WO2019161626A1

WO2019161626A1 - Corannulene-incorporated aie nanodots with highly suppressed nonradiative decay for boosted cancer phototheranostics in vivo

Info

Publication number: WO2019161626A1
Application number: PCT/CN2018/091220
Authority: WO
Inventors: Benzhong Tang; Xinggui GU
Original assignee: The Hong Kong University Of Science And Technology
Priority date: 2018-02-21
Filing date: 2018-06-14
Publication date: 2019-08-29
Also published as: CN111742034A; CN111742034B

Abstract

The present subject matter relates to fluorescent compounds that have aggregation-induced emission (AIE) characteristics and exhibit near infrared absorption. Compositions including the present compounds can include a corannulene-modified polyethylene glycol encapsulation matrix. The compositions can be in nanoparticle form. Encapsulating the AIE compounds within a corannulene matrix provides intraparticle rigidity and restricts intramolecular rotation of the encapsulated AIE compound, which results in enhanced fluorescence and ROS generation capacity of the compositions in vivo. Accordingly, the compositions can be useful in NIR imaging-guided cancer surgery and photodynamic cancer therapy.

Description

Corannulene-Incorporated AIE Nanodots with Highly Suppressed Nonradiative Decay for Boosted Cancer Phototheranostics in Vivo

CROSS-REFERENCE

The present application claims priority to provisional United States Patent Application No. 62/710,470, filed February 21, 2018, 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 compounds with aggregation-induced emission characteristics and near infrared absorption and their applications in bioimaging and phototheranostics.

BACKGROUND

Optical agents for cancer phototheranostics allow for real-time molecular diagnosis and concurrent light-triggered treatment. Among various phototheranostic agents, fluorescent nanoparticles (NPs) are particularly desirable due to their associated high sensitivity and temporal resolution of fluorescence imaging, on-demand and in situ signature of photodynamic therapy (PDT) , as well as unique enhanced permeability and retention (EPR) effects. To meet the requirements of ideal cancer phototheranostics, the fluorescent NPs must have several qualities, including sufficiently high near-infrared (NIR) emission (> 650 nm) ; reactive oxygen species (ROS) generation efficiency of the fluorescent component within the NPs; strong resistance to photobleaching; negligible cytotoxicity and in vivo toxicity; and suitable NP size and surface chemistry, permitting prominent EPR effect.

Compared with other extensively investigated fluorescent NPs, organic fluorophore-doped NPs hold the advantages of tunable photophysical properties, flexible structural tailoring, and good biocompatibility. However, π-conjugated fluorophores tend to aggregate within NPs. For conventional small-molecule fluorescent dyes with planar molecular structures, such aggregation within NPs often causes significant quenching of light emission and ROS production, owing to intramolecular interactions, such as π-π stacking and other nonradiative decays, which tremendously limit their application as cancer phototheranostics. Much effort has been devoted to overcome the aggregation-caused quenching (ACQ) effect within fluorescent NPs through the introduction of bulky side groups and hydrophobic counterions into fluorophores. Apparently, these prior efforts have not produced the desired output due to difficulties experienced in blocking the strong π-π stacking.

Aggregation-induced emission luminogens (AIEgens) have recently emerged as an alternative fluorescent material to construct fluorescent NPs, which perfectly address the challenge of ACQ and exhibit low in vivo side toxicities. AIEgens are often non-emissive in solution due to the consumption of the excited state energy via non-radiative relaxation by intramolecular motion. Upon aggregation, such relaxation from the lowest excited singlet state (S ₁) to the ground state (S ₀) is largely restricted due to steric hindrance, leading to the energy of S ₁ going through the fluorescence pathway to S ₀. This uncommon feature makes AIEgens ideal for fabrication of fluorescent NPs (also referred to as AIE dots) with ultrahigh brightness and photobleaching threshold. Prior studies, however, failed to reveal how to control and optimize the fluorescence and ROS generation ability of AIE dots.

Accordingly, advanced fluorescent NPs with both high fluorescence and ROS generation capacity for use as 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. Compositions including the present compounds can include a corannulene-modified polyethylene glycol encapsulation matrix. The compositions can be in nanoparticle form. Encapsulating the AIE compounds within a corannulene matrix provides intra-particle rigidity and restricts intramolecular rotation of the encapsulated AIE compound, which results in enhanced fluorescence and ROS generation capacity of the compositions in vivo. Accordingly, the compositions can be useful in NIR imaging-guided cancer surgery and photodynamic cancer therapy.

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

wherein each R ₁, R ₁′, R ₁”and R ₁”’is independently selected from the group consisting of

wherein each R ₂, R ₃, R ₄, R ₅, R ₆, and R ₇ is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, C _nH _2n+1, C ₁₀H ₇, C ₁₂H ₉, alkoxy, OC ₆H ₅, OC ₁₀H ₇ and OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nSH, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, N (C _nH _m) ₂, and SC _nH _m;

wherein n and m are each independently an integer from 0-10;

wherein A is a monovalent counter ion; and

wherein the monovalent counter ion is always present in the compound.

In a further embodiment, the compound is:

In yet another embodiment, the present subject matter relates to a fluorescent nanoparticle composition, comprising a fluorescent compound exhibiting aggregation induced emission properties and corannulene-modified polyethylene glycol, wherein the corannulene-modified polyethylene glycol encapsulates the fluorescent compound, and the fluorescent compound has a backbone structural formula selected from the group consisting of:

wherein each R ₂, R ₃, R ₄, R ₅, R ₆, and R ₇ is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, C _nH _2n+1, C ₁₀H ₇, C ₁₂H ₉, alkoxy, OC ₆H ₅, OC ₁₀H ₇ and OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nSH, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, N (C _nH _m) ₂, and SC _nH _m, ; and

wherein n and m are each independently an integer from 0-10;

wherein A is a monovalent counter ion selected from the group consisting of I ^-, Cl ^-, Br ^-, PF ₆ ^-, ClO ₄ ^-, BF ₄ ^-, BPh ₄ ^-, and CH ₃PhSO ₃ ^-; and

wherein the monovalent counter ion is always present in the compound.

