WO2017080449A1

WO2017080449A1 - Red fluorescent aiegens

Info

Publication number: WO2017080449A1
Application number: PCT/CN2016/105157
Authority: WO
Inventors: Benzhong Tang; Yee Yung Yu
Original assignee: Hong Kong University of Science and Technology
Current assignee: Hong Kong University of Science and Technology
Priority date: 2015-11-10
Filing date: 2016-11-09
Publication date: 2017-05-18
Anticipated expiration: 2018-05-10
Also published as: WO2017080413A1; CN108138043B; CN108138043A

Abstract

The present subject matter relates to the synthesis and application of red fluorescent AIEgens. The present subject matter relates to mitochondrial targeting AIEgens, which boost the radiosensitivity of lung carcinoma. The present AIEgens can also target cell membranes, lipid droplets, or lysosomes, as well as serve as a radio sensitizer in radiotherapy. The present subject matter further relates to two-photon imaging with AIEgens.

Description

Red Fluorescent AIEgens

RELATED APPLICATIONS

The present patent application claims priority to provisional U.S. Patent Application No. 62/285,826 filed November 10, 2015 and International Patent Application No. PCT/CN2016/089911 filed July 13, 2016, which were filed by the inventors hereof and are incorporated by reference herein in their entirety.

TECHNICAL FIELD

BACKGROUND

Fluorescent dyes have been used widely in modern biological studies and have facilitated the development of fluorescent microscopes. Fluorescent imaging is a powerful tool to look beyond tissue and observe single cells and, nowadays, has an important role in the progression and noninvasive study of gene expression, protein function, protein-protein interactions, and many other cellular processes.

Furthermore, fluorescent imaging has proved to be a powerful tool in examining the microscopic structures of polymer blends. Particularly, far-red to near-infrared (FR/NIR) fluorescent dyes are beneficial for in vivo imaging, as the effects of optical absorption and intrinsic auto-fluorescence may be minimized. Higher degrees of tissue penetration can be achieved due to a longer fluorescent wavelength. For the same reasons, the imaging of microscopic polymer blend structures utilizing FR/NIR fluorescent dyes allows for obtaining insight not only of the surface pattern, but also of deeper layers.

Regarding the molecular design of fluorogens, the aggregation-caused quenching (ACQ) effect has always given rise to photo-bleaching and attenuation in fluorescence intensity upon aggregation, thus resulting in restrictions of long-time monitoring of organelles and lower performance.

In 2001, molecules exhibiting aggregation induced emission (AIE) characteristics were discovered. AIE active molecules are highly emissive in aggregated and/or crystalline states due to restriction of intra-molecular motions (RIM) , allowing applications in various areas, such as in OLEDs and bioprobes. AIE active molecules have already been applied successfully as bioprobes, proving to possess high photostability and high bio-compatibility.

SUMMARY

In an embodiment, the present subject matter is directed to a method for preparing red fluorescent AIEgens for biological applications having aggregation induced emission characteristics comprising combining a donor selected from the group consisting of tertiary amino, alkoxy, and imidazole groups and an acceptor selected from the group consisting of cyano, pyridium, and indolium.

In an embodiment, the present subject matter is directed to a method of preparing AIEgens, comprising constructing a donor-acceptor AIE derivative compound, wherein the donor-acceptor AIE derivative comprises a backbone structure of a formula selected from the group consisting of:

wherein R, R’ , R” , and R” ’ are independently selected from the group consisting of:

wherein n is an integer from 0 to 20.

In an embodiment, the present subject matter is directed to an AIEgen for use as a dye comprising a donor-acceptor AIE derivative compound, wherein the donor-acceptor AIE derivative comprises a backbone structure of a formula selected from the group consisting of:

wherein n is an integer from 0 to 20.

In an embodiment, the present subject matter is directed to an AIEgen having a structure of:

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows (A) absorption spectra and (B) PL spectra of ASCP in different solvents. Concentration: 10 μM； λ_ex ＝ 460 nm.

FIG. 2 shows (A) PL spectra of ASCP in toluene/DMSO mixtures with different toluene fractions (f_t) . (B) Plot of relative emission intensity (I/I₀) at 650 nm versus the composition of the toluene/DMSO mixture of ASCP. I₀ ＝ emission intensity of ASCP in pure DMSO solution. Concentration: 10 μM； λ_ex ＝ 460 nm.

FIG. 3 shows viability of HeLa cells in the presence of different concentrations of ASCP for 8 h. Data is expressed as mean value for five separate trials.

FIG. 4 shows (Aand B) fluorescent and (C) bright-field images of HeLa cells stained with ASCP (5 μM) for 30 min with focus at mitochondria (A) and nucleolus (B) , respectively. λ_ex ＝ 460-490 nm； scale bar ＝ 30 μm. (D and E) Confocal images of HeLa cells stained with (D) ASCP (5 μM) and (E) MitoTracker green (MTG； 200 nM) . (F) The merged image of (D) and (E) . Conditions: λ_ex ＝ 405 nm and λ_em ＝ 600-700 nm for ASCP； λ_ex ＝ 488 nm and λ_em ＝ 500-540 nm for MTG； scale bar ＝ 20 μm.

FIG. 5 shows (A) absorption spectra and (B) PL spectra of ASCP mixed with different phospholipid vesicles (22 μM) , DNA (100 μg/mL) and RNA (100 μg/mL) in HEPES (pH 7.4) buffer solutions with 1％DMSO. Concentration: 10 μM； λ_ex ＝ 460 nm.

