WO2020253756A1

WO2020253756A1 - Fluorescent red-emissive compounds for cellular organelle imaging

Info

Publication number: WO2020253756A1
Application number: PCT/CN2020/096689
Authority: WO
Inventors: Benzhong Tang; Parvej ALAM; Guangle NIU; We HE; Nelson Lik Ching Leung
Original assignee: The Hong Kong University Of Science And Technology
Priority date: 2019-06-18
Filing date: 2020-06-18
Publication date: 2020-12-24
Also published as: CN114040962B; CN114040962A

Abstract

Small molecule fluorescent compounds having aggregation-induced emission (AIE) characteristics are disclosed. The compounds exhibit red to near-infrared solid-state emission, large Stokes shift, high fluorescence quantum yield and good two-photon absorption cross section. The compounds can provide specific organelle staining in live cells and deep tissues. The compounds also exhibit high biocompatibility and high photostability under one-photon and two-photon continuous irradiation.

Description

Fluorescent Red-Emissive Compounds for Cellular Organelle Imaging

FIELD

The present subject matter relates generally to use of a series of fluorescent red-emissive compounds having aggregation-induced emission (AIE) characteristics for specific organelle staining, and particularly, for imaging of mitochondria, lysosomes, and endoplasmic reticulum.

BACKGROUND

Cells are fundamental building blocks for many living organisms. The human body is made of trillions of cells each with their own specific functionality. Each cell is made up of cellular organelles, vital structures essential for cellular operation and health. Each organelle, such as the plasma membrane, mitochondria, lysosomes, lipid droplets, Golgi apparatus and endoplasmic reticulum (ER) , plays an important role to support the normal functions of cells and, consequently, the whole body. The plasma membrane is a biological membrane that separates the interior of all cells from the outside environment. The membrane controls the movement of substances in and out of cells and organelles. In this way, it is selectively permeable to ions and organic molecules. In addition, cell membranes are involved in a variety of cellular processes such as cell adhesion, ion conductivity and cell signaling.

Although the predominant physiological function of mitochondria is the generation of ATP by oxidative phosphorylation, additional functions include the generation and detoxification of reactive oxygen species, involvement in some forms of apoptosis, regulation of cytoplasmic and mitochondrial matrix calcium, synthesis and catabolism of metabolites, and the transport of the organelles themselves to correct locations within the cell. Abnormality in any of these processes can cause mitochondrial dysfunction. The Golgi apparatus is an array of cisternal membrane structures arranged in a stack. The Golgi apparatus is essential for biogenesis, secretion, and intracellular distribution of a wide range of biomacromolecules.

Lysosomes are membrane-bound organelles that are present in animal cells and contain acid hydrolases. Lysosomes are dynamic organelles that receive and degrade macromolecules from the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. The ER is a large membrane-bound compartment spread throughout the cytoplasm of eukaryotic cells, which is composed of one completely continuous membrane bilayer and has a single continuous lumen. The ER plays a key role in cellular metabolism, protein synthesis, and transport of intermediates and signaling molecules. Characterization of the ER structure in living cells is challenging due to a wide 3D interconnected network of flattened, membrane-enclosed sacks or tube-like cisterns and tubules with different thicknesses. The dysfunctions of these organelles are the cause of many serious diseases such as cancer, Parkinson’s diseases, Alzheimer’s disease and diabetes

Fluorescence techniques are powerful, non-invasive analytical tools for visualizing, monitoring, and studying different organelles with superb sensitivity, contrast, signal-to-noise ratio, and in-situ workability. There are quite a few series of commercial dyes for organelle staining, such as Nile Red, BODIPY 493/503, monodansylpentane, AFN, and NPBDP. Though many of these dyes are commonly used, there is still room for improving these systems as they can have unwanted side effects such as small Stokes shift leading to self-absorption and reducing efficiency. Additionally, many of these conventional organic fluorophores suffer from fluorescence quenching in higher concentrations or in aggregated state.

Fluorescence quenching caused by aggregate formation is known as aggregation-caused quenching (ACQ) and mostly occurs due to formation of π–π stacking interactions resulting in a reduction of emission efficiency. Aggregation-induced emissive (AIE) materials have demonstrated fantastic properties capable of overcoming this problem. AIEgens are mostly non-emissive/weakly emissive in the solution state but become strongly emissive in aggregate/solid state. Because of these properties, AIE luminogens (AIEgens) have found great success in applications such as biosensors, bioimaging, theranostic agents, fluorescence-guided surgery, and so forth.

During the past decade, extensive developments and significant advances have been made in the use of alkenes as key building blocks for synthesis of diverse functionalized structures in organic chemistry and material science. The cyano group is one of the best electron-withdrawing groups and its introduction to the π-conjugated structures of alkenes leads to acrylonitriles (shown below) with distinct property changes such as conformation, packing mode, stability, solubility and processability.

Acrylonitriles represent a common structural motif and are commonly found in herbicides, pharmaceuticals, agrochemicals and natural products. In recent years, considerable effort has been made to synthesize acrylonitriles with varied substitutions and functions. Jiao et al. reported that allyl halides or esters reacted with NaN ₃ or TMSN ₃ catalysed by Pd (PPh ₃) ₄ to form allyl azides, which were transformed to alkenyl nitriles (or acrylonitriles) by a subsequent oxidative rearrangement process using DDQ as oxidant, shown in the reaction scheme above. On the other hand, the strategy of direct cyanation of alkenes has also been adopted to develop various acrylonitriles. A novel oxidative cyanation of terminal and internal alkenes using a homogeneous copper catalyst to access acrylonitriles was reported by Engle et al. Based on the above-mentioned organic synthetic methods, various fascinating acrylonitriles have been synthesized. However, these routes involve the use of expensive transition metal complexes, hazardous and toxic agents, harsh reaction conditions and low atom economy. Therefore, direct functionalities of acrylonitriles remains a challenging task and there is still room for further improvement.

Recently, many research groups discovered that ketones or aldehydes and propane nitriles were reactants for direct production of acrylonitriles using a conventional base (NaOH, t-BuOK, etc. ) via a transition metal-free, non-hazardous, non-toxic and atom-economic nucleophilic reaction. Thus, such nucleophilic reactions can offer great potential for the direct and facile synthesis of versatile acrylonitriles with multiple functionalities.

Previous studies demonstrated that the cyano groups render acrylonitriles with twisted structures that result in AIE properties and avoid aggregation-caused quenching (ACQ) in the aggregate state in aqueous environment. Based on acrylonitriles, many multi-color brightly emissive AIE luminogens (AIEgens) have been synthesized and applied in various fields such as fluorescence sensing and one-and two-photon bioimaging. Among these AIEgens, acrylonitriles with bright red emission are particularly preferred in bioimaging because of the reduced photodamage, minimal background auto-fluorescence and deep tissue penetration.

Unfortunately, highly red-emissive AIEgens are still rare. In addition, their structures are complicated and their synthesis involves multi-step reaction routes and time-consuming isolation or purification.

SUMMARY

The present subject matter contemplates small molecule, fluorescent compounds with red emissive aggregation-induced emission (AIE) characteristics. The compounds can provide specific organelle staining in live cells. One or more of the fluorescent compounds can penetrate cells and selectively stain an organelle selected from lysosomes, mitochondria, and endoplasmic reticulum. The present compounds also exhibit high biocompatibility and high photostability under one-photon and two-photon irradiation.

