WO2020239025A1 - An ultrabright nir-ii aiegen for bioimaging - Google Patents

An ultrabright nir-ii aiegen for bioimaging Download PDF

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WO2020239025A1
WO2020239025A1 PCT/CN2020/092916 CN2020092916W WO2020239025A1 WO 2020239025 A1 WO2020239025 A1 WO 2020239025A1 CN 2020092916 W CN2020092916 W CN 2020092916W WO 2020239025 A1 WO2020239025 A1 WO 2020239025A1
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group
compound
independently
integer ranging
nir
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Benzhong Tang
Shunjie LIU
Dan Ding
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The Hong Kong University Of Science And Technology
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0032—Methine dyes, e.g. cyanine dyes
    • A61K49/0034—Indocyanine green, i.e. ICG, cardiogreen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0097—Cells, viruses, ghosts, red blood cells, viral vectors, used for imaging or diagnosis in vivo
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy

Definitions

  • the present subject matter relates to fluorophores for imaging in the second near-infrared window (1000 nm-1700 nm) , and particularly, to fluorophores for imaging deeply located diseases with an ultrahigh signal background ratio.
  • NIR-II fluorescence imaging with high spatiotemporal resolution and sensitivity provides a powerful tool to direct visualization of dynamic biological processes. Fluorophores emitting within the second near-infrared window (NIR-II, 1000–1700 nm) explicitly show the salient advantages of deeper tissue penetration, higher spatial resolution and better signal-to-noise ratio (SNR) , owing to reduced light scattering and autofluorescence in tissues at longer wavelengths. Therefore, NIR-II fluorescence imaging holds great promise for an accurate diagnosis of deeply located diseases.
  • NIR-II near-infrared window
  • NIR-II fluorophores fluorophores emitting within the second near-infrared window
  • QY quantum yield
  • NIR-II fluorophores there are mainly two methods for designing NIR-II fluorophores: conjugation length enhancement and donor-acceptor (D-A) engineering.
  • conjugation length enhancement increases conjugation length can redshift both absorption and emission of polymethine cyanines (such as IR-26) to the NIR-II region, whereas fluorescence attenuates efficiently due to the solvatochromism-caused quenching.
  • Polymerizing small molecules into the corresponding conjugated polymer offers another strategy; however, strong intermolecular interactions and entanglement compromises the fluorescence.
  • D-A engineering offers an effective way of reducing the bandgap and red-shifting of the absorption and emission maxima.
  • D-A based fluorophores In order to emit fluorescence in the NIR-II window, D-A based fluorophores typically adopt extensively conjugated backbones. The resultant strong intermolecular interactions often give rise to aggregation-caused quenching (ACQ) issues due to the excimer formation. In addition to that, the nonradiative decay in the dark twisted intramolecular charge transfer (TICT) state, commonly observed in polar environments such as water, can damage fluorescence QY.
  • CTI dark twisted intramolecular charge transfer
  • AIE aggregation-induced emission
  • the present subject matter contemplates small molecule, fluorescent compounds having aggregation-induced emission (AIE) characteristics.
  • the compounds can exhibit emission in the second near-infrared window (NIR-II) (1000 nm-1700 nm) .
  • NIR-II near-infrared window
  • the compounds can provide imaging for deeply located diseases with ultrahigh signal background ratio. For example, neutrophils carrying the present compounds can penetrate the brain and allow visualization of inflammation deeply located within the brain tissue through intact scalp and skull.
  • the present AIE compounds can be synthesized by constitutional isomerization of NIR-II fluorophores exhibiting aggregation-caused quenching (ACQ) .
  • ACQ aggregation-caused quenching
  • shifting the alkyl chain of an exemplary ACQ NIR-II fluorophore from the meta to the ortho position drives the NIR-II fluorophore from aggregation-caused quenching to aggregation-induced emission.
  • the fluorescent compounds can have a backbone structural formula selected from the group consisting of
  • each R 1 is independently selected from the group consisting of
  • each R 2 is independently selected from the group consisting of
  • each x is independently an integer ranging from 0 to 20;
  • each m is independently an integer ranging from 6 to 22;
  • each y is independently an integer ranging from 10 to 22.
  • each R 2 is independently selected from the group consisting of
  • the fluorescent compounds include a compound having the following backbone structural formula:
  • each R 1 is independently selected from the group consisting of
  • each R 2 is independently selected from the group consisting of
  • each x is independently an integer ranging from 0 to 20.
  • the compound is:
  • the fluorescent compounds can include a polymeric compound having a backbone structural formula selected from the group consisting of
  • each R 1 is independently selected from the group consisting of
  • each R 2 is independently selected from the group consisting of
  • each x is independently an integer ranging from 0 to 20;
  • n is an integer
  • each m is independently an integer ranging from 6 to 22;
  • each y is independently an integer ranging from 10 to 22.
  • each R 2 is independently selected from the group consisting of
  • a method of detecting inflammation in living tissue of a patient including treating a cell with the present compound to provide a labeled cell, administering the labeled cell to the patient; and identifying an area of tissue inflammation with the labeled cell using an imaging method.
  • the tissue can be brain tissue.
  • the labeled cell can be an immune cell, such as a neutrophil, with a tendency to migrate to a site of inflammation.
  • the imaging method can include NIR-II fluorescence microscopy.
  • the site of inflammation is at a depth of about 3 mm in the living tissue.
  • Fig. 1A depicts chemical structures of 2TT-oC6B and 2TT-mC6B.
  • Fig. 1B depicts optimized ground-state (S0) geometries of 2TT-oC6B and 2TT-mC6B.
  • Fig. 1C depicts calculated HOMOs and LUMOs of 2TT-oC6B and 2TT-mC6B.
  • Fig. 1D depicts absorption and emission spectra of 2TT-oC6B and 2TT-mC6B.
  • Fig. 1E depicts ⁇ AIE curves of 2TT-oC6B and 2TT-mC6B with water fraction.
  • Fig. 1F depicts a presentation of the calculated reorganization energy versus the normal mode wavenumbers of 2TT-oC6B and 2TT-mC6B.
  • Fig 1G depicts the contribution to the total reorganization energy from the bond length, bond angle, and dihedral angle of 2TT-oC6B and 2TT-mC6B.
  • Fig. 1H depicts DFT minimum energy geometries calculated for the S0 (black) and S1 (red) electronics states of of 2TT-oC6B and 2TT-mC6B.
  • Fig 2A depicts a schematic of the NE-mediated NIR-II AIE dots for brain inflammation imaging.
  • Fig. 2B depicts the DLS profile of the AIE dots. (inset represents TEM images, scale bar 100 nm) .
  • Fig. 2C depicts the normalized absorption and emission spectra of the AIE dots.
  • Fig. 3A depicts NIR-II fluorescence images of AIE@NEs and ICG@NE under 808 nm irradiation (20.6 mW/cm 2 ) with different cell number (1000 nm LP, 50 ms) .
  • Fig. 3B depicts the average fluorescence signals at cell number of 5 ⁇ 10 5 .