FIG. 6 (A and B) confocal and (C) bright-field images of HeLa cells stained with ASCP (5 μM) for 30 min. Conditions: (A) λ_ex ＝ 405 nm； λ_em ＝ 500-650 nm； (B) λ_ex ＝ 560 nm； λ_em ＝ 650-750 nm.

FIG. 7 shows fluorescent images of HeLa cells stained with (A-C) ASCP (10 μM) for 2 h and (D-F) SYTO RNASelect (5 μM) for 2 h with or without treatment with RNase or DNase.

FIG. 8 shows confocal images of HeLa cells stained with (A and C) ASCP and (B and D) SYTO RNASelect taken under continuous excitation. (E) Signal (％) of fluorescent emission of (black) ASCP and (red) SYTO RNASelect of different numbers of scan. Conditions: λ_ex ＝ 560 nm and λ_em ＝ 650-750 nm for ASCP； λ_ex ＝ 488 nm, λ_em ＝ 500-600 nm for SYTO RNASelect.

FIG. 9 shows absorption spectra of ASCP-2P in DMSO solution.

FIG. 10 shows (A) PL spectra of ASCP-2P in toluene/DMSO mixtures with different toluene fractions (f_t) and (B) plot of relative emission intensity (I/I₀) at 640 nm versus the composition of the toluene/DMSO mixture of ASCP-2P. I₀ ＝ emission intensity of ASCP-2P in pure DMSO solution. Concentration: 10 μM； λ_ex ＝ 460 nm.

FIG. 11 shows PL spectra of ASCP-2P (10 μM) with and without lipid vesicle (22 μM) in PBS solution. λex ＝ 460 nm.

FIG. 12 shows (A) PL spectra of H2DCFDA (5 μM) with ASCP-2P (10 μM) in PBS solutions under different irradiation of white light and (B) change in fluorescent intensity at 534 nm of PBS solutions containing different AIEgens (10 μM) and H2DCFDA (5 μM) with different irradiation time of white light. λ_ex ＝ 495 nm.

FIG. 13 shows (A) PL spectra of H2DCFDA (5 μM) with different AIEgens (10 μM) (B: ASCP； C: TPE-PY； and D: TPE-IQ) in PBS solutions under different irradiation of white light. λ_ex ＝ 495 nm.

FIG. 14 shows (A and B) confocal images and (C) merged image with bright-field of A549 cancer cells co-stained with ASCP-2P (5 μM) and MitoTracker Deep Red (MTDR, 50 nM) . Confocal images show the intracellular ROS levels of A549 cancer cells received different treatments by using H2DCFDA as the ROS indicator. (D) Probe +, Light -； (E) Probe +, Light +； (F) Probe +, Light +, NAC+. Conditions: (A) ASCP-2P: λ_ex ＝ 488 nm, λ_em ＝ 620-640 nm； (B) MTDR: λ_ex ＝ 633 nm, λ_em ＝ 655-675 nm； (D-F) H2DCFDA: λ_ex ＝ 488 nm, λ_em ＝ 510-530 nm.

FIG. 15 shows cell viability of A549 cells incubated with ASCP-2P in dark (Black) , ASCP-2P pretreated with white light irradiation for 1 min and followed by in dark (Red) and ASCP-2P with NAC pretreated with white light irradiation for 1 min and followed by in dark (Blue) .

FIG. 16 shows (A) clonogenic formation upon different treatments and (B) quantitative data for clonogenic assay of (A) . **represents P < 0.01.

FIG. 17 shows (A) clonogenic formation after treatment with different popularly used radiosensitizer and (B) quantitative data for clonogenic assay of (A) . **represents P < 0.01.

FIG. 18 shows Western blot analysis of (A) p-ERK, ERK, p-Akt and Akt； (B) Bcl-XL, Bcl-2, BAD, and Caspase-3； and (D) p-ERK, p-Akt, Bcl-2, Bax and BAD from A549 cells with various treatments indicated. (C) shows an illustration of the pathway that indicates how ASCP-2P serves as an effective radiosensitizer to irradiation.

FIG. 19 shows absorption spectra of 3” in THF solution.

FIG. 20 shows absorption spectra of 5” in THF solution.

FIG. 21 shows (A) PL spectra of 3” in THF/water mixture with different water fractions (f_w) and (B) plot of relative PL intensities versus f_w. I₀ are the PL intensities at 580 nm of the dyes in THF； Dye concentration: 10 μM； excitation wavelength: 410 nm.

FIG. 22 shows (A) PL spectra of 5” in THF/water mixture with different water fractions (f_w) and (B) plot of relative PL intensities versus f_w. I₀ are the PL intensities at 680 nm of the dyes in THF； Dye concentration: 10 μM； excitation wavelength: 525 nm.

FIG. 23 shows fluorescent images of HeLa cells co-stained with 3” (5 μM) and Lyso-tracker red for 15 min. (A) Lyso-tracker red: Ex. : 520-560 nm； (B) 16: Ex. : 400-440 nm； (C and D) merge imaging without daylight and image of daylight.

FIG. 24 shows confocal images of HeLa cell stained with 3” (5 μM) for 15 min and excited by 442 nm and 840 nm. Em: 500-580 nm.

FIG. 25 shows confocal images of HeLa cell stained with 5” (5 μM) for 30 min and excited by 512 nm and 1000 nm. Em: 520-630 nm.

FIG. 26 shows PL spectra in confocal images ○: PL signals in lipid droplets； △: PL signals outside lipid droplets.

