WO2020015700A1 - Photothermal agents - Google Patents

Photothermal agents Download PDF

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WO2020015700A1
WO2020015700A1 PCT/CN2019/096536 CN2019096536W WO2020015700A1 WO 2020015700 A1 WO2020015700 A1 WO 2020015700A1 CN 2019096536 W CN2019096536 W CN 2019096536W WO 2020015700 A1 WO2020015700 A1 WO 2020015700A1
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group
alkyl
alkyl chain
2tpe
photothermal agent
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PCT/CN2019/096536
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French (fr)
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Benzhong Tang
Zheng Zhao
Shunjie LIU
Dan Ding
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The Hong Kong University Of Science And Technology
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Priority to CN201980040309.4A priority Critical patent/CN112566911B/en
Publication of WO2020015700A1 publication Critical patent/WO2020015700A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/593—Polyesters, e.g. PLGA or polylactide-co-glycolide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
    • A61K49/222—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by a special physical form, e.g. emulsions, liposomes
    • A61K49/227—Liposomes, lipoprotein vesicles, e.g. LDL or HDL lipoproteins, micelles, e.g. phospholipidic or polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/06—Peri-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D495/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms
    • C07D495/22—Heterocyclic compounds containing in the condensed system at least one hetero ring having sulfur atoms as the only ring hetero atoms in which the condensed system contains four or more hetero rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D513/00—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00
    • C07D513/02—Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for in groups C07D463/00, C07D477/00 or C07D499/00 - C07D507/00 in which the condensed system contains two hetero rings
    • C07D513/04—Ortho-condensed systems

Definitions

  • the present subject matter relates generally to a series of organic, small molecule compounds and conjugated polymers and their applications in photoacoustic imaging (PAI) and photothermal therapy (PTT) .
  • PAI photoacoustic imaging
  • PTT photothermal therapy
  • PAI photoacoustic imaging
  • PTT photothermal therapy
  • NIR near-infrared
  • TICT twisted intramolecular charge transfer
  • the dark TICT state returns to the ground state mainly through nonradiative relaxation, accompanying red-shifted emission.
  • the prerequisite to the formation of TICT state relies on active molecular rotations.
  • the susceptibility of this state favors various non-radiative quenching processes. Accordingly, small molecules having a stronger TICT character may favor enhanced heat generation.
  • the photothermal agent can include small molecule, organic compounds with absorption in the near-infrared (NIR) interrogation window (700-900 nm) . Certain compounds that are thus useful can be biocompatible organic nanoparticles (ONPs) . The nanoparticles can exhibit intramolecular motion in an aggregate state.
  • the photothermal agent can include a conjugated polymer.
  • the photothermal agent can be administered to a patient to locate a tumor site in the patient using photoacoustic imaging. Once the tumor site has been determined, the tumor site can be irradiated with near-infrared light which, when combined with the present compounds, can stop or inhibit the growth of the tumor.
  • the photothermal agent comprises a compound having a donor-acceptor-donor structure:
  • each donor unit (D) being selected from the group consisting of:
  • the acceptor unit (A) being selected from the group consisting of:
  • R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C n H 2n+1 alkyl chain and a branched C n H 2n+1 alkyl chain;
  • n is an integer from 4 to 12 when the alkyl chain is linear;
  • n is an integer from 6 to 24 when the alkyl chain is branched
  • R 2 is selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group, sulfonic group, alkylthio, alkoxy group, alkyl-NCS, alkyl-N 3, alkyl-NH 2 , and alkyl-Br.
  • the compound has one of the following structural formulae:
  • the acceptor is selected from the group consisting of:
  • D is the donor group
  • R 1 is selected from the group consisting of
  • the compound has the following structural formula:
  • R is selected from the group consisting of
  • an exemplary compound further comprises poly ( ⁇ -amino ester) conjugated thereto.
  • the present subject matter relates to a photothermal agent, comprising a conjugated polymer having a donor-acceptor-donor structure
  • each donor unit (D) being selected from the group consisting of:
  • the acceptor unit (A) being selected from the group consisting of:
  • R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C n H 2n+1 alkyl chain and a branched C n H 2n+1 alkyl chain;
  • n is an integer from 4 to 12 when the alkyl chain is linear;
  • n is an integer from 6 to 24 when the alkyl chain is branched
  • p is an integer from 2 to 1000.
  • R 2 is H.
  • Fig. 1 depicts 1 H NMR spectra of 2TPE-NDTA.
  • Fig. 2 depicts the 13 C NMR spectra of 2TPE-NDTA.
  • Fig. 3 depicts high resolution mass spectrum (MALDI-TOF) of 2TPE-NDTA.
  • Fig. 4 depicts 1 H NMR spectra of 2TPE-2NDTA.
  • Fig. 5 depicts 13 C NMR spectra of 2TPE-2NDTA.
  • Fig. 6 depicts mass spectrum of 2TPE-2NDTA.
  • Fig. 7 depicts 1 H NMR spectrum of compound 2TPE-PDI-C 6 .
  • Fig. 8 depicts 13 C NMR spectra of 2TPE-PDI-C 6 .
  • Fig. 9 depicts high resolution mass spectrum (MALDI-TOF) of 2TPE-PDI-C 6 .
  • Fig. 10 depicts 1 H NMR spectrum of compound 2TPE-PDI-C 16 in CDCl 3 .
  • Fig. 11 depicts 13 C NMR spectra of 2TPE-PDI-C 16 in CDCl 3 .
  • Fig. 12 depicts high resolution mass spectrum of compound 2TPE-PDI-C 16 in CDCl 3 .
  • Fig. 13 depicts the PL spectra of NDTA, 2TPE-NDTA, and 2TPE-2NDTA in (a) THF solution (b) the encapsulated NPs and (c) water and thin films.
  • Inset of (b) shows the contrast of the PL intensities of the as-prepared NPs in water and the THF solution of NDTA.
  • Inset of (c) shows the contrast of the PL intensities of the thin films and the THF solution of NDTA.
  • Fig. 14 depicts UV-vis-NIR absorption spectra of (a) NDTA (b) 2TPE-NDTA and (c) 2TPE-2NDTA in THF solution, films and their doped organic NPs in water.
  • the insets depict photographs of the as-prepared NPs of NDTA, 2TPE-NDTA, 2TPE-2NDTA in water.
  • Fig. 15 depicts (a) the chemical structure of the semiconducting polymer: poly (cyclopentadithiophene-alt-benzothiadiazole) and (b) the schematic illustration of the semiconducting polymer nanoparticle (SPN) .
  • Fig. 16 depicts the particle size distribution and morphology of (a) NDTA-doped (b) 2TPE-NDTA-doped and (c) 2TPE-2NDTA-doped NPs studied by dynamic light scattering (DLS) and transmission electron microscopy (TEM) .
  • DLS dynamic light scattering
  • TEM transmission electron microscopy
  • Fig. 17 depicts (a) molar absorptivity of NDTA (b) molar absorptivity of 2TPE-NDTA (b) , and molar absorptivity of 2TPE-2NDTA in dilute THF.
  • Fig. 18 depicts (a) the optimized molecular structure and (b) the HOMO and LUMO orbital distributions, energy level, and band gaps of NDTA, 2TPE-NDTA and 2TPE-2NDTA.
  • Fig. 19 depicts (a) 13 C CPMAS at 5 kHz with toss and varied contact time (CT) to assign the TPE peaks of 2TPE-NDTA, (b) 13 C relaxation measurements of 2TPE-NDTA with a relaxation time of about 5577s, and (c) 13 C relaxation measurements of 2TPE-NDTA and 2TPE-2NDTA with relaxation times of 10.5 s and 7.1 s, respectively.
  • Fig. 20 depicts (a) IR thermal images of various NPs in aqueous solution (100 ⁇ M based on 2TPE-2NDTA, 2TPE-NDTA and the repeat unit of semiconducting polymer) upon exposure to 808 nm (0.8 W cm -2 ) laser irradiation for different times and (b) the temperature changes of the solutions of various NPs as a function of time.
  • the solutions were irradiated with 808 nm laser (0.8 W cm -2 ) for 300 s, followed by naturally cooling down for another 300 s.
  • Fig. 22 depicts (a) molecular design and chemical structure of NIR12 and NIR6 for PAI-guided PTT, (b) calculated HOMOs and LUMOs, (c) optimized ground-state (S 0 ) geometries, (d) schematic illustration of TICT state, (e) aggregation state and (f) PA imaging guided PTT of the NIR12 and NIR6.
  • R 1 2-hexyldecyl
  • R 2 1-hexyl.
  • Fig. 23 depicts, for NIR12 and NIR6, (a) normalized absorption spectrum in THF, (b) ⁇ em in different solvents. (c) correlation of solvent polarity parameter with stokes shift, (d) change in PL intensity with water fraction in THF/water mixutures, (e) change in PL intensity with DMSO fraction in DMF/DMSO mixtures, (f) PL spectra of NPs self-assembled by DSPE-PEG, (g) powder XRD spectra, and (h) Comparison of the photothermal conversion behavior of NIR12, NIR6 and ICG NPs in PBS solution at the same concentration (100 ⁇ M) .
  • Fig. 24 depicts (a) IR thermal images of the NIR12, and ICG NPs in PBS solutions (100 ⁇ M) under 808 nm laser irradiation for different times, (b) Photothermal conversion behavior of NIR12 NPs at different concentrations (5-100 ⁇ M) under 808 nm light irradiation, (c) antiphotobleaching property of NIR12 and ICG NPs (100 ⁇ M) during five circles of heating-cooling processes, (d) PA images of NIR12 and ICG NPs upon excitation at 780 nm at different concentrations, and (e) PA amplitudes of NIR12 and ICG NPs at 780 nm as a function of concentration.
  • Fig. 25 depicts (a) Photographs of the NIR12 and ICG NPs in PBS solutions after 808 nm light irradiation for different time (0.8 W/cm 2 ) and (b) Plot of I/I 0 versus various irradiation time.
  • I and I 0 are the maximal NIR absorption intensity of NIR12 and ICG NPs in PBS solutions after and before laser irradiation, respectively.
  • Fig. 26 depicts a schematic of NIR12 nanoparticles to prolong circulation time and enhance cellular uptake based on the pH-responsive properties of PAE.
  • Fig. 27 depicts (a) photoacoustic imaging in a cell, (b) photoacoustic intensity, (c) photoacoustic imaging in live mice, (d) temperature in tumor, (e) relative tumor volume, and (f) the body weight of mice treated with a PAE nanoparticle, a PEG nanoparticle or saline.
  • Fig. 28 depicts (a) histological H&E, fluorescence TUNEL, and PCNA staining of tumor slices at day 16 after different treatments and (b) histological H&E staining for livers and spleens on day 16 after different treatments.
  • compositions of the present teachings can also consist essentially of, or consist of, the recited components, and that the processes of the present teachings can also consist essentially of, or consist of, the recited process steps.
  • heteroaryl refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from oxygen (O) , nitrogen (N) , sulfur (S) , silicon (Si) , and selenium (Se) or a polycyclic ring system where at least one of the rings present in the ring system is aromatic and contains at least one ring heteroatom.
  • Polycyclic heteroaryl groups include two or more heteroaryl rings fused together and monocyclic heteroaryl rings fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkyl rings.
  • a heteroaryl group as a whole, can have, for example, 5 to 22 ring atoms and contain 1 -5 ring heteroatoms (i.e., 5-20 membered heteroaryl group) .
  • the heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O-O, S-S, or S-O bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S, S-dioxide) .
  • Examples of heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below:
  • T is O, S, NH, N-alkyl, N-aryl, N- (arylalkyl) (e.g., N-benzyl) , SiH 2 , SiH (alkyl) , Si (alkyl) 2 , SiH (arylalkyl) , Si (arylalkyl) 2 , or Si (alkyl) (arylalkyl) .
  • N-alkyl N-aryl
  • N- (arylalkyl) e.g., N-benzyl
  • SiH 2 SiH (alkyl) , Si (alkyl) 2 , SiH (arylalkyl) , Si (arylalkyl) 2 , or Si (alkyl) (arylalkyl) .
  • heteroaryl rings examples include pyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinox-alyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, lH-indazolyl, 2H-ind
  • heteroaryl groups include 4, 5, 6, 7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups, and the like.
  • heteroaryl groups can be substituted as described herein.
  • halo or halogen refers to fluoro, chloro, bromo, and iodo.
  • alkyl refers to a straight-chain or branched saturated hydrocarbon group.
  • alkyl groups include methyl (Me) , ethyl (Et) , propyl (e.g., n-propyl and z′-propyl) , butyl (e.g., n-butyl, z′-butyl, sec-butyl, tert-butyl) , pentyl groups (e.g., n-pentyl, z′-pentyl, -pentyl) , hexyl groups, and the like.
  • an alkyl group can have 1 to 40 carbon atoms (i.e., C1-40 alkyl group) , for example, 1-30 carbon atoms (i.e., C1-30 alkyl group) .
  • an alkyl group can have 1 to 6 carbon atoms, and can be referred to as a "lower alkyl group. " Examples of lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl and z′-propyl) , and butyl groups (e.g., n-butyl, z′-butyl, sec-butyl, tert-butyl) .
  • alkyl groups can be substituted as described herein.
  • An alkyl group is generally not substituted with another alkyl group, an alkenyl group, or an alkynyl group.
  • alkenyl refers to a straight-chain or branched alkyl group having one or more carbon-carbon double bonds.
  • alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl groups, and the like.
  • the one or more carbon-carbon double bonds can be internal (such as in 2-butene) or terminal (such as in 1-butene) .
  • an alkenyl group can have 2 to 40 carbon atoms (i.e., C2-40 alkenyl group) , for example, 2 to 20 carbon atoms (i.e., C2-20 alkenyl group) .
  • alkenyl groups can be substituted as described herein.
  • An alkenyl group is generally not substituted with another alkenyl group, an alkyl group, or an alkynyl group.
  • a "fused ring” or a “fused ring moiety” refers to a polycyclic ring system having at least two rings where at least one of the rings is aromatic and such aromatic ring (carbocyclic or heterocyclic) has a bond in common with at least one other ring that can be aromatic or non-aromatic, and carbocyclic or heterocyclic.
  • aromatic ring or heterocyclic
  • These polycyclic ring systems can be highly p-conjugated and optionally substituted as described herein.
  • heteroatom refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, silicon, sulfur, phosphorus, and selenium.
  • aryl refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system in which two or more aromatic hydrocarbon rings are fused (i.e., having a bond in common with) together or at least one aromatic monocyclic hydrocarbon ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings.
  • An aryl group can have 6 to 24 carbon atoms in its ring system (e.g., C6-24 aryl group) , which can include multiple fused rings.
  • a polycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ring position of the aryl group can be covalently linked to the defined chemical structure.
  • aryl groups having only aromatic carbocyclic ring include phenyl, 1-naphthyl (bicyclic) , 2-naphthyl (bicyclic) , anthracenyl (tricyclic) , phenanthrenyl (tricyclic) , pentacenyl (pentacyclic) , and like groups.
  • polycyclic ring systems in which at least one aromatic carbocyclic ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings include, among others, benzo derivatives of cyclopentane (i.e., an indanyl group, which is a 5, 6-bicyclic cycloalkyl/aromatic ring system) , cyclohexane (i.e., a tetrahydronaphthyl group, which is a 6, 6-bicyclic cycloalkyl/aromatic ring system) , imidazoline (i.e., a benzimidazolinyl group, which is a 5, 6-bicyclic cycloheteroalkyl/aromatic ring system) , and pyran (i.e., a chromenyl group, which is a 6, 6-bicyclic cycloheteroalkyl/aromatic ring system) .
  • aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl groups, and the like.
  • aryl groups can be substituted as described herein.
  • an aryl group can have one or more halogen substituents, and can be referred to as a "haloaryl” group.
  • Perhaloaryl groups i.e., aryl groups where all of the hydrogen atoms are replaced with halogen atoms (e.g., -C6F5) , are included within the definition of "haloaryl.
  • an aryl group is substituted with another aryl group and can be referred to as a biaryl group. Each of the aryl groups in the biaryl group can be substituted as disclosed herein.
  • a "donor” material refers to an organic material, for example, an organic nanoparticle material, having holes as the majority current or charge carriers.
  • an "acceptor" material refers to an organic material, for example, an organic nanoparticle material, having electrons as the majority current or charge carriers.
  • a "photothermal agent” refers to an organic material, for example, an organic nanoparticle material, that can convert light radiation to heat and, thereby, provide an ultrasonic emission.
  • a photothermal agent can include at least one small molecule organic compound with absorption in the near-infrared (NIR) interrogation window (700-900 nm) .
  • the compound can be an organic nanoparticle (ONP) .
  • the photothermal agent can include a conjugated polymer.
  • the photothermal agents described herein can provide ideal contrast agents for light triggered diagnostic/therapeutic techniques, such as photoacoustic imaging (PAI) associated with photothermal therapy (PTT) .
  • PAI photoacoustic imaging
  • PTT photothermal therapy
  • the compounds are provided as nanoparticles. In an embodiment, the compounds are non-emissive in solution and in the solid state.
  • the present compounds can include NIR absorbing organic molecules with a donor-acceptor (D-A-D) structure and long alkyl side chains.
  • the present compounds can include molecular rotors and bulky alkyl chains grafted to a central D-A-D core to restrict intermolecular interaction and enhance intramolecular motion in aggregates.
  • the enhanced molecular motion favors the formation of dark twisted intramolecular charge transfer (TICT) state, whose nonradiative decay enhances photothermal properties.
  • TCT dark twisted intramolecular charge transfer
  • one or more polymers can be conjugated to certain of the present compounds.
  • the present compounds include a poly ( ⁇ -amino ester) conjugated thereto.
  • the poly ( ⁇ -amino ester) is poly (cyclopentadithiophene-alt-benzothiadiazole) .
  • the present photothermal agent can include a compound having a donor-acceptor-donor structure:
  • each donor unit (D) being selected from the group consisting of:
  • the acceptor unit (A) being selected from the group consisting of:
  • R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C n H 2n+1 alkyl chain and a branched C n H 2n+1 alkyl chain;
  • n is an integer from 4 to 12 when the alkyl chain is linear;
  • n is an integer from 6 to 24 when the alkyl chain is branched
  • R 2 is selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group, sulfonic group, alkylthio, alkoxy group, alkyl-NCS, alkyl-N 3 , alkyl-NH 2 , and alkyl-Br.
  • R 1 is a C n H 2n+1 alkyl chain branched at the second carbon and n is 6-24. In another embodiment, R 1 is a linear C n H 2n+1 alkyl chain and n is 4-12.
  • the compound has one of the following structural formulae:
  • 2TPE-NDTA and 2TPE-2NDTA These compounds can be referred to as 2TPE-NDTA and 2TPE-2NDTA, respectively.
  • the acceptor is selected from:
  • D is the donor group
  • R 1 is selected from the group consisting of
  • the compound has the following structural formula:
  • R is selected from the group consisting of
  • NIR12 and NIR6 These compounds can be referred to as NIR12 and NIR6, respectively.
  • these compounds can further include a poly ( ⁇ -amino ester) conjugated thereto, as described herein.
  • the present subject matter relates to a photothermal agent, comprising a compound having a donor-acceptor-donor structure:
  • each donor unit (D) being selected from the group consisting of:
  • the acceptor unit (A) being selected from the group consisting of:
  • R 1 is hydrogen or an alkyl chain selected from the group consisting of a linear C n H 2n+1 alkyl chain and a branched C n H 2n+1 alkyl chain;
  • n is an integer from 4 to 12 when the alkyl chain is linear;
  • n is an integer from 6 to 24 when the alkyl chain is branched
  • R 2 is unsubstituted or substituted and is selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group, sulfonic group, alkylthio, alkoxy group, alkyl-NCS, alkyl-N 3, alkyl-NH 2 , and alkyl-Br.
  • the present subject matter relates to a photothermal agent, comprising a compound having a donor-acceptor-donor structure, each donor unit (D) being selected from the group consisting of:
  • the acceptor unit (A) being selected from the group consisting of:
  • R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C n H 2n+1 alkyl chain and a branched C n H 2n+1 alkyl chain;
  • n is an integer from 4 to 12 when the alkyl chain is linear;
  • n is an integer from 6 to 24 when the alkyl chain is branched
  • R 2 is H.
  • the photothermal agent may further comprise a poly ( ⁇ -amino ester) conjugated thereto.
  • the present compounds can include a D-A-D structure with typical AIE units (compounds which exhibit aggregation-induced emission) as donor units and compounds with large ⁇ -conjugation and a strong electron-withdrawing ability as acceptor units.
  • Exemplary compounds include triphenylethylene and tetraphenylethylene derivatives, as donor units, and NDI and/or PDI derivatives as acceptor units.
  • certain non-limiting embodiments of the present compounds include 2TPE-NDTA and 2TPE-2NDTA.
  • the 2TPE-NDTA and 2TPE-2NDTA compounds include tetraphenylethylene (TPE) .
  • TPE is a prototype molecule with active excited-state molecular motion.
  • the acceptor includes one or more naphthalene diimide fused 2- (1, 3-dithiol-2-ylidene) acetonitrile moieties (NDTA and 2NDTA) , with large ⁇ -conjugation and strong electron-withdrawing ability.
  • the acceptor contributes to long wavelength absorption, high molar absorptivity and strong TICT effect.
  • 2TPE-NDTA and 2TPE-2NDTA can conduct highly effective intramolecular motion in the solid state and aggregated state within NPs, leading to increased energy absorbance for heat production through the boosted non-radiative decay process.
  • 2TPE-2NDTA-doped NPs exhibit superior capability in generating a photoacoustic (PA) signal when compared with several well-established, high-performing PA imaging agents, including semiconducting polymer NPs and MB. As described herein, in vivo studies verified that 2TPE-2NDTA-doped NPs achieves excellent performance in visualizing tumors in a high-contrast manner via PA imaging.
  • PA photoacoustic
  • the present compounds include molecular rotors introduced into planar D-A based small molecules, which facilitates both molecular motion and stabilizing the dark TICT state.
  • the compounds include long alkyl chains which can provide shielding units to restrain intermolecular interaction, and most importantly to maintain the intramolecular rotations in aggregates.
  • the compounds include D-A or D-A-D conjugated small molecules based on low-bandgap benzo [1, 2-c: 4, 5-c′] bis ( [1, 2, 5] thiadiazole) (BBTD) as an acceptor, thiophene as ⁇ -conjugation unit and donor, triphenylamine (TPA) as molecular rotor and as a second donor, and long alkyl chains as shielding units.
  • BBTD low-bandgap benzo
  • exemplary compound NIR12 with long alkyl chains branched at the second carbon exhibits enhanced photothermal properties when compared to compound NIR6 with short linear alkyl chains and the commercial dye indocyanine green.
  • NIR12 nanoparticles can be used as nano-agents for photoacoustic imaging-guided photothermal therapy.
  • charge reversal with poly ( ⁇ -amino ester) makes NIR12 specifically accumulate at a tumor site.
  • the present compounds can be administered to a patient as a contrast agent for locating a tumor site in the patient using in vivo imaging techniques, e.g., photoacoustic imaging.
  • the compounds can be administered by intravenous injection, for example.
  • in vivo imaging studies demonstrate that the compounds can serve as an effective probe for PAI in a high-contrast manner.
  • the tumor site can be irradiated with light, e.g., near-infrared light which, when combined with the present compounds, can stop or inhibit the growth of the tumor.
  • the present methods can relate, in certain embodiments, to a method of locating a tumor site in a patient, comprising administering a photothermal agent as described herein to the patient; and locating the tumor site using photoacoustic imaging.
  • the present methods can relate to a method of stopping or inhibiting tumor growth in a patient, comprising administering a photothermal agent as described herein to the patient; locating a tumor site using photoacoustic imaging; and subjecting the tumor site to light irradiation while the photothermal agent is present at the tumor site to stop or inhibit the growth of the tumor.
  • the light irradiation can be irradiation by near-infrared light.
  • the photothermal agent when practicing the present methods, is administered to the patient in nanoparticle (NP) form.
  • NP nanoparticle
  • the present compounds can be co-precipitated with a poly ( ⁇ -amino ester) (PAE) to prolong circulation time in blood and enhance interaction with tumor cells.
  • PAE poly ( ⁇ -amino ester)
  • NIR12 co-precipitated with poly ( ⁇ -amino ester) (PAE) can rapidly and reversibly change surface properties and as well as provide an effective imaging probe and tumor treatment.
  • manipulation of TICT property using molecular rotors and alkyl chains may provide a useful platform for designing new photothermal agents.
  • the present compounds may demonstrate rapid temperature elevation in a tumor site under NIR light irradiation, which results in heat-caused tumor inhibition.
  • the 2TPE-2NDTA-doped NPs exhibit better capability in generating PA signal when compared with several well-established, high-performing PA imaging agents, including semiconducting polymer NPs and MB.
  • PA imaging agents including semiconducting polymer NPs and MB.
  • in vivo studies verified that 2TPE-2NDTA-doped NPs give excellent performance in visualizing tumors in a high-contrast manner via PA imaging.
  • the compounds demonstrate good photothermal/photoacoustic performance, making them promising candidates for in vivo diagnosis and therapy applications.
  • Quantum yield was determined by a integrating sphere. Particle size analyses were implemented using a wetaPlus Potential Analyzer (Brookhaven, ZETAPLUS) . Transmission electron microscopy (TEM) investigations were carried out on a JEOL-6390 instrument.
  • Femtosecond time-resolved fluorescence (fs-TRF) experiment Femtosecond time-resolved fluorescence (fs-TRF) measurements were performed on the same setup as fs-TA.
  • the output 800 nm laser pulse (200 mw) is used as gate pulse while the 400 nm laser pulse (10 mw) (second harmonic) is used as the pump laser.
  • the sample fluorescence is focused into the nonlinear crystal (BBO) mixing with the gate pulse to generate the sum frequency signal. Broadband fluorescence spectra are obtained by changing the crystal angles, and the spectra are detected by the air-cooled CCD.
  • the compound ND in THF solution were excited by a 400 nm pump beam (the second harmonic of the fundamental 800 nm from the regenerative amplifier) .
  • the 1 mL solutions were studied in a 2 mm path-length cuvette with an absorbance of 0.5 at 400 nm throughout the data acquisition.
  • NIR-12, NIR-6, and intermediates 1 H and 13 C NMR spectra were recorded at room temperature on a Unity-400 NMR spectrometer using CDCl 3 as solvent and tetramethylsilane (TMS) as reference.
  • TMS tetramethylsilane
  • the UV-vis-NIR absorption spectra were performed using a PerkinElmer Lambda 365 spectrophotometer.
  • Mass spectra (MS) were measured with a GCT premier CAB048 mass spectrometer in MALDI-TOF mode.
  • the photoluminescence (PL) spectra were conducted on a Horiba Fluorolog-3 spectrofluorometer. Dynamic light scattering (DLS) was measured on a 90 plus particle size analyzer.
  • TEM images were acquired from a JEM-2010F transmission electron microscope with an accelerating voltage of 200 kV.
  • Laser confocal scanning microscope images were collected on Zeiss laser scanning confocal microscope (LSM7 DUO) and analyzed using ZEN 2009 software (Carl Zeiss) .
  • mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) .
  • murine 4T1 breast cancer cells (1 ⁇ 10 6 ) suspended in 50 ⁇ L of RPMI-1640 medium were injected subcutaneously into the right axillary space of the mouse. After about 10 days, mice with tumor volumes at about 80-120 mm 3 were used subsequently.
  • Fs-TA experiments were done employing an experimental setup and methods detailed previously and only a brief description is provided here.
  • Fs-TA measurements were done using a 1000 Hz femtosecond regenerative amplified Ti: sapphire laser system (Maitai) in which the amplifier was seeded with the 120 fs laser pulses from an oscillator laser system.
  • the laser probe pulse was produced by utilizing ⁇ 5%of the amplified 800 nm laser pulses to generate a white-light continuum (430-750 nm) in a saphire crystal and then this probe beam was split into two parts before traversing the sample.
  • One probe laser beam goes through the sample while the other probe laser beam goes to the reference spectrometer in order to monitor the fluctuations in the probe beam intensity.
  • the compounds in THF solution were excited by a 400 nm pump beam (the second harmonic of the fundamental 800 nm from the regenerative amplifier) .
  • the 1 mL solutions were studied in a 2 mm path-length cuvette with an absorbance of 0.5 at 400 nm throughout the data acquisition.
  • NMR experiments were performed on a Varian Infinitplus-400 wide-bore (89 mm) NMR spectrometer at room temperature (25 °C) at frequencies of 399.72, 100.52 MHz for 1 H and 13 C, respectively.
  • T3 probe with a rotor diameter of 4 mm was used, and samples with a volume of 52 ⁇ L were placed in a zirconia PENCIL rotor.
  • the 90° pulse length was approximately 3 ⁇ s, corresponding to a radio frequency (RF) field strength of 83 kHz.
  • the magic angle spinning (MAS) at 5 kHz was automatically controlled with a speed controller, and the total suppression of sidebands (TOSS) sequence was also used before the signal acquisition to suppress the spinning sidebands.
  • RF radio frequency
  • the 13 C chemical shifts were referenced to external HMB (hexamethylbenzene, 17.3 ppm of CH 3 ) .
  • the ramped cross polarization (CP) was used for the 1 H- 13 C polarization transfer, and the CP contact time was 0.1ms and 1ms, respectively.
  • NDTA, 2TPE-NDTA, or 2TPE-2NDTA (1 mg) was dissolved. Then, DSPE-PEG 2000 (2 mg) was also added and dissolved in the THF solution. After that, the obtained THF solution was added into water (9 mL) accompanied with sonication using a microtip probe sonicator (XL2000, Misonix Incorporated, NY) , followed by continuative sonication of the mixture for another 60 s. The THF in the mixture was evaporated by stirring in the fume hood for 12 h. The resultant NP suspension was purified by ultrafiltration (molecule weight cutoff 100 kDa) at 3000 ⁇ g for 0.5 h, which was subsequently filtered with a 0.2 ⁇ m syringe driven filter.
  • ultrafiltration molecule weight cutoff 100 kDa
  • Murine 4T1 breast cancer cells were purchased from American Type Culture Collection (ATCC) .
  • the 4T1 cancer cells were cultured in Dulbecco′s Modified Eagle’s Medium (DMEM) containing 10%fetal bovine serum (FBS) and 1%penicillin-streptomycin at 37 °C in a humidified environment containing 5%CO 2 , respectively. Before experiments, the cells were pre-cultured until confluence was reached.
  • DMEM Dulbecco′s Modified Eagle’s Medium
  • FBS fetal bovine serum
  • penicillin-streptomycin 1%penicillin-streptomycin
  • mice Female BALB/c mice (6-week-old) were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China) .
  • the xenograft 4T1 tumor-bearing mouse model was used in the study.
  • 30 ⁇ L of cell culture medium containing 1 ⁇ 10 6 murine 4T1 breast cancer cells were injected subcutaneously into the right auxiliary space of the BALB/c mouse. After about 10 days, mice with tumor volumes of about 80-120 mm 3 were used subsequently.
