Amphiphilic polymers having multiple chromophores, compositions comprising the same, and methods of making and using the same

Abstract

Provided herein are compounds (e.g., polymers) that include a polymer backbone comprising one or more hydrophobic units and one or more hydrophilic units and one or more dyes and / or biomolecules attached to the hydrophobic units and / or the hydrophilic units. Also provided are compositions including such compounds, methods of making such compounds, and methods of using such compounds.

Description

[0001] Priority Statement This patent application claims the benefit and priority of U.S. Provisional Patent Application Serial No. 63 / 604,557, filed November 30, 2023, the contents of which are incorporated herein by reference as if fully described herein.

[0002] Government Support Statement This invention was completed with government support, pursuant to grants AI112302 and GM131501 from the National Institutes of Health (NIH). The government holds certain rights to this invention. Technical Field

[0003] This invention generally relates to polymer chromophores. It also relates to compositions comprising polymer chromophores and methods of their preparation and use. Background Technology

[0004] Many applications of chromophores are carried out in aqueous solutions, but most organic chromophores are hydrophobic or only slightly polar. Numerous methods exist for encapsulating chromophores, but chromophores must meet criteria such as simple synthesis, absence of fluorophores (fluorophore quenching), and the presence of a bioconjugatable group. Summary of the Invention

[0005] A first aspect of the invention relates to a compound (e.g., a polymer) comprising: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end of the polymer backbone, optionally wherein the first end group comprises a major dye (e.g., a luminescent group (e.g., a fluorophore) or a non-luminescent molecular entity), optionally wherein the major dye has a molecular weight in the 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 bulk group, and / or a charged group; a second end group attached to a second end of the polymer backbone, optionally wherein the second end group comprises a bioconjugated group or wherein the second end group comprises a dye; and a major dye side-attached to the polymer backbone, optionally wherein the compound has a molecular weight in the range of about 5,000 Da, or 10,000 Da to about 350,000 Da.

[0006] One aspect of the present invention relates to a compound (e.g., a polymer) comprising: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end of the polymer backbone, wherein the first end group comprises (i) a major dye and a biomolecule; (ii) a major dye and a minor dye, and optionally a biomolecule, or (iii) two or more major dyes, and optionally a biomolecule; and a second end group attached to a second end of the polymer backbone, wherein the second end group optionally comprises a bioconjugating group; and a major dye side-attached to the polymer backbone, wherein optionally the compound has a molecular weight in the range of about 5,000 Da to about 350,000 Da.

[0007] Another aspect of the invention relates to a compound (e.g., a polymer) comprising: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end of the polymer backbone, wherein the first end group optionally comprises (i) a major dye, or (ii) a hydrophobic group (e.g., a hydrophobic monomer), a charged group, and / or a bulky group; a second end group attached to a second end of the polymer backbone, wherein the second end group optionally comprises a bioconjugating group; and one or more additional major dyes (e.g., luminescent (e.g., fluorophores) or nonluminescent molecular entities) side-attached to the polymer backbone, wherein optionally the major dye has a molecular weight in the range of about 150 Da to about 3,000 Da and / or optionally the compound has a molecular weight in the range of about 5,000 Da to about 350,000 Da. In some embodiments, the compound does not contain a dye as part of the first and second end groups and includes a bulky group and / or a charged group at the first end group. In some embodiments, the compound is free of dye as part of the first and second end groups and contains a hydrophobic group (e.g., a hydrophobic monomer) at the first end group. In some embodiments, the compound is free of dye as part of the first and second end groups and contains a biomolecule at the second end group.

[0008] A further aspect of the invention relates to a compound (e.g., a polymer) comprising: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end of the polymer backbone, wherein the first end group comprises a hydrophobic group (e.g., a hydrophobic monomer), a bulk group, and / or a charged group; a second end group attached to a second end of the polymer backbone, wherein the second end group optionally comprises a bioconjugating group; and a major dye (e.g., a luminescent group (e.g., a fluorophore)) or nonluminescent molecular entity side-attached to the polymer backbone, optionally wherein the major dye has a molecular weight in the range of about 150 Da to about 3,000 Da and / or optionally wherein the compound has a molecular weight in the 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. The molecular weight is in the range of Da or at most about 350,000 Da. In some embodiments, the compound does not contain dyes from the first and second end groups.

[0009] In one aspect, a compound (e.g., a polymer) contains one or more charged groups attached to the polymer backbone.

[0010] Another aspect of the invention relates to a compound (e.g., a polymer) comprising a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end of the polymer backbone, wherein the first end group comprises a bulky group (e.g., cyclodextrin or polyhedral oligosilsesquioxane (POSS)) and / or a charged group (e.g., sulfonate or carboxylic acid); and a second end group attached to a second end of the polymer backbone, optionally wherein the second end group comprises a bioconjugating group; and a major dye (e.g., a luminescent group (e.g., a fluorophore)) or nonluminescent molecular entity side-attached to the polymer backbone, optionally wherein the major dye has a molecular weight in the range of about 150 Da to about 3,000 Da and / or optionally wherein the compound has a molecular weight in the 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. Molecular weight in the range of 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 groups are side-attached to the polymer backbone.

[0011] A further aspect of the invention relates to a method for preparing the compounds of the invention. Such a method includes the step of copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers, for example by polymerizing via living radical polymerization in the presence of an initiator (e.g., a free radical initiator) and a chain transfer agent (e.g., a trithiocarbamate) to provide a copolymer.

[0012] Another aspect of the invention relates to compounds prepared according to the method of the invention.

[0013] According to another aspect of the invention, the use of the compounds of the invention and / or the compositions of the invention is also provided, for example, in flow cytometry, imaging, photodynamic therapy, photodynamic inactivation, photoimmunotherapy and / or fluorescence-guided surgery.

[0014] A further aspect of the invention relates to a method for detecting cells and / or particles using flow cytometry, the method comprising labeling cells and / or particles with a compound of the invention or a compound comprising a biomolecule; and detecting the compound by flow cytometry, thereby detecting the cells and / or particles.

[0015] A further aspect of the invention relates to a method for detecting a compound in a subject, the method comprising labeling cells and / or particles with the compound or a biomolecule of the compound of the invention; and detecting the compound by flow cytometry; and administering the labeled cells and / or particles to the subject; and detecting the compound in the subject, thereby detecting the cells and / or particles in the subject.

[0016] Another aspect of the invention relates to a method for detecting tissues and / or reagents (e.g., cells, infectious agents, etc.) in a subject, the method comprising: administering a compound, biomolecule, or composition of the invention to the subject, optionally wherein the compound or biomolecule binds to the tissue and / or reagent; and detecting the compound or biomolecule in the subject, thereby detecting the tissue and / or reagent. In some embodiments, a major dye on the compound is detected by a first detection method (e.g., a first imaging method), and a minor 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.

[0017] Another aspect of the invention relates to a method of treating cells and / or tissues (e.g., diseased cells and / or tissues) of a subject in need, comprising administering a compound, biomolecule, or composition of the invention to the subject; and irradiating the subject or a portion thereof with light of a wavelength and intensity sufficient to treat the cells and / or tissue, optionally wherein the light activates one or more dyes on the compound. In some embodiments, such a method further comprises detecting the compound (e.g., detecting the compound in the subject using imaging techniques). In some embodiments, a primary dye is used to treat the cells and / or tissue (e.g., by photodynamic therapy), while a secondary dye is used to detect the compound, for example by magnetic resonance imaging and photoacoustic imaging.

[0018] A further aspect of the invention relates to biomolecules (e.g., antibodies, etc.) comprising one or more (e.g., 1, 2, 3, 4, 5, 6 or more) of the compounds of the invention.

[0019] It should be noted that aspects of the invention described with respect to one embodiment may be incorporated into different embodiments, although no specific description is made with respect to them. That is, features of all and / or any embodiment may be combined in any manner and / or combination. The applicant reserves the right to amend any originally filed claim and / or accordingly file any new claim, including the right to modify any originally filed claim to rely on and / or incorporate any feature of any other one or more claims, even if not initially claimed in this manner. These and other objects and / or aspects of the invention are explained in detail in the description set forth below. Other features, advantages, and details of the invention will be apparent to those skilled in the art upon reading the accompanying drawings and the following detailed description of preferred embodiments, which are merely illustrative of the invention. Attached Figure Description

[0020] Figure 1A Exemplary embodiments of chain-end functionalization of compounds according to some embodiments of the present invention are shown, wherein at least one substituent is present at one end of the polymer and may be a dye and / or a biomolecule.

[0021] Figure 1B Exemplary embodiments of chain-end functionalization of compounds according to some embodiments of the present invention are shown, comprising one or more dye molecules attached to a polymer backbone. Substituents may or may not be present at one end of the polymer, and if present, substituents may be dyes, charged groups, or bulky groups.

[0022] Figure 2 Exemplary hydrophobic units are provided according to some embodiments of the present invention.

[0023] Figures 3A-3B Exemplary hydrophobic units are provided according to some embodiments of the present invention.

[0024] Figure 4 Exemplary connectors are provided according to some embodiments of the present invention.

[0025] Figures 5A-5B Exemplary dye P1 is provided according to some embodiments of the invention, which can be used in magnetic resonance imaging (MRI) (5A) and photoacoustic imaging (PAI) (5B).

[0026] Figure 6 Charts illustrating certain dual-use applications of some compounds of the present invention are provided.

[0027] Figure 7 This is an exemplary diagram illustrating the preparation of exemplary compounds of the present invention via copolymerization of hydrophilic and hydrophobic monomers, wherein terminal functional groups (see, for example, Table 2) and bioconjugated groups are added after copolymerization. Next, modification is performed by polymerization of at least one polymer end group using, for example, a dye molecule.

[0028] Figure 8A An exemplary method is provided for preparing exemplary compounds of the present invention by homopolymerization of activated ester monomers (generating a backbone comprising bioconjugated groups and terminal functional groups). Subsequent post-polymerization modification of the polymer backbone can be performed by functionalization with hydrophobic side groups, hydrophilic side groups, and one or more dye molecules.

[0029] Figure 8B Exemplary embodiments of post-polymerization modification of the polymer ends of exemplary compounds of the present invention are provided, comprising hydrophilic side groups, hydrophobic side groups, and dye molecules connected to the polymer backbone.

[0030] Figure 9 Fluorescence spectra of certain compounds of the present invention are provided.

[0031] Figures 10A-10C Flow cytometry data are provided for cell samples using staining solutions containing exemplary antibody-fluorescent polymer compounds of the present invention, wherein two dyes are present at the ends (10A); multiple dyes are present on the backbone of bang beads (10B); and multiple dyes are present on the backbone of peripheral blood monoclonal cells (PBMCs) stained with CD8 monoclonal antibody (10C).

[0032] Figure 11A-11D Provide (11A) UV-VIS spectrum of FP, (11B) fluorescence spectrum of FP under 405 nm excitation, (11C) UV-VIS spectrum of Ab-FP conjugate, and (11D) fluorescence spectrum of the dual dye construct under 405 nm excitation.

[0033] Figure 12 Quantum yield (QY) (left) and brightness (right) vs. dye number of exemplary compounds of the present invention, measured in THF and PBS.

[0034] Figure 13 Microscopic images of peripheral blood mononuclear cells stained with compounds containing folds linked to a CD8 monoclonal antibody and containing porphyrin-dihydroporphyrin dimeric groups and charged groups with a total DP of less than 200. The stained cells were imaged using a UV light source on an Olympus IX51 inverted fluorescence microscope.

[0035] Figure 14 Microscopic images of exemplary multidye folded antibody conjugates on polystyrene beads; the exemplary multidye folded conjugates contain 2.6 660 dimers per polymer and have a total CDA weight percentage of 7%.

[0036] Figure 15 Microscopic images of exemplary multidye folded antibody conjugates included on polystyrene beads; the exemplary multidye folded conjugates contain 2.4 660 dimers per polymer and have a total CDA weight percentage of 7%. Detailed Implementation

[0037] The invention will now be described more fully below with reference to the accompanying drawings, in which embodiments of the invention are shown. However, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided to make this disclosure sufficient and complete, and to fully convey the scope of the invention to those skilled in the art.

[0038] The terminology used in this description of the invention is for the purpose of describing particular embodiments only and is not intended to limit 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.

[0039] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. It should be further understood that terms as defined in common dictionaries should be interpreted as having the meaning consistent with their meaning in the context of this application and the relevant field, and should not be interpreted in an idealized or overly formal sense, unless expressly 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 limit the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of a conflict of terminology, this specification shall prevail.

[0040] As used in this article, “and / or” means and covers any and all possible combinations of one or more related enumerations, as well as combinations that do not exist when interpreted as alternative (“or”).

[0041] Unless the context otherwise requires, the various features of the invention described herein are particularly intended to be used in any combination. Furthermore, the invention contemplates that in some embodiments of the invention, any feature or combination of features set forth herein may be excluded or omitted. For illustrative purposes, if the specification indicates that a complex comprises components A, B, and C, it is particularly intended that any one or a combination of A, B, or C may be omitted and discarded.

[0042] As used herein, the transitional phrase “consisting essentially of…” (and grammatical variations) should be interpreted as including “the listed materials or steps, as well as those that do not materially affect the essential and novel features of the claimed invention.” See also In re Herz , 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original text); see also MPEP § 2111.03. Therefore, the term “essentially composed of” as used herein should not be interpreted as equivalent to “containing”.

[0043] It should also be understood that, as used herein, the terms “instance,” “exemplary,” and their grammatical variations are intended to refer to non-limiting instance and / or variant implementations discussed herein, and are not intended to indicate that one or more implementations discussed herein are preferred over one or more other implementations.

[0044] As used herein, the term "approximately" when referring to measurable values ​​such as amount or concentration is intended to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, in addition to the specified value itself. For example, "approximately X" where X is a measurable value is intended to include X as well as variations of X of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. The ranges provided herein for measurable values ​​may include any other ranges and / or individual values ​​therein.

[0045] When used herein with respect to chemical molecules, "derivative" means a chemical molecule in which one or more atoms (e.g., hydrogen), functional groups, and / or bonds are modified (e.g., removed, substituted, etc.) compared to a parent molecule. For example, a derivative of a dye may refer to a parent dye compound in which one or more atoms (e.g., hydrogen) and / or functional groups are modified (e.g., removed) to promote covalent bonding with another group or portion (e.g., to promote covalent bonding with a polymer). In some embodiments, the derivative may include functional groups (e.g., substituents and / or auxochromes) that alter the absorption spectrum of the parent molecule.

[0046] As used herein, "alkyl" alone or as part of another group refers to a straight-chain or branched hydrocarbon containing 1 to 20 carbon atoms, which may be referred to as a C1-C20 alkyl group. Representative examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, etc. As used herein, "low-carbon alkyl" is a subset of alkyl groups and, in some embodiments, refers to a straight-chain or branched hydrocarbon group containing 1 to 4 carbon atoms. Representative examples of low-carbon alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, etc. Unless otherwise stated, the term "alkyl" or "lower alkyl" is intended to include substituted and unsubstituted alkyl or lower alkyl groups, and these groups may be substituted with groups selected from the following: halogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclic, heterocyclic, hydroxyl, alkoxy (thereby forming polyalkoxy groups, such as polyethylene glycol), alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkoxy, aryloxy, arylalkoxy, heterocyclic alkoxy, heterocyclic alkoxy, mercapto, alkyl-S(O) m alkyl haloide-S(O) m Alkenyl-S(O) m , alkynyl-S(O) m cycloalkyl-S(O) m , cycloalkylalkyl-S(O) m aryl-S(O) m arylalkyl-S(O) m Heterocyclic -S(O) m Heterocyclic alkyl-S(O) m Amino, carboxyl, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocyclic amino, heterocyclic alkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, wherein m = 0, 1, 2 or 3.

[0047] As used alone or as part of another group herein, “alkenyl” refers to a straight-chain or branched hydrocarbon containing 2 to 20 carbon atoms (or 2 to 4 carbon atoms in lower alkenyl groups), which may include 1 to 8 double bonds in the positive chain and may be referred to as a C2-C20 alkenyl. Representative examples of alkenyl groups include, but are not limited to, vinyl, 2-propenyl, 3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl, 2,4-heptadiene, etc. Unless otherwise stated, the terms “alkenyl” or “lower alkenyl” are intended to include substituted and unsubstituted alkenyl or lower alkenyl groups, and these groups may be substituted with groups as described above with respect to alkyl and lower alkyl groups.

[0048] As used alone or as part of another group herein, "alkynyl" refers to a straight-chain or branched hydrocarbon containing 2 to 20 carbon atoms (or 2 to 4 carbon atoms in a low-carbon alkynyl group), comprising one triple bond in the positive chain, and may be referred to as a C2-C20 alkynyl. Representative examples of alkynyl groups include, but are not limited to, 2-propynyl, 3-butynyl, 2-butynyl, 4-pentynyl, 3-pentynyl, etc. Unless otherwise stated, the terms "alkynyl" or "low-carbon alkynyl" are intended to include substituted and unsubstituted alkynyl or low-carbon alkynyl groups, and these groups may be substituted with the same groups described above with respect to alkyl and low-carbon alkyl groups.

[0049] As used in this article, “halogen” refers to any suitable halogen, including -F, -Cl, -Br and -I.

[0050] As used in this article, "thiol group" refers to the -SH group.

[0051] As used in this article, "azido group" refers to the -N3 group.

[0052] As used in this article, "cyano" refers to the -CN group.

[0053] As used in this article, "hydroxyl group" refers to the -OH group.

[0054] As used in this article, "nitro" refers to the -NO2 group.

[0055] As used herein alone or as part of another group, “alkoxy” refers to an alkyl or lower alkyl group as defined herein (and therefore includes substituted forms such as polyalkoxy) attached to a parent molecule via an oxygen-O- group. Representative examples of alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentoxy, hexoxy, etc.

[0056] When used alone or as part of another group, “acyl” refers to the -C(O)R group, where R is any suitable substituent, such as aryl, alkyl, alkenyl, ynyl, cycloalkyl, or other suitable substituent as described herein.

[0057] As used herein alone or as part of another group, “haloalkyl” means at least one halogen as defined herein attached to a parent molecule via an alkyl group. Representative examples of haloalkyl groups include, but are not limited to, chloromethyl, 2-fluoroethyl, trifluoromethyl, pentafluoroethyl, 2-chloro-3-fluoropentyl, etc.

[0058] When used alone or as part of another group, "alkathio" refers to an alkyl group as defined herein, attached to a parent molecule via a thio group as defined herein. Representative examples of alkathio groups include, but are not limited to, methylthio, ethylthio, tert-butylthio, and hexylthio.

[0059] As used herein, "cycloalkyl" alone or as part of another group refers to a saturated or partially unsaturated cyclic hydrocarbon group containing 3 to 20 carbon atoms (optionally, as discussed below, carbon atoms in the heterocyclic group are substituted). A cycloalkyl group may include 0, 1, 2, or more double or triple bonds. Representative examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and cyclododecyl. These rings may optionally be substituted with other substituents as described herein, such as halogens or lower carbon alkyl groups. Unless otherwise stated, the term "cycloalkyl" is generic and intended to include heterocyclic groups discussed below.

[0060] As used alone or as part of another group herein, "heterocyclic group" or "heterocyclic group" refers to aliphatic (e.g., fully or partially saturated heterocyclic group) or aromatic (e.g., heteroaryl) monocyclic or bicyclic ring systems. Examples of monocyclic ring systems are any 5- or 6-membered rings containing 1, 2, 3, or 4 heteroatoms independently selected from oxygen, nitrogen, and sulfur. 5-membered rings have 0-2 double bonds, while 6-membered rings have 0-3 double bonds. Representative examples of monocyclic systems include, but are not limited to, azacyclobutane, azacyclohexane, aziridine, diazacyclohexane, 1,3-dioxolane, dioxacyclohexane, dithiazide, furan, imidazole, imidazoline, imidazoline, isothiazole, isothiazolin, isothiazolin, isoxazole, isoxazoline, isoxazoline, morpholine, oxadiazole, oxadiazolin, oxadiazolin, oxazole, oxadiazolin, oxadiazolin, piperazine, piperidine, pyran, pyrazine, pyrazole, pyrazolin, pyrazolidine, pyridine, pyrimidine, pyridazine, pyrrole, pyrroleline, pyrrole, tetrahydrofuran, tetrahydrothiophene, tetrazine, tetrazolium, thiadiazole, thiadiazolin, thiadiazolin, thiazolidine, thiazoline, thiazoline, thiazoline, thiophene, thiomorpholine, thiomorpholine sulfone, thiaran, triazine, triazole, trithiazide, etc. Examples of bicyclic systems are any of the aforementioned monocyclic systems fused with an aryl group, a cycloalkyl group, or another monocyclic system as defined herein. Representative examples of bicyclic systems include, but are not limited to, benzimidazole, benzothiazole, benzothiadiazole, benzothiophene, benzoxadiazole, benzoxazole, benzofuran, benzopyran, benzothiaran, benzodioxin, 1,3-benzodioxane, cyclopentene, cyclophosphine, indazole, indole, dihydroindole, inazine, naphthidine, isobenzofuran, isobenzothiaphene, isoindole, isoindoleline, isoquinoline, phthalazine, purine, pyranopyridine, quinoline, quinazine, quinoxaline, quinazoline, tetrahydroisoquinoline, tetrahydroquinoline, thiaranopyridine, etc. These rings include their quaternized derivatives and may optionally be substituted with groups selected from: halogen, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heterocyclic, heterocyclic, hydroxyl, alkoxy, alkenyloxy, alkynyloxy, haloalkoxy, cycloalkoxy, cycloalkylalkoxy, aryloxy, arylalkoxy, heterocyclic alkoxy, heterocyclic alkoxy, mercapto, alkyl-S(O) m alkyl haloide-S(O) m Alkenyl-S(O) m , alkynyl-S(O) m cycloalkyl-S(O) m , cycloalkylalkyl-S(O) m aryl-S(O) m arylalkyl-S(O) m Heterocyclic -S(O) m Heterocyclic alkyl-S(O) mAmino, alkylamino, alkenylamino, alkynylamino, haloalkylamino, cycloalkylamino, cycloalkylalkylamino, arylamino, arylalkylamino, heterocyclic amino, heterocyclic alkylamino, disubstituted amino, acylamino, acyloxy, ester, amide, sulfonamide, urea, alkoxyacylamino, aminoacyloxy, nitro or cyano, wherein m = 0, 1, 2 or 3.

[0061] As used alone or as part of another group herein, “aryl” refers to a monocyclic carbocyclic system or a bicyclic carbocyclic fused-ring system having one or more aromatic rings. Representative examples of aryl groups include, but are not limited to, azulel, indanyl, indenyl, naphthyl, phenyl, tetrahydronaphthyl, etc. Unless otherwise stated, the term “aryl” is intended to include substituted and unsubstituted aryl groups, and these groups may be substituted with the same groups described above with respect to alkyl and lower alkyl groups.

[0062] When used alone or as part of another group, “arylalkyl” means an aryl group as defined herein that is attached to the parent molecule via an alkyl group as defined herein. Representative examples of arylalkyl groups include, but are not limited to, benzyl, 2-phenylethyl, 3-phenylpropyl, 2-naphth-2-ylethyl, etc.

[0063] As used in this article, "amino" refers to the group –NH2.

[0064] When used alone or as part of another group, “alkylamino” refers to the group –NHR, where R is alkyl.

[0065] When used alone or as part of another group, “ester” refers to the -C(O)OR group, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0066] As used in this article, "formyl group" refers to the -C(O)H group.

[0067] As used in this article, "carboxylic acid" refers to the –C(O)OH group.

[0068] As used herein, “sulfonyl” refers to a compound of the formula –S(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0069] As used herein, “sulfonyl” refers to a compound of the formula –S(O)(O)R, where R is any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0070] As used herein, “sulfonate” 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.

[0071] As used in this article, "sulfonic acid" refers to compounds of the formula –S(O)(O)OH.

[0072] When used alone or as part of another group, "amide" refers to -C(O)NR. a R b Group, wherein R a and R b It can be any suitable substituent, such as alkyl, cycloalkyl, alkenyl, alkynyl, or aryl.

[0073] When used alone or as part of another group, "sulfonamide" refers to -S(O)2NR. a R b Group, wherein R a and R b It can be any suitable substituent, such as H, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, heteroalkyl, or heteroaryl.

[0074] As used herein, "polymer backbone" refers to a polymer formed by the polymerization of monomers comprising one or more hydrophobic monomers and one or more hydrophilic monomers. In some embodiments, one or more additional monomers may be included to form the polymer backbone, including monomers containing dyes, bulky groups, or charged groups, and / or monomers having reactive groups that can be used to attach dyes after polymerization. However, the polymer backbone does not include the first or second end groups of the compounds of the present invention, which may be formed during the polymerization of the one or more hydrophobic monomers and one or more hydrophilic monomers (e.g., by atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT)) and / or by subsequent modification of the polymer backbone.

[0075] As used herein, "terminal group" refers to a structural portion that terminates at one end (e.g., an end) of the polymer backbone. The compounds of the present invention comprise two terminal groups located at opposite ends of the polymer backbone, and each terminal group may comprise one or more dyes, bioconjugating groups, linkers, bulk groups, charged groups, and / or biomolecules. In some embodiments, one or both terminal groups of the compound comprise reactive groups (e.g., in telechelic polymers), and in some embodiments, one or both terminal groups are formed during polymerization using living polymerization techniques such as ATRP or RAFT. In some embodiments, the reactive terminal group may react to covalently link one or more dyes, bioconjugating groups, linkers, bulk groups, charged groups, and / or biomolecules, whereby the one or more dyes, bioconjugating groups, linkers, bulk groups, charged groups, and / or biomolecules become part of the terminal group. In some embodiments, the second terminal group is hydrogen, and the final portion of the compound is a unit of the polymer backbone (e.g., a hydrophilic or hydrophobic unit). In some embodiments, the second terminal group comprises a non-reactive end-capped terminal group attached to a unit of the polymer backbone. Exemplary non-reactive end-capped groups include, but are not limited to, isobutyronitrile groups. In some embodiments, the end group, such as a charged group, may comprise a non-reactive end-capped group.

[0076] A "side-linked functional group" is a functional group that is directly connected to the polymer backbone or directly connected to a portion of the polymer backbone. Side-linked functional groups can be part of hydrophobic units and / or monomers and / or hydrophilic units and / or monomers during polymerization, or they can be added to hydrophobic units and / or hydrophilic units after polymerization.