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.

“Aggregation-induced emission” means the fluorescence/phosphorescence is turned on upon aggregation formation or in the solid state. When molecularly dissolved, the material is nonemissive. However, the emission is turned on when the intramolecular rotation is restricted.

“Emission intensity” means the magnitude of fluorescence/phosphorescence normally obtained from a fluorescence spectrometer or fluorescence microscopy measurement.

“Fluorophore” means a molecule which exhibits fluorescence.

“Luminogen” means a molecule which exhibits luminescence.

“AIEgen” means a molecule exhibiting AIE characteristics.

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.

Abbreviations

ACQ aggregation-caused quenching

AEE aggregation enhanced emission

AIE aggregation induced emission

ASCP (Z) -4- (4- (1-cyano-2- (4- (dimethylamino) phenyl) vinyl) phenyl) -

1-methylpyridin-1-ium hexafluorophosphate (V)

ASCP-2P (Z) -4- (4- (1-cyano-2- (4- (diphenylamino) phenyl) vinyl) phenyl) -

1-methylpyridin-1-ium hexafluorophosphate (V)

DCM dichloromethane

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

DNase deoxyribonuclease

FR/NIR far-red to near-infrared

GNP gold nanoparticles

H2DCFDA 2', 7'-dichlorodihydrofluorescein diacetate

HEPES 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid

HRMS high-resolution mass spectroscopy

MALDI-TOF matrix assisted laser desorption ionization time-of-flight

MEM minimum essential medium

MTDR MitoTracker Deep Red

MTG MitoTracker Green

MTT 3- (4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyltetrazolium bromide

NAC N-acetylcysteine

NMR nuclear magnetic resonance

OLED organic light emitting diode

PBS phosphate buffer saline

PDT photodynamic therapy

PL photoluminescence

RNA ribonucleic acid

RNase ribonuclease

ROS reactive oxygen species

RIM restriction of intra-molecular motions

THF tetrahydrofuran

TICT twisted intramolecular charge transfer

XTT 2, 3-bis- (2-methoxy-4-nitro-5-sulfophenyl) -2H-tetrazolium-5-

carboxanilide

The present subject matter is directed to different approaches used to prepare red fluorescent AIEgens. For example, one possible approach is based on the designed donor-acceptor structure.

Several groups of red-emitting AIEgens are designed and their biological applications are demonstrated by selective staining of polymers in blends. For cyano-substituted stilbene derivatives, different donors are incorporated into the molecular backbone and donor-acceptor systems are formed, leading to a tunable emission from yellow to red. Furthermore, targeting moieties are attached to these molecules for organelle-specific imaging. Their biological applications are also explored, including mitochondria-specific imaging and radiotherapy.

In an embodiment, the present subject matter is directed to a method for preparing red fluorescent AIEgens for biological applications having aggregation induced emission characteristics comprising combining a donor selected from the group consisting of tertiary amino, alkoxy, and imidazole groups and an acceptor selected from the group consisting of cyano, pyridium, and indolium. Accordingly, the present compounds, sometimes referred to as donor-acceptor compounds, have a donor-acceptor structure in one embodiment herein.

In an embodiment, the fluorescent signals of the AIEgens of the present subject matter are around 600 nm.

In an embodiment, the present subject matter is directed to method of preparing AIEgens, comprising constructing a donor-acceptor AIE derivative compound, wherein a donor-acceptor AIE derivative comprises a backbone structure of a formula selected from the group consisting of:

wherein n is an integer from 0 to 20.

In an embodiment, the AIEgen of the present subject matter exhibits red fluorescence.

In an embodiment, the AIEgen of the present subject matter is used for fluorescent cell imaging of lung carcinoma cells.

In an embodiment, the donor-acceptor AIE derivatives of the present subject matter can target specific organelles selected from the group consisting of mitochondria, nucleolus, lysosomes, cell membranes, and lipid droplets.

In an embodiment, the present subject matter is directed to an AIEgen for use as a dye comprising a donor-acceptor AIE derivative, wherein a donor-acceptor AIE derivative comprises a backbone structure of a formula selected from the group consisting of:

wherein n is an integer from 0 to 20.

In an embodiment, the AIEgen of the present subject matter has a structure of:

In an embodiment, the AIEgen of the present subject matter is a dye for targeting mitochondria and nucleolus.

In an embodiment, the AIEgen of the present subject matter has a structure of:

In an embodiment, the AIEgen of the present subject matter is a dye for targeting mitochondria. In an embodiment, the AIEgen of the present subject matter can be a radio sensitizer in radiotherapy.

The AIEgen of the present subject matter is a dye for targeting lysosomes. The AIEgen of the present subject matter can be used for two-photon imaging.

The AIEgen of the present subject matter is a dye for targeting lipid droplets. The AIEgen of the present subject matter can be used for two-photon imaging.

Mitochondrial Targeting ASCP Boosts Radiosensitivity of Lung Carcinoma Cells

The specific AIEgen ASCP is a dual-color organelle-specific probe with AIE features for targeting the mitochondria and nucleolus. Due to different interactions with the mitochondrial membrane and nucleic acids, distinct emission colors from the mitochondria and nucleolus are observed under fluorescence microscopy. Owing to high brightness, excellent biocompatibility, and superior photostability, the AIE fluorescent probe ASCP is a promising candidate for simultaneous mitochondria and nucleolus imaging.

