Amphiphilic polymers with multiple chromophores, compositions comprising the same, and methods of preparing and using the same

Abstract

Provided herein are compounds (e.g., polymers) that include a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s) and one or more dye(s) and / or biomolecule(s) attached to a hydrophobic unit and / or hydrophilic unit. Compositions including such compounds, methods of making such compounds, and methods of using such compounds are also provided.

Description

[0001] AMPHIPHILIC POLYMERS WITH MULTIPLE CHROMOPHORES, COMPOSITIONS COMPRISING THE SAME, AND METHODS OF PREPARING AND USING THE SAME

[0002] Statement of Priority

[0003] This patent application claims the benefit of and priority to U.S. Provisional Patent Application Serial Number 63 / 604,557, filed November 30, 2023, the contents of which are hereby incorporated by reference as if recited in full herein.

[0004] Statement of Government Support

[0005] This invention was made with government support under grant number All 12302 and GM13 1501 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0006] Field

[0007] The present invention relates generally to polymeric chromophores. The present invention also relates to compositions comprising the polymeric chromophores and methods of preparing and using the same.

[0008] Background

[0009] Many applications of chromophores take place in aqueous solution yet most organic chromophores are hydrophobic or only modestly polar. Numerous approaches abound for encapsulating chromophores yet there is a need for chromophores meeting the criteria of synthetic simplicity, absence of fluorophore-fluorophore quenching, and presence of a bioconjugatable group.

[0010] Summary

[0011] A first aspect of the invention is directed to a compound (e.g., a polymer) comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, optionally wherein the first end group comprises a primary dye (e.g., a luminophore (e.g., a fluorophore) or a non-luminescent molecular entity), optionally wherein the primary dye has a molecular weight in a range of about 150 Daltons (Da) to about 3,000 Da or wherein the first end group comprises a hydrophobic group (e.g., a hydrophobic monomer), a bulky group and / or a charged group; a second end group attached to a second terminus of the polymer backbone, optionally wherein the second end group comprises a bioconjugate group or wherein the second end group comprises a dye; and a primary dye pendant from the polymer backbone, optionally wherein the compound has a molecular weight in a range of about 5,000 Da, or 10,000 Da to about 350,000 Da.

[0012] An aspect of the invention is directed to a compound (e.g., a polymer) comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, wherein the first end group comprises (i) a primary dye and a biomolecule; (ii) a primary dye and a secondary dye, and optionally a biomolecule, or (iii) two or more primary dyes, and optionally a biomolecule; and a second end group attached to a second terminus of the polymer backbone, wherein the second end group optionally comprises a bioconjugate group; and a primary dye pendent from the polymer backbone, optionally wherein the compound has a molecular weight in a range of about 5,000 Da to about 350,000 Da.

[0013] Another aspect of the invention is directed to a compound (e.g., a polymer) comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, wherein the first end group optionally comprises (i) a primary dye, or (ii) a hydrophobic group (e.g., a hydrophobic monomer), a charged and / or bulky group; a second end group attached to a second terminus of the polymer backbone, wherein the second end group optionally comprises a bioconjugate group; and one or more additional primary dyes pendant from the polymer backbone (e.g., a luminophore (e.g., a fluorophore) or a non-luminescent molecular entity), optionally wherein the primary dye has a molecular weight in a range of about 150 Da to about 3,000 Da and / or optionally wherein the compound has a molecular weight in a range of about 5,000 Da to about 350,000 Da. In some embodiments, the compound is devoid of a dye as a part of the first and second end groups and comprises a bulky group and / or a charged group at the first end group. In some embodiments, the compound is devoid of a dye as a part of the first and second end groups and comprises a hydrophobic group (e.g., a hydrophobic monomer) at the first end group. In some embodiments, the compound is devoid of a dye as a part of the first and second end groups and comprises a biomolecule at a second end group.

[0014] A further aspect of the invention is directed to a compound (e.g., a polymer) comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, wherein the first end group comprises a hydrophobic group (e.g., a hydrophobic monomer), a bulky group and / or a charged group; a second end group attached to a second terminus of the polymer backbone, wherein the second end group optionally comprises a bioconjugate group; and a primary dye pendant from the polymer backbone (e.g., a luminophore (e.g., a fluorophore) or a non-luminescent molecular entity), optionally wherein the primary dye has a molecular weight in a range of about 150 Da to about 3,000 Da and / or optionally wherein the compound has a molecular weight in a range of about 5,000 Da, or 10,000 Da to about 175,000 Da, up to about 175,000 Da, up to about 225,000 Da, up to about 250,000 Da, up to about 275,000 Da, up to about 300,000 Da, up to about 325,000 Da or up to about 350,000 Da. In some embodiments, the compound is devoid of a dye from the first and second end groups.

[0015] In an aspect, a compound (e.g., a polymer) comprises one or more charged groups pendant from the polymer backbone.

[0016] Another aspect of the invention is directed to a compound (e.g., a polymer) comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, wherein the first end group comprises a bulky group (e.g., a cyclodextrin or a polyhedral oligomeric silsesquioxane (POSS)) and / or a charged group (e.g., a sulfonate or carboxylic acid); and a second end group attached to a second terminus of the polymer backbone, optionally wherein the second end group comprises a bioconjugate group; and a primary dye pendant from the polymer backbone (e.g., a luminophore (e.g., a fluorophore) or a non- luminescent molecular entity), optionally wherein the primary dye has a molecular weight in a range of about 150 Da to about 3,000 Da and / or optionally wherein the compound has a molecular weight in a range of about 5,000 Da, or 10,000 Da to about 175,000 Da, up to about 175,000 Da, up to about 225,000 Da, up to about 250,000 Da, up to about 275,000 Da, up to about 300,000 Da, up to about 325,000 Da or up to about 350,000 Da. In some embodiments, one or more charged group(s) is pendant from the polymer backbone.

[0017] A further aspect of the present invention is directed to a method of preparing a compound of the invention. Such methods include the step of copolymerizing one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s), e.g., by polymerizing via a living radical polymerization in the presence of an initiator (e.g., a radical initiator) and a chain-transfer agent (e.g., trithiocarbamate) to provide the copolymer. Another aspect of the present invention is directed to a compound prepared according to a method of the present invention.

[0018] Also provided according to another aspect of the present invention is the use of a compound of the present invention and / or the use of a composition of the present invention, such as, for example, in flow cytometry, imaging, photodynamic therapy, photodynamic inactivation, photoimmunotherapy, and / or fluorescent guided surgery.

[0019] A further aspect of the present invention is directed to a method of detecting cells and / or particles using flow cytometry, the method comprising labeling cells and / or particles with a compound or a compound comprising a biomolecule of the present invention; and detecting the compound by flow cytometry, thereby detecting the cells and / or particles.

[0020] A further aspect of the present invention is directed to a method of detecting a compound within a subject, the method comprising labeling cells and / or particles with a compound or biomolecule of the compound of the present invention; and detecting the compound by flow cytometry; and administering the labelled cells and / or particles to the subject; and detecting the compound within the subject, thereby detecting the cells and / or particles within the subject.

[0021] Another aspect of the present invention is directed to a method of detecting a tissue and / or agent (e.g., a cell, infecting agent, etc.) in a subject, the method comprising: administering to the subject a compound, biomolecule, or composition of the present invention, optionally wherein the compound or biomolecule associates with the tissue and / or agent; and detecting the compound or the biomolecule within the subject, thereby detecting the tissue and / or agent. In some embodiments, a primary dye on the compound is detected by a first detection method (e.g., a first imaging method) and a secondary dye on the compound is detected by a second detection method (e.g., a second imaging method). Imaging methods include, for example, magnetic resonance imaging and photoacoustic imaging.

[0022] An additional aspect of the present invention is directed to a method of treating a cell and / or tissue (e.g., a diseased cell and / or tissue) in a subject in need thereof comprising administering to the subject a compound, biomolecule, or composition of the invention; and irradiating the subject or a portion thereof with light of a wavelength and intensity sufficient to treat the cell and / or tissue, optionally wherein the light activates one or more dyes on the compound. In some embodiments, such methods further include detecting the compound (e.g., detecting the compound within the subject using an imaging technique). In some embodiments, a primary dye is used to treat the cell and / or tissue (e.g., by photodynamic therapy) and a secondary dye is used for detection of the compound, for example, by magnetic resonance imaging and photoacoustic imaging.

[0023] A further aspect of the present invention is directed to a biomolecule (e.g., an antibody, etc.) comprising one or more (e.g., 1, 2, 3, 4, 5, 6, or more) compound(s) of the present invention.

[0024] It is noted that aspects of the invention described with respect to one embodiment, may be incorporated in a different embodiment although not specifically described relative thereto. That is, all embodiments and / or features of any embodiment can be combined in any way and / or combination. Applicant reserves the right to change any originally filed claim and / or file any new claim accordingly, including the right to be able to amend any originally filed claim to depend from and / or incorporate any feature of any other claim or claims although not originally claimed in that manner. These and other objects and / or aspects of the present invention are explained in detail in the specification set forth below. Further features, advantages and details of the present invention will be appreciated by those of skill in the art from a reading of the figures and the detailed description of the preferred embodiments that follow, such description being merely illustrative of the present invention.

[0025] Brief Description of the Drawings

[0026] FIG. 1A illustrates example embodiments of chain end functionalization for compounds according to some embodiments of the present invention where at least one substituent is present at one end of the polymer terminus and may be a dye and / or a biomolecule.

[0027] FIG. 1B illustrates example embodiments of chain end functionalization for compounds according to some embodiments of the present invention that include one or more dye molecules attached to the polymer backbone. A substituent may or may not be present at one end of the polymer terminus and, if present, a substituent may be a dye, charged group, or bulky group.

[0028] FIG. 2 provides exemplary hydrophobic units according to some embodiments of the invention.

[0029] FIGS. 3A-3B provide exemplary hydrophobic units according to some embodiments of the invention.

[0030] FIG. 4 provides exemplary linkers according to some embodiments of the invention. FIGS. 5A-5B provides exemplary dyes, Pl, that may be useful for magnetic resonance imaging (MRI) (5A) and photoacoustic imaging (PAI) (5B) according to some embodiments of the invention.

[0031] FIG. 6 provides a chart illustrating certain dual use applications for certain compounds of the invention.

[0032] FIG. 7 is an example scheme for preparing an exemplary compound of the present invention by copolymerization of hydrophilic monomers and hydrophobic monomers with addition of the terminal functional group (see, e.g., Table 2) and bioconjugation group following copolymerization. Next, post-polymerization modification of at least one polymer terminal group is performed with, for example, a dye molecule.

[0033] FIG. 8A provides an example method for preparing an exemplary compound of the present invention by homopolymerization of an activated ester monomer generating a backbone comprising the bioconjugation group and terminal functional group. Subsequent post-polymerization modification of the polymer backbone can be performed by functionalization with hydrophobic pendant groups, hydrophilic pendant groups, and one or more dye molecules.

[0034] FIG. 8B provides an example embodiment of post polymerization modification of a polymer terminus of an exemplary compound of the present invention that comprises hydrophilic pendant groups, hydrophobic pendant groups, and dye molecules attached to the polymer backbone.

[0035] FIG. 9 provides a fluorescence spectrum for certain compounds of the invention.

[0036] FIGS. 10A-10C provides flow cytometry data of cellular samples with an example antibody-fluorescent polymer compound of the present invention containing staining solution with two dyes on the terminal end (10A); with multiple dyes on the backbone on bang beads (10B); and with multiple dyes on the backbone on CDS monoclonal antibody staining Peripheral blood monoclonal cells (PBMCs) (10C).

[0037] FIGS. 11A-11D provide (11A) UV-VIS spectrum of FP, (11B) Fluorescence spectrum of FP with excitation of 405 nm, (11C) UV-VIS spectrum of Ab-FP conjugation, (11D) Fluorescence spectrum of Ab-FP conjugation with excitation of 405 nm of a dual-dye construct.

[0038] FIG. 12 provides Quantum yield (QY) (left) and brightness (right) for the number of dyes for an example compound of the present invention measured in THF and in PBS. FIG. 13 Includes a microscopy image of peripheral blood mononuclear cells stained with a compound comprising foldamers attached to CDS monoclonal antibody and including porphyrin-chlorin dyads as well as charged groups with a total DP of less than 200. Stained cells were imaged on Olympus IX51 Inverted Fluorescence Microscope using violet light source.

[0039] FIG. 14 includes microscopic images of an example multi-dye foldamer antibody conjugate on polystyrene beads; the example multi-dye foldamer contained 2.6 660 dyad per polymer with a 7% weight percent of total CDA.

[0040] FIG. 15 includes microscopic images of an example multi-dye foldamer antibody conjugate on polystyrene beads; the example multi-dye foldamer contained 2.4 660 dyad per polymer with a 7% weight percent of total CDA.

[0041] Detailed Description of Example Embodiments

[0042] The present invention is now described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. This invention may, however, 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.

[0043] 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 context clearly indicates otherwise.

[0044] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the present application and relevant art and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. 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. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. In case of a conflict in terminology, the present specification is controlling.

[0045] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0046] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Moreover, the present invention also contemplates that in some embodiments of the invention, any feature or combination of features set forth herein can be excluded or omitted. To illustrate, if the specification states that a complex comprises components A, B and C, it is specifically intended that any of A, B or C, or a combination thereof, can be omitted and disclaimed.

[0047] As used herein, the transitional phrase "consisting essentially of' (and grammatical variants) is to be interpreted as encompassing the recited materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term "consisting essentially of' as used herein should not be interpreted as equivalent to "comprising."

[0048] It will also be understood that, as used herein, the terms "example," "exemplary," and grammatical variations thereof are intended to refer to non-limiting examples and / or variant embodiments discussed herein, and are not intended to indicate preference for one or more embodiments discussed herein compared to one or more other embodiments.

[0049] The term "about," as used herein when referring to a measurable value such as an amount or concentration and the like, 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 the measurable value, is meant to include X as well as variations of ± 10%, ± 5%, ± 1%, ± 0.5%, or even ± 0.1% of X. A range provided herein for a measurable value may include any other range and / or individual value therein.

[0050] "Derivative", when used herein in reference to a chemical molecule, refers to a chemical molecule with one or more atoms (e.g., hydrogen), functional groups, and / or bonds modified (e.g., removed, substituted, etc.) compared to the parent molecular entity. For example, a derivative of a dye may refer to the parent dye compound that has one or more atoms (e.g., hydrogen) and / or functional groups modified (e.g., removed) to facilitate covalent binding to another group or moiety (e.g., to facilitate covalent binding to a polymer). In some embodiments, a derivative may include a functional group (e.g., a substituent and / or auxochrome) that alters the absorption spectrum of the parent molecular entity.

[0051] "Alkyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 1 to 20 carbon atoms, which can be referred to as a Cl- C20 alkyl. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n- propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, 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. "Lower alkyl" as used herein, is a subset of alkyl, and, in some embodiments, refers to a straight or branched chain hydrocarbon group containing from 1 to 4 carbon atoms. Representative examples of lower alkyl include, but are not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, and the like. The term "alkyl" or "lower alkyl" is intended to include both substituted and unsubstituted alkyl or lower alkyl unless otherwise indicated and these groups may be substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy (thereby creating a 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, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m= 0, 1, 2 or 3.

[0052] "Alkenyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 2 to 20 carbon atoms (or in lower alkenyl 2 to 4 carbon atoms) that can include 1 to 8 double bonds in the normal chain, and can be referred to as a C2-C20 alkenyl. Representative examples of alkenyl include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3 -pentenyl, 2-hexenyl, 3 -hexenyl, 2,4- heptadiene, and the like. The term "alkenyl" or "lower alkenyl" is intended to include both substituted and unsubstituted alkenyl or lower alkenyl unless otherwise indicated and these groups may be substituted with groups as described in connection with alkyl and lower alkyl above. "Alkynyl" as used herein alone or as part of another group, refers to a straight or branched chain hydrocarbon containing from 2 to 20 carbon atoms (or in lower alkynyl 2 to 4 carbon atoms) which include 1 triple bond in the normal chain, and can be referred to as a C2- C20 alkynyl. Representative examples of alkynyl include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, and the like. The term "alkynyl" or "lower alkynyl" is intended to include both substituted and unsubstituted alkynyl or lower alkynyl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and lower alkyl above.

[0053] "Halo" as used herein refers to any suitable halogen, including -F, -Cl, -Br, and -I.

[0054] "Mercapto" as used herein refers to an -SH group.

[0055] "Azido" as used herein refers to an -N3group.

[0056] "Cyano" as used herein refers to a -CN group.

[0057] "Hydroxyl" as used herein refers to an -OH group.

[0058] "Nitro" as used herein refers to an -NO2group.

[0059] "Alkoxy" as used herein alone or as part of another group, refers to an alkyl or lower alkyl group, as defined herein (and thus including substituted versions such as polyalkoxy), appended to the parent molecular moiety through an oxy group, -O-. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert- butoxy, pentyloxy, hexyloxy and the like.

[0060] "Acyl" as used herein alone or as part of another group refers to a -C(O)R radical, where R is any suitable substituent such as aryl, alkyl, alkenyl, alkynyl, cycloalkyl or other suitable substituent as described herein.

[0061] "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.

[0062] "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, methylthio, ethylthio, tert-butylthio, hexylthio, and the like.

[0063] "Cycloalkyl" as used herein alone or as part of another group, refers to a saturated or partially unsaturated cyclic hydrocarbon group containing from 3 to 20 carbon atoms (optionally with a carbon atom replaced in a heterocyclic group as discussed below). A cycloalkyl group may include 0, 1, 2, or more double or triple bonds. Representative examples of cycloalkyl include, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclododecyl. These rings may optionally be substituted with additional substituents as described herein such as halo or lower alkyl. The term "cycloalkyl" is generic and intended to include heterocyclic groups as discussed below unless specified otherwise.

[0064] "Heterocyclic group" or “heterocyclo” as used herein alone or as part of another group, refers to an aliphatic (e.g., fully or partially saturated heterocyclo) or aromatic (e.g., heteroaryl) monocyclic- or a bicyclic-ring system. Monocyclic ring systems are exemplified by any 5 or 6 membered ring containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen and sulfur. The 5 membered ring has from 0-2 double bonds and the 6 membered ring has from 0-3 double bonds. Representative examples of monocyclic ring systems include, but are not limited to, azetidine, azepine, aziridine, diazepine, 1,3-dioxolane, dioxane, dithiane, furan, imidazole, imidazoline, imidazolidine, isothiazole, isothiazoline, isothiazolidine, isoxazole, isoxazoline, isoxazolidine, morpholine, oxadiazole, oxadiazoline, oxadiazolidine, oxazole, oxazoline, oxazolidine, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazoline, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroline, pyrrolidine, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazole, thiadiazole, thiadiazoline, thiadiazolidine, thiazole, thiazoline, thiazolidine, thiophene, thiomorpholine, thiomorpholine sulfone, thiopyran, triazine, triazole, trithiane, and the like. Bicyclic ring systems are exemplified by any of the above monocyclic ring systems fused to an aryl group as defined herein, a cycloalkyl group as defined herein, or another monocyclic ring system as defined herein. Representative examples of bicyclic ring systems include but are not limited to, for example, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiopyran, benzodioxine, 1,3 -benzodi oxole, cinnoline, indazole, indole, indoline, indolizine, naphthyridine, isobenzofuran, isobenzothiophene, isoindole, isoindoline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinolizine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiopyranopyridine, and the like. These rings include quaternized derivatives thereof and may be optionally substituted with groups selected from halo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclo, heterocycloalkyl, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkyloxy, aryloxy, arylalkyloxy, heterocyclooxy, heterocyclolalkyloxy, 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-

[0065] S(O)m, heterocycloalkyl-S(O)m, amino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocycloamino, heterocycloalkylamino, disubstituted-amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano where m = 0, 1, 2 or 3.

[0066] "Aryl" as used herein 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. The term "aryl" is intended to include both substituted and unsubstituted aryl unless otherwise indicated and these groups may be substituted with the same groups as set forth in connection with alkyl and lower alkyl above.

[0067] "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.

[0068] "Amino" as used herein means the radical -NH2.

[0069] "Alkylamino" as used herein alone or as part of another group means the radical -NHR, where R is an alkyl group.

[0070] "Ester" as used herein alone or as part of another group refers to a -C(O)OR radical, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0071] "Formyl" as used herein refers to a -C(O)H group.

[0072] "Carboxylic acid" as used herein refers to a -C(O)OH group.

[0073] "Sulfoxyl" as used herein refers to a compound of the formula -S(O)R, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0074] "Sulfonyl” as used herein refers to a compound of the formula -S(O)(O)R, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0075] "Sulfonate" as used herein refers to a salt (e.g., a sodium (Na) salt) of a sulfonic acid and / or a compound of the formula -S(O)(O)OR, where R is any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0076] "Sulfonic acid” as used herein refers to a compound of the formula -S(O)(O)OH. "Amide" as used herein alone or as part of another group refers to a -C(O)NRaRbradical, where Raand Rbare any suitable substituent such as alkyl, cycloalkyl, alkenyl, alkynyl or aryl.

[0077] "Sulfonamide" as used herein alone or as part of another group refers to a -S(O)2NRaRbradical, where Raand Rbare any suitable substituent such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.

[0078] “Polymer backbone” as used herein refers to the polymer formed by polymerization of monomers including one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s). In some embodiments, one or more additional monomers may be included to form the polymer backbone, including monomers that include a dye, a bulky group or charged group, and / or monomers having a reactive group that can be used to attach a dye after polymerization. However, the polymer backbone does not include the first or second end groups of a compound of the present invention, which may be formed during polymerization (e.g., via atom-transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT)) of the one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s) and / or by subsequently modifying the polymer backbone.

[0079] “End group” as used herein refers to a structural moiety that terminates one end (e.g., terminus) of a polymer backbone. The compounds of the invention include two end groups that are at opposing ends of the polymer backbone and each end group may include one or more dye(s), bioconjugate group(s), linker(s), bulky group(s), charged group(s), and / or biomolecule(s). In some embodiments, one or both of the end groups of the compound comprise a reactive group (e.g., in telechelic polymers), and, in some embodiments, one or both end groups are formed during polymerization using living polymerization techniques such as ATRP or RAFT. In some embodiments, a reactive end group may react to covalently attach one or more dye(s), bioconjugate group(s), linker(s), bulky group(s), charged group(s), and / or biomolecule(s), whereby the one or more dye(s), bioconjugate group(s), linker(s), bulky group(s), charged group(s), and / or biomolecule(s) become part of the end group. In some embodiments, the second end group is a hydrogen, and the final moiety of the compound is a unit of the polymer backbone (e.g., a hydrophilic or hydrophobic unit). In some embodiments, the second end group comprises a non-reactive capping end group that is attached to a unit of the polymer backbone. Exemplary non-reactive capping end groups include, but are not limited to, an isobutyronitrile group. In some embodiments, an end group, for example a charged group, can comprise a non-reactive capping end group. A "pendant functional group" is a functional group directly attached to the polymer backbone or directly attached to a moiety attached to the polymer backbone. A pendant functional group may be part of a hydrophobic unit and / or monomer and / or a hydrophilic unit and / or monomer at the time of polymerization or may be added to the hydrophobic unit and / or hydrophilic unit after polymerization.

[0080] "Dye" and "chromophore" are used interchangeably herein to refer to a luminophore (e.g., a fluorescent and / or phosphorescent molecular entity) and / or a non-luminescent molecular entity (e.g., a non-fluorescent and / or non-phosphorescent molecular entity). The term "non-luminescent molecular entity" as used herein refers to a molecular entity that has no or negligible luminescence. In some embodiments, a non-luminescent molecular entity does not form excited states of any significant lifetime and / or relaxes to the ground state rapidly and essentially quantitatively. In some embodiments, a non-luminescent molecular entity has an excited-state lifetime of less than about 100, 75, 50, 25, 10, 5, 1, 0.5, or 0.1 picoseconds. In some embodiments, a non-luminescent molecular entity has a quantum yield of internal conversion of greater than about 0.8, 0.85, 0.9, 0.95, 0.99, 0.999, 0.9999, or 0.99999, where a quantum yield of 1.0 corresponds to 100%. In some embodiments, a non-luminescent molecular entity has a luminescence quantum yield of less than about 0.2, 0.15, 0.1, 0.05, 0.01, 0.001, 0.0001, or 0.00001, where a quantum yield of 1.0 corresponds to 100%. It is known that the luminescence quantum yield derives from a competitive process of radiative decay versus the sum of all processes for depopulating the excited-state manifold. Such compounds are often referred to as "non-luminescent" although sensitive detection techniques can often detect tiny amounts of residual luminescence as expected with such low luminescence quantum yields. A small amount of luminescence may not be adverse to some applications such as, e.g., a photoacoustic imaging method, although the maximum possible conversion of the optical input to the thermal output is desired. Thus, the term “non-luminescent” is used herein to indicate a molecular entity with no or negligible luminescence. In some embodiments, a compound of the present invention comprises a dye and the dye is a non-luminescent molecular entity (e.g., a non-fluorescent and / or non-phosphorescent molecular entity). In some embodiments, a compound of the present invention comprises a dye and the dye is a luminophore (e.g., a fluorescent and / or phosphorescent molecular entity). A "fluorescent molecular entity" and "fluorophore" are used interchangeably herein to refer to a molecular entity that emits fluorescence. A dye of the present invention may have certain spectroscopic features and / or properties such as, e.g., spectroscopic features and / or properties suitable for use in a method of the present invention. In some embodiments, the dye has a molecular weight in a range of about 150 Da to about 3,000 Da, about 400 Da to about 1100 Da, or about 300 Da to about 1,000 Da. In some embodiments, the dye has a molecular weight of about 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, or 3000 Da. Exemplary dyes include, but are not limited to, the dyes described in Table 12. Exemplary dyes include, but are not limited to, tetrapyrroles; rylenes such as perylene, terrylene, and quarterrylene; fluoresceins such as TET (Tetramethyl fluorescein), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyfluorescein (HEX) and 5-carboxyfluorescein (5-FAM); phycoerythrins; 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 such as indocyanine green; phthalocyanines; boron-dipyrromethene (BODIPY) dyes; quinolines; pyrenes; acridine; stilbene; as well as 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;

[0081] 6,603,070; 6,849,730; 7,005,237; 6,916,982; 6,944,047; 7,884,280; 7,332,599; 7,148,361;

[0082] 7,022,862; 6,924,375; 7,501,507; 7,323,561; 7,153,975; 7,317,108; 7,501,508; 7,378,520;

[0083] 7,534,807; 7,919,770; 7,799,910; 7,582,751; 8,097,609; 8,187,824; 8,207,329; 7,633,007;

[0084] 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 / US 17 / 63251. In some embodiments, the dye is hydrophobic. In some embodiments the dye is hydrophilic. In some embodiments, the dye may be attached and / or bound to a monomer that is polymerized with one or more different monomers (e.g., polymerized with a hydrophobic monomer and / or hydrophilic monomer). In some embodiments, the dye is a luminophore (i.e., a material and / or compound that can emit light and does not specify the nature of the originating state (e.g., singlet, triplet, and / or another state)). Exemplary luminophores include, but are not limited to, phosphors and / or fluorophores, which afford phosphorescence and / or fluorescence, respectively. In some embodiments, the dye or dyes may be chosen based on end-use application(s). Exemplary applications for dyes include, but are not limited to, the applications exemplified in Table 12. Similarly, the number of dyes can be tuned based on a variety of factors, including, for example, targeted and non- targeted methods, polymer size, quantum yield, overall polymer charge, and performance in the multi-dye foldamer of equivalents of monomers per dye. Studies described in the working examples provide further discussion and direction enabling one of skill in the art to make and use dye-loaded polymers.