In an embodiment, the fluorescent compounds can include a compound having a backbone structural formula selected from the group consisting of:

wherein each Ar ₁ and Ar ₂ is independently a substituted or unsubstituted aromatic ring, the aromatic ring being selected from the group consisting of phenyl, benzene, thiophene, furan, pyran and thiadiazole, and the aromatic ring substituents being selected from the group consisting of hydrogen, alkyl, hydroxyl groups, halogen groups, carboxy groups, cyano groups, and substituted and unsubstituted aromatic and heterocyclic groups;

each R ₁ R ₂, R ₃, and R ₄ is independently selected from the group consisting of C _nH _2n+1, C ₆H ₅, C ₁₀H ₇, C ₁₂H ₉, OC ₆H ₅, C ₆H ₅OH, C ₆H ₅OC _nH _2n+1, OC ₁₀H ₇, OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, CN, and H;

each Z is independently selected from the group consisting of F, Cl, Br, I, PF ₆, BPh ₄, N (CN) ₂, and BF ₄;

each Y ₁ is independently selected from the group consisting of C _nH _2nC ₆H _5-mX _m, C _nH _2nC ₁₀H _7-mX _m, C _nH _2nC ₁₂H _9-mX _m, C _nH _2n, C _nH _2nCO ₂H, and C _nH _2nSO ₃H;

each Y ₂ ^-is independently selected from the group consisting of C _nH _2nSO ₃ ^- and C _nH _2nCO ₂ ^-;

each X is halogen;

each n is independently an integer ranging from 0 to 20; and

each m is independently an integer ranging from 0 to 9.

In an embodiment, the compound comprises one or more compounds selected from the group consisting of:

In an embodiment, a method of cellular imaging is contemplated, including contacting a target cell with the present compound and identifying a target of interest in the target cell using an imaging method. The imaging method can include one-photon fluorescence microscopy or two-photon fluorescence microscopy. In an embodiment, identifying a target of interest can include visualizing organelles in live cells. In an embodiment, the cellular organelle is mitochondria. In an embodiment, the cellular organelle is a lysosome. In an embodiment, the cellular organelle is endoplasmic reticulum. In an embodiment, the target cell is a tumor cell. In an embodiment, the target cell is a live cell. In an embodiment, the live cell is in live tissue.

In an embodiment, the compound has a backbone structural formula selected from the group consisting of:

wherein each R ₁ R ₂, R ₃, and R ₄ is independently selected from the group consisting of C _nH _2n+1, C ₆H ₅, C ₁₀H ₇, C ₁₂H ₉, OC ₆H ₅, C ₆H ₅OH, C ₆H ₅OC _nH _2n+1, OC ₁₀H ₇, OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, CN, and H;

each X is halogen;

each n is independently an integer ranging from 0 to 20; and

each m is independently an integer ranging from 0 to 9.

wherein each Ar ₁ and Ar ₂ is independently a substituted or unsubstituted aromatic ring, the aromatic ring being selected from the group consisting of phenyl, benzene, thiophene, furan, pyran and thiadiazole, and the aromatic ring substituents being selected from the group consisting of hydrogen, alkyl, hydroxyl groups, halogen groups, carboxy groups, cyano groups, and substituted and unsubstituted aromatic and heterocyclic groups.

In an embodiment, the cellular organelle is endoplasmic reticulum which is stained by contact with the compound and the compound comprises at least one of

In an embodiment, the cellular organelle is mitochondria which is stained by contact with the compound and the comprises at least one of

In an embodiment, the cellular organelle is a lysosome stained by contact with the compound and the compound comprises at least one of:

BRIEF DESCRIPTION OF DRAWINGS

Various embodiments will now be described in detail with reference to the accompanying drawings.

Fig. 1 depicts ¹H NMR spectrum of compound 1 in CDCl ₃.

Fig. 2 depicts ¹³C NMR spectrum of compound 1 in CDCl ₃.

Fig. 3 depict HRMS spectrum of compound 1.

Fig. 4 depicts ¹H NMR spectrum of 2TPAT-AN in THF-d ₈.

Fig. 5 depicts ¹³C NMR spectrum of 2TPAT-AN in CDCl ₃.

Fig. 6 depicts HRMS spectrum of 2TPAT-AN.

Fig. 7 depicts ¹H NMR spectrum of TPAT-AN-XF in CDCl ₃.

Fig. 8 depicts ¹³C NMR spectrum of TPAT-AN-XF in CDCl _3.

Fig. 9 depicts ¹⁹F NMR spectrum of TPAT-AN-XF in CDCl ₃.

Fig. 10 depicts HRMS spectrum of TPAT-AN-XF.

Fig. 11A depicts normalized absorption spectra of 2TPAT-AN and TPAT-AN-XF (10 μM) in THF.

Fig. 11B depicts FL spectra of 2TPAT-AN.

Fig. 11C depicts FL spectra of TPAT-AN-XF (10 μM) in THF and THF/water mixtures with different water fractions (f _w) .

Fig. 11D depicts plots of α _AIE (fluorescence intensity I/I ₀) versus the composition of the THF/water mixtures of 2TPAT-AN and TPAT-AN-XF.

Fig. 11E depicts normalized FL spectra of 2TPAT-AN and TPAT-AN-XF in the solid state (inset: fluorescent photos of solids of 2TPAT-AN and TPAT-AN-XF taken under 365 nm UV irradiation from a hand-held UV lamp) .

Fig. 11F depicts the XRD pattern of the pristine sample of 2TPAT-AN and TPAT-AN-XF.

Fig. 11G depicts molecular packing in the crystal of 2TPAT-AN at different directions.

Fig. 11H depicts two-photon absorption (TPA) cross sections of 2TPAT-AN and TPAT-AN-XF in THF. 1 GM ≡ 10 ^-50 cm ⁴ s/photon.

Fig. 12 depicts the dynamic light scattering data of 2TPAT-AN and TPAT-AN-XF (10 μM) in water containing 30%and 5%THF, respectively (hydrated diameter: 399 nm (2TPAT-AN, right) and 210 nm (TPAT-AN-XF, left) .

Fig. 13 depicts intermolecular packing interactions in the crystal of 2TPAT-AN.

Fig. 14 depicts normalized fluorescence spectra of the crystal of 2TPAT-AN before and after grinding.

Figs. 15A-15B depict normalized fluorescence spectra of (Fig. 15A) 2TPAT-AN and (Fig. 15B) TPAT-AN-XF in different polar solvents.

Figs. 16A-16B depict spatial orbital distributions of HOMOs and LUMOs of (Fig. 16A) 2TPAT-AN and (Fig. 16B) TPAT-AN-XF in the optimized ground states and excited states at the B3LYP/6-31G (d, p) level.

Figs. 17A-17B depict the DFT optimized structures of (Fig. 17A) 2TPAT-AN and (Fig. 17B) TPAT-AN-XF in the ground states and excited states.

