WO2019057196A1

WO2019057196A1 - Fluorescent compounds for lipid droplet imaging and image-guided photodynamic therapy

Info

Publication number: WO2019057196A1
Application number: PCT/CN2018/107283
Authority: WO
Inventors: Benzhong Tang; Dong Wang
Original assignee: The Hong Kong University Of Science And Technology
Priority date: 2017-09-25
Filing date: 2018-09-25
Publication date: 2019-03-28

Abstract

Fluorescent compounds that have aggregation-induced emission (AIE) characteristics and exhibit near infrared absorption. The compounds can be synthesized using a one-pot synthetic approach. The compounds can be utilized as LD-specific bio-probes in cell imaging and in vivo zebrafish-imaging, with high photostability and brightness. In addition, the compounds can efficiently generate reactive oxygen species (ROS) in vivo when irradiated with white light. As such, the compounds can be effective in killing cancer cells through photodynamic therapy (PDT) processes.

Description

Fluorescent Compounds for Lipid Droplet Imaging and Image-Guided Photodynamic Therapy

CROSS-REFERENCE

The present application claims priority to provisional United States Patent Application No. 62/606,440, filed September 25, 2017, which was filed by the inventors hereof and is incorporated herein by reference in its entirety.

FIELD

The present subject matter relates generally to a series of fluorescent compounds with aggregation-induced emission characteristics and near infrared absorption and their applications in bioimaging and phototheranostics.

BACKGROUND

Fluorescence imaging has proven to be a highly sensitive and non-invasive technology that offers researchers a very useful tool for analytical sensing and optical imaging. By utilizing fluorescence imaging, direct visualization of bioanalytes can be achieved on site and in real-time to provide useful insight into complex biological structures and processes. In particular, fluorophores with intense emission in the red/near infrared (NIR) region are useful in biological applications, owing to their capacity to overcome the interference of optical absorption, reduce light scattering, avoid auto-fluorescence of physiological environments, and minimize photo-damage to biological structures.

Although many types of NIR-emissive fluorophores have been commercialized, they have been far from ideal. Generally, these fluorophores are associated with complicated synthetic schemes. In addition, conventional red/NIR-emissive fluorophores generally suffer from a common photophysical phenomenon called aggregation-caused quenching (ACQ) . Specifically, these conventional fluorophores typically emit strongly when in solution, but experience emission quenching upon aggregate formation due to intermolecular π-π stacking and other non-radiative pathways. For example, some commercial bioimaging dyes, including

Red, ER-Tracker ^TM Red,

Deep Red and Nile Red, are all brightly red-emissive in diluted dimethyl sulfoxide solutions, but the emissions are partially or totally quenched upon the formation of aggregates after adding water. In particular, Nile Red is almost non-emissive in the solid state. ACQ phenomenon leads to low photobleaching resistance, greatly impeding practical applications in the area of biomedical research since conventional fluorophores tend to form aggregates in physiological environments or aqueous media due to the high hydrophobicity of their emitting centers with planar conformations.

A novel class of fluorophores with twisted conformations exhibit extraordinary aggregation-induced emission (AIE) , which is completely opposite to ACQ dyes. These compounds with AIE characteristics are non-emissive or weakly emissive in solution, but are typically induced to fluoresce intensely once aggregated through restriction of intramolecular motions (RIM) . As a result, AIE fluorophores can be effective at high concentrations and in the aggregated state, emitting bright fluorescence and having a high photobleaching threshold. Therefore, AIE has opened a venue to an array of possibilities with great potential for high-tech innovations.

In view of the great significance of both AIE and red/NIR-emission, some red/NIR-emissive AIEgens have been constructed and utilized in various biological applications. In general, however, preparation of red/NIR fluorescent molecules is extremely complicated irrespective of whether the compounds are also AIEgens or not. Common synthetic strategies of red/NIR fluorophores include connection of strong electron-donating (D) and electron-accepting (A) units by π-bridge (s) , expansion of π-conjugation, or a combination of the two strategies. These synthesis methods usually require several step reactions and inconvenient purifications, which are extremely time-, cost-and energy-consuming, tedious, and harmful to the environment. Moreover, their application is often restricted due to the inferior solubility resulting from their bulky structures. Therefore, further fabrication or modifications with surfactants, proteins or other materials are required before biological use. For example, TTB (Adv. Funct. Mater. 2014, 24, 635) and TPE-TPA-DCM (Adv. Funct. Mater. 2012, 22, 771) are desirable in biological applications because of their AIE characteristics, bright red region emissions and high photostabilities. Synthesis of TTB and TPE-TPA-DCM, however, currently involves seven-and six-step reactions, with cumbersome and iterative purifications. In addition, efficient cellular uptake of TTB and TPE-TPA-DCM requires pre-fabrication with PEG-containing amphiphilic polymers or bovine serum albumin.

Lipid droplets (LDs) , which are mainly located in adipocytes, hepatocytes, the adrenal cortex and myocytes, have proven to be considerably important in various bio-functions, such as regulation of the storage and metabolism of neutral lipids, protein degradation, construction and maintenance of membranes, and signal transduction. The abnormality of LDs in cells is a critical biomarker for various diseases including cancer, obesity, fatty liver disease, hyperlipidemia, atherosclerosis, inflammation, virus infection, type II diabetes, and neurodegeneration in Alzheimer’s disease. Indeed, LDs can be an ideal target organelle in therapeutic applications due to their fluidity and relevance to various bio-functions.