[0085] The terms “primary dye” and “secondary dye” are used to distinguish between two different dyes and are not intended to denote any order or preference. In some embodiments, a compound of the invention may include more than two different dyes so that tertiary, quaternary, etc. dyes may be present in an end group and / or pendant from the polymer backbone. The modifiers “first,” “second,” “third” and the like, may also be used to distinguish between elements (e.g., end groups of the compound) and this is also not intended to denote any order or preference but only to distinguish between the different elements.

[0086] A “biomolecule” as used herein may be any biologically useful molecule or moiety, including, for example, a single-stranded DNA, double-stranded DNA, RNA, oligonucleotide, protein (e.g., antibody) and / or peptide, or a functional portion and / or fragment thereof. A biomolecule may be attached (e.g., covalently bonded directly or indirectly) to a terminus or end group of the polymer backbone of a compound of the present invention to become part of the end group. In some embodiments, a first end group and / or second end group comprises a biomolecule. In some embodiments, an end group of the polymer backbone of a compound of the present invention comprises a bioconjugate group and a biomolecule may react with the bioconjugate group to thereby attach (e.g., covalently bond) the biomolecule to the polymer backbone optionally via portion of the bioconjugate group.

[0087] A “linker” as used herein refers to a chemical moiety that connects two moieties such as bulky group(s), charged group(s), dye(s) and / or biomolecule(s). With reference to the first end group of some compounds of the invention, a “linear linker” refers to a linker that attaches a first moiety (e.g., dye, charged group, bulky group, or biomolecule) with a second moiety (e.g., the same or a different charged, group, bulky group, dye or a biomolecule) in a linear fashion as shown in Figure 1, and the linear orientation may be further extended by linking one or more additional dyes, charged groups, bulky groups, and / or biomolecules (e.g., in a linear manner). A “bifunctional linker” refers to a linker that comprises a branching point with two moieties attached thereto (e.g., the same or different dyes, or a dye and biomolecule, or a bulky group and a charged group) such that the branching point is attached (either directly or indirectly) to the polymer backbone, as shown in Figure 1. One or more additional dyes, charged groups, and / or biomolecules can be attached to the bifunctional linker and / or moieties attached thereto (e.g., with a linear linker). A “trifunctional linker” refers to a linker that comprises a branching point with three moieties (e.g., the same or different dyes and / or biomolecules) attached thereto such that the branching point is attached (either directly or indirectly) to the polymer backbone. One or more additional dyes, charged groups, and / or biomolecules may further be attached to the linker and / or to the moieties attached thereto (e.g., using a linear linker). A “multi-functional linker” refers to a linker that comprises a branching point with two, three, four or more moieties (e.g., the same or different dyes, charged groups, and / or biomolecules) attached thereto. In some embodiments, an exemplary multi-functional linker is a dendron linker.

[0088] A “bulky group” as used herein refers to a moiety that, when attached to a compound of the present invention, prevents or reduces reactivity of the compound, or a portion thereof, due to the steric hindrance of the moiety. In an example embodiment, the bulky group is a group of two or more atoms that has higher substitutions such that one or more atoms are closer together on the bulky group and / or produce steric effects on a compound, or a portion thereof. In some embodiments, the bulky group is a polysaccharide, for example, a cyclodextrin (CD), including, but not limited to α-CD, β-CD, and / or γ-CD. See, e.g., Wang et al., ACS Cent. Sci. 2022, 8, 5, 663-669; doi: 10.1021 / acscentsci.2c00478. In some embodiments, the bulky group is a three-dimensional cage molecule comprising a silicon-oxygen framework bonded to one or more different organic groups, for example, a polyhedral oligomeric silsesquioxane (POSS). In some embodiments, the bulky group is a POSS or a derivative thereof, for example, the POSS has a structure of: wherein each R is independently selected from a substituted or unsubstituted C1-C8 alkyl (e.g., methyl, ethyl, propyl, butyl, (e.g., isobutyl)), alkylamino, alkoxy, and -O-Si-(R1), wherein R1is a substituted or unsubstituted C1-C8 alkyl (e.g., isobutyl, alkylamino, alkoxy).

[0089] A “charged group” as used herein refers to a chemical moiety with electropositivity (e.g., has a tendency to donate electrons) and / or electronegativity (e.g., has a tendency to attract electrons toward itself). In some embodiments, the charged group has a positive or negative charge, optionally wherein the charged group has an overall positive charge or an overall negative charge. In some embodiments, the charged group has a tendency to donate electrons. Exemplary charged groups include, but are not limited to, electronegative sulfonates (e.g., 2- Acrylamido-2-methylpropane sulfonic acid (AMPS)), succinates, carboxylic acids, halogen containing groups (e.g., F, Cl, Br, and / or I-containing groups)), O, N, and / or S-containing groups. In some embodiments, the charged group is a cationic polymer, for example, poly 2- (dimethylamino)ethyl methacrylate (DMAEMA) that can optionally be quartemized. See, e.g., Yanez-Macias, et al., (2017), Macromol Chem and Phys, 218, 10; doi: 10.1002 / macp.201700065.

[0090] In some embodiments, the charged group is an alkylsulfoxide, for example, an alkylsulfoxide having a structure of:

[0091] In some embodiments, the charged group is a carboxylic acid, for example, a carboxylic acid having a structure of:

[0092] In some embodiments, the charged group is a sulfonate, for example, a sulfonate having a structure of: Compounds, Compositions, and Biomolecules

[0093] Compounds of the present invention include polymeric fluorophores. Compounds of the invention comprise a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone and a second end group attached to a second terminus of the polymer backbone. In some embodiments, the first end group comprises one or more dyes and optionally a biomolecule. In some embodiments, the second end group comprises a bioconjugate group. In some embodiments, one or more additional dyes are pendant from the polymer backbone.

[0094] Provided according to embodiments of the present invention are compounds (e.g., polymers) comprising a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, wherein the first end group optionally comprises a primary dye (e.g., a luminophore (e.g., a fluorophore) or a non-luminescent molecular entity), optionally wherein the dye has a molecular weight in a range of about 150 Da to about 3,000 Da; a second end group attached to a second terminus of the polymer backbone, wherein the second end group optionally comprises a bioconjugate group; and one or more additional primary dyes pendant from the polymer backbone; wherein at least the first end group or the second end group comprises a primary dye, wherein the compound optionally has a molecular weight in a range of about 5,000 Da, or 10,000 Da to about 175,000 Da, up to about 175,000 Da, up to about 225,000 Da, up to about 275,000 Da, or up to about 350,000 Da, e.g., 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 60,000 Da, 70,000 Da, 80,000 Da, 90,000 Da, 100,000 Da, 150,000 Da, 200,000 Da, 250,000 Da, 300,000 Da, or about 350,000 Da. In some embodiments, the molecular weight varies according to polymer backbone and number of dye molecules attached. The term primary dye is used to indicate that each of the primary dyes is the same dye. One or more secondary (or tertiary, etc.) dyes (i.e., different dye molecules) may also be pendant from the polymer backbone.

[0095] In some embodiments, the first end group comprises one or more bulky and / or charged groups. In some embodiments, the first end group comprises one or more hydrophobic groups. In some embodiments, the polymer backbone comprises one or more bulky and / or charged groups. In some embodiments, the second end group comprises a bioconjugate group. In some embodiments, one or more dyes are pendant from the polymer backbone. In some embodiments, one or more dyes are pendant from the first end of the polymer, which may also comprise any one of the aforementioned groups (e.g., charged group, hydrophobic group).

[0096] Provided according to embodiments of the present invention are compounds (e.g., polymers) comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, optionally wherein the first end group comprises a hydrophobic group (e.g., a hydrophobic monomer), a bulky group and / or a charged group; a second end group attached to a second terminus of the polymer backbone, optionally wherein the second end group comprises a bioconjugate group; and one or more primary dyes pendant from the polymer backbone, optionally wherein each primary dye (e.g., a luminophore (e.g., a fluorophore) or a non-luminescent molecular entity) independently has a molecular weight in a range of about 150 Da to about 3,000 Da; wherein at least the first end group or the second end group comprises a bulky and / or charged group and wherein at least one primary dye is pendant from the polymer backbone, optionally wherein the compound has a molecular weight in a range of about 5,000 Da, or 10,000 Da to about 175,000 Da, up to about 175,000 Da, up to about 225,000 Da, up to about 250,000 Da, up to about 275,000 Da, up to about 300,000 Da, up to about 325,000 Da or up to about 350,000 Da, e.g., 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 60,000 Da, 70,000 Da, 80,000 Da, 90,000 Da, 100,000 Da, 150,000 Da, 200,000 Da, 250,000 Da, 300,000 Da, or about 350,000 Da. The term primary dye is used to indicate that each of the primary dyes present in the compound is the same dye, e.g., more than one of the same dye is pendant from the polymer backbone. One or more secondary (or tertiary, etc.) dyes (which are different dyes than the primary dye) may also be pendant from the polymer backbone.

[0097] For the additional primary dye(s) pendant from the polymer backbone, in some embodiments, one or more of the hydrophobic unit(s) comprises a dye and / or a charged group. In some embodiments, one or more of the hydrophilic unit(s) comprises a dye. In some embodiments, one or more additional monomers is copolymerized with the hydrophobic monomer(s) and hydrophilic monomer(s) and in some embodiments, such additional unit(s) formed therefrom comprises a dye and / or a charged group. In some embodiment, an additional monomer produces a unit that comprises a functional group that is able to react with a dye molecule to attach the dye to the polymer backbone after polymerization.

[0098] In some embodiments of the invention, the compound comprises between 2 and 80 dye molecules or any range therein, for example, between 2 and 70 dye molecules, between 2 and 60 dye molecules, between 2 and 50 dye molecules, between 2 and 40 dye molecules, between 2 and 30 dye molecules, between 2 and 20 dye molecules, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more dye molecules. In some embodiments, the first end group of the compound comprises a dye and the polymer backbone has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more dyes pendant therefrom. In some embodiments, the first end group and / or polymer backbone of the compound comprises a bulky group and / or a charged group and the polymer backbone has 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, 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, or 80 dyes pendant therefrom, which may be primary dyes, or a combination of primary, secondary and / or tertiary dyes. The hydrophobic units and hydrophilic units in the compound may be any of those described herein.

[0099] In some embodiments, the average number of monomers between dyes in a compound is between about 10 and 150, about 20 and 130, about 40 and 120, or about 50 and 115, or any range therein. In some embodiments, the average number of monomers between dyes is maximized to retain fluorescence quantum yield in water relative to the fluorescence quantum yield of the dye when the compound is present in a hydrophobic solvent, as described in the working examples of the present invention.

[0100] Provided according to some embodiments of the invention are compounds (e.g., polymers) comprising a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, wherein the first end group comprises (i) a primary dye (e.g., a luminophore or a non-luminescent molecular entity) and a biomolecule (e.g., DNA, RNA, protein, or peptide); (ii) a primary dye and a secondary dye (e.g., a luminophore or a non-luminescent molecular entity), and optionally a biomolecule, or (iii) two or more primary dyes, and optionally a biomolecule, optionally wherein the primary dye and / or the secondary dye has a molecular weight in a range of about 150 Da to about 3,000 Da; and a second end group attached to a second terminus of the polymer backbone, wherein the second end group optionally comprises a bioconjugate group, wherein the compound optionally has a molecular weight in a range of about 5,000 Da, or 10,000 Da to about 175,000 Da, up to about 175,000 Da, up to about 225,000 Da, up to about 250,000 Da, up to about 275,000 Da, up to about 300,000 Da, up to about 325,000 Da or up to about 350,000 Da, e.g., 5,000 Da, 10,000 Da, 15,000 Da, 20,000 Da, 30,000 Da, 40,000 Da, 50,000 Da, 60,000 Da, 70,000 Da, 80,000 Da, 90,000 Da, 100,000 Da, 150,000 Da, 200,000 Da, 250,000 Da, 300,000 Da, or about 350,000 Da. Provided according to some embodiments of the invention are compounds (e.g., polymers) comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, wherein the first end group comprises (i) a bulky and / or charged group and a biomolecule (e.g., DNA, RNA, protein, or peptide); or (ii) two or more bulky groups, charged groups, or combination thereof. In some embodiments, the invention provides compounds (e.g., polymers) that do not comprise (e.g., devoid of) both an acceptor dye and a donor dye, e.g., an acceptor dye on a terminal end and one or more donor luminophores on the polymer backbone.

[0101] In some embodiments, the primary and / or secondary dye is a Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole (e.g., a porphyrin). In some embodiments, the primary dye and the secondary dye are each fluorescent dyes. In some embodiments, the primary dye and the secondary dye are each non-fluorescent dyes. In some embodiments, the primary dye is a fluorescent dye and the secondary dye is a non-fluorescent dye.

[0102] In some embodiments of the invention, the compound comprises a linear linker between the hydrophobic group, the bulky and / or charged group and the polymer backbone, between the primary dye and the polymer backbone, between the primary dye and the biomolecule, between the primary dye and the secondary dye, or between two primary dyes. In some embodiments, the compound comprises a bifunctional branched linker between the polymer backbone and a hydrophobic group, a bulky and / or charged end group, between the polymer backbone and a primary dye and / or biomolecule, between a primary dye and a biomolecule, between a primary dye and a secondary dye, or between two primary dyes. Further, in some embodiments, the compound comprises a trifunctional branched linker between the polymer backbone and two hydrophobic groups or two bulky end groups and a charged end group, between the polymer backbone and a hydrophobic group or bulky end group and two charged end groups, between the polymer backbone and the primary dye, secondary dye and / or biomolecule; between the primary dye, the secondary dye, and the biomolecule; between two primary dyes and the biomolecule; between two primary dyes and the secondary dye; between the primary dye, the secondary dye, and a tertiary dye; or between three primary dyes. In some embodiments, the compound comprises a multifunctional branched linker between the polymer backbone and the primary dye, secondary dye and / or biomolecule; between the primary dye, the secondary dye, and the biomolecule; between two primary dyes and the biomolecule; between two primary dyes and the secondary dye; between the primary dye, the secondary dye, and a tertiary dye; between three primary dyes; between polymer backbone and the primary dye, secondary dye, tertiary dye and / or biomolecule; between the primary dye, the secondary dye, the tertiary dye and the biomolecule; between three primary dyes and the biomolecule; between three primary dyes and the secondary dye; between the primary dye, the secondary dye, the tertiary dye and the quaternary dye; or between four primary dyes.

[0103] In some embodiments, a linker is between a primary dye and the polymer backbone or the optional biomolecule, between a primary dye and a secondary dye, or between two or more primary dyes, the linker comprising or consisting of a moiety comprising a structure of: -(CH2)mR(CH2)nwherein: m is an integer of 0 to 3 (e.g., 0, 1, 2, or 3), n is an integer of 0 to 3, and

[0104] R is selected from -OC(O)-, C(O)O-,

[0105] -CH(OH)CH2R1-, , or -C(O)NH-, wherein R1is N or O; or

[0106] -(CH)mR(CH2)nwherein: m is an integer of 0 to 3, n is an integer of 0 to 3, and

[0107] R is -N-, -NNH-, -S-, or or

[0108] -Ph-C(O)NH(CH2)xC(O)NH-, -Ph-C(O)NH(CH2)xC(O)-, -Ph-C(O)NH(CH2)xC(O)-, or -Ph-N(CH3)C(O)(CH2)xC(O), wherein Ph=Phenyl and x is between 0 and 10.

[0109] In some embodiments, a linear linker, bifunctional linker, or trifunctional linker according to the invention comprises a PEG moiety and a second moiety having a structure of: -(CH2)mR(CH2)nwherein: m is an integer of 0 to 3, n is an integer of 0 to 3, and R is selected from -OC(O)-, C(O)O-,

[0110] -CH(OH)CH2R1-, , or -C(O)NH-, wherein R1is N or O; or

[0111] -(CH2)mR(CH2)nwherein: m is an integer of 0 to 3, n is an integer of 0 to 3, and

[0112] R is -N-, -NNH-, -S-, or or

[0113] -Ph-C(O)NH(CH2)xC(O)NH-, -Ph-C(O)NH(CH2)xC(O)-, -Ph-C(O)NH(CH2)xC(O)-, or -Ph- N(CH3)C(O)(CH2)xC(O), wherein Ph=Phenyl and x is between 0 and 10; optionally wherein the PEG moiety is covalently attached at one attachment point of the second moiety.

[0114] In some embodiments, the linker comprises or consists of a moiety having a structure of:

[0115] -(CH2)mR1(CH2)nwherein m is an integer of 1 to 3; n is an integer of 1 to 3; and R1is selected from -

[0116] OC(O)-, -C(O)O-, , -CH(OH)CH2R2-, , and

[0117] -C(O)NH, R2is N or O. In some embodiments, the linker comprises or consists of a moiety having a structure of: -(CH)mR1(CH2)nwherein m is an integer of 0 to 2; n is an integer of 1 to 3; and R1is -N-, -NNH-, -S-, and In some embodiments, the linker is substituted with a PEG molecule. Additional exemplary linkers are shown in FIG. 4.

[0118] Exemplary linkers that may be present in a compound of the present invention include, but are not limited to, a hydrocarbon moiety, a peptoid moiety, an amino acid (e.g., lysine) moiety, an oligoethylene glycol group, triazine (e.g., 1,3,5-triazine), 1,3, 5 -tri substituted benzene, self-immolative linkers, and / or a polyethylene glycol (PEG) group. A linker may be selected to provide an attachment to another portion of the compound via a carbon-carbon bond or a carbon-heteroatom (e.g., oxygen, sulfur, or nitrogen) bond. In some embodiments, the linker may be a linear or branched hydrocarbon moiety (e.g., an alkyl moiety) and / or a carrier protein. In some embodiments, a linker may be substituted with one or more substituents such as, but not limited to, an unsubstituted or substituted aryl, alkylamino, alkoxy, or heterocycle. Further exemplary linkers are shown in Scheme I.

[0119] Scheme I: Exemplary linkers that may be used in a compound of the present invention.

[0120] Linear:

[0121] Branched:

[0122]

[0123] 8-arm PEG N-hydroxysuccinimidyl ester (structure not shown)

[0124] 4- or 8-arm PEG maleimide (structure not shown)

[0125] A linker of the present invention may optionally be substituted with a PEG molecule. In some embodiments, a linker may have a PEG molecule on one or both ends of the linker, optionally wherein the linker is between a primary dye and the polymer backbone or a biomolecule, between a primary dye and a secondary dye, or between two or more primary dyes. In some embodiments, a compound of the present invention comprises a linker comprising -(CH2CH2O)x- , wherein x is an integer of 1, 5, 10, 25, or 50 to 55, 75, or 100.

[0126] A "hydrophilic unit" as used herein refers to the section or unit of the polymer backbone that comprises a hydrophilic (e.g., ionic and / or polar) functional group (e.g., a hydrophilic pendant functional group), optionally wherein the hydrophilic functional group is at a terminal portion of a moiety. As one of skill in the art would understand, a portion of a hydrophilic unit may be hydrophobic such as, e.g., the portion that forms a polymer backbone when polymerized with other monomers and / or the portion (e.g., hydrocarbon chain) of a functional group including an ionic moiety but is still referred to as a hydrophilic monomer if it comprises a hydrophilic functional group. In some embodiments, a hydrophilic unit is formed from a hydrophilic monomer (e.g., a monomer having the same hydrophilic functional group(s)). However, in some embodiments, a unit is modified after polymerization to add a hydrophilic functional group, thereby forming the hydrophilic unit.

[0127] A "hydrophobic unit" as used herein refers to the section or unit of a polymer that comprises a hydrophobic functional group (e.g., a hydrophobic pendant functional group), optionally wherein the hydrophobic functional group is at a terminal portion of a moiety and / or monomer. In some embodiments, the hydrophobic functional group is a hydrocarbon moiety (e.g., an alkyl, aryl, or arylalkyl). In some embodiments, a hydrophobic unit is formed from a hydrophobic monomer (e.g., a monomer having the same hydrophobic functional group(s)). However, in some embodiments, a unit is modified after polymerization to add a hydrophobic functional group, thereby forming the hydrophobic unit. In some embodiments, a hydrophobic unit or hydrophobic functional group of the hydrophobic unit, or a derivative thereof, referred to herein as a hydrophobic group, can be comprised on a first end of a polymer as described herein. For example, a polymer comprising a thiol-containing first end group can be further modified by attaching the hydrophobic group.

[0128] A hydrophobic unit and / or a hydrophilic unit of the polymer may comprise a pendant functional group. A pendant functional group may be part of the hydrophobic unit and / or monomer and / or hydrophilic unit and / or monomer at the time of polymerization or may be added to the hydrophobic unit and / or hydrophilic unit after polymerization. In some embodiments, a pendant functional group may be added to a hydrophobic unit and / or hydrophilic unit after polymerization (e.g., post-polymerization functionalization). In some embodiments, a pendant functional group comprises a charged group. In some embodiments, the polymer can comprise a charged group (positive or negative) at one or more locations along the backbone and / or terminal end. In some embodiments, the pendant functional group can comprise a positive charge, for example, at a hydrophobic unit or pendant functional group (e.g., PFP) of the backbone. In some embodiments, the pendant functional group can comprise a negative charge, for example, an AMPS appended at a hydrophobic unit or pendant functional group (e.g., PFP) of the backbone. In an example embodiment, one or more pendant functional groups along the backbone can comprise a negative charge, for example, an AMPS appended at a hydrophobic unit or pendant functional group (e.g., PFP) of the backbone with multiple dyes, e.g., 2, 3, 4, 5, 6, 7, 8 or more dyes along the backbone. In some embodiments, the pendant functional group can comprise a negative charge, for example, a sulfonate group (e.g,, AMPS) at the terminal end of the polymer. In an example embodiment, a compound comprises pendant functional group can comprise one or more negative charges, for example, a sulfonate group (e.g,, AMPS) at the terminal end of the polymer and multiple dyes, e.g., 2, 3, 4, 5, 6, 7, 8 or more dyes along the backbone. In some embodiments, a pendant functional group is a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., an active ester such as a pentafluorophenyl ester, succinimido ester, 2,4-dinitrophenyl ester, etc.), azido, pentafluorophenyl, succinimido, fluorophenyl, maleimido, isocyanato, or isothiocyanato group. In some embodiments, the pendant functional group is a hydrophilic group comprising a terminal cationic (e.g., ammonium), anionic (e.g., sulfonate, phosphate, carboxylate), or zwitterionic (e.g., a choline or choline-like group (e.g., a derivative of a choline)) group and optionally a polyethylene glycol) moiety and / or unit. In some embodiments, the hydrophilic group is attached to the poly(ethylene glycol) moiety and / or unit, optionally attached to a terminal portion of the poly(ethylene glycol) moiety and / or unit.

[0129] In some embodiments, a hydrophobic unit comprises a pendant functional group comprising an alkyl (e.g., dodecyl), aryl, or arylalkyl, and / or a hydrophilic unit comprises a pendant functional group comprising a glycol (e.g., poly(ethylene glycol)), sulfonic acid, and / or a sulfonate. In some embodiments, the hydrophobic unit is prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer and / or the hydrophilic unit is prepared from a glycol acrylate (e.g., PEG acrylate) monomer. In some embodiments, a compound of the present invention comprises at least one hydrophobic unit prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer and at least two different hydrophilic units, which include a first hydrophilic unit prepared from a glycol acrylate (e.g., PEG acrylate) monomer and a second repeating hydrophilic unit prepared from a sulfonic acid acrylamide monomer (e.g., 2- acrylamido-2-methylpropane sulfonic acid) and / or a sulfonate acrylate monomer.

[0130] In some embodiments, one or more of the hydrophobic unit(s) and / or one or more of the hydrophilic unit(s) may comprise a charge (e.g., a positive or negative charge) and / or a charged group (e.g., a cationic or anionic group), and the charge may suppress non-specific binding to the compound or a portion thereof (e.g., to a portion of the polymer backbone). In some embodiments, the overall charge of the polymer comprising charged groups and / or charged hydrophobic units and / or charged hydrophilic units is neutral. In some embodiments, the overall charge of the polymer comprising charged groups and / or charged hydrophobic units and / or charged hydrophilic units is negative.

[0131] In some embodiments of the invention, the polymer backbone comprises one or more of the hydrophobic units shown in Figure 2. In some embodiments of the invention, the polymer backbone comprises one or more hydrophobic units that comprise an aryl or arylalkyl functional group, including, but not limited to, one or more of the units shown in Figures 3A and 3B. In some embodiments, the polymer backbone comprises polyfluorophenyl ester acrylate.

[0132] In some embodiments, the polymer backbone may comprise one or more (e.g., 1, 5, 10, 50, 100, or more) hydrophobic unit(s) and one or more (e.g., 1, 5, 10, 50, 100, or more) hydrophilic unit(s). The polymer backbone may be prepared from one or more (e.g., 1, 5, 10, 50, 100, or more) hydrophobic monomer(s) and one or more (e.g., 1, 5, 10, 50, 100, or more) hydrophilic monomer(s) using any type of polymerization to provide the polymer comprising the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s). In some embodiments, the polymer backbone may be prepared from two or more (e.g., 2, 3, 4, 5, or more) hydrophobic monomers that are different from each other and / or two or more (e.g., 2, 3, 4, 5, or more) hydrophilic monomers that are different from each other. For example, in some embodiments, a compound of the present invention may be prepared from at least one hydrophobic monomer, at least one of a first hydrophilic monomer, and at least one of a second hydrophilic monomer, wherein the first hydrophilic monomer and the second hydrophilic monomer are different from each other.

[0133] The one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) may be randomly distributed in the polymer. In some embodiments, the polymer is a random copolymer. The polymer backbone may be an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer. In some embodiments, the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) may be 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 (hydrophobic units: hydrophilic units). In some embodiments, the ratio of hydrophobic units to hydrophilic units is about 1 :4 to about 1 :6. The length of the polymer backbone may be varied and / or controlled. In some embodiments, the polymer backbone has a molecular weight in a range of about 1,000 Da to about 175,000 Da, about 5,000 Da to about 350,000 Da, about 10,000 Da to about 350,000 Da, about 20,000 Da to about 350,000 Da, about 35,000 Da to about 350,000 Da, about 35,000 Da to about 300,000 Da, about 10,000 Da to about 175,000 Da, about 20,000 Da to about 175,000 Da, about 28,000 Da to about 175,000 Da, about 28,000 Da to about 35,000 Da, about 28,000 Da to about 50,000 Da, about 100,000 Da to about 150,000 Da, about 50,000 Da to about 130,000 Da, or about 10,000 Da to about 100,000 Da. In some embodiments, the polymer backbone 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, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 kiloDaltons (kDa). In some embodiments, the polymer backbone has a molecular weight greater than 28 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa or 350 kDa. In some embodiments, the polymer backbone has a molecular weight of about 28 kDa to about 175 kDa. In some embodiments, the polymer backbone has a molecular weight of about 28 kDa to about 35 or 50 kDa.

[0134] In some embodiments, the polymer has a degree of polymerization (DP) between 10 and 500, e.g., 30 and 450, 40 and 400, 50 and 350, 60 and 325, 70 and 300, or any range therein. In some embodiments, the number of dye molecules per polymer can depend, in part on the degree of polymerization of the polymer.