  • In vivo tumor PA imaging was carried out by a commercial small-animal opt-acoustic tomography system (MOST, iTheraMedical, Germany) . The PA images were acquired at 730 nm before administration and at 4, 8, 16, 24 h post-injection.
  • Fig. 21C depicts the time-dependent in vivo PA tumor imaging of 2TPE-2NDTA-doped NP-injected mice.
  • intense PA signal can be clearly observed at the tumor site at 4 h post injection of 2TPE-2NDTA-doped NPs.
  • the PA intensity at 4 h is around 2.7-fold higher than that at 0 h (Fig. 26D) .
  • Such high-contrast PA tumor imaging benefits from not only the excellent EPR effect of NPs but also the prominent PA effect of 2TPE-2NDTA (Figs. 21A-21B) .
  • the superb passive tumor targeting rooting in the EPR effect highlights the advantage and importance of our design that enables MEPT to occur within NPs.
  • the live animal result demonstrates that MEPT NPs are capable of serving as effective PA imaging probes for tumor diagnosis in vivo.
  • FIG. 1 depicts 1 H NMR spectra of 2TPE-NDTA.
  • Fig. 2 depicts the 13 C NMR spectra of 2TPE-NDTA.
  • Fig. 3 depicts high resolution mass spectrum (MALDI-TOF) of 2TPE-NDTA.
  • Fig. 7 depicts 1 H NMR spectrum of compound 2TPE-PDI-C 6 .
  • Fig. 8 depicts 13 C NMR spectra of 2TPE-PDI-C 6 .
  • Fig. 9 depicts high resolution mass spectrum (MALDI-TOF) of 2TPE-PDI-C 6 .
  • Fig. 10 depicts 1 H NMR spectrum of compound 2TPE-PDI-C 16 in CDCl 3 .
  • Fig. 11 depicts 13 C NMR spectra of 2TPE-PDI-C 16 in CDCl 3 .
  • Fig. 12 depicts high resolution mass spectrum of compound 2TPE-PDI-C 16 in CDCl 3 .
  • optical properties of 2TPE-NDTA and 2TPE-2NDTA were characterized by photoluminescence (PL) and UV-vis-NIR spectroscopies and compared with that of NDTA (Figs. 13A-13C and 14A-14C) .
  • nanoprecipitation strategy was employed to formulate these molecules into water-soluble NPs using 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy- (polyethylene glycol) -2000] (DSPE-PEG 2000 ) as the encapsulation matrix (Figs. 15A-15B) .
  • NDTA-doped, 2TPE-NDTA-doped, and 2TPE-2NDTA-doped NPs possess an average diameter of about 125, 152, and 156 nm, respectively (Figs. 16A-16C) .
  • Fig. 13A NDTA emits brightly at around 630 nm in dilute THF solution, whereas both its NPs and film show much decreased PL intensity but largely red-shifted emission peaks (around 810 nm) (Fig. 13B and 13C) , suggesting the aggregation-caused quenching (ACQ) and J-aggregation characteristics of NDTA in aggregates.
  • ACQ aggregation-caused quenching
  • both the dilute THF solution and the aggregates (NPs and thin film) of 2TPE-NDTA and 2TPE-2NDTA display no emission, indicating that the non-radiative relaxation process is dominated for the exciton relaxation of 2TPE-NDTA and 2TPE-2NDTA, even in the solid state. This is quite different from the typical design of AIE molecules where TPE is usually introduced to transform the ACQ molecules to AIE ones.
  • the molar absorptivities of 2TPE-NDTA (50600) and 2TPE-2NDTA (67800) are comparable to that of NDTA (67822) , revealing their superb light-harvesting ability (Figs. 17A-17B) .
  • the absorption profile and maximum peak of NDTA exhibit large red-shifts, supporting its J-aggregation stacking pattern.
  • the absorption profile of 2TPE-NDTA is broadened with the absorption maximum changing slightly after aggregation, indicating that the introduction of TPE hinders the strong ⁇ - ⁇ stacking of 2TPE-NDTA.
  • NDTA exhibits a planar structure, with the largest coefficients in the highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) both located along the whole ⁇ -backbone and overlapping well with each other, which contributes to its emission in solution.
  • HOMO highest occupied molecular orbitals
  • LUMO lowest unoccupied molecular orbitals
  • 2TPE-NDTA presents a dumbbell shape molecular conformation owing to the twisted structure of TPE, which disfavors the strong intermolecular ⁇ - ⁇ stacking.
  • the HOMO of 2TPE-NDTA is mainly distributed on the TPE moieties, whereas its LUMO is distributed on the NDTA moiety, in accordance with the ICT effect.
  • 2TPE-2NDTA shows an even more twisted conformation.
  • the central acceptor unit of 2NDTA is also highly twisted with a dihedral angle of 120°.
  • the HOMO electron density distribution of 2TPE-2NDTA is located on one of the TPE moieties while its LUMO is distributed on the 2NDTA moiety, suggestive of the ICT effect.
  • the twisted structure and the ICT effect of 2TPE-NDTA and 2TPE-2NDTA are beneficial to active intramolecular motion since they not only hinder the intermolecular ⁇ - ⁇ interaction to facilitate the spatial isolation of the molecules, but also enhance the TICT effect, finally contributing to the non-radiative relaxation of the excitons.
  • Solid-state nuclear magnetic resonance was employed to study the molecular motion behaviors of 2TPE-NDTA and 2TPE-2NDTA in the solid state (Figs. 19A-19C) . Since both the 2TPE-NDTA and 2TPE-2NDTA have no other aromatic hydrogen atom except those of TPE, it is easy to identify the 13 C signal of TPE moieties by 13 C CPMAS NMR spectra and probe their intramolecular motion behavior 33 . As shown in Fig. 19A, 13 C CPMAS NMR spectra at 5 kHz with toss and varied contact time (CT) was implemented to assign the TPE peaks of 2TPE-NDTA.
  • CT contact time
  • alkyl chain parts show ever shorter relaxation time than the TPE moiety, indicating its flowing characteristic which benefits to the molecular motion of the TPE moiety.
  • TPE substituted perylenediimides PDIs
  • 2TPE-PDI-C 6 and 2TPE-PDI-C 16 with different alkyl side chain lengths were synthesized and characterized.
  • the 2TPE-PDI-C 6 and 2TPE-PDI-C 16 derivatives were synthesized as depicted below
  • TriPE-3PDIs with longer alkyl chain of 2-hexyloctyl show much lower quantum yield (6%) in the film than the one with shorter alkyl chain of 2-ethylhexyl (30%) . Therefore, introduction of long alkyl chain into the backbone of molecular rotors is a general and effective strategy to promote the intramolecular motion and non-radiative relaxation of the excitons in the aggregates. As it has been established that the photophysical mechanisms of fluorescence and photothermy show opposite characteristics, the absorbed light energy could reasonably incline to generate heat accompanied with reduced fluorescence.
  • THF solution containing 1 mg of NIR12 compound, and 2 mg of 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy- (polyethylene glycol) -2000 (DSPE-PEG2000) was poured into 10 mL of deionized water. This step was followed by sonication with a microtip probe sonicator (XL2000, Misonix Incorporated, NY) for 2 min. The residue THF solvent was evaporated by violent stirring of the suspension in fumehood overnight, and a colloidal solution was obtained and used directly.
  • a microtip probe sonicator XL2000, Misonix Incorporated, NY
  • THF solution containing 1 mg of NIR12 compound, 1 mg of DSPE-PEG2000 and 1 mg of poly ( ⁇ -amino ester) (PAE) was poured into 10 mL of deionized water. This step was followed by sonication with a microtip probe sonicator (XL2000, Misonix Incorporated, NY) for 2 min. The residue THF solvent was evaporated by violent stirring the suspension in fumehood overnight, and a colloidal solution was obtained and used directly (Fig. 26) .
  • XL2000 microtip probe sonicator
  • the PBS solutions (pH 7.4) of NIR12-PEG NPs, and ICG NPs were irradiated under an 808 nm laser (0.8 W/cm 2 ) , and the absorption spectra were measured at different time points (Figs. 23A-23H) .
  • the temperatures of the sample solutions were recorded during five cycles of heating and cooling processes. In one heating-cooling cycle, the NIR laser first irradiated the samples for 5 min to reach a steady state, then the laser was removed, and the samples were naturally cooled down to ambient temperature in 6 min (Figs. 24A-24E) .
  • the PBS solutions (pH 7.4) of NIR12-PEG NPs, and ICG NPs were continuously exposed to an 808 nm NIR laser (0.8 W/cm 2 ) for 5 min. The temperature was measured every 20 s and stopped until the temperature nearly reached to a plateau. The corresponding IR thermal images of the sample tubes were also recorded (Fig. 25A-25B) .
  • PA signals or images were acquired on a commercial small-animal opt-acoustic tomography system (MOST, iTheraMedical, Germany) .
  • PA images were subsequently acquired at 700 nm before administration and at designated time intervals post NPs injection.
  • Figs. 27A depicts photoacoustic imaging in a cell.
  • FIG. 27B is a graph depicting photoacoustic intensity.
  • Fig. 27C depicts photoacoustic imaging in the live mice.
  • Fig. 27D is a graph depicting the temperature in the tumor.
  • Fig. 27E is a graph depicting tumor volume.
  • Fig. 27F is a graph depicting the body weight of mice treated with a PAE nanoparticle, a PEG nanoparticle, or saline.
  • NIR12-PEG NPs On day 0, for “Only Saline” , “NIR12-PEG NPs” and “NIR12-PAE NPs” groups, saline, 150 ⁇ L of NIR12-PEG NPs and NIR12-PAE NPs (600 ⁇ M based on NIR12) were injected into 4T1 tumor-bearing mice through tail vein, respectively, without subsequent laser irradiation.
  • mice After sixteen days of photothermal treatment, the above-mentioned six groups of mice were sacrificed. The liver, spleen, and tumor were excised, fixed in 4%formalin solution, and sectioned at 5 ⁇ m thickness. After conventional H&E staining, the slices were examined with a digital microscope (Leica QWin) . The fluorescent proliferating cell nuclear antigen (PCNA) staining was conducted following common immunohistochemical steps. The fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was conducted following manual instruction of DeadEnd fluorometric TUNEL system kit (Promega, USA) . The nuclei were counterstained with 40, 6-diamidino-2-phenylindole (DAPI) containing mounting solution (Dapi-Fluoromount-G, Southern Biotech, England) (Figs. 28A-28B) .
  • PCNA fluorescent proliferating cell nuclear antigen
  • Murine 4T1 breast cancer cells were cultured in Dulbecco′s Modified Eagle’s Medium (DMEM) containing 10%fetal bovine serum (FBS) and 1%penicillin-streptomycin at 37 °C in a humidified environment containing 5%CO 2 , respectively. Before experiments, the cells were pre-cultured until confluence was reached.
  • DMEM Dulbecco′s Modified Eagle’s Medium
  • FBS fetal bovine serum
  • penicillin-streptomycin 37 °C in a humidified environment containing 5%CO 2
  • an aggregation-induced emission luminogen (AIEgen) was incorporated into the inner core of the NPs with NIR12 molecule in the ratio of 1: 1, thus endowing the NPs with fluorescence.
  • Murine 4T1 breast cancer cells were seeded in confocal imaging chambers at a density of 1 ⁇ 10 5 cells. After an incubation of 24 hours, the culture medium of each well was replaced with 1 mL of fresh medium with different pH values (pH 7.4 and 6.5) , then the PEG-NIR12-AIEgen NPs and PAE-NIR12-AIEgen were added to each chamber.
  • NPs NPs were washed three times with 1 ⁇ PBS buffer and fixed using 4%paraformaldehyde for 20 min at 0 °C.
  • the cellular uptake of NPs was observed with confocal laser scanning microscopy (Zeiss LSM710) upon excitation at 405 nm and a collection of fluorescence signals above 580 nm.
  • Compound 4a Compound 3a (0.3 g, 0.33 mmol) was dissolved in a mixture of 10 mL CHCl 3 and 10 mL acetic acid under argon atmosphere, NBS (117 mg, 6.6 mmol) was added slowly over the course of 30 mins in a mixture of 5 mL CHCl 3 and 5 mL acetic acid at room temperature under the exclusion of light. The mixture was stirred overnight and was then dried by condensed air. The crude product was purified by silica gel column to obtain the product (yield: 80%) .

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Abstract

A photothermal agent can be used in both photoacoustic imaging (PAI) and photothermal therapy (PTT) applications. The photothermal agent can include a small molecule, organic compound and/or polymers with absorption in the near-infrared (NIR) interrogation window (700-900 nm). The compound can be a biocompatible organic nanoparticle (ONP). The photothermal agent can be administered to a patient to locate a tumor site in the patient using in vivo imaging techniques. Once the tumor site has been determined, the tumor site can be irradiated with near-infrared light to stop or inhibit the growth of the tumor.

Description

PHOTOTHERMAL AGENTS FIELD
The present subject matter relates generally to a series of organic, small molecule compounds and conjugated polymers and their applications in photoacoustic imaging (PAI) and photothermal therapy (PTT) .
BACKGROUND
Among versatile light-triggered diagnostic/therapeutic techniques, photoacoustic imaging (PAI) associated with photothermal therapy (PTT) is particularly effective in accurately probing tumor location and effectively inhibiting tumor growth, with minimal side effect to normal tissue. PAI is a very promising noninvasive molecular imaging approach that combines deep tissue penetration and high resolution of ultrasound imaging with high contrast of optical imaging. The therapeutic technique that typically accompanies PAI is PTT, as PAI is used primarily to detect the photothermally generated ultrasound signal. The most vital prerequisite of PAI/PTT applications is to employ efficient contrast agents with strong absorption in the near-infrared (NIR) interrogation window (700-900 nm) , since NIR light is known to penetrate much deeper tissue and cause less photodamage to a living body.
Among the existing contrast agents, organic dyes have received considerable attention owing to their outstanding biocompatibility, potential biodegradability, and processability. However, it has also been reported that anticancer efficacy of organic contrast agents can be limited by their low photothermal properties. Most existing systems simply construct strong donor (D) and acceptor (A) units into coplanar structures, as evidenced by small molecules and semiconducting polymer nanoparticles. The resulting strong intermolecular interactions between molecules inside the nanoparticle (NP) cores may block other channels of heat generation.
Certain previous studies have focused on exploring new approaches to further enhance photothermal properties. Generally, however, these studies have been based on designing planar structures to enhance intermolecular interactions in aggregates. Moreover, the complexity of polymer structures has hindered a clear mechanistic study.
More recent studies have revealed an excited state electron transfer process observed in small molecules, termed twisted intramolecular charge transfer (TICT) (Org. Sensor Actuat B-Chem, 2018, 267, 448) . In this process, after photoexcitation, the dark TICT state returns to the ground state mainly through nonradiative relaxation, accompanying red-shifted emission.  Notably, the prerequisite to the formation of TICT state relies on active molecular rotations. In the TICT process, the susceptibility of this state favors various non-radiative quenching processes. Accordingly, small molecules having a stronger TICT character may favor enhanced heat generation.
The capacity of transforming a low-density energy source of light radiation to heat makes photothermal materials promising candidates for many advanced applications such as seawater desalination, photothermal-electrical and photothermal-mechanical converters, PAI, and PTT. In the biomedical field, PAI that detects the photothermally generated ultrasound signal has recently received considerable attention as it surpasses the optical diffusion limit, permitting disease diagnosis in deeper tissue with higher spatial resolution. The PA effect of materials rooted in heat generation is positively associated with the non-radiative decay of the excited states after light absorption. Among various PAI agents, great interest has been focused on organic NPs based on organic π-conjugated molecules or polymers due to their excellent biocompatibility, easily tunable band gap and approachable structural-property relationship, in comparison with their inorganic counterparts. Some popularly used molecules, such as indocyanine green (ICG) and methylene blue (MB) , have been approved by the Food and Drug Administration for clinical use. However, these traditional planar organic dyes generally show bright emission in the solution state, while their non-radiative decay depends strongly on their aggregated state.