Taking the intrinsic advantages of fluorescence imaging, the development of efficient fluorescent probes of LDs is highly desirable. However, currently available commercial fluorophores (such as BODIPY dyes, Nile Red and Oil Red O) for LDs imaging have their respective and collective drawbacks. BODIPY dyes typically require a relatively long incubation time, and their small Stokes shifts affect the collection of imaging signals. The specificity of Nile Red to LDs is unsatisfactory. For example, pre-fixation of cells is necessary when Oil Red O is employed. In addition, commercial fluorophores exhibiting ACQ property typically show low photobleaching resistance. While a handful of LD-specific targeting AIEgens have been developed and have proven to be powerful fluorescent probes of LDs, those AIEgens are only useful as LD-specific probes and do not provide therapeutic advantages. Development of AIEgens with dual functions of simultaneous LD-imaging and therapy would, therefore, be efficient and useful.

Accordingly, fluorescent, red/NIR AIEgens which can be facilely synthesized and used both as LD-specific bio-probes and in cancer phototheranostics are highly desirable.

SUMMARY

The present subject matter relates to fluorescent compounds that have aggregation-induced emission (AIE) characteristics and exhibit near infrared absorption. The compounds can be synthesized using a one-pot synthetic approach. The compounds can be utilized as Lipid Droplet (LD) -specific bio-probes in cell imaging and in vivo zebrafish-imaging, with high photostability and brightness. In addition, the compounds can efficiently generate reactive oxygen species (ROS) in vivo when irradiated with white light. As such, the compounds can be effective in killing cancer cells through photodynamic therapy (PDT) processes.

In an embodiment, the compounds have a backbone structural formula selected from the group consisting of:

wherein each R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃ alkyl-NH ₂, and alkoxy; and

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.

In a further embodiment, the compound includes one or more compounds selected from the group consisting of :

BRIEF DESCRIPTION OF DRAWINGS

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

Fig. 1A depicts the single crystal structure of TTMN. Fig. 1B depicts a side-view of the crystal structure of TTMN. Fig. 1C depicts various inter-and intramolecular interactions in crystals of TTMN.

Fig. 2A depicts normalized absorption spectra of TPMN, TTMN, MeTTMN and MeOTTMN in an ACN solution. Fig. 2B depicts PL spectra of TPMN (10 μM) in ACN/water mixtures with different water fractions (fw) ; λex: 441 nm. Fig. 2C depicts the plot of the emission maximum and the relative emission intensity (I/I0) versus the composition of the aqueous mixture of TPMN, TTMN, MeTTMN, and MeOTTMN. Inset: Fluorescence photographs of TPMN in the dilute ACN solution and in ACN/water mixtures with 95%water fractions taken under 365 nm UV irradiation. Fig. 2D shows normalized PL spectra of TPMN (λex: 441 nm) , TTMN (λex: 483 nm) , MeTTMN (λex: 492 nm) and MeOTTMN (λex: 499 nm) in the solid state. Inset: Fluorescence photographs of TPMN, TTMN, MeTTMN and MeOTTMN in the solid state taken under 365 nm UV irradiation.

Fig. 3 depicts HOMO and LUMO energy levels of these AIEgens. Molecular orbital amplitude plots of HOMO and LUMO energy levels of TPMN, TTMN, MeTTMN and MeOTTMN are shown. Eg (energy gap) = LUMO -HOMO.

Fig. 4A depicts particle size distributions of TPMN aggregates. Fig. 4B depicts particle size distributions of TTMN aggregates. Fig. 4C depicts particle size distributions of MeTTMN aggregates. Fig. 4D depicts particle size distributions of MeOTTMN aggregates. All values were measured in ACN/water mixtures with a 95%water fraction, and at a concentration of 10 μM.

Fig. 5 depicts fluorescence decay curves of TPMN, TTMN, MeTTMN and MeOTTMN in the solid state.

Fig. 6A depicts PL spectra of TTMN (10 μM) in ACN/water mixtures with different water fractions (f _w) . Fig. 6B depicts a plot of the emission maximum and the relative emission intensity (I/I ₀) versus the composition of the aqueous mixture of TTMN.

Fig. 7A depicts UV-vis spectra of TTMN in various solvents with different polarities. Concentration: 10 μM; excitation wavelength: 483 nm. Fig. 7B depicts PL spectra of TTMN in solvents with different polarities. Concentration: 10 μM; excitation wavelength: 483 nm.

Fig. 8A depicts cell viability assessed by MTT assay for Hela cells incubated with TPMN for 24 h.

Fig. 8B depicts cell viability assessed by MTT assay for Hela cells incubated with TTMN for 24 h.

Fig. 8C depicts cell viability assessed by MTT assay for Hela cells incubated with MeTTMN for 24 h.

Fig. 8D depicts cell viability assessed by MTT assay for Hela cells incubated with MeOTTMN for 24 h.

Fig. 9A depicts bright-field images of living HeLa cells after incubation with 2 μM of TPMN for 30 min (λex: 488 nm (1%laser power) ; Scale bar = 20 μm) .

Fig. 9B depicts bright-field images of living HeLa cells after incubation with 1 μM of TPMN for 30 min (λex: 488 nm (1%laser power) ; Scale bar = 20 μm) .

Fig. 9C depicts bright-field images of living HeLa cells after incubation with 500 nM of TPMN for 30 min (λex: 488 nm (1%laser power) ; Scale bar = 20 μm) .

Fig. 9D depicts bright-field images of living HeLa cells after incubation with 200 nM of TPMN for 30 min (λex: 488 nm (1%laser power) ; Scale bar = 20 μm) .

Fig. 9E depicts confocal images of living HeLa cells after incubation with 2 μM of TPMN for 30 min (λex: 488 nm (1%laser power) ; Scale bar = 20 μm) .

Fig. 9F depicts confocal images of living HeLa cells after incubation with 1 μM of TPMN for 30 min (λex: 488 nm (1%laser power) ; Scale bar = 20 μm) .

Fig. 9G depicts confocal images of living HeLa cells after incubation with 500 nM of TPMN for 30 min (λex: 488 nm (1%laser power) ; Scale bar = 20 μm) .