BRIEF DESCRIPTION OF DRAWINGS

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

Fig. 1 (A) depicts PL spectra of TPP-TPA in DMSO-H ₂O mixtures with different water fractions (f _w) . Fig. 1 (B) depicts plot of the relative PL intensity (I/I ₀) at 680 nm versus fw of the DMSO-H ₂O mixture of TPP-TPA. Excitation wavelength: 440 nm (insets show the fluorescent photos of TPP-TPA in DMSO solution and DMSO-H2O mixture with the f _w of 99%taken under 365 nm UV lamp; concentration of TPP-TPA: 1 × 10 ^-5M) . Fig. 1 (C) depicts molecular orbital amplitude plots of HOMO and LUMO for TPP-TPA in ground states based on density functional theory (DFT) calculation under the method of opt wB97XD/6-31g**.

Fig. 2 depicts high-resolution mass spectrum of compound 2.

Fig. 3 depicts ¹H NMR spectrum of compound 2 in CD ₂Cl ₂.

Fig. 4 depicts ¹³C NMR spectrum of compound 2 in CD ₂Cl ₂.

Fig. 5 depicts high-resolution mass spectrum of TPP-TPA.

Fig. 6 depicts ¹H NMR spectrum of TPP-TPA in CD ₂Cl ₂.

Fig. 7 depicts ¹³C NMR spectrum of TPP-TPA in CD ₂Cl ₂.

Fig. 8 depicts UV-vis spectrum of TPP-TPA in DMSO solution (concentration of TPP-TPA: 1 × 10 ^-5 M) .

Fig. 9 depicts PL spectra of TPP-TPA in DMSO/H ₂O mixtures with the water fractions (f _w) of 80%, 90%and 99% (excitation wavelength: 440 nm) .

Fig. 10 depicts density functional theory (DFT) calculation for TPP-TPA based on the method of opt wB97XD/6-31g**: (A and B) (molecular orbital amplitude plots of HOMO (A) and LUMO (B) for TPP-TPA in the excited states) .

Fig. 11 depicts scheme for the preparation of Cor-AIE dots and DSPE-AIE dots using nanoprecipitation method.

Fig. 12 depicts UV-vis spectrum of Cor-AIE dots and DSPE-AIE dots in aqueous solution (concentration: 0.01 mg/mL) .

Figs. 13 (A) and 13 (B) depict DLS analysis and Figs. 13 (C) and 13 (D) depict TEM images of Cor-AIE dots (A and C) and DSPE-AIE dots (B and D) , respectively.

Fig. 14 (A) depicts PL and Fig. 14 (B) depicts fluorecence lifetime spectra of Cor-AIE dots and DSPE-AIE dots (excitation wavelength: 500 nm; inset shows the fluorescent photo of Cor-AIE dots taken under 365 nm UV lamp) . Fig. 14 (C) depicts absorption spectra and Fig. 14 (D) depicts decomposition rate of ABDA for Cor-AIE dots (Up) and DSPE-AIE dots (Down) under white light irradiation (60 mW/cm ², 400-1000 nm) , where A ₀ and A are the absorbance at 378 nm before and after irradiation, respectively (concentrations of nanoparticles (Cor-AIE dots and DSPE-AIE dots) and ABDA are 0.01 mg/mL and 100 μM, respectively. Figs. 14 (E) and 14 (F) depict Jablonski diagrams showing the non-radiative, radiative and intersystem crossing (ISC) processes for AIEgens in flexible (DSPE-AIE dots) and rigid (Cor-AIE dots) matrixes (S ₀: the ground state, S ₁: the lowest excited singlet state, T ₁: the lowest excited triplet state; k _nr, k _r and k _ISC are the rate constants of the non-radiative relaxation, the radiative decay and the ISC process, respectively; FL: fluorescence) .

Figs. 15 (A) – (E) relate to ¹H NMR titration experiment with corannulene gradually added into TPP-TPA solution; Fig. 15 (A) depicts structures of TPP-TPA and corannulene with featured protons labeled with H _a, H _b, H _c, H _d, and H _e. Figs. 15 (B) - (E) depict changes of chemical shifts for TPP-TPA (the aromatic protons of 1-methylpyridinium (B and D) , the methyl protons in 1-methylpyridinium and anisole (E) ) and corannulene (C) , as indicated with the dotted lines and evaluated by the related values (concentrations of TPP-TPA was 1 × 10 ^-2 M and corannulene were 1 × 10 ^-2 M (1 : 1) , 2 × 10 ^-2 M (1 : 2) , and 6 × 10 ^-2 M (1 : 6) in CD ₂Cl ₂ solution) .

Fig. 16 (A) depicts theoretical positions between corannulene and TPP-TPA; and Fig. 16 (B) depicts optimized molecular geometries of S ₀, S ₁ and T ₁ states for TPP-TPA at M06-2X/6-31G (d) level in the absence and presence of corannulene.

