solution. When PEG groups were introduced, the percentage of unimers was higher when the PEG to sulfonic acid group ratio was reduced from 1:1 to 1:5. At a ratio of AMPS:PEGA:LA = 5:1:1, unimers appeared to be the predominant species in aqueous solution.

[0185] [Table 7]

[0186] Synthesis of polymer-fluorophore conjugates via RAFT polymerization. Living radical polymerization of the monomers PEGA, LA, and AMPS was carried out in a 1:1:5 ratio (i.e., hydrophilic / hydrophobic ratio = 6:1) using the RAFT agent 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid 2 in the presence of the radical initiator 2,2'-azobis(2-methylpropionitrile) (AIBN) (Scheme 13). The resulting polymer 3 is heterotelechelic and contains a carboxyl group at one end and a thiocarbonylthio group at the other end. Aminolysis of polymer 3 with ethanolamine cleaved the thiocarbonyl group, revealing a free thiol group. In situ coupling of the latter with hydrophobic maleimide-substituted bacteriochlorin D1 afforded the target polymer-fluorophore conjugate F-2.

[0187] [ka]

[0188] Dynamic Light Scattering (DLS) Size Analysis of Polymer-Fluorophore Conjugates. Polymer-fluorophore samples were dissolved in 1.0 M aqueous NaCl 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 various polymers is summarized in Table 8. Polymer-fluorophore sample F-2 exhibited a unimeric form across a range of concentrations (Figure 4).

[0189] [Table 8]

[0190] Measurement of absorbance and emission spectra and fluorescence quantum yield of F-2. The absorbance and emission spectra of the target polymer-fluorophore conjugate F-2 were measured at room temperature in both water and aqueous buffer solution (Figures 5 and 6). The spectroscopic data and fluorescence quantum yield data are summarized in Table 9.

[0191] The absorbance and emission spectra of F-2 in aqueous solution are comparable to those of D1 in toluene, and Q y There is minimal broadening and decrease in absorbance. The fluorescence yield of F-2 in aqueous media is 93% (in buffer) and 80% (in water) of that of D1 in toluene. These data are consistent with slight fluorophore aggregation in aqueous media. A single fluorophore is encapsulated in an amphiphilic polymer, which maintains its intrinsic fluorescence when immersed in an aqueous environment.

[0192] [Table 9]

[0193] The foregoing is illustrative of the invention and is not to be construed as limiting thereof. The invention is defined by the following claims, including equivalents of the claims. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

Claims

1. A-B-C, or C-A-B [In the formula, A is a dye (e.g., a fluorophore), which may have a molecular weight ranging from about 150 Daltons (Da) to about 3,000 Da; B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units, said polymer optionally having a molecular weight ranging from about 1,000 Da, 5,000 Da, or 10,000 Da to about 175,000 Da; Optionally, C comprises a bioconjugate group. A compound having a structure represented by:

2. The compound of claim 1 , wherein the dye (e.g., a tetrapyrrolic macrocycle) is covalently attached to a portion (e.g., a terminal end) of the polymer.

3. 10. The compound according to any of the preceding claims, wherein the one or more hydrophobic units and the one or more hydrophilic units are randomly distributed in the polymer.

4. 10. The compound of claim 1, wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10, and optionally the one or more hydrophobic units and the one or more hydrophilic units are present in the polymer in a ratio of about 1:6 (hydrophobic units:hydrophilic units).

5. 10. A compound according to any of the preceding claims, which has conformational flexibility.

6. 10. A compound according to any of the preceding claims, which undergoes self-folding in aqueous solution, optionally into a unimeric micellar structure.

7. 10. The compound according to any of the preceding claims, wherein the polymer is an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer.

8. 10. A compound according to any of the preceding claims, which is cross-linked, optionally cross-linked when in the folded conformation.

9. 10. A compound according to any of the preceding claims, which may be folded to provide particles, said particles having diameters ranging from about 1 nm or 3 nm to about 30 nm or 40 nm.

10. 10. A compound according to any of the preceding claims, wherein at least a portion of said one or more hydrophobic units is present in the core of said particle and / or at least a portion of said one or more hydrophilic units is present at the periphery (e.g. shell) of said particle.

11. 10. The compound of any of the preceding claims, wherein the dye is encapsulated by a portion of the compound (e.g. a portion of a polymer) when the compound is in a folded conformation.

12. 10. The compound of any of the preceding claims, wherein the polymer is a telechelic polymer or a heterotelechelic polymer.