Figs. 18A-18F depict (Fig. 18A) Schematic preparation of 2TPAT-AN NPs via nanoprecipitation method by using amphiphilic block copolymer PEG-PLGA as the encapsulation materials; (Fig. 18B) TEM image of 2TPAT-AN NPs; (Fig. 18C) DLS data of 2TPAT-AN NPs in water; (Fig. 18 D) Normalized absorption and fluorescence spectra of 2TPAT-AN NPs in water (Inset: Photos of 2TPAT-AN NPs in water taken under room light (left) and 365 nm UV irradiation (right) from a hand-held UV lamp) ; (Fig. 18E) Confocal laser scanning microscopy images of HeLa cells incubated with 2TPAT-AN NPs (5 μg/mL) . Scale bar: 20 μm; (Fig. 18F) Confocal laser scanning microscopy images of HeLa cells incubated with 2TPAT-AN NPs (5 μg/mL) and LysoTraker Green DND-26 (200 nM) . Scale bar: 20 μm.

Figs. 19A-19B (Fig. 19A) The dynamic light scattering data and (Fig. 19B) absorption spectra of 2TPAT-AN NPs in water at different time points.

Fig. 20 depicts the cytotoxicity of 2TPAT-AN NPs in HeLa cells.

Fig. 21 depicts one-photon (λ _ex = 488 nm) and two-photon (λ _ex = 880 nm) fluorescent microscopic images of tumor tissues incubated with 2TPAT-AN NPs (Scale bar: 20 μm) .

Figs. 22A-22D depict ex vivo two-photon and one-photon imaging in live deep tissues. (Fig. 22A) One-photon (λ _ex = 488 nm) and (Fig. 22C) two-photon (λ _ex = 880 nm) fluorescent microscopic images of the mouse tumor tissue stained with 2TPAT-AN NPs at different penetration depths along z-axis. Scale bar: 50 μm. Reconstructed 3D (Fig. 22B) one-photon and (Fig. 22D) two-photon fluorescent microscopic images.

Figs. 23A-23B depict (Fig. 23A) in vivo imaging in 4T1 tumor-bearing nude mice at different time points after intratumor injection of 2TPAT-AN NPs (2 mg/mL, 100 μL) and (Fig. 23B) H&E staining of major organ sections (heart, liver, spleen, lung, and kidney) from mice intratumorally injected with or without 2TPAT-AN NPs.

Fig. 24 depicts normalized mean fluorescence intensity (MFI) of 2TPAT-AN NPs in tumor at different time points after intratumor injection.

Figs 25A-25B depict (Fig. 25A) ¹H NMR and (Fig. 25B) ¹³C NMR spectrum of CDPBr in CDCl ₃.

Fig. 26 depicts high resolution mass spectrum (MALDI-TOF) of CDPBr.

Figs. 27A-27B depict (Fig. 27A) ¹H NMR and (Fig. 27B) ¹³C NMR spectrum of CDPP in CDCl ₃.

Fig. 28 depicts high resolution mass spectrum (MALDI-TOF) of CDPP.

Figs. 29A-29B depict (Fig. 29A) ¹H NMR and (Fig. 29B) ¹³C NMR spectrum of CDPP-3SO ₃ in d ₆-DMSO.

Fig. 30 depicts high resolution mass spectrum (MALDI-TOF) of CDPP-3SO ₃.

Figs. 31A-31B depict (Fig. 31A) ¹H NMR and (Fig. 31B) ¹³C NMR spectrum of CDPP-4SO ₃ in d ₆-DMSO.

Fig. 32 depicts high resolution mass spectrum (MALDI-TOF) of CDPP-4SO ₃.

Figs. 33A-33B depict (Fig. 33A) ¹H NMR and (Fig. 33B) ¹³C NMR spectrum of CDPP-Bz in CDCl ₃.

Fig. 34 depicts high resolution mass spectrum (MALDI-TOF) of CDPP-Bz.

Figs. 35A-35B depict (Fig. 35A) ¹H NMR and (Fig. 35B) ¹³C NMR spectrum of CDPP-BzBr in CDCl ₃.

Fig. 36 depicts high resolution mass spectrum (MALDI-TOF) of CDPP-BzBr.

Figs. 37A-37B depict (Fig. 37A) ¹H NMR and (Fig. 37B) ¹³C NMR spectrum of CDPP-MeI in CDCl ₃.

Fig. 38 depicts high resolution mass spectrum (MALDI-TOF) of CDPP-MeI.

Figs. 39A-39B depict (Fig. 39A) ¹H NMR and (Fig. 39B) ¹³C NMR spectrum of CDPP-F ₂Ph in CDCl ₃.

Fig. 40 depicts high resolution mass spectrum (MALDI-TOF) of CDPP-F ₂Ph.

Figs. 41A-41C depict (Fig. 41A) crystal structure of CDPP-3SO ₃ and (Fig. 41B) packing showing CH···π and π···π interactions, and (Fig. 41C) electrostatic interactions.

Fig. 42A-42B depict (Fig. 42A) Normalized absorption and (Fig. 42B) emission spectra of CDPP-3SO ₃, CDPP-4SO ₃ and CDPP-BzBr in DMSO; concentration = 10 μM; λ _ex = 480 nm.

Figs. 43A-43G depict (Fig. 43A) molecular structure of CDPP derivatives and photo of their powders under 365 nm UV excitation; (Fig. 43B) PL spectra of CDPP-3SO ₃, CDPP-4SO ₃ and CDPP-BzBr. (Fig. 43C) PL spectra of CDPP-3SO ₃ in DMSO/water mixtures with different water fractions (f _w) ; concentration = 10 μM; (Fig. 43D) plots of PL maximum and relative PL intensity (α _AIE = I/I ₀) versus the composition of the DMSO/water mixture of CDPP-3SO ₃, CDPP-4SO ₃ and CDPP-BzBr, where I0 was the PL intensity at f _w = 0%; concentration = 10 μM; λ _ex = 470 nm; the fluorescence photograph at f _w = 0%and 90%and the corresponding SEM image of the nanoaggregates collected at f _w = 90%of (Fig. 43E) CDPP-3SO ₃, (Fig. 43F) CDPP-4SO ₃ and (Fig. 43G) CDPP-BzBr; concentration = 10 μM; scale bar: 1 μm (the photographs were taken under 365 nm UV irradiation from a hand-held UV lamp [CDPP= (Z) -4- (4- (1-cyano-2- (4- (diphenylamino) phenyl) vinyl) phenyl) pyridin-1-ium] ) .

Fig. 44 depicts two-photon absorption cross-section of CDPP-3SO ₃, CDPP-4SO ₃ and CDPP-BzBr; condition: DMSO/water (1: 9) , concentration: 100 μM.

Fig. 45 depicts confocal microscopy imaging of HeLa cells labeled with CDPP-3SO ₃ (1 μM) and its colocalization with (top) ER-Tracker Red (1 μM) (Pearson’s coefficient (R) = 0.85) , and (bottom) MitoTracker Deep Red (250 nM) (R = 0.55) ; scale bar = 5 μm.

Fig. 46 depicts confocal microscopy imaging of HeLa cells labeled with CDPP-4SO ₃ (1 μM) and its colocalization with (top) ER-Tracker Red (1 μM) (R = 0.86) , and (bottom) MitoTracker Deep Red (250 nM) (R = 0.57) ; scale bar = 10 μm.