Figs. 17 (A) – (D) depict confocal images of HeLa cells after incubation with Cor-AIE dots for 1 h at 37 ℃ (red, λ _ex = 560 nm, λ _em = 570–720 nm) (the nucleus was stained by Hoechst 33342 (blue, λ _ex = 405 nm, λ _em = 430–470 nm) (concentration: 1×10 ^-5 M; release of ROS monitored by H ₂DCF-DA) . Fig. 17 (E) depicts change in fluorescent intensity at 525 nm of Cor-AIE dots, H ₂DCF-DA, and their mixture in PBS upon white light (36 mW) for different times; (excitation wavelength: 488 nm; concentrations of Cor-AIE dots and H ₂DCF-DA are 0.01 mg/mL and 1 μM, respectively) . Figs. 17 (F-I) exhibit merged bright-field and fluorescent images of HeLa cells stained with (F, G) H ₂DCF-DA (1 μM) only and (H, I) Cor-AIE dots (0.01 mg/mL) and H2DCF-DA (1 μM) for 30 min (F, H) before and (G, I) after exposure to white light for 2 min. Excitation wavelength: 488 nm.

Fig. 18 (A) depicts bright field, fluorescence, bioluminescence, and H&E staining images of the tumor nodules on the surface of the intraperitoneal intestines. Fig. 18 (B) depicts bright field, fluorescence, bioluminescence, and H&E staining images of the tumor nodules on the surface of the peritoneum in peritoneal carcinomatosis-bearing mice after intravenous injection of Cor-AIE dots for 24 h.

Fig. 19 (A) depicts representative fluorescence images before operation. Fig. 19 (B) depicts representative fluorescence images after operation under white light. Fig. 19 (C) depicts representative fluorescence images after re-operation with the aid of Cor-AIE dots image-guidance. Fig. 19 (D) depicts the extracted nodules from unguided groups and Cor-AIE dots guided groups examined with a fluorescence imaging system (Left) and a bioluminescence imaging system (Right) . Fig. 19 (E) depicts a histogram of nodule diameters extracted from unguided and Cor-AIE dots-guided groups.

Fig. 20 depicts a Kaplan–Meier survival curve of tumor-free survival rate after surgery versus time (days) showing improved long-term tumor-free survival with Cor-AIE dots fluorescence image-guided surgery (blue) compared to sham surgery and standard surgery (red) without Cor-AIE dots fluorescence guidance.

Fig. 21 (A) depicts the time-dependent bioluminescence imaging of the peritoneal carcinomatosis-bearing mice after intravenous injection of Saline, DSPE-AIE dots, and Cor-AIE dots. Fig. 21 (B) depicts the average bioluminescence intensities of intraperitoneal tumors on

days

0, 1, 3, 5, and 9. Fig. 21 (C) depicts the curve of survival rate after different treatments (all of the experiment groups are “Saline” , “Cor-AIE dots” , “Light (L) ” , “DSPE-AIE dots + L” , and “Cor-AIE dots + L” . “L” is under the white light (0.4 W cm ^-2) for 10 min; concentrations of DSPE-AIE dots and Cor-AIE dots are 1 mg mL ^-1 based on TPP-TPA; volume of injection is 150 μL) .

Fig. 22 depicts time-dependent bioluminescence imaging of the peritoneal carcinomatosis-bearing mice in the groups of “Light (L) ” and “Cor-AIE dots” ( “L” is under white light (0.4 W cm ^-2) for 10 min; concentrations of Cor-AIE dots are 1 mg mL ^-1 based on TPP-TPA; volume of injection is 150 μL) .

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 and Compositions

The present subject matter contemplates a fluorescent compound having aggregation-induced emission (AIE) characteristics and exhibiting near infrared absorption. The compound can have a rotor-rich skeleton and an inherent charge. The compound can be in nanoparticle form.

Also provided are compositions including the fluorescent compound and corannulene-modified polyethylene glycol encapsulating the fluorescent compound. The compositions can be in nanoparticle form. The fluorescent compound can be encapsulated by the corannulene-modified polyethylene glycol using nanoprecipitation under ultrasonic conditions. Compositions including the nanoparticle form of the fluorescent compound and the corannulene-modified polyethylene glycol are also referred to herein as “theranostic agents” or “Cor-AIE dots” .

Encapsulating the fluorescent compound in a corannulene encapsulation matrix can enhance the intra-particle microenvironment and thereby provide enhanced fluorescence and ROS generation capacity in vivo. The corannulene matrix can provide intra-particle rigidity and restrict intramolecular rotation of the encapsulated compound, leading to highly suppressed non-radiative decay. The absorbed energy can flow to both the fluorescence pathway and the intersystem crossing (ISC) process. Intersystem crossing (ISC) from S ₁ to the lowest excited triplet state (T ₁) can result from the small S ₁-T ₁ energy gap, and result in ROS production via energy transfer (ET) from T ₁ to ambient oxygen (O ₂) .

Accordingly, the present compounds and compositions can be beneficial in diagnostic and phototheranostic applications, particularly with respect to NIR imaging-guided cancer surgery and photodynamic cancer therapy.

wherein n and m are each independently an integer from 0-10;

wherein A is a monovalent counter ion; and

wherein the monovalent counter ion is always present in the compound.

In a further embodiment, the compound is:

In yet another embodiment, the present subject matter relates to a fluorescent nanoparticle composition, comprising a fluorescent compound exhibiting aggregation induced emission properties and corannulene-modified polyethylene glycol, the corannulene-modified polyethylene glycol encapsulating the fluorescent compound, the fluorescent compound having a backbone structural formula selected from the group consisting of:

wherein each R ₂, R ₃, R ₄, R ₅, R ₆, and R ₇ is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, C _nH _2n+1, C ₁₀H ₇, C ₁₂H ₉, alkoxy, OC ₆H ₅, OC ₁₀H ₇ and OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nSH, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, N (C _nH _m) ₂, and SC _nH _m, ;

wherein n and m are each independently an integer from 0-10;

wherein the monovalent counter ion is always present in the compound.