  • Fig 3C depicts subcutaneous fluorescence images with different cell number.
  • Fig. 3D depicts the average fluorescence signals of the data provided in Fig. 3C.
  • Fig. 3E depicts noninvasive time-dependent in vivo NIR-fluorescence images of brain inflammation through intact scalp and skull (1000 nm LP, 100 ms) .
  • Fig. 3F depicts the average fluorescence signals in the infected region at different time points.
  • Fig. 3G depicts the average signal-to-background ratio in the inflamed brain at 12 h (scale bar: 5 mm) .
  • ⁇ ex refers to excitation wavelength
  • aggregation caused quenching or “ACQ” as used herein refers to the phenomenon wherein the aggregation of ⁇ -conjugated fluorophores significantly decreases the fluorescence intensity of the fluorophores.
  • the aggregate formation is said to “quench” light emission of the fluorophores.
  • AIE aggregation induced emission
  • Emission intensity refers to the magnitude of fluorescence/phosphorescence normally obtained from a fluorescence spectrometer or fluorescence microscopy measurement
  • fluorophore or “fluorogen” as used herein refer to a molecule which exhibits fluorescence
  • luminogen or “luminophore” as used herein refer to a molecule which exhibits luminescence
  • AIEgen as used herein refers to a molecule exhibiting AIE characteristics.
  • 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” .
  • 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.
  • 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., -C 6 F 5 ) , 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.
  • 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 those having two or more heteroaryl rings fused together, as well as those having at least one monocyclic heteroaryl ring 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 24 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-0 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) .
  • heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below: 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) .
  • 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) .
  • 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, quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl, cinnolinyl, lH-indazolyl, 2H-indazo
  • heteroaryl groups include 4, 5, 6, 7-tetrahydroindolyl, tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups, and the like.
  • heteroaryl groups can be substituted as described 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.
  • the present subject matter contemplates small molecule, fluorescent compounds having aggregation-induced emission (AIE) characteristics.
  • the compounds can exhibit emission in the second near-infrared window (NIR-II) (1000 nm-1700 nm) .
  • NIR-II second near-infrared window
  • the compounds can provide imaging for deeply located diseases with ultrahigh signal background ratio. For example, neutrophils carrying the present compounds can penetrate the brain and visualize inflammation deeply located within the brain tissue through intact scalp and skull.
  • the present AIE compounds can be constitutional isomers of NIR-II fluorophores exhibiting aggregation-caused quenching (ACQ) .
  • ACQ aggregation-caused quenching
  • the fluorescent compounds can have a backbone structural formula selected from the group consisting of
  • each R 1 is independently selected from the group consisting of
  • each R 2 is independently selected from the group consisting of
  • each x is independently an integer ranging from 0 to 20;
  • each m is independently an integer ranging from 6 to 22;
  • each y is independently an integer ranging from 10 to 22.
  • each R 2 is independently selected from the group consisting of
  • the fluorescent compounds include a compound having the following backbone structural formula:
  • each R 1 is independently selected from the group consisting of
  • each R 2 is independently selected from the group consisting of
  • each x is independently an integer ranging from 0 to 20.
  • the compound is:
  • the compound can be in a nanoparticle (NP) form.
  • the nanoparticle can be encapsulated in a 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) -2000] matrix to provide a composition.
  • the composition can further include a cell-penetrating peptide, such as a transactivator of transcription (TAT) protein.
  • TAT transactivator of transcription
  • Certain of the compounds described herein can display a fluorescence emission peak at 1048 nm with an optimized Quantum Yield (QY) of 5.3%in water
  • a small infrared molecule displaying typical and aggregation-caused quenching (ACQ) effect can be transformed into an aggregation induced emission (AIE) molecule with constitutional isomerization.
  • AIE aggregation induced emission
  • moving an alkyl unit of the molecule from the meta to the ortho position can result in an AIE compound with enhanced dihedral angle and more twisted structure.
  • the fluorescent compounds can include a polymeric compound having a backbone structural formula selected from the group consisting of
  • each R 1 is independently selected from the group consisting of
  • each R 2 is independently selected from the group consisting of
  • each x is independently an integer ranging from 0 to 20;
  • n is an integer
  • each m is independently an integer ranging from 6 to 22;
  • each y is independently an integer ranging from 10 to 22.
  • each R 2 is independently selected from the group consisting of
  • the photothermal contrast agent, 2TT-mC6B (Fig. 1A) with NIR-I absorption ( ⁇ 808 nm) and NIR-II emission (1064 nm) , is highly fluorescent in solution but is almost non-emissive in aggregates (dominated non-radiative decay) , displaying typical aggregation-caused quenching (ACQ) effect. It is believed that the ACQ effect in 2TT-mC6B comes from its coplanar thiophene-BBTD-thiophene (TBT) core (Fig. 1B) , which can hardly restrict the strong intermolecular interactions (Fig. 1C) even in the presence of the molecular rotor triphenylamine (TPA) .
  • TBT coplanar thiophene-BBTD-thiophene
  • the resulting 2TT-oC6B molecule shows a significantly enhanced dihedral angle of TBT (48°) (Fig. 1G) and a more twisted structure:
  • 2TT-oC6B is weakly emissive in solution but is highly fluorescent in aggregates, displaying a typical AIE attribute (Fig. 1E) .
  • Low frequency vibration modes are predominant in AIE-active 2TT-oC6B molecules, indicating the dynamic twisting motion of the distorted TBT backbone and twisted TPA rotor (Fig. 1F) .
  • the high frequency modes dominate over the total reorganization energy, which ascribes to the stretching and bending motion of bonds.
  • 2TT-oC6B shows fluorescence emission maximum at 1014 nm with a QY of 11%, one of the highest so far (Fig.
  • a method of detecting inflammation in living tissue of a patient including treating a cell with the present compound to provide a labeled cell, adminstering the labeled cell to the patient; and identifying an area of tissue inflammation with the labeled cell using an imaging method.
  • the tissue can be brain tissue.
  • the labeled cell can be an immune cell, such as a neutrophil, with a tendency to migrate to a site of inflammation.
  • the imaging method can include NIR-II fluorescence microscopy.
  • the site of inflammation is at a depth of about 3 mm in the living tissue.
  • the present compound is an ultrabright AIEgen that can provide accurate diagnosis of deeply located inflammation in living tissue, e.g., brain tissue.
  • NEs neutrophils
  • Fig. 2A-2C brain tissues
  • AIE@NE can easily migrate into the inflamed brain
  • Fig. 2A a mouse brain through the intact scalp and skull at a depth of about 3 mm
  • AIE@NE displays high emission intensity with a 4.8-fold increase in PL intensity than that of ICG@NE.
  • subcutaneous imaging was carried out.
  • AIE@NE with 1000 cells display strong fluorescence signal, which is particularly valuable for bioimaging.
  • 500 AIE@NE cells show much higher brightness than that of ICG@NE with even 2000 cells. Quantitative data from PL support these results (Fig. 3D) .