Fig. 47 depicts confocal microscopy imaging of 143B cells labeled with CDPP-3SO ₃ (1 μM) and its colocalization with ER-Tracker Deep Red (250 nM) (R = 0.86) ; scale bar = 5 μm.

Fig. 48 depicts confocal microscopy imaging of HeLa cells labeled with CDPP-BzBr (10 μM) and its colocalization with MitoTracker Deep Red (250 nM) , Pearson’s coefficient (R) = 0.89; scale bar = 10 μm.

Figs. 49A-49B depict the plot of the fluorescence signal loss of HeLa cells co-stained with (Fig. 49A) CDPP-3SO ₃ (1μM) , CDPP-4SO ₃ (1μM) , and ER Tracker Red (250 nm) , and (Fig. 49B) CDPP-BzBr (1μM) and Mito Tracker Red (250 nM) against increasing laser irradiation scans by using confocal laser scanning microscopy (CLSM) .

Figs. 50A-50B depict confocal images of HeLa cells stained with CDPP-3SO ₃ with (Fig. 50A) λ _ex = 480 nm for 1PM and (Fig. 50B) λ _ex = 820 nm for 2PM; scale bar = 25 μm.

Figs. 51A-51C depict confocal images of HeLa cells stained with CDPP-4SO ₃ with (Fig. 51A) λ _ex = 480 nm for 1PM and (Fig. 51B) λ _ex = 820 nm for 2PM and (Fig. 51C) its bright field; scale bar = 25 μm.

Figs. 52A-52C depict confocal images of HeLa cells stained with CDPP-BzBr with (Fig. 52A) λ _ex = 480 nm for 1PM ; (Fig. 52B) λ _ex = 820 nm for 2PM; and (Fig. 52C) its bright field; scale bar =50 μm.

Figs. 53A-53B depicts cytotoxicity of AIEgens on (Fig. 53A) COS-7 cells incubated with different concentrations of AIEgens and (Fig. 53B) Hela cells incubated with different concentrations of AIEgens) .

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

The present subject matter contemplates small molecule, fluorescent compounds having aggregation-induced emission (AIE) characteristics. One or more of the fluorescent compounds exhibit unique properties such as red emission in the solid state, large Stokes shift, and large two-photon absorption cross section. One or more of the fluorescent compounds can selectively stain cellular organelles selected from mitochondria, endoplasmic reticulum, and lysosomes of live cells. The cells can be in living tissue, such as living tumor tissue. The present compounds can exhibit high biocompatibility and high photostability under one-photon and two-photon irradiation.

In an embodiment, the fluorescent compounds can include one or more compounds selected from:

each Y ₂-is independently selected from the group consisting of C _nH _2nSO ₃- and C _nH _2nCO ₂ ^-;

each X is halogen;

each n is independently an integer ranging from 0 to 20; and

each m is independently an integer ranging from 0 to 9.

In an embodiment, the compound is selected from the group consisting of

The fluorescent compounds can include donor--π-acceptor (D-π-A) structures. One or more of the present compounds can include AIE-active acrylonitriles. The acrylonitriles can be synthesized by a transition metal-free, non-hazardous, non-toxic and atom-economic synthetic method. Acrylonitriles with different functionalities can be produced by simply varying the reaction temperature, as illustrated below:

wherein the Ar ₁ and Ar ₂ are selected from substituted aromatic ring and unsubstituted aromatic ring. The aromatic ring can include, for example, benzene, thiophene, furan, pyran and thiadiazole. The aromatic ring substituents can be selected from the group consisting of hydrogen, alkyl, hydroxyl groups, halogen groups, carboxy groups, cyano groups, and substituted and unsubstituted aromatic and heterocyclic groups; In an embodiment, the aromatic ring subsituents can be selected from trifluoromethyl and N, N-diphenylaniline.

The AIE-active acrylonitriles can exhibit bright solid-state red emission with high fluorescence quantum yield of up to about 37.6%. They can also display a large two-photon absorption cross section of up to about 508 GM because of their D-π-A structure and high π-conjugation.

In an embodiment, the fluorescent compounds can include one or more AIE-active acrylonitriles selected from:

and

Nanoparticles (NPs) of TPAT-AN-XF and 2TPAT-AN can be prepared by nanoprecipitation. The NPs are biocompatibile and can serve as a contrast agent for two-photon imaging. The NPs can provide specific organelle staining in lysosomes in live cells, e.g., live HeLa cells, and two-photon deep-tissue imaging with high resolution in tumor tissues. Additionally, the NPs can realize in vivo long-term imaging of tumors with high signal-to-noise ratio. Thus, these compounds show great potential for two-photon deep-tissue bioimaging and long-term dynamic tracking of tumor metastasis. It should be understood that other acrylonitrile-based fluorescent materials with diverse functions and desired properties can be prepared based on the present teachings. The compounds can be useful for biomedical imaging and other applications, such as luminescent devices and organic field-effect transistors.

One or more of the compounds can include a propeller shaped triphenylamine (TPA) segment, as a D unit, an AIE active core, such as α-cyanostilbene, as a π-bridge, and an electron-accepting unit, such as pyridinium, as an A-unit. The AIE active core can include (Z) -4- (4- (1-cyano-2- (4- (diphenylamino) phenyl) vinyl) phenyl) pyridin-1-ium (CDDP) . The UV-vis absorption for the compounds including the CDDP core can be in the visible region (approximately 470 nm) with emission in the range of about 620 nm to about 690 nm. In an embodiment, the fluorescent compounds including the CDDP core can penetrate cells and target organelles based on their functional groups. For example, the compounds with the CDDP core having a sulfonated functional group and zwitterionic property can be used for endoplasmic reticulum imaging. Exemplary fluorescent compounds including CDDP core molecules include a compound selected from the group consisting of:

Bio-imaging Applications

The present fluorescent compounds can be used for in vitro and ex vivo cellular imaging. In an embodiment, a method of cellular imaging can include contacting a target cell with one or more of the present compounds and identifying a target of interest in the target cell using an imaging method. The imaging method can include one-photon fluorescence microscopy or two-photon fluorescence microscopy. In an embodiment, identifying a target of interest can include visualizing organelles in live cells. In an embodiment, the cellular organelle is mitochondria. In an embodiment, the cellular organelle is a lysosome. In an embodiment, the cellular organelle is endoplasmic reticulum. In an embodiment, the target cell is a tumor cell. In an embodiment, the target cell is a live cell. In an embodiment, the target cell is in live tissue.

In an embodiment, the fluorescent compound has a backbone structural formula selected from the group consisting of:

each Y ₂ ^- is independently selected from the group consisting of C _nH _2nSO ₃ ^- and C _nH _2nCO ₂ ^-;

each X is halogen;

each n is independently an integer ranging from 0 to 20; and

each m is independently an integer ranging from 0 to 9.

The imaging method can include one-photon fluorescence microscopy (confocal laser scanning microscopy) or two-photon fluorescence microscopy. One-photon fluorescence microscopy uses a single photon to excite fluorescent dyes using mainly visible excitation wavelengths (390-700 nm) . Two-photon fluorescence imaging technology has been widely used for bio-imaging applications due to its high penetration depth with near-infrared (NIR) excitation, high spatial resolution and signal-to-noise ratio, and low tendency for photobleaching. Two-photon absorption (2PA) cross section (δ _2PA) is used to predict whether a luminogen is suitable for 2PM.