[0077] The terms “dye” and “chromophore” are used interchangeably herein to refer to luminescent (e.g., fluorescent and / or phosphorescent molecular entities) and / or non-luminescent (e.g., non-fluorescent and / or non-phosphorescent molecular entities). As used herein, the term “non-luminescent molecular entity” refers to a molecular entity that does not emit light or has negligible luminescence. In some embodiments, the non-luminescent molecular entity does not form excited states with any significant lifetime and / or relaxes rapidly and substantially quantitatively to the ground state. In some embodiments, the non-luminescent molecular entity has excited-state lifetimes of less than about 100, 75, 50, 25, 10, 5, 1, 0.5, or 0.1 picoseconds. In some embodiments, the non-luminescent molecular entity has quantum yields of internal transitions greater than about 0.8, 0.85, 0.9, 0.95, 0.99, 0.999, 0.9999, or 0.99999, where a quantum yield of 1.0 corresponds to 100%. In some embodiments, the non-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 originates from the competing processes of radiative decay versus the sum of all processes used to reduce the number of excited-state manifolds. Such compounds are often referred to as “non-luminescent,” although sensitive detection techniques can typically detect trace amounts of residual luminescence at such low luminescence quantum yields as expected. A small amount of luminescence may not be detrimental to some applications, such as photoacoustic imaging methods, although it is desirable to convert light input into thermal output as much as possible. Therefore, the term “non-luminescent” is used herein to indicate a molecular entity that does not emit light or has negligible luminescence. In some embodiments, the compounds of the present invention comprise 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, the compounds of the present invention comprise a dye, and the dye is a luminescent group (e.g., a fluorescent and / or phosphorescent molecular entity). The terms "fluorescent molecular entity" and "fluorophore" are used interchangeably herein to refer to a molecular entity that emits fluorescence.

[0078] The dyes of the present invention may have certain spectral characteristics and / or properties, such as those applicable to the methods of the present invention. In some embodiments, the dye has a molecular weight in the range of about 150 Da to about 3,000 Da, about 400 Da to about 1,100 Da, or about 300 Da to about 1,000 Da. In some embodiments, the dye has 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, tetrapyrrole; rylenes such as perylene, terrylene, and quarterrylene; fluoresceins such as TET (tetramethylfluorescein), 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE), 6-carboxyfluorescein (HEX), and 5-carboxyfluorescein (5-FAM); phycoerythrin; halogen dyes; coumarin dyes; rhodamine dyes such as 6-carboxy-X-rhodamine (ROX), Texas Red, and N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); cyanide dyes such as indocyanine green; phthalocyanine; boron-dipyrrole methylene (BODIPY) dyes; quinoline; pyrene; acridine; stilbene; and their derivatives. In some embodiments, the dye is tetrapyrrole, which includes porphyrin, dihydroporphyrin, and chlorophyll and their derivatives.Exemplary tetrapyrroles include, but are not limited to, U.S. Patent Nos. 6,272,038; 6,451,942; 6,420,648; 6,559,374; 6,765,092; 6,407,330; 6,642,376; 6,946,552; 6,603,070; 6,849,730; 7,005,237; 6,916,982; 6,944,047; 7,884,280; 7,332,599; 7,148,361; 7,022,862; 6,924,375; 7,501,507; and 7,323,5 61; 7,153,975; 7,317,108; 7,501,508; 7,378,520; 7,534,807; 7,919,770; 7,799,910; 7,582,751; 8,097,609; 8,187,824; 8,207,329; 7,633,007; 7,745,618; 7,994,312; 8,278,340; 9,303,165; and 9,365,722; and those described in International Applications PCT / US17 / 47266 and PCT / US17 / 63251. In some embodiments, the dye is hydrophobic. In some embodiments, the dye is hydrophilic. In some embodiments, the dye may be linked to and / or incorporated to a monomer that polymerizes with one or more different monomers (e.g., with hydrophobic and / or hydrophilic monomers). In some embodiments, the dye is a luminescent group (i.e., a material and / or compound that can emit light and does not specify the nature of its initial state (e.g., singlet, triplet, and / or another state)). Exemplary luminescent groups include, but are not limited to, phosphorescent and / or fluorophores that respectively provide phosphorescence and / or fluorescence. In some embodiments, the dye may be selected based on the end-use application. Exemplary applications of the dye include, but are not limited to, the applications exemplified in Table 12. Similarly, the amount of dye may be adjusted based on a variety of factors, including, for example, targeted and untargeted methods, polymer size, quantum yield, total polymer charge, and the performance of the monomer equivalent per dye in a multi-dye fold. The studies described in the operational examples provide further discussion and guidance to enable those skilled in the art to manufacture and use dye-loaded polymers.

[0079] The terms "primary dye" and "secondary dye" are used to distinguish two different dyes and are not intended to indicate any order or preference. In some embodiments, the compounds of the present invention may include more than two different dyes such that a third dye, a fourth dye, etc., may be present in the end groups and / or side-attached to the polymer backbone. Modifiers such as "first," "second," "third," etc., may also be used to distinguish elements (e.g., the end groups of the compound), and this is also not intended to indicate any order or preference, but only to distinguish different elements.

[0080] As used herein, "biomolecule" can be any biologically useful molecule or part, including, for example, single-stranded DNA, double-stranded DNA, RNA, oligonucleotides, proteins (e.g., antibodies), and / or peptides, or functional portions and / or fragments thereof. Biomolecules can be linked (e.g., directly or indirectly covalently bound) to the ends or end groups of the polymer backbone of the compounds of the present invention to become part of the end group. In some embodiments, the first and / or second end groups comprise biomolecules. In some embodiments, the end groups of the polymer backbone of the compounds of the present invention comprise bioconjugating groups, and the biomolecules can react with the bioconjugating groups, thereby optionally linking (e.g., covalently bound) the biomolecules to the polymer backbone via a portion of the bioconjugating groups.

[0081] As used herein, a “connector” refers to a chemical part that connects two parts, such as a bulk group, a charged group, a dye, and / or a biomolecule. Regarding the first end group of some compounds of the present invention, a “linear connector” refers to a connector that connects a first part (e.g., a dye, charged group, bulk group, or biomolecule) to a second part (e.g., the same or different charged group, bulk group, dye, or biomolecule) in a linear manner as shown in FIG. 1, and the linear orientation can be further extended by connecting one or more additional dyes, charged groups, bulk groups, and / or biomolecules (e.g., in a linear manner). A “bifunctional connector” refers to a connector, as shown in FIG. 1, that includes a branch point thereon on which two parts (e.g., the same or different dyes, or a dye and a biomolecule, or a bulk group and a charged group) are connected, such that the branch point is (directly or indirectly) connected to the polymer backbone. One or more additional dyes, charged groups, and / or biomolecules can be connected to the bifunctional connector and / or the part connected thereto (e.g., with a linear connector). A "trifunctional connector" is a connector that includes a branch point thereon to which three parts (e.g., the same or different dyes and / or biomolecules) are attached, such that the branch point is (directly or indirectly) connected to the polymer backbone. One or more additional dyes, charged groups, and / or biomolecules may be further attached to the connector and / or the parts thereto (e.g., using linear connectors). A "multifunctional connector" is a connector that includes a branch point thereon to which two, three, four, or more parts (e.g., the same or different dyes, charged groups, and / or biomolecules) are attached. In some embodiments, an exemplary multifunctional connector is a dendron linker.

[0082] As used herein, a “bulky group” refers to a portion that, when attached to a compound of the present invention, prevents or reduces the reactivity of the compound or a portion thereof due to steric hindrance. In one exemplary embodiment, a bulky group is a group having two or more atoms with higher substitutions such that one or more atoms are brought closer together on the bulky group and / or have a steric hindrance effect on the compound or a portion thereof. In some embodiments, the bulky group is a polysaccharide, such as a cyclodextrin (CD), including but not limited to α-CD, β-CD, and / or α-CD. See, for example, Wang et al., ACSCent. Sci. 2022, 8, 5, 663-669; doi:10.1021 / acscentsci.2c00478. In some embodiments, the bulky group is a three-dimensional cage-like molecule comprising a silicon-oxygen framework bonded to one or more different organic groups, such as a polyhedral oligomeric silsesquioxane (POSS). In some embodiments, the bulky group is a POSS or a derivative thereof, for example, a POSS having the following structure: Each R is independently selected from substituted or unsubstituted C1-C8 alkyl groups (e.g., methyl, ethyl, propyl, butyl (e.g., isobutyl)), alkylamino, alkoxy, and -O-Si-(R) groups. 1 ), where R 1 It is a substituted or unsubstituted C1-C8 alkyl group (e.g., isobutyl, alkylamino, alkoxy).

[0083] As used herein, a “charged group” refers to a chemical moiety that is positively charged (e.g., has an electron-donating tendency) and / or negatively charged (e.g., has an electron-attracting tendency). In some embodiments, the charged group has a positive or negative charge, optionally wherein the charged group has a total positive or total negative charge. In some embodiments, the charged group has an electron-donating tendency. Exemplary charged groups include, but are not limited to, negatively charged sulfonates (e.g., 2-acrylamido-2-methylpropanesulfonic acid (AMPS)), succinates, carboxylic acids, halogen-containing groups (e.g., groups containing F, Cl, Br and / or I), and groups containing O, N and / or S. In some embodiments, the charged group is a cationic polymer, such as poly(dimethylamino)ethyl methacrylate (DMAEMA), which may optionally be quaternized. See, for example, Yañez-Macias et al., (2017), Macromol Chem and Phys, 218, 10; doi:10.1002 / macp.201700065.

[0084] In some embodiments, the charged group is an alkyl sulfoxide, such as an alkyl sulfoxide having the following structure: .

[0085] In some embodiments, the charged group is a carboxylic acid, such as a carboxylic acid having the following structure: .

[0086] In some embodiments, the charged group is a sulfonate, such as a sulfonate having the following structure: .

[0087] Compounds, compositions and biomolecules The compounds of this invention include polymeric fluorophores. The compounds of this invention comprise: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end of the polymer backbone; and a second end group attached to a second end 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 bioconjugating group. In some embodiments, one or more additional dyes are side-attached to the polymer backbone.

[0088] According to embodiments of the invention, a compound (e.g., a polymer) is provided comprising: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end of the polymer backbone, wherein the first end group optionally comprises a major dye (e.g., a luminescent group (e.g., a fluorophore) or a non-luminescent molecular entity), wherein optionally the dye has a molecular weight in the range of about 150 Da to about 3,000 Da; a second end group attached to a second end of the polymer backbone, wherein the second end group optionally comprises a bioconjugating group; and one or more additional major dyes side-attached to the polymer backbone; wherein at least the first end group or the second end group comprises a major dye, wherein the compound optionally has a molecular weight in the 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, for example 5,000 Da, 10,000 Da, 15,000 Da. Molecules of 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 approximately 350,000 Da. In some embodiments, the molecular weight varies depending on the polymer backbone and the number of dye molecules attached. The term "primary dye" is used to indicate that each primary dye is the same dye. One or more secondary (or tertiary, etc.) dyes (i.e., different dye molecules) may also be side-attached to the polymer backbone.

[0089] 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 bioconjugated group. In some embodiments, one or more dyes are side-attached to the polymer backbone. In some embodiments, one or more dyes are side-attached to the first end of the polymer, and may also comprise any of the aforementioned groups (e.g., charged groups, hydrophobic groups).

[0090] According to embodiments of the present invention, a compound (e.g., a polymer) is provided comprising: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end 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 end of the polymer backbone, optionally wherein the second end group comprises a bioconjugating group; and one or more major dyes side-attached to the polymer backbone, optionally wherein each major dye (e.g., a luminescent group (e.g., a fluorophore) or a nonluminescent molecular entity) independently has a molecular weight in the 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 major dye is side-attached to the polymer backbone, optionally wherein the compound has a molecular weight in the range of about 5,000 Da, or 10,000 Da to about 175,000 Da, at most about 175,000 Da, at most about 225,000 Da. Molecular weights in the range of up to approximately 250,000 Da, up to approximately 275,000 Da, up to approximately 300,000 Da, up to approximately 325,000 Da, or up to approximately 350,000 Da, for example, 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 approximately 350,000 Da. The term "major dye" is used to indicate that each major dye present in the compound is the same dye; for example, more than one identical dye is side-attached to the polymer backbone. One or more secondary (or tertiary, etc.) dyes (which are different from the primary dyes) may also be side-attached to the polymer backbone.

[0091] For additional primary dyes side-attached to the polymer backbone, in some embodiments, one or more hydrophobic units comprise the dye and / or charged groups. In some embodiments, one or more hydrophilic units comprise the dye. In some embodiments, one or more additional monomers copolymerize with both hydrophobic and hydrophilic monomers, and in some embodiments, such additional units formed therefrom comprise the dye and / or charged groups. In some embodiments, additional monomers generate units comprising functional groups capable of reacting with dye molecules after polymerization to attach the dye to the polymer backbone.

[0092] In some embodiments of the invention, the compound comprises 2 to 80 dye molecules or any range thereof, such as 2 to 70 dye molecules, 2 to 60 dye molecules, 2 to 50 dye molecules, 2 to 40 dye molecules, 2 to 30 dye molecules, 2 to 20 dye molecules, for example 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 side-attached thereto. In some embodiments, the first end group and / or polymer backbone of the compound comprises bulky groups and / or charged groups, and the polymer backbone has the following order: 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, 4 0, 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 are side-attached thereto, which may be a primary dye, or a combination of a primary dye, a secondary dye, and / or a tertiary dye. The hydrophobic and hydrophilic units in the compound may be any of those described herein.

[0093] In some embodiments, the average number of monomers among the dyes in the compound is between about 10 and 150, about 20 and 130, about 40 and 120, or about 50 and 115, or any range thereof. In some embodiments, as described in the operational examples of the invention, the average number of monomers among the dyes is maximized to maintain the fluorescence quantum yield in water relative to the fluorescence quantum yield of the dye when the compound is present in a hydrophobic solvent.

[0094] According to some embodiments of the present invention, a compound (e.g., a polymer) is provided comprising: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; a first end group attached to a first end of the polymer backbone, wherein the first end group comprises (i) a primary dye (e.g., a luminescent or nonluminescent molecular entity) and a biomolecule (e.g., DNA, RNA, protein, or peptide); (ii) a primary dye and a secondary dye (e.g., a luminescent or nonluminescent molecular entity), and optionally a biomolecule, or (iii) two or more primary dyes, and optionally a biomolecule, wherein optionally the primary dye and / or the secondary dye has a molecular weight in the range of about 150 Da to about 3,000 Da; and a second end group attached to a second end of the polymer backbone, wherein the second end group optionally comprises a bioconjugating group, wherein the compound optionally has a molecular weight in the 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. Molecular weights in the range of up to approximately 275,000 Da, up to approximately 300,000 Da, up to approximately 325,000 Da, or up to approximately 350,000 Da, such as 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 approximately 350,000 Da. According to some embodiments of the invention, compounds (e.g., polymers) are provided comprising: a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; and a first end group attached to a first end 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 combinations thereof. In some embodiments, the invention provides compounds (e.g., polymers) that do not contain (e.g., lack) acceptor dyes and donor dyes, for example, acceptor dyes at the ends and one or more donor luminescent groups on the polymer backbone.

[0095] In some embodiments, the primary and / or secondary dyes are Fe(II)-chelate tetrapyrrole or Cu(II)-chelate tetrapyrrole (e.g., porphyrin). In some embodiments, the primary and secondary dyes are each fluorescent dyes. In some embodiments, the primary and secondary dyes are each non-fluorescent dyes. In some embodiments, the primary dye is a fluorescent dye and the secondary dye is a non-fluorescent dye.

[0096] In some embodiments of the invention, the compound comprises a linear linker between a hydrophobic group, a bulky and / or charged group, and the polymer backbone; between a primary dye and the polymer backbone; between a primary dye and a biomolecule; between a primary dye and a 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; between a bulky and / or charged end group; between the polymer backbone and the primary dye and / or biomolecule; between the primary dye and the biomolecule; between the primary dye and the secondary dye; or between two primary dyes. Furthermore, in some embodiments, the compound comprises a trifunctional branched linker between the polymer backbone and two hydrophobic groups or two bulky end groups and charged end groups, between the polymer backbone and hydrophobic groups or bulky end groups and two charged end groups, between the polymer backbone and a primary dye, a secondary dye, and / or a biomolecule; between a primary dye, a secondary dye, and a biomolecule; between two primary dyes and a biomolecule; between two primary dyes and a secondary dye; between a primary dye, a 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, secondary dye, and biomolecule; between two primary dyes and biomolecules; between two primary dyes and secondary dyes; between the primary dye, secondary dye, and tertiary dye; between three primary dyes; between the polymer backbone and the primary dye, secondary dye, tertiary dye, and / or biomolecule; between the primary dye, secondary dye, tertiary dye, and biomolecule; between three primary dyes and biomolecules; between three primary dyes and secondary dyes; between the primary dye, secondary dye, tertiary dye, and quaternary dye; or between four primary dyes.

[0097] In some embodiments, the connector is located between the primary dye and the polymer backbone or optionally the biomolecule, between the primary dye and the secondary dye, or between two or more primary dyes, and the connector comprises or is composed of portions having the following structures: -(CH2) m R(CH2) n in: m is an integer from 0 to 3 (e.g., 0, 1, 2, or 3). n is an integer from 0 to 3, and R is selected from -OC(O)-, C(O)O-, , -CH(OH)CH2R 1 -、 , Or -C(O)NH-, where R 1 It is N or O; or -(CH) m R(CH2) n in: m is an integer from 0 to 3. n is an integer from 0 to 3, and R is -N-, -NNH-, -S-, or ;or -Ph-C(O)NH(CH2) x C(O)NH-、-Ph-C(O)NH(CH2) x C(O)-、-Ph-C(O)NH(CH2) x C(O)- or -Ph-N(CH3)C(O)(CH2) x C(O), where Ph = phenyl and x is between 0 and 10.

[0098] In some embodiments, the linear connector, bifunctional connector, or trifunctional connector according to the present invention comprises a PEG portion and a second portion having the following structure: -(CH2) m R(CH2) n in: m is an integer from 0 to 3. n is an integer from 0 to 3, and R is selected from -OC(O)-, C(O)O-, , -CH(OH)CH2R 1 -、 , Or -C(O)NH-, where R 1 It is N or O; or -(CH2) m R(CH2) n in: m is an integer from 0 to 3. n is an integer from 0 to 3, and R is -N-, -NNH-, -S-, or ;or -Ph-C(O)NH(CH2) x C(O)NH-、-Ph-C(O)NH(CH2) x C(O)-、-Ph-C(O)NH(CH2) x C(O)- or -Ph-N(CH3)C(O)(CH2) x C(O), where Ph = phenyl and x is between 0 and 10; Optionally, the PEG portion is covalently connected at a connection point of the second portion.

[0099] In some embodiments, the connector comprises or is composed of portions having the following structures: -(CH2) m R1(CH2) n Where m is an integer from 1 to 3; n is an integer from 1 to 3; and R 1 Selected from -OC(O)-, -C(O)O-, -CH(OH)CH2R2- , and -C(O)NH,R 2 It is N or O. In some embodiments, the connector comprises or is composed of portions having the following structures: -(CH) m R1(CH2) n Where m is an integer from 0 to 2; n is an integer from 1 to 3; and R1 is -N-, -NNH-, -S-, and .

[0100] In some implementations, the connector is replaced by a PEG molecule. Other exemplary connectors are shown... Figure 4 middle.

[0101] Exemplary linkers that may be present in the compounds of the present invention include, but are not limited to, hydrocarbon moieties, peptide-like moieties, amino acid moieties (e.g., lysine), oligomeric polyethylene glycol groups, triazine (e.g., 1,3,5-triazine), 1,3,5-trisubstituted benzene, self-immolative linkers, and / or polyethylene glycol (PEG) groups. Linkers may be selected to provide connection to another moiety of the compound via carbon-carbon or carbon-heteroatom (e.g., oxygen, sulfur, or nitrogen) bonds. In some embodiments, the linker may be a straight-chain or branched hydrocarbon moieties (e.g., alkyl moieties) and / or a carrier protein. In some embodiments, the linker may be substituted with one or more substituents, such as, but not limited to, unsubstituted or substituted aryl, alkylamino, alkoxy, or heterocyclic groups. Additional exemplary linkers are shown in Figure I.

[0102] Figure I: Exemplary connector that can be used in compounds of the present invention The linker of the present invention may optionally be replaced by a PEG molecule. In some embodiments, the linker may have PEG molecules at one or both ends, optionally wherein the linker is between the primary dye and the polymer backbone or biomolecule, between the primary dye and a secondary dye, or between two or more primary dyes. In some embodiments, the compounds of the present invention comprise –(CH2CH2O). x – the connector, where x is an integer from 1, 5, 10, 25, or 50 to 55, 75, or 100.

[0103] As used herein, a “hydrophilic unit” refers to a segment or unit of a polymer backbone containing hydrophilic (e.g., ionic and / or polar) functional groups (e.g., hydrophilic side-attached functional groups), optionally wherein the hydrophilic functional groups are located at the end of a moiety. As those skilled in the art will understand, a portion of a hydrophilic unit may be hydrophobic, such as a portion that forms the polymer backbone when polymerized with other monomers and / or a portion containing ionic functional groups (e.g., a hydrocarbon chain), but if it contains hydrophilic functional groups, it is still referred to as a hydrophilic monomer. In some embodiments, the hydrophilic unit is formed from a hydrophilic monomer (e.g., a monomer having the same hydrophilic functional groups). However, in some embodiments, the unit is modified post-polymerization to add hydrophilic functional groups, thereby forming the hydrophilic unit.

[0104] As used herein, a “hydrophobic unit” refers to a segment or unit of a polymer containing a hydrophobic functional group (e.g., a hydrophobic side-attached functional group), optionally wherein the hydrophobic functional group is located at the end of a moiety and / or monomer. In some embodiments, the hydrophobic functional group is a hydrocarbon moiety (e.g., alkyl, aryl, or arylalkyl). In some embodiments, the hydrophobic unit is formed from a hydrophobic monomer (e.g., a monomer having the same hydrophobic functional group). However, in some embodiments, the unit is modified post-polymerization to add a hydrophobic functional group, thereby forming the hydrophobic unit. In some embodiments, the hydrophobic unit or the hydrophobic functional group of the hydrophobic unit or a derivative thereof (referred to herein as a hydrophobic group) may be contained on a first end of the polymer as described herein. For example, a polymer containing a first end group containing a thiol can be further modified by attaching a hydrophobic group.

[0105] The hydrophobic and / or hydrophilic units of the polymer may contain side functional groups. These side functional groups may be part of the hydrophobic units and / or monomers and / or hydrophilic units and / or monomers during polymerization, or may be added to the hydrophobic and / or hydrophilic units after polymerization. In some embodiments, the side functional groups may be added to the hydrophobic and / or hydrophilic units after polymerization (e.g., post-polymerization functionalization). In some embodiments, the side functional groups contain charged groups. In some embodiments, the polymer may contain charged groups (positive or negative) at one or more locations along the main chain and / or at the ends. In some embodiments, the side functional groups may contain a positive charge, for example, at the hydrophobic units of the main chain or at the side functional groups (e.g., PFPs). In some embodiments, the side functional groups may contain a negative charge, for example, at AMPS attached to the hydrophobic units of the main chain or at the side functional groups (e.g., PFPs). In one exemplary embodiment, one or more side functional groups along the main chain may contain a negative charge, such as AMPS attached to hydrophobic units or side functional groups (e.g., PFP) of the main chain, and have multiple dyes along the main chain, such as 2, 3, 4, 5, 6, 7, 8, or more dyes. In some embodiments, the side functional groups may contain a negative charge, such as sulfonate groups (e.g., AMPS) at the ends of the polymer. In one exemplary embodiment, the compound containing side functional groups may contain one or more negative charges, such as sulfonate groups (e.g., AMPS) at the ends of the polymer, and multiple dyes along the main chain, such as 2, 3, 4, 5, 6, 7, 8, or more dyes. In some embodiments, the side functional group is a halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., active esters such as pentafluorophenyl ester, succinimidyl ester, 2,4-dinitrophenyl ester, etc.), azide, pentafluorophenyl, succinimidyl, fluorophenyl, maleimide, isocyanate, or isothiocyanate. In some embodiments, the side functional group is a hydrophilic group comprising a terminal cation (e.g., ammonium), anion (e.g., sulfonate, phosphate, carboxyl), or zwitterionic group (e.g., choline or choline-like group (e.g., choline derivatives)) group and optionally a poly(ethylene glycol) moiety and / or unit. In some embodiments, the hydrophilic group is attached to the poly(ethylene glycol) moiety and / or unit, optionally to the terminal portion of the poly(ethylene glycol) moiety and / or unit.

[0106] In some embodiments, the hydrophobic unit comprises a side functional group containing an alkyl (e.g., dodecyl), aryl, or arylalkyl group, and / or the hydrophilic unit comprises a side functional group containing a glycol (e.g., polyethylene glycol), a sulfonic acid, and / or a sulfonate. In some embodiments, the hydrophobic unit is prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer, and / or the hydrophilic unit is prepared from a glycol acrylate (e.g., PEG acrylate) monomer. In some embodiments, the compounds of the present invention comprise at least one hydrophobic unit prepared from an alkyl acrylate (e.g., dodecyl acrylate) monomer and at least two different hydrophilic units, including a first hydrophilic unit prepared from a glycol acrylate (e.g., PEG acrylate) monomer and a second repeating hydrophilic unit prepared from a sulfonate acrylamide monomer (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or a sulfonate acrylate monomer.

[0107] In some embodiments, one or more hydrophobic units and / or one or more hydrophilic units may contain charges (e.g., positive or negative charges) and / or charged groups (e.g., cationic or anionic groups), and the charges may suppress non-specific binding to the compound or a portion thereof (e.g., a portion of the polymer backbone). In some embodiments, the total charge of the polymer containing charged groups and / or charged hydrophobic units and / or charged hydrophilic units is neutral. In some embodiments, the total charge of the polymer containing charged groups and / or charged hydrophobic units and / or charged hydrophilic units is negative.

[0108] In some embodiments of the present invention, the polymer backbone comprises one or more Figure 2 The hydrophobic unit shown is illustrated. In some embodiments of the invention, the polymer backbone includes one or more hydrophobic units comprising aryl or arylalkyl functional groups, including but not limited to one or more... Figure 3A and 3B The unit shown. In some embodiments, the polymer backbone comprises polyfluorophenyl acrylate.

[0109] In some embodiments, the polymer backbone may comprise one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophobic units and one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophilic units. The polymer backbone may be prepared by any type of polymerization using one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophobic monomers and one or more (e.g., 1, 5, 10, 50, 100 or more) hydrophilic monomers to provide a polymer comprising the one or more hydrophobic units and the one or more hydrophilic units. In some embodiments, the polymer backbone may be prepared by two or more (e.g., 2, 3, 4, 5 or more) hydrophobic monomers that are different from each other and / or two or more (e.g., 2, 3, 4, 5 or more) hydrophilic monomers that are different from each other. For example, in some embodiments, the compounds of the present invention may be prepared by at least one hydrophobic monomer, at least one first hydrophilic monomer, and at least one second hydrophilic monomer, wherein the first hydrophilic monomer and the second hydrophilic monomer are different from each other.