[0135] In some embodiments, the polymer is a random polymer, comprising at least two monomers with comparable reactivity ratios (e.g., two or more different acrylate-based monomers). In some embodiments, the polymer is a random polymer comprising a single functional monomer (e.g., pentafluorophenyl acrylate (PFPA)) that is further modified post- polymerization to introduce further functional groups randomly. See, e.g., Example 2G.

[0136] In some embodiments, the polymer is a gradient polymer, comprising at least two monomers with different reactivity ratios. In some embodiments, the polymer comprises a segment or a portion of the polymer that is a gradient polymer; thus the polymer may comprise a portion or segment with one monomer or two or more monomers with comparable reactivity ratios followed by a portion or segment comprising a gradient, comprising at least two monomers with different reactivity ratios.

[0137] In some embodiments, the polymer backbone comprises at least one hydrophobic unit having a structure represented by Formula III: wherein:

[0138] R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);

[0139] R1is absent or is -O-, -NH-, -CH2-;

[0140] R’ is absent or a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or a C5-C25 arylalkyl;

[0141] R2is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azido, maleimido, isocyanato, or isothiocyanato group, or R2is a dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

[0142] In some embodiments, R2in the compound of Formula III is a hydroxyl, carboxyl, amino, formyl, or ester group. In some embodiments, R2in the compound of Formula III is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimido group, which may optionally be added and / or provided before polymerization and / or by post-polymerization functionalization. In some embodiments, R2in the compound of Formula III is hydrogen. In some embodiments, R’ in the compound of Formula III is a C2-C4 alkyl, a C2-C6 alkyl, a C4-C20 alkyl, a C6-C20 alkyl, a C8-C16 alkyl, a C8-C18 alkyl, a C10-C14 alkyl, or a C10- C12 alkyl. In some embodiments, R’ in the compound of Formula III is a C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl, alkenyl, or alkynyl. In some embodiments, R’ in the compound of Formula III is a Cl, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl. In some embodiments, R’ in the compound of Formula III is a C3-C5 cycloalkyl, a C3-C6 cycloalkyl, a C4-C20 cycloalkyl, a C6-C20 cycloalkyl, a C8-C16 cycloalkyl, a C8-C18 cycloalkyl, a C10- C14 cycloalkyl, or a C10-C12 cycloalkyl. In some embodiments, R’ in the compound of Formula III is a C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 cycloalkyl. In some embodiments, R’ in the compound of Formula III is a C5-C20 aryl, a C5-C16 aryl, a C5-C14 aryl, or a C5-C10 aryl. In some embodiments, R’ in the compound of Formula III is a C6-C25 arylalkyl, a C6-C20 arylalkyl, or a C8-C18 arylalkyl. In some embodiments, a compound of the present invention comprises at least one hydrophobic unit having a structure of Formula III, wherein R2is a dye and the compound comprises a bulky group and / or a charged group at one terminus of the compound.

[0143] In some embodiments, a hydrophilic unit may have a structure represented by Formula

[0144] IV: wherein:

[0145] R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);

[0146] R1is absent or is -O-, -NH-, or -CH2-; R3is selected from the group consisting of -(CH2CH2R5)n-, -C1-C6alkyl, -C1-C6alkyl- O-, and -C1-C6alkyl-SO3- or a salt thereof, wherein R5is -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;

[0147] R4is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-

[0148] (trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl or ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group, or optionally R4is a dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

[0149] In some embodiments, R4in the compound of Formula IV is a hydroxyl, carboxyl, amino, formyl, or ester group, optionally when R3is -(CH2CH2R5)n-, -C1-C6alkyl, or -C1-C6alkyl-O-. In some embodiments, when R3in the compound of Formula IV is -C1- C6alkyl-O- or -(CH2CH2R5)n- with R5being -O-, then R4may be a hydrogen, alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), or phosphoryl group. In some embodiments, when R3in the compound of Formula IV is -C1-C6alkyl or -(CH2CH2R5)n- with R5being -CH2-, then R4may be a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group. In some embodiments, R4in the compound of Formula IV is a hydrogen, alkyl, phosphono, sulfono, phosphatidyl choline, phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or ester group. In some embodiments, R4in the compound of Formula IV is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimido group, which may optionally be added and / or provided before polymerization and / or by post-polymerization functionalization. In some embodiments, when R3in the compound of Formula IV is -C1-C6alkyl-SO3- or a salt thereof, then R4is hydrogen or is absent. In some embodiments, R3in the compound of Formula IV is a salt (e.g., a sodium salt) of -C1-C6alkyl-SO3- and R4is absent. In some embodiments, R3in the compound of Formula IV is -(CH2CH2R5)n-. In some embodiments a compound of the present invention comprises at least one hydrophilic unit having a structure of Formula IV, wherein R4is a dye and the compound comprises a bulky group and / or a charged group at one terminus of the compound.

[0150] In an example embodiment, a compound of the present invention may comprise a PFP ester acrylate polymer, and may further comprise dodecylamine and a PEG (e.g., a poly etheramine). In some embodiments, the PFP A polymer can further comprise charged groups on the backbone and / or a terminal end of the polymer, which may be positively charged and / or negatively charged. In some embodiments, the charged groups on the backbone are positive and the charged groups on the terminal end are negative. In some embodiments, the charged groups on the backbone are negative and the charged groups on the terminal end are positive.

[0151] In some embodiments, a compound of the present invention may comprise and / or be a telechelic polymer, which is a polymer or prepolymer that is capable of entering into further polymerization or other reactions through one or more of its reactive end-groups. In some embodiments, a compound of the present invention may comprise and / or be a heterotelechelic polymer, which is a polymer or prepolymer that is capable of entering into further polymerization or other reactions through a reactive end-group at each terminus of the polymer or prepolymer, and the two reactive end groups are not identical to each other. In some embodiments, a compound of the present invention may comprise and / or be a homotelechelic polymer, which is a polymer or prepolymer that is capable of entering into further polymerization or other reactions through a reactive end-group at each terminus of the polymer or prepolymer, and the two reactive end groups are identical to each other. In some embodiments, a compound of the present invention may comprise and / or be a semitelechelic polymer, which is a polymer or prepolymer that is capable of entering into further polymerization or other reactions through a reactive end-group at one terminus of the polymer or prepolymer.

[0152] In some embodiments, a compound of the present invention may be attached to a single biomolecule via a bioconjugate group in the second end group. In some embodiments, such biomolecules may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) compound(s) of the present 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) compound(s) of the present invention.

[0153] A bioconjugate group may optionally be present in a compound of the present invention (e.g., in the second end group of the compound). "Bioconjugatable group", "bioconjugatable site", or "bioconjugate group" and grammatical variations thereof, refer to a moiety and / or functional group that may be used to bind or is bound to a biomolecule (e.g., a protein, peptide, DNA, RNA, etc.). Thus, "bioconjugatable group", "bioconjugatable site", or "bioconjugate group" and grammatical variations thereof do not comprise a biomolecule. However, in some embodiments, a bioconjugate group is used to bind to a biomolecule or a bioconjugate group or derivative thereof is bound to a biomolecule (e.g., a protein, peptide, DNA, RNA, etc.). Exemplary bioconjugatable groups include, but are not limited to, amines (including amine derivatives) such as isocyanates, isothiocyanates, iodoacetamides, azides, diazonium salts, etc.; acids or acid derivatives such as N-hydroxysuccinimide esters (more generally, active esters derived from carboxylic acids, e.g., p-nitrophenyl ester), acid hydrazides, etc.; and other linking groups such as aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiol groups, maleimides, aziridines, acryloyls, halo groups, biotin, 2 -iminobiotin, etc. Linking groups such as the foregoing are known and described in U.S. Patent Nos. 6,728,129; 6,657,884; 6,212,093; and 6,208,553. For example, a compound of the present invention may comprise a bioconjugate group that comprises a carboxylic acid and the carboxylic acid may be used for bioconjugation to a biomolecule (e.g., via carbodiimide-activation and coupling with an amino-substituted biomolecule).

[0154] In some embodiments, a biomolecule may comprise and / or be a protein (e.g., an antibody and / or a carrier protein), peptide, DNA, RNA, etc. In some embodiments, a biomolecule may comprise a moiety (e.g., a polymer) that optionally may include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) binding sites for a compound of the present invention. In some embodiments, the biomolecule may be a member of a specific binding pair. "Specific binding pair" and "ligand-receptor binding pair" are used interchangeably herein and refer to two different molecules, where one of the molecules has an area on the surface or in a cavity of the molecule that specifically attracts or binds to a particular spatial or polar organization of the other molecule, causing both molecules to have an affinity for each other. The members of the specific binding pair can be referred to as ligand and receptor (anti-ligand). The terms ligand and receptor are intended to encompass the entire ligand or receptor or portions thereof sufficient for binding to occur between the ligand and the receptor. Examples of ligand-receptor binding pairs include, but are not limited to, hormones and hormone receptors, for example epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor-a and tumor necrosis factor-receptor, and interferon and interferon receptor; avidin and biotin or antibiotin; antibody and antigen pairs; enzymes and substrates; drug and drug receptor; cell- surface antigen and lectin; two complementary nucleic acid strands; nucleic acid strands and complementary oligonucleotides; interleukin and interleukin receptor; and stimulating factors and their receptors such as granulocyte-macrophage colony stimulating factor (GMCSF) and GMCSF receptor and macrophage colony stimulating factor (MCSF) and MCSF receptor. In some embodiments, a compound of the present invention or a portion thereof has a non-rigid backbone (e.g., a non-rigid polymer backbone) and / or has conformational flexibility. Conformational flexibility of molecular chains can be described and quantitated by the "persistence length" of the compound or portion thereof (e.g., the polymer portion). In some embodiments, the persistence length of a compound of the present invention may be on the order of the length of a given carbon-carbon bond.

[0155] A compound of the present invention may be self-folding such as, for example, self- folding in water and / or an aqueous solution. " Self-folding" as used herein refers to a compound transitioning from a partially or completely extended or unfolded structure to a structure wherein at least a portion of the extended or unfolded structure becomes folded upon contact with a solution (e.g., an aqueous solution) or compound, and the folding is innate as it occurs spontaneously (i.e., without external control or forces) upon contact with a solution. In some embodiments, a compound of the present invention self-folds upon contact with water and / or an aqueous solution. A compound of the present invention may self-fold into a unimer micellar structure, optionally upon contact with water and / or an aqueous solution. The aqueous solution in which a compound of the present invention folds may be a buffer such as a phosphate buffer (e.g., phosphate buffered saline). In some embodiments, the aqueous solution (e.g., an aqueous buffer) in which a compound of the present invention folds may have a low ionic strength; for example, the aqueous solution may have a mu value of about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 160 mM to about 180 mM, or about 160 mM to about 170 mM. In some embodiments, the aqueous solution in which a compound of the present invention folds may have a mu value of less than about 100 mM. In some embodiments, the aqueous solution in which a compound of the present invention folds may comprise IM NaCl. In some embodiments, the aqueous solution in which a compound of the present invention folds may comprise less than IM NaCl such as less than about 0.75M, 0.5M, or 0.25M NaCl. In some embodiments, the aqueous solution in which a compound of the present invention folds comprises 10 mM NaH2PO4and 150 mM NaCl, and has a pH of about 7.35.

[0156] In some embodiments, a compound of the present invention may be in the form of a particle. A compound of the present invention may form a particle such as, e.g., upon contact with a solution (e.g., an aqueous solution). In some embodiments, a single (i.e., 1) compound may form the particle. Thus, the compound and the particle are present in a ratio of about 1 : 1 (i.e., there is one compound per particle). A compound of the present invention may comprise a portion of the one or more hydrophobic unit(s) in the core or interior region of the particle and / or a portion of the one or more hydrophilic unit(s) at the periphery or exterior region (e.g., shell) of the particle. In some embodiments, the particle has a micellar structure (e.g., a unimer micellar structure). A compound of the present invention may comprise one or more dyes that may be encapsulated by a portion of the compound (e.g., a portion of the polymer) when the compound is in a folded structure and / or in the form of a particle (e.g., a unimer micellar structure). In some embodiments, one or more dyes or a portion thereof and one or more hydrophobic unit(s) may be present in the core or interior region of the particle and one or more hydrophilic unit(s) may surround the dye and / or one or more hydrophobic unit(s).

[0157] In some embodiments, one or more of the hydrophilic units comprise a non-ionic (i.e., neutral / uncharged) pendant functional group (e.g., PEG) and / or are formed from a non-ionic monomer (e.g., PEG acrylate (PEGA)). In some embodiments, one or more of the hydrophilic units comprise 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-methylpropane sulfonic acid)). In some embodiments, the hydrophilic units are formed from at least two different monomers such as, for example, a non-ionic (i.e., neutral / uncharged) hydrophilic monomer (e.g., PEG acrylate (PEGA)) and an ionic (e.g., anionic, charged) hydrophilic monomer (e.g., sulfonic acid acrylate (e.g., 2-acrylamido-2- methylpropane sulfonic acid)). As one of skill in the art understands, a monomer comprising an acid such as, e.g., sulfonic acid, may be present in the form of the acid and / or in its ionic form. In some embodiments, a monomer comprising an acid is predominantly (i.e., greater than 50%) in its ionic form. In some embodiments, the ionic hydrophilic monomer is an acid in deprotonated form (e.g., deprotonated sulfonic acid acrylate) and / or in a salt form, e.g., a sodium sulfonate acrylate (e.g., 2-acrylamido-2-methylpropane sulfonic acid as the sodium salt).

[0158] In some embodiments, where two or more different hydrophilic units are present in a polymer of the present invention the ratio of the two or more different hydrophilic units can vary such as, for example from about 10: 1 to about 1 : 10. For example, in some embodiments, a polymer comprises non-ionic (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-m ethylpropane sulfonic 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 (non-ionic units:ionic units).

[0159] In some embodiments, a polymer of the present invention comprises about 1% to about 40% hydrophobic units based on the total molar amount of monomers used to prepare the polymer and about 60% to about 99% hydrophilic units based on the total molar amount of monomers used to prepare the polymer. In some embodiments, a polymer of the present invention comprises 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 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 greater than about 70% (e.g., greater than about 75%, 80%, 85%, 90%, or 95%) hydrophilic units based on the total molar amount of monomers used to prepare the polymer.

[0160] A polymer of the present invention may have a weight fraction of hydrophobic units of about 1%, 5%, 10%, 15% or 20% to about 25%, 30%, 35%, or 40% based on the total weight of the polymer. In some embodiments, the polymer may have a weight fraction of hydrophobic units of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, or 40% based on the total weight of the polymer. In some embodiments, the polymer may have a weight fraction of hydrophobic units of less than about 30% (e.g., less than about 25%, 20%, 15%, 10%, or 5%) based on the total weight of the polymer. A polymer 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, a polymer of the present invention may have a weight fraction of hydrophilic units of about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98, or 99% based on the total weight of the polymer. In some embodiments, the polymer may have a weight fraction of hydrophilic units of greater than about 70% (e.g., greater than about 75%, 80%, 85%, 90%, or 95%) based on the total weight of the polymer.

[0161] In some embodiments, the amount of unimer micellar structures formed upon contact with a solution is about 50% to about 100% of the total polymer added to solution, about 75% to about 100%, about 85% to about 100%, or about 95% to about 100%, optionally as measured using sizing methods (e.g., dynamic light scattering (DLS) spectroscopy). In some embodiments, the amount of unimer micellar structures formed upon contact of the polymer with a 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 sizing methods (e.g., dynamic light scattering (DLS) spectroscopy). The solution in which a unimer is present may be an aqueous solution as described herein such as an aqueous buffer. In some embodiment, the aqueous solution in which a unimer is present is a phosphate buffer (e.g., phosphate buffered saline). In some embodiments, the aqueous solution (e.g., an aqueous buffer) in which a unimer is present has a low ionic strength (e.g., may have a mu value of about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 160 mM to about 180 mM, or about 160 mM to about 170 mM). In some embodiments, the aqueous solution in which a unimer is present comprises 10 mM NaH2PO4and 150 mM NaCl, and has a pH of about 7.35.

[0162] In some embodiments, dilution of a solution containing a compound of the present invention in the form of a unimer micellar structure results in no loss or a loss of less than about

[0163] 20% of the unimer micellar structures present in the solution compared to the amount of unimer micellar structures present in the solution prior to dilution. In some embodiments, the amount of unimer micellar structures present in a 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 unimer micellar structures present in the solution prior to dilution.

[0164] In some embodiments, a solution comprising a compound of the present invention in the form of a unimer micellar structure comprises 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 unimer micellular structure. In some embodiments, dilution of a solution comprising a compound of the present invention in the form of a unimer micellar structure results in no or minimal additional aggregate formation compared to the amount of aggregates present in the solution prior to dilution. In some embodiments, the amount of aggregates present in a solution comprising a compound of the present invention does not change upon dilution or changes by less than about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7% 6%, 5%, 4%, 3%, 2%, 1%, or 0.1% compared to the amount of aggregates present in the solution prior to dilution. In some embodiments, the diluted solution comprises 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%).

[0165] A compound of the present invention may have a diameter (e.g., when folded such as in a unimer micellar structure) in a range of about 1 nm to about 50 nm or about 3 nm to about 30 nm in water and / or an aqueous solution. In some embodiments, the compound is utilized in a non-targeted application, and has a diameter of about 20 nm or less. In some embodiments, the compound may have a diameter (e.g., when folded such as in a unimer micellar structure) 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 an aqueous solution. In some embodiments, a compound of the present invention may be in the form of a particle (i.e., an at least partially folded structure).

[0166] In some embodiments, a compound of the present invention is cross-linked, optionally wherein the compound is cross-linked when the compound is in a folded structure. In some embodiments, a compound of the present invention may be in a solution (e.g., an aqueous solution) and / or may be cross-linked with a cross-linking agent. Cross-linking a compound of the present invention may comprise linking together two or more moieties and / or functional groups (e.g., pendant functional groups) of the hydrophobic unit(s) and / or hydrophilic unit(s). Cross-linking may provide the compound in a folded structure that cannot be unfolded without breaking one or more of the linkages formed by cross-linking. The degree or amount of cross- linking may be controlled, modified, and / or tuned, for example, by the amount of cross-linking agent reacted with the compound. In some embodiments, the step of cross-linking the compound may comprise a reaction and / or reactive entity (e.g., functional group) as listed in Table 1

[0167] Table 1: Exemplary cross-linker reactions and functional groups.

[0168] In some embodiments of a compound of the present invention, the fluorescence quantum yield of the dye when the compound is present in water and / or an aqueous solution may decrease by about 20%, 15%, or 10% or less (e.g., 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 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., in toluene). Upon bioconjugation of a compound of the present invention to 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 a hydrophobic solvent. In some embodiments, if the fluorescence quantum yield of the dye is 1.00 (theoretical maximum), then a decrease of 10-fold or less (e.g., about 10, 9, 8, 7, 6, 5, 4, 3, 2-fold or less) may be acceptable. However, in some embodiments, the fluorescence quantum yield of the dye when the compound is present in water and / or an aqueous solution may decrease by about 30%, 40%, 50% or more relative to the fluorescence quantum yield of the dye in water and / or a hydrophobic solvent while retaining desirable characteristics, e.g., brightness, because if the high-performance of compounds made according to the disclosure herein.

[0169] In some embodiments, a compound of the present invention is water soluble. The compound may have a solubility in water at room temperature of at least 1 mg / mL, e.g., about 1 mg / mL to about 100 mg / mL. In some embodiments, the compound has a solubility in water at room temperature of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg / mL. In some embodiments, a composition of the present invention comprises a compound of the present invention in a solution such as, e.g., water, an aqueous solution, and / or a hydrophobic solvent.

[0170] In some embodiments, a compound of the present invention comprises a gradient structure. In a compound with a gradient structure, the gradient structure can comprise a portion of the backbone, e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or any range therein, or substantially all of the backbone, e.g., 95%, 96%, 97%, 98%, 99% or more. In some embodiments, the gradient structure of the polymer comprises two or more monomers, wherein at least two of the monomers comprise different copolymerization reactivity ratios. In some embodiments where the gradient comprises a portion of the backbone, the backbone comprises a gradual transition between units, for example between hydrophobic and hydrophilic units, or between two different hydrophobic units or two different hydrophilic units in the polymer backbone.

[0171] In some embodiments, the polymer backbone comprises two or more segments. In some embodiments, the polymer backbone comprises a first segment and a second segment. In some embodiments, the first segment comprises one or more monomer units, for example hydrophilic units (e.g., PEGMA) or a combination of hydrophilic and hydrophobic units (e.g., CD A and PEGMA). In some embodiments, the degree of polymerization for the first segment is between about 10 and 100, between about 20 and 80 monomer units, or any range therein. In some embodiments, the second segment comprises hydrophilic units (e.g., PEGMA) or a combination of hydrophilic and hydrophobic units (e.g., CD A and PEGMA) present in the first segment, and may further comprise a hydrophobic unit (e.g., PFPA). In some embodiments, the degree of polymerization for the second segment is between about 30 to 300, between about 40 to 280, between about 60 to 240 monomer units, or any range therein.

[0172] In some embodiments, a method of producing a polymer with two distinct segments is provided. In some embodiments, an initial monomer feedstock consisting of one or two species is provided, and the initial monomer feedstock is reacted. After proceeding with the reaction of the initial monomer feedstock, additional, different monomers are charged into the reaction flask after an amount of time, for example between 30 minutes and 50 hours, e.g., about 30 minutes or 1, 2, 5, 10, 25, or 50 hours, or any range therein. Advantageously, the method achieves separate segments with varying compositions within a single reaction vessel.

[0173] In some embodiments, a compound and / or particle of the present invention is resistant to dilution. "Resistant to dilution" as used herein refers to the compound and / or particle retaining its structure and / or a property. In some embodiments, resistant to dilution refers to the compound and / or particle retaining a folded structure (e.g., a unimer micellar structure), which may be determined by measuring the diameter of the particle before and after dilution, and the diameter after dilution may remain within ± 50%, 40%, 30%, 20%, 10% or less of the diameter prior to dilution. In some embodiments, resistant to dilution refers to the compound and / or particle 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 prior to dilution. In some embodiments, a compound and / or particle of the present invention remains in a folded structure when diluted up to 25x, 50x, 75x, or 100x or when diluted to sub-micromolar concentrations.

[0174] Also provided according to embodiments of the invention are compositions that include a compound of the invention. In some embodiments, the compositions are aqueous solutions, including any of the aqueous compositions described herein. In some embodiments, the composition is devoid of organic solvent.

[0175] Methods of Making Compounds

[0176] Provided according to some embodiments of the present invention are methods of preparing compounds and / or compositions of the present invention. Example methods for copolymerizing and end group modification may be found in WO 2019 / 126144 (PCT / US2018 / 066195) and WO 2021 / 118782 (PCT / 2020 / 061285), the disclosures of each of which are incorporated by reference in their entirety. In some embodiments, methods of preparing a compound comprise copolymerizing one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s) to provide a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s); optionally attaching a primary dye (e.g., a luminophore (e.g., a fluorophore) or a non- luminescent molecular entity) to a first end group of the compound; and attaching at least one additional primary dye to a functional group pendant from the polymer backbone, optionally attaching a bioconjugate group to a second end group of the compound; and optionally cross- linking the compound. In some embodiments, the primary dye is attached to the first end group after the at least one additional primary dye is attached to a functional group pendant from the polymer backbone.

[0177] In some embodiments of the invention, methods of preparing a compound comprise copolymerizing one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s) to provide a compound comprising a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s), a first end group and a second end group; attaching a first primary dye (e.g., a luminophore (e.g., a fluorophore) or a non- luminescent molecular entity) to the first end group to become part of the first end group; attaching a second primary dye, a secondary dye, and / or a biomolecule to the first primary dye and / or another portion of the first end group; optionally attaching a bioconjugate group to the second end group; and / or optionally cross-linking the compound. The attachment of the dyes and / or biomolecules may be direct or indirect such that a linker may be present between dye(s), biomolecule(s), and end group(s). The order of attachment of the dyes and / or biomolecules in the first end group may also be varied such that a biomolecule may, in some embodiments, be attached to the first end group before the primary dye.

[0178] In some embodiments, a method of preparing a compound of the present invention comprises copolymerizing one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s) to provide a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s); optionally attaching a bulky group (e.g., cyclodextrin (CD)) or a charged group (e.g., sulfonate) to a first end group of the compound; and attaching at least one primary dye to a functional group pendant from the polymer backbone, optionally attaching a bioconjugate group to a second end group of the compound; and optionally cross-linking the compound. In some embodiments, the bulky group (e.g., CD) or charged group (e.g., sulfonate) is attached to the first end group after the at least one additional primary dye is attached to a functional group pendant from the polymer backbone. In some embodiments, a method of preparing a compound of the present invention comprises providing a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s); optionally attaching a charged group (e.g., sulfonate) to one or locations along the backbone of the compound; and attaching at least one primary dye to a functional group, optionally a charged group, pendant from the polymer backbone, optionally attaching a bioconjugate group to a second end group of the compound; and optionally cross- linking the compound. In some embodiments, a charged group (e.g., sulfonate) can be attached to the first end group.

[0179] In some embodiments, a method of preparing a compound of the present invention comprises copolymerizing one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s) to provide a compound comprising a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s), a first end group and a second end group; attaching a bulky group (e.g., CD) or charged group (e.g., sulfonate) to the first end group such that the bulky group or charged group becomes part of the first end group; optionally attaching a bioconjugate group to the second end group and / or optionally cross- linking the compound. The bulky and / or charged group and / or biomolecule may be directly attached to a portion of the compound or indirectly attached such that a linker is present between the bulky group, charged group, and / or biomolecule and a portion of the compound. The order of attachment of the bulky groups and / or charged groups in the first end group may be varied.

[0180] In some embodiments, one or more of the compounds of the present invention can be prepared according to the exemplary methods described in the working examples. While the working examples describe attachment of dyes with, for example, dendron or bifunctional linkers, bulky and / or charged groups can be used in place of dyes.

[0181] The hydrophobic monomer and hydrophilic monomer may be polymerized using any method known to those of skill in the art such as, but not limited to, via a condensation reaction (e.g., reaction with a diol and a diacid) and / or living radical polymerization (e.g., atom-transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT)). In some embodiments, polymerizing the hydrophobic monomer and the hydrophilic monomer is performed with a method that provides a copolymer with one or both end groups of the copolymer that are reactive (i.e., one or both of the end groups of the copolymer are capable of entering into further polymerization or reactions), and the two end groups may be the same or different. In some embodiments, polymerizing the hydrophobic monomer and the hydrophilic monomer is achieved via a living radical polymerization (e.g., ATRP) in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally a co- catalyst to provide a copolymer. In some embodiments, polymerizing the hydrophobic monomer and the hydrophilic monomer is achieved via a living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT agent (e.g., thiocarb onylthio compound).

[0182] In some embodiments, the polymer backbone is formed by copolymerizing a hydrophobic monomer (which may be used to provide a hydrophobic unit of a polymer backbone as described herein) which may have a structure represented by Formula I: wherein:

[0183] R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);

[0184] R1is absent or is -O-, -NH-, -CH2-;

[0185] R’ is absent or a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or a C6-C25 arylalkyl;

[0186] R2is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azido, maleimido, isocyanato, or isothiocyanato group, or R2is a dye. The specific R, R1, R’ and R2groups described above with respect to the hydrophobic unit may also be used for the hydrophobic monomer. Generally, R2will not be a dye as it is typically added to a unit after polymerization, but in some embodiments, a dye could be present on the hydrophobic monomer.