In an embodiment, the present compounds can stain cellular organelles in live tissue with deep tissue penetration. Deep tissue penetration can include a depth ranging from about 50 μm to about 100 μm in live tissue. For example, 2TPAT-AN was successfully used for staining in live tumor-tissue (e.g., at a depth of about 60 μm) under two-photon excited imaging mode.

The present teachings are illustrated by the following examples.

EXAMPLE 1

Synthesis (2TPAT-AN and TPAT-AN-XF)

An exemplary synthesis route for preparing 2TPAT-AN and TPAT-AN-XF is provided below

As shown below, a Suzuki coupling reaction of

compounds

3 and 4 in the presence of catalyst Pd (PPh ₃) ₄ resulted in compound 1.

The reaction of

compounds

1 and 2 in refluxed anhydrous EtOH with t-BuOK produced 2TPAT-AN, which was successfully confirmed by the single crystal structure analysis. TPAT-AN-XF was prepared by the reaction of

compounds

1 and 2 in the presence of t-BuOK in anhydrous EtOH at room temperature. It is believed that this is the first time different functionalized acrylonitriles have been prepared by simply tuning the reaction temperature. The structures of the intermediate compound 1 and the final products (2TPAT-AN and TPAT-AN-XF) were well characterized by ¹H NMR, ¹³C NMR, ¹⁹F NMR and HRMS spectroscopy (Figs. 1-10) .

EXAMPLE 2

Photophysical Properties (2TPAT-AN and TPAT-AN-XF)

The photophysical properties of 2TPAT-AN and TPAT-AN-XF were investigated. The absorption and fluorescence (FL) spectra are shown in Figs. 11A-15B and the corresponding data are summarized in Table 1.2TPAT-AN showed a red-shifted absorption maximum (λ _abs) at 482 nm, which was more than that of TPAT-AN-XF (λ _abs of 439 nm) in dilute THF solution (Fig. 11A) . 2TPAT-AN exhibited an emission maximum (λ _em) at 572 nm in dilute THF solution, and the emission showed a slight red shift after addition of water to the THF solution, while the FL intensity decreased initially and then increased with the water fraction (f _w) exceeding 50% (Figs. 11B and 11D) . 2TPAT-AN showed a strong FL intensity (λ _abs of 610 nm) at f _w = 70%due to the formation of aggregates, demonstrating the aggregation-enhanced emission (AEE) property. Further increasing f _w to 90%and 99%cause a slight drop of the FL intensity. This phenomenon is generally observed for AIEgens and is probably due to the change of the morphology and size of the aggregates formed at high water fractions in aqueous mixtures.

It should be pointed out that a shoulder peak of about 650 nm appeared at f _w = 90%and 99%, indicating different aggregation modes do exist in such aqueous mixtures. Similarly, TPAT-AN-XF also shows AEE property as proved by the fluorescence analysis (Figs. 11C and 11D) . In comparison with the emission in THF, the FL intensity of 2TPAT-AN in aqueous suspensions is only slightly increased in comparison with the several-fold enhancement of TPAT-AN-XF (Fig. 11D) , which is possibly ascribed to the loose aggregates of 2TPAT-AN and dense aggregates of TPAT-AN-XF in aqueous media. The dynamic light scattering data present the hydrated diameters of 399 nm and 210 nm in aqueous suspensions for 2TPAT-AN and TPAT-AN-XF, respectively, supporting the existence of aggregates (Fig. 12) . 2TPAT-AN and TPAT-AN-XF exhibited low emission efficiencies in THF but remarkably high fluorescence quantum yields of 34.3%and 37.6%, respectively, in the solid state (Table 1) , due to the active intramolecular motion induced energy loss in THF and restriction of intramolecular motion (RIM) in the solid state.

Table 1. Photophysical properties of 2TPAT-AN and TPAT-AN-XF ^a.

^aAbbreviation: λ _abs = absorption maximum; λ _em = emission maximum; Φ _F, Sand Φ _F, P =fluorescence quantum yield in solution and solid powder, respectively; α _AIE = Φ _F, S/Φ _F, P.

Remarkably, 2TPAT-AN showed a higher degree of red shifted fluorescence of 635 nm than that of TPAT-AN-XF (591 nm, Fig. 11E) , probably due to the existence of much stronger interaction in the pristine sample (Fig. 11F) . The intermolecular interaction and packing of 2TPAT-AN in crystal state were further investigated (Figs. 13 and 11G) . 2TPAT-AN exhibits a strong intramolecular π-π interaction and C–H··π interaction to restrain the molecular motion (Fig. 12) , resulting in low non-radiative energy loss and high fluorescence quantum yield. The isolated dimer was formed between two adjacent molecules because of the existence of π-πinteraction (Fig. 11G) , contributing to the red-shifted emission of 2TPAT-AN in aggregates (the shoulder peak of ～650 nm, Fig. 11B) and the solid state (630 nm, Figure 11F) . It should be noted that the π-π interaction existing in the crystal of 2TPAT-AN is particularly stable even upon strong grinding treatment (Fig. 14) . In addition, these two AIEgens exhibit typical positive solvatochromism due to the typical D-π-A structures and intramolecular charge transfer effect, as confirmed by the gradually red-shifted emission in organic solvents with different polarities (Figs. 15A-15B) .

Previous studies have demonstrated that donor-π-acceptor (D-π-A) based fluorescent materials show strong two-photon absorption. Given their good D-π-Aconjugated structures, the two-photon absorption properties of 2TPAT-AN and TPAT-AN-XF in THF were investigated via two-photon excited fluorescence by using a femtosecond pulsed laser as excitation source. These two AIEgens show strong two-photon fluorescence signals excited at 800-980 nm. The two-photon absorption cross section was also determined by using Rhodamine B in MeOH as the standard. As shown in Fig. 11H, they exhibit remarkably high two-photon absorption cross sections especially at 880 nm (508 GM and 366 GM for 2TPAT-AN and TPAT-AN-XF, respectively) . Interestingly, 2TPAT-AN shows larger two-photon absorption cross sections especially from 800-880 nm than those of TPAT-AN-XF, probably because the D-π-A-π-D conjugated 2TPAT-AN has better conjugation than the D-π-Abased TPAT-AN-XF. These excellent two-photon absorption properties with large two-photon absorption cross sections have tremendous potential in biomedical imaging.

The photophysical properties of 2TPAT-AN and TPAT-AN-XF were further studied by using density functional theory (DFT) calculation performed at the B3LYP/6-31G level of theory via the Gaussian 09 program package. The spatial orbital distributions of HOMOs and LUMOs in the optimized ground states and excited states are depicted in Figs. 16A-16B. The orbitals of HOMOs of 2TPAT-AN are basically delocalized on the whole molecules, while those of LUMOs are mainly distributed at the part of thiophene substituted acrylonitrile. The orbitals of HOMOs of TPAT-AN-XF are generally delocalized on the conjugated parts of triphenylamine and thiophene in the ground state, while those of HOMOs are mainly distributed at the triphenylamine part in excited state.