  • AIE@NE is then used to pinpoint brain inflammation through intact skull and scalp of mice.
  • NIR-II imaging of AIE@NE gave a SNR of 30.6 for in vivo diagnosis, which was 22.5-fold higher than that of ICG, allowing accurate brain inflammation diagnostics (Fig. 3E) .
  • UV-Vis-NIR absorption spectra were performed using a PerkinElmer Lambda 365 spectrophotometer. 1 H and 13 C spectra were recorded at room temperature on a Unity-400 NMR spectrometer using CDCl 3 as solvent and tetramethylsilane (TMS) as a reference. 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 Transmission electron microscopy
  • DFT Density functional theory
  • a 10 mL tube was charged with organotin (0.7 g, 1 mmol) , Dibromo-BBT (87 mg, 0.25 mmol) , Pd2 (dba) 3 (22 mg, 0.025 mmol) , P (o-tol) 3 (66 mg, 0.21 mmol) , and degassed dry toluene (1.5 mL) , and sealed with a Teflon cap.
  • the reaction mixture was heated with stirring to 130 °C for 48 h under N 2 atmosphere. Upon cooling, the crude product was quenched with KF solution and extracted with DCM. The combined organic phase was dried with Na 2 SO 4 .
  • IR-26 dissolved in 1, 2-dichloroethane (DCE)
  • DCE 1, 2-dichloroethane
  • a total of five IR-26 solutions in DCE with linearly spaced concentrations were transferred into 10-mm path fluorescence cuvette at a time.
  • the excitation source was an 808-nm diode laser.
  • the emission was collected in the transmission geometry with a 900-nm long-pass filter to reject the excitation light, and the emission spectrum was taken in the 900 nm to 1500 nm region.
  • the same procedures were carried out for ACQ and AIE dyes in DCE and H 2 O solutions.
  • all emission spectra of both the reference and the samples were integrated into the 900 to 1500 nm NIR-II region.
  • the integrated NIR-II fluorescence intensity was plotted against absorbance at the excitation wavelength of 808 nm and fitted into a linear function. Two slopes, one obtained from the reference of IR-26 in DCE and the other from the sample (ACQ or AIE dyes) , were employed in the calculation of the quantum yield of the sample, based on equation (1) as follows:
  • n sample and n ref are the refractive indices of H 2 O and DCE, respectively.
  • a mixture of 2TT-oC6B (1 mg) , DSPE-PEG2000-maleimide (1.5 mg) and THF (1mL) was sonicated (12 W output, XL2000, Misonix Incorporated, NY) to obtain a clear solution.
  • the mixture was quickly injected into 9 mL of water, which was sonicated vigorously in water for 2 min.
  • the mixture was stirred in fume food for 12 h to remove the THF.
  • AIE dot suspension was performed for ultrafiltration (molecule weight cutoff 100 kDa) at 3000 g for 30 min.
  • the amount of 2TT-oC6B aggregates successfully encapsulated into the DSPE-PEG2000-maleimide matrix was estimated by the absorption spectra utilizing a calibration curve of THF solutions of 2TT-oC6B as the reference.
  • Imaging was carried out on a home-built imaging set-up consisting of a 2D InGaAs camera (Princeton Instruments, 2D OMA-V) .
  • the excitation source was an 808 nm laser.
  • the power density of the excitation laser at the imaging plane was 20.6 mW/cm 2 , which is significantly lower than the reported safe exposure limit of 329 mW cm -2 at 808 nm.
  • the emitted fluorescence was allowed to pass through an 810-, a 880-, a 1000-, a 1250-nm long pass (LP) filter to embody the advantage of NIR-II fluorescence imaging. While, for in vitro and in vivo NIR-II fluorescence imaging, the LP filter was fixed at 1000 nm.
  • the NIR-II fluorescence imaging system was applied to collect the NIR-II fluorescent signals (1000LP) under 808 nm excitation (20.6 mW/cm 2 ) with an exposure time of 50 ms
  • mice 6-8 weeks old were anesthetized with Avertin (2, 2, 2-Tribromoethanol, 250 mg/kg, IP) and positioned on a stereotaxic apparatus (Stoelting co. ) .
  • mice were intravenously injected with AIE dots and ICG, respectively, the NIR-II fluorescence imaging at a specific site was used to collect images at different time points.
  • 808 nm continuous irradiation (20.6 mW/cm 2 ) was used as the light source in the NIR-II fluorescence imaging system with an exposure time of 50 ms equipped with 1000 nm long-pass filter (1000LP) .
  • the PL fluorescence intensity was averaged at a specific blood vessel.
  • mice were intravenously injected with AIE dots and ICG, respectively, the NIR-II fluorescence imaging of blood vessels in the brain was used to collect images at different time points.
  • 808 nm continuous irradiation (20.6 mW/cm 2 ) was used as the light source in the NIR-II fluorescence imaging system with an exposure time of 50 ms equipped with 1000 nm long-pass filter (1000 LP) .
  • the PL fluorescence intensity was averaged at a specific blood vessel.
  • the skull of the mouse was opened for better visualization.
  • mice Female mice (6-8 weeks old) were anesthetized with Avertin (2, 2, 2-Tribromoethanol, 250 mg/kg, IP) and positioned on a stereotaxic apparatus (Stoelting co. ) .
  • Escherichia coli (E. coli) LPS (Serotype O111: B4, S-form. Enzo Life Sciences, ALX581-M005) was administered into the right hemisphere (AP 0.0 mm, ML +2.5 mm, DV -4.0 mm from bregma) to induce acute neuro-inflammation.
  • Each animal received 3 ⁇ g of E. coli LPS in 2 ⁇ L of PBS over 5 minutes.
  • the un-injected contra-lateral hemispheres were used as controls.
  • Mature NEs were isolated from murine bone marrow using a modified method. Briefly, the bones were immersed in RPMI 1640 medium after removal of the muscle and sinew. Bone marrow was flushed from the bone with phosphate-buffered saline (PBS) , centrifuged at 200 g for 3 min and resuspended in PBS. The unicellular suspension was added into a Percoll mixture solution consisting of 55%, 65%and 78% (v: v) Percoll in PBS, followed by centrifugation at 500g for 30 min. The mature NEs were recovered at the interface of the 65%and 78%fractions and washed by ice-cold PBS thrice.
  • PBS phosphate-buffered saline
  • the yield was quantified using a haemacytometer (Bright-Line, Sigma-Aldrich) .
  • the viability of the obtained mature NEs was calculated by trypan blue exclusion, and the purity was determined using immunofluorescence double staining with fluorescein isothiocyanate (FITC) -conjugated Ly-6G/Ly-6C (Gr-1) antibody (250 ng mL-1) (BioLegend) and phycoerythrin (PE) -conjugated MAIR-IV (CLM-5) antibody (1 ⁇ g ml-1) (BioLegend) .