Only dyes with strong face-to-face π-π stacking such as H-aggregation exhibit efficient non-radiative decay according to kasha’s exciton model, which unfortunately is uncontrollable. In most cases, the aggregates formed in the organic NPs are randomly aligned and amorphous, which thus exhibit both insufficient radiative decay and non-radiative decay. Consequently, they are not ideal agents for either PAI or fluorescence imaging.
Accordingly, the development of photothermal agents for effective PAI/PTT applications is desired.
SUMMARY
The present subject matter relates to a photothermal agent that can be used in both photoacoustic imaging (PAI) and photothermal therapy (PTT) applications. According to some embodiments, the photothermal agent can include small molecule, organic compounds with absorption in the near-infrared (NIR) interrogation window (700-900 nm) . Certain compounds  that are thus useful can be biocompatible organic nanoparticles (ONPs) . The nanoparticles can exhibit intramolecular motion in an aggregate state. According to some embodiments, the photothermal agent can include a conjugated polymer.
The photothermal agent can be administered to a patient to locate a tumor site in the patient using photoacoustic imaging. Once the tumor site has been determined, the tumor site can be irradiated with near-infrared light which, when combined with the present compounds, can stop or inhibit the growth of the tumor.
In an embodiment, the photothermal agent comprises a compound having a donor-acceptor-donor structure:
D-A-D
with each donor unit (D) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000001
the acceptor unit (A) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000002
wherein R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
n is an integer from 4 to 12 when the alkyl chain is linear;
n is an integer from 6 to 24 when the alkyl chain is branched; and
R 2 is selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group, sulfonic group,  alkylthio, alkoxy group, alkyl-NCS, alkyl-N 3, alkyl-NH 2, and alkyl-Br.
In a further embodiment, the compound has one of the following structural formulae:
Figure PCTCN2019096536-appb-000003
wherein R is
Figure PCTCN2019096536-appb-000004
In an embodiment, the acceptor is selected from the group consisting of:
Figure PCTCN2019096536-appb-000005
Figure PCTCN2019096536-appb-000006
wherein D is the donor group, and
wherein R 1 is selected from the group consisting of
Figure PCTCN2019096536-appb-000007
In an embodiment, the compound has the following structural formula:
Figure PCTCN2019096536-appb-000008
wherein R is selected from the group consisting of
Figure PCTCN2019096536-appb-000009
In an embodiment, an exemplary compound further comprises poly (β-amino ester) conjugated thereto.
In another embodiment, the present subject matter relates to a photothermal agent, comprising a conjugated polymer having a donor-acceptor-donor structure
D-A-D
each donor unit (D) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000010
the acceptor unit (A) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000011
Figure PCTCN2019096536-appb-000012
wherein R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
n is an integer from 4 to 12 when the alkyl chain is linear;
n is an integer from 6 to 24 when the alkyl chain is branched;
p is an integer from 2 to 1000; and
R 2 is H.
BRIEF DESCRIPTION OF DRAWINGS
Various embodiments will now be described in detail with reference to the accompanying drawings.
Fig. 1 depicts  1H NMR spectra of 2TPE-NDTA.
Fig. 2 depicts the  13C NMR spectra of 2TPE-NDTA.
Fig. 3 depicts high resolution mass spectrum (MALDI-TOF) of 2TPE-NDTA.
Fig. 4 depicts  1H NMR spectra of 2TPE-2NDTA.
Fig. 5 depicts  13C NMR spectra of 2TPE-2NDTA.
Fig. 6 depicts mass spectrum of 2TPE-2NDTA.
Fig. 7 depicts  1H NMR spectrum of compound 2TPE-PDI-C 6.
Fig. 8 depicts  13C NMR spectra of 2TPE-PDI-C 6.
Fig. 9 depicts high resolution mass spectrum (MALDI-TOF) of 2TPE-PDI-C 6.
Fig. 10 depicts  1H NMR spectrum of compound 2TPE-PDI-C 16 in CDCl 3.
Fig. 11 depicts  13C NMR spectra of 2TPE-PDI-C 16 in CDCl 3.
Fig. 12 depicts high resolution mass spectrum of compound 2TPE-PDI-C 16 in CDCl 3.
Fig. 13 depicts the PL spectra of NDTA, 2TPE-NDTA, and 2TPE-2NDTA in (a) THF solution (b) the encapsulated NPs and (c) water and thin films. Inset of (b) shows the contrast of the PL intensities of the as-prepared NPs in water and the THF solution of NDTA. Inset of (c) shows the contrast of the PL intensities of the thin films and the THF solution of NDTA.
Fig. 14 depicts UV-vis-NIR absorption spectra of (a) NDTA (b) 2TPE-NDTA and (c) 2TPE-2NDTA in THF solution, films and their doped organic NPs in water. The insets depict photographs of the as-prepared NPs of NDTA, 2TPE-NDTA, 2TPE-2NDTA in water.
Fig. 15 depicts (a) the chemical structure of the semiconducting polymer: poly (cyclopentadithiophene-alt-benzothiadiazole) and (b) the schematic illustration of the semiconducting polymer nanoparticle (SPN) .
Fig. 16 depicts the particle size distribution and morphology of (a) NDTA-doped (b) 2TPE-NDTA-doped and (c) 2TPE-2NDTA-doped NPs studied by dynamic light scattering (DLS) and transmission electron microscopy (TEM) .
Fig. 17 depicts (a) molar absorptivity of NDTA (b) molar absorptivity of 2TPE-NDTA (b) , and molar absorptivity of 2TPE-2NDTA in dilute THF.
Fig. 18 depicts (a) the optimized molecular structure and (b) the HOMO and LUMO orbital distributions, energy level, and band gaps of NDTA, 2TPE-NDTA and 2TPE-2NDTA.
Fig. 19 depicts (a)  13C CPMAS at 5 kHz with toss and varied contact time (CT) to assign the TPE peaks of 2TPE-NDTA, (b)  13C relaxation measurements of 2TPE-NDTA with a  relaxation time of about 5577s, and (c)  13C relaxation measurements of 2TPE-NDTA and 2TPE-2NDTA with relaxation times of 10.5 s and 7.1 s, respectively.
Fig. 20 depicts (a) IR thermal images of various NPs in aqueous solution (100 μM based on 2TPE-2NDTA, 2TPE-NDTA and the repeat unit of semiconducting polymer) upon exposure to 808 nm (0.8 W cm -2) laser irradiation for different times and (b) the temperature changes of the solutions of various NPs as a function of time. The solutions were irradiated with 808 nm laser (0.8 W cm -2) for 300 s, followed by naturally cooling down for another 300 s.
Fig. 21 depicts (a) PA spectra of various NPs, (b) PA signal (excited by 680 nm pulsed laser) comparison of different agents at the same concentration of 100 μM based on 2TPE-2NDTA, 2TPE-NDTA, MB and the repeat unit of semiconducting polymer, (c) representative PA images of tumors from 2TPE-2NDTA-doped NP-administrated mice, and (d) PA intensity of tumor as a function of time after intravenous injection of 2TPE-2NDTA-doped NPs. Error bars, mean ± s.d. (n = 3 mice) . Before (0 h) and after 2TPE-2NDTA-doped NPs (300 μM based on 2TPE-2NDTA) were intravenously injected into xenograft 4T1 tumor-bearing mice for designated time intervals, PA images were taken by excitation at 730 nm.
Fig. 22 depicts (a) molecular design and chemical structure of NIR12 and NIR6 for PAI-guided PTT, (b) calculated HOMOs and LUMOs, (c) optimized ground-state (S 0) geometries, (d) schematic illustration of TICT state, (e) aggregation state and (f) PA imaging guided PTT of the NIR12 and NIR6. Note: R 1 = 2-hexyldecyl, R 2 = 1-hexyl.
Fig. 23 depicts, for NIR12 and NIR6, (a) normalized absorption spectrum in THF, (b) λ em in different solvents. (c) correlation of solvent polarity parameter with stokes shift, (d) change in PL intensity with water fraction in THF/water mixutures, (e) change in PL intensity with DMSO fraction in DMF/DMSO mixtures, (f) PL spectra of NPs self-assembled by DSPE-PEG, (g) powder XRD spectra, and (h) Comparison of the photothermal conversion behavior of NIR12, NIR6 and ICG NPs in PBS solution at the same concentration (100 μM) .
Fig. 24 depicts (a) IR thermal images of the NIR12, and ICG NPs in PBS solutions (100 μM) under 808 nm laser irradiation for different times, (b) Photothermal conversion behavior of NIR12 NPs at different concentrations (5-100 μM) under 808 nm light irradiation, (c) antiphotobleaching property of NIR12 and ICG NPs (100 μM) during five circles of heating-cooling processes, (d) PA images of NIR12 and ICG NPs upon excitation at 780 nm at different concentrations, and (e) PA amplitudes of NIR12 and ICG NPs at 780 nm as a function  of concentration.
Fig. 25 depicts (a) Photographs of the NIR12 and ICG NPs in PBS solutions after 808 nm light irradiation for different time (0.8 W/cm 2) and (b) Plot of I/I 0 versus various irradiation time. I and I 0 are the maximal NIR absorption intensity of NIR12 and ICG NPs in PBS solutions after and before laser irradiation, respectively.
Fig. 26 depicts a schematic of NIR12 nanoparticles to prolong circulation time and enhance cellular uptake based on the pH-responsive properties of PAE.
Fig. 27 depicts (a) photoacoustic imaging in a cell, (b) photoacoustic intensity, (c) photoacoustic imaging in live mice, (d) temperature in tumor, (e) relative tumor volume, and (f) the body weight of mice treated with a PAE nanoparticle, a PEG nanoparticle or saline.
Fig. 28 depicts (a) histological H&E, fluorescence TUNEL, and PCNA staining of tumor slices at day 16 after different treatments and (b) histological H&E staining for livers and spleens on day 16 after different treatments.
DETAILED DESCRIPTION
The following definitions are provided for the purpose of understanding the present subject matter and for constructing the appended patent claims.
Definitions
It should be understood that the drawings described above or below are for illustration purposes only. The drawings are not necessarily to scale, with emphasis generally being placed upon illustrating the principles of the present teachings. The drawings are not intended to limit the scope of the present teachings in any way.
Throughout the application, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings can also consist essentially of, or consist of, the recited components, and that the processes of the present teachings can also consist essentially of, or consist of, the recited process steps.
In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or  components. Further, it should be understood that elements and/or features of a composition, an apparatus, or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present teachings, whether explicit or implicit herein
The use of the terms "include, " "includes" , "including, " "have, " "has, " or "having" should be generally understood as open-ended and non-limiting unless specifically stated otherwise.
The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise. In addition, where the use of the term "about" is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10%variation from the nominal value unless otherwise indicated or inferred.
It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Moreover, two or more steps or actions may be conducted simultaneously.
As used herein, "heteroaryl" refers to an aromatic monocyclic ring system containing at least one ring heteroatom selected from oxygen (O) , nitrogen (N) , sulfur (S) , silicon (Si) , and selenium (Se) or a polycyclic ring system where at least one of the rings present in the ring system is aromatic and contains at least one ring heteroatom. Polycyclic heteroaryl groups include two or more heteroaryl rings fused together and monocyclic heteroaryl rings fused to one or more aromatic carbocyclic rings, non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkyl rings. A heteroaryl group, as a whole, can have, for example, 5 to 22 ring atoms and contain 1 -5 ring heteroatoms (i.e., 5-20 membered heteroaryl group) . The heteroaryl group can be attached to the defined chemical structure at any heteroatom or carbon atom that results in a stable structure. Generally, heteroaryl rings do not contain O-O, S-S, or S-O bonds. However, one or more N or S atoms in a heteroaryl group can be oxidized (e.g., pyridine N-oxide, thiophene S-oxide, thiophene S, S-dioxide) . Examples of heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below:
Figure PCTCN2019096536-appb-000013
where T is O, S, NH, N-alkyl, N-aryl, N- (arylalkyl) (e.g., N-benzyl) , SiH 2, SiH (alkyl) , Si (alkyl)  2, SiH (arylalkyl) , Si (arylalkyl)  2, or Si (alkyl) (arylalkyl) . Examples of such heteroaryl rings include pyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl, triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl, thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl, quinox-alyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, lH-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl, naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl, thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl, pyridopyrimidinyl, pyridopyrazinyl, pyridopyridazinyl, thienothiazolyl, thienoxazolyl, thienoimidazolyl groups, and the like. Further examples of heteroaryl groups include 4, 5, 6, 7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups, and the like. In some embodiments, heteroaryl groups can be substituted as described herein.
As used herein, "halo" or "halogen" refers to fluoro, chloro, bromo, and iodo.
As used herein, "alkyl" refers to a straight-chain or branched saturated hydrocarbon group. Examples of alkyl groups include methyl (Me) , ethyl (Et) , propyl (e.g., n-propyl and z′-propyl) , butyl (e.g., n-butyl, z′-butyl, sec-butyl, tert-butyl) , pentyl groups (e.g., n-pentyl, z′-pentyl, -pentyl) , hexyl groups, and the like. In various embodiments, an alkyl group can have 1 to 40 carbon atoms (i.e., C1-40 alkyl group) , for example, 1-30 carbon atoms (i.e., C1-30 alkyl group) . In some embodiments, an alkyl group can have 1 to 6 carbon atoms, and can be referred to as a "lower alkyl group. " Examples of lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl and z′-propyl) , and butyl groups (e.g., n-butyl, z′-butyl, sec-butyl, tert-butyl) . In some embodiments, alkyl groups can be substituted as described herein. An alkyl group is generally not substituted with another alkyl group, an alkenyl group, or an alkynyl group.
As used herein, "alkenyl" refers to a straight-chain or branched alkyl group having one or more carbon-carbon double bonds. Examples of alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, butadienyl, pentadienyl, hexadienyl groups, and the like. The one or more carbon-carbon double bonds can be internal (such as in 2-butene) or terminal (such as in 1-butene) . In various embodiments, an alkenyl group can have 2 to 40 carbon atoms (i.e., C2-40 alkenyl group) , for example, 2 to 20 carbon atoms (i.e., C2-20 alkenyl group) . In some embodiments, alkenyl groups can be substituted as described herein. An alkenyl group is generally not substituted with another alkenyl group, an alkyl group, or an alkynyl group.
As used herein, a "fused ring" or a "fused ring moiety" refers to a polycyclic ring system having at least two rings where at least one of the rings is aromatic and such aromatic ring (carbocyclic or heterocyclic) has a bond in common with at least one other ring that can be aromatic or non-aromatic, and carbocyclic or heterocyclic. These polycyclic ring systems can be highly p-conjugated and optionally substituted as described herein.
As used herein, "heteroatom" refers to an atom of any element other than carbon or hydrogen and includes, for example, nitrogen, oxygen, silicon, sulfur, phosphorus, and selenium.