As for the LUMOs of TPAT-AN-XF, they are mainly located on parts of the molecule other than the triphenylamine moiety. The spatial orbital distributions of HOMOs and LUMOs reveal that these two AIEgens, especially TPAT-AN-XF show obvious orbital separation due to the strong intramolecular charge transfer (ICT) effect. In ground states, 2TPAT-AN displays higher HOMO levels than TPAT-AN-XF due to its excellent π-conjugation, while TPAT-AN-XF shows slightly lower LUMO levels than 2TPAT-AN, owing to its strong ICT effect. Such spatial orbital distributions of HOMOs and LUMOs in the optimized ground states lead to narrower energy band gap of 2TPAT-AN than that of TPAT-AN-XF, resulting in red-shifted absorption of 2TPAT-AN. In excited states, however, the LUMO levels of TPAT-AN-XF are greatly decreased in comparison to those of 2TPAT-AN due to strong ICT effect as well as highly twisted structure (Figs. 17A-17B) , resulting in narrow energy gap in excited states and red-shifted emission of TPAT-AN-XF in solution. These DFT data further demonstrate that 2TPAT-AN exhibits red-shifted absorption but blue-shifted emission in solution compared with TPAT-AN-XF, which is in good accordance with the photophysical data measured in THF (Table 1) .

Example 3

Biomedical Imaging (2TPAT-AN and TPAT-AN-XF)

Encouraged by the excellent photophysical properties of 2TPAT-AN, biological imaging was then investigated. Water-soluble 2TPAT-AN nanoparticles (NPs) were prepared via a typical nanoprecipitation method using amphiphilic block copolymer PEG-PLGA (Mw: 1000-1000) as the encapsulation materials (Fig. 18A) . Transmission electron microscopy (TEM) data in Fig. 18B shows that the particle diameters of 2TPAT-AN NPs are distributed in the range of 30-65 nm. The 2TPAT-AN NPs have a hydrated diameter of about 102 nm verified by dynamic light scattering (DLS) data (Fig. 18C) . The transparent solution of 2TPAT-AN NPs shows a comparable absorption maximum at about 479 nm and an emission maximum at 608 nm with a shoulder peak of about 650 nm (Fig. 18D) , and such fluorescence spectrum is very similar to that of 2TPAT-AN in aggregates in THF with f _w = 90%or 99% (Fig. 11B) , presumably resulting from the formation of different packing modes. In addition, 2TPAT-AN NPs exhibit good colloid stability as the DLS size and absorption were almost unchanged after seven days (Figs. 19A-19B) .

Before bioimaging was performed in live cells, the cytotoxicity of 2TPAT-AN NPs in HeLa cells by standard MTT assay was investigated. After incubation of 2TPAT-AN NPs for 24 h, the cell viabilities were still very high (over 85%) even at a concentration of 80 μg/mL (Fig. 20) , confirming the low cytotoxicity of 2TPAT-AN NPs towards live cells. Then, 2TPAT-AN NPs were applied for in vitro live cell imaging by using confocal laser scanning microscopy. After incubation for 30 min, strong red emission was observed in the cytoplasm of HeLa cells, and z-stack imaging data further confirmed the localization of 2TPAT-AN NPs in the cytoplasm rather than being bound or adsorbed on the cell surface (Fig. 18E) . Generally, fluorescent organic NPs tend to locate in lysosomes due to endocytosis. Co-staining imaging studies were carried out using commercial lysosome dye LysoTraker Green DND-26 to verify the specific localization of 2TPAT-AN NPs in live HeLa cells. As shown in Fig. 18F, 2TPAT-AN NPs and LysoTraker Green DND-26 display a very similar staining pattern with the Pearson's coefficient of 0.89. This data confirmed that 2TPAT-AN NPs exhibit good cell permeability and selectively locate in lysosomes in live cells.

Compared with one-photon imaging, two-photon imaging excited by near-infrared pulsed laser displays much better performance in terms of lower photodamage, higher signal-to-noise ratio and deeper tissue penetration. Some recent works also successfully demonstrated the deep-tissue penetration advantage of two-photon bioimaging. To further confirm such merit of two-photon microscopy, ex vivo two-photon imaging was carried out in live tumor tissues. Given the large two-photon absorption cross section at 880 nm as well as strong two-photon excited fluorescence, two-photon imaging was performed using a NIR pulsed laser at 880 nm. Compared with one-photon imaging, two-photon excited fluorescence with much better resolution and higher signal-to-noise ratio could be distinctly seen in live tumor tissues (Fig. 21) . Such two-photon fluorescence signals show very similar distribution with that excited by one-photon laser, demonstrating that 2TPAT-AN NPs show high potential in two-photon imaging in live tissues. Then, the fluorescent images were scanned at different depths and the fluorescent images were captured every 2 μm along the z-axis (Figs. 22A-22D) . One-photon fluorescent signals excited by 488 nm laser could only be obtained at a depth of less than 40 μm (Fig. 22A) , which was distinctly revealed by the reconstructed 3D one-photon fluorescent microscopic image (Fig. 22B) . In sharp contrast, the two-photon fluorescence signals with high signal-to-noise ratio could be clearly detected at a depth of up to 60 μm and 3D two-photon fluorescent microscopic image was successfully reconstructed (Figs. 22C and 22D) . This excellent ex vivo imaging data revealed that 2TPAT-AN NPs hold great potential in in vivo two-photon deep-tissue imaging for cancer diagnosis.

Considering the wide emission spectrum including the deep-red to near-infrared region, it was anticipated that 2TPAT-AN NPs could exhibit good imaging performance in live animals. To demonstrate this, in vivo imaging was carried out by intratumor injection of 2TPAT-AN NPs in 4T1 tumor-bearing nude mice and the normalized mean fluorescence intensity from the tumor was also recorded at different time points (Figs 23A-23B and 24) . After 30 min post-injection, strong in vivo fluorescence of 2TPAT-AN NPs from the 4T1 tumor could be dramatically collected with remarkably high signal-to-noise ratio. However, only some background auto-fluorescence signal throughout the whole mouse was obtained in the control group without injection of 2TPAT-AN NPs.

It should be noted that some of the 2TPAT-AN NPs were already metabolized by the mouse, which was verified by the slightly higher fluorescence around the tumor than that from the background auto-fluorescence. The fluorescence signal from the tumor region was gradually decreased with increased time after intra-tumor injection and reached a plateau at about 12 h post-injection, which was obviously confirmed by the normalized mean fluorescence intensity data (Fig. 24) . Interestingly, tumor fluorescence only showed a slight decrease even after 72 h post-injection (Fig. 23A) , indicating the tremendous potential application of 2TPAT-AN NPs for long-term tumor tracking. Furthermore, the cytotoxicity of 2TPAT-AN NPs to live mice was evaluated by hematoxylin and eosin (H&E) staining for histological analysis at 72 h post-injection. In the experimental group treated with 2TPAT-AN NPs, no noticeable abnormality was found in the major organs (heart, liver, spleen, lung, and kidney) of mice (Fig. 23B) , demonstrating the high biocompatibility of 2TPAT-AN NPs at the tested conditions. These data indicated that biocompatible 2TPAT-AN NPs exhibit promising potential in in vivo bioimaging.