Fig. 9H depicts confocal images of living HeLa cells after incubation with 200 nM of TPMN for 30 min (λex: 488 nm (1%laser power) ; Scale bar = 20 μm) .

Figs. 10 (A) , 10 (E) , 10 (I) , and 10 (M) depict bright-field images of HeLa cells stained with TPMN (200 nM) , TTMN (200 nM) , MeTTMN (200 nM) , and MeOTTMN (2 μM) , respectively (Scale bar = 20 μm) .

Figs. 10 (B) , 10 (F) , 10 (J) , and 10 (N) depict confocal images of HeLa cells stained with TPMN (200 nM) , TTMN (200 nM) , MeTTMN (200 nM) , MeOTTMN (2 μM) , respectively (Scale bar = 20 μm) .

Figs. 10 (C) , 10 (G) , 10 (K) , and 10 (O) depict confocal images of HeLa cells stained with BODIPY493/503 Green (100 nM) (Scale bar = 20 μm) .

Figs. 10 (D) , 10 (H) , 10 (L) , and 10 (P) depict merged images of Fig. 10 (B) and Fig. 10 (C) , Fig. 10 (F) and Fig. 10 (G) , Fig. 10 (J) and Fig. 10 (K) , and Fig. 10 (N) and Fig. 10 (O) , respectively (Scale bar = 20 μm) .

Figs. 11 (A) , 11 (B) , 11 (C) and 11 (D) depict a scatter plot indicating a correction coefficient between each AIEgen ( (A) TPMN, (B) TTMN, (C) MeTTMN, (D) MeOTTMN) and BODIPY493/503 Green (Pearson correlation coefficients Rr = 0.97 (TPMN) , 0.96 (TTMN) , 0.95 (MeTTMN) , and 0.91 (MeOTTMN) ) .

Figs. 12 (A) , 12 (B) , 12 (C) and 12 (D) depict confocal images of HeLa cells before laser irradiation (0 min, upper panel) and Figs. 12 (E) , 12 (F) , 12 (G) and 12 (H) depict confocal images of HeLa cells after laser irradiation for 15 min (lower panel) of cells stained with (A, E) TPMN, (B, F) TTMN, (C, G) MeTTMN, and (D, H) MeOTTMN (Concentration: 200 nM (TPMN, TTMN, MeTTMN) , 2 μM (MeOTTMN) and 100 nM (BODIPY493/503 Green) ) ; λex: 488 nm; Scanning rate: 22.4 s per frame; laser power of confocal fluorescence microscope: 0.3 μW; Scale bar = 20 μm) . Fig. 12 (I) is a graph depicting the loss in fluorescence of HeLa cells stained with AIEgens and BODIPY493/503 Green with the number of scans of laser irradiation.

Fig. 13 (A) is a graph depicting relative change in fluorescent intensity (I/I ₀-1) at 534 nm of H2DCF-DA, TPMN, TTMN, MeTTMN, MeOTTMN, and mixtures of each AIEgen and H2DCF-DA in PBS upon white light irradiation for different times. Concentrations: 10 μM (AIEgens) and 5 μM (H2DCF-DA) . Fig. 13 (B) and Fig. 13 (C) are graphs depicting cell viability of HeLa cells stained with different concentrations of (B) MeTTMN or (C) MeOTTMN in the absence or presence of white light irradiation.

Figs. 14 (A) –14 (E) depict bright-field microscope images and Figs. 14 (F) -14 (J) depict fluorescence microscope images of living zebrafish embryos stained with (B, G) TPMN, (C, H) TTMN, (D, I) MeTTMN, (E, J) MeOTTMN, as well as (A, F) images of zebrafish embryo without staining (concentration: 5 μM; staining time: 30 min) .

DETAILED DESCRIPTION

The following definitions are provided for the purpose of understanding the present subject matter and for constructing the appended patent claims.

Definitions

It should be understood that the drawings described above or below are for illustration purposes only. The drawings are not necessarily to scale, with emphasis generally being placed upon illustrating the principles of the present teachings. The drawings are not intended to limit the scope of the present teachings in any way.

Throughout the application, where compositions are described as having, including, or comprising specific components, or where processes are described as having, including, or comprising specific process steps, it is contemplated that compositions of the present teachings can also consist essentially of, or consist of, the recited components, and that the processes of the present teachings can also consist essentially of, or consist of, the recited process steps.

In the application, where an element or component is said to be included in and/or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components. Further, it should be understood that elements and/or features of a composition, an apparatus, or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present teachings, whether explicit or implicit herein

The use of the terms "include, " "includes" , "including, " "have, " "has, " or "having" should be generally understood as open-ended and non-limiting unless specifically stated otherwise.

The use of the singular herein includes the plural (and vice versa) unless specifically stated otherwise.

It should be understood that the order of steps or order for performing certain actions is immaterial so long as the present teachings remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

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 refers to a molecule which exhibits fluorescence; “luminogen” or “luminophore” as used herein refers 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 Noxide 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) , SiH2, 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.

As used herein, a "theranostic agent" refers to an organic material, for example, an organic nanoparticle material, having both diagnostic and therapeutic capabilities.

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. In addition, where the use of the term "about" is before a quantitative value, the present teachings also include the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term "about" refers to a ±10%variation from the nominal value unless otherwise indicated or inferred.

Fluorescent Compounds

The present subject matter contemplates a fluorescent compound having aggregation-induced emission (AIE) characteristics and exhibiting red/near-infrared (NIR) absorption. The compounds can be synthesized using a one-pot synthetic approach. The compounds can be utilized as LD-specific bio-probes in cell imaging and in vivo zebrafish-imaging, with high photostability and brightness. In addition, the compounds can efficiently generate reactive oxygen species (ROS) in vivo when irradiated with white light. As such, the compounds can be effective in killing cancer cells through photodynamic therapy (PDT) processes.