[0110] The one or more hydrophobic units and the one or more hydrophilic units may be randomly distributed in the polymer. In some embodiments, the polymer is a random copolymer. The polymer backbone may be an amphiphilic random copolymer, optionally a linear amphiphilic random copolymer. In some embodiments, the one or more hydrophobic units and the one or more hydrophilic units may be present in the polymer at a ratio of approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (hydrophobic unit: hydrophilic unit). In some embodiments, the ratio of hydrophobic units to hydrophilic units is approximately 1:4 to approximately 1:6. The length of the polymer backbone may be varied and / or controlled. In some embodiments, the molecular weight of the polymer backbone is in the range of approximately 1,000 Da to approximately 175,000 Da, approximately 5,000 Da to approximately 350,000 Da, approximately 10,000 Da to approximately 350,000 Da, approximately 20,000 Da to approximately 350,000 Da, approximately 35,000 Da to approximately 350,000 Da, approximately 35,000 Da to approximately 300,000 Da, approximately 10,000 Da to approximately 175,000 Da, approximately 20,000 Da to approximately 175,000 Da, approximately 28,000 Da to approximately 175,000 Da, approximately 28,000 Da to approximately 35,000 Da, approximately 28,000 Da to approximately 50,000 Da, approximately 100,000 Da to approximately 150,000 Da, and approximately 50,000 Da. The molecular weight ranges from about 130,000 Da to about 10,000 Da, or from 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 from about 28 kDa to about 175 kDa. In some embodiments, the polymer backbone has a molecular weight from about 28 kDa to about 35 or 50 kDa.

[0111] In some embodiments, the degree of polymerization (DP) of the polymer is between 10 and 500, for example, between 30 and 450, 40 and 400, 50 and 350, 60 and 325, 70 and 300, or any range thereof. In some embodiments, the number of dye molecules per polymer may depend in part on the degree of polymerization of the polymer.

[0112] In some embodiments, the polymer is a random polymer comprising at least two monomers having comparable polymerization rates (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)) which is further modified after polymerization to randomly introduce additional functional groups. See, for example, Example 2G.

[0113] In some embodiments, the polymer is a gradient polymer comprising at least two monomers with different reactivity ratios. In some embodiments, the polymer comprises segments or portions of the polymer as a gradient polymer; thus, the polymer may comprise portions or segments having one monomer or two or more monomers with comparable reactivity ratios, followed by portions or segments comprising a gradient comprising at least two monomers with different reactivity ratios.

[0114] In some embodiments, the polymer backbone comprises at least one hydrophobic unit having a structure represented by Formula III: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R' is absent or is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or C5-C25 arylalkyl; R 2 It is hydrogen or halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azide, maleimide, isocyanate, or isothiocyanate, or R 2 It is a dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

[0115] In some embodiments, R in the compound of formula III 2It is a hydroxyl, carboxyl, amino, formyl, or ester group. In some embodiments, R in the compound of formula III... 2 It is a vinyl, epoxy, mercapto, azide, isocyanate, isothiocyanate, or maleimide group, which may optionally be added and / or provided before polymerization and / or through post-polymerization functionalization. In some embodiments, R in the compound of formula III 2 It is hydrogen. In some embodiments, R' in the compound of formula III is C2-C4 alkyl, C2-C6 alkyl, C4-C20 alkyl, C6-C20 alkyl, C8-C16 alkyl, C8-C18 alkyl, C10-C14 alkyl, or C10-C12 alkyl. In some embodiments, R' in the compound of formula III is C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, or C20 alkyl, alkenyl, or alkynyl. In some embodiments, R' in the compound of formula III is a C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 alkyl. In some embodiments, R' in the compound of formula III is a C3-C5 cycloalkyl, C3-C6 cycloalkyl, C4-C20 cycloalkyl, C6-C20 cycloalkyl, C8-C16 cycloalkyl, C8-C18 cycloalkyl, C10-C14 cycloalkyl, or C10-C12 cycloalkyl. In some embodiments, R' in the compound of formula III is a C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, 19, or C20 cycloalkyl. In some embodiments, R' in the compounds of Formula III is a C5-C20 aryl, C5-C16 aryl, C5-C14 aryl, or C5-C10 aryl. In some embodiments, R' in the compounds of Formula III is a C6-C25 arylalkyl, C6-C20 arylalkyl, or C8-C18 arylalkyl. In some embodiments, the compounds of the present invention comprise at least one hydrophobic unit having the structure of Formula III, wherein R... 2 It is a dye, and the compound contains a bulky group and / or a charged group at one end of the compound.

[0116] In some implementations, the hydrophilic unit may have a structure represented by Formula IV: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1It does not exist or is either –O–, –NH–, or –CH2–; R 3 Choose freely – (CH2CH2R) 5 ) n The group consisting of -, -C1-C6 alkyl, -C1-C6 alkyl-O- and –C1-C6 alkyl-SO3- or their salts, wherein R 5 It is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000; R 4 The absence of hydrogen, alkyl, phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester (e.g., pentafluorophenyl ester, succinimidyl ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) groups, or optionally R 4 It is a dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

[0117] In some embodiments, R in the compound of formula IV 4 It is a hydroxyl, carboxyl, amino, formyl, or ester group, optionally when R 3 Yes – (CH2CH2R) 5 ) n -, -C1-C6 alkyl or -C1-C6 alkyl-O-. In some embodiments, when R in the compound of formula IV... 3 It is a -C1-C6 alkyl-O- or -(CH2CH2R) 5 ) n - (where R) 5 When it is -O-), R 4 It can be hydrogen, alkyl (e.g., methyl or ethyl), phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), or phosphoryl. In some embodiments, when R in the compound of formula IV... 3 It is a -C1-C6 alkyl group or -(CH2CH2R) 5 ) n - (where R) 5 When it is –CH2–, R 4 It can be hydroxyl, carboxyl, amino, ammonium, formyl, ester, phosphonyl, or sulfonyl. In some embodiments, R in the compound of formula IV... 4It is hydrogen, alkyl, phosphonoyl, sulfonyl, phosphatidylcholine, phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester group. In some embodiments, R in the compound of formula IV 4 It is a vinyl, epoxy, mercapto, azide, isocyanate, isothiocyanate, or maleimide group, which may optionally be added and / or provided before polymerization and / or through post-polymerization functionalization. In some embodiments, when R in the compound of formula IV... 3 When it is a –C1-C6 alkyl-SO3- or its salt, R 4 It is hydrogen or absent. In some embodiments, R in the compound of formula IV 3 It is a salt of –C1-C6 alkyl-SO3- (e.g., a sodium salt), and R 4 It does not exist. In some embodiments, R in the compound of formula IV 3 Yes – (CH2CH2R) 5 ) n - In some embodiments, the compounds of the present invention comprise at least one hydrophilic unit having the structure of formula IV, wherein R 4 It is a dye, and the compound contains a bulky group and / or a charged group at one end of the compound.

[0118] In one exemplary embodiment, the compounds of the present invention may comprise a PFP ester acrylate polymer and may further comprise dodecylamine and PEG (e.g., polyetheramine). In some embodiments, the PFPA polymer may further comprise charged groups on the main chain and / or ends of the polymer, which may be positively and / or negatively charged. In some embodiments, the charged groups on the main chain are positive, while the charged groups at the ends are negative. In some embodiments, the charged groups on the main chain are negative, while the charged groups at the ends are positive.

[0119] In some embodiments, the compounds of the present invention may comprise and / or a telechelic polymer, which is a polymer or prepolymer capable of further polymerization or other reactions via one or more of its reactive end groups. In some embodiments, the compounds of the present invention may comprise and / or a heterotelechelic polymer, which is a polymer or prepolymer capable of further polymerization or other reactions via reactive end groups at each end of the polymer or prepolymer, and these two reactive end groups are different from each other. In some embodiments, the compounds of the present invention may comprise and / or a homotelechelic polymer, which is a polymer or prepolymer capable of further polymerization or other reactions via reactive end groups at each end of the polymer or prepolymer, and these two reactive end groups are identical to each other. In some embodiments, the compounds of the present invention may comprise and / or a semi-telechelic polymer, which is a polymer or prepolymer capable of further polymerization or other reactions via a reactive end group at one end of the polymer or prepolymer.

[0120] In some embodiments, the compounds of the present invention can be linked to a single biomolecule via a bioconjugating group in a second end group. In some embodiments, such a biomolecule may comprise one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the compounds of the present invention. Therefore, in some embodiments, the biomolecule and / or a portion thereof comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of the compounds of the present invention.

[0121] Bioconjugating groups may optionally be present in the compounds of the present invention (e.g., in the second end group of the compound). The terms "bioconjugable group," "bioconjugable site," or "bioconjugating group" and their grammatical variations refer to a portion and / or functional group that can be used to bind to or conjugate to a biomolecule (e.g., protein, peptide, DNA, RNA, etc.). Therefore, "bioconjugable group," "bioconjugable site," or "bioconjugating group" and their grammatical variations do not contain a biomolecule. However, in some embodiments, a bioconjugating group is used to bind to a biomolecule, or a bioconjugating group or its derivatives bind to a biomolecule (e.g., protein, peptide, DNA, RNA, etc.). Exemplary bioconjugable groups include, but are not limited to, amines (including amine derivatives), such as isocyanates, isothiocyanates, iodoacetamide, azides, diazonium salts, etc.; acids or acid derivatives, such as N-hydroxysuccinimide esters (more generally, active esters derived from carboxylic acids, such as p-nitrophenyl esters), hydrazides, etc.; and other linking groups, such as aldehydes, sulfonyl chlorides, sulfonyl hydrazides, epoxides, hydroxyl groups, thiols, maleimides, aziridines, acryloyl groups, halogen groups, biotin, 2-iminobiotin, etc. Linking groups, such as those mentioned above, are known and described in U.S. Patent Nos. 6,728,129; 6,657,884; 6,212,093; and 6,208,553. For example, the compounds of the present invention may contain a bioconjugable group containing a carboxylic acid, and the carboxylic acid may be used for bioconjugation to biomolecules (e.g., by activation with carbodiimide and coupling with amino-substituted biomolecules).

[0122] In some embodiments, the biomolecule may comprise and / or proteins (e.g., antibodies and / or carrier proteins), peptides, DNA, RNA, etc. In some embodiments, the biomolecule may comprise portions (e.g., polymers) that optionally include one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more) binding sites to the compounds of the present invention. In some embodiments, the biomolecule may be a member of a specific binding pair. The terms "specific binding pair" and "ligand-receptor binding pair" are used interchangeably herein and refer to two different molecules, one of which has a specific spatial or polar region on its surface or in a cavity that specifically attracts or binds to the other molecule, such that the two molecules have an affinity for each other. Members of a specific binding pair may 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 receptor. Examples of ligand-receptor binding pairs include, but are not limited to, hormones and hormone receptors, such as epidermal growth factor and epidermal growth factor receptor, tumor necrosis factor-α and tumor necrosis factor receptor, and interferon and interferon receptor; avidin and biotin or antibiotin; antibody and antigen pairs; enzymes and substrates; drugs and drug receptors; cell surface antigens and lectins; two complementary nucleic acid chains; nucleic acid chains and complementary oligonucleotides; interleukins and interleukin receptors; and stimulatory factors and their receptors, such as granulocyte-macrophage colony-stimulating factor (GMCSF) and GMCSF receptor, and macrophage colony-stimulating factor (MCSF) and MCSF receptor.

[0123] In some embodiments, the compounds of the present invention, or portions thereof, have a non-rigid backbone (e.g., a non-rigid polymer backbone) and / or exhibit conformational flexibility. The conformational flexibility of the molecular chain can be described and quantified by the "persistence length" of the compound or portions thereof (e.g., polymer portions). In some embodiments, the persistence length of the compounds of the present invention can be approximately equal to the length of a given carbon-carbon bond.

[0124] The compounds of the present invention may be self-folding, for example, self-folding in water and / or aqueous solutions. As used herein, "self-folding" means that a compound transforms from a partially or fully extended or unfolded structure into a structure in which at least a portion of the extended or unfolded structure folds upon contact with a solution (e.g., an aqueous solution) or the compound, and that the folding is innate because it occurs spontaneously upon contact with the solution (i.e., without external control or force). In some embodiments, the compounds of the present invention self-fold upon contact with water and / or aqueous solutions. The compounds of the present invention may optionally self-fold into a monomeric micelle structure upon contact with water and / or aqueous solutions. The aqueous solution in which the compounds of the present invention fold can be a buffer solution, such as a phosphate buffer (e.g., phosphate-buffered saline). In some embodiments, the aqueous solution (e.g., an aqueous buffer) in which the compounds of the present invention fold are folded may have a low ionic strength; for example, the aqueous solution may have a μ value of about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 160 mM to about 180 mM, or about 160 mM to about 170 mM. In some embodiments, the aqueous solution in which the compounds of the present invention fold are folded may have a μ value of less than about 100 mM. In some embodiments, the aqueous solution in which the compounds of the present invention fold are folded may contain 1 M NaCl. In some embodiments, the aqueous solution in which the compounds of the present invention fold are folded may contain less than 1 M NaCl, such as less than about 0.75 M, 0.5 M, or 0.25 M NaCl. In some embodiments, the aqueous solution in which the compounds of the present invention fold are folded may contain 10 mM NaH2PO4 and 150 mM NaCl, and have a pH of about 7.35.

[0125] In some embodiments, the compounds of the present invention may be in particulate form. The compounds of the present invention may form particles, for example, upon contact with a solution (e.g., an aqueous solution). In some embodiments, a single (i.e., one) compound may form particles. Thus, the compounds and particles exist in an approximately 1:1 ratio (i.e., one compound per particle).

[0126] The compounds of the present invention may be contained in a portion of one or more hydrophobic units in the core or internal region of a particle and / or in a portion of one or more hydrophilic units in the outer periphery or external region (e.g., shell) of the particle. In some embodiments, the particle has a micellar structure (e.g., a monomeric micellar structure). The compounds of the present invention may contain one or more dyes, which, when the compound is in a folded structure and / or particle form (e.g., a monomeric micellar structure), may be encapsulated by a portion of the compound (e.g., a portion of a polymer). In some embodiments, one or more dyes or a portion thereof and one or more hydrophobic units may be present in the core or internal region of the particle, and one or more hydrophilic units may surround the dye and / or one or more hydrophobic units.

[0127] In some embodiments, one or more hydrophilic units comprise nonionic (i.e., neutral / uncharged) side-joint functional groups (e.g., PEG) and / or are formed from nonionic monomers (e.g., PEG acrylate (PEGA)). In some embodiments, one or more hydrophilic units comprise ionic (e.g., anionic, charged) side-joint functional groups (e.g., sulfonic acids and / or sulfonates) and / or are formed from ionic monomers (e.g., sulfonate acrylates (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). In some embodiments, the hydrophilic unit is formed from at least two different monomers, such as nonionic (i.e., neutral / uncharged) hydrophilic monomers (e.g., PEG acrylate (PEGA)) and ionic (e.g., anionic, charged) hydrophilic monomers (e.g., sulfonate acrylates (e.g., 2-acrylamido-2-methylpropanesulfonic acid)). As those skilled in the art will understand, monomers containing acids (e.g., sulfonic acids) may be present in acidic form and / or in their ionic form. In some embodiments, the monomers containing acids are predominantly (i.e., greater than 50%) in their ionic form. In some embodiments, the ionic hydrophilic monomer is an acid in a deprotonated form (e.g., a deprotonated sulfonate acrylate) and / or an acid in a salt form, such as sodium sulfonate acrylate (e.g., 2-acrylamido-2-methylpropanesulfonic acid as a sodium salt).

[0128] In some embodiments, when two or more different hydrophilic units are present in the polymer of the present invention, the ratio of the two or more different hydrophilic units can vary, for example, from about 10:1 to about 1:10. For example, in some embodiments, the polymer comprises nonionic (i.e., neutral / uncharged) hydrophilic units (e.g., formed from polyethylene glycol-modified methyl acrylate (PEGA)) and ionic (e.g., anionic, charged) hydrophilic units (e.g., formed from sulfonated acrylates (e.g., 2-acrylamido-2-methylpropanesulfonic acid)) in a ratio of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (nonionic unit: ionic unit) to ionic units.

[0129] In some embodiments, the polymer of the present invention comprises approximately 1% to approximately 40% hydrophobic units and approximately 60% to approximately 99% hydrophilic units based on the total molar amount of the monomers used to prepare the polymer. In some embodiments, the polymer of the present invention comprises approximately 1%, 5%, 10%, 15%, or 20% to approximately 25%, 30%, 35%, or 40% hydrophobic units and approximately 60%, 65%, 70%, 75%, or 80% to approximately 85%, 90%, 95%, or 99% hydrophilic units based on the total molar amount of the monomers used to prepare the polymer. In some embodiments, the polymer comprises approximately 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 approximately 30% (e.g., less than approximately 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 approximately 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% hydrophilic units based on the total molar amount of monomers used to prepare the polymer. In some embodiments, the polymer comprises more than about 70% (e.g., more than about 75%, 80%, 85%, 90%, or 95%) hydrophilic units based on the total molar amount of monomers used to prepare the polymer.

[0130] Based on the total weight of the polymer, the polymer of the present invention may have a weight fraction of hydrophobic units ranging from approximately 1%, 5%, 10%, 15%, or 20% to approximately 25%, 30%, 35%, or 40%. In some embodiments, based on the total weight of the polymer, the polymer may have a weight fraction of hydrophobic units ranging from approximately 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%. In some embodiments, based on the total weight of the polymer, the polymer may have a weight fraction of hydrophobic units less than approximately 30% (e.g., less than approximately 25%, 20%, 15%, 10%, or 5%).

[0131] Based on the total weight of the polymer, the polymer of the present invention may have a weight fraction of hydrophilic units ranging from approximately 60%, 65%, 70%, 75%, or 80% to approximately 85%, 90%, 95%, or 99%. In some embodiments, based on the total weight of the polymer, the polymer of the present invention may have a weight fraction of hydrophilic units ranging from approximately 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%. In some embodiments, based on the total weight of the polymer, the polymer may have a weight fraction of hydrophilic units greater than approximately 70% (e.g., greater than approximately 75%, 80%, 85%, 90%, or 95%).

[0132] In some embodiments, the amount of monomeric micelle structures formed upon contact with the solution is approximately 50% to approximately 100%, approximately 75% to approximately 100%, approximately 85% to approximately 100%, or approximately 95% to approximately 100% of the total polymer added to the solution, optionally measured using a size determination method such as dynamic light scattering (DLS) spectroscopy. In some embodiments, the amount of monomeric micelle structures formed when the polymer contacts the solution is approximately 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, optionally measured using a size determination method (e.g., dynamic light scattering (DLS) spectroscopy). The solution in which the monomers are present can be an aqueous solution as described herein, such as an aqueous buffer solution. In some embodiments, the aqueous solution in which the monomers are present is a phosphate buffer solution (e.g., phosphate-buffered saline). In some embodiments, the aqueous solution (e.g., an aqueous buffer) in which the monomers are present has a low ionic strength (e.g., may have a μ value of about 100 mM to about 250 mM, about 100 mM to about 200 mM, about 150 mM to about 250 mM, about 160 mM to about 180 mM, or about 160 mM to about 170 mM). In some embodiments, the aqueous solution in which the monomers are present contains 10 mM NaH2PO4 and 150 mM NaCl and has a pH of about 7.35.

[0133] In some embodiments, dilution of a solution containing a compound of the invention in the form of a monomeric micelle structure results in no loss or a loss of less than about 20% of the monomeric micelle structure present in the solution compared to the amount of monomeric micelle structure present in the solution before dilution. In some embodiments, the amount of monomeric micelle structure present in the solution remains unchanged 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% after dilution compared to the amount of monomeric micelle structure present in the solution before dilution.

[0134] In some embodiments, solutions of the compounds of the invention comprising a monomeric micelle structure contain 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%). Therefore, at least 50% or more of the compound is unaggregated and may be in the form of a monomeric micelle structure. In some embodiments, dilution of a solution of the compounds of the invention comprising a monomeric micelle structure results in little or no additional aggregate formation compared to the amount of aggregates present in the solution before dilution. In some embodiments, the amount of aggregates present in a solution containing the compound of the present invention remains unchanged 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% after dilution, compared to the amount of aggregates present in the solution before dilution. In some embodiments, the diluted solution contains less than about 50% of the 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%).

[0135] The compounds of the present invention may have a diameter in water and / or aqueous solutions ranging from about 1 nm to about 50 nm or from about 3 nm to about 30 nm (e.g., when folded, as in a monomeric micelle structure). In some embodiments, the compound is used for non-targeted applications and has a diameter of about 20 nm or less. In some embodiments, the compound may have a diameter of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nm (e.g., when folded, as in a monomeric micelle structure). In some embodiments, the compound of the present invention may be in the form of particles (i.e., at least partially folded structures).

[0136] In some embodiments, the compounds of the present invention are cross-linked, optionally wherein the compound is cross-linked when it has a folded structure. In some embodiments, the compounds of the present invention can be cross-linked in a solution (e.g., an aqueous solution) and / or can be cross-linked with a cross-linking agent. Cross-linking of the compounds of the present invention may include linking two or more portions and / or functional groups (e.g., side-attached functional groups) of hydrophobic and / or hydrophilic units together. Cross-linking can provide the compound with a folded structure that cannot unfold without breaking one or more bonds formed by cross-linking. The degree or amount of cross-linking can be controlled, altered, and / or adjusted, for example, by the amount of cross-linking agent reacting with the compound. In some embodiments, the cross-linking step of the compound may include reactive and / or reactive entities (e.g., functional groups) as listed in Table 1.

[0137] Table 1: Exemplary crosslinking agent reactions and functional groups reaction Functional groups polymerization Olefins polymerization acrylate Thiol-ene reaction Thiol group + olefin azide-acetylene reaction Azide group + alkyne Thiol-maleimide reaction Thiol group + maleimide Hydroxyl group + glutaraldehyde Hydroxyl + Aldehyde Amine + glutaraldehyde Amino + Aldehyde Disulfide formation Thiol + Thiol amide formation amine + carboxylic acid ester formation Hydroxyl + Carboxylic Acid Acetourethrogenesis Carbodiimide + Carboxylic Acid hydrazone formation Acylhydrazine + aldehyde Alkoxy alcohol formation Epoxide + Alcohol .

[0138] In some embodiments of the compounds of the present invention, the fluorescence quantum yield of the dye in water and / or aqueous solutions may be reduced by approximately 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 in a hydrophobic solvent (e.g., toluene). After bioconjugation of the compounds of the present invention with a biomolecule (e.g., a protein), the fluorescence quantum yield of the dye may be the same as or substantially the same as the fluorescence quantum yield of the dye in water and / or hydrophobic solvents (e.g., within ±20%). In some embodiments, if the fluorescence quantum yield of the dye is 1.00 (theoretical maximum), a reduction of 10-fold or less (e.g., approximately 10, 9, 8, 7, 6, 5, 4, 3, 2-fold or less) may be acceptable. However, in some embodiments, due to the high performance of the compounds manufactured according to the disclosure herein, the fluorescence quantum yield of the dye can be reduced by about 30%, 40%, 50% or more relative to the fluorescence quantum yield of the dye in water and / or hydrophobic solvents when the compound is present in water and / or aqueous solutions, while maintaining desirable properties, such as brightness.

[0139] In some embodiments, the compounds of the present invention are water-soluble. The compound may have a solubility in water at room temperature of at least 1 mg / mL, for example, from 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, the compositions of the present invention are contained in a solution, such as water, an aqueous solution, and / or a hydrophobic solvent.

[0140] In some embodiments, the compounds of the present invention comprise a gradient structure. In compounds having a gradient structure, the gradient structure may comprise a portion of the main chain, such as 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or any range thereof, or substantially the entire main chain, such as 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 monomers comprise different copolymerization rates. In some embodiments where the gradient comprises a portion of the main chain, the main chain comprises a gradual transition between units, such as between hydrophobic and hydrophilic units in the polymer main chain, or between two different hydrophobic units or two different hydrophilic units.

[0141] 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 monomeric units, such as hydrophilic units (e.g., PEGMA), or a combination of hydrophilic and hydrophobic units (e.g., CDA and PEGMA). In some embodiments, the degree of polymerization of the first segment is about 10 to 100, about 20 to 80 monomeric units, or any range thereof. In some embodiments, the second segment comprises hydrophilic units (e.g., PEGMA) or a combination of hydrophilic and hydrophobic units (e.g., CDA and PEGMA) present in the first segment, and may further comprise hydrophobic units (e.g., PFPA). In some embodiments, the degree of polymerization of the second segment is about 30 to 300, about 40 to 280, about 60 to 240 monomeric units, or any range thereof.

[0142] In some embodiments, a method for producing polymers having two distinct segments is provided. In some embodiments, an initial monomer feedstock consisting of one or two classes of substances is provided, and the initial monomer feedstock is reacted. After the initial monomer feedstock has reacted, after a period of time, such as 30 minutes to 50 hours, for example about 30 minutes or 1, 2, 5, 10, 25, or 50 hours or any range thereof, another distinct monomer is loaded into a reaction flask. Advantageously, this method achieves separate segments with different compositions within a single reaction vessel.

[0143] In some embodiments, the compounds and / or particles of the present invention are dilution-resistant. As used herein, “dilution-resistant” means that the compound and / or particles retain their structure and / or properties. In some embodiments, dilution-resistant means that the compound and / or particles retain a folded structure (e.g., a monomeric micelle structure), which can be determined by measuring the diameter of the particles before and after dilution, and the diameter after dilution can be maintained within ±50%, 40%, 30%, 20%, 10% or less of the diameter before dilution. In some embodiments, dilution-resistant means that the compound and / or particles maintain the fluorescence quantum yield of the diluted dye within ±50%, 40%, 30%, 20%, 10% or less of the fluorescence quantum yield of the dye before dilution. In some embodiments, the compounds and / or particles of the present invention retain a folded structure when diluted to 25x, 50x, 75x, or 100x or when diluted to submicromolar concentrations.

[0144] According to embodiments of the invention, compositions comprising the compounds of the invention are also provided. In some embodiments, the composition is an aqueous solution, including any aqueous compositions described herein. In some embodiments, the composition does not contain organic solvents.

[0145] Methods for manufacturing compounds According to some embodiments of the present invention, methods for preparing the compounds and / or compositions of the present invention are provided. Exemplary methods for copolymerization and end-group modification can be found in WO 2019 / 126144 (PCT / US2018 / 066195) and WO 2021 / 118782 (PCT / 2020 / 061285), the disclosures of which are incorporated herein by reference in their entirety.

[0146] In some embodiments, the method of preparing the compound includes copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers to provide a polymer backbone comprising one or more hydrophobic units and one or more hydrophilic units; optionally attaching a major dye (e.g., a luminescent group or a non-luminescent molecular entity) to a first end group of the compound; and optionally attaching at least one additional major dye to a functional group side-attached to the polymer backbone, optionally attaching a bioconjugating group to a second end group of the compound; and optionally crosslinking the compound. In some embodiments, the major dye is attached to the first end group after the at least one additional major dye is attached to the functional group side-attached to the polymer backbone.