Example 4

Synthesis (CDPP-3SO ₃, CDPP-4SO ₃, CDPP-Bz, CDPP-BzBr, CDPP-MeI and CDPP- F ₂Ph)

Electron-donating groups, such as the propeller shaped triphenylamine (TPA) segment were chosen as the donor (D) unit, AIE active cores, such as (Z) -4- (4- (1-cyano-2- (4- (diphenylamino) phenyl) vinyl) phenyl) pyridin-1-ium (CDPP) were chosen as the π-bridge, and electron accepting units such as pyridinium were accepted as acceptor (A) units. The D-π-A compounds were synthesized (as shown below) by a two-step reaction: i) a Knoevenagel condensation between 4- (diphenylamino) benzaldehyde and 2- (4-bromophenyl) acetonitrile followed by ii) a Suzuki coupling with 4-pyridine boronic acid, yielding yellow powder with a total yield of 73%:

Finally, the targeted fluorophores CDPP-3SO ₃, CDPP-4SO ₃, CDPP-Bz, CDPP-BzBr, CDPP-MeI and CDPP-F ₂Ph were synthesized by a simple reaction between CDPP and 1, 3-propane sultone, 1, 4-butane sultone, benzyl bromide, 4 bromobenzyl bromide, methyl iodide, and 1- (bromomethyl) -3, 5-difluorobenzene, respectively. The chemical structures of all the synthesized compounds were characterized by standard spectroscopic techniques such as ¹H NMR, ¹³C NMR, and HRMS (Figs. 25A-40) .

Example 5

Photophysical Properties (CDPP-3SO ₃, CDPP-4SO ₃, CDPP-Bz, CDPP-BzBr, CDPP-MeI and CDPP-F ₂Ph)

One of the synthesized compounds, CDPP-3SO ₃ was successfully characterized by single crystal X-ray diffraction (SXRD) (Figs. 41A-41C) . The propeller conformation of TPA showed dihedral angles of 54.1°, 68.1°, and 74.2° among the three phenyl rings. The crystal packing showed various CH···π, π···π, and CH···O interactions, which were measured to be2.72

3.98

and 2.58

respectively. The centroid to centroid distance between two neighboring molecules were measured to be 3.74

It is speculated that the propeller conformation allows for molecular motions which lead to non-irradiative decay pathways in the solution state. However, the short interactions suppress molecular motions inhibiting non-radiative decay and open new radiative channels in aggregate/solid state.

The compounds in general exhibited poor solubility in many polar solvents and were non-soluble in non-polar solvents. Optical properties, and the absorption and emission spectra, of CDPP-3SO ₃, CDPP-4SO ₃, and CDPP-BzBr were investigated in DMSO at room temperature (Figs 42A-42B) . The absorption spectra of CDPP-3SO3, CDPP-4SO3, and CDPP-BzBr showed similar absorption maxima at ～480 nm (Fig. 42A) . The emission spectra of CDPP-3SO ₃, CDPP-4SO ₃, and CDPP-BzBr were found in the range of 585 nm to 620 nm in dimethyl sulfoxide (DMSO) (Fig. 42B) . The solid-state fluorescence efficiency of CDPP-3SO ₃, CDPP-4SO ₃, and CDPP-BzBr was found to be 39.3%, 5.3%and 8.3%, respectively (Figs. 43A-43G) . The solid-state emission of CDPP-3SO and CDPP-4SO ₃ showed similar emission maxima at ～620 nm; however, CDPP-BzBr showed red shifted emission at 665 nm compared to CDPP-3SO and CDPP-4SO ₃ (Fig. 43B) .

Furthermore, the emission behavior of CDPP-3SO ₃, CDPP-4SO ₃, and CDPP-BzBr were studied in DMSO/water solvent mixtures to evaluate their aggregation properties. For CDPP-3SO ₃, increasing the water fraction (f _w) in DMSO/water mixtures from 0%to 60%resulted in no visible changes in the PL intensity (Figs. 43C-43D) . When f _w ≥ 70%, the emission intensity became enhanced alongside a bathochromic shift (from 620 nm to 640 nm) . This signaled the formation of nanoaggregates. The maximum PL intensity was observed at f _w = 90%and α _AIE or I/I ₀ was found to be ～14. Similar AIE behavior was observed for CDPP-4SO ₃ and CDPP-BzBr in the presence of different DMSO/water mixtures and their α _AIE was found to be ～17 and 30, respectively (Fig. 43D) . Additionally, scanning electron microscopy (SEM) was performed to validate the aggregate formation when f _w = 90% (Figs. 43E-43G) . SEM revealed that the CDPP-3SO ₃ and CDPP-4BzBr formed spherical shaped nanoaggregates. CDPP-4SO ₃, however, formed wire-shaped nanoaggregates. Fluorescence decay measurements of CDPP-3SO ₃, CDPP-4SO ₃, and CDPP-BzBr nanoaggregates at f _w = 90%revealed that their lifetimes were 2.1 ns, 2.0 ns, and 2.0 ns, respectively.

The synthesized CDPP derivatives were anticipated to have strong two photon absorption (2PA) because of their strong electron donating and withdrawing groups. The 2PA measurements of these compounds were carried out using two-photon excited fluorescence (TPEF) technique with a femtosecond pulsed laser source, and comparative TPEF intensity at f _w = 90%were measured using Rhodamine 6G as the standard. TPEF intensities were scanned between 820 to 1000 nm at an interval of 30 nm and respective δ _2PA values were calculated. The highest δ _2PA value of 163 GM was calculated for CDPP-4SO ₃ at 820 nm. The δ _2PA values of the two other molecules, CDPP-3SO ₃ and CDPP-BzBr, were also calculated and found to be 122 GM and 71 GM at 820 nm and 970 nm, respectively (Fig. 44) . The obtained 2PA values are much higher than most fluorescence proteins such as EGFP (39 GM) . Hence, the CDPP derivatives may be utilized as good two-photon imaging probes.

Example 6

Endoplasmic Reticulum Staining (CDPP-3SO ₃, CDPP-4SO ₃, CDPP-Bz, CDPP-BzBr, CDPP-MeI and CDPP-F ₂Ph)

The specific ER targeting ability of CDPP-3SO ₃ and CDPP-4SO ₃ was demonstrated by co-staining HeLa cells with ER-Tracker Red, one of the frequently adopted probes for ER-staining. Both CDPP-3SO ₃ and CDPP-4SO ₃ efficiently stained the cell within 1 h at 1 μM. The fluorescent area matched very well with those of ER-Tracker Red with a high Pearson’s correlation factor at 0.85 for CDPP-3SO ₃, and 0.86 for CDPP-4SO ₃, indicating that these AIEgens can selectively target ER (Figs. 45-46) . In addition, MitoTracker Deep Red was employed to investigate the subcellular localization pattern of CDPP-3SO ₃ and CDPP-4SO ₃, with the resultant images clearly indicating that their staining region only partially overlapped with MitoTracker Deep Red. The Pearson’s correlation values between MitoTracker Deep Red and CDPP-3SO ₃ was 0.55, and 0.57 for CDPP-4SO ₃, suggesting that these AIEgens did not stain mitochondria in HeLa cells. Moreover, the ER staining property of both AIEgens were successfully demonstrated when employed to stain 143B cells (Fig. 47) .