  • FITC fluorescein isothiocyanate
  • Gr-1 antibody 250 ng mL-1) (BioLegend)
  • PE phycoerythrin
  • CLM-5 phycoerythrin
  • the morphology of NEs stained with Wright-Giemsa (Jiancheng Bio) was observed by an optical microscope (Ts2R
  • mice were used during parallel experiments to collect all the in vivo data. According to the animal ethics approved by Shenzhen Institutes of Advanced Technology, the anesthesia time of mice used in experiments should not exceed 24 h. Therefore, in this study, the in vivo imaging was monitored from before NPs injection to 24 h post-injection. After mice were intravenously injected with AIE@NEs (2*106 Neutrophils) , the NIR-II fluorescence imaging was used to collect images over different time points.
  • AIE@NEs 2*106 Neutrophils

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Abstract

The present subject matter contemplates small molecule, fluorescent compounds having aggregation-induced emission (AIE) characteristics. The compounds can exhibit emission in the second near-infrared window (NIR-II) (1000 nm-1700 nm). The compounds can provide imaging for deeply located diseases with ultrahigh signal background ratio. For example, neutrophils carrying the present compounds can penetrate the brain and visualize inflammation deeply located within the brain tissue through intact scalp and skull

Description

An Ultrabright NIR-II AIEgen for Bioimaging FIELD
The present subject matter relates to fluorophores for imaging in the second near-infrared window (1000 nm-1700 nm) , and particularly, to fluorophores for imaging deeply located diseases with an ultrahigh signal background ratio.
BACKGROUND
Fluorescence imaging with high spatiotemporal resolution and sensitivity provides a powerful tool to direct visualization of dynamic biological processes. Fluorophores emitting within the second near-infrared window (NIR-II, 1000–1700 nm) explicitly show the salient advantages of deeper tissue penetration, higher spatial resolution and better signal-to-noise ratio (SNR) , owing to reduced light scattering and autofluorescence in tissues at longer wavelengths. Therefore, NIR-II fluorescence imaging holds great promise for an accurate diagnosis of deeply located diseases.
Although deeply located diseases such as brain tumors could be visualized using fluorophores emitting within the second near-infrared window (herein, “NIR-II fluorophores” ) , achieving an excellent imaging quality with high SNR can be challenging. This requires the exploitation of NIR-II fluorophores with high quantum yield (QY) . To date, the majority of NIR-II fluorophores derived from organic molecules have exhibited low QY (around 2%) in aqueous dispersion or solution, owing to the dominated non-radiative decay pathways, which decreases contrast and raises demand for the sensitivity and imaging of optical detectors during in vivo imaging.
Currently, there are mainly two methods for designing NIR-II fluorophores: conjugation length enhancement and donor-acceptor (D-A) engineering. In the former case, increasing conjugation length can redshift both absorption and emission of polymethine cyanines (such as IR-26) to the NIR-II region, whereas fluorescence attenuates efficiently due to the solvatochromism-caused quenching. Polymerizing small molecules into the corresponding conjugated polymer offers another strategy; however, strong intermolecular interactions and entanglement compromises the fluorescence. Alternatively, D-A engineering offers an effective way of reducing the bandgap and red-shifting of the absorption and emission maxima. In order to emit fluorescence in the NIR-II window, D-A based fluorophores typically adopt extensively  conjugated backbones. The resultant strong intermolecular interactions often give rise to aggregation-caused quenching (ACQ) issues due to the excimer formation. In addition to that, the nonradiative decay in the dark twisted intramolecular charge transfer (TICT) state, commonly observed in polar environments such as water, can damage fluorescence QY.
To address these problems, electron-donating groups with steric hindrance are often grafted to a strong acceptor (such as benzobisthiadiazole, BBTD) to distort the conjugated backbones, which can reduce the intermolecular interactions and thereby the formation of excimers. Further, dialkyl substituted fluorene is employed as a shielding unit introduced into the central DAD core to prevent the interaction with water. These strategies open-up new avenues for developing a series of high bright NIR-II fluorophores. Despite long side chains being capable of reducing intermolecular interactions, they are found to also trigger molecular motions in aggregates, whose nonradiative decay spoils fluorescence QY. Therefore, efficiently increasing the radiative decay presents a significant bottleneck for high bright NIR-II fluorophores.
Molecules with aggregation-induced emission (AIE) character hold great potential to solve this problem. AIE luminogens (AIEgens) are typically full of propeller-like molecular rotors, so that the profoundly twisted structures can effectively reduce intermolecular interactions. It is for this reason that AIEgens are significantly more emissive than other molecules as nanoparticles (NPs) . According to the AIE molecular design philosophy, many NIR-II emissive AIEgens have already been developed. The optimized ones possess a slightly enhanced QY of 6.2%, while compromising the fluorescent emission (975 nm < 1000 nm) . Thus far, it remains challenging to obtain high QY for NIR-II fluorophores using the AIE molecular design philosophy.
SUMMARY
In an embodiment, the present subject matter contemplates small molecule, fluorescent compounds having aggregation-induced emission (AIE) characteristics. The compounds can exhibit emission in the second near-infrared window (NIR-II) (1000 nm-1700 nm) . The compounds can provide imaging for deeply located diseases with ultrahigh signal background ratio. For example, neutrophils carrying the present compounds can penetrate the brain and allow visualization of inflammation deeply located within the brain tissue through intact scalp  and skull.
The present AIE compounds can be synthesized by constitutional isomerization of NIR-II fluorophores exhibiting aggregation-caused quenching (ACQ) . For example, shifting the alkyl chain of an exemplary ACQ NIR-II fluorophore from the meta to the ortho position drives the NIR-II fluorophore from aggregation-caused quenching to aggregation-induced emission.
In an embodiment, the fluorescent compounds can have a backbone structural formula selected from the group consisting of
Figure PCTCN2020092916-appb-000001
wherein each R 1 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000002
wherein each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000003
Figure PCTCN2020092916-appb-000004
wherein each x is independently an integer ranging from 0 to 20;
wherein each m is independently an integer ranging from 6 to 22; and
wherein each y is independently an integer ranging from 10 to 22.
In an embodiment, each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000005
In an embodiment, the fluorescent compounds include a compound having the following backbone structural formula:
Figure PCTCN2020092916-appb-000006
wherein each R 1 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000007
wherein each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000008
wherein each x is independently an integer ranging from 0 to 20.
In an embodiment, the compound is:
Figure PCTCN2020092916-appb-000009
In an embodiment, the fluorescent compounds can include a polymeric compound having a backbone structural formula selected from the group consisting of
Figure PCTCN2020092916-appb-000010
wherein each R 1 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000011
wherein each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000012
wherein each x is independently an integer ranging from 0 to 20;
wherein each n is an integer;
wherein each m is independently an integer ranging from 6 to 22; and
wherein each y is independently an integer ranging from 10 to 22.
In an embodiment, each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000013
In an embodiment, a method of detecting inflammation in living tissue of a patient is contemplated, including treating a cell with the present compound to provide a labeled cell, administering the labeled cell to the patient; and identifying an area of tissue inflammation with the labeled cell using an imaging method. The tissue can be brain tissue. The labeled cell can be an immune cell, such as a neutrophil, with a tendency to migrate to a site of inflammation. The imaging method can include NIR-II fluorescence microscopy. In an embodiment, the site of inflammation is at a depth of about 3 mm in the living tissue.