[0147] In some embodiments of the invention, the method of preparing the compound includes copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers to provide a compound comprising a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units, a first end group, and a second end group; attaching a first major dye (e.g., a luminescent group or a non-luminescent molecular entity) to the first end group to become part of the first end group; attaching a second major dye, a minor dye, and / or a biomolecule to another portion of the first major dye and / or the first end group; optionally attaching a bioconjugating group to the second end group; and / or optionally crosslinking the compound. The attachment of the dye and / or biomolecule can be direct or indirect, such that a linker can be present between the dye, the biomolecule, and the end group. The order in which the dye and / or biomolecule are attached in the first end group can also be changed, such that in some embodiments, the biomolecule can be attached to the first end group before the major dye.

[0148] In some embodiments, a method for preparing the compounds of the present invention includes copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers to provide a polymer backbone comprising one or more hydrophobic units and one or more hydrophilic units; optionally attaching a bulk group (e.g., cyclodextrin (CD)) or a charged group (e.g., sulfonate) to a first end group of the compound; and optionally attaching at least one major dye to a functional group side-attached to the polymer backbone, optionally attaching a bioconjugating group to a second end group of the compound; and optionally crosslinking the compound. In some embodiments, the bulk group (e.g., CD) or the charged group (e.g., sulfonate) is attached to the first end group after the at least one additional major dye is attached to a functional group side-attached to the polymer backbone.

[0149] In some embodiments, a method for preparing the compounds of the present invention includes providing a polymer backbone comprising one or more hydrophobic units and one or more hydrophilic units; optionally attaching a charged group (e.g., a sulfonate) to one or more sites along the backbone of the compound; and attaching at least one major dye to a functional group, optionally a charged group, optionally a bioconjugating group to a second end group of the compound; and optionally crosslinking the compound. In some embodiments, the charged group (e.g., a sulfonate) may be attached to a first end group.

[0150] In some embodiments, a method for preparing the compounds of the present invention includes copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers to provide a compound comprising a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units, a first end group, and a second end group; attaching a bulk group (e.g., CD) or a charged group (e.g., sulfonate) to the first end group such that the bulk group or charged group is part of the first end group; optionally attaching a bioconjugating group to the second end group and / or optionally crosslinking the compound. The bulk and / or charged groups and / or biomolecules may be directly or indirectly attached to a portion of the compound such that a connector exists between the bulk group, charged group, and / or biomolecule and a portion of the compound. The order in which the bulk group and / or charged group are attached in the first end group can be varied.

[0151] In some embodiments, one or more compounds of the present invention may be prepared according to the exemplary methods described in the operational examples. Although the operational examples describe the connection of the dye to, for example, dendrons or bifunctional junctions, bulky and / or charged groups may be used instead of the dye.

[0152] Hydrophobic and hydrophilic monomers can be polymerized using any method known to those skilled in the art, such as, but not limited to, condensation reactions (e.g., reactions with diols and diacids) and / or living radical polymerization (e.g., atom transfer radical polymerization (ATRP) or reversible addition-fragmentation chain transfer (RAFT)). In some embodiments, the polymerization of hydrophobic and hydrophilic monomers is carried out using a method for providing copolymers in which one or both end groups are reactive (i.e., one or both end groups of the copolymer are capable of entering further polymerization or reaction), and the two end groups may be the same or different. In some embodiments, the polymerization of hydrophobic and hydrophilic monomers is achieved by living radical polymerization (e.g., ATRP) in the presence of an initiator (e.g., a bromide initiator), a catalyst (e.g., a ruthenium catalyst), and optionally a co-catalyst to provide copolymers. In some embodiments, the polymerization of hydrophobic and hydrophilic monomers is achieved by living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT reagent (e.g., a thiocarbonyl thio compound).

[0153] In some embodiments, the polymer backbone is formed by copolymerizing hydrophobic monomers (which can be used to provide hydrophobic units for the polymer backbone as described herein), the hydrophobic monomers having a structure represented by Formula I: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R' is absent or is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or C6-C25 arylalkyl; R 2 It is hydrogen or halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azide, maleimide, isocyanate or isothiocyanate, or R 2 It is a dye. The specific R and R' values ​​described above regarding the hydrophobic unit... 1 , R' and R 2 The group is also used in this hydrophobic monomer. Typically, R... 2 It is not a dye, because it is usually added to the unit after polymerization, but in some implementations, the dye may be present on the hydrophobic monomer.

[0154] In some embodiments, the hydrophilic monomer (which can be used to provide the hydrophilic unit of the polymer as described herein) may have a structure represented by Formula II: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R 3 Choose freely – (CH2CH2R) 5 ) n The group consisting of -, -C1-C6 alkyl, -C1-C6 alkyl-O- and –C1-C6 alkyl-SO3- or their salts, wherein R 5 It is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; and R 4 The following groups are absent or do not contain hydrogen, alkyl, phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) groups, or R. 4 It is a dye. In some embodiments, the compounds of the present invention comprise at least one hydrophobic unit having the structure of formula IV, wherein R 4 It is a dye, and the compound contains a bulky group and / or a charged group at one end of the compound. The specific R, R... described above regarding the hydrophilic unit... 1 R3 and R 4 The group is also used in this hydrophobic monomer. Typically, R... 4 It is not a dye, because it is usually added to the unit after polymerization, but in some implementations, the dye may be present on the hydrophilic monomer.

[0155] Exemplary reactive end groups of compounds that may allow dye linkage or bioconjugation include, but are not limited to, those described in Table 2. These terminal functional groups are not side-attached functional groups, but may be present at either end of the copolymer. Exemplary reactive end groups of compounds that may allow bulk groups and / or charged groups to be linked include, but are not limited to, thiol functional groups and maleimide groups on bulk groups and / or charged groups on the copolymer, and succinimide functional groups and amino groups on bulk groups and / or charged groups on the copolymer.

[0156] Table 2: Exemplary terminal functional groups (FGs) and exemplary linkages and chemistry on compounds, dyes, or biomolecules. .

[0157] Some functional groups may be unstable under certain polymerization conditions. Therefore, in some embodiments, functional groups may be introduced in a protected form. Thus, these functional groups can be used for dye linking or bioconjugation after deprotection. Exemplary protected forms of certain functional groups include, but are not limited to, those listed in Table 3.

[0158] Table 3: Exemplary Protected Forms of Certain Functional Groups .

[0159] In some embodiments, a portion of the compound (e.g., an end group) may comprise a halogen group (e.g., Cl, Br, I). The halide moiety of the compound may be derivatized with a nucleophile or end-capping reagent to generate a functional group for dye linking or bioconjugation. In some embodiments, a portion of the compound (e.g., an end group) may comprise a thiol group, which may be derivatized with a reagent containing a thiol reactive group to generate a functional group for dye linking or bioconjugation. Examples of thiol reactive groups include, but are not limited to, halides (e.g., bromine, chlorine, iodine), alkynes, aldehydes, vinyl ketones, and / or maleimide functional groups. All functional groups listed in Tables 2 and 3 are compatible with these strategies, and additional exemplary functional groups include, but are not limited to, those listed in Table 4.

[0160] Table 4: Exemplary terminal functional groups (FGs) and exemplary linkages and chemistry on derivatized compounds, dyes, or biomolecules. .

[0161] The polymerization of hydrophobic and hydrophilic monomers (optionally via ATRP or RAFT) may include polymerizing hydrophobic and hydrophilic monomers at a ratio of approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (hydrophobic monomer: hydrophilic monomer). In some embodiments, this ratio may be from approximately 1:1 to approximately 1:3 or approximately 1:6. In some embodiments, the hydrophobic monomer is an alkyl acrylate (e.g., dodecyl acrylate), and / or the hydrophilic monomer is a glycol acrylate (e.g., PEG acrylate). In some embodiments, one or more hydrophobic monomers are polymerized with two or more different hydrophilic monomers (optionally via RAFT or ATRP) at a ratio of approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (hydrophobic monomer: hydrophilic monomer). For example, in some embodiments, the first hydrophilic monomer is ionic (e.g., sulfonate acrylate monomers (e.g., 2-acrylamido-2-methylpropanesulfonic acid) and / or sulfonate monomers), and the second hydrophilic monomer can be nonionic (e.g., glycol acrylates (e.g., polyethylene glycol-modified methyl acrylate)). The ratio of the first hydrophilic monomer to the second hydrophilic monomer can vary (e.g., the ratio of the first hydrophilic monomer to the second hydrophilic monomer can be approximately 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5, or 1:6).

[0162] Exemplary catalysts that can be used in the methods of the present invention include, but are not limited to, ruthenium complexes, iron complexes, copper complexes, nickel complexes, palladium complexes, rhodium complexes, and rhenium complexes. Exemplary ruthenium complexes include, but are not limited to, dichlorotris(triphenylphosphine)ruthenium(II)[RuCl2(PPh3)3], pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II)chloride[RuCp*Cl(PPh3)2], chloro(cyclopentadienyl)bis(triphenylphosphine)ruthenium[RuCpCl(PPh3)2], dihydrotetrahydrotetra(triphenylphosphine)ruthenium(II)[RuH2(PPh3)4], and dichloro(p-cymene)ruthenium(II) dimer. Exemplary iron complexes include, but are not limited to, dichlorobis(triphenylphosphine)fer(II) [FeCl2(PPh3)2], bromo(cyclopentadienyl)dicarbonylfer(II) [FeCpBr(CO)2], and cyclopentadienylferric 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 trifluoromethanesulfonate, cuprous hexafluorophosphate, and cuprous acetate. Exemplary nitrogen-based ligands include, but are not limited to, 2,2'-bipyridine and its derivatives, 1,10-phenanthroline and its derivatives, sparteine, and other diamines, as well as terpyridine and its derivatives. Exemplary nickel complexes include, but are not limited to, dibromobis(triphenylphosphine)nickel(II) [NiBr2(PPh3)2] and tetra(triphenylphosphine)nickel [Ni(PPh3)4]. An exemplary palladium complex is tetra(triphenylphosphine)palladium [Pd(PPh3)4]. An exemplary rhodium complex is rhodium tris(triphenylphosphine)bromide. An exemplary rhenium complex is rhenium dioxobis(triphenylphosphine)iodide. In some embodiments, the catalyst is pentamethylcyclopentadienylbis(triphenylphosphine)ruthenium(II)chloride.

[0163] The co-catalyst may optionally be present in the method of the present invention, for example in the polymerization step of the hydrophobic monomer and the hydrophilic monomer. In some embodiments, the co-catalyst may be present and may be 4-(dimethylamino)-1-butanol.

[0164] In some embodiments, the method of the present invention includes optionally hydrolyzing the compound in the presence of trifluoroacetic acid and water to provide a formyl group at a first portion (e.g., a first terminal group) of the compound. The method may include reacting a dye with the formyl group of the compound to form an hydrazone bond between the dye and the compound, optionally via aldehyde-hydrazide chemistry, thereby attaching the dye to the first terminal group of the compound, thus making it part of the first terminal group. In some embodiments, biomolecules can be linked by reacting the formyl group with an amino group on a bioconjugating group via reductive amination.

[0165] In some embodiments, the method of the present invention includes reacting the compound with thioglycolic acid and triethylamine to provide a carboxymethyl thioether group at a second end group (e.g., a second terminal) of the compound. The carboxymethyl thioether group may be derivatized to provide an N-hydroxysuccinimide ester at a 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.

[0166] In some embodiments, the method of the present invention includes reacting the compound with sodium azide to provide an azide group, and optionally attaching the dye to the azide group via copper-catalyzed azide-alkyne chemistry.

[0167] In some embodiments, the method of the present invention includes RAFT polymerization. In some embodiments, RAFT polymerization occurs in the presence of a free radical initiator (e.g., AIBN) and a RAFT reagent (e.g., a thiocarbonyl thio compound). Other examples of RAFT reagents include, but are not limited to, dithioesters, dithiocarbamates, trithiocarbonates, dithiobenzoates, and / or xanthates.

[0168] In some embodiments, the method of the present invention includes cleaving a thiocarbonyl thio group present at the end of a copolymer obtained using RAFT polymerization. This cleavage can be performed using any common method known in the art. For example, in some embodiments, the thiocarbonyl thio group is cleaved by ammonolysis, for example, in the presence of ethanolamine, to produce a free thiol. In some embodiments, the free thiol can be coupled to a dye containing a maleimide functional group, thereby linking the dye to a first portion (e.g., a first end group) of the compound. In some embodiments, a biomolecule can be attached to the free thiol group of the first portion (e.g., the end group). In some embodiments, a biomolecule can be attached to the opposite end group of the compound.

[0169] In some embodiments, the method of the present invention includes generating a random structure through copolymerization of two monomers (e.g., two or more different acrylate-based monomers) having comparable reactivity ratios. In some embodiments, the method of the present invention includes generating a gradient structure through copolymerization of two monomers with different reactivity ratios. In some embodiments, the method of the present invention includes generating a random structure through polymerization of a single type of functional monomer (e.g., pentafluorophenyl acrylate, PFPA), followed by further post-polymerization modification to randomly introduce desired functional groups.

[0170] In some embodiments, a method for producing polymers having two distinct segments is provided. In some embodiments, an initial monomer feedstock composed of one or two classes of substances is provided, and the initial monomer feedstock is reacted. After the initial monomer feedstock has reacted, another distinct monomer is loaded into a reaction flask after a period of time. Advantageously, this method achieves individual segments with different compositions within a single reaction vessel. In some embodiments, this method avoids emulsion formation to provide advantages over using block copolymers that would form emulsions.

[0171] In some embodiments, multiple dyes, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more dyes, are attached to the polymer backbone via post-polymerization (directly or indirectly). In some embodiments, one or more of the multiple dyes are each attached to the polymer backbone via a covalent bond between the dye (which optionally includes a connector) and a side group attached to the polymer backbone. In some embodiments, each of the multiple dyes is attached to a portion of a polymer backbone containing PFPA via post-polymerization (see, for example...). Figure 8A ).

[0172] How to use The compounds of this invention can be used for both single-purpose and dual-purpose life science applications. For example, in some embodiments, specific compounds of this invention can be selected and optimized for various dual-purpose applications and / or independently optimized for single-purpose applications, which involve and generate 1) Fluorescence 2) Selection and optimization to maximize photoacoustic imaging 3) Selection and optimization for generating reactive oxygen species, such as singlet oxygen species, for photodynamic and photoimmunotherapy. 4) Selection and optimization for PET imaging applications 5) Selection and optimization for magnetic resonance imaging (MRI) applications. For example, certain dyes may be particularly useful for MRI applications, and some instances of them have been shown in [the following text is incomplete and requires further context]. Figure 5A Examples of certain dyes that can be used for photoacoustic imaging are shown in the image. Figure 5B middle.

[0173] In some embodiments of the invention, the compounds of the invention enable a single compound to be used in dual-purpose applications, including, for example... 1. Fluorescent materials used in applications such as fluorescence-guided surgery, flow cytometry, microscopy, and immunoassay. 2. Photoacoustic imaging 3. Photodynamic / Photoimmunotherapy 4. PET imaging 5. MRI imaging Certain application combinations are displayed Figure 6 However, various combinations of dyes can be used for the application, and are described in further detail below, including, for example, in Table 12, to allow one or two or more different dyes to be used for a single application, or two or more different dyes to be used for a dual-purpose application.

[0174] Dual-purpose application The strategies described in Examples 6 (Synthesis of polymers with different dyes at the ends and on the main chain) and 7 (Synthesis of polymers with different dyes at the ends and on the main chain) provide an exemplary method for producing dual-purpose folded bodies, which includes a combination of two dyes that produce two different signals (e.g., fluorescence or ultrasound) and / or outputs (e.g., reactive oxygen species (e.g., singlet oxygen) or heat).

[0175] In one embodiment, a composition suitable for flow cytometry and photoacoustic imaging (PAI) can be synthesized. In some embodiments, a mixture of fluorescent and ultrasonic dyes can be attached to the polymer backbone as described herein (see, for example, Example 6) to enable flow cytometry for fluorescence-based sorting of desired cells. In some embodiments, fluorescent dyes can be attached to the polymer backbone or the ends of the polymer, and dyes that generate ultrasonic signals can be attached to different sites, i.e., the polymer backbone or the ends of the polymer (see, for example, Example 7). The desired cells can then be injected back into an animal or patient, and the same labeled cells can be tracked and analyzed in vivo using photoacoustic ultrasound imaging. Additionally, this dual-purpose fold can be used with a photoacoustic-based flow cytometer to sort cells, and fluorescence or chemiluminescence imaging modalities can be used to track and analyze the labels in vivo.

[0176] In one embodiment, compositions suitable for both optical and fluorescence microscopy applications can be synthesized. In some embodiments, a mixture of fluorescent dyes and chromophore dyes can be linked to the polymer backbone using the description provided herein (see, for example, Example 6). In some embodiments, the fluorescent dye can be linked to the polymer backbone or the end of the polymer, and the chromophore dye can be linked to different sites (e.g., to the polymer backbone or the end of the polymer) (see, for example, Example 7). This mixture allows the user to analyze the same sample using both optical and fluorescence-based microscopes. In some embodiments, the fluorescent dye can be linked to the polymer backbone or the end of the polymer, and the chromophore dye can be linked to different sites (e.g., the polymer backbone or the end of the polymer) to allow the user to analyze the same sample in two different modes.

[0177] In one embodiment, a composition suitable for PAI imaging combined with photodynamic therapy (PDT) dye application can be synthesized. In some embodiments, a mixture of an ultrasound-generating dye and a singlet oxygen-generating PDT dye can be linked to a polymer backbone (see, for example, Example 6) to allow a user to image targeted or untargeted folds in vivo using the PAI dye to confirm appropriate locations. In some embodiments, an ultrasound-generating dye can be linked to the polymer backbone or the end of the polymer, and a singlet oxygen-generating PDT dye can be linked to different sites (e.g., to the polymer backbone or the end of the polymer) (see, for example, Example 7) to allow a user to image targeted or untargeted folds in vivo using the PAI dye to confirm appropriate locations. Once appropriate localization is confirmed, the same or different light sources can be used to generate a cell-killing response from the PDT dye.

[0178] In one embodiment, a composition suitable for use in conjunction with a PDT dye for fluorescence imaging can be synthesized. In some embodiments, a mixture of a fluorescently signaling dye and a singlet oxygen-generating PDT dye can be linked to the polymer backbone, allowing a user to image targeted or untargeted folds in vivo to confirm appropriate locations (see, for example, Example 6). In some embodiments, a fluorescently signaling dye can be linked to the polymer backbone or the end of the polymer, and a singlet oxygen-generating PDT dye can be linked to different sites (e.g., to the polymer backbone or the end of the polymer) (see, for example, Example 7), allowing a user to image targeted or untargeted folds in vivo to confirm appropriate locations. Once appropriate localization is confirmed, the same or different light sources can be used to generate a cell-killing response from the PDT dye.

[0179] According to some embodiments, the compounds and / or compositions of the present invention can be used in flow cytometry. Flow cytometry is known and described, for example, in U.S. Patent Nos. 5,167; 5,915,925; 6,248,590; 6,589,792; and 6,890,487. In some embodiments, the particles to be detected (e.g., cells) are labeled with a luminescent compound (such as the compounds of the present invention) for detection. Labeling can be performed by any suitable technique, such as binding a luminescent compound (e.g., the compounds of the present invention) to particles or cells, for example by an antibody that specifically binds to particles or cells, by taking up or internalizing the luminescent compound into cells or particles, by non-specific adsorption of the luminescent compound onto cells or particles, and so on. The compounds described herein can be used as such luminescent compounds in flow cytometry techniques (including fluorescence-activated cell sorting or FACS) that can be performed based on techniques known to those skilled in the art based on this disclosure or variations thereof.

[0180] In some embodiments, a method for detecting cells and / or particles using flow cytometry is provided, the method comprising labeling cells and / or particles with 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 labeled cells and / or particles to a subject, and detecting the compound in the subject, thereby detecting the cells and / or particles in the subject. Detection of the compound in the subject can be performed, for example, by using imaging techniques such as photoacoustic imaging and / or magnetic resonance imaging. Additional dyes (third dyes, fourth dyes, etc.) can be used, and each can (or may not) be used for different detection methods.

[0181] In some embodiments, a method is provided for detecting tissues and / or reagents (e.g., cells, infection reagents, etc.) in a subject, the method comprising: administering to a subject a compound, biomolecule, and / or composition of the present invention, optionally wherein said compound or biomolecule binds to said tissue and / or reagent; and detecting said compound in the subject, thereby detecting said tissue and / or reagent. Examples of detection methods include, but are not limited to, imaging techniques such as MRI, PAI, and fluorescence spectroscopy. In some embodiments, the compound of the present invention has a primary dye and a secondary dye, and a first detection method (e.g., a first imaging method) is used to detect the primary dye, and a second detection method (e.g., a second imaging method) is used to detect the secondary dye. For example, in some embodiments, MRI is used to detect the primary dye, and PAI is used to detect the secondary dye (i.e., a different dye). The terms "primary" and "secondary" are used only to distinguish different dye molecules, so that the secondary dye can be used for detection by MRI, and the primary dye can be used for detection by PAI.

[0182] In some embodiments, a method of using the compounds of the present invention in photoacoustic imaging is provided. According to some embodiments, the method of the present invention includes a method of performing photoacoustic imaging. The appeal of PAI lies in its detection independent of light emission (Haisch, C., Quantitative analysis in medicine using photoacoustictomography. Anal. Bioanal. Chem. 2009, 393, 473-479; Cox, B.; Laufer, JG; Arridge, SR; Beard, PC Quantitative spectroscopic photoacoustic imaging: a review. J. Biomed. Opt. 2012, 17, 061202). Light emission is affected by light scattering. In PAI, thermoelastic expansion and ultrasonic pressure waves follow laser irradiation (e.g., optionally with a non-ionizing laser pulse). The detection of ultrasonic pressure waves can be achieved via a conventional ultrasonic detector. Essentially, ultrasonic imaging can be performed using laser input. Notably, PAI is independent of ionizing radiation compared to X-ray imaging methods.

[0183] The method of the present invention may include administering the compounds, biomolecules and / or compositions of the present invention to a subject, optionally wherein the compounds or biomolecules bind to tissues and / or cells in the subject; irradiating at least a portion or site of the subject with a laser, optionally wherein the portion or site of the subject contains the compounds of the present invention; and imaging at least the portion or site of the subject, optionally wherein the imaging includes ultrasound imaging.

[0184] PAI can be performed without the application of any exogenous contrast agents or chemical probes. In this case, different absorptions of endogenous chromophores in the native tissue produce different signals. For example, absorption by hemoglobin helps to depict the presence of blood vessels. However, hemoglobin has a low molar absorption coefficient and may not be sufficient for clear depiction in deep tissues. In this case, the use of contrast agents is very attractive. In some embodiments, the compounds of the present invention are used as contrast agents in PAI and / or contain dyes that can be used as contrast agents in PAI.

[0185] A wide variety of substances have been examined for use as contrast agents in photoacoustic imaging (PAI). Exemplary dyes for PAI include, but are not limited to, gold nanomaterials, carbon nanotubes, porphyrins in liposomes, semiconductor polymers, and naphthalocyanines (Chitgupi, U.; Lovell, JF Naphthalocyanines as contrast agents for photoacoustic and multimodal imaging. Biomed. Eng. Lett. 2018, 8, 215–221; de la Zerda, A. et al., Advanced contrast nanoagents for photoacoustic molecular imaging, cytometry, blood test and photothermal theranostics. Contrast Media Mol. Imaging 2011, 6, 346-369). In some embodiments, the dyes present in the compounds of the present invention and / or the compounds of the present invention have the following photophysical properties: after absorbing light, the dye / compound immediately and quantitatively relaxes to the ground state without emitting light or forming any metastable state with a significant lifetime. In other words, the yield of internal conversion (i.e., without radiative decay) should be quantifiable, and ideally, the rate of internal conversion should be exceptionally fast, with excited-state lifetimes less than 1 picosecond. This description essentially expresses "photo-acoustic conversion efficiency" in molecular photophysics (Cheng, K.; Cheng, Z. Near infrared receptor-targeted nanoprobes for early diagnosis of cancers. Curr. Med. Chem. 2012, 19, 4767-4785). The appeal of this rapid and quantifiable internal conversion is the conversion of all absorbed light into heat, i.e., thermal expansion to induce ultrasound. One research group has termed such contrast agents "sonochromes" (Duffy, MJ et al., Towards optimized naphthalocyanines as sonochromes for photoacoustic imaging in vivo. Photoacoustics 2018, 9, 49-61) to distinguish them from the more generally known photopigments or fluorescent pigments or luminescent groups, all of which imply the emission of light after absorbing incident light. In some embodiments, the compounds of the present invention are and / or contain sonochrome.

[0186] In some embodiments, the dyes present in the compounds of the present invention and / or the compounds of the present invention absorb red or near-infrared (NIR) light. For example, in some embodiments, the compounds of the present invention can be used for deep tissue imaging, where absorption in red or NIR is required because this region provides an optical window that allows light to pass through. At shorter wavelengths, absorption by endogenous chromophores (e.g., hemoglobin, melanin) can occur; at longer wavelengths, light scattering caused by the overtone vibration bands of water can be observed. In some embodiments, the dyes present in the compounds of the present invention and / or the compounds of the present invention absorb in red or NIR, and the molar absorptivity is as large as possible to produce high sensitivity, for example, a molar absorptivity of 1,000 M. -1 cm -1 10,000 M -1 cm -1 100,000 M -1 cm -1 Or even higher. In some embodiments, the dye is anthocyanin, such as indocyanine green, with a molar absorptivity of approximately 156,000 M. -1 cm -1 – 223000 M -1 cm -1 Within a certain range. In some embodiments, dihydroporphyrin exhibits a range of approximately 10,000 M. -1 cm -1 Up to approximately 100,000 M - 1 cm -1 Q within the range y Molar absorptivity of the bands. In some embodiments, chlorophyll exhibits an absorptivity of approximately 50,000 M. - 1 cm -1 Up to approximately 200,000 M -1 cm -1 Q within the range y Molar absorption coefficient of the spectral band.

[0187] In some embodiments, the method of the present invention provides multi-wavelength multiplexing. Multi-wavelength multiplexing can be achieved by using two or more absorbers as PAI contrast agents, all of which exhibit quantitative (or near-quantitative) internal switching, wherein the two or more absorbers are two or more different compounds of the present invention. The two or more different compounds of the present invention may have largely non-overlapping absorption bands. Multiplexing can be achieved by scanning the NIR and red spectral regions with an incident light source (e.g., a laser), wherein the generated ultrasound is detected after successive absorption of different contrast agents in each spectrum. Alternatively, a set of multiple lasers can be used, each dedicated to a different PAI contrast agent.

[0188] In some embodiments, the dye present in the compounds of the present invention comprises dihydroporphyrin or chlorophyll, optionally said compound being used in the method of the present invention for PAI. Considering the strong and sharp long wavelength (Q... y The absorption bands of dihydroporphyrin and / or chlorophyll are ideal for photoacoustic imaging. Dihydroporphyrin and / or chlorophyll can be modified to produce high internal conversion yields and / or packaged in a way that allows for solubilization in aqueous media.