Synthesis of ASCP

ASCP (Compound 6) was prepared according to the synthetic route shown according to the below scheme:

Synthesis of 3: 4-bromophenylacetonitrile (1； 0.69 g, 3.50 mmol) and 4- (dimethylamino) benzaldehyde (2； 0.81 g, 3.00 mmol) were dissolved in 40 mL ethanol in a 100 mL round bottom flask. Sodium hydroxide (0.14 g, 3.50 mmol) in 5 mL ethanol was then added slowly into the mixture. After stirring for 2 h, the pale yellow precipitates were filtered, washed with ethanol, and dried under reduced pressure. Yield: 80％. ¹H NMR (400 MHz, CDCl₃) , δ (ppm) : 7.86 (d, 2H, J ＝ 8.4 Hz) , 7.54–7.48 (m, 4H) , 7.38 (s, 1H) , 6.73 (d, 2H, J ＝ 8 Hz) , 3.07 (s, 6H) . HRMS (MALDI-TOF) : m/z 326.0217 (M⁺, calcd. 326.0419) .

Synthesis of 5: 3 (0.10 g, 0.306 mmol) , 4-pyridinylboronic acid (4； 45 mg, 0.387 mmol) , potassium carbonate (0.422 g, 3.06 mmol) , and Pd (PPh₃) ₄ (10 mg, 0.01 mmol) were added in 20 mL THF and 3 mL water under nitrogen in a 100 mL two-necked round bottom flask equipped with a condenser. The mixture was stirred and heated to reflux overnight. After cooling to room temperature, the mixture was extracted with dichloromethane (DCM) three times. The organic phase was collected, washed with water, and dried over anhydrous sodium sulfate. After solvent evaporation, the crude product was purified by silica-gel column chromatography using DCM/ethyl acetate (v/v ＝ 99: 1) as eluent to furnish an orange solid as product. Yield: 74％. ¹H NMR (400 MHz, CDCl₃) , δ (ppm) : 8.68 (d, 2H, J ＝ 4.4 Hz) , 7.76-7.68 (m, 4H) , 7.55 (d, 2H, J ＝ 4.4 Hz) , 7.26 (s, 1H) , 6.74 (d, 2H, J ＝ 8.4 Hz) , 3.08 (s, 6H) . ¹³C NMR (100 MHz, CDCl₃) , δ (ppm) : 150.7, 149.4, 149.3, 144.6, 142.4, 137.4, 137.5, 131.2, 130.9, 129.2, 127.0, 126.8, 125.4, 120.9, 120.9, 120.7, 111.0, 110.6, 106.0, 39.4, 39.3. HRMS (MALDI-TOF) : m/z 325.1575 (M⁺, calcd. 325.1579) .

Synthesis of Compound 6 (ASCP) : 5 (50 mg, 0.154 mmol) was dissolved in 5 mL acetonitrile in a 100 mL two-necked round bottom flask equipped with a condenser. Iodomethane (0.1 mL) was then added, and the mixture was heated to reflux for 8 h. After cooling to room temperature, the mixture was poured into diethyl ether. The dark red precipitates formed were filtered by suction filtration. The precipitates were re-dissolved in acetone and mixed with saturated KPF₆ solution (5 mL) . After stirring for 1 h, acetone was evaporated by compressed air. The dark red precipitates were filtered again, washed with water, and dried under reduced pressure. Yield: 95％. ¹H NMR (400 MHz, DMSO-d₆) , δ (ppm) : 8.98 (d, 2H, J ＝ 6.8 Hz) , 8.53 (d, 2H, J ＝ 6.8 Hz) , 8.18 (d, 2H, J ＝ 8.4 Hz) , 8.03 (s, 1H) , 7.93–7.40 (m, 4H) , 6.83 (d, 2H, J ＝ 8.8 Hz) , 4.29 (s, 3H) , 3.03 (s, 6H) . ¹³C NMR (100 MHz, DMSO-d₆) , δ (ppm) : 153.0, 152.0, 145.3, 144.4, 138.3, 132.0, 131.6, 128.5, 125.6, 123.5, 120.3, 118.9, 111.4, 100.3, 46.8. HRMS (MALDI-TOF) : m/z 340.1826 (M⁺, calcd. 340.1814) .

Optical Properties

The optical properties of ASCP were studied. Due to the hydrophilic nature of the Py salt, ASCP is soluble in polar solvents, slightly soluble in water, but insoluble in nonpolar solvents such as dioxane and toluene. ASCP exhibits an absorption band at around 450 nm, irrespective of the type of solvent used (FIG. 1A) . On the contrary, it shows obvious different emission colors and intensities when the measurement was carried out in different solvents (FIG. 1B) .

ASCP emits a strong orange light in dilute dioxane solution. Owing to the twisted intramolecular charge transfer (TICT) effect, the emission of the dye molecule was weakened and red-shifted by increasing the solvent polarity. In dilute DMSO solution, ASCP shows a faint red fluorescence. In contrast, gradual addition of toluene into its DMSO solution has enhanced the light emission and changed the emission color to orange due to the gradual decrement of the solvent polarity (FIG. 2) . At a high toluene fraction, a much more rapid fluorescence enhancement was observed, which was due to the formation of ASCP aggregates along with the activation of the AIE process.

Cell Imaging

The strong emission of ASCP in the aggregated state encourages utilization as a fluorescent visualizer for mitochondrion imaging. To examine whether the dye is suitable for bio-imaging, the cytotoxicity of ASCP on HeLa cells was first evaluated using MTT assay. As depicted in FIG. 3, the cell viability remains high at ASCP concentrations as high as 10 μM, suggesting that ASCP possesses a good biocompatibility.