Accordingly, the present compounds can be beneficial in both diagnostic and phototheranostic applications, particularly with respect to detecting abnormalities in lipid droplets in cells and photodynamic cancer therapy.

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.

One-Pot Synthesis

A method of preparing the fluorescent compounds according to the present teachings includes a simple one-pot synthesis. An exemplary reaction scheme for preparing the fluorescent compounds is provided below:

Cell Imaging and Cancer Treatment

An abnormality in lipid droplets (LD or LDs) of a cell is a critical biomarker for diseases, such as cancer. The fluorescent compounds described herein can specifically target lipid droplets in the cell and provide efficient fluorescent probes of the lipid droplets, permitting detection of certain abnormalities therein. The fluorescent compounds are lipophilic compounds. Without being limited to any specific mechanism of action, it is believed that the lipid droplet staining specificity of the fluorescent compounds can be attributed to like-like interactions resulting from accumulation of the fluorescent compounds in the hydrophobic lipid droplets.

The fluorescent compounds can be contacted with a cell and an imaging method can then be used to visualize the lipid droplets in the cell for diagnostic purposes. The imaging method can include, for example, fluorescence microscopy or confocal laser scanning microscopy. The fluorescent compounds can be efficiently introduced into a cell without any encapsulation or modification.

The fluorescent compounds can also be useful as photosensitizers in ROS generation. As described in detail herein, the fluorescent compounds can efficiently generate ROS under white light irradiation to kill cancer cells. An ROS indicator, such as H2DCF-DA, can be used as an indicator of ROS generation.

In addition, the fluorescent compounds demonstrate low cytotoxicity in dark conditions. As such, the fluorescent compounds can be successfully used in photodynamic therapy (PDT) applications. PDT is a promising approach to cancer treatment because of the precise controllability, minimal invasive nature, and high spatiotemporal accuracy it offers. The fluorescent compounds can be contacted with a target cancer cell and an imaging method can be used to visualize the cancer cell. The imaging method can include, for example, fluorescence microscopy or confocal laser scanning microscopy. The target cancer cell can be subjected to white light irradiation while the compound is contacting the target cancer cell to generate ROS in the cell and, thereby, kill the cell.

Advantageously, the present fluorescent compounds typically do not damage cell viability at lower imaging concentrations, e.g., concentrations ranging from about 200 nM to about 2.5 μM. As such, the present compounds are ideal for dual applications of cell imaging and photodynamic therapy.

In vivo imaging

As cellular imaging in vitro by bio-probes often does not properly reflect circumstances in vivo, visualization of biological structures and processes in vivo is of great importance. The fluorescent compounds described herein can be used for in vivo imaging of an animal. The present compounds can effectively provide fluorescent images with bright red emission. In an embodiment, the fluorescent compounds can be used for in vivo imaging of zebrafish.

The present teachings are illustrated by the following examples.

EXAMPLES

Materials and Instruments

Dulbecco’s Modified Essential Medium (DMEM) and RPMI-1640 were purchased from Gibco (Life Technologies) . Ultra-pure water was supplied by Milli-Q Plus System (Millipore Corporation, United States) . Phosphate buffered saline (PBS) , fetal bovine serum (FBS) , penicillin, streptomycin, and BODIPY 493/503 were purchased from Thermo Fisher Scientific. H2DCF-DA was purchased from Sigma-Aldrich and used as received. Other reagents used in this work were purchased from Sigma-Aldrich and used as received without further purification. All the chemicals used in the synthesis of AIEgens were purchased from Sigma-Aldrich.

¹H and ¹³C NMR spectra were measured on Bruker ARX 400 NMR spectrometers using CDCl ₃ as the deuterated solvent. High-resolution mass spectra (HRMS) were recorded on a Finnegan MAT TSQ 7000 Mass Spectrometer System operating in a MALDI-TOF mode. UV absorption spectra were taken on a Milton Ray Spectronic 3000 array spectrophotometer. Steady-state fluorescence spectra were recorded on a Perkin Elmer LS 55 spectrometer. Fluorescence images were collected on Olympus BX 41 fluorescence microscope. Laser confocal scanning microscope images were collected on Zeiss laser scanning confocal microscope (LSM7 DUO) and analyzed using ZEN 2009 software (Carl Zeiss) .

For cell culturing, HeLa cells were cultured in the MEM containing 10%FBS and antibiotics (100 units/mL penicillin and 100 g/mL streptomycin) in a 5%CO ₂ humidity incubator at 37 ℃.

For cell imaging, cells were grown in a 35 mm Petri dish with a coverslip at 37 ℃. The live cells were incubated with a specific dye at a certain concentration for a certain time (by adding 2 μL of a stock solution in DMSO solution to 2 mL of cell culture medium, DMSO < 0.1 vol%) . After incubation with AIEgens, the cells were washed with PBS three times. The AIEgen-labelled cells were mounted and imaged using a laser scanning confocal microscope (LSM7 DUO) at 488 nm with 1%laser power (the scanning rate was 22.4 s per frame) . The emission filter was 600–744 nm.

For co-staining with lipid dye BODIPY493/503 Green, cells were first incubated with AIEgens and BODIPY493/503 Green (100 nM) at 37 ℃ for 30 min. The medium was then removed and the cells were rinsed with PBS three times and then imaged under confocal microscope. For probe 1, its fluorescence was first photoactivated by irradiation at 405 nm (1%laser power) for designated time intervals and then the fluorescence images were taken. For AIEgens, the emission filter was 600–740 nm. For BODIPY493/503 Green, the excitation was 488 nm and the emission filter was 510–553 nm.