In a further embodiment, the fluorescent compound of the composition can be:

An exemplary reaction scheme for preparing the inherently-charged TPP-TPA compound is as provided below:

To extend the emission spectrum of the compound to NIR range, two electron-donating diphenylamine groups and one electron-withdrawing 1-methylpyridinium unit is incorporated into triphenylethene. Strong electron donor-acceptor interactions endow TPP-TPA with a large dipole moment. The large number of rotatable aryl rings makes the skeleton of TPP-TPA flexible. Compound 1 is obtained with a high yield of 95%according to a known procedure, and subsequently reacted with (4-methoxy-phenyl) -pyridin-4-yl-methanone undergoing McMurry coupling to yield compound 2 in 70%. Reaction of compound 2 with iodomethane followed by ion-exchange reaction with potassium hexafluorophosphate gives the desirable product TPP-TPA in a high yield of 99%. All intermediates and product of this reaction were characterized by NMR and mass spectroscopies, from which satisfactory data corresponding to their structures were obtained (Figs. 2-8) .

Cancer diagnostics and/or cancer therapy

The theranostic agents described herein can be beneficial in cancer diagnostic and phototheranostic applications, particularly with respect to NIR imaging-guided cancer surgery and photodynamic cancer therapy. Image-guided cancer surgery using NIR fluorescence has been verified to be feasible during clinical cancer surgery, and holds great promise for successful outcomes in cancer surgery. The theranostic agents described herein can be used as efficient NIR fluorescent probes that meet the necessary requirements of image-guided cancer surgery.

As described herein, compositions including TPP-TPA and lipid-PEG (DSPE-PEG: 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] ) and compositions including corannulene-modified PEG (Cor-PEG) , respectively, afforded two types of AIE dots with different intra-particle rigid microenvironments. Corannulene is a polyaromatic hydrocarbon from a part of C ₆₀ and is well-known in organic optoelectronics due to its uneven electron distribution, electron-negative core, and electron-positive periphery. Corannulene possesses a bowl shape with large curvature that inhibits bowl-to-bowl inversion at room temperature, owing to the large energy barrier of 11.5 kcal/mol. Hence, corannulene possesses a large dipole moment of 2.1D, superhydrophobicity, and hyper-rigidity. Corannulene typically has a chemical formula of C ₂₀H ₁₀. The molecule consists of a cyclopentane ring fused with 5 benzene rings, so another name for it is [5] circulene. Corannulene has a typical structure of:

As compared to TPP-TPA-loaded DSPE-PEG nanodots (DSPE-AIE dots) with relatively low NIR fluorescence and weak ROS generation capability, the TPP-TPA-loaded Cor-PEG nanodots (Cor-AIE dots) show 4.0-fold amplified fluorescence quantum yield and 5.4-fold enhanced ROS production. ¹H NMR titration and theoretical calculations essentially demonstrate that the corannulene provides intra-particle rigidity and strong interactions with TPP-TPA, which restrict intramolecular rotation of the encapsulated AIEgens, leading to highly suppressed nonradiative decay. The absorbed energy thus flows to both the fluorescence pathway and ISC process. As described herein, this highly amplified NIR emission and ROS generation capacity demonstrated significant phototheranostic efficacies in terms of NIR imaging-guided cancer surgery and photodynamic cancer therapy using a peritoneal carcinomatosis-bearing mouse model. Since, to date, DSPE-PEG has been the most widely used encapsulation matrix for building AIE dots, the comparative studies described herein not only provide a new strategy and molecular guideline to prepare superior AIE dots, but also bring new insights into the design of advanced fluorescent NPs for biomedical applications.

According to an embodiment, the present subject matter relates to a method of killing cancer cells, which can include contacting the theranostic agent with a target cancer cell, imaging the target cancer cell while the theranostic agent contacts the target cancer cell, and subjecting the target cancer cell to near infrared light irradiation while the theranostic agent contacts the target cancer cell to kill the target cancer cell. The imaging method can be selected from fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy. The theranostic agent can be combined with a buffer solution prior to contacting the target cancer cell.

According to an embodiment, the present subject matter relates to a method of locating a tumor site in a patient, which can include administering the theranostic agent to the patient, contacting a tumor site with the theranostic agent, and locating the tumor site using an imaging method after the tumor site is contacted with the theranostic agent. The theranostic agent can be administered by intravenous injection. The theranostic agent can be combined with a buffer solution prior to administering the theranostic agent to the patient. The imaging method can include at least one of fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy. Luciferin can be administered to the patient prior to use of bioluminescence imaging. Once the tumor site has been determined, the tumor site can be irradiated with near-infrared light radiation which, when combined with the present compounds, can stop or inhibit the growth of the tumor. In an embodiment, the compounds can be administered to the patient six hours prior to imaging and treatment of the tumor.

According to an embodiment, the present subject matter relates to a method of stopping or inhibiting tumor growth in a patient, which can include administering the theranostic agent to the patient; contacting a tumor site with the theranostic agent; locating the tumor site using an imaging method after the tumor site is contacted with the theranostic agent; and subjecting the tumor site to near-infrared light irradiation while the theranostic agent is present at the tumor site to stop or inhibit the growth of the tumor. Subjecting the tumor site to near-infrared light irradiation while the theranostic agent is present at the tumor site generates reactive oxygen species to stop or inhibit the growth of the tumor. The theranostic agent can be administered by intravenous injection. The theranostic agent can be combined with a buffer solution prior to administering the theranostic agent to the patient. The imaging method can include at least one of fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy. Luciferin can be administered to the patient prior to use of bioluminescence imaging.

The present teachings are illustrated by the following examples.