[0187] In some embodiments, a hydrophilic monomer (which may be used to provide a hydrophilic unit of a polymer as described herein) may have a structure represented by Formula II: wherein:

[0188] R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);

[0189] R1is absent or is -O-, -NH-, or -CH2-;

[0190] R3is selected from the group consisting of a -(CH2CH2R5)n-, -C1-C6alkyl, -C1-C6alkyl-O-, and -C1-C6alkyl-SO3- or a salt thereof, wherein R5is -O- or -CH2- and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; and

[0191] R4is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-

[0192] (trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl or ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group, or R4is a dye. In some embodiments, a compound of the present comprises at least one hydrophobic unit having a structure of Formula IV, wherein R4is a dye and the compound comprises a bulky group and / or a charged group at one terminus of the compound. The specific R, R1, R3 and R4groups described above with respect to the hydrophilic unit may also be used for the hydrophobic monomer. Generally, R4will not be a dye as it is typically added to a unit after polymerization, but in some embodiments, a dye could be present on the hydrophilic monomer.

[0193] Exemplary reactive end groups of the compound that may allow for dye-attachment or bioconjugation include, but are not limited to, those described in Table 2. These terminal functional groups are not pendant functional groups but may be present at either end of the copolymer. Exemplary reactive end groups of the compound that may allow for bulky and / or charge group attachment include, but are not limited to, mercapto functional group on the copolymer and maleimide group on the bulky and / or charge group, and succinimido functional group on the copolymer and amino group on the bulky and / or charge group.

[0194] Table 2: Exemplary terminal functional group (FG) on the compound and on the dye or biomolecule and exemplary linkage and chemistry.

[0195]

[0196] Some functional groups may be labile under certain polymerization conditions. Hence, in some embodiments, a functional group may be introduced in a protected form. As a result, these functional groups may be 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.

[0197] Table 3: Exemplary protected forms of certain functional groups.

[0198]

[0199] In some embodiments, a portion (e.g., an end group) of the compound may comprise a halo group (e.g., Cl, Br, I). The halide portion of the compound may be derivatized with nucleophiles or end-capping reagents to generate a functional group for dye attachment or bioconjugation. In some embodiments, a portion (e.g., an end group) of the compound may comprise a thiol group, which may be derivatized with reagents comprising a thiol reactive group to generate a functional group for dye attachment or bioconjugation. Examples of thiol reactive groups include, but are not limited to, halides (e.g., bromo, chloro, iodo), alkynes, aldehydes, vinyl ketones, and / or maleimido functional groups. All of the functional groups listed in Tables 2 and 3 are compatible with these strategies, and additional exemplary functional groups include, but are not limited to, those listed in Table 4.

[0200] Table 4: Exemplary terminal functional group (FG) on the compound after derivatization and on the dye or biomolecule and exemplary linkage and chemistry.

[0201]

[0202] Polymerizing the hydrophobic monomer and the hydrophilic monomer (optionally via ATRP or RAFT) may comprise 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 (hydrophobic monomer(s):hydrophilic monomer(s)). In some embodiments, the ratio may be about 1:1 to about 1:3 or about 1:6. In some embodiments, the hydrophobic monomer is an alkyl acrylate (e.g., dodecyl acrylate) and / or the hydrophilic monomer is a glycol acrylate (e.g., PEG acrylate). In some embodiments, one or more hydrophobic monomers are polymerized with two or more different hydrophilic monomers (optionally via RAFT or ATRP) 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(s):hydrophilic monomer(s)). For example, in some embodiments, a first hydrophilic monomer may be ionic (e.g., a sulfonic acid acrylate monomer (e.g., 2-acrylamido-2 -methylpropane sulfonic acid) and / or a sulfonate monomer) and a second hydrophilic monomer may be non-ionic (e.g., a glycol acrylate (e.g., PEGylated methyl acrylate)). The ratio of the first hydrophilic monomer and the second hydrophilic monomer may vary (e.g., the ratio of the first hydrophilic monomer: second hydrophilic monomer may be about 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, or 1:6. Exemplary catalysts that may be used in a method of the present invention include, but are not limited to, a ruthenium complex, iron complex, copper complex, nickel complex, palladium complex, rhodium complex, and rhenium complex. 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

[0203] [RuCpCl(PPh3)2], dihydridotetrakis(triphenylphosphine)ruthenium(II) [RuH2(PPh3)4], and dichloro(p-cymene)ruthenium(II) dimer. Exemplary iron complexes include, but are not limited to, dichlorobis(triphenylphosphine)iron (II) [FeCl2(PPh3)2], bromo(cyclopentadienyl)dicarbonyliron(II) [FeCpBr(CO)2], and cyclopentadienyliron dicarbonyl dimer. In some embodiments, copper complexes generated in-situ with copper salts and ligands may be used and exemplary copper salts include, but are not limited to, cuprous chloride, cuprous bromide, cuprous triflate, cuprous hexafluorophosphate, and cuprous acetate, etc. 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 a pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II) chloride.

[0204] A co-catalyst may optionally be present in a method of the present invention such as, e.g., in the step of polymerizing the hydrophobic monomer and the hydrophilic monomer. In some embodiments, a co-catalyst may be present and may be 4-(dimethylamino)-l -butanol.

[0205] In some embodiments, a method of the present invention comprises hydrolyzing the compound, optionally in the presence of trifluoroacetic acid and water, to provide a formyl group at the first portion (e.g., the first end group) of the compound. The method may comprise reacting the dye and the formyl group of the compound to form a hydrazone bond between the dye and the compound, optionally via aldehyde-hydrazide chemistry, to thereby attach the dye to the first end group of the compound, thus becoming part of the first end group. In some embodiments, a biomolecule may be attached by reacting the formyl group with an amine group on the bioconjugate group via reductive amination. In some embodiments, a method of the present invention comprises reacting the compound with mercaptoacetic acid and triethylamine to provide a carboxymethylthioether group at the second end group (e.g., the second terminus) of the compound. The carboxymethylthioether group may be derivatized to provide a N-hydroxysuccinimide ester at the second portion of the copolymer. A biomolecule (e.g., avidin) may be attached to the N- hydroxysuccinimide ester at the second end group of the compound.

[0206] In some embodiments, a method of the present invention comprises reacting the compound with sodium azide to provide an azido group, and optionally attaching a dye to the azido group via copper-catalyzed azide-alkyne chemistry.

[0207] In some embodiments, a method of the present invention comprises a RAFT polymerization. In some embodiments, RAFT polymerization occurs in the presence of a radical initiator (e.g., AIBN) and a RAFT agent such as, for example, a thiocarb onylthio compound. Additional examples of RAFT agents include, but are not limited to, dithioesters, dithiocarbamates, trithiocarbonates, dithiobenzoates and / or xanthates.

[0208] In some embodiments, a method of the present invention comprises cleaving the thiocarb onylthio functionality present on a terminal end of the copolymer obtained using RAFT polymerization. Such cleavage may occur using any general methods known in the art. For example, in some embodiments, the thiocarbonylthio functionality is cleaved via aminolysis, e.g., in the presence of ethanolamine, to render the free thiol. In some embodiments, the free thiol may be coupled to a dye comprising a maleimido functionality thereby attaching the dye to a first portion (e.g., first end group) of the compound. In some embodiments, a biomolecule may be attached to the free thiol group of the first portion (e.g., terminal end). In some embodiments, a biomolecule may be attached to the opposite end group of the compound.

[0209] In some embodiments, a method of the present invention comprises producing a random structure via copolymerization of two monomers with comparable reactivity ratios (e.g., two or more different acrylate-based monomers). In some embodiments, a method of the present invention comprises producing a gradient structure via copolymerization of two monomers with differing reactivity ratios. In some embodiments, a method of the present invention comprises producing a random structure through polymerization of a single type of functional monomer (e.g., pentafluorophenyl acrylate, PFPA) with further post-polymerization modification introducing desired functional groups randomly.

[0210] In some embodiments, a method of producing a polymer with two distinct segments is provided. In some embodiments, an initial monomer feedstock consisting of one or two species is provided, and the initial monomer feedstock is reacted. After proceeding with the reaction of the initial monomer feedstock, additional, different monomers are charged into the reaction flask after an amount of time. Advantageously, the method achieves separate segments with varying compositions within a single reaction vessel. In some embodiments, the method avoids the formation of an emulsion, providing an advantage over use of block copolymers where an emulsion would form.

[0211] In some embodiments, a plurality of dyes, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more dyes are attached (directly or indirectly) to the polymer backbone via post-polymerization. In some embodiments, one or more dyes of the plurality of dyes are each attached to the polymer backbone through a covalent bond between the dye (that optionally includes a linker) and a pendant group attached to the polymer backbone. In some embodiments, the plurality of dyes is each attached to a portion of the polymer backbone comprising PFPA via post-polymerization (see, e.g., FIG. 8A).

[0212] Methods of Use

[0213] The compounds of the invention may be useful for single and dual-use life sciences applications. For example, in some embodiments, particular compounds of the invention can be chosen and optimized for various dual-use applications and / or independently optimized for single use applications involving and generating

[0214] 1) Fluorescence

[0215] 2) Chosen and optimized for maximizing photoacoustic imaging

[0216] 3) Chosen and optimized for generating reactive oxygen species, for example, singlet oxygen species, for photodynamic and photoimmunotherapy

[0217] 4) Chosen and optimized for PET imaging applications

[0218] 5) Chosen and optimized for magnetic resonance imaging (MRI) imaging applications

[0219] For example, certain dyes may be particularly useful for MRI applications, certain examples of which are shown in Figure 5A. Examples of certain dyes that may be useful in photoacoustic imaging are shown in Figure 5B.

[0220] In some embodiments, of the invention, the compounds of the invention allow for a single compound to be used in dual use applications including, for example,

[0221] 1. Fluorescence for, e.g., fluorescence guided surgery, flow cytometry, microscopy, immunoassays 2. Photoacoustic imaging

[0222] 3. Photodynamic / photoimmunotherapy

[0223] 4. PET Imaging

[0224] 5. MRI Imaging

[0225] Certain particular combinations of applications are shown in Figure 6, but a multitude of combinations of dyes for applications can be utilized and are described in further detail below, including, for example at Table 12, allowing for one or two or more different dyes used for a single application, or two or more different dyes used in dual use applications.

[0226] Dual Use Applications

[0227] The strategies described in Example 6, synthesis of a polymer with different dyes on the terminal end and backbone, and Example 7, synthesis of a polymer with different dyes on the terminal end and backbone, provide exemplary approaches to produce dual-purpose foldamers including a combination of two dyes that would produce two distinct signals (e.g., fluorescence or ultrasound) and / or outputs (e.g., reactive oxygen species (e.g., singlet oxygen) or heat).

[0228] In an embodiment, a composition useful for both flow cytometry and photoacoustic imaging (PAI) can be synthesized. In some embodiments, a mixture of fluorescent dye(s) and ultrasound dye(s) can be attached to the polymer backbone as described herein (see, e.g., Example 6), enabling flow cytometry to be used to sort the desired cells based on fluorescence. In some embodiments, a fluorescent dye(s) can be attached to either the polymer backbone or terminal end of the polymer and an ultrasound signal producing dye can be attached to a different site, either the polymer backbone or terminal end of the polymer (see, e.g., Example 7). The desired cells then could be injected back into the animal or patient and the same labeled cells could be tracked and analyzed in vivo via photoacoustic ultrasound imaging. Additionally, the dual-purpose foldamer could be utilized with a photoacoustic-based flow cytometer to sort the cells and a fluorescence or chemiluminescence imaging modality could be used to track and analyze the label in vivo.

[0229] In an embodiment, a composition useful for both light microscopy and fluorescence microscopy applications can be synthesized. In some embodiments, a mixture of fluorescent dye(s) and chromophore dye(s) can be attached to the polymer backbone using the description provided herein (see, e.g., Example 6). In some embodiments, a fluorescent dye(s) can be attached to either the polymer backbone or terminal end of the polymer and a chromophore dye can be attached to a different site (e.g., to either the polymer backbone or terminal end of the polymer) (see, e.g., Example 7). The mixture can enable a user to analyze the same sample on both a light-based microscope and fluorescence-based microscope. In some embodiments, fluorescent dye can be attached to either the polymer backbone or terminal end of the polymer and a chromogenic dye can be attached to a different site (e.g., the polymer backbone or terminal end of the polymer), enabling the user to analyze the same sample in two different modalities.

[0230] In an embodiment, a composition useful for both PAI imaging combined with a photodynamic therapy (PDT) dye application can be synthesized. In some embodiments, a mixture of ultrasound signal producing dye(s) and singlet oxygen producing PDT dye(s) can be attached to the polymer backbone (see, e.g., Example 6), enabling the user to image the targeted or non-targeted foldamer to confirm proper location using the PAI dye in the organism. In some embodiments, an ultrasound signal producing dye(s) can be attached to either the polymer backbone or terminal end of the polymer and a singlet oxygen producing PDT dye can be attached at a different site (e.g., to either the polymer backbone or terminal end of the polymer) (see, e.g., Example 7) enabling the user to image the targeted or non-targeted foldamer to confirm proper location using the PAI dye in the organism. Once proper localization is confirmed, the same or different light source can be used to generate a cell killing response from the PDT dye(s).

[0231] In an embodiment, a composition useful for both fluorescence imaging combined with a PDT dye application can be synthesized. In some embodiments, a mixture of fluorescent signal producing dye(s) and singlet oxygen producing PDT dye(s) can be attached to the polymer backbone, enabling the user to image the targeted or non-targeted foldamer to confirm proper location using the fluorescent dye in the organism (see, e.g., Example 6). In some embodiments, a fluorescent signal producing dye(s) can be attached to either the polymer backbone or terminal end of the polymer and a singlet oxygen producing PDT dye can be attached to a different site (e.g., to either the polymer backbone or terminal end of the polymer) (see, e.g., Example 7), enabling the user to image the targeted or non-targeted foldamer to confirm proper location using the fluorescent dye in the organism. Once proper localization is confirmed, the same or different light source could be used to generate a cell killing response from the PDT dye(s).

[0232] According to some embodiments, a compound and / or composition of the present invention may be used in flow cytometry. Flow cytometry is known and described in, for example, U.S. Patents Nos. 5,167; 5,915,925; 6,248,590; 6,589,792; and 6,890,487. In some embodiments the particle being detected, such as a cell, is labeled with a luminescent compound, such as a compound of the present invention, for detection. Labeling can be carried out by any suitable technique such as, e.g., binding the luminescent compound (e.g., a compound the present invention) to the particle or cell such as through an antibody that specifically binds to the particle or cell, by uptake or internalization of the luminescent compound into the cell or particle, by non-specific adsorption of the luminescent compound to the cell or particle, etc. The compounds described herein may be useful in flow cytometry as such luminescent compounds, which flow cytometry techniques (including fluorescent activated cell sorting or FACS) may be carried out in accordance with known techniques or variations thereof which will be apparent to those skilled in the art based upon the instant disclosure.

[0233] In some embodiments, provided is a method of detecting cells and / or particles using flow cytometry, the method comprising labeling cells and / or particles with a compound or biomolecule of the present invention and detecting the compound or biomolecule by flow cytometry, thereby detecting the cells and / or particles. In some embodiments, the method further comprises administering the labelled cells and / or particles to a subject and detecting the compound within the subject, thereby detecting the cells and / or particles within the subject. Detecting the compound within the subject may be performed, for example, by using an imaging technique such as photoacoustic imaging and / or magnetic resonance imaging. Additional dyes (tertiary, quaternary dyes, etc.) may be used and each may (or may not) be used for a different detection method.

[0234] In some embodiments, provided is a method of detecting a tissue and / or agent (e.g., a cell, infecting agent, etc.) in a subject, the method comprising: administering to the subject a compound, biomolecule, and / or composition of the present invention, optionally wherein the compound or biomolecule associates with the tissue and / or agent; and detecting the compound within the subject, thereby detecting the tissue and / or agent. Examples of detection methods include but are not limited to imaging techniques such as MRI, PAI, and fluorescence spectroscopy. In some embodiments, a compound of the invention has a primary dye and a secondary dye, and the primary dye is detected using a first detection method (e.g., a first imaging method) and the secondary dye is detected using a second detection method (e.g., a second imaging method). For example, in some embodiments, a first dye (primary dye) is detected using MRI and a second dye (i.e., a different dye) is detected using PAI. Primary and secondary designations are only provided to distinguish between different dye molecules so the secondary dye could thus be used to detect via MRI and the primary dye could be used to detect via PAI.

[0235] In some embodiments, provided is a method of using a compound of the present invention in photoacoustic imaging. According to some embodiments, a method of the present invention comprises a method of performing photoacoustic imaging. PAI is attractive in not relying on optical emission for detection (Haisch, C., Quantitative analysis in medicine using photoacoustic tomography. Anal. Bioanal. Chem. 2009, 393, 473-479; Cox, B.; Laufer, J. G.; Arridge, S. R.; Beard, P. C. Quantitative spectroscopic photoacoustic imaging: a review. J. Biomed. Opt. 2012, 17, 061202). Optical emission can be affected by light-scattering. In PAI, laser irradiation (e.g., optionally carried out with non-ionizing laser pulses) is followed by thermoelastic expansion and an ultrasonic pressure wave. Detection of the ultrasonic pressure wave can be achieved via a conventional ultrasound detector. In essence, ultrasound imaging can be carried out with laser input. It is noteworthy that in contrast to X-ray imaging methods, PAI does not rely on ionizing radiation.

[0236] A method of the present invention may comprise administering a compound, biomolecule, and / or composition of the present invention to a subject, optionally wherein the compound or biomolecule associates with a tissue and / or cell in the subject; irradiating at least a portion or part of the subj ect using a laser, optionally wherein the portion or part of the subj ect contains the compound of the present invention; and imaging at least the portion or part of the subject, optionally wherein the imaging comprises ultrasound imaging.

[0237] PAI can be performed without application of any exogenous contrast agent or chemical probe. In such cases, the distinct absorption of endogenous chromophores in native tissues engenders distinct signals. Absorption by hemoglobin, for example, facilitates delineation of the presence of blood vessels. However, the molar absorption coefficient of hemoglobin is low and may be insufficient for clear delineation in deep tissue. In such cases, the use of a contrast agent is very attractive. In some embodiments, a compound of the present invention is used as a contrast agent in PAI and / or comprises a dye that can be used as a contrast agent in PAI.

[0238] Diverse substances have been examined for use as contrast agents in PAI. Example dyes for use in PAI include, but are not limited to, gold nanomaterials, carbon nanotubes, porphyrins in liposomes, semiconducting polymers, and naphthalocyanines (Chitgupi, U.; Lovell, J. F. Naphthalocyanines as contrast agents for photoacoustic and multimodal imaging. Biomed. Eng. Lett. 2018, 8, 215-221; de la Zerda, A., et al., Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics. Contrast Media Mol. Imaging 2011, 6, 346-369). In some embodiments, a dye present in a compound of the present invention and / or a compound of the present invention has the following photophysical characteristic, which is that following absorption of light, the dye / compound relaxes to the ground state immediately and quantitatively, without emission of light or formation of metastable states of any significant lifetime. In other words, the yield of internal conversion (i.e., radiationless decay) should be quantitative, and ideally, the rate of internal conversion should be exceptionally fast, with an excited-state lifetime of less than 1 picosecond. This description essentially couches the “optical-to-acoustic conversion efficiency” (Cheng, K.; Cheng, Z. Near infrared receptor-targeted nanoprobes for early diagnosis of cancers. Curr. Med. Chem. 2012, 19, 4767-4785) in terms of molecular photophysics. The attraction for such rapid and quantitative internal conversion is to convert all of the absorbed light into heat, namely, the thermal expansion that engenders the ultrasonic wave. One research group has referred to such contrast agents as “sonochromes” (Duffy, M. J., et al., Towards optimized naphthalocyanines as sonochromes for photoacoustic imaging in vivo. Photoacoustics 2018, 9, 49-61) to distinguish them from more commonly known lumichromes or fluorochromes or luminophores, all of which imply the emission of light following absorption of incident light. In some embodiments, a compound of the present invention is and / or comprises a sonochrome.

[0239] In some embodiments, a dye present in a compound of the present invention and / or a compound of the present invention absorbs light in the red or near-infrared region (NIR). For example, in some embodiments, a compound of the present invention may be used for imaging deep tissue, where absorption in the red or NIR is desired as this region presents an optical window allowing penetration of light. At shorter wavelengths, absorption by endogenous chromophores (e.g., hemoglobin, melanin) can occur; at longer wavelengths, scattering of light by the overtone vibrational band of water can be observed. In some embodiments, a dye present in a compound of the present invention and / or a compound of the present invention absorbs in the red or NIR and the molar absorption coefficient is as large as possible to engender great sensitivity such as, e.g., molar absorption coefficient values of 1,000 M-1cm-1, 10,000 M-1cm-1100,000 M-1cm-1or greater. In some embodiments, the dye is a cyanine such as indocyanine green, with a molar absorption coefficient in the range of about 156000 M-1cm-1- 223000 M-

[0240] 1cm-1. In some embodiments, a chlorin exhibits a Qyband molar absorption coefficient in the range from about 10,000 M-1cm-1to about 100,000 M-1cm-1. In some embodiments, a bacteriochlorin exhibits a Qyband molar absorption coefficient in the range from about 50,000 M-1cm-1to about 200,000 M-1cm-1.

[0241] In some embodiments, a method of the present invention provides for multi wavelength multiplexing. Multi wavelength multiplexing may be achieved by using two or more absorbers as PAI contrast agents, all of which exhibit quantitative (or near-quantitative) internal conversion, wherein the two or more absorbers are two or more different compounds of the present invention. The two or more different compounds of the present invention may have largely non-overlapping absorption bands. Multiplexing may be achieved by sweeping the incident light source (e.g., a laser) across the NIR and red spectral regions, with detection of the resulting ultrasound wave upon successive absorption of each spectrally distinct contrast agent. Alternatively, a set of multiple lasers may be used with each laser dedicated to a different PAI contrast agent.

[0242] In some embodiments, the dye present in a compound of the present invention comprises a chlorin or bacteriochlorin, optionally wherein the compound is used in a method of the present invention for PAI. Chlorins and / or bacteriochlorins can be ideal for photoacoustic imaging given the strong and sharp long- wavelength (Qy) absorption band. Chlorins and / or bacteriochlorins may be modified to engender a high yield of internal conversion and / or packaged in a manner to achieve solubilization in aqueous media.

[0243] For example, a tetrapyrrole macrocycle that is fluorescent in its free base form can be rendered non-fluore scent by metalation with an appropriate metal. Tetrapyrroles include porphyrins and hydroporphyrins; the latter includes chlorins and bacteriochlorins. There exists a veritable "periodic chart of metallotetrapyrroles" given extensive work on the preparation and study of metallotetrapyrroles over nearly a century. Metals that afford a non-luminescent tetrapyrrole chelate are well known (see, e.g., Gouterman, M. Optical spectra and electronic structure. In The Porphyrins; Dolphin, D. (Ed.), Vol. Ill, Academic Press: New York, 1978, pp 1-165). Examples of metals that can afford a non-luminescent tetrapyrrole chelate (valencies not shown for clarity) include, but are not limited to, Fe, Co, Ni, Cu, Zr, Ru, and the lanthanides. In some embodiments, the dye present in a compound of the present invention is a tetrapyrrole macrocycle that comprises iron. Iron may be particularly attractive given the presence of iron as a native constituent in human metabolism, the immense study that has been devoted to iron tetrapyrroles (given the fact that heme is the iron chelate of protoporphyrin IX), and the extraordinarily short excited-state lifetime of iron porphyrins. In some embodiments, a compound of the present invention comprises an iron chlorin or an iron bacteriochlorin. In some embodiments, a method of the present invention comprises administering to a subject a compound of the present invention that comprises an iron chlorin or an iron bacteriochlorin as a PAI contrast agent and performing photoacoustic imaging. In some embodiments, the dye present in a compound of the present invention is a tetrapyrrole macrocycle that comprises copper (e.g., Cu(II)). In some embodiments, the dye present in a compound of the present invention comprises copper (e.g., Cu(II)) and is optionally used for photoacoustic imaging. In some embodiments, the dye present in a compound of the present invention comprises iron (e.g., Fe(II)) and is optionally used for oxygen sensing.

[0244] In some embodiments, a compound of the present invention comprises a Fe(II) tetrapyrrole that is sterically hindered and / or does not form a mu-oxo dimer of Fe(III) tetrapyrroles. In some embodiments, a compound of the present invention comprises a Fe(III) tetrapyrrole. It warrants mention that Fe(II) tetrapyrroles can coordinate to molecular oxygen, and if not sterically hindered, can cause a chemical reaction leading to the mu-oxo dimer of Fe(III) tetrapyrroles. In contrast, Fe(III) tetrapyrroles do not coordinate to molecular oxygen, and do not undergo mu-oxo dimer formation. Fe(III) tetrapyrroles are the preferred oxidation state of iron tetrapyrroles upon formation under aerobic conditions. Diverse methods of longstanding establishment are available for formation of Fe(III) tetrapyrroles, and for conversion of Fe(II) tetrapyrroles to the corresponding Fe(III) tetrapyrroles.

[0245] Free base tetrapyrroles can afford a certain amount of fluorescence (e.g., quantum yield of up to ~10%), a certain amount of triplet-state formation (e.g., quantum yield of up to ~70%), and the remainder is internal conversion (e.g., quantum yield of up to ~20%). As stated above, a convenient way to achieve a quantum yield of ~100% for internal conversion (i.e., radiationless decay) is to metalate the tetrapyrrole with a metal that, by one or more mechanisms, causes the excited state to relax promptly and essentially quantitatively to the ground state. An alternative approach to promote internal conversion versus radiative decay (i.e., fluorescence) and intersystem crossing (i.e., triplet-state formation) is to attach appropriate substituents to the tetrapyrrole. Typical substituents are those that cause spin-orbit coupling such as the heavier halogens, including bromo, iodo, and astatine. Thus, in some embodiments, introduction of one or more halogens in a dye and / or compound of the present invention can be employed alone, or together with a metal that itself alone affords limited luminescence, thereby affording rapid and essentially quantitative relaxation to the ground state. Such metals include many of the metals in the periodic chart. Methods of metalation of tetrapyrroles are well known (Buehler, J. W. Static coordination chemistry of metalloporphyrins. In Porphyrins and Metalloporphyrins; Smith, K. M. (Ed.), 1975, Elsevier Scientific Publishing Co. : Amsterdam, pp 157-231 ; Sanders, J. K. M., et al., Axial coordination chemistry of metalloporphyrins. In The Porphyrin Handbook; Kadish, K. M.; Smith, K. M.; Guilard, R. (Eds.), Vol. 3, 2000, Academic Press: San Diego, pp 1-48). Because heavy atoms attached to arenes are well known to cause rapid relaxation of the excited state, a wide variety of heavy-atom substituted arenes are excellent candidates for use in PAI in accordance with methods of the present invention. In some embodiments, a compound of the present invention comprises a tetrapyrrole (e.g., a tetrapyrrole bearing a heavy atom substituent at the macrocycle periphery and / or a centrally chelated metal that affords non-luminescence). Such a tetrapyrrole (e.g., a chlorin or bacteriochlorin) may provide a number of possible narrow-band absorptions across the red and NIR spectral regions.