ASCP was first assessed for its capability to stain specific organelles in live HeLa cells. The HeLa cells were cultured and incubated in MEM with 5 μM ASCP for 30 min. The cells were washed with fresh PBS and then observed under fluorescence microscope. Thanks to the high specificity of the Py unit in ASCP, the reticulum structures of mitochondria are stained with an intense orange emission (FIG. 4A) .

To further validate the specificity of ASCP, MitoTracker Green (MTG) , a commercial mitochondrial imaging agent, was used to co-stain the HeLa cells. The cell images taken on confocal microscopy illustrate that the orange fluorescence from ASCP has an excellent correlation (96.4％) with the green emission of MTG (FIG. 4D-F) .

Surprisingly, by altering the focus, red fluorescence was observed in the nucleolus (FIG. 4B) . Thus, it seems that the two distinct fluorescences observed from the mitochondria and nucleolus correlate with the specific interactions of ASCP with different biomolecules.

The most abundant components in the mitochondria and nucleolus are phospholipids and nucleic acids (DNAs and RNAs) , respectively. For examination, phospholipids found in the mitochondrial membrane and nucleic acids were chosen for mimicking the actual intracellular environment. Different lipid vesicles were first fabricated as models of mitochondria by mixing desired ratio of phospholipids. The absorption and emission spectra of ASCP in the presence of lipid vesicles and nucleic acids in HEPES were then recorded. ASCP exhibits an absorption maximum at 435 nm in HEPES, showing no or little wavelength shift when treated with lipid vesicles. On the other hand, the absorption maximum of ASCP is red-shifted by 20 nm in the presence of nucleic acids (FIG. 5A) . Similarly, while a 50 nm bathochromic shift in the emission maximum was observed when ASCP was mixed with nucleic acids, no change on the ASCP emission was observed by lipid vesicles (FIG. 5B) . These results are consistent with the observations from fluorescent images as shown in FIG. 4A-B.

ASCP may be used to collect individual fluorescence from mitochondria and nucleolus without cross contamination. Confocal images of dye-labelled HeLa cells were collected by changing the excitation wavelengths and the emission filters. After optimizing the conditions, mitochondria can be visualized individually with orange fluorescence under 405 nm light excitation (FIG. 6A) . On the other hand, only red fluorescence was observed in nucleoli at an excitation wavelength of 560 nm (FIG. 6B) .

Origin of Fluorescence in Nucleolus

Intercalation and electrostatic attraction are possible interactions between ASCP and nucleic acids. When ASCP enters the cavities of nucleic acids, it may adopt a more co-planar and conjugated conformation, and hence shows a redder emission. On the other hand, the hydrogen bonds between the nucleotides in nucleic acids may provide a relative polar environment for ASCP to emit at the longer wavelength region.

In order to gain a better understanding of the origin of red fluorescence, fluorescence imaging experiments were performed after the ASCP-labelled cells were fixed and treated with deoxyribonuclease (DNase) and ribonuclease (RNase) . From the fluorescent images shown in FIG. 7, the specificity of ASCP to the nucleolus was lost when RNase was applied (FIG. 7B) . However, the dye-labelled cells are still emissive after being treated with DNase (FIG. 7C) .

The performance of ASCP was further verified by using SYTO RNASelect, a commercial fluorescent probe for the nucleolus. As shown in FIG. 7E-F, SYTO RNASelect performed similar to ASCP. Since RNA contributes the major constituent in the nucleolus, both ASCP and SYTO RNASelect tend to accumulate in the nucleolus due to the strong electrostatic attraction. When the dye-labelled cells are treated with RNase, the binding sites for intercalation are collapsed, and the dye molecules no longer bind to the RNA fragments. Thus, the fluorescent emission of ASCP and SYTO RNASelect in RNA-rich nucleolus is decreased dramatically.

Photostability

Photostability is a critical parameter for a fluorescent probe for finding promising applications in organelle imaging and tracking. To quantitatively investigate the photo-bleaching resistance of ASCP and SYTO RNASelect, continuous scanning of the dye-labelled cells by laser irradiation was carried out, and the fluorescent signal at each scan was recorded. The dye-labelled cells were irradiated at 560 and 488 nm, respectively, with the same power. As shown in FIG. 8, 5％fluorescence loss was observed in the ASCP-stained cells after 50 scans. On the contrary, almost no fluorescent signal was detected from the cells stained with SYTO RNASelect after 15th scans. This result suggests that ASCP possesses a higher photo-bleaching resistance or photostability than SYTO RNASelect.

Mitochondrial Targeting ASCP-2P Boosts Radiosensitivity of Lung Carcinoma Cells

ASCP-2P is a red-emissive AIEgen, designed and synthesized with a strong donor-acceptor structure. ASCP-2P is AIE-active and mitochondrial targeting, and its ROS generation ability was studied and verified by a commercial ROS sensor. ASCP-2P was utilized as a photosensitizer to increase the radiosensitivity of lung carcinoma cells in radiotherapy, obtaining an ultra-high value of SER10 compared with paclitaxel and gold nanoparticles. Apoptotic death path was identified by Western blot analysis. The first demonstration of the photosensitizer in radiotherapy showed high potential for cancer treatment.