For photostability studies, the dye-labelled HeLa cells were imaged by a confocal microscope (Zeiss laser scanning confocal microscope LSM7 DUO) using ZEN 2009 software (Carl Zeiss) . For AIEgens, excitation wavelength was 488 nm. For BODIPY 493/503, excitation wavelength was 488 nm. Laser powers were unified at 6 μW (TPPCN) and 2.16 μW (MTR) . For TPE-CP and BODIPY 493/503, excitation wavelength was 489 nm, and laser powers were unified at 0.55 μW.

Quantitative data were expressed as mean ± standard deviation. Statistical comparisons were made by ANOVA analysis and Student’s t-test. P value < 0.05 was considered statistically significant.

Example 1

Synthesis of TPMN

A dried Schlenk tube equipped with a magnetic stirring bar was charged under a nitrogen atmosphere with 4-Bromo-N, N-diphenylaniline (162 mg, 0.5 mmol) , (4-formylphenyl) boronic acid (112.5 mg, 0.75 mmol) , K ₃PO ₄ (530 mg, 2.5 mmol) , Pd (OAc) ₂ (5.6 mg, 5 mol%) , and EtOH (8 mL) . The mixture was stirred at 78 ℃ for 12 h, then CNCH ₂CN (66 mg, 1 mmol) was added. The resulting mixture was stirred at 78℃ for another 72 hours. After cooling down to room temperature, the solvent was removed under vacuum, then water (20 mL) was added into the mixture, which was extracted with CH ₂Cl ₂ (5 mL h 3) . The combined organic phase was dried over Na ₂SO ₄ and filtered. The filtrate was removed under reduced pressure in order to obtain the crude product, which was further purified by silica-gel chromatography (petroleum ether/CH ₂Cl ₂ as eluent) to provide TPMN with a yield of 42%. ¹H NMR (400 MHz, CDCl ₃) : 7.96 (d, J = 8.4Hz, 2H) , 7.73 (t, J = 6.6Hz, 3H) , 7.52 (d, J = 8.8Hz, 2H) , 7.28-7.32 (m, 4H) , 7.07-7.16 (m, 8H) . ESI HRMS: calcd. for C ₂₈H ₁₉N ₃ [M] ⁺: 397.1579, found: 397.1557.

Example 2

Synthesis of TTMN

A dried Schlenk tube equipped with a magnetic stirring bar was charged under a nitrogen atmosphere with 4-Bromo-N, N-diphenylaniline (162 mg, 0.5 mmol) , (5-formylthiophen-2-yl) boronic acid (117 mg, 0.75 mmol) , K ₃PO ₄ (530 mg, 2.5 mmol) , Pd (OAc) ₂ (5.6 mg, 5 mol%) , and EtOH (8 mL) . The mixture was stirred at 78 ℃ for 12 h, then CNCH ₂CN (66 mg, 1 mmol) was added. The resulting mixture was stirred at 78℃ for another 72 hours. After cooling down to room temperature, the solvent was removed under vacuum, then water (20 mL) was added into the mixture, which was extracted with CH ₂Cl ₂ (5 mL h 3) . The combined organic phase was dried over Na ₂SO ₄ and filtered. The filtrate was removed under reduced pressure in order to obtain the crude product, which was further purified by silica-gel chromatography (petroleum ether/CH ₂Cl ₂ as eluent) to provide the TTMN with a yield of 49%. ¹H NMR (400 MHz, CDCl ₃) : 7.75 (s, 1H) , 7.68 (d, J = 4.0Hz, 1H) , 7.53 (d, J = 8.8Hz, 2H) , 7.29-7.33 (m, 5H) , 7.10-7.16 (m, 6H) , 7.04 (d, J = 8.8Hz, 2H) . ESI HRMS: calcd. for C ₂₆H ₁₇N ₃S [M] ⁺: 403.1143, found: 403.1150.

Example 3

Synthesis of MeTTMN

A dried Schlenk tube equipped with a magnetic stirring bar was charged, under a nitrogen atmosphere, with 4-bromo-N, N-di-p-tolylaniline (176 mg, 0.5 mmol) , (5-formylthiophen-2-yl) boronic acid (117 mg, 0.75 mmol) , K ₃PO ₄ (530 mg, 2.5 mmol) , Pd (OAc) ₂ (5.6 mg, 5 mol%) , and EtOH (8 mL) . The mixture was stirred at 78 ℃ for 12 h, then CNCH ₂CN (66 mg, 1 mmol) was added. The resulting mixture was stirred at 78℃ for another 72 hours. After cooling down to room temperature, the solvent was removed under vacuum. Then, water (20 mL) was added into the mixture, which was extracted with CH ₂Cl ₂ (5 mL h 3) . The combined organic phase was dried over Na ₂SO ₄ and filtered. The filtrate was removed under reduced pressure in order to obtain the crude product, which was further purified by silica-gel chromatography (petroleum ether/CH ₂Cl ₂ as eluent) to provide MeTTMN with a yield of 58%. ¹H NMR (400 MHz, CDCl ₃) : 7.73 (s, 1H) , 7.66 (s, 1H) , 7.49 (d, J = 8.8 Hz, 2H) , 7.30 (s, 1H) , 6.96-7.13 (m, 10H) , 2.34 (s, 6H) . ESI HRMS: calcd. for C ₂₈H ₂₁N ₃S [M] ⁺: 431.5570, found: 403.1431.