EXAMPLES

Materials and Instruments

THF (Labscan) was purified by simple distillation from sodium benzophenone ketyl under nitrogen immediately before use. Zinc dust, titanium (IV) chloride, 4, 4'-difluorobnzophenone, diphenylamine, potassium tert-butoxide (t-BuOK) , 4-methoxyphenyl-4-pyridylketon, iodomethane (CH ₃I) , potassium hexafluorophosphate (KPF ₆) , H2DCF-DA, dimethyl sulfoxide (DMSO) , and other reagents were all purchased from Aldrich and used as received. Milli-Q water was supplied by Milli-Q Plus System (Millipore Corporation, United States) . Minimum essential medium (MEM) , Dulbecco’s modified eagle medium (DMEM) , fetal bovine serum (FBS) , penicillin and streptomycin, and phosphate buffered saline (PBS) were purchased from Invitrogen. ¹H and ¹³C NMR spectra were measured on Bruker ARX 400 NMR spectrometers using CD ₂Cl ₂ as the deuterated solvent. High-resolution mass spectrometry (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) . The observation of nanoparticle morphology was investigated using transmission electron microscopy (TEM, JEM-2010F, JEOL, Japan) . Size distribution of nanoparticles was conducted on dynamic light scattering (DLS) with a particle size analyzer (90 Plus, Brookhaven Instruments Co. USA) at a fixed angle of 90° at room temperature.

For cell culturing, luciferase-expressed 4T1 breast cancer cells and human HeLa cancer cells were cultured in the DMEM containing 10%FBS and antibiotics (100 units/mL penicillin and 100 g/mL streptomycin) in a 5%CO ₂ humidity incubator at 37 ℃. MDCK-II, U87 cells were cultured in the DMEM 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 overnight on a 35 mm petri dish with a cover slip or a plasma-treated 25 mm round cover slip mounted at the bottom of a 35 mm petri dish with an observation window. The live cells were incubated with Cor-AIE dots at certain concentration for certain time. The dye-labelled cells were mounted and imaged under Zeiss laser scanning confocal microscope (LSM7 DUO) . Conditions were as follows: for Cor-AIE dots, excitation laser: 560 nm, emission collection: 570-720 nm; for Hoechst 33342, excitation laser: 405 nm, emission collection: 430-470 nm.

For the histological study, the excised tumors of the mice were fixed in 4%formalin, processed into paraffin, sectioned at 5 μm thickness, and stained with hematoxylin and eosin (H&E) . The slices were examined by a digital microscope (Leica QWin) .

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 Compound 2

Compound 2: TiCl ₄ (1 mL, 9.0 mmol) was slowly added into a suspension of Zn dust (1.17 g, 18.0 mmol) in dry THF (50 mL) under -78 ℃. After reflux for 2 h, a mixture of compound 1 (2.325 g, 4.5 mmol) and 4-methoxyphenyl-4-pyridylketon (0.640 g, 3 mmol) in dry THF (20 mL) was added to the reaction. The mixture was allowed to continue reflux for another 5 h. After removal of the solvent by compressed air, the residue was extracted by DCM and dried over anhydrous Na ₂SO ₄. The crude product was purified on a silica-gel column using DCM as eluent. Compound 2 was isolated as a yellow solid in 70%yield. ¹H NMR (400 MHz, CD ₂Cl ₂, δ) : 8.33 (d, 2H, J= 0.011) , 7.27-7.22 (m, 8H) , 7.05-7.03 (m, 9H) , 7.02-7.01 (m, 2H) , 7.00-6.99 (m, 1H) , 6.97 (d, 2H, J= 0.013) , 6.95 (d, 2H, J=0.011) , 6.93 (d, 2H, J=0.004) , 6.92 (d, 2H, J =0.007) , 6.82 (d, 2H, 0.004) , 6.79 (d, 2H, 0.005) , 6.71 (d, 2H, J= 0.021) , 3.77 (s, 3H) ; ¹³C NMR (100 MHz, CD ₂Cl ₂, δ) : 158.50, 152.44, 148.92, 147.55, 147.50, 146.80, 146.49, 142.31, 137.21, 136.89, 136.76, 135.15, 132.52, 132.14, 132.07, 129.22, 129.17, 126.13, 124.45, 124.35, 122.99, 122.91, 122.51, 113.19, 55.12; HRMS (m/z) calcd. for C ₅₀H ₃₉NO ₃ [M] ⁺: 697.3093; found, 697.3121.

Example 2

Synthesis of TPP-TPA

Compound 2 (0.174g, 0.25 mmol) was dissolved in 20 mL toluene, after which 0.1 mL of CH ₃I (large overdose) was added to form a mixture. The reaction mixture was refluxed overnight. After cooling to room temperature, the precipitates were filtered and washed with cold toluene three times. The obtained solid was dissolved in 20 mL acetone and 100 mg of KPF ₆ was added for ion-exchange for 2 h. The solvent was removed and the solid was washed with water. Pure yellow product of TPP-TPA was obtained by recrystallization from DCM/hexane mixture (1: 5 by volume) in 99%yield. ¹H NMR (400 MHz, CD ₂Cl ₂, δ) : 8.11 (d, 2H, J = 0.016) , 7.45 (d, 2H, J = 0.016) , 7.33-7.30 (m, 4H) , 7.28-7.24 (m, 4H) , 7.13-7.08 (m, 7H) , 7.07-7.04 (m, 5H) , 6.98-6.93 (m, 4H) , 6.90-6.85 (m, 4H) , 6.80-6.75 (m, 4H) , 4.20 (s, 3H) , 3.79 (s, 3H) ; ¹³C NMR (100 MHz, CD ₂Cl ₂, δ) : 162.95, 159.34, 151.19, 148.96, 148.05, 147.03, 146.84, 142.76, 134.81, 133.82, 133.40, 132.92, 132.75, 132.60, 129.64, 129.51, 129.33, 125.53, 125.10, 124.10, 123.69, 121.12, 120.89, 114.17, 55.26, 47.43; HRMS (m/z) calcd. for C ₂₉H ₂₈NO ₃ ⁺ [M-PF ₆] ⁺: 712.3322; found, 712.3315.