Synthesis of ASCP-2P

ASCP-2P (Compound 4’ ) was prepared according to the synthetic route shown according to the below scheme:

Synthesis of 3’ : 1’ (0.14 g, 0.31 mmol) , (4-hydroxylphenyl) boronic acid (2’ ； 45 mg, 0.39 mmol) , potassium carbonate (0.42 g, 3.06 mmol) , and Pd (PPh₃) ₄ (10 mg, 0.01 mmol) were added in 20 mL THF and 3 mL water in a 100 mL two-necked round bottom flask equipped with a condenser under nitrogen. The mixture was heated to reflux overnight with stirring. After cooling to room temperature, the mixture was extracted with DCM three times. The organic phase was collected, washed with water, and then dried over anhydrous sodium sulfate. After solvent evaporation, the crude product was purified by silica-gel column chromatography using DCM/ethyl acetate (v/v ＝ 99: 1) as eluent to furnish an orange solid as the product. Yield: 72％. ¹H NMR (400 MHz, CDCl₃) , δ (ppm) : 8.68 (d, 2H, J ＝ 4.4 Hz) , 7.81-7.70 (m, 3H) , 7.66 (d, 2H, J ＝ 7.6 Hz) , 7.58 (d, 2H, J ＝ 8.0 Hz) , 7.52 (d, 2H, J ＝ 4.8 Hz) , 7.33 (t, 4H, J ＝ 7.2 Hz) , 7.26-7.14 (m, 2H) , 7.11-7.04 (m, 4H) , 6.83 (d, 2H, J ＝ 8.4 Hz) . ¹³C NMR (100 MHz, CDCl₃) , δ (ppm) : 149.6, 146.8, 145.8, 143.7, 141.6, 137.3, 135.3, 130.6, 130.2, 129.1, 129.0, 128.9, 127.0, 126.9, 125.7, 125.1, 125.0, 123.9, 123.8, 120.9, 120.8, 120.0, 119.6, 105.9. HRMS (MALDI-TOF) : m/z 449.1916 (M⁺, calcd. 449.1892) .

Synthesis of ASCP-2P (Compound 4’ ) : 3’ (50 mg, 0.082 mmol) was dissolved in 5 mL acetonitrile in a 50 mL two-necked round bottom flask equipped with a condenser. Iodomethane (0.1 mL) was then added, and the mixture was heated to reflux for 8 h. After cooling to room temperature, the mixture was poured into diethyl ether. The dark red precipitates formed were filtered by suction filtration. The precipitates were re-dissolved in acetone and mixed with saturated KPF₆ solution (5 mL) . After stirring for 1 h, acetone was evaporated by compressed air. The red precipitates were filtered again, washed with water, and dried under reduced pressure. Yield: 96％. ¹H NMR (400 MHz, DMSO-d₆) , δ (ppm) : 8.95 (d, 2H, J ＝ 6.8 Hz) , 8.50 (d, 2H, J ＝ 6.8 Hz) , 8.17 (d, 2H, J ＝ 8.4 Hz) , 8.07 (s, 1H) , 7.93 (d, 2H, J ＝ 8.4 Hz) , 7.89 (d, 2H, J ＝ 8.8 Hz) , 7.39 (t, 4H, J ＝ 8.0 Hz) , 7.20–7.13 (m, 4H) , 6.93 (d, 2H, J ＝ 8.8 Hz) , 4.30 (s, 3H) . ¹³C NMR (100 MHz, DMSO-d₆) , δ (ppm) : 145.6, 145.3, 143.7, 137.5, 131.1, 129.8, 128.6, 126.2, 125.7, 124.9, 124.7, 119.2, 104.5. HRMS (MALDI-TOF) : m/z 464.2133 (M⁺, calcd. 464.2127) .

Optical Properties and Cell Imaging

The photophysical properties of ASCP-2P were studied. The maximum absorption of ASCP-2P was at 460 nm (FIG. 9) . The fluorescent property was studied in mixtures of DMSO and toluene. There was nearly no emission in pure DMSO solution. Upon addition of toluene to the DMSO solution, the fluorescent intensities gradually increased and blue-shifted from 660 nm to 620 nm, but there were further dramatic enhancements after 80％toluene fraction (FIG. 10) .

Since the ASCP-2P structure is a strong donor-acceptor design similar to ASCP, a strong TICT property was revealed. ASCP-2P was molecularly dissolved in DMSO solution, and the emission was weaken by the TICT effect and discouraged by the free intramolecular motions through non-radiative decay. Toluene served as a poor solvent to induce aggregate formation of ASCP-2P. At the beginning, the slight enhancement and a blue shift was due to the TICT effect because of the low polarity of toluene. After 80％toluene fraction, the significant enhancement was due to aggregate formation, activating the RIM process and relaxing in the radiative channel.

It is noted that ASCP-2P reveals AIE characteristics, but also TICT effects from the strong donor-acceptor structure. Py group has been reported to be a mitochondria targeting group. The mitochondrial membrane environment was mimicked by preparing a lipid vesicle and mixing with ASCP-2P in PBS solution (FIG. 11) . The weak emission in PBS solution was due to the TICT effect in a highly polar aqueous solution. After binding to phospholipids, the motions of ASCP-2P were restricted, activating the RIM process and blocking non-radiative decay.

Recently, AIEgens have been used as photosensitizers to generate ROS and were developed for image-guided PDT. Inspired by this idea, the ability of ROS generation of ASCP-2P was investigated and compared with several AIEgens. A commercial ROS indicator, 2', 7'-dichlorodihydrofluorescein diacetate (H2DCFDA) , was used and is a fluorescein derivative and can recover its green fluorescence through oxidation by ROS.