[0189] For example, tetrapyrrole macrocycles that fluoresce in their free base form can be made non-fluorescent by metallization with a suitable metal. Tetrapyrroles include porphyrins and hydroporphyrins; the latter include dihydroporphyrins and chlorophyll. A true “periodic chater of metallotetrapyrroles” exists, thanks to extensive work on the preparation and study of metallotetrapyrroles over the past century. Metals that provide non-luminescent tetrapyrrole chelates are well known (see, for example, Gouterman, M. Optical spectra and electronic structure. In The Porphyrins; Dolphin, D. (Ed.), Vol. III, Academic Press: New York, 1978, pp. 1-165). Examples of metals that can provide non-luminescent tetrapyrrole chelates (valence not shown for clarity) include, but are not limited to, Fe, Co, Ni, Cu, Zr, Ru, and the lanthanides. In some embodiments, the dye present in the compounds of the present invention is a tetrapyrrole macrocycle containing iron. Iron may be particularly attractive given its presence as a native component of human metabolism, the extensive research already focused on tetrapyrrole iron (considering the fact that heme is an iron chelate of protoporphyrin IX), and the particularly short excited-state lifetime of iron porphyrins. In some embodiments, the compounds of the present invention comprise ferric dihydroporphyrin or ferric chlorophyll. In some embodiments, the method of the present invention comprises administering the compounds of the present invention comprising ferric dihydroporphyrin or ferric chlorophyll as a PAI contrast agent to a subject and performing photoacoustic imaging. In some embodiments, the dye present in the compounds of the present invention is a tetrapyrrole macrocycle comprising copper (e.g., Cu(II)). In some embodiments, the dye present in the compounds of the present invention comprises copper (e.g., Cu(II)) and is optionally used for photoacoustic imaging. In some embodiments, the dye present in the compounds of the present invention comprises iron (e.g., Fe(II)) and is optionally used for oxygen sensing.

[0190] In some embodiments, the compounds of the present invention comprise Fe(II) tetrapyrrole, which is sterically hindered and / or does not form a μ-oxodimer of Fe(III) tetrapyrrole. In some embodiments, the compounds of the present invention comprise Fe(III) tetrapyrrole. It should be noted that Fe(II) tetrapyrrole can coordinate with molecular oxygen and, if there is no steric hindrance, can undergo a chemical reaction to yield a μ-oxodimer of Fe(III) tetrapyrrole. Conversely, Fe(III) tetrapyrrole does not coordinate with molecular oxygen and does not undergo μ-oxodimer formation. Fe(III) tetrapyrrole is the preferred oxidation state of iron tetrapyrrole when formed under aerobic conditions. Various long-established methods can be used to form Fe(III) tetrapyrrole and to convert Fe(II) tetrapyrrole into the corresponding Fe(III) tetrapyrrole.

[0191] Free base tetrapyrrole can provide a certain amount of fluorescence (e.g., up to ~10% quantum yield), a certain amount of triplet formation (e.g., up to ~70% quantum yield), and the remainder is internal conversion (e.g., up to ~20% quantum yield). As mentioned above, a convenient way to achieve ~100% quantum yield of internal conversion (i.e., no radiative decay) is to metallize tetrapyrrole with a metal, which rapidly and substantially quantitatively relaxes the excited state to the ground state through one or more mechanisms. An alternative method to promote internal conversion relative to radiative decay (i.e., fluorescence) and intersystem crossing (i.e., triplet formation) is to attach a suitable substituent to tetrapyrrole. Typical substituents are those that cause spin-orbit coupling, such as heavier halogens, including bromine, iodine, and astatine. Thus, in some embodiments, the introduction of one or more halogens into the dyes and / or compounds of the present invention can be used alone or in conjunction with a metal that alone provides limited luminescence, thereby providing rapid and substantially quantitative relaxation to the ground state. Such metals include many metals in the periodic table. The metallization method of tetrapyrrole is well known (Buchler, JW Static coordination chemistry of metalloporphyrins. In Porphyrins and Metalloporphyrins; Smith, KM (ed.), 1975, Elsevier Scientific Publishing Co.: Amsterdam, pp. 157–231; Sanders, JKM et al., Axialcoordination chemistry of metalloporphyrins. In The Porphyrin Handbook; Kadish, KM; Smith, KM; Guilard, R. (ed.), Vol. 3, 2000, Academic Press: San Diego, pp. 1–48). Since heavy atoms attached to aromatics are known to induce rapid relaxation of excited states, a wide variety of heavily substituted aromatics are excellent candidates for PAI using the method according to the invention. In some embodiments, the compounds of the invention comprise tetrapyrrole (e.g., tetrapyrrole with heavy atom substituents on the periphery of the macrocycle and / or providing a non-luminescent central chelate metal). Such tetrapyrroles (e.g., dihydroporphyrin or chlorophyll) can provide many possible narrowband absorptions in the red and NIR spectral regions.

[0192] Although various mechanisms have been described for the rapid and substantially quantitative recovery of a compound's excited state to its ground state, the invention is not limited thereto, and other mechanisms known in the art may be used. For example, such mechanisms may arise from (1) a high rate of internal transition relative to the rate of radiative decay and intersystem crossing; (2) a high rate of intersystem crossing relative to the rate of radiative decay and internal transition, followed by immediate and nonradiative decay from the excited multivariate to the ground state; and / or (3) a high rate of charge transfer relative to all other rates of depopulation of the excited state, followed by charge recombination to quantitatively lead to the ground state. Another example is the twisting of the macrocycle from a fundamentally planar structure. Other mechanisms are known to those skilled in the art. Regardless of the mechanism, tetrapyrrole exhibiting an excited state, with an extremely short lifetime and substantially quantitative relaxation to the ground state, can be formed using established methods known to those skilled in the art. Rapid and near-quantitative relaxation to the ground state can provide what is referred to herein as a "non-luminescent" molecular entity, which can be used for PAIs.

[0193] The compounds of the present invention can package a metal tetrapyrrole, optionally for use in PAI. The metal tetrapyrrole may have a biocompatible conjugating group, which can be used to link the metal tetrapyrrole to a polymer as described herein to provide the compounds of the present invention. Therefore, the compounds of the present invention may comprise a single metal tetrapyrrole. In some embodiments, the compounds of the present invention can maintain the inherent spectral characteristics (e.g., absorption spectrum, fluorescence spectrum, fluorescence quantum yield, etc.) of the dye by packaging the dye within a portion of the compound (e.g., within a polymer portion), optionally without alteration by interaction with external entities, such as other dyes and / or biological substances (e.g., cellular components, proteins, etc.). Containing a single dye (e.g., Fe(III) tetrapyrrole) in the compounds of the present invention can maintain the inherent absorption spectrum of the dye.

[0194] According to some embodiments, the dye present in the compounds of the present invention can be a non-luminescent molecular entity (e.g., a non-fluorescent and / or non-phosphorescent molecular entity), optionally wherein the compound is used for PAI. The dye may have a rapid optical-to-acoustic transition. 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 irradiation, the excited state can immediately revert to the ground state to release heat. The heat generates "sound waves," which can be detected by a microphone. The structure of the compounds of the present invention can protect the dye from physiological environmental influences and / or can be adapted to methods for performing PAI.

[0195] In some embodiments, the compounds of the present invention can be used in fluorescence-guided surgery (FGS). FGS is an intraoperative medical technique used to generate real-time fluorescence images of the surgical area and guide surgical procedures. Therefore, in some embodiments, the detection method can be fluorescence spectroscopy. In some embodiments, the primary dye can absorb and / or emit light of a first wavelength, while the secondary dye can absorb and / or emit light of a second wavelength. Additional dyes (third dyes, fourth dyes, etc.) can be used, and each can (or may not) absorb and / or emit light at different wavelengths.

[0196] According to embodiments of the invention, a method for treating cells and / or tissues (e.g., diseased cells and / or tissues) of a subject in need is also provided, the method comprising: administering to the subject a compound, biomolecule, and / or composition of the invention, optionally wherein the compound or biomolecule binds to the cells and / or tissue; and irradiating the subject or a portion thereof (e.g., the location of the cells and / or tissue) with light of a wavelength and intensity sufficient to treat the cells and / or tissue, optionally wherein the light activates the compound. In some embodiments, in addition to treating cells and / or tissues, the compounds of the invention can also be used to detect the compounds and thus the cells and / or tissues. In some embodiments, an imaging technique can be used to detect a first dye (major dye) on the compound; and a second, different dye (minor dye) on the compound can be used to treat the cells and / or tissues—the use of which includes methods of activating the minor dye (e.g., to release reactive oxygen species into diseased tissues) thereby treating the cells and / or tissues. Examples of imaging techniques include MRI and PAI. Examples of treatment methods include photodynamic therapy and photoimmunotherapy. Since the terms "primary" and "secondary" are used only to distinguish different dyes, imaging techniques can also be used to detect secondary dyes, while primary dyes can be used to treat cells and / or tissues. Additional dyes (third dyes, fourth dyes, etc.) can be used, and each may (or may not) be used for different detection or treatment methods.

[0197] In some embodiments, the therapeutic methods using the compounds of the present invention are photodynamic therapy (PDT) and / or photodynamic inactivation (PDI). PDT is a form of phototherapy involving light and photosensitizing chemicals (e.g., the compounds of the present invention) used in combination with molecular oxygen to induce cell death (phototoxicity). PDT can be used to kill microbial cells, including bacteria, fungi, and viruses. PDT can also be used to treat cancer. When light energy is applied in PDT to destroy tumors, various forms of energy are within the scope of the present invention, as those skilled in the art will understand. Such forms of energy include, but are not limited to, heat, sound, ultrasound, chemical, light, microwaves, ionization (such as X-rays and gamma rays), mechanical, and / or electrical energy. For example, sonodynamic inducing or activating agents include, but are not limited to, gallium-porphyrin complexes (see Yumita et al., Cancer Letters 112: 79-86 (1997)), other porphyrin complexes such as protoporphyrin and hematoporphyrin (see Umemura et al., UltrasonicsSonochemistry 3: S187-S191 (1996)); and other cancer drugs used in the presence of ultrasound therapy, such as daunorubicin and doxorubicin (see Yumita et al., Japan J. Hyperthermic Oncology 3(2):175-182 (1987)).

[0198] Examples of therapeutic areas for PDT and / or PDI include, but are not limited to, the following: (i) Treatment of opportunistic infections The compounds, compositions, and / or methods of the present invention can be used for opportunistic infections, particularly soft tissue PDT. For antimicrobial treatment (via PDT) of infections, particularly wound infections, the infectious organism may include (as a non-limiting example) Staphylococcus aureus (… Staphylococcus aureus ), Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) and / or Escherichia coli ( Escherichia coli In hospital-acquired infections, *Pseudomonas aeruginosa* is the cause of 8% of surgical wound infections and 10% of bloodstream infections. In some implementations, the subjects are immunocompromised, such as those with AIDS and / or undergoing immunosuppressant therapy.

[0199] (ii) Treatment of burns. Infections with Staphylococcus aureus and Gram-positive bacteria are particularly prominent in burns (Lambrechts, 2005). Multidrug resistance in Staphylococcus aureus poses a significant medical challenge. In this regard, the compounds, compositions, and / or methods of the present invention can be used to treat opportunistic infections or burns.

[0200] (iii) SepsisThe compounds, compositions, and / or methods of the present invention can be used to treat patients infected with Vibrio vulnificus (Vibrio vulnificus). Vibrio vulnificus PDT treatment for subjects with opportunistic infections. Vibrio vulnificus is a Gram-negative bacterium that causes primary sepsis, wound infections, and / or gastrointestinal diseases in humans.

[0201] (iv) ulcer The compounds, compositions, and / or methods of the present invention can be used against bacteria that cause ulcers (Helicobacter pylori). Helicobacter pylori PDT treatment. In clinical practice, treatment can be achieved in any suitable manner, such as by inserting a fiber optic cable (similar to an endoscope but with a device for delivering red or near-infrared light) into the stomach and / or the affected area.

[0202] (v) Periodontal disease The compounds, compositions, and / or methods of the present invention can be used to treat periodontal disease, including periodontal disease treatment (PDT) for gingivitis. Periodontal disease is caused by bacteria such as Gram-negative anaerobic bacteria *Porphyromonas gingivalis* (Porphyromonas gingivalis). Porphyromonas gingivalis This is caused by the overgrowth of *Porphyromonas gingivalis*. As with many PDT treatments, a targeting or solubilizing entity bound to the photosynthetic class is essential for the proper delivery of the photosynthetic class to the desired cells. Targeted oral pathogens of interest include, but are not limited to, *Porphyromonas gingivalis*. Porphyromonas gingivalis ), Actinobacillus consanguineus ( Actinobacillus actinomycetemcomitans ), Bacteroides forsythia Bacteroides forsythus Campylobacter rectum ( Campylobacter rectus ), Eikenella corrosa ( Eikenella corrodens ), Fusobacterium nucleatum polymorphic subspecies ( Fusobacterium nucleatum subsp. Polymorphum ), sticky actinomycetes ( Actinomyces viscosus ( ) and streptococci. For such applications, the compounds and / or compositions of the present invention can be applied topically (e.g., as a mouthwash or rinse), and then light can be applied using an external device, an intraoral instrument, or a combination thereof.

[0203] (vi) Atherosclerosis The compounds, compositions, and / or methods of this invention can be used for photodynamic therapy (PDT) of vulnerable atherosclerotic plaques. While not wishing to be bound by any particular theory, it is believed that invading inflammatory macrophages secrete metalloproteinases that degrade the thin layer of collagen in the coronary arteries, leading to thrombosis, which is often fatal (Demidova and Hamblin, 2004). Chlorophyll targeting such inflammatory macrophages may be useful for PDT of vulnerable plaques.

[0204] (vii) Cosmetic and Dermatological ApplicationsThe compounds, compositions, and / or methods of the present invention can be used in phototherapy (PDT) to treat a wide range of cosmetic dermatological problems, such as hair removal, treatment of psoriasis, and / or treatment of hypopigmentation. Ruby lasers are currently used for hair removal; in many laser treatments, melanin is the photosensitive chromophore. Such treatments are quite effective for individuals with light skin and dark hair. The compounds, compositions, and / or methods of the present invention can be used as near-infrared sensitizers for hair removal, enabling targeting of chromophores with more specific and / or sharp absorption bands.

[0205] (viii) Acne The compounds, compositions, and / or methods of the present invention can be used for phototherapy-induced treatment (PDT) of acne. Acne vulgaris is caused by Propionibacterium acnes, which infects the sebaceous glands; approximately 80% of young people are affected. Here, the increasing resistance of the bacteria to antibiotic treatment leads to a surge in difficult-to-treat acne. Current PDT treatment for acne typically relies on the addition of aminolevulinic acid, which is converted into free basal porphyrin in the hair follicle or sebaceous gland. Depending on the specific circumstances, the compounds and / or compositions of the present invention can be administered to the subject topically or parenterally (e.g., by subcutaneous injection).

[0206] (ix) Infectious diseases The compounds, compositions, and / or methods of the present invention can be used for phototherapy (PDT) in the treatment of infectious diseases. For example, cutaneous leishmaniasis and subcutaneous leishmaniasis, which are prevalent in the Mediterranean and Middle East, are currently treated with arsenic-containing compounds. Recently, PDT has been used to produce reasonable effects in human subjects in at least one case. The use of the compounds and / or compositions of the present invention is equally useful and potentially offers advantages such as ease of synthesis and better spectral absorption properties.

[0207] (x) tissue sealant The compounds, compositions, and / or methods of the present invention can be used as tissue sealants for subjects in need during phototherapy (PDT). Photoactivated tissue sealants are attractive for sealing wounds, adhesive tissues, and / or closing defects in tissues. In many applications, sutures and / or staples are undesirable, and the use of such mechanical sealing methods often leads to infection and / or scarring.

[0208] (xi) Tumor diseases The compounds, compositions, and / or methods of the present invention can be used for the treatment of tumor diseases and / or cancers via PDT, including skin cancer, lung cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, ovarian cancer, basal cell carcinoma, leukemia, lymphoma, squamous cell carcinoma, melanoma, plaque-stage cutaneous T-cell lymphoma, and / or Kaposi's sarcoma.

[0209] In photodynamic therapy, the compounds of the present invention are administered to a subject in need (e.g., a subject suffering from any of the aforementioned diseases). The administered compound can bind to diseased tissue present in the subject's body, and exposure of the subject to a suitable light source emitting appropriate wavelength and intensity can activate the compound (e.g., release reactive oxygen species (ROS)) into the diseased tissue, thereby treating the diseased tissue, optionally without affecting healthy tissue. For example, in some embodiments, the diseased tissue is hyperproliferative tissue (e.g., a tumor).

[0210] In some embodiments of the invention, the compound comprises a biomolecule, and the biomolecule targets the compound to a specific cell, tissue, or biological site and / or facilitates the transport of the compound across the cell membrane. For example, in some embodiments, the biomolecule includes a biomarker at a specific site in vivo, and its presence guides the compound to the cell, tissue, or other biological site. In some embodiments, the biomolecule guides the compound to overproliferating cells and / or tissues.

[0211] In some embodiments, the compound is prepared for targeted application. The targeting compound may comprise a biomolecule, for example, that specifically attracts or binds to a particular spatial or polar tissue of another molecule. Thus, the targeting compound may target, for example, cells containing specific surface molecules that can be used to diagnose or detect biological sites, such as growth factor receptors, tumor necrosis factor, or interleukin receptors. The targeting compound may comprise a biomolecule as a binding pair member, wherein the binding member is present in the tissue, cell, or target biological site.

[0212] In some embodiments, the compound is non-biomolecule-free and can be used in non-targeting methods. For example, in some embodiments, the compound contains a dye capable of imaging to confirm the presence of the compound in a region of interest. In some embodiments, the non-targeting method comprises a non-fluorescent dye, including, for example, a dye that generates an ultrasound signal and / or a dye that generates reactive oxygen species. In some embodiments, the non-targeting method comprises topically applying the compound or injecting it into a specific tissue, cavity, or region of interest. In some embodiments, the compound is prepared for non-targeting methods and may, for example, contain a mixture of a dye that generates a fluorescent signal and a PDT dye that generates singlet oxygen, so that a user can use the fluorescent dye to image the non-targeted fold in a biological body to confirm the appropriate location. Exemplary dyes for non-targeting applications are described in detail in the operational embodiments of this application and in Table 12.

[0213] The invention is explained in more detail in the following non-limiting embodiments. Example

[0214] Example 1: Connector design for additional portions of multiple dyes and / or end groups A. Linear connector design A dual-purpose design can be achieved using linear linkers, where one dye or biomolecule is directly linked to another, and the two dyes or biomolecules thus linked are connected to the polymer backbone via the linker (see Scheme 1). This design can be achieved, for example, by using a dye (compound I in Scheme 1) that retains a functional group for coupling to another dye, such as an aryl halogen, which can be used in metal-mediated cross-coupling reactions such as the Suzuki, Sonogashira, or Stille reactions. The second dye molecule has two reactive functional groups, one for cross-coupling (e.g., boric acid, pinacol ester of borate, alkyne) to the first dye molecule, and the other for connection to the polymer backbone (e.g., carboxylic acid, amine, maleimide). In the example shown in Scheme 1, the first chlorophyll (compound I) is prepared with an aryl halogen, which undergoes Sonogashira coupling with the alkyne of dihydroporphyrin (compound II). The methyl ester of compound II is then converted to a maleimide, which can then be linked to the free thiol end of the polymer backbone (also called the folded body). In this implementation, the connector is designed to provide rigidity, thereby providing spatial separation of the molecule to minimize interactions (such as energy transfer) across space. This can help maintain the individual properties of the two probes.

[0215] Figure 1. Example of a linear connector design method for a probe based on a dual-purpose folded body. B. Dual-functional branched connector design Dual-purpose probes can also be prepared by functionalizing the central linker molecule in two steps, followed by attaching the dual-modified linker to the polymer in a subsequent step. A biotargeting molecule may or may not be introduced in another subsequent step after this method.

[0216] For bifunctional reagents, this method can be practically accomplished using a trifunctional linker such as N-Boc-L-lysine methyl ester hydrochloride (Scheme 2). This type of linker can first react with probe 1 (P1), which contains a carboxylic acid group. P1 can consist of a fluorescent dye, a non-fluorescent dye, or a biomolecule such as a peptide or oligonucleotide. Coupling can be achieved under many other conditions known in the art (e.g., including but not limited to DCC, DIC, EDAC, HATU, HBTU, PyBop, oxaloyl chloride), or as shown by in-situ activation with a reagent such as TSTU in the presence of a base such as triethylamine in a solvent such as dimethylformamide that provides sufficient solubility for the reaction components. The resulting P1-linker compound is treated with a strong base such as sodium hydroxide to expose the reactive carboxylic acid, which can then be coupled with probe 2 (P2) under the conditions previously described for P1. Similarly, P2 can consist of a fluorescent dye, a non-fluorescent dye, a small molecule drug, or a biomolecule such as a peptide or oligonucleotide. Finally, the lysine side-chain amine Boc group is deprotected under acidic conditions (e.g., HCl, trifluoroacetic acid) to yield a final reactive amine group, which can be used to link the P1-P2-linker to a folded body appropriately functionalized with a carboxylic acid. The coupling conditions can be as described previously for the linking of P1 and P2. The resulting folded body contains P1 and P2, as well as reactive thiol groups that can be further derivatized at opposite ends of the polymer. If further characterization is not required, the thiol can be retained as is or capped with a simple maleimide group such as benzylmaleimide. Alternatively, the folded body can be prepared by initial reaction of its terminal thiol with the maleimide group of an iso-bifunctional linker (e.g., 4-(maleimidemethyl)cyclohexanecarboxylic acid N-succinimide ester (SMCC)). The SMCC contains an N-hydroxysuccinimide group, which can then be used to link biomolecules containing amine groups (T1), such as peptides, oligonucleotides, antibodies, or proteins.

[0217] Figure 2. A diagram showing two distinct probe molecules and an optional third group for molecular targeting attached to the fold body. The synthesis of bifunctional branched molecules can be carried out using the following synthetic procedure: Lys-P Intermediate 1. Fmoc-Lys(Boc)-OH (11.5 mg, 27.61 µmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (31.8 mg, 165.67 µmol), 4-dimethylaminopyridine (2.0 mg, 16.6 µmol), and 4 Å molecular sieve (2.0 mg) were added to an RBF equipped with a stir bar. The flask was sealed with a diaphragm, evacuated, purged with argon, and DCM (7.5 mL) was added. The mixture was stirred at room temperature for 1 hour, and then porphyrin aryl alcohol (11.5 mg, 27.61 µmol) solid was added in bulk, and the reaction was stirred at room temperature for 22 hours. The reaction mixture was diluted with DCM and washed with saturated sodium bicarbonate aqueous solution, water, and brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography on silica media and eluted with 0–50% ethyl acetate / hexane. The major product was isolated as a 20.3 mg (85%) red solid after drying. [M + H] + = 867.6, calculated value 867.4.

[0218] 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 temperature for 30 minutes. The solvent was removed, and the residue was purified by column chromatography on silica using 0–10% MeOH / DCM. The desired product was separated as a 13.9 mg (92%) deep red solid. [M+ H] + = 645.2, calculated value 645.3.

[0219] 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 an RBF equipped with a stir bar. The flask was evacuated, purged with argon, and DCM (2.0 mL) was added, followed by triethylamine (3.8 µL, 27.45 µmol). The reaction was stirred at room temperature for 4 hours, and then the solvent was removed. The residue was dissolved in DCM; washed with saturated aqueous sodium bicarbonate solution, water, and brine; dried over sodium sulfate; filtered, and concentrated. The residue was purified by column chromatography on silica using 0–5% MeOH / DCM. 8.3 mg (70%) of a deep red solid was separated. [M + H] + =1294.6, calculated value 1294.6.

[0220] Lys-P-dye II. Prepared as described for compound Lys-P-dye I. [M + H]+ = 1218.9, calculated value 1218.6.

[0221] Lys-P-dye III. Prepared as described for compound Lys-P-dye I. [M + H] + = 1232.9, calculated value 1232.6.

[0222] Lys-P-dye IV. Prepared as described for compound Lys-P-dye I. [M + H] + = 1310.9, calculated value 1310.6.

[0223] C. Trifunctional Branched Connector Design The fold-based reagent can also be prepared containing three distinct probes, each with a different function. This reagent can be rationally prepared starting with a trifunctional linker N-Boc-L-lysine methyl ester hydrochloride in a manner similar to that previously described for bifunctional reagents (Scheme 3). The initial introduction of probe 1 (P1) is achieved using standard amide coupling chemistry known in the art. P1 can consist of a fluorescent dye, a non-fluorescent dye, a small molecule drug, or a biomolecule such as a peptide or oligonucleotide. Saponification of this methyl ester provides a carboxylic acid, which can be coupled to a commercially available L-cysteine ​​methyl ester hydrochloride under standard amide conditions. The resulting linker now contains a reactive thiol group, which can be used to connect probe 2 (P2), which has been modified to contain a terminal maleimide group. The methyl ester containing the cysteine ​​residue is then saponified with a strong base as previously described. This provides a unique carboxylic acid, which can undergo amide coupling with probe 3 (P3), functionalized to contain a terminal amine. The Boc group of the side-chain amine used to protect the initiating lysine molecule is stable under all the foregoing conditions and can be removed upon treatment with an acid (e.g., HCl, trifluoroacetic acid) after the introduction of P3. The removal of the Boc group provides a terminal amine, which can be used to attach the P1 / P2 / P3 trifunctional linker to the folded body. The opposite end of the folded body containing the free thiol can remain intact, capped with a simple maleimide group such as benzylmaleimide, or further reacted with a reagent, such as the SMCC reaction previously described for the design of bifunctional linkers, to attach to another amine-containing functional molecule.

[0224] Figure 3. A diagram showing two distinct probe molecules and an optional third group for molecular targeting attached to the fold. Example 2: Multiple dyes on the polymer backbone Polymers containing orthogonal reactive functional groups at each end and including a third orthogonal reactive group along the polymer length are described. This polymer design allows for selective modification via three different reaction chemistry methods and can be used as template molecules for covalent chemical linkages to dyes, biomolecules, or other selected molecules. For example, dyes can be added to one or more ends of the polymer backbone, and additional dyes can be incorporated via post-polymerization reactions with suitable functional groups side-attached to the polymer backbone (see [link to relevant documentation]). Figure 7 , Figures 8A-8B The total number of dyes per chain can be controlled by selecting the initial stoichiometric ratio of the dye to the polymer. The total number of potential dyes that can be added to each polymer is limited only by the degree of polymerization of the polymer (the number of monomers containing reactive groups on the polymer).