BRIEF DESCRIPTION OF DRAWINGS
Various embodiments will now be described in detail with reference to the accompanying drawings.
Fig. 1A depicts chemical structures of 2TT-oC6B and 2TT-mC6B.
Fig. 1B depicts optimized ground-state (S0) geometries of 2TT-oC6B and 2TT-mC6B.
Fig. 1C depicts calculated HOMOs and LUMOs of 2TT-oC6B and 2TT-mC6B.
Fig. 1D depicts absorption and emission spectra of 2TT-oC6B and 2TT-mC6B.
Fig. 1E depicts αAIE curves of 2TT-oC6B and 2TT-mC6B with water fraction.
Fig. 1F depicts a presentation of the calculated reorganization energy versus the normal mode wavenumbers of 2TT-oC6B and 2TT-mC6B.
Fig 1G depicts the contribution to the total reorganization energy from the bond length, bond angle, and dihedral angle of 2TT-oC6B and 2TT-mC6B.
Fig. 1H depicts DFT minimum energy geometries calculated for the S0 (black) and S1 (red) electronics states of of 2TT-oC6B and 2TT-mC6B.
Fig 2A depicts a schematic of the NE-mediated NIR-II AIE dots for brain inflammation  imaging.
Fig. 2B depicts the DLS profile of the AIE dots. (inset represents TEM images, scale bar 100 nm) .
Fig. 2C depicts the normalized absorption and emission spectra of the AIE dots.
Fig. 3A depicts NIR-II fluorescence images of AIE@NEs and ICG@NE under 808 nm irradiation (20.6 mW/cm 2) with different cell number (1000 nm LP, 50 ms) .
Fig. 3B depicts the average fluorescence signals at cell number of 5×10 5.
Fig 3C depicts subcutaneous fluorescence images with different cell number.
Fig. 3D depicts the average fluorescence signals of the data provided in Fig. 3C.
Fig. 3E depicts noninvasive time-dependent in vivo NIR-fluorescence images of brain inflammation through intact scalp and skull (1000 nm LP, 100 ms) .
Fig. 3F depicts the average fluorescence signals in the infected region at different time points.
Fig. 3G depicts the average signal-to-background ratio in the inflamed brain at 12 h (scale bar: 5 mm) .
DETAILED DESCRIPTION
Definitions
The following definitions are provided for the purpose of understanding the present subject matter and for constructing the appended patent claims.
It is noted that, as used in this specification and the appended claims, the singular forms “a” , “an” , and “the” include plural references unless the context clearly dictates otherwise.
The term “λ ex” as used herein refers to excitation wavelength.
The phrase “aggregation caused quenching” or “ACQ” as used herein refers to the phenomenon wherein the aggregation of π-conjugated fluorophores significantly decreases the fluorescence intensity of the fluorophores. The aggregate formation is said to “quench” light emission of the fluorophores.
The phrase “aggregation induced emission” or “AIE” as used herein refers to the phenomenon manifested by compounds exhibiting significant enhancement of light-emission upon aggregation in the amorphous or crystalline (solid) states whereas they exhibit weak or  almost no emission in dilute solutions.
“Emission intensity” as used herein refers to the magnitude of fluorescence/phosphorescence normally obtained from a fluorescence spectrometer or fluorescence microscopy measurement; “fluorophore” or “fluorogen” as used herein refer to a molecule which exhibits fluorescence; “luminogen” or “luminophore” as used herein refer to a molecule which exhibits luminescence; and “AIEgen” as used herein refers to a molecule exhibiting AIE characteristics.
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, “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., -C 6F 5) , 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, “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 those having two or more heteroaryl rings fused together, as well as those having at least one monocyclic heteroaryl ring 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 24 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-0 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: 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, quinoxalyl, 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, 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.
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.
Fluorescent Compounds
In an embodiment, the present subject matter contemplates small molecule, fluorescent compounds having aggregation-induced emission (AIE) characteristics. The compounds can exhibit emission in the second near-infrared window (NIR-II) (1000 nm-1700 nm) . The compounds can provide imaging for deeply located diseases with ultrahigh signal background ratio. For example, neutrophils carrying the present compounds can penetrate the brain and visualize inflammation deeply located within the brain tissue through intact scalp and skull.
The present AIE compounds can be constitutional isomers of NIR-II fluorophores exhibiting aggregation-caused quenching (ACQ) . For example, shifting the alkyl chain of the ACQ photothermal contrast agent, 2TT-mC6B (Fig. 1A) from the meta to the ortho position drives the NIR-II fluorophore from aggregation-caused quenching to aggregation-induced emission.
In an embodiment, the fluorescent compounds can have a backbone structural formula selected from the group consisting of
Figure PCTCN2020092916-appb-000014
wherein each R 1 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000015
wherein each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000016
wherein each x is independently an integer ranging from 0 to 20;
wherein each m is independently an integer ranging from 6 to 22; and
wherein each y is independently an integer ranging from 10 to 22.
In an embodiment, each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000017
Figure PCTCN2020092916-appb-000018
In an embodiment, the fluorescent compounds include a compound having the following backbone structural formula:
Figure PCTCN2020092916-appb-000019
wherein each R 1 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000020
wherein each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000021
wherein each x is independently an integer ranging from 0 to 20.
In an embodiment, the compound is:
Figure PCTCN2020092916-appb-000022
In an embodiment, the compound can be in a nanoparticle (NP) form. The nanoparticle can be encapsulated in a 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) -2000] matrix to provide a composition. In an embodiment, the composition can further include a cell-penetrating peptide, such as a transactivator of transcription (TAT) protein.
Certain of the compounds described herein can display a fluorescence emission peak at 1048 nm with an optimized Quantum Yield (QY) of 5.3%in water
As described herein, a small infrared molecule displaying typical and aggregation-caused quenching (ACQ) effect can be transformed into an aggregation induced emission (AIE) molecule with constitutional isomerization. For example, moving an alkyl unit of the molecule from the meta to the ortho position can result in an AIE compound with enhanced dihedral angle and more twisted structure.
While small molecules are mainly described herein, it should be understood that an ACQ property of a conjugated polymer can be similarly transformed. In an embodiment, the fluorescent compounds can include a polymeric compound having a backbone structural formula selected from the group consisting of
Figure PCTCN2020092916-appb-000023
Figure PCTCN2020092916-appb-000024
wherein each R 1 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000025
wherein each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000026
wherein each x is independently an integer ranging from 0 to 20;
wherein each n is an integer
wherein each m is independently an integer ranging from 6 to 22; and
wherein each y is independently an integer ranging from 10 to 22.