As used herein, "aryl" refers to an aromatic monocyclic hydrocarbon ring system or a polycyclic ring system in which two or more aromatic hydrocarbon rings are fused (i.e., having a bond in common with) together or at least one aromatic monocyclic hydrocarbon ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings. An aryl group can have 6 to 24 carbon atoms in its ring system (e.g., C6-24 aryl group) , which can include multiple fused rings. In some embodiments, a polycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ring position  of the aryl group can be covalently linked to the defined chemical structure. Examples of aryl groups having only aromatic carbocyclic ring (s) include phenyl, 1-naphthyl (bicyclic) , 2-naphthyl (bicyclic) , anthracenyl (tricyclic) , phenanthrenyl (tricyclic) , pentacenyl (pentacyclic) , and like groups. Examples of polycyclic ring systems in which at least one aromatic carbocyclic ring is fused to one or more cycloalkyl and/or cycloheteroalkyl rings include, among others, benzo derivatives of cyclopentane (i.e., an indanyl group, which is a 5, 6-bicyclic cycloalkyl/aromatic ring system) , cyclohexane (i.e., a tetrahydronaphthyl group, which is a 6, 6-bicyclic cycloalkyl/aromatic ring system) , imidazoline (i.e., a benzimidazolinyl group, which is a 5, 6-bicyclic cycloheteroalkyl/aromatic ring system) , and pyran (i.e., a chromenyl group, which is a 6, 6-bicyclic cycloheteroalkyl/aromatic ring system) . Other examples of aryl groups include benzodioxanyl, benzodioxolyl, chromanyl, indolinyl groups, and the like. In some embodiments, aryl groups can be substituted as described herein. In some embodiments, an aryl group can have one or more halogen substituents, and can be referred to as a "haloaryl" group. Perhaloaryl groups, i.e., aryl groups where all of the hydrogen atoms are replaced with halogen atoms (e.g., -C6F5) , are included within the definition of "haloaryl. " In certain embodiments, an aryl group is substituted with another aryl group and can be referred to as a biaryl group. Each of the aryl groups in the biaryl group can be substituted as disclosed herein.
As used herein, a "donor" material refers to an organic material, for example, an organic nanoparticle material, having holes as the majority current or charge carriers.
As used herein, an "acceptor" material refers to an organic material, for example, an organic nanoparticle material, having electrons as the majority current or charge carriers.
As used herein, a "photothermal agent" refers to an organic material, for example, an organic nanoparticle material, that can convert light radiation to heat and, thereby, provide an ultrasonic emission.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the presently described subject matter pertains.
Where a range of values is provided, for example, concentration ranges, percentage ranges, or ratio ranges, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within  the described subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the described subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the described subject matter.
Throughout the application, descriptions of various embodiments use “comprising” language. However, it will be understood by one of skill in the art, that in some specific instances, an embodiment can alternatively be described using the language “consisting essentially of” or “consisting of” .
For purposes of better understanding the present teachings and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about” . Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
Photothermal agents
The present subject matter contemplates photothermal agents, or agents that can convert a delivered light energy to heat and, thereby, provide an ultrasonic emission. The photothermal agents can be useful for diagnostic and/or therapeutic purposes. According to some embodiments, a photothermal agent, as contemplated herein, can include at least one small molecule organic compound with absorption in the near-infrared (NIR) interrogation window (700-900 nm) . The compound can be an organic nanoparticle (ONP) . In some embodiments, the photothermal agent can include a conjugated polymer. The photothermal agents described herein can provide ideal contrast agents for light triggered diagnostic/therapeutic techniques, such as photoacoustic imaging (PAI) associated with photothermal therapy (PTT) .
In an embodiment, the compounds are provided as nanoparticles. In an embodiment, the compounds are non-emissive in solution and in the solid state.
The present compounds can include NIR absorbing organic molecules with a donor-acceptor (D-A-D) structure and long alkyl side chains. The present compounds can include  molecular rotors and bulky alkyl chains grafted to a central D-A-D core to restrict intermolecular interaction and enhance intramolecular motion in aggregates. The enhanced molecular motion favors the formation of dark twisted intramolecular charge transfer (TICT) state, whose nonradiative decay enhances photothermal properties.
According to an embodiment, one or more polymers can be conjugated to certain of the present compounds. In an embodiment, the present compounds include a poly (β-amino ester) conjugated thereto. In certain, non-limiting, embodiments, the poly (β-amino ester) is poly (cyclopentadithiophene-alt-benzothiadiazole) .
In an embodiment, the present photothermal agent can include a compound having a donor-acceptor-donor structure:
D-A-D
each donor unit (D) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000014
the acceptor unit (A) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000015
wherein R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
n is an integer from 4 to 12 when the alkyl chain is linear;
n is an integer from 6 to 24 when the alkyl chain is branched; and
R 2 is selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group, sulfonic group, alkylthio, alkoxy group, alkyl-NCS, alkyl-N 3, alkyl-NH 2, and alkyl-Br.
In an embodiment, R 1 is a C nH 2n+1 alkyl chain branched at the second carbon and n is 6-24.In another embodiment, R 1 is a linear C nH 2n+1 alkyl chain and n is 4-12.
In an embodiment, the compound has one of the following structural formulae:
Figure PCTCN2019096536-appb-000016
wherein R is
Figure PCTCN2019096536-appb-000017
These compounds can be referred to as 2TPE-NDTA and 2TPE-2NDTA, respectively.
In an embodiment, the acceptor is selected from:
Figure PCTCN2019096536-appb-000018
Figure PCTCN2019096536-appb-000019
wherein D is the donor group, and
wherein R 1 is selected from the group consisting of
Figure PCTCN2019096536-appb-000020
In an embodiment, the compound has the following structural formula:
Figure PCTCN2019096536-appb-000021
wherein R is selected from the group consisting of
Figure PCTCN2019096536-appb-000022
These compounds can be referred to as NIR12 and NIR6, respectively. In an embodiment, these compounds can further include a poly (β-amino ester) conjugated thereto, as described herein.
In a further embodiment, the present subject matter relates to a photothermal agent, comprising a compound having a donor-acceptor-donor structure:
D-A-D
each donor unit (D) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000023
the acceptor unit (A) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000024
wherein R 1 is hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
n is an integer from 4 to 12 when the alkyl chain is linear;
n is an integer from 6 to 24 when the alkyl chain is branched; and
R 2 is unsubstituted or substituted and is selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group, sulfonic group, alkylthio, alkoxy group, alkyl-NCS, alkyl-N 3, alkyl-NH 2, and alkyl-Br.
In another embodiment, the present subject matter relates to a photothermal agent, comprising a compound having a donor-acceptor-donor structure, each donor unit (D) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000025
the acceptor unit (A) being selected from the group consisting of:
Figure PCTCN2019096536-appb-000026
wherein R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
n is an integer from 4 to 12 when the alkyl chain is linear;
n is an integer from 6 to 24 when the alkyl chain is branched; and
R 2 is H.
According to this embodiment, the photothermal agent may further comprise a poly (β-amino ester) conjugated thereto.
The present compounds can include a D-A-D structure with typical AIE units (compounds which exhibit aggregation-induced emission) as donor units and compounds with large π-conjugation and a strong electron-withdrawing ability as acceptor units. Exemplary compounds include triphenylethylene and tetraphenylethylene derivatives, as donor units, and  NDI and/or PDI derivatives as acceptor units. For example, certain non-limiting embodiments of the present compounds include 2TPE-NDTA and 2TPE-2NDTA. The 2TPE-NDTA and 2TPE-2NDTA compounds include tetraphenylethylene (TPE) . TPE is a prototype molecule with active excited-state molecular motion. TPE containing compounds having a D-A, or D-A-D, structure lead to twisted intramolecular charge transfer (TICT) , which facilitates the non-radiative decay with the help of intramolecular motion. According to these embodiment, the acceptor includes one or more naphthalene diimide fused 2- (1, 3-dithiol-2-ylidene) acetonitrile moieties (NDTA and 2NDTA) , with large π-conjugation and strong electron-withdrawing ability. The acceptor contributes to long wavelength absorption, high molar absorptivity and strong TICT effect. The long alkyl chain in the molecular backbone can enable intermolecular spatial isolation when aggregated, thus producing some necessary space to promote free intramolecular motion. Based on this rational molecular design, 2TPE-NDTA and 2TPE-2NDTA can conduct highly effective intramolecular motion in the solid state and aggregated state within NPs, leading to increased energy absorbance for heat production through the boosted non-radiative decay process.
2TPE-2NDTA-doped NPs exhibit superior capability in generating a photoacoustic (PA) signal when compared with several well-established, high-performing PA imaging agents, including semiconducting polymer NPs and MB. As described herein, in vivo studies verified that 2TPE-2NDTA-doped NPs achieves excellent performance in visualizing tumors in a high-contrast manner via PA imaging.
According to some embodiments, the present compounds include molecular rotors introduced into planar D-A based small molecules, which facilitates both molecular motion and stabilizing the dark TICT state. In some embodiments, the compounds include long alkyl chains which can provide shielding units to restrain intermolecular interaction, and most importantly to maintain the intramolecular rotations in aggregates. In some embodiments, the compounds include D-A or D-A-D conjugated small molecules based on low-bandgap benzo [1, 2-c: 4, 5-c′] bis ( [1, 2, 5] thiadiazole) (BBTD) as an acceptor, thiophene as π-conjugation unit and donor, triphenylamine (TPA) as molecular rotor and as a second donor, and long alkyl chains as shielding units. It is believed that emission of the AIE unit is weakened by activation of molecular motion in aggregates and that photothermal conversion is achieved owing to the stabilization of the dark TICT state and restriction of fluorescence decay.
The presence of molecular rotors and long alkyl chains can be important to produce the  TICT state in aggregates. As described in detail herein, exemplary compound NIR12 with long alkyl chains branched at the second carbon exhibits enhanced photothermal properties when compared to compound NIR6 with short linear alkyl chains and the commercial dye indocyanine green. Both in vitro and in vivo experiments demonstrated that NIR12 nanoparticles can be used as nano-agents for photoacoustic imaging-guided photothermal therapy. Moreover, charge reversal with poly (β-amino ester) makes NIR12 specifically accumulate at a tumor site.
Identifying Tumors and Stopping or Inhibiting Tumor Growth
The present compounds can be administered to a patient as a contrast agent for locating a tumor site in the patient using in vivo imaging techniques, e.g., photoacoustic imaging. The compounds can be administered by intravenous injection, for example. As set forth in detail herein, in vivo imaging studies demonstrate that the compounds can serve as an effective probe for PAI in a high-contrast manner. Once the tumor site has been determined, the tumor site can be irradiated with light, e.g., near-infrared light which, when combined with the present compounds, can stop or inhibit the growth of the tumor.
In particular, the present methods can relate, in certain embodiments, to a method of locating a tumor site in a patient, comprising administering a photothermal agent as described herein to the patient; and locating the tumor site using photoacoustic imaging.
In certain further embodiments, the present methods can relate to a method of stopping or inhibiting tumor growth in a patient, comprising administering a photothermal agent as described herein to the patient; locating a tumor site using photoacoustic imaging; and subjecting the tumor site to light irradiation while the photothermal agent is present at the tumor site to stop or inhibit the growth of the tumor. In some non-limiting embodiments, the light irradiation can be irradiation by near-infrared light.
In certain embodiments, when practicing the present methods, the photothermal agent is administered to the patient in nanoparticle (NP) form.
According to some embodiments, the present compounds can be co-precipitated with a poly (β-amino ester) (PAE) to prolong circulation time in blood and enhance interaction with tumor cells. For example, NIR12 co-precipitated with poly (β-amino ester) (PAE) can rapidly and reversibly change surface properties and as well as provide an effective imaging probe and tumor treatment. Collectively, manipulation of TICT property using molecular rotors and alkyl chains may provide a useful platform for designing new photothermal agents.
The present compounds may demonstrate rapid temperature elevation in a tumor site under NIR light irradiation, which results in heat-caused tumor inhibition. For example, the 2TPE-2NDTA-doped NPs exhibit better capability in generating PA signal when compared with several well-established, high-performing PA imaging agents, including semiconducting polymer NPs and MB. As described herein, in vivo studies verified that 2TPE-2NDTA-doped NPs give excellent performance in visualizing tumors in a high-contrast manner via PA imaging.
The compounds demonstrate good photothermal/photoacoustic performance, making them promising candidates for in vivo diagnosis and therapy applications.
The present teachings are illustrated by the following examples.
EXAMPLES
Materials and Instruments
All chemicals were commercially available and used as supplied without further purification. Deuterated solvents were purchased from J&K. TEP-B (OH)  2 was purchased from AIEgen Biotech Co., Ltd. In at least some cases, the solvents for chemical reactions were distilled before use. Tetrahydrofuran (THF) was dried by distillation using sodium as drying agent and benzophenone as indicator. Benzo [1, 2-c: 4, 5-c′] bis ( [1, 2, 5] thiadiazole) was purchased from Derthon Optoelectronic Materials Science Technology Co LTD. Poly (β-amino ester) (PAE) was synthesized according to the previous report (Chem. Commun., 2015, 51, 14985) . All air and moisture sensitive reactions were carried out in flame-dried glasswares under a nitrogen atmosphere.
For characterization of 2TPE-NDTA and 2TPE-2NDTA derivatives,  1H and  13C NMR spectra were recorded on a Bruker ARX 400 NMR spectrometer using the deuterated solvent as the lock and tetramethylsilane (TMS; δ = 0) as internal reference. High-resolution mass spectra (HRMS) were obtained on a Finnigan MAT TSQ 7000 Mass Spectrometer System operated in a MALDI-TOF mode. Absorption spectra were measured on a JASCO V-570 UV-vis-NIR spectrophotometer. Steady-state photoluminescence (PL) spectra were recorded on an Edinburgh FLS980 fluorescence spectrophotometer. Quantum yield was determined by a
Figure PCTCN2019096536-appb-000027
integrating sphere. Particle size analyses were implemented using a wetaPlus Potential Analyzer (Brookhaven, ZETAPLUS) . Transmission electron microscopy (TEM) investigations were carried out on a JEOL-6390 instrument.
Femtosecond time-resolved fluorescence (fs-TRF) experiment. Femtosecond time-resolved fluorescence (fs-TRF) measurements were performed on the same setup as fs-TA. The output 800 nm laser pulse (200 mw) is used as gate pulse while the 400 nm laser pulse (10 mw) (second harmonic) is used as the pump laser. After excitation by the pump laser, the sample fluorescence is focused into the nonlinear crystal (BBO) mixing with the gate pulse to generate the sum frequency signal. Broadband fluorescence spectra are obtained by changing the crystal angles, and the spectra are detected by the air-cooled CCD. For the present experiments, the compound ND in THF solution were excited by a 400 nm pump beam (the second harmonic of the fundamental 800 nm from the regenerative amplifier) . The 1 mL solutions were studied in a 2 mm path-length cuvette with an absorbance of 0.5 at 400 nm throughout the data acquisition.
For characterization of NIR-12, NIR-6, and intermediates,  1H and  13C NMR spectra were recorded at room temperature on a Unity-400 NMR spectrometer using CDCl 3 as solvent and tetramethylsilane (TMS) as reference. The UV-vis-NIR absorption spectra were performed using a PerkinElmer Lambda 365 spectrophotometer. Mass spectra (MS) were measured with a GCT premier CAB048 mass spectrometer in MALDI-TOF mode. The photoluminescence (PL) spectra were conducted on a Horiba Fluorolog-3 spectrofluorometer. Dynamic light scattering (DLS) was measured on a 90 plus particle size analyzer. Transmission electron microscopy (TEM) images were acquired from a JEM-2010F transmission electron microscope with an accelerating voltage of 200 kV. Laser confocal scanning microscope images were collected on Zeiss laser scanning confocal microscope (LSM7 DUO) and analyzed using ZEN 2009 software (Carl Zeiss) .