Example 7

Mitochondria Staining (CDPP-3SO ₃, CDPP-4SO ₃, CDPP-Bz, CDPP-BzBr, CDPP-MeI and CDPP-F ₂Ph)

CDPP-BzBr containing only one positive charge was successfully designed to target mitochondria instead of ER (Fig. 48) . The staining ability of CDPP-BzBr was investigated by co-staining with MitoTracker Deep Red. It showed excellent overlap co-localized pattern with MitoTracker Deep Red in Hela cells with a high Pearson’s correlation factor of 0.89. This further demonstrated that the zwitterionic property of the functional group was critical for ER imaging.

Example 8

Photostability (CDPP-3SO ₃, CDPP-4SO ₃, CDPP-Bz, CDPP-BzBr, CDPP-MeI and CDPP-F ₂Ph)

The photostability of CDPP-3SO3, CDPP-4SO3, and ER-Tracker Red were assessed in parallel, with continuous excitation and sequential scanning with a confocal microscope. The result showed that the emission intensity of CDPP-3SO ₃ and CDPP-4SO ₃ only slightly decreased within 80 irradiation scans. In contrast, the fluorescence loss of ER-Tracker Red was very obvious upon irradiation under the same conditions, demonstrating the superior photostability of CDPP-3SO ₃ and CDPP-4SO ₃ to that of ER-Tracker Red (Figs. 49A-49B) . Moreover, two-photon cell imaging was conducted. Sufficient signals were obtained for CDPP-3SO ₃, CDPP-4SO ₃, and CDPP-BzBr under both one-photon and two-photon excitation (Figs. 50A-52C) , indicating both AIEgens was suitable for two-photon imaging as well as one-photon imaging.

Example 9

Cytotoxicity (CDPP-3SO ₃, CDPP-4SO ₃, CDPP-Bz, CDPP-BzBr, CDPP-MeI and CDPP- F ₂Ph)

The cytotoxicity of CDPP series AIEgens were evaluated by the method of 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) assay. As shown in Figs 53A-53C, CDPP series AIEgens only showed significant toxicity to both COS-7 and HeLa cells when staining concentration was at 40 μM. At the working concentration for cell imaging of 1 μM, the dyes exhibited minimal toxicity. Therefore, CDPP series AIEgens are suitable for long-term tracking 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

A fluorescent compound exhibiting aggregation induced emission properties, the compound having a backbone structural formula selected from the group consisting of:

wherein each Ar ₁ and Ar ₂ is independently a substituted or unsubstituted aromatic ring, the aromatic ring being selected from the group consisting of phenyl, benzene, thiophene, furan, pyran and thiadiazole, and the aromatic ring substituents being selected from the group consisting of hydrogen, alkyl, hydroxyl groups, halogen groups, carboxy groups, cyano groups, and substituted and unsubstituted aromatic and heterocyclic groups;

each R ₁ R ₂, R ₃, and R ₄ is independently selected from the group consisting of C _nH _2n+1, C ₆H ₅, C ₁₀H ₇, C ₁₂H ₉, OC ₆H ₅, C ₆H ₅OH, C ₆H ₅OC _nH _2n+1, OC ₁₀H ₇, OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, CN, and H;

each Z is independently selected from the group consisting of F, Cl, Br, I, PF ₆, BPh ₄, N (CN) ₂, and BF ₄;

each Y ₁ is independently selected from the group consisting of C _nH _2nC ₆H _5-mX _m, C _nH _2nC ₁₀H _7-mX _m, C _nH _2nC ₁₂H _9-mX _m, C _nH _2n, C _nH _2nCO ₂H, and C _nH _2nSO ₃H;

each Y ₂ ^- is independently selected from the group consisting of C _nH _2nSO ₃ ^- and C _nH _2nCO ₂ ^-;

each X is halogen;

each n is independently an integer ranging from 0 to 20; and

each m is independently an integer ranging from 0 to 9.
The compound according to claim 1, wherein the compound comprises one or more compounds selected from the group consisting of:
A method of cellular imaging, comprising:

contacting a target cell with the compound of claim 1; and

identifying a target of interest in the target cell using an imaging method.
The method of claim 3, wherein the compound has a backbone structural formula selected from the group consisting of:

wherein each R ₁ R ₂, R ₃, and R ₄ is independently selected from the group consisting of C _nH _2n+1, C ₆H ₅, C ₁₀H ₇, C ₁₂H ₉, OC ₆H ₅, C ₆H ₅OH, C ₆H ₅OC _nH _2n+1, OC ₁₀H ₇, OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, CN, and H;

each Z is independently selected from the group consisting of F, Cl, Br, I, PF ₆, BPh ₄, N (CN) ₂, and BF ₄;

each Y ₁ is independently selected from the group consisting of C _nH _2nC ₆H _5-mX _m, C _nH _2nC ₁₀H _7-mX _m, C _nH _2nC ₁₂H _9-mX _m, C _nH _2n, C _nH _2nCO ₂H, and C _nH _2nSO ₃H;

each Y ₂ ^- is independently selected from the group consisting of C _nH _2nSO ₃ ^- and C _nH _2nCO ₂ ^-;

each X is halogen;

each n is independently an integer ranging from 0 to 20; and

each m is independently an integer ranging from 0 to 9.
The method of claim 3, wherein the compound has a backbone structural formula selected from the group consisting of:

wherein each Ar ₁ and Ar ₂ is independently a substituted or unsubstituted aromatic ring, the aromatic ring being selected from the group consisting of phenyl, benzene, thiophene, furan, pyran and thiadiazole, and the aromatic ring substituents being selected from the group consisting of hydrogen, alkyl, hydroxyl groups, halogen groups, carboxy groups, cyano groups, and substituted and unsubstituted aromatic and heterocyclic groups.
The method of claim 3, wherein the target of interest is a cellular organelle selected from the group consisting of endoplasmic reticulum, mitochondria, and lysosome.
The method of claim 6, wherein the cellular organelle is endoplasmic reticulum which is stained by contact with the compound and the compound comprises at least one of
The method of claim 6, wherein the cellular organelle is mitochondria which is stained by contact with the compound and the compound comprises at least one of
The method of claim 6, wherein the cellular organelle is a lysosome stained by contact with the compound and the compound comprises at least one of:
The method of claim 6, wherein the target of interest is in a tumor cell.
The method of claim 6, wherein the target cell is a live cell.
The method of claim 6, wherein the target cell is in live tissue.
The method of claim 6, wherein the imaging method is selected from the group consisting of one-photon fluorescence microscopy and two-photon fluorescence microscopy.
The method of claim 13, wherein the imaging method is two-photon fluorescence microscopy and the target cell is in live tissue.
A fluorescent compound comprising a compound selected from the group consisting of:
A method of cellular imaging, comprising:

contacting a target cell with the compound of claim 15; and

identifying a target of interest in the target cell using an imaging method.
The method of claim 16, wherein the target of interest is a cellular organelle selected from the group consisting of endoplasmic reticulum, mitochondria, and lysosome.
The method of claim 16, wherein the cellular organelle is endoplasmic reticulum or mitochondria which is stained by contact with the compound and the compound comprises at least one of
The method of claim 16, wherein the cellular organelle is a lysosome and the compound comprises
The method of claim 16, wherein the compound exhibits bright solid-state red emission and large two-photon cross section.