In an embodiment, each R 2 is independently selected from the group consisting of
Figure PCTCN2020092916-appb-000027
The photothermal contrast agent, 2TT-mC6B (Fig. 1A) with NIR-I absorption (～808 nm) and NIR-II emission (1064 nm) , is highly fluorescent in solution but is almost non-emissive in aggregates (dominated non-radiative decay) , displaying typical aggregation-caused quenching (ACQ) effect. It is believed that the ACQ effect in 2TT-mC6B comes from its coplanar thiophene-BBTD-thiophene (TBT) core (Fig. 1B) , which can hardly restrict the strong intermolecular interactions (Fig. 1C) even in the presence of the molecular rotor triphenylamine (TPA) . By merely moving the hexyl unit from the meta to the ortho position, as shown below, the resulting 2TT-oC6B molecule shows a significantly enhanced dihedral angle of TBT (48°) (Fig. 1G) and a more twisted structure:
Figure PCTCN2020092916-appb-000028
As expected, 2TT-oC6B is weakly emissive in solution but is highly fluorescent in aggregates, displaying a typical AIE attribute (Fig. 1E) . Low frequency vibration modes are predominant in AIE-active 2TT-oC6B molecules, indicating the dynamic twisting motion of the distorted TBT backbone and twisted TPA rotor (Fig. 1F) . Whereas, in ACQ-active 2TT-mC6B, despite the presence of inherent twisted TPA, the high frequency modes dominate over the total reorganization energy, which ascribes to the stretching and bending motion of bonds. Most importantly, 2TT-oC6B shows fluorescence emission maximum at 1014 nm with a QY of 11%, one of the highest so far (Fig. 1D) . In addition, the conformational overlap between S0 and S1 in 2TT-oC6B is lower than that in 2TT-mC6B (Fig. 1H) . These results confrm that the low-frequency twisting motions in 2TT-oC6B contributes signifcantly to the nonradiative decay channels and, thus, results in low fluorescence QY of 2TT-oC6B (1.1%) in solution (Fig. 1G) . In the aggregate state, the presence of the distorted backbone and twisted TPA rotor in 2TT-oC6B reduces intermolecular interactions and gives a high fluorescence QY. Moreover, the NIR-II  imaging quality with 2TT-oC6B NPs is far superior to that of indocyanine green (ICG) for imaging mouse hindlimb and scalp vasculature (Figs. 3A-3G) .
Bio-Imaging Applications
In an embodiment, a method of detecting inflammation in living tissue of a patient is contemplated, including treating a cell with the present compound to provide a labeled cell, adminstering the labeled cell to the patient; and identifying an area of tissue inflammation with the labeled cell using an imaging method. The tissue can be brain tissue. The labeled cell can be an immune cell, such as a neutrophil, with a tendency to migrate to a site of inflammation. The imaging method can include NIR-II fluorescence microscopy. In an embodiment, the site of inflammation is at a depth of about 3 mm in the living tissue.
The present compound is an ultrabright AIEgen that can provide accurate diagnosis of deeply located inflammation in living tissue, e.g., brain tissue. For example, to endow targeting ability to deeply located diseases, neutrophils (NEs) , the most abundant type of immune cells with a tendency towards inflammation areas, can be used to penetrate brain tissues (Fig. 2A-2C) . NEs carrying 2TT-oC6B NPs (AIE@NE) can easily migrate into the inflamed brain (Fig. 2A) . For example, AIE@NE can non-invasively identify the inflammation site within a mouse brain through the intact scalp and skull at a depth of about 3 mm (Figs. 2B-2C) . In order to assess the NIR-II fluorescence imaging quality of AIE@NE, cell imaging (1000 nm LP, 50 ms) was frst conducted. As shown in Fig. 3A, the NIR-II fluorescence intensity increases with enhanced cell number of AIE@NE, demonstrating the successful internalization of AIE dots by NEs. The NIR-II fluorescence intensity in 500 AIE@NE cells is even stronger than 5000 of ICG@NE, indicating the excellent sensitivity and brightness garnered by AIE dots. To more intuitively compare the fluorescence emission of the cargo-carrying NEs, we measured the PL intensity of 5 × 10 5 NE cells. As shown in Fig. 3B, AIE@NE displays high emission intensity with a 4.8-fold increase in PL intensity than that of ICG@NE. To evaluate the imaging performance of AIE@NE in vivo, subcutaneous imaging was carried out. As shown in Fig. 3C, at a depth of ≈1 mm, AIE@NE with 1000 cells display strong fluorescence signal, which is particularly valuable for bioimaging. Moreover, 500 AIE@NE cells show much higher brightness than that of ICG@NE with even 2000 cells. Quantitative data from PL support these results (Fig. 3D) . AIE@NE is then used to pinpoint brain inflammation through intact skull and scalp of mice. No fluorescence signal (1000 nm LP, 100 ms) is detected at 1 h post-injection of AIE@NE (2 × 106  cells) , whereas, weak fluorescent delineation of the inflammatory region is achieved at 4 h (Fig. 3E) . The fluorescence signal at the inflamed site increases over time and becomes the strongest and clearest at 12 h post-injection. However, in mice treated with ICG@NE, the inflamed site is hardly differentiated from healthy tissue, owing to its weak fluorescence. These results suggest the deeper penetration depth and higher brightness of AIE dots than that of ICG. The quantitative study of the inflamed site in the brain reveals that the fluorescence intensity in mice treated with AIE@NE is 6.5-fold higher than that of ICG@NE (Fig. 3F) . Furthermore, during a 24 h period of study, the NIR-II fluorescence intensity in the inflamed site reaches maxima at 12 h post-injection and the SBR value in AIE@NE treated group is as high as 30.6, while it is only 5.6 for the group treated with ICG@NE (Fig. 3G) .
Notably, NIR-II imaging of AIE@NE gave a SNR of 30.6 for in vivo diagnosis, which was 22.5-fold higher than that of ICG, allowing accurate brain inflammation diagnostics (Fig. 3E) .
The present teachings are illustrated by the following examples.
EXAMPLES
MATERIALS AND METHODS
All the chemicals and reagents were purchased from chemical sources, and the solvents for chemical reactions were distilled before use.
The UV-Vis-NIR absorption spectra were performed using a PerkinElmer Lambda 365 spectrophotometer.  1H and  13C spectra were recorded at room temperature on a Unity-400 NMR spectrometer using CDCl 3 as solvent and tetramethylsilane (TMS) as a reference. 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. Density functional theory (DFT) calculations were carried out by the B3LYP/6G (d) , Gaussian 09 package.