13. 10. The compound of any of the preceding claims, wherein the dye (e.g., a tetrapyrrolic macrocycle) is hydrophobic.

14. 10. The compound of any of the preceding claims, which is water-soluble and may have a solubility in water at room temperature ranging from about 1 mg / mL to about 10 mg / mL.

15. 10. The compound of claim 1, wherein at least one of the one or more hydrophobic units and / or the one or more hydrophilic units comprises a pendant functional group, which may be a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, or fluorophenyl ester), azido, maleimide, isocyanato, isothiocyanato, phosphono, sulfono, ammonio, or phosphatidylcholine group, and / or the pendant functional group may be a hydrophilic group comprising a terminal cationic (e.g., ammonium), anionic (e.g., sulfonic acid, phosphate, carboxylic acid, or phosphonic acid), or zwitterionic (e.g., choline-like) group, and optionally a poly(ethylene glycol) moiety.

16. 10. The compound of claim 1, wherein at least one of the one or more hydrophobic units comprises an alkyl pendant group (e.g., dodecylmethyl) and / or at least one of the one or more hydrophilic units comprises a glycol pendant group (e.g., poly(ethylene glycol)).

17. 10. The compound of any of the preceding claims, wherein the polymer is attached to one dye and optionally one bioconjugate group.

18. 10. The compound of any of the preceding claims, wherein the hydrophobic unit has a structure represented by Formula III: [In the formula, R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl); R １ is absent or is —O—, —NH—, —CH ２ - and A is a C1-C20 alkyl, a C2-C20 alkenyl, or a C2-C20 alkynyl; R ２ is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azido, maleimide, isocyanato, or isothiocyanato group; p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000.

19. R in the hydrophobic unit ２ 19. The compound of claim 18, wherein is hydrogen or a hydroxyl, carboxyl, amino, formyl, or ester group.

20. R in the hydrophobic unit ２ is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimide group.

21. 10. The compound of any of the preceding claims, wherein the hydrophilic unit has a structure represented by Formula IV: [In the formula, R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl); R １ is absent or is —O—, —NH—, or —CH ２ - and R ３ is -(CH ２ CH ２ R ５ ) ｎ -, -C １ -C ６ Alkyl, —C １ -C ６ Alkyl-O-, and -C １ -C ６ Alkyl-SO ３ -, or a salt thereof, wherein R ５ is -O- or -CH ２ and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; R ４ is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group; p is an integer from 1 to 10, 100, 1,000, 5,000, 10,000, 50,000, or 100,000.

22. R in the hydrophilic unit ４ 22. The compound of claim 21, wherein is hydrogen, alkyl, phosphono, sulfono, phosphatidylcholine, phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or an ester group.

23. R in the hydrophilic unit ４ is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimide group.

24. R ３ But, -C １ -C ６ Alkyl-O-, or -(CH ２ CH ２ R ５ ) ｎ - and R ５ is —O—, and R ４ is hydrogen, alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), or phosphoryl group.

25. R ３ is C １ -C ６ Alkyl or -(CH ２ CH ２ R ５ ) ｎ - and R ５ Ga-CH ２ -, and R in the hydrophilic unit ４ 23. The compound of claim 21 or 22, wherein is a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group.

26. R ３ Ga-C １ -C ６ Alkyl-SO ３ 23. The compound according to claim 21 or 22, which is - or a salt thereof.

27. A-B-C, or C-A-B [In the formula, A is a dye (e.g., a fluorophore); B is a polymer comprising one or more hydrophobic units and one or more hydrophilic units; Optionally, C comprises a bioconjugate group. a particle including a core and a shell, the particle including a compound having a structure represented by Water and A composition comprising:

28. 28. The composition of claim 27, wherein the compound forms the particles.

29. 29. The composition of claim 27 or 28, wherein the compound and the particles are present in the composition in a ratio of about 1:1 (e.g., one compound per particle).

30. 30. The composition of any one of claims 27 to 29, wherein at least a portion of the one or more hydrophobic units are present in the core of the particle.

31. 31. The composition of any one of claims 27 to 30, wherein at least a portion of the one or more hydrophilic units are present in (e.g., at the periphery of) the shell of the particle.

32. 32. The composition of any one of claims 27 to 31, wherein the particles are resistant to dilution and may remain in a folded structure when the composition is diluted up to 100x or to submicromolar concentrations.

33. 33. The composition of any one of claims 27 to 32, wherein the dye is present in the core of the particle and / or is encapsulated by at least a portion of the polymer.