[0225] For example, under standard reversible addition-fragmentation chain transfer (RAFT) conditions, polymers containing azido, thiol, and N-hydroxysuccinimide reactive groups can be generated using a suitable initiator (e.g., 2,2′-azobis(2-methylpropionitrile) (AIBN)) and a chain transfer agent (CTA, e.g., 2-(dodecylthiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester). This template enables selective reactions of molecules containing alkyne, maleimide, and amine groups. One embodiment of this polymer system can use N-succinimide p-vinylbenzoate (NSVB) as a monomer. Another embodiment can use N-succinimide acrylate as a monomer under RAFT conditions to produce a polymer system with the same reactive groups but lower hydrophobicity. Polymers containing azido, thiol, and amine reactive groups can be produced by using a monomer such as ethyl methacrylate (2-Boc-amino) ester for RAFT, followed by removal of the Boc protecting group under suitable conditions (e.g., TFA or HCl).

[0226] A. Synthesis of amine monomer dyes The synthesis of monomeric dyes can be carried out using the following synthetic procedure: BC11a. Compound BC11 (279.2 mg, 500.0 µmol, prepared as described in J. Org. Chem., 2010, 75, 1016–1039), pinacol 4-methoxycarbonylphenylboronic acid (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 a stir bar. The flask was sealed with a diaphragm, evacuated, and purged three times with argon for 30 minutes. Toluene (66 mL) and DMF (33 mL) were added, and the mixture was stirred. The system was evacuated for 2 minutes and then purged twice with argon. The flask was added to a preheated oil bath at 80 °C and stirred under an argon atmosphere. After 16 hours, the flask was cooled, and the reaction mixture was diluted with ethyl acetate (100 mL). The organic layer was washed with deionized water (3 × 125 mL) without mixing, then washed with a mixture (3 × 125 mL). The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in a minimal amount of dichloromethane and dried on diatomaceous earth. The filter cake was eluted on a 24 g silica column with 0–65% dichloromethane / hexane for 15 minutes. The desired product was separated as 207.9 mg (68%) of dark solid.

[0227] BC11b. BC11a (199.7 mg, 325.5 µmol), pinacol phenylborate (79.7 mg, 390.6 µmol), cesium 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 container with a stir bar. The flask was sealed with a diaphragm, evacuated, and purged three times with argon for 30 minutes. Toluene (22 mL) and DMF (11 mL) were added, and the mixture was stirred. The system was evacuated for 2 minutes and then purged twice with argon. The flask was added to a preheated oil bath at 80 °C and stirred under an argon atmosphere. After 18 hours, the flask was cooled, and the reaction mixture was diluted with ethyl acetate (50 mL). The organic layer was washed with deionized water (3 × 50 mL) without mixing, and then washed with a mixture (3 × 50 mL). The organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated. The residue was dissolved in a minimal amount of dichloromethane and eluted on an 80 g silica column with 20% ethyl acetate / hexane. The desired product was separated as 160 mg (80%) dark solid.

[0228] BC11c. BC11b (157.0 mg, 257.1 µmol) was added to 100 mL of RBF with a stir bar and dissolved in tetrahydrofuran (13.0 mL). Methanol (6.5 mL) was added and the solution was stirred vigorously. 5 M NaOH aqueous solution (6.5 mL) was added to the stirred solution. The reaction was stirred at room temperature under a foil cover. After 16 hours, the reaction mixture was gradually added to 1 M HCl aqueous solution (40 mL) cooled in an ice-water bath with stirring. The mixture was diluted with ethyl acetate. The aqueous layer was removed, and the organic layer was washed with water (2 × 50 mL unmixed, 2 × 50 mL mixed) and brine (50 mL), dried over sodium sulfate, filtered, and concentrated. The product was dried under high vacuum to give 140.5 mg (92%) of green solid.

[0229] BC11d. BC11c (49.5 mg, 83.0 µmol) and N,N,N′,N′-tetramethyl-O-(N-succinimide)ureon tetrafluoroborate (30.0 mg, 99.5 µmol) were added to a flame-dried 25 mL RBF with a stir bar. The flask was sealed with a diaphragm, evacuated, and purged with argon. Dimethylformamide (8.3 mL) was added, followed by triethylamine (69.3 µL, 497.7 µmol), and the mixture was stirred at room temperature. After 30 minutes, N-boc-ethylamine diamine (19.7 µL, 124.4 µmol) was added, and the reaction was stirred under an argon atmosphere. After 24 hours, the reaction mixture was diluted with ethyl acetate (20 mL) and washed with water (4 × 25 mL, unmixed), washed with a semi-saturated ammonium chloride aqueous solution (2 × 25 mL), and then washed with brine. The organic layer was dried over sodium sulfate, filtered, and concentrated. The residue was diluted in a minimal amount of dichloromethane and eluted on a 24 g silica column with 20%–60% ethyl acetate / hexane for 15 minutes. The product was separated and dried to give 44.9 mg (73%) of solid.

[0230] Amine monomer dye. BC11d (44.9 mg, 60.8 µmol) was dissolved in dichloromethane (3.5 mL) under argon atmosphere. A 4M HCl / dioxane solution (0.5 mL) was added dropwise at room temperature. After 2 hours, the dioxane was removed by purging with argon. The residue was suspended in ethyl acetate, and a saturated aqueous sodium bicarbonate solution was added. The mixture was stirred vigorously for 10 minutes. The aqueous layer was removed. The organic layer was washed with a semi-saturated aqueous sodium bicarbonate solution, then washed with brine, dried over sodium sulfate, rinsed with dichloromethane, filtered, and concentrated. Drying under high vacuum gave 25.4 mg (60%) of green film.

[0231] B. Synthesis of NSVB polymers N-succinimide p-vinylbenzoate (NSVB) (1.00 g, 4.08 mmol, see synthesis details) J. Poly. Sci. A Add 2-(dodecylthiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester CTA (317.5 μL in 103.54 µmol / g dioxane solution), then add AIBN (114 μL in 16.9 µmol / g dioxane solution), and mesitylene (200 μL) as a standard. Seal the flask with a diaphragm, evacuate for 30 seconds, and then purge with argon. Repeat the evacuation / argon purging sequence twice. Bubble argon through the reaction mixture for 8 minutes using the needle vent in the diaphragm. Remove the vent and stir the reaction under low flow of argon. Add the flask to a preheated oil bath at 70 °C and heat for 18 hours. The flask was cooled, the stir bar removed, and DMF removed by rotary evaporation to obtain a yellow oil. The oil was diluted in dichloromethane (3 mL) and transferred dropwise to methanol (400 mL) stirred in a 500 mL beaker. The resulting precipitate was allowed to settle, and most of the methanol was removed by decantation. The remaining precipitate / methanol mixture was filtered through a 30 mL fine ground glass filter and dried under vacuum for 1 hour to obtain a pale yellow, free-flowing granular solid. The solid was dissolved in dichloromethane (15 mL) while gently heated to 40 °C. The clear amber solution was added dropwise to methanol to cause secondary precipitation of the polymer. The solution was allowed to settle, then decanted, filtered, and dried under vacuum as previously described. The solid was further dried under high vacuum to obtain 0.569 g of a pale yellow solid. The degree of polymerization was calculated to be 56 based on NMR analysis.

[0232] C. Synthesis of poly(PEGA-co-2-(N-Boc-ethylidene)acrylate) copolymer 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-(dodecylthiocarbonylthio)-2-methylpropanoate 3-azido-1-propanol ester (420 mg in 133.1 µmol / g dioxane solution), AIBN (225 mg in 28.1 µmol / g dioxane solution), 0.17 g mesitylene, and 8.7 g dioxane were added to a 25 mL round-bottom flask (RBF). The flask was sealed with a diaphragm, and argon gas was bubbled through the reaction mixture for 45 minutes using a needle vent in the diaphragm. The flask was then placed in a preheated oil bath at 75 °C and heated for 90 minutes. The reaction was quenched by purging the flask with air. The reaction mixture was cooled to room temperature and poured into 500 mL of Et₂O:hexane in a 1:1 ratio with vigorous stirring. The liquid polymer was allowed to settle at 4 °C for 2 hours, and then the Et₂O:hexane was removed by decantation. The polymer was then dried under vacuum at 60 °C for 4 hours. The dried polymer (a very viscous yellow liquid) was dissolved in 12 mL of DI water and transferred to a 3.5 kDa filtration membrane, and dialyzed with methanol for 24 hours, exchanging methanol three times during the dialyzing process. After dialyzing, the polymer solution was passed through a 0.2 μm PTFE syringe filter, and methanol was removed using a rotary evaporator. The polymer was dried under vacuum at 60 °C for 24 hours to obtain 1.67 g of a very viscous yellow liquid. The degree of polymerization was calculated to be 54 based on NMR analysis.

[0233] D. Synthesis of PFPA polymers Pentafluorophenyl acrylate (PFPA) (800 mg, 3.36 mmol) was added to a 25 mL Schlenk flask and dissolved in dioxane (2.7 mL). 2-(dodecylthiocarbonylthio)-2-methylpropionic acid 3-azido-1-propanol ester CTA (122.1 μL in 123.89 µmol / g dioxane solution) was added, followed by AIBN (41.4 μL in 18.26 µmol / g dioxane solution) and mesitylene (50 μL) as an NMR internal standard. The flask was sealed, and argon gas was bubbled through the reaction mixture for 8 minutes using the needle vent in the diaphragm. The vent was removed, the flask was sealed, and the mixture was added to a preheated oil bath at 80 °C and heated with stirring for 3 hours. The flask was cooled, the stir bar was removed, and the mixture was transferred dropwise to methanol (500 mL) stirred in a 1000 mL beaker. The precipitate formed was allowed to settle, and most of the methanol was removed by decantation. The remaining precipitate / methanol mixture was filtered through a 30 mL fine-ground glass filter and dried under vacuum for 1 hour to obtain a white powder. The solid was further dried under high vacuum to obtain 0.629 g of white solid. The degree of polymerization was calculated to be 162 based on NMR analysis.

[0234] E. Main-chain dye labeling of NSVB polymers The amine monomer dye was prepared as a tetrahydrofuran solution (0.97 mg / 200 μL). Four mL amber vials were flame-dried and cooled under high vacuum, then purged with argon. Dye fractions (80 μL = 4 equivalents, 120 μL = 6 equivalents, 160 μL = 6 equivalents, 200 μL = 10 equivalents) were added to each vial. THF was removed with an argon flow. The NSVB polymer was prepared as a DMF solution (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 hours. Each reaction was concentrated and redissolved in 1:1 toluene / dimethylformamide (200 μL). Unreacted excess dye was removed by size exclusion chromatography using a 1:1 toluene / DMF solvent system on SX-1 resin. The photophysical properties of the samples were analyzed (Table 5).

[0235] Table 5: Photophysical characterization data of NSVB polymers labeled with variable equivalent dyes. Measurements were performed in dimethylformamide at room temperature.

[0236] F. Further polymerization modification of dye-labeled folded bodies The dye-labeled NSVB polymer (0.012 µmol), dodecylamine (variable equivalent range), and PEG8-NH2 (variable equivalent) prepared as DMF solutions were added together to 1.5 mL Eppendorf tubes. The total volume of each reaction was adjusted to 0.2 mM in a 1:1 toluene / DMF solution. The reactions were covered with foil and set on a fixed-track shaker at 90 rot / min. After 16 hours, the toluene was removed, and each sample was dissolved in PBS (pH 7.2) to a final concentration of 100 µM. The photophysical properties of the samples were analyzed.

[0237] Figure 9 This displays emission data for dye-labeled NSVB polymer samples after reaction with hydrophilic groups (PEG8-NH2) and hydrophobic groups (dodecylamine). The ratios mentioned are the relative equivalences of hydrophilic and hydrophobic groups used in the reaction. In this case, the emission intensity is clearly correlated with the increased hydrophilicity in the polyethylene glycol-modified NSVB polymer.

[0238] Post-polymerization modification of G. PFPA polymers 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 tightly sealed. The reaction mixture was stirred and heated to 50 °C. After 2 hours, 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 another 2 hours. Then, Jeffamine M-1000 (8.75 μmol, 8.75 mg) (as a stock solution in THF) was added. The vial was sealed and the reaction was heated at 50 °C for another 16 hours. The solvent was removed by evaporation. The residue was dissolved in toluene and purified by size exclusion chromatography (1 × 20 cm column, Biorad S-X1 resin, toluene mobile phase). The sample was vacuum dried to obtain 4.7 mg (59%) of deep red viscous oil.

[0239] Table 6. Photophysical characterization data of PFPA-based polymer folds as the amount of dye used increases. The dye equivalent number used is specified in the fold name.

[0240] Terminal / terminal dye labeling Poly(PEGA-co-2-(N-Boc-ethyleneamine)acrylate) copolymer (24.0 mg, 1.13 µmol) was added to a 5 mL RBF container fitted with a small stir bar. The flask was sealed with a diaphragm, evacuated, and purged three times with argon. The polymer was diluted in DMF (anhydrous, degassed, 280.4 µL, 4 mM). The system was briefly disrupted by adding hydrazine hydrate (HH, 80% w / v, 0.17 µL, 2.82 µmol) (prepared as a stock solution in DMF). The flask was then capped and stirred at room temperature under an argon atmosphere for 10 minutes. The stir bar was removed, and the reaction mixture was concentrated. The stir bar was then reinserted, the flask was capped, and placed under high vacuum.

[0241] After 10 minutes, the system was purged with argon, and dye (3.07 mg, 2.82 µmol, 2.5 equivalents) was added in bulk. The system was sealed with a diaphragm, evacuated, and purged with argon three times. DMF (1121.5 µL) was added, and stirring was initiated. The system was briefly disrupted, and trimethyl phosphite (0.66 µL, 1.23 µmol) was added directly to the solution. Then, triethylamine (0.17 µL, 1.23 µmol), prepared as a stock solution in DMF, was added directly to the reaction solution. The flask was sealed with a diaphragm, protected from light with foil, and stirred at room temperature under an argon atmosphere.

[0242] After 16 hours, the stir bar was removed, the mixture was rinsed with toluene, and the reaction was concentrated. The residue was redissolved 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 the reaction flask also rinsed (100 µL). The tube was rotated at 12 KG for 5 minutes. The supernatant was carefully removed, and SEC was performed using SX-1 media (2 x 20 cm column, toluene solvent). The desired dye-labeled product was separated as 21.7 mg (87%) of dark membrane.

[0243] Example 3: Dual-purpose method The following is a list of the proposed dual-use combinations. Molecules designated as P1, P2, and / or T1 according to Synthetic Formula 2 for these functions are enclosed in parentheses and can 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 also possible.

[0244] 1) MRI and PAI (e.g., P1 = manganese porphyrin, P2 = copper chlorophyll, T1 = n / a) Objective: MRI provides volume detection, while PAI is used for edge detection. MRI is used to provide rapid screening of the general location of tumors, which can be supplemented by the more sensitive detection using PAI.

[0245] 2) MRI and PDT / PIT (e.g., P1 = manganese porphyrin, P2 = copper chlorophyll, T1 = target peptide) Objective: MRI is used to provide rapid screening of the general location of tumors, which can then be used for local PDT.

[0246] 3) FGS and PDT / PIT (e.g., P1 = Alexa Fluor) TM 790, P2 = platinum chlorophyll, T1 = targeted peptide) Objective: To target peptides to specific cancer cell sites that can be visualized using FGS probes. The PDT cargo can then be photoactivated at the cancer cell site.

[0247] 4) FC and PAI (e.g., P1 = porphyrin dimer, P2 = copper chlorophyll, T1 = no or no targeting peptide) Objective: First, cells were phenotypically analyzed and sorted using FC, and then reintroduced into the organism and localized using PAI imaging.

[0248] 5) PAI and PDT / PIT (e.g., P1 = copper chlorophyll, abs < 750 nm, P2 = platinum chlorophyll, abs > 800 nm, T1 = targeted peptide) Objective: To utilize the penetration depth of PAI as an imaging modality to identify diseased tissues, and then initiate cell killing through photoactivation of PDT reagent.

[0249] Example 4: Dendron Linker Design for Connecting Multiple Parts to the Ends of a Polymer Main Chain Synthesis of Gen2 dendron linkers. The following exemplary dendrons were synthesized using an improved Fmoc solid-phase peptide synthesis procedure, as described below.

[0250] A low-loading Rink amide resin was used as the solid phase, with amino acids Fmoc-Gly-OH and Fmoc-Lys(alloc)-OH as amino acid structural units, and N,N-bis(N'-Fmoc-3-aminopropyl)-glycine hemisulfate (APG) as the branched amino acid. The immobilized Gly-Lys(alloc)-Gly sequence was synthesized to serve as the starting point for dendration units for further modification. Glycine units were added between each branched amino acid to improve flexibility and yield. As the starting point of each coupling cycle, the Fmoc groups on the solid resin were deprotected for 15 min in the presence of 20% piperidine / DMF, followed by washing 6 times with DMF. For amino acid addition, coupling was performed for 40 min in the presence of 4.0 equivalents of amino acid, 4.0 equivalents of HBTU, and 8 equivalents of diisopropylethylamine (DIPEA), followed by washing 3.0 times with DMF. For branched-chain amino acids, coupling was performed overnight in the presence of 3.0 equivalents of branched-chain amino acids, 3.0 equivalents of HBTU, and 6.0 equivalents of DIPEA, followed by washing three times with DMF. A Kaiser test was performed after each coupling to verify completion. After synthesis, alloc groups were removed by washing six times (2 mL; 30 min per cycle) with 0.1 equivalents of tetrakis(triphenylphosphine)palladium(0) and 20.0 equivalents of phenylsilane / CH₂Cl₂. The resulting resin was washed sequentially with 0.5% DIPEA / DMF, 0.5% sodium diethyldithiocarbamate / DMF, and 50% CH₂Cl₂ / DMF. The resin was then coupled overnight in the presence of 4.0 equivalents of 6-maleimide hexanoic acid, 4.0 equivalents of HBTU, and 8.0 equivalents of DIPEA, followed by washing three times with DMF. After final deprotection, the dendritic units were cleaved in 2 mL of trifluoroacetic acid (TFA). After 3 hours, the cutting solution was collected and the resin was washed twice with pure TFA. The TFA was evaporated under air, and the residual solution was precipitated with cold diethyl ether. The resulting precipitate was collected by centrifugation at 5000 rpm for 5 minutes and washed three times with cold diethyl ether.

[0251] Gen2-Dendron-660DD - The synthesized Gen2-dendritic moiety (2.6 mg, 2 µmol) and 660DD-NHS (9.9 mg, 9.6 µmol) were added to a 10 mL round-bottom flask (RBF) equipped with a stir bar, followed by 1 mL of DMF. The flask was sealed with a rubber diaphragm, evacuated, and then purged with argon. 5% DIPEA (50 µL) was rapidly added. The reaction mixture was stirred at room temperature under an argon atmosphere and protected 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 / CH2Cl2. 3.2 mg (34%) of the dendritic moiety product was isolated, with a net conjugation of 2.5 dimers per dendritic moiety.

[0252] Synthesis of fluorescent folded molecules from dendritic motifs-dimers Gen2-Dentalization Element-660DD-P57. The P57 folded form (5.5 mg, 0.22 µmol) was added to a 5 mL RBF container with a small stir bar. The flask was sealed with a diaphragm, evacuated, and purged three times with argon. The folded form was diluted in DMF (anhydrous, degassed, 54 µL). The system was briefly disrupted, and a hydrazine solution (2.0 µL stock solution in DMF, 0.54 µmol, 2.5 equivalents) was added to the reaction solution via pipette. The flask was then capped and stirred at room temperature under an argon flow. After 10 minutes, the stir bar was removed, and the reaction mixture was concentrated. The stir bar was then reinserted, the flask was capped, and placed under high vacuum. After 1 hour under vacuum, the system was purged with argon, and 660D-BFL dye (3.2 mg, 0.89 µmol, 4.0 equivalents) was added in bulk. The system was sealed with a diaphragm, evacuated, and purged three times with argon. Add DMF (217 µL) and start stirring. Briefly disrupt the system and add trimethyl phosphite (0.13 µL, 1.1 µmol, 5.0 equivalents) directly to the solution via micropipette. Then add triethylamine (2.0 µL stock solution in DMF, 0.24 µmol, 1.1 equivalents) directly. Seal the flask with a diaphragm, protect from light, and stir at room temperature under a low flow rate of argon. After 16 hours, remove the stir bar, rinse with toluene, and concentrate the reaction. Redissolve the residue in toluene and concentrate again. Then place the residue under high vacuum for 1 hour. Then dissolve the residue in toluene (250 µL) and transfer to a 1.5 mL low-adhesion microcentrifuge tube, and rinse the reaction flask again with toluene (125 µL). Rotate the tube at 12 KG for 5 minutes. Decant the supernatant into a second tube. The precipitate (pellet) was carefully washed with toluene (2 x 50 µL), and the wash solution was transferred to a second tube. This tube was rotated at 12 KG for 5 min. The supernatant was removed, and size exclusion chromatography was performed using SX-1 media and toluene elution (1 x 20 cm column, flow rate 1 mL / min). The product fraction was concentrated and dried under high vacuum. 5.7 mg (91%) was isolated, with a dendritic unit-dimer conjugation rate of 20%. The photophysical properties of the sample were analyzed (Table 7).

[0253] Table 7. Photophysical characterization data of dyes, multifunctional junctions, and folded bodies .

[0254] Example 5: A combinable bifunctional connector design for attaching multiple parts to the ends of a polymer backbone. A. Synthesis of linker molecules with ends replaced by two identical parts 660M Dual Functional Connector (660M-BFL-Mal). [The text abruptly ends here, likely due to an incomplete sentence or a formatting error.] N -Mal- N -Bis(PEG4-amine) TFA salt linker (7.7 mg, 9.22 µmol) and 660M-NHS dye (14.4 mg, 18.45 µmol) were added to an RBF containing a stir bar. The flask was sealed with a diaphragm, evacuated, and purged with argon. DMF (0.92 mL) was added, followed by rapid addition of triethylamine (5.1 µL, 36.88 µmol). The reaction mixture was stirred at room temperature under an argon atmosphere and protected from light. After 16 hours, the reaction mixture was diluted with ethyl acetate, washed with water and brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography using 0–10% MeOH / DCM. 10.7 mg (60%) of the desired product was isolated. [M+H] + = 1939.1, calculated value 1937.9.

[0255] 660D dual-functional connector. N -Mal- N -Bis(PEG4-amine) TFA salt linker (4.5 mg, 5.39 µmol) and 660D-NHS (11.7 mg, 11.32 µmol) were added together to an RBF equipped with a stir bar. The flask was sealed with a diaphragm, evacuated, and purged with argon. DMF (0.57 mL) was added, followed by rapid addition of triethylamine (3.0 µL, 21.56 µmol). The reaction mixture was stirred at room temperature under an argon atmosphere and protected from light. After 16 hours, 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 with brine, dried over sodium sulfate, filtered, and concentrated. The residue was purified by column chromatography using 0–10% MeOH / DCM. 3.5 mg (27%) of the desired product was isolated.

[0256] Synthesis of folded fluorescent molecules substituted by two identical molecules using a bifunctional linker 660M-BFL-P57. The P57 folded body (15.8 mg, 0.62 µmol) was added to a 5 mL RBF container fitted with a small stir bar. The flask was sealed with a diaphragm, evacuated, and purged three times with argon. The folded body was diluted in DMF (anhydrous, degassed, 156 µL). The system was briefly disrupted, and a hydrazine solution (2.0 µL stock solution in DMF, 0.10 µL, 1.55 µmol, 2.5 equivalents) was added to the reaction solution via pipette. The flask was then capped and stirred under an argon flow at room temperature. After 10 minutes, the stir bar was removed, and the reaction mixture was concentrated. The stir bar was then reinserted, the flask capped, and placed under high vacuum. After 10 minutes under vacuum, the system was purged with argon, and 660M-BFL dye (4.82 mg, 2.49 µmol, 4.0 equivalents) was added in bulk. The system was sealed with a diaphragm, evacuated, and purged three times with argon. DMF (622 µL) was added and stirring was initiated. The system was briefly disrupted, and trimethyl phosphite (0.37 µL, 3.11 µmol, 5.0 equivalent) was added directly to the solution via micropipette. Triethylamine (2.0 µL stock solution in DMF, 0.10 µL, 0.68 µmol, 1.1 equivalent) was then added directly. The flask was sealed with a diaphragm, protected from light, and stirred at room temperature under a low flow rate of argon. After 16 hours, the stir bar was removed, the flask was rinsed with toluene, and the reaction was concentrated. The residue was redissolved in toluene and concentrated a second time. The residue was then placed under high vacuum for 1 hour. The residue was then dissolved in toluene (250 µL) and transferred to a 1.5 mL low-adhesion microcentrifuge tube, and the reaction flask was rinsed again with toluene (125 µL). The tube was rotated at 12 KG for 5 minutes. The supernatant was decanted into a second tube. The precipitate (pellet) was carefully washed with toluene (2 x 50 µL), and the washings were transferred to a second tube. This tube was rotated at 12 KG for 5 min. The supernatant was removed, and size exclusion chromatography was performed using SX-1 media and toluene elution (1 x 20 cm column, flow rate 1 mL / min). The product fraction was concentrated and dried under high vacuum. 16.8 mg (99%) of the membrane was separated.

[0257] 660D-BFL-P57. The P57 folded body (11.3 mg, 0.45 µmol) was added to a 5 mL RBF container fitted with a small stir bar. The flask was sealed with a diaphragm, evacuated, and purged three times with argon. The folded body was diluted in DMF (anhydrous, degassed, 111 µL). The system was briefly disrupted, and a hydrazine solution (2.0 µL stock solution in DMF, 1.11 µmol, 2.5 equivalents) was added to the reaction solution via pipette. The flask was then capped and stirred at room temperature under an argon flow. After 10 minutes, the stir bar was removed, and the reaction mixture was concentrated. The stir bar was then reinserted, the flask was capped, and placed under high vacuum. After 1 hour under vacuum, the system was purged with argon, and 660D-BFL dye (3.3 mg, 1.35 µmol, 4.0 equivalents) was added in bulk. The system was sealed with a diaphragm, evacuated, and purged three times with argon. Add DMF (445 µL) and begin stirring. Briefly disrupt the system and add trimethyl phosphite (0.26 µL, 2.23 µmol, 5.0 equivalents) directly to the solution via micropipette. Then add triethylamine (2.0 µL stock solution in DMF, 0.49 µmol, 1.1 equivalents) directly. Seal the flask with a diaphragm, protect from light, and stir at room temperature under a low flow rate of argon. After 16 hours, remove the stir bar, rinse with toluene, and concentrate the reaction. Redissolve the residue in toluene and concentrate again. Then place the residue under high vacuum for 1 hour. Then dissolve the residue in toluene (250 µL) and transfer to a 1.5 mL low-adhesion microcentrifuge tube, and rinse the reaction flask again with toluene (125 µL). Rotate the tube at 12 KG for 5 minutes. Decant the supernatant into a second tube. The precipitate (pellet) was carefully washed with toluene (2 x 50 µL), and the washings were transferred to a second tube. This tube was rotated at 12 kg for 5 min. The supernatant was removed, and size exclusion chromatography was performed using SX-1 media and toluene elution (1 x 20 cm column, flow rate 1 mL / min). The product fraction was concentrated and dried under high vacuum. 11.3 mg (92%) of the membrane was separated. The photophysical properties of the sample are analyzed in Table 8.