Example 3

Preparation and Characterization of Cor-AIE dots and DSPE-AIE dots

Cor-PEG or DSPE-PEG (1 mg) powder and TPP-TPA (0.2 mg) were completely dissolved in THF (1 mL) . After that, the THF solution was added into 9 mL Milli-Q water (18.2 MU) slowly under continuous ultrasound (125 W) . The mixed solution was further kept in ultrasound for another 1 minute, then the THF was removed by evaporation while stirring under N ₂ at room temperature. Finally, the clear solution was obtained for use.

Example 4

Photophysical properties

UV-vis absorption and photoluminescence (PL) spectra of TPP-TPA are reflected in Figs. 8 and 1A. TPP-TPA absorbs at 440 nm with the absorption tail extended to 600 nm in DMSO, covering most of visible light range. Such solution emits almost no light even when increasing the water fraction (f _w) in the DMSO-H ₂O mixture up to 50%, which can be ascribed to the active intramolecular rotation of the aryl rings (Fig. 1C) . The emission of TPP-TPA was enhanced dramatically when the f _wwas over 50%. Fig. 1B shows the plot of emission intensity at 680 nm against f _w and the inset fluorescent photos of the red emission in the 99%aggregated solution compared to the negligible emission in DMSO. As shown in Fig. 1B, TPP-TPA exhibited typical AIE characteristics. Notably, the emission intensity slightly decreased after the f _w exceeded 80%, which is mainly attributed to the serious twisted intramolecular charge transfer (TICT) effect in the polar solvent water. Such effect can be further supported by the emission red shift of 10 nm from 80%to 99% (Fig. 9) and can also be indicated by the typical electron distribution of HOMO and LUMO in both ground and excited states (Figs. 1C and Figs. 10A-10B) . Additionally, the Stokes shift for TPP-TPA was evaluated to be 220 nm, which is much larger than the small Stokes shifts of less than 50 nm of most commercial NIR fluorophores, avoiding the light contamination of excitation light and self-absorption of emission during biomedical imaging.

Example 5

Preparation, photophysical properties, and ROS production of DSPE-AIE dots and Cor-AIE dots

The TPP-TPA-loaded NPs, were prepared by a nanoprecipitation method as shown in Fig. 11. TPP-TPA was formulated using corannulene-modified polyethylene glycol ( “Cor-PEG” ) having a bowl-shaped corannulene and 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) -2000] ( “DSPE-PEG” ) with linear alkyl chain as the encapsulation matrix, respectively, obtaining Cor-AIE dots and DSPE-AIE dots, which possess similar absorption to TPP-TPA itself in aqueous media (Fig. 12) . The sizes of Cor-AIE dots and DSPE-AIE dots were recorded by dynamic light scattering (DLS) with the values of 46.9 nm and 49.1 nm, respectively (Figs. 13A and 13B) . TEM was further used to confirm these nanoparticles bearing the spherical shape (Figs. 13C and 13D) . As suggested in Figs. 14A and 14B, Cor-AIE dots exhibited stronger emission with the quantum yield of 26.8%, which is four times larger than 6.7%for DSPE-AIE dots. In addition, the average fluorescence lifetime of Cor-AIE dots was measured to be 4.34 ns, which is about four times that of DSPE-AIE dots. The possibility of fluorescence resonance energy transfer (FRET) from corannulene to TPP-TPA can be completely ruled out because the whole absorption spectrum of corannulene is located on the UV range and its emission cannot reach the excitation of 500 nm. Therefore, it is likely that such enhanced emission and lengthened fluorescence lifetime originates from the enhanced radiative pathway of TPP-TPA.

Furthermore, 9, 10-anthracenediyl-bis (methylene) dimalonic acid (ABDA) , a commercially available ROS indicator, was used to evaluate the ROS production capacities of Cor-AIE dots and DSPE-AIE dots. Noteworthy, the absorbance of ABDA in water dramatically decreased in the presence of Cor-AIE dots (Up in Fig. 14C) under white light irradiation, whereas faint decrease of ABDA absorbance was observed for DSPE-AIE dots (Down in Fig. 14C) , suggesting that Cor-AIE dots were capable of producing more ROS to decompose ABDA more quickly than DSPE-AIE dots under the same experimental conditions. Plotting of the decomposition rate of ABDA in Fig. 14D quantitatively reveals that the ROS generation ability of Cor-AIE dots is around 5.4 times better than that of DSPE-AIE dots, indicative of the far higher ROS production efficiency of Cor-AIE dots.