EXAMPLE 1
Synthesis
Compound 2TT-oC6B:
For synthesizing compound 2TT-oC6B, a 10 mL tube was charged with organotin (0.7 g, 1 mmol) , Dibromo-BBT (87 mg, 0.25 mmol) , Pd2 (dba) 3 (22 mg, 0.025 mmol) , P (o-tol) 3 (66 mg, 0.21 mmol) , and degassed dry toluene (1.5 mL) , and sealed with a Teflon cap. The reaction mixture was heated with stirring to 130 ℃ for 48 h under N 2 atmosphere. Upon cooling, the crude product was quenched with KF solution and extracted with DCM. The combined organic phase was dried with Na 2SO 4. After removing the solvent, the product was purified with silica column to obtain a dark green solid (yield: 35%) .  1H NMR (400 MHz, CDCl 3) , δ (ppm) = 7.59-7.56 (4H, m) , 7.37 (2H, s) , 7.31-7.26 (8H, m) , 7.16-7.12 (8H, m) , 7.11-7.03 (8H, m) , 2.61-2.57 (4H, t, J = 8Hz) , 1.63, (4H, m) , 1.15-1.10 (12H, m) , 0.73 (6H, m) .  13C NMR (100 MHz, CDCl 3) , δ (ppm) : 152.6, 146.9, 146.8, 146.3, 145.0, 128.7, 127.5, 126.1, 124.1, 124.0, 122.7, 122.5, 115.4, 99.3, 30.8, 29.8, 29.6, 28.4, 21.8, 13.3. MS: m/z: [M] + calcd for C 62H 56N 6S 4: 1012.3, found: 1012.3.
EXAMPLE 2
Determination of fluorescence quantum yield (QY) of the dyes.
The QY of the dyes was measured using NIR-II fluorescent IR-26 dye as the reference (QY = 0.5%) . For reference calibration, IR-26, dissolved in 1, 2-dichloroethane (DCE) , was diluted to a DCE solution to prepare five samples with their absorbance value at 808 nm of ～0.1, ～0.08, ～0.06, ～0.04, and ～0.02, given that these highly diluted samples can minimize the second optical processes such as re-absorption and re-emission effects. Then, a total of five IR-26 solutions in DCE with linearly spaced concentrations were transferred into 10-mm path fluorescence cuvette at a time. The excitation source was an 808-nm diode laser. The emission was collected in the transmission geometry with a 900-nm long-pass filter to reject the excitation light, and the emission spectrum was taken in the 900 nm to 1500 nm region. The same procedures were carried out for ACQ and AIE dyes in DCE and H 2O solutions. Then, all emission spectra of both the reference and the samples were integrated into the 900 to 1500 nm NIR-II region. The integrated NIR-II fluorescence intensity was plotted against absorbance at the excitation wavelength of 808 nm and fitted into a linear function. Two slopes, one obtained from the reference of IR-26 in DCE and the other from the sample (ACQ or AIE dyes) , were employed in the calculation of the quantum yield of the sample, based on equation (1) as follows:
Figure PCTCN2020092916-appb-000029
where n sample and n ref are the refractive indices of H 2O and DCE, respectively.
Example 3
Fabrication of AIE Dots
A mixture of 2TT-oC6B (1 mg) , DSPE-PEG2000-maleimide (1.5 mg) and THF (1mL) was sonicated (12 W output, XL2000, Misonix Incorporated, NY) to obtain a clear solution. The mixture was quickly injected into 9 mL of water, which was sonicated vigorously in water for 2 min. The mixture was stirred in fume food for 12 h to remove the THF. AIE dot suspension was performed for ultrafiltration (molecule weight cutoff 100 kDa) at 3000 g for 30 min. The amount of 2TT-oC6B aggregates successfully encapsulated into the DSPE-PEG2000-maleimide matrix was estimated by the absorption spectra utilizing a calibration curve of THF solutions of 2TT-oC6B as the reference.
Example 5
Fabrication of AIE-dots-TAT
A mixture of 2TT-oC6B (1 mg) , DSPE-PEG2000-maleimide (1.5 mg) and THF (1mL) was sonicated (12 W output, XL2000, Misonix Incorporated, NY) to obtain a clear solution. The mixture was quickly injected into 9 mL of water, which was sonicated vigorously in water for 2 min. To conjugate cell-penetrating peptides (derived from HIV-1 transactivator of transcription (TAT) protein) on the AIE dots, 1 μmol of the peptide was added into the above AIE dot suspension, and allowed to react for 12 h. The free Tat peptide was subsequently removed by ultrafiltration.
Example 6
In vitro and in vivo NIR-II fluorescence imaging
Imaging was carried out on a home-built imaging set-up consisting of a 2D InGaAs camera (Princeton Instruments, 2D OMA-V) . The excitation source was an 808 nm laser. The power density of the excitation laser at the imaging plane was 20.6 mW/cm 2, which is significantly lower than the reported safe exposure limit of 329 mW cm -2 at 808 nm. The emitted fluorescence was allowed to pass through an 810-, a 880-, a 1000-, a 1250-nm long pass (LP) filter to embody the advantage of NIR-Ⅱ fluorescence imaging. While, for in vitro and in vivo NIR-II fluorescence imaging, the LP filter was fixed at 1000 nm.
Example 7
In vitro NIR-II fluorescence Imaging of AIE dots
The NIR-II fluorescence imaging system was applied to collect the NIR-II fluorescent signals (1000LP) under 808 nm excitation (20.6 mW/cm 2) with an exposure time of 50 ms
Example 8
In vivo penetration depth of NIR II Fluorescence Imaging of AIE dots
Before conducting experiments, adult female mice (6-8 weeks old) were anesthetized with Avertin (2, 2, 2-Tribromoethanol, 250 mg/kg, IP) and positioned on a stereotaxic apparatus (Stoelting co. ) . To study the imaging ability, AIE dots and ICG were directly subjected to subcutaneous injection in two mouse hindlimbs (500 μM) , respectively. Mice were imaged immediately after dots and ICG injection from a prone position (thickness = 8 mm) and supine position (thickness = 0.5 mm) .
Example 9
In vivo NIR II Fluorescence Imaging of blood vessels in hindlimb and scalp
After mice were intravenously injected with AIE dots and ICG, respectively, the NIR-II fluorescence imaging at a specific site was used to collect images at different time points. 808 nm continuous irradiation (20.6 mW/cm 2) was used as the light source in the NIR-II fluorescence imaging system with an exposure time of 50 ms equipped with 1000 nm long-pass filter (1000LP) . The PL fluorescence intensity was averaged at a specific blood vessel.
Example 10
In vivo NIR II Fluorescence Imaging of blood vessels in the brain
After mice were intravenously injected with AIE dots and ICG, respectively, the NIR-II fluorescence imaging of blood vessels in the brain was used to collect images at different time points. 808 nm continuous irradiation (20.6 mW/cm 2) was used as the light source in the NIR-II fluorescence imaging system with an exposure time of 50 ms equipped with 1000 nm long-pass filter (1000 LP) . The PL fluorescence intensity was averaged at a specific blood vessel. The skull of the mouse was opened for better visualization.
Example 11
Preparation of brain inflammation mouse model
Female mice (6-8 weeks old) were anesthetized with Avertin (2, 2, 2-Tribromoethanol, 250 mg/kg, IP) and positioned on a stereotaxic apparatus (Stoelting co. ) . Escherichia coli (E. coli) LPS (Serotype O111: B4, S-form. Enzo Life Sciences, ALX581-M005) was administered into the right hemisphere (AP 0.0 mm, ML +2.5 mm, DV -4.0 mm from  bregma) to induce acute neuro-inflammation. Each animal received 3 μg of E. coli LPS in 2 μL of PBS over 5 minutes. The un-injected contra-lateral hemispheres were used as controls.