Example 4

Synthesis of MeOTTMN

A dried Schlenk tube equipped with a magnetic stirring bar was charged, under a nitrogen atmosphere, with 4-bromo-N, N-bis (4-methoxyphenyl) aniline (192 mg, 0.5 mmol) , (5-formylthiophen-2-yl) boronic acid (117 mg, 0.75 mmol) , K ₃PO ₄ (530 mg, 2.5 mmol) , Pd (OAc) ₂ (5.6 mg, 5 mol%) , and EtOH (8 mL) . The mixture was stirred at 78℃ for 12 h. Then CNCH ₂CN (66 mg, 1 mmol) was added. The resulting mixture was stirred at 78℃ for another 72 hours. After cooling down to room temperature, the solvent was removed under vacuum, then water (20 mL) was added into the mixture, which was extracted with CH ₂Cl ₂ (5 mL h 3) . The combined organic phase was dried over Na ₂SO ₄ and filtered. The filtrate was removed under reduced pressure in order to obtain the crude product, which was further purified by silica-gel chromatography (petroleum ether/CH ₂Cl ₂ as eluent) to provide MeOTTMN with a yield of 55%. ¹H NMR (400 MHz, CDCl ₃) : 7.72 (s, 1H) , 7.65 (d, J = 4 Hz, 1H) , 7.47 (d, J = 8.8 Hz, 2H) , 7.28 (d, J = 4 Hz, 1H) , 7.09-7.11 (m, 4H) , 6.86-6.89 (m, 6 H) , 3.81 (s, 6H) . ESI HRMS: calcd. for C ₂₈H ₂₁N ₃O ₂S [M] ⁺: 463.1354, found: 463.1381.

Example 5

Single Crystal Structure of TTMN

Single crystals of TTMN were obtained by slow evaporation of its solution in mixed solvents of CHCl ₃ and hexane. As illustrated in Figs. 1A and 1B, TTMN is comprised of a triphenylamine segment (D) , thiophene fragment (D and π-bridge) , carbon-carbon double bond (π-bridge) , and two cyano units (A) . The molecular geometry, excluding the two phenyl rings at the end, is relatively coplanar, thus allowing good electron delocalization of the whole molecule. The ingenious combination of strong electron donor-acceptor (D-A) interaction and extended π- conjugation with good electron delocalization in this structure not only indicates great potential for red emission, but also is likely to result in an excellent push-pull system and strong excited-state ICT, endowing large Stokes shift. In contrast, all the moieties of TTMN could serve as freely rotated molecular rotators that consume the energy of the excited state upon photo-excitation, thus ensuring that TTMN is weakly emissive in solution. Twisted conformation of the triphenylamine segment extends the intermolecular distance

between two parallel planes (Fig. 1C) , remarkably reducing the intermolecular π–π interaction, and essentially preventing emission quenching in its aggregate state. In addition, abundant inter-and intramolecular interactions (such as C-H···π, C-H···C, S···N and S···C) in the crystal lattice strongly rigidify the molecular conformation and restrict molecular motions, resulting in the possibility of bright emission in the crystal state. Interestingly, the existence of intramolecular S···N interactions may weaken any undesirable twisted intramolecular charge transfer (TICT) effect, which is one of the major non-radiative pathways for the excited state to relax and deactivate, thus eventually enhancing its emissive ability.

Example 6

Photophysical Properties

Compounds TPMN, TTMN, MeTTMN and MeOTTMN possess good solubilities in common organic solvents, such as toluene, dichloromethane, chloroform, tetrahydrofuran, acetonitrile (ACN) , methanol and dimethyl sulfoxide. Their UV-vis spectra measured in ACN are peaked at 441, 483, 492 and 499 nm, respectively, as shown in Fig. 2A. Their absorption maximums are located in the range of visible light, which causes less damage to biological systems than UV light. The gradually red-shifted absorption wavelengths can be attributed to the orderly enhanced D-A effect from TPMN to MeOTTMN. On the other hand, as depicted in Fig. 3, the orderly decreased values of calculated energy gaps (2.565, 2.555, 2.498 and 2.405 eV) are in good accord with experimental data of absorption maximums.

The AIE property of TPMN, TTMN, MeTTMN and MeOTTMN was studied in ACN/water mixtures with different water fractions (f _w) , which enabled solute aggregation to a certain extent. As shown in Table 1, in ACN solution, TPMN, TTMN and MeTTMN weakly emit red photoluminescences (PL) at 635, 664 and 673 nm, respectively, with 0.1%to 0.32%of quantum yields, while MeOTTMN is non-emissive in solution.

Table 1. Optical properties of AIEgens TPMN, TTMN, MeTTMN and MeOTTMN.

^a) Absorption maximum in ACN solutions; ^b) Emission maximum in ACN (10 μM) ; ^c) Fluorescence quantum yield determined by a calibrated integrating sphere; ^d) Emission maximum in solid state; ^e) Fluorescence lifetime, measured under ambient conditions. ^f) The quantum yield of TTMN in aggregation state is 11.7%.

All four compounds showed gradual increase in PL intensity with raising fraction of water starting from 70%, due to the formation of nanoaggregates that were measured and confirmed by dynamic light scattering analysis. The average hydrodynamic diameters of these nanoaggregates that formed in corresponding suspensions containing 95%fraction of water ranged from 76.6 to 101 nm with polydispersity indexes from 0.1 to 0.22 (Figs. 4A-4D) . The strongest PL intensities were found with 90%or 95%fraction of water upon aggregation, in which their PL intensities were enhanced to about 266-, 12-, 68-and 34-fold, respectively, compared with those of ACN solutions (Figs. 2B-2C) . Their quantum yields (18.6%, 15.8%, 7.4%and 1.1%) in the solid state also dramatically increased compared with those in the solution state. The remarkable enhancements of PL intensities in both aggregated and solid states clearly demonstrate their AIE characteristics. Their maximum emissions in the aggregation state were located at 637, 672, 681 and 701 nm, indicating their red-, far red-and NIR-emissive properties. In addition, red-shifts of emissions were observed from aggregate to solid state, in which PL spectra peaked at 648, 690, 719 and 715 nm (Fig. 2D) . As illustrated in Table 1, these AIEgens have extremely large Stokes shifts, even more than 200 nm. Moreover, the fluorescence decay curves of TPMN, TTMN, MeTTMN and MeOTTMN in the solid state reveal that their lifetimes range from 0.95 to 8.32 ns (Fig. 5) . Apparently, both the long emission wavelength and the bright emission of the AIEgen TTMN in aggregates perfectly match the results and hypotheses that were obtained from the analysis of its single crystal. The collected photophysical data suggest that in this developed system, emissions in the red/NIR region are easily tunable by fine-varying their molecular structures and substituents having different extent of D-A effect, demonstrating excellent controllability of this system in terms of emission.