Example 6

¹H titration and theoretical calculation

The underlying principle of the significantly enhanced fluorescence and ROS production described in Example 6 was studied. ¹H NMR titration experiment was first conducted to investigate the interactions between corannulene and TPP-TPA (Fig. 15A) . Upon stepwise addition of corannulene into the CD ₂Cl ₂ solution of TPP-TPA, the signals of the aromatic protons in 1-methylpyridinium of TPP-TPA gradually shifted upfield by 0.08 ppm (H _ain Fig. 15B) and 0.05 ppm (H _b in Fig. 15D) , while those of the protons in corannulene shifted by about 0.03 ppm (H _e) (Fig. 15C) to the higher field. Meanwhile, the methyl protons in 1-methylpyridinium (H _c) and methoxyphenyl (H _d) moieties of TPP-TPA were also shifted by around 0.07 ppm and 0.02 ppm upfield, respectively (Fig. 15E) . These chemical shifts evidently imply the unique interactions and the probably-relative positions between corannulene and TPP-TPA, with the positively-charged 1-methylpyridinium of TPP-TPA close to the electronegative bottom of corannulene and the TPA and methoxyphenyl units of TPP-TPA outside the bowl of corannulene. Some possible theoretical positions of corannulene and TPP-TPA are shown in Fig. 16A, which can result in the shielding effect of corannulene for 1-methylpyridinium of TPP-TPA. Density functional theory (DFT) calculation based on TD-DFT/M06-2X/6-31G (d) method was also used to study the ISC process (Fig. 16B) . The related optimized molecular geometries of S ₀, S ₁ and T ₁ states for TPP-TPA at M06-2X/6-31G (d) level were calculated in the absence and presence of corannulene. The gaps between the S ₁ and T ₁ states (ΔE _ST) for TPP-TPA upon the interaction with corannulene were significantly decreased compared with that for TPP-TPA alone, and the respective SOC constants (ξ (S ₁, T ₁) ) between S ₁ and T ₁ were also increased, suggesting the more accessible ISC process in the presence of corannulene.

The bowl-shaped corannulene possesses a super-hydrophobic skeleton and an ultra-rigid curvature compared with the flexible alkyl-chained DSPE, and hence constructs a more confined microenvironment in aqueous solution. Corannulene bears a large dipole moment and the bottom of the corannulene bowl is electronegative with the periphery being electropositive, which attracts inherent positively-charged TPP-TPA by dipole-dipole and electrostatic interactions. Compared to the intra-particle microenvironment of DSPE-AIE dots, the more confined and rigid micro-cavity within Cor-AIE dots (resulting from the uniqueness of corannulene structure as well as the strong interactions between corannulene and TPP-TPA) restricts the intramolecular rotation of the phenyl rings in TPP-TPA to a much greater extent than that of DSPE-AIE dots, and thus inhibits nonradiative relaxation more effectively (Figs. 14E-14F) . As the absorbed energy of TPP-TPA is fixed, the highly suppressed nonradiative decay for Cor-AIE dots reasonably makes its absorbed energy flow to both the fluorescence pathway and ISC process, achieving significantly amplified emission and ROS generation. This is supported by the theoretical formulas of Φ _F = k _r/ (k _r+k _nr+k _ISC) and Φ _ISC = k _ISC/ (k _r+k _nr+k _ISC) , where the dramatic decrease in nonradiative rate k _nr undoubtedly induces the extensive increase of the fluorescence emission efficiency Φ _F and the ISC efficiency Φ _ISC. In addition, as TPP-TPA has strong electron donor-acceptor structures in the backbone, the fast process of charge transfer-induced fluorescence quenching in aqueous media competes with ROS production. Cor-AIE dots can also provide a more isolated hydrophobic environment to reduce the polar-solvent disruption (such as TICT) for TPP-TPA, bringing about further enhancement of emission efficiency and ROS production. Indeed, the reduced TICT effect in Cor-AIE dots was reflected by the slight blue shift of about 10 nm in the emission spectrum compared to DSPE-AIE dots (Fig. 14A) .

Example 7

Cancer phototheranostics

Due to the excellent NIR emission output and ROS production, the utility and strength of Cor-AIE dots in cancer phototheranostics were investigated. After it was demonstrated that Cor-AIE dots could be internalized in cancer cells and generate ROS within cells effectively (Figs. 17A-17I) , in vivo studies were carried out using a peritoneal carcinomatosis-bearing mouse model, which was established by intraperitoneal inoculation of murine 4T1 cancer cells, as described below. All animal studies were performed in compliance with the guidelines set by Tianjin Committee of Use and Care of Laboratory Animals and the overall project protocols were approved by the Animal Ethics Committee of Nankai University. All the mice were obtained from Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China) .

To establish peritoneal carcinomatosis-bearing mouse model, a total of 300,000 luciferase-expressed 4T1 cancer cells in 0.1 mL of PBS buffer were intraperitoneally injected into the Balb/c mice. After about 5 days, small tumor nodules were formed and scattered in the mouse peritoneal cavity, which could be detected by bioluminescence imaging upon injection of a solution of D-luciferin (150 mg/kg) . It is worthy to note that the in vivo inoculated 4T1 cancer cells express luciferase. As such, the living cancer cells emitted bioluminescence when the tumor-bearing mice were administered with the substrate of luciferase (D-luciferin) , allowing for precise tracking of the tumor nodules in the mouse peritoneal cavity.

NIR fluorescence image-guided surgery

An experiment was conducted to determine whether the highly boosted NIR fluorescence in Cor-AIE dots can be used in image-guided cancer surgery. In this experiment, the peritoneal carcinomatosis-bearing mouse model was selected because of the large number of tumor nodules, especially those with diameters < 1 mm, present in the mouse peritoneal cavity. In practice, the submillimeter tumor nodules are generally difficult for a surgeon to spot and are often missed. These missed small tumors are the major culprits for in situ cancer recurrence. Prior NIR fluorescent probes have generally been incapable of accurately locating the submillimeter tumors, mainly because of the low NIR emission output of these probes.

150 μL of Cor-AIE dots (1 mg mL ^-1 based on TPP-TPA) was intravenously injected into the peritoneal carcinomatosis-bearing mice. At 24 h post-injection, the mice were anesthetized. The abdominal cavity of the mice was opened, followed by bioluminescence and fluorescence imaging during surgery. Bioluminescence imaging was performed using the Xenogen

Lumina II system post intraperitoneal injection of D-luciferin (150 mg/kg) into the mice. The bioluminescence signals were quantified in units of maximum photons per second per square centimeter per steridian. Fluorescence imaging was carried out using a Maestro EX in vivo fluorescence imaging system (CRi, Inc.; excitation: 455 nm, spectral imaging from 500 nm to 900 nm) .