ASCP-2P was compared with ASCP, TPE-PY, and TPE-IQ (FIG. 13) under irradiation of white light. Surprisingly, the fluorescent signals were saturated after 30 seconds of the irradiation in mixing with ASCP-2P. Other candidates were far away from the saturated intensity in 30 seconds of the irradiation (FIG. 12) . The main reason of the poor ROS generation abilities of TPE-IQ and TPE-PY may be due to absorption. Their absorptions were shorter than 400 nm, meaning that most of the molecules were not excited under white light and less oxygen obtained the excited energy in the triplet state. Interestingly, ASCP showed almost no ROS generation and ASCP-2P showed high ability in ROS generation (FIGS. 12-13) .

To study the oxidized effect of ASCP-2P, A549 cancer cells were incubated with ASCP-2P for 2 h. Then H2DCFDA was added into the medium, followed by exposure to white light for 1 min. After that, the cells were immediately observed under confocal imaging. As shown in FIG. 14D-F, the green fluorescence represented the level of intracellular ROS. The brighter the green fluorescence, the higher the level of intracellular ROS was. It is noted that ASCP-2P could greatly increase the level of intracellular ROS as represented by brighter fluorescence, which indicated that light was an effective trigger to the induction of ROS by ASCP-2P (FIG. 14E) . Moreover, co-treatment of an antioxidant agent, N-acetylcysteine (NAC) , with ASCP-2P substantially reversed the induction of ROS, demonstrating that the ROS-inducing effect of ASCP-2P by light could be abrogated by antioxidant NAC (FIG. 14F) .

XTT assay was employed to evaluate the anticancer effect of ASCP-2P. As shown in FIG. 15, ASCP-2P without light exposure was almost non-toxic to A549 cells. There were nearly 90％cells alive even at the highest concentration (80 μM) . However, with exposure to white light for 1 min, ASCP-2P led to dose-dependent cell death. The IC₅₀ value was about 33 μM. In addition, co-treatment of NAC significantly attenuated the cytotoxic effect of ASCP-2P with light. For instance, 80 μM of ASCP-2P led to more than 90％cell death, while more than 75％cells were alive upon NAC co-treatment.

Radiosensitization

Clonogenic assay was performed to evaluate the radiosensitization effect of ASCP-2P. Prior to irradiation, A549 cancer cells were incubated with ASCP-2P (10 μM) for 2 h to ensure the targeting delivery of ASCP-2P to mitochondria. After that, irradiation was given at a series of doses (2, 4, and 6 Gy) . Cells were then immediately seeded into 6 well plates to study the colony forming ability. As shown in FIG. 16A-B, ASCP-2P without light showed no radiosensitization effect when compared to irradiation alone. However, the exposure of ASCP-2P-treated cells to light significantly sensitized cancer cells to radiation. The calculated SER10 was 1.62.

Recent studies showed that certain drugs, such as paclitaxel and cisplatin, have a radiosensitization effect. Clinical studies also demonstrated that paclitaxel has been recommended as a standard therapy for synchronized chemotherapy and radiotherapy. In addition, several studies focused on potential nanomaterials that may possess a radiosensitization effect, and gold nanoparticles (GNP) were one of the most promising radiosensitization agents in the field of nanotechnology.

In this regard, the radiosensitization effects of ASCP-2P were compared with paclitaxel and GNP. FIG. 16-17 showed that ASCP-2P with light was the most effective agent that could sensitize lung cancer cells to irradiation. There was a significant difference between the colony forming ability in cells treated with ASCP-2P and paclitaxel or GNP. As calculated from the curve, SER10 of paclitaxel was 1.32, while that of GNP was 1.19. Both were significantly lower than SER10 of ASCP-2P, which reached 1.62, the highest among the three agents.

To investigate the underlying mechanism of the radiosensitization effect, a lower dose of ASCP-2P (10 μM) was chosen to combine with irradiation. 10 μM of ASCP-2P with light induced very little apoptosis, which can be considered as almost non-toxic. As shown in FIG. 18A, irradiation alone inhibited the phosphorylation of both Akt and ERK, whereas ASCP-2P with light barely influenced the expression of p-Akt and p-ERK. More importantly, combination of ASCP-2P with light and irradiation significantly blocked the phosphorylation process, indicating the synergistic effect of the inhibition of p-Akt and p-ERK.

Furthermore, the down-stream apoptotic pathways were also evaluated by western blotting (FIG. 18B) . ASCP-2P with light induced little apoptosis, while irradiation inhibited the expression of anti-apoptotic proteins (Bcl-2, Bcl-XL) and promoted the expression of pro-apoptotic proteins (Bax, BAD) . One of the most important apoptotic markers, caspase-3, underwent significant decrease of pro-caspase-3 and increase of cleaved caspase-3. Moreover, the combination of irradiation and ASCP-2P with light was much more effective in inducing apoptosis than irradiation alone or ASCP-2P with light.

In addition, an antioxidant agent NAC was used as a ROS scavenger to investigate if the radiosensitization effect of ASCP-2P was mainly dependent on the induction of intracellular ROS (FIG. 18) . It is obvious that NAC significantly attenuated the inhibitory effect of ASCP-2P on the expression of p-Akt and p-ERK, which reversed the induction of down-stream apoptotic pathway. For example, co-treatment of NAC substantially decreased the expression of anti-apoptotic Bcl-2 and strengthened the expression of the pro-apoptotic Bax and BAD after the exposure to ASCP-2P with light, which clearly demonstrated that the radiosensitization effect of ASCP-2P was closely related to the induction of intracellular ROS by light.