For NIR-12, NIR-6 testing, all animal studies were conducted under the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals, and overall procedures were approved by the Animal Ethics Committee in Nankai University. Six-week-old female BALB/c mice were purchased from Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) . To establish the xenograft 4T1 tumor-bearing mouse model, murine 4T1 breast cancer cells (1×10 6) suspended in 50 μL of RPMI-1640 medium were injected subcutaneously into the right axillary space of the mouse. After about 10 days, mice with tumor volumes at about 80-120 mm 3 were used subsequently.
Example 1
Femtosecond transient absorption (fs-TA) experiment
The fs-TA experiments were done employing an experimental setup and methods detailed previously and only a brief description is provided here. Fs-TA measurements were done using a 1000 Hz femtosecond regenerative amplified Ti: sapphire laser system (Maitai) in which the amplifier was seeded with the 120 fs laser pulses from an oscillator laser system. The laser probe pulse was produced by utilizing ～5%of the amplified 800 nm laser pulses to generate a white-light continuum (430-750 nm) in a saphire crystal and then this probe beam was split into two parts before traversing the sample. One probe laser beam goes through the sample while the other probe laser beam goes to the reference spectrometer in order to monitor the fluctuations in the probe beam intensity. For the present experiments, the compounds in THF solution were excited by a 400 nm pump beam (the second harmonic of the fundamental 800 nm from the regenerative amplifier) . The 1 mL solutions were studied in a 2 mm path-length cuvette with an absorbance of 0.5 at 400 nm throughout the data acquisition.
Example 2
Solid-state NMR experiments
NMR experiments were performed on a Varian Infinitplus-400 wide-bore (89 mm) NMR spectrometer at room temperature (25 ℃) at frequencies of 399.72, 100.52 MHz for  1H and  13C, respectively. T3 probe with a rotor diameter of 4 mm was used, and samples with a volume of 52 μL were placed in a zirconia PENCIL rotor. The 90° pulse length was approximately 3 μs, corresponding to a radio frequency (RF) field strength of 83 kHz. The magic angle spinning (MAS) at 5 kHz was automatically controlled with a speed controller, and the total suppression of sidebands (TOSS) sequence was also used before the signal acquisition to suppress the spinning sidebands. The  13C chemical shifts were referenced to external HMB (hexamethylbenzene, 17.3 ppm of CH 3) . The ramped cross polarization (CP) was used for the  1H- 13C polarization transfer, and the CP contact time was 0.1ms and 1ms, respectively.
Example 3
Preparation of NPs
To a THF solution (1 mL) , NDTA, 2TPE-NDTA, or 2TPE-2NDTA (1 mg) was dissolved. Then, DSPE-PEG 2000 (2 mg) was also added and dissolved in the THF solution. After that, the obtained THF solution was added into water (9 mL) accompanied with sonication using a microtip probe sonicator (XL2000, Misonix Incorporated, NY) , followed by continuative  sonication of the mixture for another 60 s. The THF in the mixture was evaporated by stirring in the fume hood for 12 h. The resultant NP suspension was purified by ultrafiltration (molecule weight cutoff 100 kDa) at 3000 × g for 0.5 h, which was subsequently filtered with a 0.2 μm syringe driven filter.
Example 4
Cell culture
Murine 4T1 breast cancer cells were purchased from American Type Culture Collection (ATCC) . The 4T1 cancer cells were cultured in Dulbecco′s Modified Eagle’s Medium (DMEM) containing 10%fetal bovine serum (FBS) and 1%penicillin-streptomycin at 37 ℃ in a humidified environment containing 5%CO 2, respectively. Before experiments, the cells were pre-cultured until confluence was reached.
Example 5
Animals and tumor-bearing mouse model
All animal studies were conducted under the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals, and the overall project protocols were approved by the Animal Ethics Committee of Nankai University. Female BALB/c mice (6-week-old) were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China) . The xenograft 4T1 tumor-bearing mouse model was used in the study. To set up the animal model, 30 μL of cell culture medium containing 1 × 10 6 murine 4T1 breast cancer cells were injected subcutaneously into the right auxiliary space of the BALB/c mouse. After about 10 days, mice with tumor volumes of about 80-120 mm 3 were used subsequently.
Example 6
Photoacoustic imaging
The 4T1 tumor-bearing mice were firstly anesthetized using 2%isoflurane in oxygen, followed by intravenous injection of 2TPE-2NDTA-doped NPs (300 μM based on 2TPE-2NDTA) using a microsyringe via the tail vein (n = 3 mice) . In vivo tumor PA imaging was carried out by a commercial small-animal opt-acoustic tomography system (MOST, iTheraMedical, Germany) . The PA images were acquired at 730 nm before administration and at 4, 8, 16, 24 h post-injection.
The feasibility of conceptual MEPT in achieving advanced practical application was also studied. In vivo PA imaging of MEPT NPs was carried out, as PA effect depends on the  photothermy. Before the live animal study, the PA properties of various agents including 2TPE-2NDTA-doped NPs, 2TPE-NDTA-doped NPs, SPNs and MB were measured and compared. Their PA spectra in the region of 680-980 nm, as displayed in Fig. 21A, illustrate that the maximum PA amplitudes of 2TPE-NDTA-doped NPs, SPNs and MB locate at 680 nm, whereas 2TPE-2NDTA-doped NPs have a PA peak at 735 nm. The comparison in PA signal (Fig. 21B) among different PA agents was then performed with 680 nm pulsed laser. At the same condition, the PA intensity of 2TPE-NDTA-doped NPs is much higher than those of SPNs and MB, while the signal of 2TPE-2NDTA-doped NPs is even higher than 2TPE-NDTA-doped NPs. Interestingly, 2TPE-2NDTA-doped NPs show the highest PA intensity at 680 nm even though 680 nm is not the optimized excitation wavelength. The PA intensity of 2TPE-2NDTA-doped NPs is about 1.6-fold and 2.1-fold higher than that of SPNs and MB, respectively. It has been reported that the SPNs are well-known as an excellent PA contrast agents that exhibit even better PA signal than single-walled carbon nanotubes. Additionally, MB is a star molecule for PA imaging. The comparison data reveal that MEPT is a desirable platform for developing superior PA imaging probes and that active intramolecular motion within NPs determines biomedical function and effectiveness.
In vivo PA imaging was subsequently conducted before and after administration of 2TPE-2NDTA-doped NPs into the xenograft 4T1 tumor-bearing mice via the tail vein. Fig. 21C depicts the time-dependent in vivo PA tumor imaging of 2TPE-2NDTA-doped NP-injected mice. As compared to the PA image before NP injection (0 h) , intense PA signal can be clearly observed at the tumor site at 4 h post injection of 2TPE-2NDTA-doped NPs. The PA intensity at 4 h is around 2.7-fold higher than that at 0 h (Fig. 26D) . Such high-contrast PA tumor imaging benefits from not only the excellent EPR effect of NPs but also the prominent PA effect of 2TPE-2NDTA (Figs. 21A-21B) . The superb passive tumor targeting rooting in the EPR effect highlights the advantage and importance of our design that enables MEPT to occur within NPs. The live animal result demonstrates that MEPT NPs are capable of serving as effective PA imaging probes for tumor diagnosis in vivo.
Example 7
2TPE-NDTA, 2TPE-2NDTA, 2TPE-PDI-C 6, and 2TPE-PDI-C 16 synthesis and  characterization
An exemplary reaction scheme for preparing 2TPE-NDTA and 2TPE-2NDTA is provided  below:
Figure PCTCN2019096536-appb-000028
Synthesis of 2, 3 and 4 Compound NDTA (117 mg, 0.1 mmol) and Br 2 (160 mg, 0.11 mmol) , were dissolved in chloroform (8 mL) under atmosphere and reacted at room temperature for 0.5 h. The reaction was terminated by adding water and extracted with dichloromethane and then purified by silica-gel column chromatography, affording  compound  2 and 3 with 73 mg and 46 mg, respectively. yield: 55%for 2 and 37%for 3.
Compound 2.  1H NMR (400 MHz, CDCl 3, 25 ℃) , δ (ppm) : 4.17 (s, 4H) , 2.04 (br, 2H) , 1.38-1.22 (br, 80H) , 0.87-0.84 (m, 12H) .  13C NMR (100 MHz, CDCl 3, 25 ℃) , δ (ppm) : 162.0,  161.9, 161.6, 147.5, 147.4, 145.4, 145.2, 125.0, 124.7, 124.5, 116.5, 116.1, 114.1, 71.2, 46.2, 36.4, 32.0, 31.5 30.1, 29.7, 29.4, 26.3, 22.7, 14.1.
Compound 3.  1H NMR (400 MHz, CDCl 3, 25 ℃) , δ (ppm) : 5.67 (s, 1H) , 4.15 (s, 4H) 1.99 (br, 2H) , 1.36-1.21 (br, 80H) , 0.88-0.84 (m, 12H) .  13C NMR (100 MHz, CDCl 3, 25 ℃) , δ (ppm) : 164.5, 162.3, 162.1, 161.8, 147.5, 147.4, 147.3, 147.2, 146.9, 146.8, 144.7, 125.02, 124.8, 124.7, 116.6, 116.2, 115.6, 114.3, 85.8, 71.2, 46.2, 46.1, 36.6, 36.5, 32.0, 31.6, 31.5, 30.2, 30.1, 29.8, 29.7, 29.5, 26.5, 26.4, 22.8, 14.2.
Synthesis of 4. To a solution of compound 3 (200 mg, 0.16 mmol) in anhydrous DMF (15 mL) and DMSO (3 mL) , Pd (PhCN)  2Cl 2 (3.1 mg, 0.008 mmol) , AgNO 3 (81.6 mg, 0.48 mmol) and KF (27.8 mg, 0.48 mmol) were added successively under N 2. The reaction mixture was stirred at 120℃ for 8h under N 2. After cooling to room temperature, saturated NH 4Cl (aq) was added to the mixture and the precipitated product was filtered and collected. The crude product was purified by column chromatography (Hex/DCM = 2/3) to afford the pure 4 (159 mg) in a yield of 80%. MS (MALDI-TOF) : m/z: [M] + calcd for C 136H 196Br 2N 8O 8S 8, 2487.4; found, 2487.3. Elemental analysis: calcd, C: 65.67%, H: 7.94%, N: 4.50%; found, C: 65.39%, H: 7.82%, N: 4.31%.
Synthesis of 2TPE-NDTA. To a solution of compound 2 (150 mg, 0.11mmol) , TPE-B (OH)  2 (213 mg, 0.57 mmol) and K 2CO 3 (125 mg, 0.91 mmol) in THF (10 mL) and H 2O (4 mL) , Pd (PPh 3)  4 (13.1 mg, 0.011 mmol) was added under N 2 protection. The mixture was stirred overnight at 100℃ under N 2, after which the mixture was extracted with CH 2Cl 2 and the organic solvent was removed under reduced pressure. The crude product was obtained by column chromatography to afford the pure 2TPE-NDTA (140 mg) in a yield of 70%. Melting point: 269-270 ℃. Fig. 1 depicts  1H NMR spectra of 2TPE-NDTA. Fig. 2 depicts the  13C NMR spectra of 2TPE-NDTA. Fig. 3 depicts high resolution mass spectrum (MALDI-TOF) of 2TPE-NDTA.
1H NMR (400 MHz, CDCl 3, 25 ℃) , δ (ppm) : 7.43-7.41 (d, 4H) , 7.18-7.17 (m, 36H) , 7.14-7.07 (m, 20H) , 4.18 (s, 2H) , 4.16 (s, 2H) , 2.03 (br, 2H) , 1.37-1.21 (br, 80H) , 0.88-0.83 (m, 12H) .  13C NMR (100 MHz, CDCl 3, 25 ℃) , δ (ppm) : 162.3, 162.2, 157.2, 147.4, 147.3, 146.0, 145.9, 145.1, 143.5, 143.3, 143.2, 142.6, 139.9, 132.4, 131.6, 131.5, 131.4, 131.0, 128.1, 128.0, 127.8, 127.1, 126.9, 126.8, 126.7, 115.8, 115.5, 101.7, 46.1, 36.4, 32.0, 31.5, 30.2, 29.8, 29.7, 29.5, 26.5, 26.4, 22.8, 14.2. HRMS (MALDI-TOF) : m/z: [M] + calcd for C 120H 136N 4O 4S 4, 1824.9444; found, 1824.9493.
Synthesis of 2TPE-2NDTA. The compounds 4 (150 mg, 0.06 mmol) , TPE-B (OH)  2 (113 mg, 0.3 mmol) , K 2CO 3 (66 mg, 0.48 mmol) and Pd (PPh 3)  4 (7 mg, 0.006 mmol) were mixed in THF (10 mL) and deoxy H 2O (3 mL) under N 2. The mixture was stirred overnight at 100℃ under N 2. After cooling to room temperature, the mixture was extracted with CH 2Cl 2 and the organic solvent was removed under reduced pressure. The crude product was obtained by column chromatography to afford the pure 2TPE-2NDTA (79 mg) in a yield of 44%. Melting point: 299.2-301.2℃. Fig. 4 depicts  1H NMR spectra of 2TPE-2NDTA. Fig. 5 depicts  13C NMR spectra of 2TPE-2NDTA. Fig. 6 depicts mass spectrum of 2TPE-2NDTA.
1H NMR (400 MHz, CDCl 3, 25 ℃) , δ (ppm) : 7.46-7.43 (m, 4H) , 7.18-7.16 (m, 14H) , 7.14-7.07 (m, 20H) , 4.24 (s, 4H) , 4.15 (s, 4H) , 2.02 (br, 4H) , 1.24-1.18 (br, H) , 0.88-0.83 (m, 24H) .  13C NMR (100 MHz, CDCl 3, 25 ℃) , δ (ppm) : 162.3, 162.2, 157.2, 147.4, 147.3, 146.0, 145.9, 145.1, 143.5, 143.3, 143.2, 142.6, 139.9, 132.4, 131.6, 131.5, 131.4, 131.0, 128.1, 128.0, 127.8, 127.1, 126.9, 126.8, 126.7, 115.8, 115.5, 101.7, 46.1, 36.4, 32.0, 31.5, 30.2, 29.8, 29.7, 29.5, 26.5, 26.4, 22.8, 14.2. MS (MALDI-TOF) : m/z: [M] + calcd for C 188H 234N 8O 8S 8, 2990.4; found, 2990.2.
Synthesis of 2TPE-PDI-C 6. The mixture of 2TPE-PDI-C 6 (143 mg, 0.2 mmol) , TPE-B (OH)  2 (188 mg, 0.5 mmol) , K 2CO 3 (221 mg, 1.6 mmol) and Pd (PPh 3)  4 (23 mg, 0.006 mmol) were mixed in THF (10 mL) and deoxy H 2O (3 mL) under N 2. The mixture was stirred overnight at 100℃ under N 2. After cooling to room temperature, the mixture was extracted with CH 2Cl 2 and the organic solvent was removed under reduced pressure. The crude product was obtained by column chromatography to afford the pure 2TPE-PDI-C 6 (160 mg) in a yield of 66%. Fig. 7 depicts  1H NMR spectrum of compound 2TPE-PDI-C 6. Fig. 8 depicts  13C NMR spectra of 2TPE-PDI-C 6.
Fig. 9 depicts high resolution mass spectrum (MALDI-TOF) of 2TPE-PDI-C 6.
1H NMR (400 MHz, CDCl 3, 25 ℃) , δ (ppm) : 8.52 (s, 2H) , 8.17-8.15 (d, J = 8Hz, 2H) , 7.80-7.78 (d, J = 8Hz, 2H) , 7.19-7.07 (m, H) , 5.06-5.00 (br, 2H) , 2.61-2.53 (m, 4H) , 1.93-1.90 (m, 4H) , 1.77-1.74 (m, 6H) , 1.50-1.46 (m, 4H) , 1.39-1.33 (m, 2H) .  13C NMR (100 MHz, CDCl 3, 25 ℃) , δ (ppm) : 144.5, 143.6, 143.4, 143.3, 142.0, 140.7, 140.1, 135.0, 134.4, 133.1, 132.1, 131.4, 131.3, 129.9, 129.0, 128.4, 128.0, 127.9, 127.7, 127.5, 127.0, 126.8, 126.7, 122.6, 122.2, 54.0, 29.1, 26.6, 25.5. HRMS (MALDI-TOF) : m/z: [M] + calcd for C 88H 66N 2O 4, 1214.5023; found, 1214.5033.