34. 34. The composition of any one of claims 27 to 33, wherein the compound is a compound of any one of claims 2 to 26.

35. polymerizing a hydrophobic monomer and a hydrophilic monomer to provide a copolymer comprising hydrophobic and hydrophilic units; providing the compound by attaching a dye to a first portion (e.g., one end or terminal portion) of the copolymer; and / or attaching a bioconjugate group to a second portion of the copolymer (e.g., the other terminus or terminal portion); A method of preparing a compound, which may include cross-linking said compound.

36. 36. The method of claim 35, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer via living radical polymerization (e.g., ATRP) in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally a co-catalyst to provide a copolymer.

37. 37. The method of claim 35 or 36, wherein the catalyst is a ruthenium complex, an iron complex, a copper complex, a nickel complex, or a rhenium complex, and optionally the catalyst is pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride.

38. 38. The method of any one of claims 35 to 37, wherein the co-catalyst is present, and optionally the co-catalyst is 4-(dimethylamino)-1-butanol.

39. 36. The method of claim 35, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer via living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT agent (e.g., a thiocarbonylthio compound) to provide a copolymer.

40. 40. The method of claim 39, wherein the RAFT agent is a dithioester, dithiocarbamate, trithiocarbonate, dithiobenzoate, and / or xanthate.

41. 41. The method of any one of claims 35 to 40, wherein the hydrophobic monomer is an alkyl acrylate (e.g., dodecyl methyl acrylate) and / or the hydrophilic monomer is a glycol acrylate (e.g., PEGylated methyl acrylate).

42. 42. The method of any one of claims 35 to 41, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer to provide a copolymer comprises polymerizing at least one hydrophobic monomer with two or more different hydrophilic monomers, and the two or more different hydrophilic monomers may comprise a non-ionic hydrophilic monomer (e.g., glycol acrylate (e.g., PEGylated methyl acrylate)) and an ionic hydrophilic monomer (e.g., sulfonic acid acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid)).

43. 43. The method of claim 42, wherein the at least one hydrophobic monomer is polymerized with the non-ionic hydrophilic monomer and the ionic hydrophilic monomer, and the non-ionic hydrophilic monomer and the ionic hydrophilic monomer are polymerized in a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, or 1:

6.

44. 44. The method of any one of claims 35 to 43, wherein polymerizing the hydrophobic monomer and the hydrophilic monomer comprises polymerizing the hydrophobic monomer and the hydrophilic monomer in a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:

10.

45. 45. The method of any one of claims 35 to 44, wherein the initiator comprises the dye (e.g., a tetrapyrrolic macrocycle).

46. 46. The method of any one of claims 35 to 45, further comprising hydrolyzing the copolymer, optionally in the presence of trifluoroacetic acid and water, to provide a formyl group at the first portion (e.g., first end) of the copolymer.

47. 47. The method of claim 46, wherein attaching the dye to the first portion of the copolymer comprises reacting the dye with the formyl group of the copolymer to optionally form a hydrazone bond between the dye and the copolymer via aldehyde-hydrazide chemistry.

48. 48. The method of any one of claims 35 to 47, further comprising reacting the copolymer with mercaptoacetic acid and triethylamine to provide a carboxymethylsulfanyl group at the second portion (e.g., second end) of the copolymer.

49. 49. The method of claim 48, further comprising derivatizing the carboxymethylsulfanyl group to provide an N-hydroxysuccinimide ester at the second portion of the copolymer.

50. 50. The method of claim 49, comprising attaching the bioconjugate to the second portion of the copolymer, wherein attaching the bioconjugate to the second portion of the copolymer comprises attaching a biomolecule (e.g., avidin) to the N-hydroxysuccinimide ester or attaching the formyl group to an amine group on the biomolecule via reductive amination.

51. 46. The method of any one of claims 35 to 45, further comprising reacting the copolymer with sodium azide to provide an azide group, and optionally attaching the dye to the azide group via copper-catalyzed azide-alkyne chemistry.

52. 52. The method of any one of claims 35 to 51, comprising cross-linking the compound, wherein cross-linking the compound comprises reacting the compound with a cross-linking group.

53. 53. The method of any one of claims 35 to 52, further comprising reacting the compound with a drug to provide the hydrophilic and / or hydrophobic units with pendant functional groups that are halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, or fluorophenyl ester), azido, maleimide, isocyanato, isothiocyanato, ammonium, phosphono, sulfono, or phosphatidylcholine groups.

54. 41. The method of claim 39 or 40, further comprising reacting the copolymer with an amine to provide a thiol group at the first portion (e.g., first end) of the copolymer.