To investigate the TICT effect, TTMN was chosen as an example. It was found that an emission maximum of TTMN slightly red-shifted from 664 to 671 nm, and the emission intensity remarkably decreased with the increase of water faction at low water content in mixed ACN/water solutions (Figs. 6A-6B) , indicating the existence of TICT effect. In fact, ACN/water is not an ideal system to study TICT effect, due to the small polarity difference. Thus, PL spectra of TTMN were recorded in different solvents with varied polarities. As depicted in Figs. 7A-7B, when the solvent was changed from nonpolar toluene to polar dimethyl sulfoxide, the emission maximum largely red-shifted from 573 to 665 nm while emission intensity was considerably reduced, suggesting a strong TICT effect. Indeed, AIE properties and the TICT effect are competitive in determining the PL intensity; nevertheless, the enhanced emission feature of AIEgens in aggregates reveals a stronger AIE feature than the TICT effect in this system, reasonably benefitting from both the rigidified molecular conformation caused by powerful inter-and intra-molecular interactions and restricted molecular motions in aggregates.

Example 7

Cell Imaging

To evaluate the cytotoxicities of the AIEgens, TPMN, TTMN, MeTTMN and MeOTTMN, in living cells, a 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide (MTT) assay was used with different concentrations of the AIEgens. Cells were seeded in 96-well plates (Costar, IL, USA) at a density of 5 × 10 ³ cells/well. After overnight culturing, medium in each well were replaced by 100 μL fresh medium containing different concentrations of the presented AIEgens. The volume fraction of DMSO was below 0.2%. 24 hours later, 10 μL MTT solution (5mg/mL in PBS) was added into each well. After 4 hours of incubation, 100 μL SDS-HCl aqueous solution (10%SDS and 0.01 M HCl) was added to each well. After incubation for 4 hours, the absorption of each well at 595 nm was recorded via a plate reader (Perkin-Elmer Victor3 ^TM) . Each trial was performed with 6 wells parallel.

As illustrated in Figs. 8A-8B, no significant decrease of cell viability was observed even when the HeLa cells were cultured in the presence of 10 μM of the AIEgens for 24 h, demonstrating good biocompatibilities of these AIEgens to HeLa cells.

Cell imaging studies employed HeLa cells as a cell model, in which the cells were pre-treated with oleic acid to induce considerable amount of neutral lipids. The influence of AIEgen concentration was investigated by the use of 2 μM, 1 μM, 500 nM and 200 nM of TPMN. As shown in Figs. 9A-9H, TPMN was strongly emissive in cells and the brightness did not significantly decline even when the concentration was as low as 200 nM, suggesting a high brightness of TPMN in cell imaging. Co-localization experiments were then conducted by incubating HeLa cells with each presented AIEgen for 30 min and BODIPY493/503 Green for 10 min. The cell imaging of AIEgens and BODIPY493/503 Green overlapped perfectly, indicating their powerful LDs-specific targeting capability (Figs. 10A-10P) . Pearson’s correlation coefficients, commonly used to determine the linear association of two variables, were calculated to be up to 97% (Figs. 11A-11D) , solidly demonstrating the high specificities of these AIEgens for staining LDs. The excellent LDs-staining specificity can be attributed to the efficient accumulation of these lipophilic AIEgens in the hydrophobic spherical LDs which contain mainly diverse neutral lipids such as triacylglycerol and cholesteryl ester, due to the “like-like” interactions. Moreover, LDs were clearly visualized with a very high signal-to-noise ratio when relatively low concentrations (200 nM for TPMN, TTMN and MeTTMN, and 2 μM for MeOTTMN) of these AIEgens were utilized. Compared with other AIEgens used for LDs-imaging, the presented AIEgens held the lowest working concentrations, almost comparative to that of BODIPY493/503 Green. Interestingly, efficient cell uptake of the presented AIEgens can be achieved without any encapsulation or modification steps.

As one of the key criteria for evaluating a fluorescent bioprobe, the photostabilities of the presented AIEgens were assessed by continuous excitation and sequential scanning with confocal microscope. As depicted in Figs. 12A-12H, for both TTMN and MeTTMN, their fluorescence intensities remained almost constant after 40 scans within 15 min irradiation, while the fluorescence intensities of TPMN and MeOTTMN retained 84%and 89%, respectively, of their initial values in this process. By contrast, BODIPY493/503 Green suffered an obvious drop of fluorescence intensity to 35%of its initial intensity under the same conditions. Evidently, the photostabilities of these AIEgens are greatly superior to that of commercial BODIPY493/503 Green.

Example 8

ROS generation

The presented AIEgens also proved to be powerful photosensitizers in ROS generation. In this experimental study, H2DCF-DA that emits fluorescence at around 534 nm triggered by ROS was employed as a ROS indicator. Considering the strong absorption of these presented AIEgens in the visible light region, white light was utilized as the excitation light source. As illustrated in Fig. 13A, in the presence of TPMN, TTMN, MeTTMN and MeOTTMN, the emission of H2DCF-DA was gradually and rapidly intensified with the increase of irradiation time. After 90 seconds exposure to white light, the emission intensities of H2DCF-DA were 17, 13, 25 and 19 times higher than the original emission intensities without light irradiation, respectively. Such change, however, was not observed in AIEgens or H2DCF-DA alone under the same conditions. These results demonstrate the high ROS generation efficiencies of these AIEgens.