[0246] While describing various mechanisms by which the excited state for a compound can revert promptly and essentially quantitatively to the ground state, the present invention is not limited thereto and other mechanisms known in the art may be used. For example, such a mechanism can stem from (1) a high rate of internal conversion versus the rates of radiative decay and intersystem crossing; (2) a high rate of intersystem crossing versus the rates of radiative decay and internal conversion followed by immediate and non-radiative decay from the excited multiplet state to the ground state; and / or (3) a high rate of charge-transfer versus all other rates for depopulation of the excited state followed by charge recombination that leads quantitatively to the ground state. Another example is to structurally distort the macrocycle from essential planarity. Other mechanisms are known to those of skill in the art. Regardless of the mechanism, established methods known to those of skill in the art can be employed to create tetrapyrroles that exhibit excited states with exquisitely short lifetimes and essentially quantitative relaxation to the ground state. The prompt and near-quantitative relaxation to the ground state can afford what are referred to herein as "non-luminescent" molecule entities, which may be used in PAI.

[0247] A compound of the present invention may package a metallotetrapyrrole, optionally for use in PAI. The metallotetrapyrrole may have a bioconjugatable group that can be used to attach the metallotetrapyrrole to a polymer as described herein to provide a compound of the present invention. Accordingly, a compound of the present invention may comprise a single metallotetrapyrrole. In some embodiments, a compound of the present invention may maintain the intrinsic spectral features (e.g., absorption spectrum, fluorescence spectrum, fluorescence quantum yield, etc.) of the dye by packaging the dye within a portion of the compound (e.g., within the polymer portion), optionally without alteration by interaction with external entities such as, e.g., other dyes and / or biological substances (e.g., cellular constituents, proteins, etc.). Inclusion of a single dye (e.g., a Fe(III) tetrapyrrole) in a compound of the present invention may preserve the intrinsic absorption spectrum of the dye.

[0248] According to some embodiments, a dye present in a compound of the present invention may be a non-luminescent molecular entity (e.g., a non-fluorescent and / or non-phosphorescent molecular entity), optionally wherein the compound is used in PAI. The dye may have a rapid optical to acoustic conversion. In some embodiments, the dye is a non-luminescent molecular entity and has a short excited-state lifetime, optionally wherein the excited-state lifetime is in the sub-picosecond range. Upon illumination, the excited state may immediately revert to the ground state, liberating heat. The heat produces an "acoustic wave", which can be detected by a microphone. The structure of a compound of the present invention may protect the dye from the physiological environment and / or may be suitable for use in a method of performing PAI.

[0249] In some embodiments, a compound of the invention may be used in fluorescence guided surgery (FGS). FGS is an intraoperative medical technique used to generate a real-time fluorescence image of the surgical region and guide the surgical procedure. As such, in some embodiments, the detection method may be fluorescence spectroscopy. In some embodiments, a primary dye may absorb and / or emit light at a first wavelength and a secondary dye may absorb and / or emit light at a second wavelength. Additional dyes (tertiary, quaternary dyes, etc.) may be used and each may (or may not) absorb and / or emit at a different wavelength.

[0250] Also provided according to embodiments of the invention is a method for treating a cell and / or tissue (e.g., a diseased cell and / or tissue) in a subject in need thereof, the method comprising: administering to the subject the compound, biomolecule, and / or composition of the invention, optionally wherein the compound or biomolecule associates with the cell and / or tissue, and irradiating the subject or a portion thereof (e.g., a location where the cell and / or tissue are present) with light of a wavelength and intensity sufficient to treat the cell and / or tissue, optionally wherein the light activates the compound. In some embodiments, in addition to treating the cell and / or tissue, the compound of the invention may be used to detect the compound and thus the cell and / or tissue. In some embodiments, a first dye (a primary dye) on the compound may be detected using an imaging technique; and a second, different dye (the secondary dye) on the compound may be used to treat the cell and / or tissue using a method comprising activating the secondary dye, e.g., to release reactive oxygen species into the diseased tissue, to thereby treat the cell and / or tissue. Examples of imaging techniques include MRI and PAI. Examples of treating methods include photodynamic therapy and photoimmunotherapy. As the designations primary and secondary are only used to distinguish between different dyes, the secondary dye could also be detected using an imaging technique and the primary dye could be used to treat the cell and / or tissue. Additional dyes (tertiary, quaternary dyes, etc.) may be used and each may (or may not) be used for a different detection or treatment method.

[0251] In some embodiments, a treatment method that uses a compound of the present invention is photodynamic therapy (PDT) and / or photodynamic inactivation (PDI). PDT is a form of phototherapy involving light and a photosensitizing chemical substance (e.g., a compound of the present invention) that is used in conjunction with molecular oxygen to elicit cell death (phototoxicity). PDT can be used to kill microbial cells, including bacteria, fungi and viruses. PDT may also be used to treat cancer. When light energy is administered in PDT to destroy tumors, various forms of energy are within the scope of this invention, as will be understood by those of skill in the art. Such forms of energy include, but are not limited to, thermal, sonic, ultrasonic, chemical, light, microwave, ionizing (such as x-ray and gamma ray), mechanical, and / or electrical. For example, sonodynamically induced or activated agents include, but are not limited to, gallium -porphyrin complex (see Yumita et al., Cancer Letters 112: 79-86 (1997)), other porphyrin complexes, such as protoporphyrin and hematoporphyrin (see Umemura et al., Ultrasonics Sonochemistry 3: S187-S191 (1996)); other cancer drugs, 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)).

[0252] Examples of treatment areas for PDT and / or PDI include, but are not limited to, the following:

[0253] (i) Treatment of opportunistic infections. Compounds, compositions and / or methods of the present invention may be useful for PDT of opportunistic infections, particularly of soft tissue. For antimicrobial treatment (via PDT) of infections, particularly wound infections, the infecting organism may include (as non-limiting examples) Staphylococcus aureus, Pseudomonas aeruginosa, and / or Escherichia coli. In nosocomial infections, P. aeruginosa is responsible for 8% of surgical -wound infections and 10% of bloodstream infections. In some embodiments, a subject is an immunocompromised subject, such as, e.g., those afflicted with AIDS and / or undergoing treatment with an immunosuppressive agent.

[0254] (ii) Treatment of burns. Infections by S. aureus and gram-positive bacteria in general are particularly pronounced in burns (Lambrechts, 2005). The multidrug resistance of S. aureus presents significant medical challenges. In this regard, compounds, compositions and / or methods of the present invention may be useful for the treatment of opportunistic infections or burns.

[0255] (Hi) Sepsis. Compounds, compositions and / or methods of the present invention may be useful for the PDT treatment of a subject afflicted with opportunistic infections of Vibrio vulnificus. V. vulnificus, a gram-negative bacterium, causes primary sepsis, wound infections, and / or gastrointestinal illness in a human.

[0256] (iv) Ulcers. Compounds, compositions and / or methods of the present invention may be useful for PDT treatment of the bacterium that causes ulcers (Helicobacter pylori). In the clinic, treatment may be effected in any suitable manner, such as, e.g., by insertion of a fiber optic cable (akin to an endoscope but with provisions for delivery of red or near-IR light) into the stomach and / or afflicted region.

[0257] (v) Periodontal disease. Compounds, compositions and / or methods of the present invention may be useful in PDT for the treatment of periodontal disease, including gingivitis. Periodontal disease is caused by the overgrowth of bacteria, such as the gram-negative anaerobe Porphyromonas gingivalis. As with many PDT treatments, targeting or solubilizing entities in conjunction with the photoactive species are essential for appropriate delivery of the photoactive species to the desired cells. The oral pathogens of interest for targeting include, but are not limited to, Porphyromonas gingivalis, Actinobacillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Eikenella corrodens, Fusobacterium nucleatum subsp. Polymorphum, Actinomyces viscosus, and the streptococci. For such applications, the compounds and / or compositions of the present invention may be topically applied (e.g., as a mouthwash or rinse) and then light administered with an external device, in-the-mouth instrument, or combination thereof.

[0258] (vi) Atherosclerosis. Compounds, compositions and / or methods of the invention may be useful in PDT to treat vulnerable atherosclerotic plaque. Without wishing to be bound to any particular theory, invading inflammatory macrophages are believed to secrete metalloproteinases that degrade a thin layer of collagen in the coronary arteries, resulting in thrombosis, which often is lethal (Demidova and Hamblin, 2004). Bacteriochlorins targeted to such inflammatory macrophages may be useful for PDT of vulnerable plaque.

[0259] (vii) Cosmetic and dermatologic applications. Compounds, compositions and / or methods of the present invention may be useful in PDT to treat a wide range of cosmetic dermatological problems, such as hair removal, treatment of psoriasis, and / or removal of skin discoloration. Ruby lasers are currently used for hair removal; in many laser treatments melanin is the photosensitized chromophore. Such treatments work reasonably well for fair-skinned individuals with dark hair. Compounds, compositions and / or methods of the present invention may be used as near-IR sensitizers for hair removal, which enables targeting a chromophore with a more specific and / or sharp absorption band.

[0260] (viii) Acne. Compounds, compositions and / or methods of the present invention may be useful in PDT to treat acne. Acne vulgaris is caused by Propionibacterium acnes. which infects the sebaceous gland; some 80% of young people are affected. Here again, the growing resistance of bacteria to antibiotic treatment is leading to an upsurge of acne that is difficult to treat. Current PDT treatments of acne typically rely on the addition of aminolevulinic acid, which in the hair follicle or sebaceous gland is converted to free base porphyrins. Compounds and / or compositions of the present invention may be administered to a subject topically or parenterally (e.g., by subcutaneous injection) depending upon the particular condition.

[0261] (ix) Infectious diseases. Compounds, compositions and / or methods of the present invention may be useful in PDT to treat infectious diseases. For example, Cutaneous leishmaniasis and sub-cutaneous leishmaniasis, which occurs extensively in the Mediterranean and Mideast regions, is currently treated with arsenic-containing compounds. PDT has been used to reasonable effect recently, at least in one case, on a human subject. The use of compounds and / or compositions of the present invention are likewise useful, and potentially offer advantages such as ease of synthesis and better spectral absorption properties.

[0262] (x) Tissue sealants. Compounds, compositions and / or methods of the present invention may be useful in PDT as tissue sealants in a subject in need thereof. Light-activated tissue sealants are attractive for sealing wounds, bonding tissue, and / or closing defects in tissue. There are many applications where sutures and / or staples are undesirable, and use of such mechanical methods of sealing often leads to infection and / or scarring.

[0263] (xi) Neoplastic disease. Compounds, compositions and / or methods of the present invention may be useful in PDT for treating 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, plaque-stage cutaneous T-cell lymphoma, and / or Kaposi sarcoma.

[0264] During photodynamic therapy a compound of the invention is administered to a subject in need thereof (e.g., a subject having any of the above mentioned diseases). The administered compound may associate with the diseased tissue present inside the subject, and exposure of the subject to a light source emitting a suitable light with the proper wavelength and intensity may activate the compound (e.g., release reactive oxygen species (ROS)) into the diseased tissue thereby treating the diseased tissue, optionally without affecting the healthy tissue. For example, in some embodiments, the diseased tissue is a hyperproliferative tissue (e.g., a tumor).

[0265] In some embodiments of the invention, the compound comprises a biomolecule and the biomolecule localizes the compound to a particular cell, tissue, or biological site and / or aids in transport of the compound across the cell membrane. For example, in some embodiments, the biomolecule includes a biomarker for a particular site in vivo and its presence will direct the compound to a cell, tissue, or other biological site. In some embodiments, the biomolecule directs the compound to a hyperproliferative cell and / or tissue.

[0266] In some embodiments, the compound is prepared for targeting applications. The targeted compound can comprise a biomolecule, for example, that specifically attracts or binds to a particular spatial or polar organization of the other molecule. Accordingly, the targeted compound may target, e.g., a cell comprising a particular surface molecule, for example, growth factor receptors, tumor necrosis factors, interleukin receptors that may be used for diagnosis or detection of the biological site. The targeted compound may comprise a biomolecule that is a member of a binding pair, with the binding member present in the tissue, cell or targeted biological site.

[0267] In some embodiments, the compound is devoid of a biomolecule and the compound can be used in non-targeting methods. For example, in some embodiments, the compound comprises a dye capable of being imaged to confirm presence of the compound in an area of interest. In some embodiments, the non-targeting methods comprise a non-fluorescent dye, including, for example, ultrasound signal producing dye(s) and / or reactive oxygen species producing dye(s). In some embodiments, non-targeting methods comprise local administration of a compound, or injection into a particular tissue, cavity, or area of interest. In some embodiments, the compound is prepared for non-targeting methods, and may, for example comprise a mixture of fluorescent signal producing dye(s) and singlet oxygen producing PDT dye(s), enabling the user to image a non-targeted foldamer to confirm proper location using the fluorescent dye in the organism. Example dyes for non-targeting applications are described in detail in the working examples and at Table 12 of the present application.

[0268] The present invention is explained in greater detail in the following non-limiting examples. EXAMPLES

[0269] EXAMPLE 1: Linker Designs for Multiple Dyes and / or Additional Moeities in End Group

[0270] A. Linear linker designs

[0271] Dual use designs may be achievable using a linear linker, wherein one dye or biomolecule is attached directly to another and the two so joined are connected to the polymer backbone by a linker (See Scheme 1). This design can be achieved for example through use of one dye that maintains functionality for coupling onto another, such as an aryl halide (Compound I in Scheme 1) that may be leveraged for metal-mediated cross coupling reactions such as Suzuki, Sonogashira, or Stille reactions. The second dye molecule has two reactive functional groups, one for cross coupling (e.g., boronic acid, boronic pinacol ester, alkyne) to attach to the first dye molecule and another for attachment to the polymer backbone (e.g., carboxylic acid, amine, maleimide). In the example shown in Scheme 1, a first bacteriochlorin (Compound I) is prepared with an aryl halide that undergoes Sonogashira coupling to the alkyne of the chlorin (Compound II). The methyl ester of Compound II is then converted into a maleimide that can then be attached to a free thiol end of the polymer backbone (also referred to as the foldamer). The linker in this embodiment is designed to provide rigidity and in so doing to impart spatial separation of the molecules to minimize through-space interactions such as energy transfer. This may help to maintain the individual properties of the two probes.

[0272]

[0273] Scheme 1. Example of linear linker design approach to dual-use foldamer-based probe.

[0274] B. Bifunctional branched linker designs

[0275] Dual use probes can also be prepared by functionalizing a central linker molecule in two steps, followed by attachment of that dually modified linker to the polymer in a subsequent step. This process may or may not be followed by incorporation of a biological targeting molecule in another following step.

[0276] For dually functional reagents, this process can reasonably be accomplished in practice through the use of a trifunctional linker such as N-Boc-L-lysine methyl ester hydrochloride (Scheme 2). A linker of this type can first be reacted with probe 1 (Pl) that contains a carboxylic acid group. Pl could consist of a fluorescent dye, non-fluorescent dye, or biomolecule such as a peptide or oligonucleotide. The coupling may be achieved under many other conditions known in the art (e.g., including but not limited to DCC, DIC, ED AC, HATU, HBTU, PyBop, oxalyl chloride) or as shown through in situ activation using a reagent such as TSTU in the presence of a base such as triethylamine in a solvent such as dimethylformamide that provides adequate solubility to the components of the reaction. Treatment of the resulting Pl -linker compound with strong base such as sodium hydroxide reveals a reactive carboxylic acid that can next be coupled with probe 2 (P2) under conditions as previously described for Pl. Similarly, P2 may consist of a fluorescent dye, non-fluorescent dye, small molecule drug, or biomolecule such as a peptide or oligonucleotide. Finally, deprotection of the lysine side chain amine Boc group under acidic conditions (e.g., HC1, trifluoroacetic acid) affords the final reactive amine group that can be used to attach the Pl-P2-linker to a foldamer suitably functionalized with a carboxylic acid. Coupling conditions may be performed as previously described for attachment of Pl and P2. The resulting foldamer now contains Pl and P2 as well as a reactive thiol group available for further derivatization at the opposite terminus of the polymer. If no further features are desired, the thiol could either be left as is or capped with a simple maleimide group such as benzyl maleimide. Alternatively, the foldamer could be prepared for attachment to a biomolecule through initial reaction of its terminal thiol with the maleimide group of aa heterobifunctional linker such aass N-Succinimidyl 4-

[0277] (maleimidomethyl)cyclohexanecarboxylate (SMCC). SMCC contains an N- hydroxy succinimide group that can then be used to attach a biomolecular targeting agent (Tl) such as a peptide, oligonucleotide, antibody, or protein, that contains an amine group.

[0278] Scheme 2. Scheme for attachment of two distinct probe molecules and optionally a third group for molecular targeting onto the foldamer.

[0279] The synthesis of bifunctional branched molecules may be performed using the following synthetic procedure:

[0280] Lys-P intermediate 1. Fmoc-Lys(Boc)-OH (11.5 mg, 27.61 μmol), l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide (31.8 mg, 165.67 μmol), 4-dimethylaminopyridine (2.0 mg, 16.6 μmol), and 4 angstrom molecular sieves (2.0 mg) were added to a RBF with stirbar. The flask was septum sealed, evacuated, argon flushed, and DCM (7.5 mL) was added. The mixture was stirred at room temp for 1 h, then porphyrin aryl alcohol (11.5 mg, 27.61 μmol) solid was added in bulk and the reaction was allowed to stir for 22 h at room temp. The reaction was diluted with DCM and washed with saturated aqueous sodium bicarbonate, water, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography with silicon dioxide medium and eluting with 0 - 50% ethyl acetate in hexanes. Main product was isolated after drying as 20.3 mg (85%) red solid. [M + H]+= 867.6, calc. 867.4.

[0281] Lys-P intermediate 2. Lys-P intermediate 1 (20.3 mg, 23.41 μmol) was dissolved in DMF (2.0 mL) and piperidine (0.11 mL) was added. The solution was stirred at room temp for 30 min. The solvent was removed, and the residue was purified by column chromatography on silicon dioxide with 0 - 10% MeOH in DCM. The desired product was isolated as 13.9 mg (92%) deep red solid. [M + H]+= 645.2, calc. 645.3.

[0282] Lys-P-dye I. Lys-P intermediate 2 (5.9 mg, 9.15 μmol) and BC740-NHS (9.8 mg, 12.81 μmol) were added to a RBF with stir bar. The flask was evacuated, argon flushed, and DMF (2.0 mL) was added, followed by tri ethylamine (3.8 μL, 27.45 μmol). The reaction was stirred at room temp for 4 h, then the solvent was removed. The residue was dissolved in DCM; washed with saturated aqueous sodium bicarbonate, water, and brine; dried over sodium sulfate; filtered; and concentrated. The residue was purified by column chromatography on silicon dioxide with 0 - 5% MeOH in DCM. Isolated 8.3 mg (70%) deep red solid. [M + H]+= 1294.6, calc. 1294.6. Lys-P-dye II. Prepared as described for compound Lys-P-dye I. [M + H]+= 1218.9, calc. 1218.6. Lys-P-dye III. Prepared as described for compound Lys-P-dye I. [M + H]+1232.9, calc. 1232.6.

[0283] Lys-P-dye IV. Prepared as described for compound Lys-P-dye I. [M + H]+1310.9, calc. 1310.6.

[0284] C. Trifunctional branched linker designs

[0285] A foldamer-based reagent may also be prepared to contain three distinct probes each with different functional roles. This reagent may reasonably be prepared in a similar fashion as previously described for bifunctional reagents starting with the trifunctional linker N-Boc-L- lysine methyl ester hydrochloride (Scheme 3). Initial introduction of probe 1 (Pl) ifs accomplished using standard amide coupling chemistry known in the art. Pl could consist of a fluorescent dye, non-fluore scent dye, small molecule drug, or biomolecule such as a peptide or oligonucleotide. Saponification of the methyl ester provides a carboxylic acid which can be coupled under standard amide conditions to commercially available L-cysteine methyl ester hydrochloride. The resulting linker now contains a reactive sulfhydryl group which can be used to attach probe 2 (P2) which has been modified to contain a terminal maleimide group. The methyl ester of the cysteine residue is then saponified using strong base as previously described. This provides a unique carboxylic acid which can undergo amide coupling with probe 3 (P3) that is functionalized to contain a terminal amine. The Boc group used to protect the side chain amine of the starting lysine molecule is stable to all preceding conditions and can be removed following incorporation of P3 upon treatment with acid (e.g., HCI, trifluoroacetic acid). Boc group removal affords a terminal amine which can be used to attach the P1 / P2 / P3 trifunctional linker to the foldamer. The opposite terminus of the foldamer containing a free thiol can be either left as is, capped with a simple maleimide group such as benzyl maleimide, or further reacted with a reagent such as SMCC as previously described for the bifunctional linker design to allow attachment to another functional molecule that contains an amine.

[0286]

[0287] Scheme 3. Scheme for attachment of two distinct probe molecules and optionally a third group for molecular targeting onto the foldamer.

[0288] EXAMPLE 2: Multiple Dyes on Polymer Backbone A polymer that contains orthogonally reactive functional groups at each terminus and includes a third orthogonal reactive group along the length of the polymer is described. This polymer design allows selective modification by three distinct reaction chemistries and can be used as a template molecule for covalent chemical attachment of dyes, biomolecules, or other molecules of choice. For example, a dye may be added to one or more ends of the polymer backbone and additional dyes may also be incorporated via post-polymerization reaction with suitable functional groups pendant from the polymer backbone (See Figure 7, Figures 8A- 8B). The total number of dyes per chain can be controlled through selection of the initial stoichiometric ratio of dye to polymer. The overall potential number of dyes that may be added per polymer is limited only by the degree of polymerization of the polymer (the number of monomers that contain reactive groups on the polymer).

[0289] For example, under standard reversible addition fragmentation chain transfer (RAFT) conditions using a suitable initiator (e.g., 2, 2'-Azobis(2 -methylpropionitrile (AIBN)) and chain transfer reagent (CTA, e.g., 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid 3- azido-1 -propanol ester), a polymer can be generated that includes azide, thiol, and N- hydroxysuccinimide reactive groups. This template allows selective reaction of molecules containing alkyne, maleimide, and amine groups. One embodiment of this polymer system can may use N-succinimidyl p-vinyl benzoate (NSVB) as a monomer. Another embodiment of the polymer system with the same reactive groups but with less hydrophobicity can be made under RAFT conditions using N-succinimidyl acrylate as the monomer. A polymer that contains azide, thiol, and amine reactive groups can be made via RAFT using a monomer such as (2- Boc-amino)ethyl methacrylate followed by Boc protecting group removal under suitable conditions (e.g., TFA, or HCl).

[0290] A. Synthesis of Amine Monomer Dye

[0291] The sythesis of a monomer dye may be performed using the following synthetic procedure:

[0292]

[0293] BClla. Compound BC11 (279.2 mg, 500.0 μmol, prepared as described in J. Org. Chem., 2010, 75, 1016-1039), 4-Methoxycarbonylphenylboronic acid pinacol ester (144.2 mg, 550.0 μmol), potassium carbonate (691.1 mg, 5000.0 μmol), and tetrakis(triphenylphosphine)palladium (57.8 mg, 50.0 μmol) were added to a flame-dried 250 mL RBF with stir bar. The flask was septum sealed, evacuated and argon flushed 3 times over 30 minutes. Toluene (66 mL) and DMF (33 mL) were added and the mixture was stirred. The system was evacuated for 2 minutes, then flushed with argon twice in succession. The flask was added to a pre-heated oil bath at 80 °C and stirred under argon atmosphere. After 16 hours, the flask was allowed to cool and the reaction mixture was diluted with ethyl acetate (100 mL). The organic layer was washed with deionized water (3 x 125 mL) without mixing, and then with mixing (3 x 125 mL). The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in a miinniimmuumm amount of dichloromethane and dried onto Celite. The cake was eluted on a 24 g silica column with 0 - 65% dichloromethane in hexanes over 15 minutes. The desired product was isolated as 207.9 mg (68%) dark solid.

[0294] BCllb. BClla (199.7 mg, 325.5 μmol), phenylboronic acid pinacol ester (79.7 mg, 390.6 μmol), cceessiiuumm carbonate (318.2 mg, 976.5 μmol), and tetrakis(triphenylphosphine)palladium (112.9 mg, 97.7 μmol) were added to a flame-dried 100 mL RBF with stir bar. The flask was septum sealed, evacuated and argon flushed 3 times over 30 minutes. Toluene (22 mL) and DMF (11 mL) were added and the mixture was stirred. The system was evacuated for 2 minutes, then flushed with argon twice in succession. The flask was added to a pre-heated oil bath at 80 °C and stirred under argon atmosphere. After 18 hours, the flask was allowed to cool and the reaction mixture was diluted with ethyl acetate (50 mL). The organic layer was washed with deionized water (3 x 50 mL) without mixing, and then with mixing (3 x 50 mL). The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in a minimum amount of di chloromethane and eluted on an 80 g silica column with 20% ethyl acetate in hexanes. The desired product was isolated as 160 mg (80%) dark solid.

[0295] BCllc. BCllb (157.0 mg, 257.1 μmol) was added to a 100 mL RBF with stir bar and dissolved in tetrahydrofuran (13.0 mL). Methanol (6.5 mL) was added and the solution was stirred vigorously. To the stirring solution was added 5 M aqueous NaOH (6.5 mL). The reaction was stirred at room temperature covered in foil. After 16 h, The reaction mixture was added gradually to a stirring solution of 1 M aqueous HC1 (40 mL) chilled in an ice-water bath. This mixture was diluted with ethyl acetate. The aqueous layer was removed, then the organic layer was washed with water (2 x 50 mL no mixing, 2 x 50 mL with mixing), brine (50 mL), dried over sodium sulfate, filtered and concentrated. The product was dried under high vacuum to give 140.5 mg (92%) green solid.

[0296] BClld BCllc (49.5 mg, 8833..00 μmol) and N,N,N',N'-Tetramethyl-O-(N- succinimidyl)uronium tetrafluoroborate (30.0 mg, 99.5 μmol) were added to a flame-dried 25 mL RBF with stir bar. The flask was septum sealed, evacuated, and argon flushed. Dimethylformamide (8.3 mL) was added, followed by triethylamine (69.3 uL, 497.7 μmol) and the mixture was allowed to stir at room temperature. After 30 minutes, N-boc- ethylaminediamine (19.7 μL, 124.4 μmol) was added and the reaction was stirred under argon atmosphere. After 24 h, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with water (4 x 25 mL, no mixing), washed with half-saturated aqueous ammonium chloride (2 x 25 mL), then brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was diluted in a minimum amount of dichloromethane and eluted on a 24 g silica column with 20 - 60% ethyl acetate in hexanes over 15 minutes. The product was isolated and dried to give 44.9 mg (73%) solid.