Synthesis of 2TPE-PDI-C 16. The mixture of 2TPE-PDI-C 16 (143 mg, 0.2 mmol) , TPE-B (OH)  2 (188 mg, 0.5 mmol) , K 2CO 3 (221 mg, 1.6 mmol) and Pd (PPh 3)  4 (23 mg, 0.006 mmol) were mixed in THF (10 mL) and deoxy H 2O (3 mL) under N 2. The mixture was stirred overnight at 100℃ under N 2. After cooling to room temperature, the mixture was extracted with CH 2Cl 2 and the organic solvent was removed under reduced pressure. The crude product was obtained by column chromatography to afford the pure 2TPE-PDI-C 16 (160 mg) in a yield of 66%. Fig. 10 depicts  1H NMR spectrum of compound 2TPE-PDI-C 16 in CDCl 3. Fig. 11 depicts  13C NMR spectra of 2TPE-PDI-C 16 in CDCl 3. Fig. 12 depicts high resolution mass spectrum of compound 2TPE-PDI-C 16 in CDCl 3.
1H NMR (400 MHz, CDCl 3, 25 ℃) , δ (ppm) : 8.47 (s, 1H) , 8.46 (s, 1H) , 8.03-8.01 (d, J = 8Hz, 2H) , 7.61-7.59 (d, J = 8Hz, 2H) , 7.30-7.29 (br, 6H) , 7.20-7.10 (m, 32H) , 4.16-4.15 (br, 4H) , 1.99 (br, 2H) , 1.26 (m, 50H) , 0.85-0.82 (m, 12H) .  13C NMR (100 MHz, CDCl 3, 25 ℃) , δ (ppm) : 163.7, 163.5, 144.6, 143.7, 143.4, 143.2, 142.0, 140.7, 140.0, 135.0, 134.3, 133.2, 132.0, 131.3, 129.9, 128.9, 128.8, 128.3, 128.1, 127.9, 127.8, 127.3, 127.1, 126.8, 126.7, 122.0, 121.6, 44.7, 36.7, 31.9, 31.8, 30.1, 29.8, 29.6, 29.3, 26.6, 22.7, 14.1. HRMS (MALDI-TOF) : m/z: [M] + calcd for C 108H 110N 2O 4, 1498.8466; found, 1498.8438.
Example 8
Photophysical properties
The optical properties of 2TPE-NDTA and 2TPE-2NDTA were characterized by photoluminescence (PL) and UV-vis-NIR spectroscopies and compared with that of NDTA (Figs. 13A-13C and 14A-14C) . To evaluate their optical characteristics in aggregated state, nanoprecipitation strategy was employed to formulate these molecules into water-soluble NPs using 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy- (polyethylene glycol) -2000] (DSPE-PEG 2000) as the encapsulation matrix (Figs. 15A-15B) . The afforded NDTA-doped, 2TPE-NDTA-doped, and 2TPE-2NDTA-doped NPs possess an average diameter of about 125, 152, and 156 nm, respectively (Figs. 16A-16C) . As shown in Fig. 13A, NDTA emits brightly at around 630 nm in dilute THF solution, whereas both its NPs and film show much decreased PL intensity but largely red-shifted emission peaks (around 810 nm) (Fig. 13B and 13C) , suggesting the aggregation-caused quenching (ACQ) and J-aggregation characteristics of NDTA in aggregates. This is reasonable since the large π-conjugation and planar structure of NDTA  benefited strong π-π stacking. However, after coupling with TPE, both the dilute THF solution and the aggregates (NPs and thin film) of 2TPE-NDTA and 2TPE-2NDTA display no emission, indicating that the non-radiative relaxation process is dominated for the exciton relaxation of 2TPE-NDTA and 2TPE-2NDTA, even in the solid state. This is quite different from the typical design of AIE molecules where TPE is usually introduced to transform the ACQ molecules to AIE ones.
Additionally, the molar absorptivities of 2TPE-NDTA (50600) and 2TPE-2NDTA (67800) are comparable to that of NDTA (67822) , revealing their superb light-harvesting ability (Figs. 17A-17B) . From solution to the aggregates, the absorption profile and maximum peak of NDTA exhibit large red-shifts, supporting its J-aggregation stacking pattern. In comparison with NDTA, the absorption profile of 2TPE-NDTA is broadened with the absorption maximum changing slightly after aggregation, indicating that the introduction of TPE hinders the strong π-πstacking of 2TPE-NDTA. The absorption feature of 2TPE-2NDTA in solution state is almost identical to that of 2TPE-NDTA, manifesting that 2TPE-2NDTA has comparable conjugation to 2TPE-NDTA in solution state. This could be attributed to the twisted structure of 2NDTA, which hampers the conjugation of the whole molecule. Upon aggregation, however, the molecular planarity of 2TPE-2NDTA is significantly improved, leading to the large red-shift of the absorption spectrum. Of note, the absorption of 2TPE-2NDTA-doped NPs can be extended to the region of 750-880 nm, with a strong absorption intensity at the wavelength of 808 nm, matching well with the excitation wavelength of commercial NIR laser source. These results further confirm that the compounds can be useful in PA imaging and photothermal applications.
Example 9
Theoretical Calculation
The electronic structures in the ground state (S 0) at the level of B3LYP/6-31G (d, p) were investigated to decipher the optical properties variation of the studied molecules (Figs. 18A-18B) . NDTA exhibits a planar structure, with the largest coefficients in the highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) both located along the whole π-backbone and overlapping well with each other, which contributes to its emission in solution. Nevertheless, 2TPE-NDTA presents a dumbbell shape molecular conformation owing to the twisted structure of TPE, which disfavors the strong intermolecular π-π stacking. In addition, the HOMO of 2TPE-NDTA is mainly distributed on the TPE moieties, whereas its  LUMO is distributed on the NDTA moiety, in accordance with the ICT effect. 2TPE-2NDTA shows an even more twisted conformation. Besides the twisted TPE moieties, the central acceptor unit of 2NDTA is also highly twisted with a dihedral angle of 120°. The HOMO electron density distribution of 2TPE-2NDTA is located on one of the TPE moieties while its LUMO is distributed on the 2NDTA moiety, suggestive of the ICT effect. The twisted structure and the ICT effect of 2TPE-NDTA and 2TPE-2NDTA are beneficial to active intramolecular motion since they not only hinder the intermolecular π-π interaction to facilitate the spatial isolation of the molecules, but also enhance the TICT effect, finally contributing to the non-radiative relaxation of the excitons.
Example 10
Solid-state nuclear magnetic resonance (SSNMR)
Solid-state nuclear magnetic resonance (SSNMR) was employed to study the molecular motion behaviors of 2TPE-NDTA and 2TPE-2NDTA in the solid state (Figs. 19A-19C) . Since both the 2TPE-NDTA and 2TPE-2NDTA have no other aromatic hydrogen atom except those of TPE, it is easy to identify the  13C signal of TPE moieties by  13C CPMAS NMR spectra and probe their intramolecular motion behavior 33. As shown in Fig. 19A,  13C CPMAS NMR spectra at 5 kHz with toss and varied contact time (CT) was implemented to assign the TPE peaks of 2TPE-NDTA. Then, the relaxation of the signals of  13C in TPE moieties with time was recorded and plotted, affording the relaxation time of the TPE moieties to evaluate the activity of the intramolecular motion. Single TPE is a typical AIEgen with strong solid emission due to the suppression of intramolecular motion. Indeed, single TPE molecule shows a long relaxation time of around 5577s in the solid state by  13C CPMAS NMR measurement, demonstrating its hampered intramolecular motion. On the contrary, the relaxation time of TPE moieties in 2TPE-NDTA is as short as 10.5 s, suggesting its more strengthened intramolecular motion. Similarly, 2TPE-2NDTA exhibits even shorter relaxation time, indicating the more active molecular motion of its TPE moieties. It is worthy to note that the alkyl chain parts show ever shorter relaxation time than the TPE moiety, indicating its flowing characteristic which benefits to the molecular motion of the TPE moiety. These data verify that even in the solid state, there is still effective intramolecular motion in both 2TPE-NDTA and 2TPE-2NDTA.
After demonstrating that active intramolecular motion is chiefly responsible for the non-emission of 2TPE-NDTA and 2TPE-2NDTA in the aggregates, the long alkyl chain in the  backbone of these molecules may play a decisive role in intermolecular spatial isolation and thus, creates some room to permit free intramolecular motion in the NP and solid states. An unsuccessful attempt was made to synthesize 2TPE-NDTA and 2TPE-2NDTA with shorter alkyl side chain to elucidate the role of long alkyl side chain. The attempt was unsuccessful due to the solubility issue. As such, analogues were synthesized to confirm this conjecture. Two TPE substituted perylenediimides (PDIs) , 2TPE-PDI-C 6 and 2TPE-PDI-C 16 with different alkyl side chain lengths were synthesized and characterized. The 2TPE-PDI-C 6 and 2TPE-PDI-C 16 derivatives were synthesized as depicted below
Figure PCTCN2019096536-appb-000029
As expected, with the alkyl chain length increasing form cyclohexyl to 2-hexyloctyl, the quantum yield of 2TPE-PDI-C 16 (6%) in the film was dramatically decreased as compared to 2TPE-PDI-C 6 (17%) due to the more active intramolecular motion of 2TPE-PDI-C 16 in the solid state. To confirm whether the hypothesis could be extended to other systems, TriPE-3PDIs, the reported semiconductors with different side chains and strong red emission were synthesized and their optical properties were compared with each other. The results indicated that TriPE-3PDIs with longer alkyl chain of 2-hexyloctyl show much lower quantum yield (6%) in the film than the one with shorter alkyl chain of 2-ethylhexyl (30%) . Therefore, introduction of long alkyl chain into the backbone of molecular rotors is a general and effective strategy to promote the  intramolecular motion and non-radiative relaxation of the excitons in the aggregates. As it has been established that the photophysical mechanisms of fluorescence and photothermy show opposite characteristics, the absorbed light energy could reasonably incline to generate heat accompanied with reduced fluorescence.
Example 11
Photothermal Conversion Properties
The photothermal conversion properties of 2TPE-NDTA-doped and 2TPE-2NDTA-doped NPs in water were investigated, as practical biomedical application requires working in aqueous media. A semiconducting polymer NP (SPN) , reported as a high-performing photothermal agent, was used as a positive control, which was prepared by formulation of poly (cyclopentadithiophene-alt-benzothiadiazole) using DSPE-PEG 2000 as the matrix (Figs. 15A-15B) . Upon laser irradiation at 808 nm (0.8 W cm -2) , the temperatures of aqueous solutions of all the three NPs elevate over time and reach a maximum at 300 s (Figs. 25A-25B) . As shown in Figs. 20A-20B, the plateau of photothermal temperatures of 2TPE-2NDTA-doped NPs, 2TPE-NDTA-doped NPs and SPNs are 81.4, 69.6, and 57.5 ℃, respectively. According to the literature, the photothermal conversion efficiency of the SPNs is as high as 27.5%. Using the same calculation method, 2TPE-2NDTA-doped NPs and 2TPE-NDTA-doped NPs possess much higher photothermal conversion efficiencies of 54.9%and 43.0%, respectively, which are considered to be ultrahigh among currently available photothermal agents. Such excellent photothermal behavior can be attributed to active intramolecular motion, which harvests absorbed light energy for heat production. This result manifests that active intramolecular motion within NPs is a highly efficient approach to enhance photothermy
Example 12
Preparation of NIR12-PEG, NIR12-PAE NPs
One mL of THF solution containing 1 mg of NIR12 compound, and 2 mg of 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy- (polyethylene glycol) -2000 (DSPE-PEG2000) was poured into 10 mL of deionized water. This step was followed by sonication with a microtip probe sonicator (XL2000, Misonix Incorporated, NY) for 2 min. The residue THF solvent was evaporated by violent stirring of the suspension in fumehood overnight, and a colloidal solution was obtained and used directly.
One mL of THF solution containing 1 mg of NIR12 compound, 1 mg of DSPE-PEG2000 and 1 mg of poly (β-amino ester) (PAE) was poured into 10 mL of deionized water. This step was followed by sonication with a microtip probe sonicator (XL2000, Misonix Incorporated, NY) for 2 min. The residue THF solvent was evaporated by violent stirring the suspension in fumehood overnight, and a colloidal solution was obtained and used directly (Fig. 26) .
Example 13
Photothermal stability studies and performance (NIR12-PEG NPs, and ICG NPs)
For photothermal stability studies, the PBS solutions (pH 7.4) of NIR12-PEG NPs, and ICG NPs were irradiated under an 808 nm laser (0.8 W/cm 2) , and the absorption spectra were measured at different time points (Figs. 23A-23H) . For antiphotobleaching studies, the temperatures of the sample solutions were recorded during five cycles of heating and cooling processes. In one heating-cooling cycle, the NIR laser first irradiated the samples for 5 min to reach a steady state, then the laser was removed, and the samples were naturally cooled down to ambient temperature in 6 min (Figs. 24A-24E) .
The PBS solutions (pH 7.4) of NIR12-PEG NPs, and ICG NPs were continuously exposed to an 808 nm NIR laser (0.8 W/cm 2) for 5 min. The temperature was measured every 20 s and stopped until the temperature nearly reached to a plateau. The corresponding IR thermal images of the sample tubes were also recorded (Fig. 25A-25B) .
Example 14
In vivo photoacoustic imaging and In vivo photothermal therapy
PA signals or images were acquired on a commercial small-animal opt-acoustic tomography system (MOST, iTheraMedical, Germany) . The xenograft 4T1 tumor-bearing mice were anesthetized using 2%isoflurane in oxygen, and then NIR12-PAE and NIR12-PEG NPs (150 μL, 600 μM based on NIR12) were injected into the tumor-bearing mice through tail vein using a microsyringe (n = 3) . PA images were subsequently acquired at 700 nm before administration and at designated time intervals post NPs injection. Figs. 27A depicts photoacoustic imaging in a cell. Fig. 27B is a graph depicting photoacoustic intensity. Fig. 27C depicts photoacoustic imaging in the live mice. Fig. 27D is a graph depicting the temperature in the tumor. Fig. 27E is a graph depicting tumor volume. Fig. 27F is a graph depicting the body weight of mice treated with a PAE nanoparticle, a PEG nanoparticle, or saline.
The xenograft 4T1 tumor-bearing mice were randomly divided into 6 groups (n = 6 per  group per group) , named “Only Saline” , “Saline + Laser” , “NIR12-PEG NPs” , “NIR12-PEG NPs + Laser” , “NIR12-PAE NPs” , and “NIR12-PAE NPs + Laser” , respectively. On day 0, for “Only Saline” , “NIR12-PEG NPs” and “NIR12-PAE NPs” groups, saline, 150μL of NIR12-PEG NPs and NIR12-PAE NPs (600 μM based on NIR12) were injected into 4T1 tumor-bearing mice through tail vein, respectively, without subsequent laser irradiation. For “Saline + Laser” , “NIR12-PEG NPs + Laser” and “NIR12-PAE NPs + Laser” groups, after intravenous injection of saline, NIR12-PEG NPs and NIR12-PAE NPs (150 μL, 600 μM based on NIR12) for 7 h, respectively, the tumors of mice in each group were continuously irradiated with an 808 nm laser (0.5 mW/cm 2) for 5 min. In the meantime, the temperature changes of the tumors were recorded every 10 s via an IR thermal camera (Fluke Shanghai Inc. ) . After a variety of treatments, the tumor volumes and mouse body weights were measured every other day for 16 days. The tumor volume was measured by caliper and calculated as follows: volume = (tumor length) × (tumor width)  2/2. Relative tumor volume was calculated as V/V O (V O was the initial tumor volume) .