Example 9

Photodynamic Therapy

Both MeTTMN and MeOTTMN served as better sensitizers for ROS generation than the other two AIEgens tested. In this context, MeTTMN and MeOTTMN were chosen as photosensitizers to investigate the therapeutic effect of these AIEgens through PDT, which was quantitatively evaluated on HeLa cells by standard MTT assay. HeLa cells were seeded in 96-well plates (Costar, IL, USA) at a density of 5 × 10 ³ cells/well. After overnight culturing, medium in each well were replaced by 100 μL fresh medium containing different concentrations of MeTTMN or MeOTTMN. The volume fraction of DMSO was below 0.2%. After 8 h incubation, plates containing HeLa cells were exposed to white light (36 mW) for 30 min, and another array of plates with cells were kept in the dark as a control. Then the plates were subjected to the same treatment as the biocompatibility test.

Both of the AIEgens exhibited very low cytotoxicity in dark conditions, regardless of the AIEgen concentration used for cell staining (Figs. 13A-13C) . Low cytotoxicity in dark condition is one of the essential features of photosensitizers for PDT application. With white light irradiation, a dose-dependent toxicity was observed for both. In the case of MeTTMN, the HeLa cell viability decreased gradually to around 50%with a concentration of 1 μM, while increasing the concentration to 2.5 μM eventually lead to almost complete cell apoptosis (Fig. 13B) . When MeOTTMN was examined, cell viability remained the same with concentrations lower than 2.5 μM; however, a sharp decline of cell viability was found with the concentration of 5 μM (Fig. 13C) . These results clearly reveal that both of the two AIEgens are remarkably effective for killing cancer cells by PDT pathway. In addition, MeTTMN shows higher efficiency than MeOTTMN, perfectly matching the outcomes of ROS generation. Meanwhile, at the imaging concentrations of 200 nM (MeTTMN) and 2 μM (MeOTTMN) , these two AIEgens did not cause obvious damage of cell viability even under light irradiation, making them ideal for dual applications of cell imaging and therapy through controlling AIEgen concentrations. It seems reasonable to infer that due to the fluidity and various bio-functions of LDs, the ROS that are generated by the AIEgens with specific-targeting towards LDs can efficiently initiate cell apoptosis.

Example 10

Zebrafish Imaging

Visualization of biological structures and processes on in vitro level is of great importance. However, imaging of cells in vitro by bio-probes often does not represent the real circumstances in vivo because of separation from native environments. Encouraged by the excellent cell imaging results produced by the present AIEgens, further investigations were done for in vivo imaging of living zebrafish embryos. Zebrafish is an ideal vertebrate model for biological in vivo imaging due to the exceptionally high optical clarity in embryonic and larval stages. In this experiment, living zebrafish embryos were stained by the present AIEgens with the concentration of 5 μM for 30 min. As depicted in Figs. 14A-14J, AIEgens TPMN (Fig. 14G) , TTMN (Fig. 14H) and MeTTMN (Fig. 14I) provided fluorescent images with bright red emission showing the contour profile of Zebrafish. In contrast, in the case of MeOTTMN, an unclear image of zebrafish was obtained, perhaps due to its long emission wavelength located in the NIR region. These preliminary results of zebrafish imaging suggest the great potential of these AIEgens for observing biological processes at in vivo levels.

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 R is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkyl-NCS, alkyl-N ₃ alkyl-NH ₂, and alkoxy; and

wherein X is selected from the group consisting of phenyl, heteroaryl, and C=C.
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 target of interest comprises lipid droplets.
The method of claim 3, wherein the imaging method is selected from the group consisting of fluorescence microscopy and confocal laser scanning microscopy.
A method of killing cancer cells, comprising:

contacting a target cancer cell with the compound according to claim 1;

imaging the target cancer cell while the compound contacts the target cancer cell using an imaging method; and

subjecting the target cancer cell to white light irradiation while the compound is contacting the target cancer cell to kill the target cancer cell.
The method of claim 6, wherein the imaging method is selected from the group consisting of fluorescence microscopy and confocal laser scanning microscopy.
A method of generating reactive oxygen species in cancer cells, comprising irradiating the compound of claim 1 with white light.
A method of in vivo imaging of an animal, comprising:

administering the compound of claim 1 to the animal; and

obtaining images of the animal while the compound is within the animal using an imaging method.
The method of claim 9, wherein the imaging method is selected from the group consisting of fluorescence microscopy and confocal laser scanning microscopy.
The method of claim 9, wherein the animal is a zebrafish
A fluorescent compound exhibiting aggregation induced emission properties, the compound comprising 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 12; and

identifying a target of interest in the target cell using an imaging method.
The method of claim 13, wherein the target of interest comprises lipid droplets.
The method of claim 13, wherein the imaging method is selected from the group consisting of fluorescence microscopy and confocal laser scanning microscopy.
A method of killing cancer cells, comprising:

contacting a target cancer cell with the compound according to claim 12;

imaging the target cancer cell while the compound contacts the target cancer cell using an imaging method; and

subjecting the target cancer cell to white light irradiation while the compound is contacting the target cancer cell to kill the target cancer cell.
A method of generating reactive oxygen species in cancer cells, comprising irradiating the compound of claim 12 with white light.
A method of in vivo imaging of an animal, comprising:

administering the compound of claim 12 to the animal; and

obtaining images of the animal while the compound is within the animal using an imaging method.
The method of claim 18, wherein the animal is a zebrafish.
A method of preparing the compound of claim 12, comprising a one-pot synthesis process represented by the following reaction scheme:

wherein R ₁ is H, alkyl, or alkoxy; and

wherein R ₂ is phenyl or thiophene.