[0258] Table 8. Photophysical characterization data of dyes, multifunctional linkers, and fluorescent folds .

[0259] Antibody / fluorescent fold conjugation Fresh sodium bicarbonate buffer (100 mM, pH 8.2) was prepared by diluting 336 mg in 40 mL of ultrapure DI and adjusting the pH. 0.5 mg of BCN-PEG12-NHS adapter was diluted in 100 mL of sodium bicarbonate (bicarb). 0.3 mg (14 mL) of UCHT-4 mouse anti-human CD8 antibody was added to a low-biding Eppendorf tube for reaction and diluted with 122 mL of sodium bicarbonate (bicarb) buffer. BCN adapter was added to achieve a 40 / 80 equivalent relative to the antibody. The adapter was allowed to react with the Ab at room temperature for at least 1 hour. A 30 Kd MWCO filter was pre-washed with PBS, pH 7.2, and the reaction was transferred to the MWCO filter with 200 mL of PBS wash buffer. The filter was rotated at 12 kg for 5 minutes. Further washing was performed with PBS (400 mL added, rotated at 12 kg for 5 minutes, for a total of 4–5 wash cycles). Dissolve 1 mg P57-BFL-660D (equivalent to 20 antibody doses) in 50 mL PBS (pH 7.2). Transfer the retained Ab-BCN reaction mixture from the MWCO tube. Allow it to react for at least 4 hours. Add 1% NaN3 / PBS to each tube to quench the reaction site at room temperature for 30 minutes. Perform ion exchange chromatography using a HiTrap ANX FF(HS) column with 1 mL media buffer: loading / wash buffer: 25 mM sodium bicarbonate; elution buffer: 1:1 25 mM sodium bicarbonate (bicarb) / 1 M NaCl. Transfer the sample to a 30 Kd MWCO UFD and wash 300 μL with IEX loading buffer. Rotate at 12 kg for 5 minutes. Wash the ANX IEX column with loading buffer (10 mL), dilute the sample to 500 mL in loading buffer, and add it to the column. Elute excess unconjugated folds with 5 mL loading buffer. Elute the conjugates with 5 mL elution buffer. Concentrate the reagent in 30 kDa MWCO UFDs (12 kg x 5 min). Dilute the osmotic residue with PBS (final volume approximately 400 mL) and rotate again. Dilute the final osmotic residue to 50 mL.

[0260] Table 9. Photophysical characterization data of antibody fluorescent fold conjugates exp ID Fluorescent fold Ab Equivalent joint Equivalent fluorescent fold QY sample F / P brightness B x F / P SY40-004_40 P57-BFL-660M aCD8, Human, UCHT-4 40 20 0.15 4.2 25,350 107,499 SY40-004_80 P57-BFL-660M aCD8, Human, UCHT-4 80 20 0.18 4.1 29,113 120,125 SY40-006_40 P57-BFL-660DD aCD8, Human, UCHT-4 40 20 0.19 3.7 49,167 179,724 SY40-006_80 P57-BFL-660DD aCD8, Human, UCHT-4 80 20 0.17 4.2 44,897 190,606 .

[0261] Preparation of flow cytometry cell staining samples PBMC cells were obtained from ZenBio and thawed in a water bath at 37°C for 2 minutes. The thawed cells were added to 50 ml conical tubes, and the vials were rinsed with 1 ml of pre-warmed lymphocyte culture medium. Then, another 14 ml of thawing buffer was added to the conical tubes. At the end, the cell suspension was gently mixed, transferred to 16 Eppendorf tubes, and centrifuged at 400 g for 10 minutes. The suspension was carefully removed without disturbing the pellet. The cells were resuspended in 1 mL of BioLegend staining medium by gentle pipetting and centrifuged at 400 g for 10 minutes. The suspension was carefully removed without disturbing the pellet. A staining solution was prepared by directly diluting the antibody-FP stock solution to the desired concentration (40 mg / mL or 100 mg / mL) in the cell staining medium at a total volume of 100 μL. The cell pellet was resuspended directly in 100 μL of cell staining medium by gentle pipetting, and then the staining solution containing antibody-FP was added and gently mixed. Incubate the cell suspension on ice and stain in the dark for 20 minutes. After staining, spin down the cells at 600 g for 10 minutes. Wash each sample with 1 ml of clean cell staining medium and spin down at 600 g for 10 minutes. Resuspend the cells in 250 μl of cell staining buffer, then add 250 μl of 1% formaldehyde to fix the cells. After 15 minutes of fixation, briefly vortex the sample to resuspend the cells and filter through a 70 mm cell filter for testing. Flow cytometry was performed on a Becton Dickinson LSR Fortessa, and the analysis data showed that... Figure 10A middle.

[0262] discuss The described bifunctional linker (BFL) method (see, for example, Table 7) can provide a smaller total number of dyes that can be added to the polymer via the linker than the dendration motif (2 vs. 4), but it exists as a smaller total molecule. BFL-dye molecules actually conjugate to the polymer much more efficiently than the dendration motif version, 78% and 89% in the two examples compared to 20% for the dendration motif Gen2. Using the same polymer backbone (P57) and the same dye (660DD), the brightness increased from 16,853 in the dendration motif case to 57,311 using the BFL method. BFL-based folds also showed greater susceptibility to antibody conjugation to obtain conjugates with fold / Ab ratios in the target range of 3–4.

[0263] The PFPA-based polymers described herein offer the opportunity to add more dye per particle compared to dendritic motifs or BFL methods. Conjugation efficiency is high (93% or higher) in the dye equivalent range of 2 to 16 (see Table 8) because there are now significantly 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 versus that of the purified isolated folded body material. The calculated values ​​match very well with the number of dye equivalents used per reaction, within the expected error range typically associated with extinction coefficient measurements. A PFP generation probe with two dye molecules exhibited a measured brightness of 76,715, which can be compared to the aforementioned BFL folded bodies prepared with the same two dye molecules, exhibiting a brightness of 57,311. Brightness data show an increase within the tested dye equivalent range. Advantageously, even at higher dye loadings, the quantum yield of folded bodies containing multiple dyes was largely maintained in PBS solvent. This indicates that the dyes are sufficiently protected from solvent and dye-dye quenching.

[0264] Example 6. Synthesis of polymers with different dyes at the ends and on the main chain This embodiment describes an exemplary method for synthesizing a dual-dye fold, wherein one type of dye is at the end of the polymer, and different types of dyes are on the polymer backbone. Exemplary compounds synthesized according to this method are depicted below. Those skilled in the art can utilize the description provided herein to attach different dyes to the backbone and ends to achieve variations of the compositions. An exemplary fold having a porphyrin-dihydroporphyrin dimer attached to the end and two porphyrin-chlorophyll dimers attached to the polymer backbone is depicted below: As described in the folded structure above, gradient and random structures can be synthesized within the folded structure. For example, gradient structures can be generated by copolymerization of two selected monomers with significantly different copolymerization reactivity ratios. Typically, copolymerization of methacrylate-based monomers (e.g., polyethylene glycol methacrylate, PEGMA) and acrylate-based monomers (e.g., cyclododecyl acrylate, CDA) results in a non-uniform monomer distribution. In this case, the repeating units of PEGMA are expected to be more concentrated near the bio-conjugated ends of the polymer, while those of CDA are more concentrated near the opposite ends of the polymer.

[0265] Random structures can be generated by copolymerization of two monomers (e.g., two or more different acrylate-based monomers) with comparable reactivity ratios or by polymerization of a single type of functional monomer (e.g., pentafluorophenyl acrylate, PFPA) and further post-polymerization modification to randomly introduce desired functional groups.

[0266] Polymers with two distinct segments can be prepared using a semi-batch process, where the initial monomer feedstock consists of one or two classes of substances. The reaction initially proceeds using the initial components, and additional different monomers can be loaded into the reaction flask after a period of time. This method achieves individual segments with different compositions within a single reaction vessel. Unlike block copolymers, the boundaries between segments exhibit a gradual transition due to the one-pot reaction. In the current example, the first segment contains PEGMA units or PEGMA and cDNA units in a gradient structure. The degree of polymerization (number of repeating units) of this segment can be from 20 to 80. The second segment consists of any PEGMA and cDNA not consumed during the first stage, plus PFPA introduced at the start of the second stage. The typical degree of polymerization of the second segment can be from 60 to 240 monomer units.

[0267] A. Modification of polymer ends by chain extension after polymerization In the exemplary procedure, a dry 10 mL Schlenk flask was loaded with a polymer (102.15 mg, 0.002 mmol of P187), monomers (2-acrylamido-2-methylpropanesulfonic 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 bubbled with argon for 10 minutes. The flask was sealed and then placed in a blue light reactor. After exposure to blue light to initiate the reaction, a first kinetic aliquot was obtained using an airless syringe. After 3 hours, the kinetic sample was removed and the reaction was quenched by exposure to air. The polymer was purified once by precipitation in a 1:1 mixture of diethyl ether and hexane. The modified polymer was separated as a membrane under vacuum. 1 H NMR confirmed the final composition.

[0268] B. The Hanger strategy of ligating porphyrin-chlorophyll duals onto the fold backbone P187 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 equivalents) was dissolved in 150 μL of anhydrous THF. The two solutions were then mixed in dry 4 mL vials. N,N- Diisopropylethylamine (10 μL of 5% v / v THF solution) was added to the solution. The vial was capped and stirred at 50 °C on an aluminum pie block. After 2 hours, the reaction was removed from the aluminum pie block and cooled to room temperature. Cyclododecylamine (0.66 mg, 3.6 μmol, 24 equivalences) was dissolved in 25 μL of anhydrous THF and added to the solution. The reaction was then placed back on the pie plate and stirred at 50 °C for another 2 hours. Jeffamine M-1000 (26 mg, 26 μM, 200 equivalences) was dissolved in 75 μL of anhydrous THF. The reaction was cooled again and the Jeffamine solution was added. After capping the reaction vial, the reaction was stirred at 50 °C for another 16 hours. The reaction mixture was purified in toluene by passing through a 1 x 20 cm S-X1 column. The product fraction was concentrated, and 9.2 mg (72%) was isolated.

[0269] C. Conjugating a porphyrin-dihydroporphyrin dual at the end of the fold body. The modified Pl 87 product from the first step (9.2 mg, 0.11 μM) was dissolved in 100 mL of anhydrous DMF and added to 5 mL of RBF. The flask was sealed with a diaphragm, evacuated, and purged three times with argon. The system was briefly disrupted, and a hydrazine solution (2.0 µL stock solution in DMF, 0.013 μL, 0.27 μmol, 2.5 equivalents) was added to the reaction solution via pipette. The flask was then capped and stirred at room temperature under an argon flow. After 10 minutes, the reaction mixture was concentrated by rotary evaporation. 660DD-MAL dye (0.43 mg, 0.43 μmol, 4.0 equivalents) was then added to the flask, which was sealed with a diaphragm, evacuated, and purged three times with argon. DMF (200 µL) was added, and stirring was initiated. The system was briefly disrupted, and trimethyl phosphite (0.63 mL stock solution in DMF, 0.063 μL, 0.067 μmol, 5.0 equivalent) was added directly to the 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 sealed with a diaphragm, protected from light, and stirred at room temperature under a low flow rate of argon. After 16 hours, the reaction was concentrated. The reaction mixture was purified using a 1 x 20 cm S-X1 column and eluted with toluene. The product fraction was concentrated and dried under high vacuum. 8.5 mg (92%) of the membrane was isolated. The photophysical properties of the sample were analyzed. Each FP contained four 743DD dyes and one 660DD dye. Absorbance and emission spectra are shown in [data missing]. Figure 11A and 11B middle.

[0270] D. Antibody / Folded Body Conjugation UCHT-4 mouse anti-human CD8 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 container, DBCO-PEG12-NHS adapter (0.5 mg) was diluted in anhydrous DMSO (50 μL). A fraction of the adapter solution (1.34 μL) was then added to the antibody solution to achieve a 10:1 adapter / antibody ratio and allowed to react at room temperature for 1 hour. Next, the reaction mixture was centrifuged (12 kg x 5 min) using a 30 kDa MWCO filter (pre-washed with PBS; pH adjusted to 7.2). It was then rinsed again (4–5 times) with 400 μL of PBS solution (rotated at 12 kg for 5 min). In a separate Eppendorf tube, dual-label FP (2.3 mg, 20 antibody equivalents) was diluted with 50 μL of PBS (pH 7.2). The antibody-adaptor mixture was then transferred to this FP solution and mixed overnight at room temperature using a shaker. Then, 1% NaN3 / PBS solution was added to each tube. After 30 minutes, ion exchange chromatography was performed using the following buffer (1 mL HiTrap ANX FF(HS)): 25 mM sodium bicarbonate (loading / washing buffer), 1:125 mM sodium bicarbonate / 1 M NaCl (elution buffer). The sample was then transferred to a 30 kDa MWCO filter tube. The column was centrifuged with IEX loading buffer (12 kg for 5 min). The 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 folds were eluted with 5 mL of loading buffer, and conjugated folds were eluted with another 5 mL of elution buffer. The conjugated folds were then centrifuged in a 30 kDa MWCO filter tube (12 kg x 5 min). The osmotic residue was diluted to 400 μL with PBS and centrifuged again. The final osmotic residue was diluted to 50 μL with PBS. Analysis of the photophysical properties of folded-antibody conjugates ( Figure 11C and 11D The results show that 6.5 folds are conjugated to each antibody.

[0271] Furthermore, flow cytometry analysis of exemplary folds with multiple dyes on the backbone was performed on bang beads. Figure 10B ); and on CD8 monoclonal antibody-stained PBMCs, multiple dyes are present on the main chain ( Figure 10C ).

[0272] Example 7. Synthesis of a polymer having two different dyes linked to the main chain This embodiment describes an exemplary method for synthesizing a bi-dye fold having two different types of dyes on the polymer backbone. Exemplary compounds synthesized according to this method are depicted below. Those skilled in the art can utilize the description provided herein to attach different dyes to the backbone to achieve variations of the compositions described herein. The exemplary fold chemical structures having two different dyes (porphyrin-chlorophyll dual and porphyrin-dihydroporphyrin dual) on the polymer backbone are depicted below: An exemplary folded form with two different dyes (porphyrin-chlorophyll dimeric and porphyrin-dihydroporphyrin dimeric) on the polymer backbone was synthesized. The PFP-containing polymer was dissolved in anhydrous THF. 743D-NH2 (2 equivalents) and 660D-NH2 (1 equivalent) were dissolved in anhydrous THF, and the two solutions were mixed in a dry 4 mL vial. N,N - Diisopropylethylamine (15 equivalents of 5% v / v THF solution) was added to the solution. The vial was capped and stirred at 50°C. After 2 hours, the reaction was removed from the plate and cooled to room temperature. The amount of cyclododecylamine was calculated to produce a side chain with a hydrophobic composition of 7% (by weight). The calculated amount of cyclododecylamine was dissolved in anhydrous THF and added to the solution. The reaction was stirred at 50°C for another 2 hours. Jeffamine M-1000 (twice the equivalent of PFP on the polymer side chain) was dissolved in anhydrous THF. The reaction was cooled again and the Jeffamine solution was added. After capping the reaction vial, the reaction was stirred at 50°C for another 16 hours. The reaction mixture was purified using a 1 x 20 cm S-X1 column and eluted with toluene. The product fraction was dried on a rotary evaporator.

[0273] Example 8. Optimization of Multi-Dye Folded Body Design Design parameters Several design parameters have been explored to optimize dye brightness on the folded bodies in PBS. Several of these have been identified as important, including: 1) the total length of the polymer; 2) the number of dyes per polymer chain; 3) the dye spacing along the chain; 4) the location of hydrophobic and hydrophilic enrichment regions within the polymer; and 5) the relative percentage of the hydrophobic component. These parameters were optimized in the examples shown in Table 9. Data analysis methodsFor each novel multi-dye fold (FP) synthesized, photophysical data, including maximum absorbance, maximum emission, extinction coefficient (ε), and quantum yield (QY), were collected in organic solvents (toluene, THF, or DMF) and aqueous solvents (PBS). Fold design optimization was guided by calculating the percentage of ε and QY retained in aqueous solution compared to their values ​​in organic solvents. These variables were interpreted as reflecting the polymer's ability to protect the dye from fluorescence quenching associated with interactions with the solvent and / or other dye molecules.

[0274] Table 9. Examples and photophysical properties of multi-dye folds entry polymer #Dyes / Polymers QY retained Retained brightness 1 P138 5.5 60% 55% 2 P156 3.9 71% 61% 3 P183 2.7 80% 56% 4 P189 2.7 89% 78% 5 P202 3.3 99% 109% .

[0275] Important component variables The data were categorized by the specific dye type used to allow for comparison of brightness values ​​based solely on polymer design characteristics rather than the dye itself. Specifically, analysis was performed on FPs containing NRV660DD dye for comparison.

[0276] Data were categorized by the amount of QY retained in PBS and grouped into percentage ranges of 0–49%, 50–69%, 70–79%, 80–89%, and 90+. The average polymer length or degree of polymerization (DP) for these groups was analyzed. A general trend of increasing QY retention with increasing average polymer length was noted (Table 10). The data suggest that a polymer length of approximately 180 units or greater provides optimal QY retention. The DP range for the exemplary FPs evaluated was 50–340.

[0277] Table 10. Average polymer DP of the multisheet, grouped by QY% retention rate in PBS vs. organic solvents. QY retention rate Average polymer DP 0-49% 69 50-69% 101 70-79% 163 80-89% 183 90+% 185 .

[0278] General biasing of hydrophobic properties in the dye-labeled environment along the polymer backbone, achieved through gradient synthesis methods, is also important. This can be seen from the increasing QY retention as one moves from the random copolymer P138 to the gradient polymers P156 and subsequent versions.

[0279] Example 9. Multi-dye loading This embodiment tested the limits of loading exemplary dyes onto each of the exemplary polymers. The P133 polymer (DP = 235) was loaded with 10, 20, 40, and 80 equivalents of 660M-NH2 dye, having cyclododecylamine as a hydrophobic group (10% of the reactive sites) and the balance Jeffamine M-1000 (a hydrophilic polyether monoamine with a molecular weight of approximately 1000).

[0280] Table 11 below includes the results of the dye loading experiments: Table 11. .

[0281] Figure 12 The quantum yield (QY) (left) and brightness (right) of exemplary compounds measured in THF and PBS are shown compared to the number of dyes per compound. Results show a maximum dye loading of 53 achieved with 80 dye equivalents. QY remains well-maintained in THF with increasing dye number, but a decrease is observed in PBS. Brightness in PBS reaches its maximum with approximately 30 660M-NH2 dyes per P133 polymer, decreasing as the dye / polymer ratio increases beyond 30. Compounds prepared with 80 dye equivalents require heating to dissolve the sample in PBS.

[0282] Figure 13 Peripheral blood mononuclear cells stained with multi-dye folds linked to a CD8 monoclonal antibody are shown. The P222 folds used included a porphyrin-dihydroporphyrin dimeric compound and charged groups with a total DP of less than 200. The stained cells were imaged using a UV light source on an Olympus IX51 inverted fluorescence microscope.

[0283] Example 10. Multidye gradient folds linked to antibodies Microscopic images of multi-dye folded antibody conjugates on polystyrene beads are shown. Figure 14-15 In. Figure 14 In this formulation, P220-AMP begins with a DP 51 PEG-CDA gradient, followed by DP 62 PFP. The total DP is 121, with 11 AMP added at the end. This folded form contains 2.6 660 dimeric units per polymer and 7% by weight of total CDA. The compound contains 6.1 folded units per antibody. Figure 14 The image below shows positive vs. negative beads. Figure 15 In this formulation, P220 begins with a DP 51 PEG-CDA gradient, followed by DP 62 PFP. The total DP is 121. This folded form contains 2.4 dimeric units per polymer with a total CDA content of 7% by weight. The compound contains 1.9 folded units per antibody. Figure 15The image below shows positive vs. negative beads.

[0284] Example 11. Confirmation of the incorporation of charged groups in the folded body Successful incorporation of the charged 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) monomer into an exemplary polymer extended using reversible addition-fragmentation chain transfer (RAFT) was confirmed by comparing the relative integral of the vinyl proton peak with that of the internal standard strobilurine. The prominent AMPS proton appeared at a chemical shift of 6.05 ppm, while the corresponding proton of strobilurine was identified at 6.75 ppm. Kinetic samples obtained at the start and end of the reaction were used to calculate the change in the relative integral of the AMPS vinyl proton during the reaction. The observed 60% decrease in the relative integral of the vinyl proton implies approximately 60% monomer conversion, thus resulting in an average incorporation of approximately 12 AMPS units per polymer terminus. Similar analytical methods have been used to confirm the incorporation of AMPS, as well as other monomers, into the polymer backbone as detailed herein.

[0285] Table 12. Exemplary dyes incorporated into the fold body, individually or in combination. FC Microscopy PAI MRI PET Fluorescence-guided surgery PDT Tetrapyrrole Bacteriochlorophyll Free base form Y Y Y Y Y Metallized form Y Y Y Y Y Y Y Chlorophyll Free base form Y Y Y Y Y Metallized form Y Y Heme Metallized form Y Y Y Bacteriochlorin Free base form Y Y Y Y Y Metallized form Y Y Y Y Y Y Y Chlorin Free base form Y Y Y Y Y Metallized form Y Y Porphyrin Free base form Y Y Y Y Metallized form Y Y Y Y Y Y Y Gem-dimethyl substituted phorbine Bacteriochlorins & Isobacteriochlorins Free base form Y Y Y Y Y Metallized form Y Y Y Y Y Y Y Chlorin Free base form Y Y Y Y Y Metallized form Y Y Chlorin-chlorin dimers Free base form Y Y Y Y Y Metallized form Y Y Bacteriochlorin-bacteriochlorin dimers Free base form Y Y Y Y Y Metallized form Y Y Y Y Y Y Y Porphyrin-chlorin dimers Free base form Y Y Y Y Y Metallized form Y Y Porphyrin-bacteriochlorin dimers Free base form Y Y Y Y Y Metallized form Y Y Chlorin-bacteriochlorin dimers Free base form Y Y Y Y Y Metallized form Y Y Other classes of dyes Cyanine dyes Y Y Y Y Y Example: Indocyanine green Y Y Y Y Y Melanin Y Phthalocyanine Free base form Y Y Y Y Y Metallized form Y Y Y Y Y Y Y Indocyanine Free base form Y Y Y Y Y Metallized form Y Y Y Y Y Y Y Linear polyenes Y Example: β-carotene Triarylmethanes Y Example: Crystal violet Malachite green Azo dyes Y Phenothiazine Y Y Example: Methylene blue Rylene dyes, including Y Y Y Example: Perylene Y Y Y Terrylene Y Y Y Quaterrylene Y Y Y Boron-dipyrromethene Y Y Y Y Example: BODIPY TR Y Y Y Y <![CDATA[BODIPY-chlorin 二联体3、6、8 > Y Y Y Y Y <![CDATA[dBODIPY-bacteriochlorin dimer 3、6、8 > Y Y Y Y Y Squaraine dyes (some squaraines are members of the cyanine dye family, others are not) Y Y Y Y Xanthenes, including Y Y Y Rhodamine Y Y Y Example: Rhodamine 6G Y Y Y Fluorescein Y Y Y Alexa Fluor 488 Y Y Y .

[0286] The following references are related to Table 12: 1. Yu, Zhanqian, and Marcin Ptaszek. Near-IR emissive chlorin–bacteriochlorin energy-transfer dyads with a common donor and acceptors with tunable emission wavelength. The Journal of organic chemistry 78. 21 (2013):10678-10691. 2. Yu, Z., Pancholi, C., Bhagavathy, GV, Kang, HS, Nguyen, JK, Ptaszek, M. (2014). Strongly conjugated hydroporphyrin dyads: extensive modification of hydroporphyrins' properties by expanding the conjugatedsystem. The Journal of organic chemistry , 79 17 (2014): 7910-7925. 3. Meares, A., Satraitis, A., Santhanam, N., Yu, Z., Ptaszek, M.Deep-red emissive BODIPY–chlorine arrays excitable with green and redwavelengths. The Journal of organic chemistry , 1999 . 80 . . . . 8 (2015): 3858–3 4. Kang, HS, Esemoto, NN, Diers, JR, Niedzwiedzki, DM,Greco, JA, Akhigbe, J, Yu, Z, Pancholi, C, Bhagavathy, GV, Nguyen, JK, Kirmajer, C, Birge, RR, Ptaszek, M, Bocian, DF bacteriochlorin dyads. The Journal of Physical Chemistry A , 1999 . 120 . . . . 3 (2016): 379–395. 5. Esemoto, NN, Yu, Z., Wiratan, L., Satraitis, A., Ptaszek, M.Bacteriochlorine dyads as solvent polarity dependent near-infrared fluorophores and reactive oxygen species photosensitizers. Organic letters 18.18 (2016): 4590–4593. 6. Meares, A., Satraitis, A., Akhigbe, J., Santhanam, N.,Swaminathan, S., Ehudin, M., Ptaszek, M. Amphiphilic BODIPY-hydroporphyrinenergy 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. 7. Esemoto, N. N., Satraitis, A., Wiratan, L., Ptaszek, M.Symmetrical and nonsymmetrical meso–meso directly linked hydroporphyrindyads: synthesis and photochemical properties. Inorganic chemistry , 57 . 6(2017): 2977-2988. 8. Meares, A., Satraitis, A., Ptaszek, M. BODIPY–bacteriochlorinenergy transfer arrays: toward near-IR emitters with broadly tunable,multiple absorption bands. The Journal of organic chemistry , 82 . 24 (2017):13068-13075. 9. Ogata, F., Nagaya, T., Maruoka, Y., Akhigbe, J., Meares, A., Lucero, M.Y., Satraitis, 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. 10. McCleese, C., Yu, Z., Esemoto, N. N., Kolodziej, C., Maiti, B., Bhandari, S., Dunjetz, 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. 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 . 16 (2018): 9076-9087. 12. Meares, A., Yu, Z., Bhagavathy, G.V., Satraitis, A., Ptaszek, M.Photoisomerization of Enediynyl Linker Leads to Slipped CofacialHydroporphyrin Dyads with Strong Through-Bond and Through-Space ElectronicInteractions. The Journal of Organic Chemistry , 84 . 12 (2019): 7851-7862. 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 Holten, D. Photophysical Properties and Electronic Structure ofHydroporphyrin Dyads Exhibiting Strong Through-Space and Through-BondElectronic Interactions. The Journal of Physical Chemistry A , 126 . 31 (2022):5107-5125. 14. Molecular Probes Handbook: a Guide to Fluorescent Probes andLabeling Technologies, 11th Edition, Life Technologies, 2010. 15. International Patent Publication No. WO2021118782A2. 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. Stablesynthetic bacteriochlorins overcome the resistance of melanoma tophotodynamic therapy. FASEB JOURNAL 24, 3160–70. doi:10.1096 / fj.09-152587 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 QuantitativeStructure-Activity Relationship Studies with Stable Synthetic Near-Infrared-Absorbing Bacteriochlorin Photosensitizers. JOURNAL OF MEDICINAL CHEMISTRY53, 4018–27. doi:10.1021 / jm901908s 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 asSelective Antimicrobial Photosensitizers. ANTIMICROBIAL AGENTS ANDCHEMOTHERAPY 54, 3834–41. doi:10.1128 / aac.00125-10 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 evaluationof cationic bacteriochlorin amphiphiles with effective in vitro photodynamicactivity against cancer cells at low nanomolar concentration. JOURNAL OFPORPHYRINS AND PHTHALOCYANINES 17, 73–85. doi:10.1142 / s108842461250126x 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. StableSynthetic Bacteriochlorins for Photodynamic Therapy: Role of DicyanoPeripheral Groups, Central Metal Substitution (2H, Zn, Pd), and Cremophor ELDelivery. ChemMedChem 7, 2155–67. doi:10.1002 / cmdc.201200351 21. Yang, E., Diers, J. R., Huang, Y.-Y., Hamblin, M. R., Lindsey, J.S., Bocian, D. F.,&Holten, D. (2013). Molecular Electronic Tuning ofPhotosensitizers to Enhance Photodynamic Therapy: SyntheticDicyanobacteriochlorins as a Case Study. PHOTOCHEMISTRY AND PHOTOBIOLOGY, 89(3), 605–618. doi:10.1111 / php.12021 22. Huang, L., Krayer, M., Roubil, JGS, Huang, Y.-Y., Holten, D.,Lindsey, JS, Hamblin, MR, 2014. Stable synthetic mono-substitutedcationic bacteriochlorins mediate selective broad-spectrum photoinactivation of drug-resistant pathogens at nanomolar concentrations. JOURNAL OFPHOTOCHEMISTRY AND PHOTOBIOLOGY B-BIOLOGY 141, 119–27. doi:10.1016 / j.jphotobiol.2014.09.016.