55. 55. The method of claim 54, wherein attaching the dye to the first portion comprises attaching the dye to the thiol group of the first portion of the copolymer.

56. 56. The method of any one of claims 35 to 55, wherein the hydrophobic monomer has a structure represented by Formula I: [In the formula, R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl); R １ is absent or is —O—, —NH—, —CH ２ - and A is a C1-C20 alkyl, a C2-C20 alkenyl, or a C2-C20 alkynyl; R ２ is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, or ester (eg, succinimide ester, 2,4-dinitrophenyl ester, pentafluorophenyl ester, fluorophenyl ester, etc.) group.

57. R in the hydrophobic monomer ２ 57. The method of claim 56, wherein is hydrogen or a hydroxyl, carboxyl, amino, formyl, or ester group.

58. 58. The method of any one of claims 35 to 57, wherein the hydrophilic monomer has a structure represented by Formula II: [In the formula, R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7, or C8 alkyl); R １ is absent or is —O—, —NH—, or —CH ２ - and R ３ is -(CH ２ CH ２ R ５ ) ｎ -, -C １ -C ６ Alkyl, —C １ -C ６ Alkyl-O-, and -C １ -C ６ Alkyl-SO ３ -, or a salt thereof, wherein R ５ is -O- or -CH ２ and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; R ４ is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group.

59. R in the hydrophilic monomer ４ 59. The method of claim 58, wherein is a hydroxyl, carboxyl, amino, formyl, or ester group.

60. R ３ Ga-C １ -C ６ alkyl-O-, and R in the hydrophilic monomer ４ 59. The method of claim 58, wherein is hydrogen, alkyl (e.g., a methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidylcholine, or phosphoryl group.

61. R ３ Ga-C １ ~C ６ Alkyl or -(CH ２ CH ２ R ５ ) ｎ - and R ５ is —O—, and R ４ 59. The method of claim 58, wherein is a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group.

62. 62. The method of any one of claims 35 to 61, wherein the compound is a compound according to any one of claims 1 to 26.

63. 63. A compound prepared according to the method of any one of claims 35 to 62.

64. 36. Use of a compound according to any one of claims 1 to 26 or 63 or a composition according to any one of claims 27 to 34 in flow cytometry.

65. 1. A method for detecting cells and / or particles using flow cytometry, comprising the steps of labeling the cells and / or particles with a compound according to any one of claims 1 to 26 or 63 or a compound prepared according to the method of any one of claims 35 to 62; detecting said cells and / or particles by detecting said compounds by flow cytometry; A method comprising:

66. 1. A method for detecting tissues and / or pathogens (e.g., cells, infectious agents, etc.) in a subject, comprising: Administering to the subject a compound of any one of claims 1-26 or 63, a composition of any one of claims 27-34, or a compound prepared according to the method of any one of claims 35-62; wherein said compound may be associated with said tissue and / or pathogen. and, detecting said tissue and / or pathogen by detecting said compound in said subject. A method comprising:

67. 1. A method of treating cells and / or tissues (e.g., diseased cells and / or tissues) in a subject in need thereof, comprising: administering to the subject a compound of any one of claims 1-26 or 63, a composition of any one of claims 27-34, or a compound prepared according to a method of any one of claims 35-62; wherein said compound may be associated with said cells and / or tissues and, irradiating the subject or a portion thereof (e.g., where the cells and / or tissue are located) with light of a wavelength and intensity sufficient to treat the cells and / or tissue; wherein the light may activate the compound or a portion thereof. A method comprising:

68. 68. The method of claim 67, wherein the cells and / or tissues are hyperproliferative tissues (e.g., tumors).

69. 1. A photodynamic therapy method for treating hyperproliferative tissue in a subject in need thereof, comprising: Administering to the subject a compound of any one of claims 1-26 or 63, a composition of any one of claims 27-34, or a compound prepared according to the method of any one of claims 35-62; wherein said compound may associate with said hyperproliferative tissue. and, irradiating said hyperproliferative tissue with light of a wavelength and intensity sufficient to activate said compound or portion thereof, thereby treating said hyperproliferative tissue. A method comprising:

70. 64. A biomolecule comprising a compound according to any one of claims 1 to 26 or 63 or a compound prepared according to the method of any one of claims 35 to 62.

71. 71. The biomolecule of claim 70, comprising two or more compounds of any one of claims 1 to 26 or 63 or two or more compounds prepared according to the method of any one of claims 35 to 62.

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