Example 12
Extraction and purification of neutrophils (NEs)
Mature NEs were isolated from murine bone marrow using a modified method. Briefly, the bones were immersed in RPMI 1640 medium after removal of the muscle and sinew. Bone marrow was flushed from the bone with phosphate-buffered saline (PBS) , centrifuged at 200 g for 3 min and resuspended in PBS. The unicellular suspension was added into a Percoll mixture solution consisting of 55%, 65%and 78% (v: v) Percoll in PBS, followed by centrifugation at 500g for 30 min. The mature NEs were recovered at the interface of the 65%and 78%fractions and washed by ice-cold PBS thrice. The yield was quantified using a haemacytometer (Bright-Line, Sigma-Aldrich) . The viability of the obtained mature NEs was calculated by trypan blue exclusion, and the purity was determined using immunofluorescence double staining with fluorescein isothiocyanate (FITC) -conjugated Ly-6G/Ly-6C (Gr-1) antibody (250 ng mL-1) (BioLegend) and phycoerythrin (PE) -conjugated MAIR-IV (CLM-5) antibody (1 μg ml-1) (BioLegend) . The morphology of NEs stained with Wright-Giemsa (Jiancheng Bio) was observed by an optical microscope (Ts2R, Nikon) .
Example 13
Evaluation of AIE-dots-TAT/ICG uptake by NEs (AIE@NEs
1×10 6 neutrophils were treated with culture media and AIE-dots-TAT (1 mg/mL) or ICG (3 mg/mL) for 1 h at 37 ℃. At the end of incubation, the NEs were washed with ice-cold PBS thrice, trypsinized and resuspended in the media. The intracellular containing of AIE dots and ICG was determined by measurement of the absorbance at a designed wavelength of 745 nm and 780 nm, respectively.
Example 14
In vivo NIR II Fluorescence Imaging of brain inflammation
Before conducting experiments, , Avertin (2, 2, 2-Tribromoethanol, 250 mg/kg, IP) was used to anesthetize mice in a prone position. Three mice were used during parallel experiments to collect all the in vivo data. According to the animal ethics approved by Shenzhen Institutes of Advanced Technology, the anesthesia time of mice used in experiments should not exceed 24 h. Therefore, in this study, the in vivo imaging was monitored from before NPs injection to 24 h  post-injection. After mice were intravenously injected with AIE@NEs (2*106 Neutrophils) , the NIR-II fluorescence imaging was used to collect images over different time points. An 808 nm continuous irradiation (20.6 mW/cm 2) was used as the light source in the NIR-II fluorescence imaging system with an exposure time of 100 ms equipped with 1000 nm long-pass filter (1000 LP) . The signal/background ratio was processed using Image J software by counting six points and obtaining the average value.
Example 15
In vivo distribution study
The in vivo biodistribution of AIE dots and ICG in healthy mice and tissue distribution of AIE@NE and ICG@NE in brain inflammation mouse model, respectively, were monitored. At designated time intervals, the sample-injected mice were sacrificed and various tissues including brain, heart, liver, spleen, lung, kidney, stomach, intestine, skin, muscle, bone, were isolated and imaged using the NIR-Ⅱ fluorescence imaging.
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 fluorescent compound exhibiting aggregation induced emission properties, wherein the compound has a backbone structural formula selected from the group consisting of
    Figure PCTCN2020092916-appb-100001
    wherein each R 1 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100002
    wherein each R 2 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100003
    wherein each x is independently an integer ranging from 0 to 20;
    wherein each m is independently an integer ranging from 6 to 22; and
    wherein each y is independently an integer ranging from 10 to 22.
  2. The compound according to claim 1, wherein the compound has the following backbone structural formula:
    Figure PCTCN2020092916-appb-100004
    wherein each R 1 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100005
    wherein each R 2 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100006
    wherein each x is independently an integer ranging from 0 to 20;
    wherein each m is independently an integer ranging from 6 to 22; and
    wherein each y is independently an integer ranging from 10 to 22.
  3. The compound according to claim 2, wherein each R 2 is independently selected from  the group consisting of
    Figure PCTCN2020092916-appb-100007
    Figure PCTCN2020092916-appb-100008
  4. The compound according to claim 3, wherein the compound is:
    Figure PCTCN2020092916-appb-100009
  5. The compound according to claim 1, wherein the compound is in nanoparticle form.
  6. A composition comprising the compound of claim 5 and a 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) -2000] matrix, the compound being encapsulated in the matrix.
  7. The composition of claim 6, further comprising a cell-penetrating peptide.
  8. The composition of claim 7, wherein the cell-penetrating peptide comprises a transactivator of transcription (TAT) protein.
  9. A method of detecting inflammation in a living tissue of a patient, comprising:
    penetrating a cell with the compound of claim 1 to provide a labeled cell;
    administering the labeled cell to the patient; and
    detecting inflammation in the living tissue by fluorescence imaging of the labeled cell.
  10. The method of claim 9, wherein the inflammation is about 3 mm within the living tissue.
  11. The method of claim 9, wherein the living tissue is brain tissue.
  12. A method of detecting inflammation in living tissue of a patient, comprising:
    penetrating a cell with a fluorescent compound to provide a labeled cell;
    administering the labeled cell to the patient; and
    detecting inflammation in the living tissue by fluorescence imaging of the labeled cell;
    wherein the fluorescent compound comprises the following backbone structural formula:
    Figure PCTCN2020092916-appb-100010
    wherein each R 1 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100011
    wherein each R 2 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100012
    Figure PCTCN2020092916-appb-100013
    wherein each x is independently an integer ranging from 0 to 20;
    wherein each m is independently an integer ranging from 6 to 22; and
    wherein each y is independently an integer ranging from 10 to 22..
  13. The method of claim 12, wherein each R 2 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100014
    Figure PCTCN2020092916-appb-100015
  14. The method of claim 13, wherein the compound is:
    Figure PCTCN2020092916-appb-100016
  15. The method of claim 12, wherein the compound is in nanoparticle form.
  16. The method of claim 15, wherein the compound is encapsulated in a 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [maleimide (polyethylene glycol) -2000] matrix,
  17. The method of claim 16, wherein the compound is conjugated with a cell-penetrating peptide.
  18. The method of claim 12, wherein the living tissue is brain tissue.
  19. The method of claim 12, wherein the inflammation is about 3 mm within the living tissue.
  20. A fluorescent polymer compound exhibiting aggregation induced emission properties, the compound having a backbone structural formula selected from the group consisting of
    Figure PCTCN2020092916-appb-100017
    wherein each R 1 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100018
    wherein each R 2 is independently selected from the group consisting of
    Figure PCTCN2020092916-appb-100019
    Figure PCTCN2020092916-appb-100020
    wherein each x is independently an integer ranging from 0 to 20;
    wherein each m is independently an integer ranging from 6 to 22; and
    wherein each y is independently an integer ranging from 10 to 22..
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