[0297] Amine monomer dye. BClld (44.9 mg, 60.8 μmol) was dissolved in dichloromethane (3.5 mL) under argon. 4M HC1 in dioxane solution (0.5 mL) was added dropwise at room temp. After 2 hours, the dioxane was removed by flushing with argon. The residue was suspended in ethyl acetate and saturated aqueous sodium bicarbonate solution was added and the mixture was stirred vigorously for 10 minutes. The aqueous layer was removed. The organic layer was washed with half-saturated aqueous sodium bicarbonate, then with brine, dried over sodium sulfate, filtered with dichloromethane rinse, and concentrated. Dried under high vacuum to give 25.4 mg (60%) green film. B. Synthesis of NSVB Polymer

[0298] N-succinimidyl p-vinyl benzoate (NSVB) (1.00 g, 4.08 mmol, see J. Poly. Sci. A, 2007, 45, 5618 for synthesis) was added to a 25 mL round bottomed flask (RBF) and dissolved in DMF (2.5 mL). 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid 3 -azido- 1 -propanol ester CTA (317.5 μL of a 103.54 μmol / g solution in dioxane) was added, followed by AIBN (114 μL of 16.9 μmol / g dioxane solution), and mesitylene (200 uL) as standard. The flask was septum sealed, evacuated 30 seconds, then argon flushed. The evacuation / argon flush sequence was repeated two times. Argon was sparged through the reaction mixture with needle outlet vent in the septum for 8 minutes. The vent was removed, and the reaction was stirred under low-flow argon. The flask was added to a pre-heated oil bath at 70 °C and heated for 18 h. The flask was cooled, stir bar removed, and DMF was removed by rotary evaporation to give a yellow oil. The oil was diluted in dichloromethane (3 mL) and transferred dropwise into methanol (400 mL) stirred in a 500 mL beaker. The precipitate formed was allowed to settle and the majority of methanol was removed by decanting. The remaining precipitate / methanol mixture was filtered thru a 30 mL fine-ground glass filter and dried under vacuum for 1 hour to give a pale yellow, free-flowing granular solid. The solid was dissolved in dichloromethane (15 mL) with mild heating to 40 °C. The clear amber solution was added dropwise to methanol to precipitate the polymer a second time. The solution was allowed to settle, then decanted, filtered, and dried under vacuum as described previously. The solid was further dried under high vacuum to give 0.569 g pale yellow solid. Calculated degree of polymerization based on NMR analysis was 56.

[0299] C. Synthesis of poly(PEGA-co-2-(N-Boc-ethyleneamine) acrylate) copolymer

[0300] Poly(ethylene glycol) methyl ether acrylate (Mn = 480 g / mol, 3.2 g, 6.67 mmol), 2-(N- Boc-ethyleneamine) acrylate (0.7 g, 3.3 mmol), 2-(Dodecylthiocarbonothioylthio)-2- m ethylpropionic acid 3 -azido- 1 -propanol ester (420 mg of a 133.1 μmol / g solution in dioxane), AIBN (225 mg of 28.1 μmol / g dioxane solution), 0.17 g mesitylene, and 8.7 g dioxane were added to a 25 mL round bottomed flask (RBF). The flask was septum sealed and argon was sparged through the reaction mixture with needle outlet vent in the septum for 45 minutes. The flask was added to a pre-heated oil bath at 75 °C and heated for 90 min. Reaction was quenched by opening the flask to the air. The reaction mixture was cooled to room temperature and poured in 500 mL Et2O:hexane=l : l mixture under vigorous stirring. The liquid polymer was allowed to settle at 4 °C for 2 hours and then the Et2O:hexane was removed by decanting. The polymer was then dried at 60 °C for 4 hours under vacuum. The dried polymer (very viscous yellow liquid) was dissolved in 12 ml of DI water and transferred to a 3.5 kDa cut-off dialysis membrane and was dialyzed against methanol for 24 hours; methanol was exchanged 3 times during dialysis. After dialysis, the polymer solution was passed through a 0.2 um PTFE syringe filter and methanol was removed on the rotary evaporator. The polymer was dried at 60 °C for 24 hours under vacuum to give 1.67 g of very viscous yellow liquid. Calculated degree of polymerization based on NMR analysis was 54.

[0301] D. Synthesis of PFPA Polymer

[0302] Pentafluorophenyl acrylate (PFPA) (800 mg, 3.36 mmol) was added to a 25 mL schlenk flask and dissolved in dioxane (2.7 mL). 2-(Dodecylthiocarbonothioylthio)-2-methylpropionic acid 3 -azido- 1 -propanol ester CTA (122.1 μL of a 123.89 μmol / g solution in dioxane) was added, followed by AIBN (41.4 μL of 18.26 μmol / g dioxane solution), and mesitylene (50 uL) as NMR internal standard. The flask was sealed and Argon was sparged through the reaction mixture with needle outlet vent in the septum for 8 minutes. The vent was removed, and the flask was sealed added to a pre-heated oil bath at 80 °C and heated for 3 h under stirring. The flask was cooled, stir bar removed, and transferred dropwise into methanol (500 mL) stirred in a 1000 mL beaker. The precipitate formed was allowed to settle and the majority of methanol was removed by decanting. The remaining precipitate / methanol mixture was filtered thru a 30 mL fine-ground glass filter and dried under vacuum for 1 hour to give a white powder. The solid was further dried under high vacuum to give 0.629 g white solid. Calculated degree of polymerization based on NMR analysis was 162.

[0303] E. Main-chain dye-labeling of NSVB Polymer

[0304] The amine monomer dye was prepared as a solution in tetrahydrofuran (0.97 mg / 200 μL). Flame dried 4 mL amber vials and cooled under high vacuum then argon flushed. Dye portions added to each vial (80 μL = 4 equiv., 120 μL = 6 equiv., 160 μL = 6 equiv., 200 μL = 10 equiv.). THF removed with argon flow. The NSVB polymer was prepared as a solution in DMF (2.0 mg / 200 μL) and 200 μL was added to each reaction vial. The vials were capped and stirred at room temperature for 24 h. Each reaction was concentrated and redissolved in 1 : 1 toluene / dimethylformamide (200 μL). Unreacted excess dye was removed by size-exclusion chromatography on SX-1 resin using a 1 : 1 toluene / DMF solvent system. Samples were analyzed for photophysical properties (Table 5).

[0305] Table 5: Photophysical characterization data for NSVB Polymer labeled with variable equivalencies of dye. The measurements were performed in dimethylformamide at room temperature.

[0306] F. Further post-polymerization modification of dye-labeled foldamer

[0307] Dye-labeled NSVB polymer (0.012 μmol), dodecylamine (variable range of equivalencies), and PEG8-NH2(variable equivalencies), all prepared as solutions in DMF, were added together to a 1.5 mL Eppendorf tube. Total volume adjusted to 0.2 mM in 1 : 1 toluene / DMF per reaction. Reactions covered in foil and set on orbital shaker at 90 rot / min. After 16 h, toluene was removed and each sample was dissolved in PBS (pH 7.2) to a final concentration of 100 uM. Samples were analyzed for photophysical properties.

[0308] Figure 9 shows the emission data for the dye-labeled NSVB polymer samples following reaction with hydrophilic ( PEG8-NH2) and hydrophobic (dodecylamine) groups. The ratios referenced are the relative equivalencies of hydrophilic to hydrophobic group used in the reaction. The emission intensity is clearly correlated in this case with increasing hydrophilic content in the PEGylated NSVB polymer.

[0309] G. Post-polymerization modification of PFPA Polymer

[0310] In a flame-dried, vacuum cooled 4 mL reaction vial, P133 polymer (0.035 μmol, 1.97 mg) and 660D dye (0.070 μmol, 0.068 mg), both prepared as stock solutions in anhydrous THF, were added. The vial was capped and sealed tightly. The reaction mixture was stirred and heated to 50 °C. After 2 h, dodecylamine (0.875 μmol, 0.16 mg), as a stock solution in THF, was added. The vial was sealed and the reaction was heated at 50 °C for an additional 2 h.

[0311] Jeffamine M-1000 (8.75 μmol, 8.75 mg), as a stock solution in THF, was then added. The vial was sealed and the reaction was heated at 50 °C for an additional 16 h. The solvent was removed by evaporation. The residue was dissolved in toluene and purified by size exclusion chromatography (1 x 20 cm column, Biorad S-Xl resin, toluene mobile phase). The sample was dried under vacuum to give 4.7 mg (59%) dark red viscous oil.

[0312] Table 6. Photophysical characterization data for PFPA polymer-based Foldamers with increasing amounts of dye used. The number of equivalents of dye used is specified in the foldamer name.

[0313] Terminus / end group dye-labeling

[0314] The poly(PEGA-co-2-(N-Boc-ethyleneamine) acrylate) copolymer (24.0 mg, 1.13 μmol) was added to a 5 mL RBF with a small stir bar. The flask was septum sealed, evacuated and argon flushed three times. The polymer was diluted in DMF (anhydrous, degassed, 280.4 μL, 4 mM). The system was broken briefly and hydrazine hydrate (HH, 80% w / v, 0.17 μL, 2.82 μmol), prepared as a stock solution in DMF, was added. The flask was then capped and stirred at room temperature under argon flow for 10 minutes. The stir bar was removed, and the reaction mixture was concentrated. The stir bar was then re-added, and the flask was capped and placed under high vacuum.

[0315] After 10 minutes, the system was argon flushed and dye (3.07 mg, 2.82 μmol, 2.5 equiv) was added in bulk. The system was septum sealed, evacuated and argon flushed 3X. DMF (1121.5 μL) was added and stirring was begun. The system was broken briefly and trimethyl phosphite (0.66 uL, 1.23 μmol) was added directly into solution. Triethylamine (0.17 μL, 1.23 μmol), prepared as a stock solution in DMF, was then added directly into the reaction solution. The flask was septum sealed, shielded from light with foil, and stirred under argon atmosphere at room temperature.

[0316] After 16 h, the stir bar was removed with toluene rinse and the reaction was concentrated. The residue was re-dissolved in toluene and concentrated a second time. The residue was then placed under high vacuum. The residue was then dissolved in toluene (200 μL) and transferred to a 1.5 mL low adhesion Eppendorf tube with additional rinsing of the reaction flask (100 μL). The tube was spun at 12 KG for 5 min. The supernatant was carefully removed and submitted to SEC using SX-1 media (2 x 20 cm column, toluene solvent). The desired dye-labeled product was isolated as 21.7 mg (87%) dark film.

[0317] EXAMPLE 3: Dual Use Methods

[0318] The following is a list of proposed dual-use combinations. Suggested molecules to be employed for these roles, designated as Pl, P2, and / or T1 per the synthesis Scheme 2, are included in parentheses and may be found in Example 2 above. Abbreviations include: MRI (magnetic resonance imaging); PAI (photoacoustic imaging); PDT / PIT (photodynamic therapy / photoimmunotherapy); FGS (fluorescence guided surgery); and FC (flow cytometry). These are exemplary embodiments and other dyes and combinations are possible.

[0319] 1) MRI and PAI (e.g., Pl = manganese porphryin, P2 = Copper bacteriochlorin, T1 = n / a) Purpose: MRI provides volume detection, whereas PAI performs edge detection. MRI serves to provide rapid screen of for general location of tumors that can be supplemented by more sensitive detection using PAI.

[0320] 2) MRI and PDT / PIT (e.g., Pl = Manganese porphyrin, P2 = Copper bacteriochlorin, T1:targeting peptide)

[0321] Purpose: MRI serves to provide rapid screen of for general location of tumors that can be followed by localized PDT.

[0322] 3) FGS and PDT / PIT (e.g,, Pl AlexaFluor™ 790, P2 Platinum bacteriochlorin, T1 targeting peptide)

[0323] Purpose: A targeting peptide localizes the reagent to the site of cancer cells which can be visualized using FGS probe. Then the PDT cargo can be photoactivated at the site of the cancer cells. 4) FC and PAI (e.g„ Pl = porphyrin dyad, P2 = Copper bacteriochlorin, T1 = none or targeting peptide)

[0324] Purpose: Cells are first phenotyped and sorted using FC, then reintroduced into an organism and imaged for specific localization using PAI.

[0325] 5) PAI and PDT / PIT (e.g., Pl = Copper bacteriochlorin, abs 750 nm, P2 Platinum bacteriochlorin, abs > 800 nm, T1 = targeting peptide)

[0326] Purpose: Leverage depth of penetration of PAI for imaging modality to identify diseased tissue then initiate cell killing through light activation of PDT reagent.

[0327] EXAMPLE 4: Dendron Linker Design for Attaching Multiple Moieties To Terminal End of Polymer Backbone

[0328] Synthesis of Genl Dendron Linker. The following example dendron was synthesized using a modified Fmoc solid-phase peptide synthesis procedure, as described below.

[0329] Low-loading Rink Amide resin was used as the solid phase, the amino acids Fmoc-Gly- OH and Fmoc-Lys(alloc)-OH were used as amino acid building blocks, and N,N-bis(N'-Fmoc- 3-aminopropyl)-glycine potassium hemisulfate (APG) was used as the branching amino acid. A fixed Gly-Lys(alloc)-Gly sequence was synthesized for use as the beginning of the dendron for further modification. Between each of the branching amino acids, a glycine unit was added to increase the flexibility and to improve the yield. As the starting point of each of coupling cycle, the Fmoc group on the solid-phase resin was deprotected in the presence 20% piperidine / DMF for 15 minutes, followed by 6 DMF washes. For the addition of the amino acids, the couplings were carried out in the presence of 4.0 equivalents amino acid, 4.0 equivalents HBTU and 8 equivalents diisopropyl ethyl amine (DIPEA) for 40 min, followed by 3.0 DMF washes. For the branching amino acids, the coupling was carried out overnight in the presence of 3.0 equivalents branching amino acid, 3.0 equivalents HBTU and 6.0 equivalents DIPEA, followed by 3 DMF washes. After each coupling, a Kaiser test was performed to verify the completion of coupling. After the completion of the synthesis, the alloc group was removed by washing with 0.1 equivalents tetrakis(triphenylphosphine)palladium(0) and 20.0 equivalents phenylsilane in CH2CI26 times (2 mL; 30 min each round). The resulting resin was sequentially washed with 0.5% DIPEA i inn DMF, 0.5% sodium diethyldithiocarbamate in DMF and 50% CH2CI2in DMF. Then the resin was coupled in the presence of 4.0 equivalents 6-maleimidohexanoic acid, 4.0 equivalents HBTU and 8.0 equivalents DIPEA overnight, followed by 3 DMF washes. After a final deprotection, the dendron was cleaved in 2 mL trifluoroacetic acid (TFA). After 3 h, the cleavage solution was collected and the resin was washed twice with neat TFA. The TFA was evaporated under air flow and the residual solution was precipitated using cold diethyl ether. The resulting precipitate was collected by centrifugation at 5000 rpm for 5 min and washed three times with cold diethyl ether.

[0330] Gen2-Dendron-660DD - The synthesized Gen2-dendron (2.6 mg, 2 μmol) and 660DD-NHS (9.9 mg, 9.6 μmol) were added to a 10 mL round bottom flask (RBF) with a stir bar, followed by the addition of 1 mL DMF. The flask was sealed with a rubber septum, evacuated and then flushed with argon. 5% of DIPEA (50 μL) was added rapidly. The reaction mixture was stirred at room temperature under argon atmosphere and shielded from light for 16 hours. The solvent was then removed by rotary evaporation. The resulting residue was then purified by column chromatography using 0 - 100% MeOH in CH2CI2. 3.2 mg (34%) of the dendron product were isolated with a net conjugate rate of 2.5 dyad per dendron.

[0331]

[0332] Synthesis of Fluorescent Foldamer molecule from Dendron-Dyad

[0333] Gen2-Dendron-660DD-P57. P57 foldamer (5.5 mg, 0.22 μmol) was added to a 5 mL RBF with a small stir bar. The flask was sealed with a septum, evacuated and flushed with argon 3 times. The foldamer was diluted in DMF (anhydrous, degassed, 54 μL). The system was broken briefly and a hydrazine solution (2.0 μL stock solution in DMF, 0.54 μmol, 2.5 equiv.) was added into the reaction solution via pipet. The flask was then capped and stirred at room temperature under argon flow. After 10 min., the stir bar was removed, and the reaction mixture was concentrated. The stir bar was then re-added, and the flask was capped and placed under high vacuum. After 1 h under vacuum, the system was argon flushed and 660D-BFL dye (3.2 mg, 0.89 μmol, 4.0 equiv) was added in bulk. The system was sealed with a septum, evacuated and argon flushed 3X. DMF (217 μL) was added and stirring begun. The system was broken briefly and trimethyl phosphite (0.13 μL, 1.1 μmol, 5.0 equiv.) was added directly into the solution via micropipette. Triethylamine (2.0 μL stock solution in DMF, 0.24 μmol, 1.1 equiv.) was then added directly. The flask was sealed with a septum, shielded from light, and stirred under low flow argon at room temperature. After 16 h, the stir bar was removed and washed with toluene and the reaction was concentrated. The residue was re-dissolved in toluene and concentrated a second time. The residue was then placed under high vacuum for 1 h. The residue was then dissolved in toluene (250 μL) and transferred to a 1.5 mL low adhesion microcentrifuge tube with additional rinsing of the reaction flask with toluene (125 μL). The tube was spun at 12 KG for 5 min. The supernatant was decanted into a second tube. The pellet was rinsed carefully with toluene (2 x 50 μL) and rinses were transferred to the second tube. This tube was spun at 12 KG for 5 min. The supernatant was removed and submitted to size exclusion chromatography using SX-1 media and eluting with toluene (1 x 20 cm column, flow rate 1 mL / min). Product fractions were concentrated and dried under high vacuum. Isolated 5.7 mg (91%), the conjugation rate of Dendron-dyad is 20%. Samples were analyzed for photophysical properties (Table 7).

[0334] Table 7. Photophysical characterization data for dyes, multi-functional linkers, and

[0335] Foldam ers

[0336] EXAMPLE 5: Conjugatable Bifunctional Linker Design for Attaching Multiple Moieties To Terminal End of Polymer Backbone

[0337] A. Synthesis of Linker Molecule Terminally Substituted with Two of the Same Moieties

[0338] 660M bifunctional linker (660M-BFL-Mal). N-Mal- N-bis(PEG4-amine) TFA salt linker (7.7 mg, 9.22 μmol) added with 660M-NHS dye (14.4 mg, 18.45 μmol) to RBF with stir bar. Flask septum sealed, evacuated, and flushed with argon. DMF (0.92 mL) was added, followed quickly by addition of triethylamine (5.1 μL, 36.88 μmol). Reaction mix was stirred at room temperature under argon atmosphere and shielded from light. After 16 h, the reaction mixture was diluted with ethyl acetate and washed with water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography using 0 - 10% MeOH in DCM. Isolated 10.7 mg (60%) desired product. [M + H]+= 1939.1, calc. 1937.9. 660D Bifunctional linker. N-Mal-N-bis(PEG4-amine) TFA salt linker (4.5 mg, 5.39 μmol) was added with 660D-NHS (11.7 mg, 11.32 μmol) to RBF with stir bar. Flask septum sealed, evacuated and flushed with argon. DMF (0.57 mL) was added, followed quickly by addition of tri ethylamine (3.0 μL, 21.56 μmol). Reaction mix was stirred at room temperature under argon atmosphere and shielded from light. After 16 h, the reaction mixture was diluted with ethyl acetate and washed with water. The organic layer was removed, concentrated, and the residue was redissolved in dichloromethane. The organic layer was washed brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography using 0 - 10% MeOH in DCM. Isolated 3.5 mg (27%) desired product.

[0339] Synthesis of Foldamer fluorescent molecule substituted with two of the same molecules from bifunctional linkers

[0340] 660M-BFL-P57. P57 foldamer (15.8 mg, 0.62 μmol) was added to 5 mL RBF with a small stirbar. The flask was septum sealed, evacuated and argon flushed 3X. Foldamer diluted in DMF (anhydrous, degassed, 156 μL). System broken briefly and hydrazine solution (2.0 μL stock solution in DMF, 0.10 μL, 1.55 μmol, 2.5 equiv.) was added into reaction solution via pipet. The flask was then capped and stirred at room temperature under argon flow. After 10 min., the stir bar was removed, and the reaction mixture was concentrated. The stir bar was then re-added, and the flask was capped and placed under high vacuum. After 10 min. under vacuum, the system was argon flushed and 660M-BFL dye (4.82 mg, 2.49 μmol, 4.0 equiv) was added in bulk. The system was septum sealed, evacuated and Ar flushed 3X. DMF (622 μL) was added and stirring begun. The system was broken briefly and trimethyl phosphite (0.37 μL, 3.11 μmol, 5.0 equiv.) was added directly into solution via micropipette. Triethylamine (2.0 μL stock solution in DMF, 0.10 μL, 0.68 μmol, 1.1 equiv.) was then added directly. The flask was septum sealed, shielded from light, and stirred under low flow argon at room temperature. After 16 h, the stir bar was removed with toluene rinse and the reaction was concentrated. The residue was re-dissolved in toluene and concentrated a second time. The residue was then placed under high vacuum for 1 h. The residue was then dissolved in toluene (250 uL) and transferred to a 1.5 mL low adhesion microcentrifuge tube with additional rinsing of the reaction flask with toluene (125 uL). The tube was spun at 12 KG for 5 min. The supernatant was decanted into a second tube. The pellet was rinsed carefully with toluene (2 x 50 uL) and rinses were transferred to the second tube. This tube was spun at 12 KG for 5 min. The supernatant was removed and submitted to size exclusion chromatography using SX-1 media and eluting with toluene (1 x 20 cm column, flow rate 1 mL / min). Product fractions were concentrated and dried under high vacuum. Isolated 16.8 mg (99%) film.

[0341] 660D-BFL-P57. P57 foldamer (11.3 mg, 0.45 μmol) was added to 5 mL RBF with a small stirbar. The flask was septum sealed, evacuated and argon flushed 3X. Foldamer diluted in DMF (anhydrous, degassed, 111 μL). System broken briefly and hydrazine solution (2.0 μL stock solution in DMF, 1.11 μmol, 2.5 equiv.) was added into reaction solution via pipet. The flask was then capped and stirred at room temperature under argon flow. After 10 min., the stir bar was removed, and the reaction mixture was concentrated. The stir bar was then re-added, and the flask was capped and placed under high vacuum. After 1 h under vacuum, the system was argon flushed and 660D-BFL dye (3.3 mg, 1.35 μmol, 4.0 equiv) was added in bulk. The system was septum sealed, evacuated and Ar flushed 3X. DMF (445 μL) was added and stirring begun. The system was broken briefly and trimethyl phosphite (0.26 μL, 2.23 μmol, 5.0 equiv.) was added directly into solution via micropipette. Triethylamine (2.0 μL stock solution in DMF, 0.49 μmol, 1.1 equiv.) was then added directly. The flask was septum sealed, shielded from light, and stirred under low flow argon at room temperature. After 16 h, the stir bar was removed with toluene rinse and the reaction was concentrated. The residue was re-dissolved in toluene and concentrated a second time. The residue was then placed under high vacuum for 1 h. The residue was then dissolved in toluene (250 μL) and transferred to a 1.5 mL low adhesion microcentrifuge tube with additional rinsing of the reaction flask with toluene (125 μL). The tube was spun at 12 KG for 5 min. The supernatant was decanted into a second tube. The pellet was rinsed carefully with toluene (2 x 50 μL) and rinses were transferred to the second tube. This tube was spun at 12 KG for 5 min. The supernatant was removed and submitted to size exclusion chromatography using SX-1 media and eluting with toluene (1 x 20 cm column, flow rate 1 mL / min). Product fractions were concentrated and dried under high vacuum. Isolated 11.3 mg (92%) film. Samples were analyzed for photophysical properties in Table 8.

[0342] Table 8. Photophysical characterization data for dyes, multi-functional linkers, and

[0343] Fluorescent foldamers

[0344]

[0345] Antibody / Fluorescent Foldamer Conjugation

[0346] Prepare fresh sodium bicarbonate buffer (100 mM, pH 8.2) by diluting 336 mg in 40 mL ultrapure DI and adjust the pH. Dilute 0.5 mg BCN-PEG12-NHS linker in 100 mL bicarb. Add 0.3 mg (14 mL) UCHT-4 mouse anti human CD8 antibody to low-biding Eppendorf tubes for reactions and dilute with 122 mL bicarb buffer. Add BCN linker to reach 40 / 80 equivalents towards antibody. Allow linker to react with Ab at room temperature for at least 1 h. Pre-rinse 30Kd MWCO filters with PBS, pH 7.2 and transfer reactions to MWCO filters with 200 mL PBS rinse. Spin at 12 Kg x 5 min. Perform additional rinses with PBS (add 400 mL, spin at 12 Kg x 5 min, 4-5 wash cycles total). Dissolve 1 mg P57-BFL-660D (20 equivalents towards antibody) in 50 mL PBS (pH 7.2). Transfer the retention Ab-BCN reaction mixture from MWCO tubes. Allow to react for at least 4 h. Add 1% NaN3 in PBS to each tube to quench reaction sites at room temp for 30 min. Perform ion exchange chromatography using HiTrap ANX FF(HS) 1 mL media Buffers: Loading / wash buffer: 25 mM sodium bicarbonate; Elution buffer: 1 : 1 25 mM bicarb / 1 M NaCl. Transfer sample to 30 Kd MWCO UFD with 300 μL rinse using IEX loading buffer. Spin at 12Kg x 5 min. Wash ANX IEX column with loading buffer (10 mL), Dilute sample to 500 mL in loading buffer and add to column. Elute off excess unconjugated foldamer with 5 mL loading buffer. Elute conjugation with 5 mL elution buffer. Concentrate reagent in 30 Kd MWCO UFDs (12 Kg x 5 min). Dilute retentate with PBS (final vol. approx. 400 mL) and spin down again. Dilute final retentate to 50 mL.

[0347] Table 9. Photophysical characterization data for Antibody Fluorescent Foldamer conjugations

[0348]

[0349] Flow Cytometry Cell Staining Sample Preparation

[0350] PBMC cells were obtained from ZenBio, cell was thawed in in water bath at 37 C for

[0351] 2 mins. The thawed cells were added to 50 ml conical tube, and 1 ml pre-warmed lymphocyte medium was used to rinse the vial, then 14 ml more thaw buffer was added to the conical tube.

[0352] The cell suspension was gently mixed by ending over and the suspension was transferred to 16

[0353] Eppendorf tubes and centrifuge at 400 g for 10 mins. The suspension is carefully removed without disturbing the pellet. The cell was resuspended in 1 mL cell staining medium (Bio legend) by gently pipetting and centrifuge at 400 g for 10 mins. The suspension is carefully removed without disturbing the pellet. The staining solution was made by directly dilute antibody -FP stock solution into cell staining medium at the desired concentration (40 mg / mL, or 100 mg / mL) at total volume of 100 uL. Cell pellet is directly resuspended into 100 μL of cell staining medium with gently pipetting, then the antibody-FP containing staining solution was added and gently mixed together. The cell suspension was allowed to stain on ice in dark for

[0354] 20 minutes. After staining, the cells were spined down at 600 g for 10 minutes. Each sample was washed with 1 ml clean cell staining medium and spined down at 600 g for 10 minutes.

[0355] Then cells were resuspended in 250 μL of cell staining buffer, then 250 μL of 1% formaldehyde was added to fix the cell. After 15 min of fixation, the sample was shortly vortexed to resuspend the cell and filtered through 70 mm cell strainer for test. Flow cytometry was tested on Becton Dickinson LSRFortessa, analyzed data is shown in Figure 10A.

[0356] Discussion

[0357] The described bifunctional linker (BFL) approach, see, e.g., Table 7, may provide fewer total number of dyes that can be added to the polymer via the linker relative to a dendron (2 vs. 4) but it exists as a smaller overall molecule. The BFL-dye molecules did in fact conjugate much more efficiently with the polymer than dendron versions, with 78% and 89% as two examples vs. 20% for the dendron Gen2. Using the same polymer backbone (P57) and the same dye (660DD), brightness increased from 16,853 for the dendron to 57,311 using the BFL approach. The BFL-based foldamers were also shown to more readily undergo antibody conjugation, giving conjugates with Foldamer / Ab ratios in the target 3-4 range.