[0287] The foregoing is an illustrative description of the invention and should not be construed as limiting it. The invention is defined by the following claims, including their equivalents. All publications, patent applications, patents, patent publications, and other references cited herein are incorporated herein in their entirety with respect to the teachings relating to the sentences and / or paragraphs in which such references are made.

Claims

1. A compound (e.g., a polymer) comprising: A polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; A first end group is attached to a first end of the polymer backbone, wherein the first end group optionally comprises a major dye (e.g., a luminescent group (e.g., a fluorophore) or a non-luminescent molecular entity), a bulk group, a hydrophobic group (e.g., a hydrophobic monomer), and / or a charged group. A second end group connected to a second end opposite to the first end of the polymer backbone, optionally wherein the second end group comprises a bioconjugating group or a dye; and The main dye is attached to the polymer backbone.

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

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

4. The compound according to any one of claims 1-3, wherein at least one of the first or second end groups comprises a major dye, optionally wherein the first end group is free of bulk groups and charged groups, and the first end and / or the second end comprises a major dye.

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

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

7. The compound according to 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 cyclodextrin or polyhedral oligomeric silsesquioxane (POSS).

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

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

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

12. The compound according to any one of the preceding claims, wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the compound at a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, optionally wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the compound at a ratio of about 1:6 (hydrophobic unit: hydrophilic unit).

13. The compound according to any one of the preceding claims, wherein the compound has conformational flexibility.

14. The compound according to any one of the preceding claims, wherein the compound self-folds in an aqueous solution, optionally self-folding into a monomeric micelle structure.

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

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

17. The compound according to any one of the preceding claims, wherein the compound is folded to provide particles, optionally wherein the particles have a diameter in the 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 units are present in the core of the particle and / or at least a portion of the one or more hydrophilic units are present at the periphery (e.g., shell) of the particle.

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

20. The compound according to any one of the preceding claims, wherein the compound is a telechelic polymer or anisotended telechelic polymer.

21. The compound according to any one of the preceding claims, wherein the primary dye (e.g., a tetrapyrrole macrocycle) is hydrophobic.

22. The compound according to any one of the preceding claims, wherein the compound is water-soluble, and optionally the compound has a solubility in water at room temperature of at least 1 mg / mL.

23. The compound according to any one of the preceding claims, wherein at least one of the one or more hydrophobic units and / or the one or more hydrophilic units comprises a side functional group, optionally wherein the side functional group is a halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, or fluorophenyl ester), azide, maleimide, isocyanate, isothiocyanate, phosphono, sulfonyl, ammonium, or phosphatidylcholine group, and / or the side functional group is a hydrophilic group comprising a terminal cation (e.g., ammonium), anion (e.g., sulfonate, phosphate, carboxylate, or phosphonate), or zwitterionic (e.g., choline-like) group and optionally a poly(ethylene glycol) moiety.

24. The compound according to any one of the preceding claims, wherein at least one of the one or more hydrophobic units comprises an alkyl side group (e.g., dodecyl), and / or at least one of the one or more hydrophilic units comprises a diol side group (e.g., poly(ethylene glycol)).

25. The compound according to any one of the preceding claims, 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 major dyes.

26. The compound according to any one of the preceding claims, wherein the compound comprises a hydrophobic unit having a structure represented by Formula III: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R' is absent or is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or C6-C25 arylalkyl; R 2 It is hydrogen or halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azide, maleimide, isocyanate, or isothiocyanate, or R 2 It is the main 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 the R of the hydrophobic unit 2 It is a hydrogen, hydroxyl, carboxyl, amino, formyl, or ester group, optionally a pentafluorophenyl ester.

28. The compound of claim 26, wherein the R of the hydrophobic unit 2 It is vinyl, epoxy, mercapto, azide, isocyanate, isothiocyanate, or maleimide.

29. The compound according to claim 26, wherein R 2 It is another primary dye, optionally said other primary dye comprising two or more dyes.

30. The compound according to any one of the preceding claims, wherein the compound comprises a hydrophilic unit having a structure represented by Formula IV: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R 3 Choose freely – (CH2CH2R) 5 ) n The group consisting of -, -C1-C6 alkyl, -C1-C6 alkyl-O- and –C1-C6 alkyl-SO3- or their salts, wherein R 5 It is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000; R 4 The following groups are absent or do not contain hydrogen, alkyl, phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) groups, or R. 4 It is another major 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 R in the hydrophilic unit 4 It is hydrogen, alkyl, phosphonoyl, sulfonyl, phosphatidylcholine, phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester.

32. The compound of claim 30, wherein R in the hydrophilic unit 4 It is vinyl, epoxy, mercapto, azide, isocyanate, isothiocyanate, or maleimide.

33. The compound according to claim 26, wherein R 3 It is a -C1-C6 alkyl-O- or –(CH2CH2R) 5 ) n -, where R 5 It is -O-, and R in the hydrophilic unit 4 It is hydrogen, alkyl (e.g., methyl or ethyl), phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), or phosphoryl.

34. The compound of claim 26, wherein R in the hydrophilic unit 3 It is a C1-C6 alkyl group or –(CH2CH2R) 5 ) n -, where R 5 It is –CH2-, and R in the hydrophilic unit 4 It can be hydroxyl, carboxyl, amino, ammonium, formyl, ester, phosphonyl, or sulfonyl.

35. The compound according to claim 26, wherein R 3 It is –C1-C6 alkyl-SO3- or its salt.

36. The compound according to any one of the preceding claims, wherein the primary dye and / or secondary dye is Fe(II)-chelate tetrapyrrole or Cu(II)-chelate tetrapyrrole (e.g., porphyrin).

37. The compound according to any one of the preceding claims, wherein the polymer backbone further comprises additional units comprising one or more additional major dyes.

38. A compound (e.g., a polymer) comprising: A polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; A first end group is attached to a first end of the polymer backbone, wherein the first end group comprises (i) Major dyes (e.g., luminescent or nonluminescent molecular entities) and biomolecules (e.g., DNA, RNA, proteins or peptides). (ii) Primary dyes, secondary dyes (e.g., luminescent or nonluminescent molecular entities), and optionally biomolecules, or (iii) Two or more primary dyes and optionally biomolecules; and A second end group is attached to a second end of the polymer backbone, wherein the second end group optionally comprises a bioconjugating group.

39. A compound (e.g., a polymer) comprising: A polymer backbone containing one or more hydrophobic units and one or more hydrophilic units; A first end group is attached to a first end of the polymer backbone, wherein the first end group comprises (i) Hydrophobic groups, bulky groups, or charged groups; or (ii) Hydrophobic groups, bulky groups and / or charged groups; A second end group connected to the second end of the polymer backbone, optionally wherein the second end group comprises a bioconjugating group; and Primary and secondary dyes are attached to 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 the range of about 150 Daltons (Da) to about 3,000 Da.

41. The compound according to any one of claims 38-40, wherein the compound has a molecular weight in the 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 according to any one of claims 38-41, wherein the one or more hydrophobic units and the one or more hydrophilic units are randomly distributed in the polymer backbone.

43. The compound according to any one of claims 38-42, wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the compound at a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, optionally wherein the one or more hydrophobic units and the one or more hydrophilic units are present in the compound at a ratio of about 1:6 (hydrophobic unit: hydrophilic unit).

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

45. The compound according to any one of claims 38-44, wherein the compound self-folds in an aqueous solution, optionally self-folding into a monomeric micelle structure.

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

47. The compound according to any one 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 according to any one of claims 38-47, wherein the compound is folded to provide particles, optionally wherein the particles have a diameter in the 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 units are present in the core of the particle and / or at least a portion of the one or more hydrophilic units are present on the periphery (e.g., shell) of the particle.

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

51. The compound according to any one of claims 38-50, wherein the compound is a telechelic polymer or anisotended telechelic polymer.

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

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

54. The compound according to any one of claims 38-53, wherein at least one of the one or more hydrophobic units and / or the one or more hydrophilic units comprises a side functional group, optionally wherein the side functional group is a halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimidyl ester, or fluorophenyl ester), azide, maleimide, isocyanate, isothiocyanate, phosphono, sulfonyl, ammonium, or phosphatidylcholine group, and / or the side functional group is a hydrophilic group comprising a terminal cation (e.g., ammonium), anion (e.g., sulfonate, phosphate, carboxylate, or phosphonate), or zwitterionic (e.g., choline-like) group and optionally a poly(ethylene glycol) moiety.

55. The compound according to any one of claims 38-54, wherein at least one of the one or more hydrophobic units comprises an alkyl side group (e.g., dodecyl), and / or at least one of the one or more hydrophilic units comprises a diol side group (e.g., poly(ethylene glycol)).

56. The compound according to any one of claims 38-55, wherein the compound comprises a hydrophobic unit having a structure represented by formula III: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R' is absent or is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or C6-C25 arylalkyl; R 2 It is hydrogen or halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azide, maleimide, isocyanate, or isothiocyanate, or R 2 It is a primary dye, a secondary dye, another primary dye, and / or another 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 R in the hydrophobic unit 2 It can be hydrogen, hydroxyl, carboxyl, amino, formyl, or ester group.

58. The compound of claim 56, wherein R in the hydrophobic unit 2 It is vinyl, epoxy, mercapto, azide, isocyanate, isothiocyanate, or maleimide.

59. The compound according to any one of claims 38-58, wherein the compound comprises a hydrophilic unit having a structure represented by formula IV: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R 3 Choose freely – (CH2CH2R) 5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl-O- and –C1-C6 alkyl-SO3- or their salts, wherein R 5 It is a group consisting of -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000; R 4 The following groups are absent or do not contain hydrogen, alkyl, phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) groups, or R. 4 It is a primary dye, a secondary dye, another primary dye, and / or another 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 R in the hydrophilic unit 4 It is hydrogen, alkyl, phosphonoyl, sulfonyl, phosphatidylcholine, phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester.

61. The compound of claim 59, wherein R in the hydrophilic unit 4 It is vinyl, epoxy, mercapto, azide, isocyanate, isothiocyanate, or maleimide.

62. The compound of claim 59, wherein R in the hydrophilic unit 3 It is a -C1-C6 alkyl-O- or –(CH2CH2R) 5 ) n -, where R 5 It is -O-, and R in the hydrophilic unit 4 It is hydrogen, alkyl (e.g., methyl or ethyl), phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), or phosphoryl.

63. The compound of claim 59, wherein R in the hydrophilic unit 3 It is a C1-C6 alkyl group or –(CH2CH2R) 5 ) n -, where R 5 It is –CH2-, and R in the hydrophilic unit 4 It can be hydroxyl, carboxyl, amino, ammonium, formyl, ester, phosphonyl, or sulfonyl.

64. The compound according to claim 63, wherein R 3 It is –C1-C6 alkyl-SO3- or its salt.

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

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

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

68. The compound according to any one 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 according to any one of claims 43-71, wherein the first end group comprises a 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 according to any one 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 according to any one 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 two or more primary dyes, wherein the bifunctional branched linker is side-attached to the polymer backbone.

72. The compound according to any one of claims 38-71, further comprising a trifunctional branching linker between the polymer backbone, one or more major dyes, one or more minor dyes, one or more charged groups, one or more bulk groups, and / or one or more biomolecules. Optionally, the first end base includes: Two primary dyes and the secondary dye, and the trifunctional branched connector between the two primary dyes and the secondary dye, or The primary dye, the secondary dye, and the third dye, and the trifunctional branched connector between the primary dye, the secondary dye, and the third dye, or Three primary dyes, and the trifunctional branched connector between each of the three primary dyes, or Two main dyes and the biomolecules, or The primary dye, the secondary dye, and the biomolecule, or Two charged groups and the bulky group, and the trifunctional branched connector is located between the two charged groups and the bulky group, or Three large volume groups, and the trifunctional branched connector is between each of the three large volume groups, or Three charged groups, and the trifunctional branched connector is between each of the three charged groups, or Two charged dye groups and the biomolecule, or Two large-volume groups and the biomolecule, or One or more hydrophobic groups (e.g., hydrophobic monomers), or The bulky group, the charged group, and the biomolecule.

73. The compound according to any one 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 two or more primary dyes, between the bulk group and the charged group, between the bulk group and the biomolecule, between two bulk groups, or between two charged groups, the linker comprising or consisting of portions having the following structure: -(CH2) m1 Ra(CH2) n1 But Where m1 is an integer from 0 to 3; n1 is an integer from 0 to 3; and Ra is selected from -OC(O)-, -C(O)O-, , -CH(OH)CH2R x -、 , or -C(O)NH-; where R x It is O or N; Or a part having the following structure: -(CH) m2 Rb(CH2) n2 Where m2 is between 0 and 3; n2 is between 0 and 3; and Rb is -N-, -NNH-, -S-. -Ph-C(O)NH(CH2) x C(O)NH-、-Ph-C(O)NH(CH2) x C(O)-、-Ph-C(O)NH(CH2) x C(O)- or -Ph-N(CH3)C(O)(CH2) x C(O), where Ph is phenyl and x is an integer between 0 and 4; optionally, the connector is replaced by a PEG molecule.

74. The compound according to any one of claims 71-73, wherein the linear connector, bifunctional connector, or trifunctional connector comprises a PEG moiety and a second moiety having the following structure: -(CH2) m1 Ra(CH2) n1 But Where m1 is an integer from 0 to 3; n1 is an integer from 0 to 3; and Ra is selected from -OC(O)-, -C(O)O-, , -CH(OH)CH2R x -、 , or -C(O)NH-; where R x It is O or N; Or a part having the following structure: -(CH) m2 Rb(CH2) n2 Where m2 is between 0 and 3; n2 is between 0 and 3; and Rb is -N-, -NNH-, -S-. -Ph-C(O)NH(CH2) x C(O)NH-、-Ph-C(O)NH(CH2) x C(O)-、-Ph-C(O)NH(CH2) x C(O)- or -Ph-N(CH3)C(O)(CH2) x C(O), where Ph is a phenyl group and x is an integer between 0 and 4. Optionally, the PEG portion is covalently connected at a connection point of the second portion.

75. A composition comprising the compound according to any one of claims 1-74.

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

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

78. The composition according to any one of claims 75-77, wherein at least a portion of the one or more hydrophobic units are present in the nucleus of the particle.

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

80. The composition according to any one of claims 75-79, wherein the particles are dilution resistant, optionally wherein the particles retain a folded structure when the composition is diluted to 100x or to a submicromolar concentration.

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

82. The composition according to any one of claims 75-77, wherein the composition is free of organic solvents.

83. A method for preparing a compound, comprising: copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers to provide a compound comprising a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units, a first end group and a second end group; A bulky group, a hydrophobic group, and / or a charged group are attached to the first end group of the compound to become part of the first end group; The main dye (e.g., luminescent (e.g., fluorophore) or nonluminescent molecular entity) is attached to a functional group side-attached to the polymer backbone; Optionally, a bioconjugating group is attached to a second end group of the compound; and Optionally, the compound may be crosslinked.

84. A method for preparing a compound, comprising: copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers to provide a compound comprising a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units, a first end group and a second end group; Optionally, a primary dye (e.g., a luminescent group (e.g., a fluorophore) or a non-luminescent molecular entity) is attached to a first end group of the compound to become part of the first end group; The additional main dye is attached to the functional group side-attached to the polymer backbone; Optionally, a bioconjugating group is attached to a second end group of the compound; and Optionally, the compound may be crosslinked.

85. A method for preparing a compound, comprising: Solution polymerization of one or more hydrophilic monomers and optionally one or more hydrophobic monomers, followed by adding one or more hydrophobic monomers to the solution and polymerizing the solution to provide a compound comprising a polymer backbone, a first end group and a second end group, the polymer backbone comprising a gradient backbone containing one or more hydrophobic units and one or more hydrophilic units; Optionally, a primary dye (e.g., a luminescent group (e.g., a fluorophore) or a non-luminescent molecular entity) is attached to a first end group of the compound to become part of the first end group; The additional main dye is attached to the functional group side-attached to the polymer backbone; Optionally, a bioconjugating group is attached to a second end group of the compound; and Optionally, the compound may be crosslinked.

86. The method according to claims 83-85, wherein one or more hydrophobic monomers and one or more hydrophilic monomers are copolymerized by 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 according to claims 83-85, wherein the one or more hydrophobic monomers and the one or more hydrophilic monomers are copolymerized by living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT reagent (e.g., a thiocarbonyl thio compound) to provide the polymer backbone.

88. The method according to any one of claims 83-86, wherein the one or more hydrophobic monomers and the one or more hydrophilic monomers are copolymerized at a ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10 (hydrophobic monomer / hydrophilic monomer ratio).

89. The method according to any one of claims 83-88, wherein at least one hydrophobic monomer has a structure represented by formula I: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R' is absent or is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or C6-C25 arylalkyl; R 2 It is hydrogen or halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azide, maleimide, isocyanate, or isothiocyanate, or R 2 It is another major 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 R in the hydrophobic monomer 2 It can be hydrogen, hydroxyl, carboxyl, amino, formyl, or ester group.

91. The method according to any one of claims 83-90, wherein at least one hydrophilic monomer has a structure represented by formula II: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R 3 Choose freely – (CH2CH2R) 5 ) n The group consisting of -, -C1-C6 alkyl, -C1-C6 alkyl-O- and –C1-C6 alkyl-SO3- or their salts, wherein R 5 It is -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000, or 10,000; and R 4 The absence of hydrogen, alkyl, phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), phosphoryl, halogen, hydroxyl, carboxyl, amino, ammonium, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) groups, or optionally R 4 It is another major dye.

92. The method according to claim 91, wherein R in the hydrophilic monomer 4 It can be a hydroxyl, carboxyl, amino, formyl, or ester group.

93. The method of claim 91, wherein R 3 It is -C1-C6 alkyl-O-, and R in the hydrophilic monomer 4 It is hydrogen, alkyl (e.g., methyl or ethyl), phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine, or phosphoryl.

94. The method of claim 91, wherein R 3 It is a -C1-C6 alkyl group or –(CH2CH2R) 5 ) n -, where R 5 It is –CH2-, and R in the hydrophilic monomer 4 It can be hydroxyl, carboxyl, amino, ammonium, formyl, ester, phosphonyl, or sulfonyl.

95. A method for preparing a compound, comprising: copolymerizing one or more hydrophobic monomers and one or more hydrophilic monomers to provide a compound comprising a polymer backbone containing one or more hydrophobic units and one or more hydrophilic units, a first end group and a second end group; A bulky group, a hydrophobic group (e.g., a hydrophobic monomer), and / or a charged group are attached to a first end group (i.e., become part of a first end group) or optionally a major dye (e.g., a luminescent group (e.g., a fluorophore) or a non-luminescent molecular entity) is attached to a first end group of the compound to become part of a first end group; A first primary dye (e.g., a luminescent group (e.g., a fluorophore) or a non-luminescent molecular entity) is attached to the polymer backbone; Attach 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, the bioconjugating group is attached to the second end group; and / or Optionally, the compound may be crosslinked.

96. The method of claim 95, wherein one or more hydrophobic monomers and one or more hydrophilic monomers are copolymerized by 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 monomers and the one or more hydrophilic monomers are copolymerized by living radical polymerization (e.g., RAFT) in the presence of an initiator (e.g., AIBN) and a RAFT reagent (e.g., a thiocarbonyl thio compound) to provide the polymer backbone.

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

99. The method according to any one of claims 95-98, wherein at least one hydrophobic monomer has a structure represented by formula I: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R' is absent or is a C1-C20 alkyl, C2-C20 alkenyl, C2-C20 alkynyl, C3-C20 cycloalkyl, C5-C20 aryl, or C6-C25 arylalkyl; R 2 It is hydrogen or halogen, hydroxyl, carboxyl, amino, formyl, vinyl, epoxy, mercapto, ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester), azide, maleimide, isocyanate, or isothiocyanate, or when the hydrophobic unit is in the polymer backbone (e.g., R...). 2 When R is not the terminal base, 2 It is another major dye; and p is an integer from 1 to 10, 100, 1,000, 5,000, or 10,000.

100. The method of claim 95, wherein R in the hydrophobic monomer 2 It can be hydrogen, hydroxyl, carboxyl, amino, formyl, or ester group.

101. The method according to any one of claims 95-100, wherein at least one hydrophilic monomer has a structure represented by formula II: in: R is hydrogen or C1-C8 alkyl (e.g., C1, C2, C3, C4, C5, C6, C7 or C8 alkyl); R 1 It does not exist or is either –O–, –NH–, or –CH2–; R 3 Choose freely – (CH2CH2R) 5 ) n -, -C1-C6 alkyl, -C1-C6 alkyl-O- and –C1-C6 alkyl-SO3- or their salts, wherein R 5 It is a group consisting of -O- or -CH2-, and n is an integer from 1 or 5 to 10, 25, 50, 75, 100, 1,000, 5,000 or 10,000; and R 4 The presence of hydrogen, alkyl, phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine (i.e., 2-(trimethylammonium)ethoxy(hydroxy)phosphonoyl), phosphoryl, halogen, hydroxy, carboxyl, amino, ammonium, formyl, or ester (e.g., pentafluorophenyl ester, succinimide ester, fluorophenyl ester, or 2,4-dinitrophenyl ester) groups, or optionally when the hydrophilic unit is in the polymer backbone and the terminal end group comprises a bulky group and / or a charged group (e.g., R...). 4 When R is not the terminal base, 4 It is another major dye.

102. The method of claim 101, wherein R in the hydrophilic monomer 4 It can be a hydroxyl, carboxyl, amino, formyl, or ester group.

103. The method of claim 101, wherein R 3 It is -C1-C6 alkyl-O-, and R in the hydrophilic monomer 4 It is hydrogen, alkyl (e.g., methyl or ethyl), phosphonoyl (e.g., dihydroxyphosphonoyl), sulfonyl (e.g., hydroxysulfonyl), phosphatidylcholine, or phosphoryl.

104. The method of claim 101, wherein R 3 It is a -C1-C6 alkyl group or –(CH2CH2R) 5 ) n -, where R 5 It is -O-, and R in the hydrophilic monomer 4 It can be hydroxyl, carboxyl, amino, ammonium, formyl, ester, phosphonyl, or sulfonyl.

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

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

107. The method according to any one 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 according to any one of claims 93-107, wherein the second primary dye, the secondary dye, and / or the biomolecule are connected to the first primary dye via a linear connector.

109. The method according to any one of claims 93-107, wherein the second primary dye, the secondary dye, and / or the biomolecule are connected to the first primary dye via a bifunctional connector.

110. The method according to any one of claims 93-107, wherein the second primary dye, the secondary dye, and / or the biomolecule are connected to the first primary dye via a trifunctional connector.

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

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

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

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

115. A method for detecting labeled cells in a subject, the method comprising performing the method of claim 114, wherein the method includes labeling and detecting cells, applying the labeled cells to the subject, and detecting the compound or the biomolecule in the subject, thereby detecting the cells in the subject.

116. The method of claim 115, wherein the cells are detected in the subject using imaging techniques (e.g., photoacoustic imaging and / or magnetic resonance imaging).

117. A method for detecting tissues and / or reagents in a subject, the method comprising: Administering to a subject the compound according to any one of claims 1-74, the composition according to any one of claims 75-82, the compound prepared by the method according to any one of claims 83-110, or the biomolecule according to claim 111 or 112, optionally wherein the compound or the biomolecule is bound to the tissue and / or reagent; and The compound or biomolecule is detected in the subject (e.g., using fluorescence, MRI, or PAI), thereby detecting the tissue and / or reagent.

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 using magnetic resonance imaging to detect the primary dye and photoacoustic imaging to detect the secondary dye, or wherein the compound is detected by using photoacoustic imaging to detect the primary dye and magnetic resonance imaging to detect the secondary dye.

120. A method for treating cells and / or tissues (e.g., diseased cells and / or tissues) of a subject in need, said method comprising: Administering to a subject the compound according to any one of claims 1-74, the composition according to any one of claims 75-82, the compound prepared by the method according to any one of claims 83-110, or the biomolecule according to claim 111 or 112, optionally wherein the compound binds to the cells and / or tissues; and The subject or a portion thereof (e.g., the location of the cells and / or tissue) is irradiated with light of a wavelength and intensity sufficient to treat the cells and / or tissue. Optionally, the light activates the compound.

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

122. The method of claim 121, wherein detecting the compound comprises detecting the primary dye using imaging techniques, and treating the cells and / or tissue comprises activating the secondary dye (e.g., to release reactive oxygen species into the diseased tissue), thereby treating the cells and / or tissue, or The detection of the compound includes using imaging techniques to detect the secondary dye, and the treatment of the cells and / or tissues includes activating the primary dye (e.g., to release reactive oxygen species into the diseased tissue), thereby treating the cells and / or tissues.

123. The method according to any one of claims 120-122, wherein treating the cells and / or tissues comprises photodynamic therapy and / or photoimmunotherapy.

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

126. The method of claim 125, wherein the cells and / or tissues are excessively proliferating.

127. A compound prepared by the method according to any one of claims 83-110.

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