It is known that radiation resistance in cancer cells has a very close relationship to the modulation of PI3k/Akt and MAPK pathways. Several studies demonstrated that constitutive expression of PI3k/Akt, which protects the cells from apoptosis, plays a vital role in chemosensitization of cancer cells. Here it was demonstrated that the efficient ROS-inducing effect of ASCP-2P was triggered by light exposure, and ASCP-2P can act as an effective radiosensitizer to irradiation through the inhibition of p-Akt and p-ERK and the following induction of apoptosis (FIG. 18C-D) .

Two-Photon Imaging with New AIEgens for Lysosome and Lipid Droplets

Synthesis

Compounds 3” and 5” were prepared according to the synthetic route shown according to the below scheme:

Compound 1” (0.5 mmol) and 2” or 4” (0.5 mmol) were added to a 25 mL two necked round-bottom flask equipped with a condenser under nitrogen. 10 mL of dried ethanol was added, and the mixture underwent reflux. 2 mL of morpholine was further added, and the reaction was carried out for 2 hours. After being cooled to room temperature, the mixture was extracted with DCM three times. The organic phase was combined and washed with water and dried over anhydrous sodium sulfate. After the evaporation of solvents, the crude product was purified by silica gel column chromatography using hexane/ethyl acetate in the volume ratio of 7: 3 as eluent. The orange solid was obtained in the yield of 56％.

The structure of compound 3” is:

The structure of compound 5” is:

Photophysical properties

For compounds 3” and 5” , the absorptions were 410 nm and 525 nm, respectively (FIG. 19-20) . Compound 3” was AEE active, and the fluorescence around 600 nm was enhanced and red shifted due to increasing water fraction (FIG. 21) . There was a slight difference in compound 5” . The fluorescence around 650 nm was increased, but decreased after 50％water fraction (FIG. 22) . It is proposed that when the distance of molecules is closed enough, pi-pi interaction occurs.

Application of 3” and 5”

Compound 3” was utilized for lysosome targeting (FIG. 23) . The selectivity was confirmed by commercial dye Lyso-tracker red. It can also be used for two photon imaging in order to give a higher resolution and high signal-to-noise ratio (FIG. 24) .

Compound 5” was used for lipid droplet imaging. In confocal images, it was found that the signals come from the whole cells. However, when the range for collection of emission is changed from 520 nm to 630 nm, the signals are found to only come from lipid droplets (FIG. 25) . This is due to the environment of lipid droplets being non-polar, which will shift the emission into more blue regions (FIG. 26) .

With the information contained herein, various departures from precise descriptions of the present subject matter will be readily apparent to those skilled in the art to which the present subject matter pertains, without departing from the spirit and the scope of the below claims. The present subject matter is not considered limited in scope to the procedures, properties, or components defined, since the preferred embodiments and other descriptions are intended only to be illustrative of particular aspects of the presently provided subject matter. Indeed, various modifications of the described modes for carrying out the present subject matter which are obvious to those skilled in chemistry, biochemistry, or related fields are intended to be within the scope of the following claims.

Claims

A method for preparing red fluorescent AIEgens for biological applications having aggregation induced emission characteristics comprising combining a donor selected from the group consisting of tertiary amino, alkoxy, and imidazole groups and an acceptor selected from the group consisting of cyano, pyridium, and indolium.
The method of claim 1, wherein fluorescent signals of the AIEgens are around 600 nm.
A method of preparing AIEgens, comprising constructing a compound in a donor-acceptor AIE derivative compound, wherein thedonor-acceptor AIE derivative comprises a backbone structure of a formula selected from the group consisting of:

wherein R, R’ , R” , and R”’ a re independently selected from the group consisting of:

wherein n is an integer from 0 to 20.
The method of claim 3, wherein the AIEgen exhibits red fluorescence.
The method of claim 3, wherein the AIEgen is used for fluorescent cell imaging of lung carcinoma cells.
The method of claim 3, wherein donor-acceptor AIE derivatives can target specific organelles selected from the group consisting of mitochondria, nucleolus, lysosomes, cell membranes, and lipid droplets.
An AIEgen for use as a dye comprising a donor-acceptor AIE derivative compound, wherein the donor-acceptor AIE derivative comprises a backbone structure of a formula selected from the group consisting of:

wherein R, R’ , R” , and R”’ a re independently selected from the group consisting of:

wherein n is an integer from 0 to 20.
The AIEgen of claim 7, wherein the AIEgen has a structure of:
The AIEgen of claim 8, wherein the AIEgen is a dye for targeting mitochondria and nucleolus.
The AIEgen of claim 7, wherein the AIEgen has a structure of:
The AIEgen of claim 10, wherein the AIEgen is a dye for targeting mitochondria.
The AIEgen of claim 10, wherein the AIEgen can be a radiosensitizer in radiotherapy.
An AIEgen, wherein the AIEgen has a structure of:
The AIEgen of claim 13, wherein the AIEgen is a dye for targeting lysosomes.
The AIEgen of claim 13, wherein the AIEgen can be used for two-photon imaging.
An AIEgen, wherein the AIEgen has a structure of:
The AIEgen of claim 16, wherein the AIEgen is a dye for targeting lipid droplets.
The AIEgen of claim 16, wherein the AIEgen can be used for two-photon imaging.