[0358] The PFPA based polymer described herein can provide the opportunity to add more dyes per particle than either the dendron or BFL approaches. The efficiency of conjugation was high (93% or higher) across a range of dyes from 2 to 16 equivalents (see Table 8), because there are now far more available reaction sites per particle. The number of dyes per particle was calculated based on a comparison of the extinction coefficient of the dye vs. that of the foldamer material isolated after purification. The calculated values matched closely to the number of equivalents of dye used per reaction, within an expected range of error generally associated with the measurement of extinction coefficients. A PFP-generated probe bearing 2 dye molecules had a measured brightness of 76,715, which may be compared to the aforementioned BFL foldamer, also prepared with 2 of the same dye molecules, that had a brightness of 57,311. Brightness was shown to increase across the range of dye equivalencies tested. Advantageously, quantum yields of the multiple-dye containing foldamers were largely maintained in PBS solvent, even at higher dye loading. This indicates that the dyes are sufficiently shielded from the effects of both solvent and dye-dye quenching.

[0359] EXAMPLE 6. Synthesis of Polymer with Different Dyes on Terminal End and Backbone

[0360] An example approach to a two dye foldamer synthesis with one type of dye on the terminal end of the polymer and a different type of dye on the polymer backbone is described in this example. An exemplary compound synthesized according to this approach is depicted below. One of skill in the art could utilize the description provided herein to append different dyes on the backbone and terminal end to achieve variations of the described composition. An example foldamer with porphyrin-chlorin dyad attached to the terminal end and two porphyrin- bacteriochlorin dyads attached to the polymer backbone is depicted below:

[0361] As depicted in the foldamer above, gradient and random structures can be synthesized in the foldamer. For example, gradient structure can result from copolymerization of two monomers chosen with significantly different copolymerization reactivity ratios. Typically, a copolymerization of a methacrylate-based (e.g., polyethylene glycol methacrylate, PEGMA) monomer and an acrylate-based monomer (e.g., cyclododecyl acrylate, CD A) results in a non- uniform monomer distribution. In such case, it is expected that PEGMA repeat units are more concentrated close to the bioconjugation terminus of the polymer and CDA is more concentrated close to the opposite terminus of the polymer.

[0362] A random structure can be produced either through copolymerization of two monomers with comparable reactivity ratios (e.g., two or more different acrylate-based monomers) or through polymerization of a single type of functional monomer (e.g., pentafluorophenyl acrylate, PFPA) and further post-polymerization modification in order to introduce the desired functional groups randomly.

[0363] A polymer with two distinct segments can be prepared using a semi-batch approach, where the initial monomer feedstock consists of one or two species. The reaction initially proceeds utilizing the initial components, and additional, different monomers can be charged into the reaction flask after a certain amount of time. This process achieves separate segments with varying compositions within a single reaction vessel. As opposed to block copolymers, boundaries between segments exhibit a gradual transition due to the one-pot reaction approach. In the current example, the first segment contains either PEGMA units or PEGMA and CD A units in the form of a gradient structure. The degree of polymerization (number of repeat units) for this segment can range from 20 to 80. The second segment consists of any PEGMA and CD A not consumed during the first stage as well as PFPA introduced at the start of the second stage. Typical degree of polymerization of the second segment can range from 60 to 240 monomer units.

[0364] A. Post polymerization modification of polymer terminus by chain extension

[0365] In an example procedure, a dry 10 mL Schlenk flask was charged with polymer (102.15 mg, 0.002 mmol of P187), monomer (2-Acrylamido-2-methylpropane sulfonic acid (AMPS, 10 mg, 0.05 mmol) or 2-(methylsulfinyl)ethyl methacrylate (MSEA, 7 mg, 0.05 mmol) or 2- methacryloyloxyethyl phosphorylcholine (MPC, 14 mg, 0.05 mmol)), mesitylene (0.05 mL) and anhydrous DMSO (0.5 mL). The solution was sparged with argon for 10 minutes. The flask was sealed and then placed in the blue light photoreactor. The first kinetic aliquot was taken using an air-free syringe after starting the reaction upon exposure to blue light. After 3 hours, a kinetic sample was taken and the reaction was quenched by exposure to air. The polymer was purified by precipitation into a 1 : 1 mixture of diethyl ether and hexane once. The modified polymer was isolated as a film under vacuum. The final composition was confirmed by1H NMR.

[0366] B. Hanger strategy to conjugate porphyrin-bacteriochlorin dyad on foldamer backbone.

[0367] Pl 87 polymer (6.4 mg, 0.15 μmol) was dissolved in 150 μL of anhydrous THF, and 743D-NH2 (0.75 mg, 0.75 μmol, 5 equivalent) was dissolved in 150 μL of anhydrous THF. The two solutions were mixed in a dried 4 mL vial. N,N-Diisopropylethylamine (10 μL of 5% v / v in THF) was then added to the solution. The vial was tightly capped and stirred at 50 °C on an aluminum pie block. After 2 h, the reaction was taken off the block to cool down to room temperature, cyclododecylamine (0.66 mg, 3.6 μmol, 24 equivalent) was dissolved in 25 μL of anhydrous THF and added to the solution. Then the reaction was put back on the pie plate and continued stirring at 50 °C for 2 h. Jeffamine M-1000 (26 mg, 26 μM, 200 equivalent) was dissolved in 75 μL of anhydrous THF. The reaction was allowed to cool down again and the Jeffamine solution was added. After tightly capping the reaction vial, the reaction continued stirring at 50 °C for 16 h. The reaction mixture was purified through a 1 x 20 cm S-Xl column in toluene. Product fractions were concentrated and 9.2 mg (72%) was isolated. c. Conjugate porphyrin-chlorin dyad on foldamer terminus

[0368] The modified Pl 87 product of the first step (9.2 mg, 0.11 μM) was dissolved in 100 mL of anhydrous DMF and added to a 5 mL RBF. The flask was septum sealed, evacuated and argon flushed 3X. The system was broken briefly and hydrazine solution (2.0 μL stock solution in DMF, 0.013 uL, 0.27 μmol, 2.5 equivalent) was added into the reaction solution via pipet. The flask was then capped and stirred at room temperature under argon flow. After 10 min., the reaction mixture was concentrated via rotary evaporation. Then 660DD-MAL dye (0.43 mg, 0.43 μmol, 4.0 equivalent) was added to the flask and the flask was septum sealed, evacuated, and Ar flushed 3X. DMF (200 μL) was added and stirring was begun. The system was broken briefly and trimethyl phosphite (0.63 mL stock solution in DMF, 0.063 μL, 0.067 μmol, 5.0 equivalent) was added directly into solution via micropipette. Triethylamine (2.0 μL stock solution in DMF, 0.012 μL, 0.016 μmol, 1.1 equivalent) was then added directly. The flask was septum sealed, shielded from light, and stirred under low flow argon at room temperature. After 16 h, the reaction was concentrated. The reaction mixture was purified using a 1 x 20 cm S-Xl column and eluting with toluene. Product fractions were concentrated and dried under high vacuum. Isolated 8.5 mg (92%) film. Samples were analyzed for photophysical properties. Each FP was determined to contain four 743DD dyes and one 660DD dye. Absorbance and emission spectra are shown in Figures 11A and 11B.

[0369] D. Antibody / Foldamer Conjugation

[0370] UCHT-4 mouse anti human CDS antibody (0.2 mg) was added to a low-binding Eppendorf tube and diluted with PBS (29.2 μL, pH 7.2). In a separate vessel, DBCO-PEG12- NHS linker (0.5 mg) was diluted in anhydrous DMSO (50 μL). A portion of the linker solution (1.34 μL) was then added to the antibody solution to achieve a 10: 1 ratio of linker to antibody and allowed to react for 1 h at room temperature. Next, the reaction mixture was centrifuged (12 Kg x 5 min) using a 30 kDa MWCO filter (prerinsed with PBS; pH adjusted to 7.2). Additional rinses (4-5) were performed using 400 μL PBS solution (spun at 12 Kg x 5 min). In a separate Eppendorf tube, duo label FP (2.3 mg, 20 equivalents toward antibody) was diluted with 50 μL PBS (pH 7.2). The antibody-linker mixture was then transferred to this FP solution and mixed via shaker at room temperature overnight. A 1% solution of NaN3 in PBS was then added to each tube. After 30 minutes, ion exchange chromatography (1 mL HiTrap ANX FF(HS)) was performed with the following buffers: 25 mM sodium bicarbonate (loading / wash buffer), 1 : 1 25 mM sodium bicarbonate / 1 M NaCl (elution buffer). The sample was then transferred to a 30 kDa MWCO filter tube. Centrifugation was performed (spun at 12 Kg x 5 min) with IEX loading buffer. An ANX column was then washed with fresh loading buffer (10 mL). The IEX loading buffer containing the sample was diluted to 400 μL with additional loading buffer and added to the column. Excess unconjugated foldamer was eluted with 5 mL loading buffer and the conjugated foldamer was eluted with an additional 5 mL loading buffer. The conjugated foldamer was then centrifuged (12 Kg x 5 min) in a 30 kDa MWCO filter tube. The retentate was diluted to 400 μL with PBS and centrifuged again. The final retentate was diluted to 50 μL with PBS. Analysis of photophysical properties of the foldamer-antibody conjugate (Figures 11C and 11D) showed that 6.5 foldamer was conjugated to each antibody.

[0371] Example foldamers with multiple dyes on the backbone were also analyzed by flow cytometry on bang beads (Figure 10B); and with multiple dyes on the backbone on CDS monoclonal antibody staining PBMCs (Figure 10C).

[0372] EXAMPLE 7. Synthesis of Polymer with Two Different Dyes attached to the Backbone

[0373] An example approach to a two dye foldamer synthesis with two different types of dye on the polymer backbone is described in this example. An exemplary compound synthesized according to this approach is depicted below. One of skill in the art could utilize the description provided herein to append different dyes on the backbone to achieve variations of the composition described herein. An example foldamer chemical structure with two different dyes (porphyrin-bacteriochlorin dyads and porphyrin-chlorin dyads on the polymer backbone is depicted below:

[0374]

[0375] Synthesis of an example foldamer with two different dyes (porphyrin-bacteriochlorin dyads and porphyrin-chlorin dyads) on the polymer backbone was conducted. A PFP containing polymer was dissolved in anhydrous THF. 743D-NH2 (2 equivalent) and 660D- NH2 (1 equivalent) were dissolved in anhydrous THF and the two solutions were mixed in a dried 4 mL vial. N,N-Diisopropylethylamine (15 equivalent of 5% v / v in THF) was added to the solution. The vial was capped tightly and stirred under 50 °C. After 2 hours, the reaction was taken off the plate to cool down to room temperature. The amount of cyclododecylamine was calculated to produce a side chain with a 7% (by weight) hydrophobic composition. The calculated amount of cyclododecylamine was dissolved in anhydrous THF and added to the solution. The reaction was allowed to continue stirring at 50 °C for 2 h. Jeffamine M-1000 (two-fold equivalents towards the PFP on polymer side chain) was dissolved in anhydrous THF. The reaction was allowed to cool down again and the Jeffamine solution was added in. After tightly capping the reaction vial, the reaction was continued stirring at 50 °C for 16 h. The reaction mixture was purified using a 1 x 20 cm S-Xl column and eluting with toluene. Product fractions were dried on rotary evaporator.

[0376] Example 8. Multi-Dye Foldamer Design Optimization

[0377] Design parameters. Multiple design parameters have been explored to optimize dye brightness on a foldamer in PBS. Several of these have been found to be important, including: 1) the overall length of the polymer, 2) the number of dyes per polymer chain, 3) the spacing of dyes along the chain, 4) the placement of hydrophobic and hydrophilic areas of enrichment within the polymer, and 5) the relative percentage of hydrophobic components. These parameters were optimized across examples in Table 9. Data analysis methods. For each new multi-dye foldamer (FP) synthesized, photophysical data, including absorbance maxima, emission maximum, extinction coefficient (ε), and quantum yield (QY), was collected in both an organic solvent (toluene, THF, or DMF) and an aqueous solvent (PBS). Foldamer design optimization was guided by calculating the percent retention of ε and QY in aqueous solution as compared to values in organic solvent. These variables were interpreted to reflect the ability of the polymer to protect the dyes from fluorescence quenching associated with interaction with solvent and / or other dye molecules.

[0378] Table 9. Multi-Dye Foldamer examples and photophysical properties.

[0379] Composition variables of importance. Data was sorted by specific dye type used to be able to compare brightness values based solely on design features of the polymer rather than the dye itself. Specifically, analysis was performed on FPs containing the NRV660DD dye to allow for comparison.

[0380] The data was sorted by QY retained in PBS and grouped into percent ranges, 0-49%, 50-69%, 70-79%, 80-89%, and 90+%. These groups were analyzed for average polymer length, or degree of polymerization (DP). A general trend of increasing QY retention with increasing average polymer length was noted (Table 10). The data implies that a polymer length of about 180 units or more provides the best QY retention. The range of DP for the exemplary FPs evaluated was 50 - 340.

[0381] Table 10. Average polymer DP of Multi-Dye Foldamers, grouped by QY % retention in PBS vs organic solvent.

[0382]

[0383] General biasing of hydrophobic character in the environment of dye labeling along the polymer backbone as achieved by the gradient synthesis method was also important. This can be seen from the trend towards increasing QY retention when moving from random co-polymer

[0384] P138 to gradient polymers Pl 56 and subsequent versions.

[0385] Example 9. Multi-dye loading

[0386] The present example tests the limit of loading an example dye per polymer in an example polymer. Pl 33 polymer (DP = 235) was loaded with 660M-NH2 dye at 10, 20, 40 and

[0387] 80 equivalents with cyclododecylamine as hydrophobic group (10% of reactive sites) and a balance of Jeffamine M- 1000 (hydrophilic poly ether monamine with molecular weight of about

[0388] 1000).

[0389] Table 11, below, includes results of the dye-loading experiment:

[0390] Table 11.

[0391] Figure 12 shows the quantum yield (QY) (left) and brightness (right) for the number of dyes per compound in the example compound as measured in THF and in PBS. The results show dye loading of a maximum 53 was achieved with 80 equivalents of dye. QY in THF maintains well with increasing dye number, but a drop is seen in PBS. Brightness in PBS reaches a maximum at approximately 30 660M-NH2 dyes per Pl 33 polymer, with brightness dropping with an increase in dyes / polymer beyond 30. Compound made with 80 equivalents of dye required heating to dissolve the sample in PBS.

[0392] Figure 13 shows peripheral blood mononuclear cells stained with multi-dye foldamers attached to CDS monoclonal antibody. The P222 foldamer used included porphyrin-chlorin dyads as well as charged groups with a total DP of less than 200. Stained cells were imaged on Olympus 1X51 Inverted Fluorescence Microscope using violet light source.

[0393] Example 10. Multi-Dye Gradient Foldamers Attached to Antibodies

[0394] Microscopic images of multi-dye foldamer antibody conjugates on polystyrene beads are shown in Figures 14-15. In Figure 14, P220-AMP was started with a DP 51 PEG-CDA gradient, followed by DP 62 PFP. The total DP was 121, with 11 AMP added on the terminal. The foldamer contained 2.6 660 dyad per polymer with a 7% weight percent of total CDA. The compound comprised 6.1 foldamers per antibody. Positive versus negative beads are shown in Figure 14, lower panel. In Figure 15, P220 was started with a DP 51 PEG-CDA gradient, following by DP 62 PFP, The total DP was 121. The foldamer contained 2.4 660 dyad per polymer with a 7% weight percent of total CDA. The compound comprised 1.9 foldamers per antibody. Positive versus negative beads are shown in Figure 15, lower panel.

[0395] Example 11. Confirmation of Charged-Group Incorporation in Foldamer

[0396] The successful incorporation of a charged group, 2-acrylamido-2-methyl-l- propanesulfonic acid (AMPS) monomer, in an example polymer utilizing reversible addition- fragmentation chain transfer (RAFT) chain extension was confirmed through comparative analysis of the relative integration of vinyl proton peaks relative to the internal standard mesitylene. Notable AMPS protons appeared at a chemical shift of 6.05 ppm, while mesitylene's relevant protons were identified at 6.75 ppm. Kinetic samples obtained at the start and the end of the reaction were utilized to calculate the change in relative integration of AMPS vinyl protons over the course of the reaction. The observed decrease of 60% in the relative integration of vinyl protons translates to approximately 60% monomer conversion and consequently the incorporation of approximately 12 units of AMPS on average per polymer terminus. Similar analytical approach has been utilized to confirm AMPS incorporation, as well as other monomers, in the backbone of polymers as detailed herein. Table 12. Exemplary dyes for incorporation into foldamers, alone or in combination.

[0397]

[0398] The following references are related to Table 12:

[0399] 1. Yu, Zhanqian, and Marcin Ptaszek. Near-IR emissive chlorin-bacteriochlorin energy- transfer dyads with a common donor and acceptors with tunable emission wavelength. lhe Journal of organic chemistry 13. 21 (2013): 10678-10691.

[0400] 2. Yu, Z., Pancholi, C., Bhagavathy, G. V., Kang, H. S., Nguyen, J. K., Ptaszek, M. (2014). Strongly conjugated hydroporphyrin dyads: extensive modification of hydroporphyrins’ properties by expanding the conjugated system. The Journal of organic chemistry, 79. 17 (2014): 7910-7925.

[0401] 3. Meares, A., Satraitis, A., Santhanam, N., Yu, Z., Ptaszek, M. Deep-red emissive BODTPY-chlorin arrays excitable with green and red wavelengths. The Journal of organic chemistry, 80. 8 (2015): 3858-3869.

[0402] 4. Kang, H. S., Esemoto, N. N., Diers, J. R., Niedzwiedzki, D. M., Greco, J. A., Akhigbe, J., Yu, Z., Pancholi, C., Bhagavathy, G. V., Nguyen, J. K., Kirmajer, C., Birge, R. R., Ptaszek, M., Bocian, D. F. Effects of strong electronic coupling in chlorin and bacteriochlorin dyads. The Journal of Physical Chemistry A, 120. 3 (2016): 379-395.

[0403] 5. Esemoto, N. N., Yu, Z., Wiratan, L., Satraitis, A., Ptaszek, M. Bacteriochlorin dyads as solvent polarity dependent near-infrared fluorophores and reactive oxygen species photosensitizers. Organic letters 18.18 (2016): 4590-4593.

[0404] 6. Meares, A., Satraitis, A., Akhigbe, J., Santhanam, N., Swaminathan, S., Ehudin, M., Ptaszek, M. Amphiphilic BODIPY-hydroporphyrin energy transfer arrays with broadly tunable absorption and deep red / near-infrared emission in aqueous micelles. The Journal of organic chemistry, 82. 12 (2017): 6054-6070.

[0405] 7. Esemoto, N. N., Satraitis, A., Wiratan, L., Ptaszek, M. Symmetrical and nonsymmetrical meso-meso directly linked hydroporphyrin dyads: synthesis and photochemical properties. Inorganic chemistry, 57. 6 (2017): 2977-2988.

[0406] 8. Meares, A., Satraitis, A., Ptaszek, M. BODIPY-bacteriochlorin energy transfer arrays: toward near-IR emitters with broadly tunable, multiple absorption bands. The Journal of organic chemistry, 82. 24 (2017): 13068-13075.

[0407] 9. Ogata, F., Nagaya, T., Maruoka, Y., Akhigbe, J., Meares, A., Lucero, M.Y., Satraitis,

[0408] A., Fujimura, D., Okada, R., Inagaki, F. and Choyke, P.L., Ptaszek, M., Kobayashi, H. Activatable near-infrared fluorescence imaging using PEGylated bacteriochlorin- based chlorin and. BODIPY-dyads as probes for detecting cancer. Bioconjugate chemistry, 30. 1 (2018): 169-183.

[0409] 10. McCleese, C., Yu, Z., Esemoto, N. N., Kolodziej, C., Maiti, B., Bhandari, S., Dunjetz,

[0410] B. D., Burda, C., Ptaszek, M. Excitonic interactions in bacteriochlorin homo-dyads enable charge transfer: A new approach to the artificial photosynthetic special pair. The Journal of Physical Chemistry B, 122. 14 (2018): 4131-4140.

[0411] 11. Meares, A., Bhagavathy, G.V., Zik, S.R., Gallagher, T. and Ptaszek, M. Expanding π- conjugation in chlorins using ethenyl linker. The Journal of Organic Chemistry, 83.

[0412] 16 (2018): 9076-9087.

[0413] 12. Meares, A., Yu, Z., Bhagavathy, G.V., Satraitis, A., Ptaszek, M. Photoisomerization of Enediynyl Linker Leads to Slipped Cofacial Hydroporphyrin Dyads with Strong Through-Bond and Through-Space Electronic Interactions. The Journal of Organic Chemistry, 84. 12 (2019): 7851-7862.

[0414] 13. Roy, A., Diers, J.R., Niedzwiedzki, D.M., Meares, A., Yu, Z., Bhagavathy, G.V., Satraitis, A., Kirmaier, C., Ptaszek, M., Bocian, D.F. and Molten, D. Photophysical Properties and Electronic Structure of Hydroporphyrin Dyads Exhibiting Strong Through-Space and Through-Bond Electronic Interactions. The Journal of Physical Chemishy A, 126. 31 (2022): 5107-5125.

[0415] 14. Molecular Probes Handbook: a Guide to Fluorescent Probes and Labeling Technologies, 11th Edition, Life Technologies, 2010.

[0416] 15. International Patent Publication No. WO2021118782A2.

[0417] 16. Mroz, P., Huang, Y.-Y., Szokalska, A., Zhiyentayev, T., Janjua, S., Nifli, A.-P., Sherwood, M.E., Ruzie, C., Borbas, K.E., Fan, D., Krayer, M., Balasubramanian, T., Yang, E., Kee, H.L., Kirmaier, C., Diers, J.R., Bocian, D.F., Holten, D., Lindsey, J.S., Hamblin, M.R., 2010. Stable synthetic bacteriochlorins overcome the resistance of melanoma to photodynamic therapy. FASEB JOURNAL 24, 3160-70. doi: 10.1096 / fj.09-152587

[0418] 17. Huang, Y.-Y., Mroz, P., Zhiyentayev, T., Sharma, S.K., Balasubramanian, T., Ruzie, C., Krayer, M., Fan, D., Borbas, K.E., Yang, E., Kee, H.L., Kirmaier, C., Diers, J.R., Bocian, D.F., Holten, D., Lindsey, J.S., Hamblin, M.R., 2010. In Vitro Photodynamic Therapy and Quantitative Structure- Activity Relationship Studies with Stable Synthetic Near-Infrared-Absorbing Bacteriochlorin Photosensitizers. JOURNAL OF MEDICINAL CHEMISTRY 53, 4018-27. doi:I0.102.l / jm901908s

[0419] 18. Huang, L., Huang, Y.-Y., Mroz, P., Tegos, G.P., Zhiyentayev, T., Sharma, S.K., Lu, Z., Balasubramanian, T., Krayer, M., Ruzie, C., Yang, E., Kee, H.L., Kirmaier, C., Diers, J.R., Bocian, D.F., Holten, D , Lindsey, J.S., Hamblin, M.R., 2010. Stable Synthetic Cationic Bacteriochlorins as Selective Antimicrobial Photosensitizers. ANTIM ICROBIAL AGENTS AND CHEMOTHERAPY 54, 3834-41. doi: 10.1128 / aac.00125-10

[0420] 19. Sharma, S.K., Krayer, M., Sperandio, F.F., Huang, L., Huang, Y.-Y., Holten, D., Lindsey, J.S., Hamblin, M.R., 2013. Synthesis and. evaluation of cationic bacteriochlorin amphiphiles with effective in vitro photodynamic activity against cancer cells at low nanomolar concentration. JOURNAL OF PORPHYRINS AND PHTHALOCYANINES 17, 73-85. doi : 10.1142 / s 108842461250126x

[0421] 20. Huang, Y.-Y., Balasubramanian, T., Yang, E., Luo, D., Diers, J.R., Bocian, D.F., Lindsey, J.S., Holten, D,, Hamblin, M.R., 2012. Stable Synthetic Bacteriochlorins for Photodynamic Therapy: Role of Dicyano Peripheral Groups, Central Metal Substitution (2H, Zn, Pd), and Cremophor EL Delivery. ChemMedChem 7, 2155-67. doi : 10.1002 / cmdc.201200351

[0422] 21. Yang, E., Diers, J. R., Huang, Y.-Y., Hamblin, M. R., Lindsey, J. S., Bocian, D. F., & Holten, D. (2013). Molecular Electronic Tuning of Photosensitizers to Enhance Photodynamic Therapy: Synthetic Dicyanobacteriochlorins as a Case Study. PHOTOCHEMISTRY AND PHOTOBIOLOGY, 89(3), 605-618. doi: 10.1111 / php.12021

[0423] 22. Huang, L., Krayer, M., Roubil, J.G.S., Huang, Y.-Y., Holten, D., Lindsey, J.S., Hamblin, M.R., 2014. Stable synthetic mono-substituted cationic bacteriochlorins mediate selective broad-spectrum photoinactivation of drug-resistant pathogens at nanomolar concentrations. JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 141, 1 19-27. doi:10.1016 / j.jphotobiol.2014.09.016 The foregoing is illustrative of the present invention and is not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated by reference in their entireties for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

Claims

THAT WHICH IS CLAIMED IS:

1. A compound (e.g., polymer) comprising: a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, optionally wherein the first end group comprises a primary dye (e.g., a luminophore (e.g., a fluorophore) or a non-luminescent molecular entity), a bulky group, a hydrophobic group (e.g., a hydrophobic monomer), and / or a charged group; a second end group attached to a second terminus of the polymer backbone that opposes the first end, optionally wherein the second end group comprises a bioconjugate group or a dye; and a primary dye pendant from the polymer backbone.

2. The compound of claim 1, wherein the primary dye has a molecular weight in a range of about 150 Daltons (Da) to about 3,000 Da.

3. The compound of claim 1 or 2, wherein the compound has a molecular weight in a range of about 5,000 Da to about 350,000 Da.

4. The compound of any one of claims 1-3, wherein at least one of the first end group or second end group comprises a primary dye, optionally wherein the first end group is devoid of a bulky group and a charged group and the first end and / or the second end comprise the primary dye.

5. The compound of claim any one of claims 1-3, wherein the first end group comprises a charged group.

6. The compound of claim 5, wherein the charged group comprises a sulfonate or carboxylic acid.

7. The compound of any one of claims 1-3, wherein the first end group comprises a bulky group.

8. The compound of claim 7, wherein the bulky group comprises a cyclodextrin or a polyhedral oligomeric silsesquioxane (POSS).

9. The compound of any one of claims 1-3, wherein the first end group comprises a hydrophobic group (e.g., a hydrophobic monomer).

10. The compound of any preceding claim, wherein the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) are randomly distributed in the polymer backbone.

11. The compound of any one of claims 1-10, wherein the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) are distributed in a gradient in substantially all or a portion of the polymer backbone.

12. The compound of any preceding claim, wherein the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) are present in the compound 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, optionally wherein the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) are present in the compound in a ratio of about 1 :6 (hydrophobic unit(s): hydrophilic unit(s)).

13. The compound of any preceding claim, wherein the compound has conformational flexibility.

14. The compound of any preceding claim, wherein the compound is self-folding in an aqueous solution, optionally self-folding into a unimer micellar structure.

15. The compound of any preceding claim, wherein the compound is an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer.

16. The compound of any preceding claim, wherein the compound is cross-linked, optionally wherein the compound is cross-linked when it is in a folded structure.

17. The compound of any preceding claim, wherein the compound is folded to provide a particle, optionally wherein the particle has a diameter in a range of about 1 nm or 3 nm to about 30 nm or 40 nm.