Example 15
Histological Studies
After sixteen days of photothermal treatment, the above-mentioned six groups of mice were sacrificed. The liver, spleen, and tumor were excised, fixed in 4%formalin solution, and sectioned at 5 μm thickness. After conventional H&E staining, the slices were examined with a digital microscope (Leica QWin) . The fluorescent proliferating cell nuclear antigen (PCNA) staining was conducted following common immunohistochemical steps. The fluorescent terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was conducted following manual instruction of DeadEnd fluorometric TUNEL system kit (Promega, USA) . The nuclei were counterstained with 40, 6-diamidino-2-phenylindole (DAPI) containing mounting solution (Dapi-Fluoromount-G, Southern Biotech, England) (Figs. 28A-28B) .
Example 16
Cell Culture and Cellular Uptake
Murine 4T1 breast cancer cells were cultured in Dulbecco′s Modified Eagle’s Medium (DMEM) containing 10%fetal bovine serum (FBS) and 1%penicillin-streptomycin at 37 ℃ in a humidified environment containing 5%CO 2, respectively. Before experiments, the cells were pre-cultured until confluence was reached.
In order to investigate the cellular uptake of NIR12-PEG NPs and NIR12-PAE NPs  visually, an aggregation-induced emission luminogen (AIEgen) was incorporated into the inner core of the NPs with NIR12 molecule in the ratio of 1: 1, thus endowing the NPs with fluorescence. Murine 4T1 breast cancer cells were seeded in confocal imaging chambers at a density of 1 × 10 5 cells. After an incubation of 24 hours, the culture medium of each well was replaced with 1 mL of fresh medium with different pH values (pH 7.4 and 6.5) , then the PEG-NIR12-AIEgen NPs and PAE-NIR12-AIEgen were added to each chamber. After 12 hours of further incubation, the cells were washed three times with 1× PBS buffer and fixed using 4%paraformaldehyde for 20 min at 0 ℃. The cellular uptake of NPs was observed with confocal laser scanning microscopy (Zeiss LSM710) upon excitation at 405 nm and a collection of fluorescence signals above 580 nm.
Example 17
Density functional theory calculations
All the calculations were performed in the gas phase using a Gaussian 09 program. The ground-state structure was optimized with B3LYP method and 6-311G (d, p) basis set. Then, a vertical excitation was carried out based on the optimized structure with the same method, from which the ground-state molecular orbital energy was obtained.
Example 18
NIR-6 and NIR-12 Synthesis
An exemplary reaction scheme for preparing NIR12 and NIR6 is provided below:
Figure PCTCN2019096536-appb-000030
Compound 3a: Dibromo-BBT 1 (0.1 g, 0.28 mmol) and 2a (0.7 mmol) , Pd (PPh 3)  4 (20 mg, 0.017 mmol) and 20 mL of toluene were added into a two-necked flask. After the mixture was refluxed for 24 h, additional 2a (0.7 mmol) and Pd (PPh 3)  4 (20 mg, 0.017 mmol) were added to the reaction system. The solution was allowed to reflux for another 24 h. After cooling down to room temperature, the solvent was removed by rotary evaporation. The crude product was purified by silica gel column (hexane) to obtain the product (yield: 55%) .  1H NMR (400 MHz, CDCl 3) , δ (ppm) : 8.83 (2H, s) , 7.28 (2H, s) , 2.73 (4H, d, J = 8 Hz) , 1.77 (2H, m) , 1.32 (80H, m) , 0.9 (12H, m) .
Compound 3b: Compound 3b was synthesized in a similar manner to 3a.  1H NMR (400 MHz, CDCl 3) , δ (ppm) : 8.85 (2H, S) , 7.32 (2H, S) , 2.79 (4H, t, J = 8 Hz) , 1.77 (4H, m) , 1.32 (12H, m) , 0.91 (6H, m) .
Compound 4a: Compound 3a (0.3 g, 0.33 mmol) was dissolved in a mixture of 10 mL CHCl 3 and 10 mL acetic acid under argon atmosphere, NBS (117 mg, 6.6 mmol) was added slowly over the course of 30 mins in a mixture of 5 mL CHCl 3 and 5 mL acetic acid at room temperature under the exclusion of light. The mixture was stirred overnight and was then dried by condensed air. The crude product was purified by silica gel column to obtain the product (yield: 80%) .  1H NMR (400 MHz, CDCl 3) , δ (ppm) : 8.73 (2H, s) , 2.67 (4H, d, J= 8Hz) , 1.84 (2H, m) , 1.3 (80H, m) , 0.89 (12H, m) .
Compound 4b: Compound 3b (0.3 g, 0.57 mmol) was dissolved in a mixture of 10 mL CHCl 3 and 10 mL acetic acid under argon atmosphere. 0.22 g NBS (1.25 mmol) was added slowly over the course of 30 mins in a mixture of 5 mL CHCl 3 and 5 mL acetic acid at room temperature under the exclusion of light. The mixture was stirred overnight and was then dried by condensed air. The crude product was purified by silica gel column (hexane) to get the product (yield= 80%) .  1H NMR (400 MHz, CDCl 3) , δ (ppm) : 8.76 (2H, s) , 2.73 (4H, t, J = 8 Hz) , 1.76 (4H, m) , 1.26 (12H, m) , 0.9 (6H, m) .
Compound 5a (NIR12) : To a solution of compounds 4a (50 mg, 0.042 mmol) and tributyl (4- (diphenylamino) phenyl) stannane (80 mg, 0.15 mmol) in toluene (10 mL) was added Pd (PPh 3)  4 (4 mg) . The mixture was stirred for 48 h at 100 ℃. After cooling down to room temperature, the mixture was poured into water and extracted with DCM. The organic layer was washed with saturated KF and brine before being dried over MgSO 4. After evaporation of the solvent, the residue was purified by column chromatography on silica gel with DCM: hexane (1: 5,  v/v) as the eluent to afford product (yield: 30%) .  1H NMR (400 MHz, CDCl 3) , δ (ppm) : 8.89 (2H, s) , 7.46 (4H, d, J=8 Hz) , 7.32-7.27 (10H, m) , 7.18-7.13 (10H, m) , 7.09-7.05 (4H, m) , 2.79 (4H, t) , 1.81 (2H, m) , 1.21 (80H, m) , 0.87 (12H, m) .  13C NMR (100 MHz, CDCl 3) , δ (ppm) : 150.5, 146.8, 144.1, 138.1, 135.5, 134.7, 129.4, 128.7, 127.8, 124.1, 122.5, 122.34, 122.31, 112.3, 38.4, 32.8, 32.5, 31.3, 29.5, 29.1, 28.74, 25.9, 22.1, 13.5. MS: m/z: [M]  + calcd for C 98H 128N 6S 4: 1518.3, found: 1518.2.
Compound 5b (NIR6) : The synthesis of 5b was similar to that of 5a (yield: 10%) .  1H NMR (400 MHz, CDCl 3) , δ (ppm) : 8.93 (2H, s) , 7.47 (2H, d, J= Hz) , 7.32-7.27 (10H, m) , 7.18-7.12 (10H, m) , 7.09-7.0 (4H, m) , 2.87 (4H, m) , 1.83 (4H, m) , 1.26 (12H, m) , 0.89 (6H, m) .  13C NMR (100 MHz, CDCl 3) , δ (ppm) : 150.6, 146.8, 146.7, 143.4, 138.9, 135.0, 132.4, 129.1, 128.7, 128.0, 125.5, 124.1, 123.9, 123.5, 122.6, 122.0, 112.3, 30.4, 29.0, 28.7, 22.1, 20.6, 13.5, 13.1. MS:m/z: [M]  + calcd for C 62H 56N 6S 4: 1012.3, found: 1012.3.
Fig. 22 depicts (a) molecular design and chemical structure of NIR12 and NIR6 for PAI-guided PTT, (b) calculated HOMOs and LUMOs, (c) optimized ground-state (S 0) geometries, (d) schematic illustration of TICT state, (e) aggregation state and (f) PA imaging guided PTT of the NIR12 and NIR6 (R 1 = 2-hexyldecyl, R 2 = 1-hexyl) .
The present subject matter being thus described, it will be apparent that the same may be modified or varied in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the present subject matter, and all such modifications and variations are intended to be included within the scope of the following claims.

Claims (20)

  1. A photothermal agent, comprising a compound having a donor-acceptor-donor structure:
    D-A-D
    each donor unit (D) being selected from the group consisting of:
    Figure PCTCN2019096536-appb-100001
    the acceptor unit (A) being selected from the group consisting of:
    Figure PCTCN2019096536-appb-100002
    Figure PCTCN2019096536-appb-100003
    Figure PCTCN2019096536-appb-100004
    wherein R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
    n is an integer from 4 to 12 when the alkyl chain is linear;
    n is an integer from 6 to 24 when the alkyl chain is branched; and
    R 2 is selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group, sulfonic group, alkylthio, alkoxy group, alkyl-NCS, alkyl-N 3, alkyl-NH 2, and alkyl-Br.
  2. The photothermal agent according to claim 1, wherein R 1 is a linear C nH 2n+1 alkyl chain and n is 4-12.
  3. The photothermal agent according to claim 1, wherein R 1 is a C nH 2n+1 alkyl chain branched at the second carbon and n is 6-24.
  4. The photothermal agent according to claim 1, wherein the compound is selected from the group consisting of:
    Figure PCTCN2019096536-appb-100005
    wherein R is
    Figure PCTCN2019096536-appb-100006
  5. The photothermal agent according to claim 1, wherein the acceptor is selected from the group consisting of:
    Figure PCTCN2019096536-appb-100007
    wherein D is the donor group, and
    wherein R 1 is selected from the group consisting of
    Figure PCTCN2019096536-appb-100008
  6. The photothermal agent according to claim 5, wherein the compound has the following structural formula:
    Figure PCTCN2019096536-appb-100009
    wherein R is selected from the group consisting of
    Figure PCTCN2019096536-appb-100010
  7. The photothermal agent according to claim 6, wherein the compound further comprises a poly (β-amino ester) conjugated thereto.
  8. The photothermal agent according to claim 1, wherein the compound is non-emissive in solution and in solid state.
  9. A method of locating a tumor site in a patient, comprising:
    administering the photothermal agent of claim 1 to the patient; and
    locating the tumor site using photoacoustic imaging.
  10. The method of claim 9, wherein the photothermal agent is administered in nanoparticle form.
  11. A method of stopping or inhibiting tumor growth in a patient, comprising:
    administering the photothermal agent of claim 1 to the patient;
    locating a tumor site using photoacousting imaging; and
    subjecting the tumor site to light irradiation while the compound is present at the tumor site to stop or inhibit the growth of the tumor.
  12. The method of claim 11, wherein the light is near-infrared light.
  13. A photothermal agent, comprising a compound having a donor-acceptor-donor structure:
    D-A-D
    each donor unit (D) being selected from the group consisting of:
    Figure PCTCN2019096536-appb-100011
    the acceptor unit (A) being selected from the group consisting of:
    Figure PCTCN2019096536-appb-100012
    wherein R 1 is hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
    n is an integer from 4 to 12 when the alkyl chain is linear;
    n is an integer from 6 to 24 when the alkyl chain is branched; and
    R 2 is unsubstituted or substituted and is selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, carboxyl group, amino group, sulfonic group, alkylthio, alkoxy group, alkyl-NCS, alkyl-N 3, alkyl-NH 2, and alkyl-Br.
  14. A method of locating a tumor site in a patient, comprising:
    administering the photothermal agent of claim 13 to the patient; and
    locating the tumor site using photoacoustic imaging.
  15. A method of stopping or inhibiting tumor growth in a patient, comprising:
    administering the photothermal agent of claim 13 to the patient;
    locating a tumor site using photoacoustic imaging; and
    subjecting the tumor site to light irradiation while the photothermal agent is present at the tumor site to stop or inhibit the growth of the tumor.
  16. A photothermal agent, comprising a compound having a donor-acceptor-donor structure, each donor unit (D) being selected from the group consisting of:
    Figure PCTCN2019096536-appb-100013
    the acceptor unit (A) being selected from the group consisting of:
    Figure PCTCN2019096536-appb-100014
    wherein R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
    n is an integer from 4 to 12 when the alkyl chain is linear;
    n is an integer from 6 to 24 when the alkyl chain is branched; and
    R 2 is H.
  17. The photothermal agent according to claim 16, wherein the photothermal agent further comprises a poly (β-amino ester) conjugated thereto.
  18. A method of locating a tumor site in a patient, comprising:
    administering the photothermal agent of claim 16 to the patient; and
    locating the tumor site using photoacoustic imaging.
  19. A method of stopping or inhibiting tumor growth in a patient, comprising:
    administering the photothermal agent of claim 16 to the patient;
    locating a tumor site using photoacoustic imaging; and
    subjecting the tumor site to light irradiation while the photothermal agent is present at the tumor site to stop or inhibit the growth of the tumor.
  20. A photothermal agent, comprising a conjugated polymer having a donor-acceptor-donor structure
    D-A-D
    each donor unit (D) being selected from the group consisting of:
    Figure PCTCN2019096536-appb-100015
    the acceptor unit (A) being selected from the group consisting of:
    Figure PCTCN2019096536-appb-100016
    Figure PCTCN2019096536-appb-100017
    wherein R 1 is a hydrogen or an alkyl chain selected from the group consisting of a linear C nH 2n+1 alkyl chain and a branched C nH 2n+1 alkyl chain;
    n is an integer from 4 to 12 when the alkyl chain is linear;
    n is an integer from 6 to 24 when the alkyl chain is branched;
    p is an integer from 2 to 1000; and
    R 2 is H.
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CN111533885A (en) * 2020-03-30 2020-08-14 威智医药有限公司 Pyrrole and tellurium-thiophene based copolymer nanoparticle and synthesis method and application thereof
CN111533885B (en) * 2020-03-30 2022-04-01 威智医药有限公司 Pyrrole and tellurium-thiophene based copolymer nanoparticle and synthesis method and application thereof
CN113292576A (en) * 2021-06-08 2021-08-24 山西大学 Triphenylamine-based photo-thermal small molecule and preparation method and application thereof
CN113292576B (en) * 2021-06-08 2023-05-30 山西大学 Triphenylamine-based photo-thermal small molecule and preparation method and application thereof
CN114231272A (en) * 2021-12-17 2022-03-25 华中科技大学 Near-infrared II-region nano probe for liver imaging and preparation and application thereof
CN114470202A (en) * 2022-01-11 2022-05-13 浙江大学 AIE-PET bimodal imaging agent and preparation method and application thereof
CN114470202B (en) * 2022-01-11 2023-09-15 浙江大学 AIE-PET bimodal imaging agent and preparation method and application thereof
CN114853787A (en) * 2022-04-13 2022-08-05 河南大学 Near-infrared two-region AIE molecular probe and preparation method and application thereof
CN114853787B (en) * 2022-04-13 2023-06-06 河南大学 Near infrared two-region AIE molecular probe and preparation method and application thereof
CN115724898A (en) * 2022-11-18 2023-03-03 山西大学 Near-infrared fluorescent nano probe with aggregation-induced emission and preparation method thereof

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