18. The compound of claim 17, wherein at least a portion of the one or more hydrophobic unit(s) are present in the core of the particle and / or at least a portion of the one or more hydrophilic unit(s) are present at the periphery (e.g. shell) of the particle.

19. The compound of any preceding claim, wherein the primary dye is encapsulated by a portion of the compound (e.g., a portion of the polymer) when it is in a folded structure.

20. The compound of any preceding claim, wherein the compound is a telechelic polymer or a heterotelechelic polymer.

21. The compound of any preceding claim, wherein the primary dye (e.g., tetrapyrrole macrocycle) is hydrophobic.

22. The compound of any preceding claim, wherein the compound is water soluble, optionally wherein the compound has a solubility in water at room temperature of at least 1 mg / mL.

23. The compound of any preceding claim, wherein at least one of the one or more hydrophobic unit(s) and / or the one or more hydrophilic unit(s) comprises a pendant functional group, optionally wherein the pendant functional group is a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimido ester, or fluorophenyl ester), azido, maleimido, isocyanato, isothiocyanato, phosphono, sulfono, ammonio, or phosphatidyl choline group and / or the pendant functional group is a hydrophilic group comprising a terminal cationic (e.g., ammonium), anionic (e.g., sulfonate, phosphate, carboxylate, or phosphonate), or zwitterionic (e.g., choline-like) group and optionally a polyethylene glycol) moiety.

24. The compound of any preceding claim, wherein at least one of the one or more hydrophobic unit(s) comprises an alkyl pendant group (e.g., dodecyl) and / or at leastone of the one or more hydrophilic unit(s) comprise a glycol pendant group (e.g., poly(ethylene glycol)).

25. The compound of any preceding claim, wherein the compound comprises 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 or 30 primary dyes.

26. The compound of any preceding claim, wherein the compound comprises a hydrophobic unit having a structure represented by Formula III:wherein:Ris hydrogen or a Cl-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);R1is absent or is -O-, -NH-, -CH2-;R’ is absent or a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or a C6-C25 arylalkyl;R2is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azido, maleimido, isocyanato, or isothiocyanato group, or R2is the primary dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

27. The compound of claim 26, wherein R2of the hydrophobic unit is hydrogen, hydroxyl, carboxyl, amino, formyl, or ester group, optionally pentafluorophenyl ester.

28. The compound of claim 26, wherein R2of the hydrophobic unit is vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimido group.

29. The compound of claim 26, wherein R2is an additional primary dye, optionally wherein the additional primary dye comprises two or more dyes.

30. The compound of any preceding claim, wherein the compound comprises a hydrophilic unit having a structure represented by Formula IV:wherein:R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);R1is absent or is -O-, -NH-, or -CH2-;R3is selected from the group consisting of -(CH2CH2R5)n-, -C1-C6alkyl, -C1-C6alkyl- O-, and -C1-C6alkyl-SO3- or a salt thereof, wherein R5is -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;R4is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl or ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group, or R4is the additional primary dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

31. The compound of claim 30, wherein R4in the hydrophilic unit is a hydrogen, alkyl, phosphono, sulfono, phosphatidyl choline, phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or ester group.

32. The compound of claim 30, wherein R4in the hydrophilic unit is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimido group.

33. The compound of claim 26, wherein R3is -C1-C6alkyl-O- or -(CH2CH2R5)n- with R5being -O-, and R4in the hydrophilic unit is a hydrogen, alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), or phosphoryl group.

34. The compound of claim 26, wherein R3in the hydrophilic unit is C1-C6alkyl or -(CH2CH2R5)n- with R5being -CH2-, and R4in the hydrophilic unit is a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group.

35. The compound of claim 26, wherein R3is -C1-C6alkyl-SO3- or a salt thereof.

36. The compound of any preceding claim, wherein the primary and / or a secondary dye is a Fe(II) -chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole (e.g., a porphyrin).

37. The compound of any preceding claim, wherein the polymer backbone further comprises an additional unit comprising one or more additional primary dye(s).

38. A compound (e.g., polymer) comprising: a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s); a first end group attached to a first terminus of the polymer backbone, wherein the first end group comprises(i) a primary dye (e.g., a luminophore or a non-luminescent molecular entity) and a biomolecule (e.g., DNA, RNA, protein, or peptide);(ii) a primary dye, a secondary dye (e.g., a luminophore or a non-luminescent molecular entity), and optionally a biomolecule, or(iii) two or more primary dyes and optionally a biomolecule; and a second end group attached to a second terminus of the polymer backbone, optionally wherein the second end group comprises a bioconjugate group.

39. A compound (e.g., polymer) comprising: a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s);a first end group attached to a first terminus of the polymer backbone, wherein the first end group comprises(i) a hydrophobic group, a bulky group, or a charged group; or(ii) a hydrophobic group, a bulky group, and / or a charged group; a second end group attached to a second terminus of the polymer backbone, optionally wherein the second end group comprises a bioconjugate group; and a primary dye and a secondary dye pendant on the polymer backbone.

40. The compound of claim 38 or 39, wherein the primary dye and / or the secondary dye has a molecular weight in a range of about 150 Daltons (Da) to about 3,000 Da.

41. The compound of any one of claims 38-40, wherein the compound has a molecular weight in a range of about 5,000 Da, 10,000 Da to about 175,000 Da, up to about 175,000 Da, up to about 225,000 Da, up to about 275,000 Da, or up to about 350,000 Da.

42. The compound of any one of claims 38-41, wherein the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) are randomly distributed in the polymer backbone.

43. The compound of any of claims 38-42, wherein the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) are present in the compound 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, optionally wherein the one or more hydrophobic unit(s) and the one or more hydrophilic unit(s) are present in the compound in a ratio of about 1 :6 (hydrophobic unit(s):hydrophilic unit(s)).

44. The compound of any of any of claims 38-43, wherein the polymer has conformational flexibility.

45. The compound of any of claims 38-44, wherein the compound is self-folding in an aqueous solution, optionally self-folding into a unimer micellar structure.

46. The compound of any of claims 38-45, wherein the compound is an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer.

47. The compound of any of claims 38-46, wherein the compound is cross-linked, optionally wherein the compound is cross-linked when it is in a folded structure.

48. The compound of any of claims 38-47, wherein the compound is folded to provide a particle, optionally wherein the particle has a diameter in a range of about 1 nm or 3 nm to about 30 nm or 40 nm.

49. The compound of claim 48, wherein at least a portion of the one or more hydrophobic unit(s) are present in the core of the particle and / or at least a portion of the one or more hydrophilic unit(s) are present at the periphery (e.g. shell) of the particle.

50. The compound of any of claims 38-49, wherein the primary dye and / or the secondary dye is encapsulated by a portion of the compound when it is in a folded structure.

51. The compound of any of claims 38-50, wherein the compound is a telechelic polymer or a heterotelechelic polymer.

52. The compound of any of claims 38-51, wherein the primary dye and / or the secondary dye (e.g., tetrapyrrole macrocycle) is hydrophobic.

53. The compound of any of claims 38-52, wherein the compound is water soluble, optionally wherein the compound has a solubility in water at room temperature of at least 1 mg / mL.

54. The compound of any of claims 38-53, wherein at least one of the one or more hydrophobic unit(s) and / or the one or more hydrophilic unit(s) comprises a pendant functional group, optionally wherein the pendant functional group is a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimido ester, or fluorophenyl ester), azido, maleimido, isocyanato, isothiocyanato, phosphono, sulfono, ammonio, or phosphatidyl choline group and / or the pendant functional group is a hydrophilic group comprising a terminal cationic (e.g., ammonium), anionic (e.g., sulfonate, phosphate, carboxylate, or phosphonate), or zwitterionic (e.g., choline-like) group and optionally a poly(ethylene glycol) moiety.

55. The compound of any of claims 38-54, wherein at least one of the one or more hydrophobic unit(s) comprises an alkyl pendant group (e.g., dodecyl) and / or at least one of the one or more hydrophilic unit(s) comprise a glycol pendant group (e.g., poly(ethylene glycol)).

56. The compound of any of claims 38-55, wherein the compound comprises a hydrophobic unit having a structure represented by Formula III:wherein:R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);R1is absent or is -O-, -NH-, -CH2-;R’ is absent or a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or a C6-C25 arylalkyl;R2is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azido, maleimido, isocyanato, or isothiocyanato group, or R2is the primary dye, the secondary dye, an additional primary dye and / or an additional secondary dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

57. The compound of claim 56, wherein R2in the hydrophobic unit is hydrogen or is a hydroxyl, carboxyl, amino, formyl, or ester group.

58. The compound of claim 56, wherein R2in the hydrophobic unit is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimido group.

59. The compound of any of claims 38-58, wherein the compound comprises a hydrophilic unit having a structure represented by Formula IV:wherein:R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);R1is absent or is -O-, -NH-, or -CH2-;R3is selected from the group consisting of -(CH2CH2R5)n-, -C1-C6alkyl, -C1-C6alkyl- O-, and -C1-C6alkyl-SO3- or a salt thereof, wherein R5is -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;R4is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl or ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group, or R4is the primary dye, secondary dye, an additional primary dye and / or an additional secondary dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

60. The compound of claim 59, wherein R4in the hydrophilic unit is a hydrogen, alkyl, phosphono, sulfono, phosphatidyl choline, phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl, or ester group.

61. The compound of claim 59, wherein R4in the hydrophilic unit is a vinyl, epoxy, mercapto, azido, isocyanato, isothiocyanato, or maleimido group.

62. The compound of claim 59, wherein R3in the hydrophilic unit is -C1-C6alkyl- O- or -(CH2CH2R5)n- with R5being -O-, and R4in the hydrophilic unit is a hydrogen, alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), or phosphoryl group.

63. The compound of claim 59, wherein R3in the hydrophilic unit is C1-C6alkyl or -(CH2CH2R5)n- with R5being -CH2-, and R4in the hydrophilic unit is a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group.

64. The compound of claim 63, wherein R3is -C1-C6alkyl-SO3- or a salt thereof.

65. The compound of any of claims 38-64, wherein the primary dye, the secondary dye, and / or one or more of the two or more primary dyes is a Fe(II)-chelated tetrapyrrole or a Cu(II)-chelated tetrapyrrole (e.g., a porphyrin).

66. The compound of any of claims 38-65, wherein the primary dye, the secondary dye, and / or one or more of the two or more primary dyes is a fluorescent dye.

67. The compound of any of claims 38-65, wherein the primary dye, the secondary dye, and / or one or more of the two or more primary dyes is a non-fluorescent dye.

68. The compound of any of claims 38 or 41-65, wherein the first end group comprises the primary dye and the secondary dye and the primary dye is a fluorescent dye and the secondary dye is a non-fluorescent dye.

69. The compound of any of claims 43-71, wherein the first end group comprises the bulky group and the bulky group comprises a cyclodextrin or a polyhedral oligomeric silsesquioxane (POSS) or wherein the first end group comprises a charged group.

70. The compound of any of claims 38-69, further comprising a linear linker between the primary dye and the polymer backbone or the optional biomolecule, between the primary dye and the secondary dye, or between the two or more primary dyes.

71. The compound of any of claims 38-69, further comprising a bifunctional branched linker between the primary dye and the polymer backbone or the optional biomolecule, between the primary dye and the secondary dye, or between the two or more primary dyes wherein the bifunctional branched linker is pendant from the polymer backbone.

72. The compound of any of claims 38-71, further comprising a trifunctional branched linker between the polymer backbone, one or more primary dye(s), one or more secondary dye(s), one or more charged groups, one or more bulky groups, and / or one or more biomolecule(s), optionally wherein the first end group comprises: two primary dyes and the secondary dye and the trifunctional branched linker is between the two primary dyes and the secondary dye, or the primary dye, the secondary dye, and a tertiary dye and the trifunctional branched linker is between the primary dye, the secondary dye, and the tertiary dye, or three primary dyes and the trifunctional branched linker is between each of the three primary dyes, or two primary dyes and the biomolecule, or the primary dye, the secondary dye, and the biomolecule, or two charged groups and the bulky group and the trifunctional branched linker is between the two charged groups and the bulky group, or three bulky groups and the trifunctional branched linker is between each of the three bulky groups, or three charged groups and the trifunctional branched linker is between each of the three charged groups, or two charged groups dyes and the biomolecule, or two bulky groups and the biomolecule, or one or more hydrophobic groups (e.g., hydrophobic monomer), or the bulky group, the charged group, and the biomolecule.

73. The compound of any of claims 38-72, further comprising a linker between the primary dye and the polymer backbone or the optional biomolecule, between the primary dye and the secondary dye, between the two or more primary dyes, between the bulky group and the charged group, between the bulky group and the biomolecule, between two bulky groups, or between two charged groups, the linker comprising or consisting of a moiety having a structure of:-(CH2)mlRa(CH2)nl-wherein ml is an integer of 0 to 3; nl is an integer of 0 to 3; and Ra is selected from -OC(O)-, -C(O)O-, -CH(0H)CH2Rx-,or -C(O)NH-; where Rxis O or N; or a moiety having a structure of: -(CH)m2Rb(CH2)n2wherein m2 is between 0 and 3; n2 is between 0 and 3; and Rb is -N-, -NNH-,-S-,-Ph-C(O)NH(CH2)xC(O)NH-, -Ph-C(O)NH(CH2)xC(O)-, -Ph-C(O)NH(CH2)xC(O)-, or -Ph-N(CH3)C(O)(CH2)xC(O), wherein Ph is phenyl and x is an integer between 0 and 4; optionally wherein the linker is substituted with a PEG molecule.

74. The compound of any of claims 71-73, wherein the linear linker, bifunctional linker, or trifunctional linker comprises a PEG moiety and a second moiety having a structure of:-(CH2)miRa(CH2)ni- wherein ml is an integer of 0 to 3; nl is an integer of 0 to 3; and Ra is selected from -5 OC(O)-, -C(O)O-,, -CH(0H)CH2Rx-,or -C(O)NH-; where Rxis O or N; or a moiety having a structure of: -(CH)m2Rb(CH2)n2wherein m2 is between 0 and 3; n2 is between 0 and 3; and Rb is -N-, -NNH-,-S-,-Ph-C(O)NH(CH2)xC(O)NH-, -Ph-C(O)NH(CH2)xC(O)-, -Ph-C(O)NH(CH2)xC(O)-, or -Ph-N(CH3)C(O)(CH2)xC(O), wherein Ph is phenyl and x is an integer between 0 and 4, optionally wherein the PEG moiety is covalently attached at one attachment point of the second moiety.

75. A composition comprising a compound of any one of claims 1-74.

76. The composition of claim 75, wherein the compound forms a particle in the composition.

77. The composition of claim 75 or 76, wherein the compound and the particle are present in a ratio of about 1 : 1 in the composition (e.g., there is one compound per particle).

78. The composition of any one of claims 75-77, wherein at least a portion of the one or more hydrophobic unit(s) is present in the core of the particle.

79. The composition of any one of claims 75-78, wherein at least a portion of the one or more hydrophilic unit(s) are present in the shell (e.g., at the periphery) of the particle.

80. The composition of any one of claims 75-79, wherein the particle is resistant to dilution, optionally wherein the particle remains in a folded structure when the composition is diluted up to 100x or is diluted to sub-micromolar concentrations.

81. The composition of any one of claims 75-80, wherein the primary dye and / or secondary dye is present in the core of the particle and / or is encapsulated by at least a portion of the compound.

82. The composition of any one of claims 75-77, wherein the composition is devoid of an organic solvent.

83. A method of preparing a compound comprising: copolymerizing one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s) to provide a compound comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s), a first end group, and a second end group;attaching a bulky group, hydrophobic group, and / or charged group to the first end group of the compound to become part of the first end group; attaching a primary dye (e.g., a luminophore (e.g., a fluorophore) or a non-luminescent molecular entity) to a functional group pendant from the polymer backbone; optionally attaching a bioconjugate group to the second end group of the compound; and optionally cross-linking the compound.

84. A method of preparing a compound comprising: copolymerizing one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s) to provide a compound comprising a polymer backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s), a first end group, and a second end group; optionally attaching a primary dye (e.g., a luminophore (e.g., a fluorophore) or a non- luminescent molecular entity) to the first end group of the compound to become part of the first end group; attaching an additional primary dye to a functional group pendant from the polymer backbone; optionally attaching a bioconjugate group to the second end group of the compound; and optionally cross-linking the compound.

85. A method of preparing a compound comprising: polymerizing a solution of one or more hydrophilic monomer(s) and optionally one or more hydrophobic monomer(s), subsequently adding one or more hydrophobic monomer(s) to the solution and polymerizing the solution to provide a compound comprising a polymer backbone comprising a gradient backbone that comprises one or more hydrophobic unit(s) and one or more hydrophilic unit(s), a first end group, and a second end group; optionally attaching a primary dye (e.g., a luminophore (e.g., a fluorophore) or a non- luminescent molecular entity) to the first end group of the compound to become part of the first end group; attaching an additional primary dye to a functional group pendant from the polymer backbone;optionally attaching a bioconjugate group to the second end group of the compound; and optionally cross-linking the compound.

86. The method of claim 83-85, wherein one or more hydrophobic monomer(s) and the one or more hydrophilic monomer(s) are copolymerized via a 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 polymer backbone.

87. The method of claim 83-85, wherein the one or more hydrophobic monomer(s) and the one or more hydrophilic monomer(s) are copolymerized via a living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN), and a RAFT agent (e.g., a thiocarb onylthio compound) to provide the polymer backbone.

88. The method of any one of claims 83-86, the one or more hydrophobic monomer(s) and the one or more hydrophilic monomer(s) are copolymerized 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 to hydrophilic monomer ratio).

89. The method of any one of claims 83-88, wherein at least one hydrophobic monomer has a structure represented by Formula I:wherein:R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);R1is absent or is -O-, -NH-, -CH2-;R’ is absent or a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or a C6-C25 arylalkyl;R2is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenylester), azido, maleimido, isocyanato, or isothiocyanato group, or R2is the additional primary dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

90. The method of claim 89, wherein R2in the hydrophobic monomer is hydrogen or is a hydroxyl, carboxyl, amino, formyl or ester group.

91. The method of any one of claims 83-90, wherein at least one hydrophilic monomer has a structure represented by Formula II:wherein:R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);R1is absent or is -O-, -NH-, or -CH2-;R3is selected from the group consisting of a -(CH2CH2R5)n-, -C1-C6alkyl, -C1-C6alkyl-O-, and -C1-C6alkyl-SO3- or a salt thereof, wherein R5is -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; andR4is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl or ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group, or optionally R4is the additional primary dye.

92. The method of claim 917, wherein R4in the hydrophilic monomer is a hydroxyl, carboxyl, amino, formyl, or ester group.

93. The method of claim 91, wherein R3is -C1-C6alkyl-O-, and R4in the hydrophilic monomer is a hydrogen, alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxy sulfonyl), phosphatidyl choline, or phosphoryl group.

94. The method of claim 91, wherein R3is -C1-C6alkyl or -(CH2CH2R5)n- with R5being -O-, and R4in the hydrophilic monomer is a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group.

95. A method of preparing a compound comprising: copolymerizing one or more hydrophobic monomer(s) and one or more hydrophilic monomer(s) to provide a compound comprising a polymer backbone comprising one or more hydrophobic unit(s) and one or more hydrophilic unit(s), a first end group, and a second end group; attaching a bulky group, a hydrophobic group (e.g., a hydrophobic monomer), and / or charged group to the first end group (i.e., to become part of the first end group) or optionally attaching a primary dye (e.g., a luminophore (e.g., a fluorophore) or a non-luminescent molecular entity) to the first end group of the compound to become part of the first end group; attaching a first primary dye (e.g., a luminophore (e.g., a fluorophore) or a non- luminescent molecular entity) to the polymer backbone; attaching a second primary dye, a secondary dye, and / or a biomolecule to the first primary dye and / or another portion of the polymer backbone; optionally attaching a bioconjugate group to the second end group; and / or optionally cross-linking the compound.

96. The method of claim 95, wherein one or more hydrophobic monomer(s) and the one or more hydrophilic monomer(s) are copolymerized via a 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 polymer backbone.

97. The method of claim 95, wherein the one or more hydrophobic monomer(s) and the one or more hydrophilic monomer(s) are copolymerized via a living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN), and a RAFT agent (e.g., a thiocarb onylthio compound) to provide the polymer backbone.

98. The method of any one of claims 95-97, the one or more hydrophobic monomer(s) and the one or more hydrophilic monomer(s) are copolymerized in a ratio of about1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, or 1 : 10 (hydrophobic monomer to hydrophilic monomer ratio).

99. The method of any one of claims 95-98, wherein at least one hydrophobic monomer has a structure represented by Formula I:wherein:R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);R1is absent or is -O-, -NH-, -CH2-;R’ is absent or a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or a C6-C25 arylalkyl;R2is hydrogen or a halo, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azido, maleimido, isocyanato, or isothiocyanato group, or R2is an additional primary dye when the hydrophobic unit is in the polymer backbone (e.g., R2is not a terminal end group); and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

100. The method of claim 95, wherein R2in the hydrophobic monomer is hydrogen or is a hydroxyl, carboxyl, amino, formyl or ester group.

101. The method of any one of claims 95-100, wherein at least one hydrophilic monomer has a structure represented by Formula II:wherein:R is hydrogen or a C1-C8 alkyl (e.g., a Cl, C2, C3, C4, C5, C6, C7, or C8 alkyl);R1is absent or is -O-, -NH-, or -CH2-;R3is selected from the group consisting of a -(CH2CH2R5)n-, -C1-C6alkyl, -C1-C6alkyl-O-, and -C1-C6alkyl-SO3- or a salt thereof, wherein R5is -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; andR4is absent or is a hydrogen, alkyl, phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxysulfonyl), phosphatidyl choline (i.e., 2-(trimethylammonio)ethoxy(hydroxy)phosphoryl), phosphoryl, halo, hydroxyl, carboxyl, amino, ammonio, formyl or ester (e.g., pentafluorophenyl ester, succinimido ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) group, or optionally R4is an additional primary dye when the hydrophilic unit is in the polymer backboneand the terminal end group comprises a bulky group and / or charged group (e.g., R4is not a terminal end group).

102. The method of claim 101, wherein R4in the hydrophilic monomer is a hydroxyl, carboxyl, amino, formyl, or ester group.

103. The method of claim 101, wherein R3is -C1-C6alkyl-O-, and R4in the hydrophilic monomer is a hydrogen, alkyl (e.g., methyl or ethyl group), phosphono (e.g., dihydroxyphosphoryl), sulfono (e.g., hydroxy sulfonyl), phosphatidyl choline, or phosphoryl group.

104. The method of claim 101, wherein R3is -C1-C6alkyl or -(CH2CH2R5)n- with R5being -O-, and R4in the hydrophilic monomer is a hydroxyl, carboxyl, amino, ammonio, formyl, ester, phosphono, or sulfono group.

105. The method of any of claims 97-104, wherein the first primary dye and the secondary dye are each fluorescent dyes.

106. The method of any of claims 95-104, wherein the first primary dye and the secondary dye are each non-fluorescent dyes.

107. The method of any of claims 95-104, wherein the first primary dye is a fluorescent dye and the secondary dye is a non-fluorescent dye.

108. The method of any of claims 93-107, wherein the second primary dye, the secondary dye, and / or the biomolecule is attached to the first primary dye via a linear linker.

109. The method of any of claims 93-107, wherein the second primary dye, the secondary dye, and / or the biomolecule is attached to the first primary dye via a bifunctional linker.

110. The method of any of claims 93-107, wherein the second primary dye, the secondary dye, and / or the biomolecule is attached to the first primary dye via a trifunctional linker.

111. A biomolecule comprising at least one compound (e.g., 1, 2 or more) of any of claims 1-74.

112. The biomolecule of claim 108, further comprising a polypeptide (e.g., a protein such as an antibody) attached (e.g., covalently bonded) to the at least one compound.

113. Use of the compound of any one of claims 1-74, the composition of any one of claims 75-82, the compound prepared according to the method of any one of claims 83-110, or the biomolecule of claim 111 or 112 in flow cytometry, imaging, photodynamic therapy, photodynamic inactivation, photoimmunotherapy, and / or fluorescent guided surgery.

114. A method of detecting cells and / or particles using flow cytometry, the method comprising labelling cells and / or particles with the compound of any one of claims 1-74, the composition of any one of claims 75-82, the compound prepared according to the method of any one of claims 83-110, or the biomolecule of claim 111 or 112; and detecting the compound or the biomolecule by flow cytometry, thereby detecting the cells and / or particles.

115. A method of detecting labelled cells in a subject, the method comprising, performing the method of claim 114, wherein the method comprises labelling and detecting cells, and administering the labelled cells to a subject and detecting the compound or the biomolecule within the subject, thereby detecting the cells within the subject.

116. The method of claim 115, wherein the cells are detected within the subj ect using an imaging technique (e.g., photoacoustic imaging and / or magnetic resonance imaging).

117. A method of detecting a tissue and / or agent in a subj ect, the method comprising: administering to the subject the compound of any one of claims 1-74, the composition of any one of claims 75-82, the compound prepared according to the method of any one of claims 83-110, or the biomolecule of claim 111 or 112, optionally wherein the compound or the biomolecule associates with the tissue and / or agent; and detecting (e.g., using fluorescence, MRI, or PAI) the compound or the biomolecule within the subject, thereby detecting the tissue and / or agent.

118. The method of claim 117, wherein the compound comprises the primary dye and the secondary dye and the method comprises detecting the primary dye using a first detection method (e.g., a first imaging method) and detecting the secondary dye using a second detection method (e.g., a second imaging method).

119. The method of claim 118, wherein the compound is detected by detecting the primary dye using magnetic resonance imaging and detecting the secondary dye using photoacoustic imaging, or wherein the compound is detected by detecting the primary dye using photoacoustic imaging and detecting the secondary dye using magnetic resonance imaging.

120. A method for treating a cell and / or tissue (e.g., a diseased cell and / or tissue) in a subject in need thereof, the method comprising: administering to the subject the compound of any one of claims 1-74, the composition of any one of claims 75-82, the compound prepared according to the method of any one of claims 83-110, or the biomolecule of claim 111 or 112, optionally wherein the compound associates with the cell and / or tissue, and irradiating the subject or a portion thereof (e.g., a location where the cell and / or tissue are present) with light of a wavelength and intensity sufficient to treat the cell and / or tissue, optionally wherein the light activates the compound.

121. The method of claim 120, further comprising detecting the compound (e.g., by an imaging technique).

122. The method of claim 121, wherein detecting the compound comprises detecting the primary dye using an imaging technique, and treating the cell and / or tissue comprises activating the secondary dye (e.g., to release reactive oxygen species into the diseased tissue) to thereby treat the cell and / or tissue, or wherein detecting the compound comprises detecting the secondary dye using an imaging technique, and treating the cell and / or tissue comprises activating the primary dye (e.g., to release reactive oxygen species into the diseased tissue) to thereby treat the cell and / or tissue.

123. The method of any of claims 120-122, wherein treating the cell and / or tissue comprises photodynamic therapy and / or photoimmunotherapy.

124. The method of any of claims 121-123, wherein the primary dye is detected using fluorescence, magnetic resonance imaging, and / or photoacoustic imaging and / or wherein the secondary dye is detected using fluorescence, magnetic resonance imaging, and / or photoacoustic imaging.

125. The method of claim 121, wherein the compound further comprises a biomolecule and the biomolecule localizes the compound to the cell and / or tissue.

126. The method of claim 125, wherein the cell and / or tissue is hyperproliferative.

127. A compound prepared by a method of any of claims 83-110.

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