In vivo NIR fluorescence imaging during surgery indicated that bright Cor-AIE dot fluorescence clearly lighted up many tissues as well as their boundaries with rather high signal-to-background ratio (Fig. 18A) . As luciferase-expressed 4T1 tumors have bioluminescence, the scattering intraperitoneal tumor nodules were totally and specifically displayed upon bioluminescence imaging post injection with D-luciferin. As shown in Fig. 18A, the bioluminescence signal from luciferase and NIR fluorescence signal from Cor-AIE dots were colocalized perfectly on the surface of the intestines, suggesting that the Cor-AIE dots-visualized tissues are indeed tumors, which are further confirmed by the hematoxylin and eosin (H&E) histological staining. Noteworthy, from in vivo fluorescence imaging, the fluorescence intensity ratio of tumor to normal intestine is as high as 5.2 for Cor-AIE dots, which exceeds the Rose criterion. As depicted in Fig. 18B, the tumor nodules on the peritoneum are also roughed out with an even larger tumor-to-peritoneum ratio of about 8.0. These results quantitatively demonstrate that the Cor-AIE dots possess remarkable EPR effect, permitting high tumor uptake via passive targeting and thus leading to visualization of intraperitoneal tumor nodules and their boundaries in a specific and high-contrast manner. The fluorescence intensity ratios of tumor to normal tissues achieved by Cor-AIE dots are significantly higher than those of many reported NIR fluorescent probes including methylene blue and indocyanine green. More importantly, it is also found that the Cor-AIE dots can distinctly delineate the tumor nodules with sizes < 1 mm in the peritoneal cavity (indicated by the red arrows in Fig. 18) , revealing that Cor-AIE dots are efficacious in sharply visualizing submillimeter tumors due to their highly boosted NIR emission.

As Cor-AIE dots can serve as an extremely effective NIR fluorescent probe to precisely visualize tumors and their boundaries against normal tissues, its application in guidance for surgical tumor removal was studied. To this end, a surgeon from Tianjin First Central Hospital (Tianjin, China) was invited to conduct the operation. 150 μL of Cor-AIE dots (1 mg mL ^-1 based on TPP-TPA) was injected into the peritoneal carcinomatosis-bearing mice via the tail vein. After 24 h, tumor resection surgery was first performed by a surgeon from Tianjin First Central Hospital (Tianjin, China) without imaging guidance (unguided) . This was followed by a second surgery on the same mice by the guidance of Cor-AIE dots fluorescence. The excised tumor nodules were analyzed by both fluorescence imaging and bioluminescence imaging. The tumor sizes resected from the first and second surgery were also quantified.

As shown in Figs. 19A and 19B, when the surgeon was blinded to the NIR fluorescence imaging by Cor-AIE dots, he removed many intraperitoneal tumors with relatively large diameters (> 1 mm) . However, after the unguided surgery, there were a number of residual tumor nodules remaining in the peritoneal cavity indicated by Cor-AIE dots, which are mainly the ones with diameters < 1 mm (Fig. 19B) . The surgeon then performed a second operation under the guidance of Cor-AIE dots fluorescence, which achieved almost complete removal of the remaining small tumors (Figs. 19C-E) , confirmed by the negligible intraperitoneal bioluminescence signal. It is noted that all of the harvested tumor nodules (mainly submillimeter ones) in the second operation have bioluminescence signals (Fig. 19D) , validating the precise cancer surgery assisted by Cor-AIE dots. After unguided and Cor-AIE dot fluorescence image-guided surgery, respectively, the survival of the mice was monitored over time with each group containing 10 mice. Due to the high malignancy and fast growth of intraperitoneal 4T1 tumors, all of the 10 mice in the unguided surgery group died within 2 weeks. Encouragingly, 7 of 10 mice in the cohort with Cor-AIE dot fluorescence image-guided surgery were able to survive during a 2 week-monitoring duration (Fig. 20) . These results illustrate that Cor-AIE dots greatly enhance cancer surgery outcome and considerably prolong the lifetimes of tumor-bearing mice post operation by accurately lighting up submillimeter tumor nodules.

Cor-AIE dots in photodynamic tumor therapy

In many cases, surgeons in practice cannot perform tumor-removal operations after opening their patients’abdomen because there are so many small tumors that are difficult to excise manually. As a result, surgeons are forced to close the abdominal wall and choose a treatment strategy other than surgery.

Since in addition to high NIR emission, strong ROS generation ability is the other signature of Cor-AIE dots by virtue of the important contribution of corannulene, the feasibility of Cor-AIE dots in photodynamic tumor therapy was examined in the aforementioned cases in which surgical resection was not feasible after the abdomen was opened. To this end, peritoneal carcinomatosis-bearing mice were randomly divided into 5 groups (n = 8 per group) . which were designated as "Saline" , "Light (L) " , "DSPE-AIE dots + L" , "Cor-AIE dots + L" , and "Cor-AIE dots" . On day 0, bioluminescence imaging was first carried out for all of the mice in the 5 groups. Subsequently, Cor-AIE dots (1 mg mL ^-1 based on TPP-TPA; 150 μL) were intravenously injected into the mice in "Cor-AIE dots" and "Cor-AIE dots + L" groups. At 24 h post-injection (day 1) , the mouse abdomen was opened for each mouse in these 2 groups. Then, for each mouse in "Cor-AIE dots + L" group, the whole peritoneal cavity was irradiated with white light (0.4 W cm ^-2) for 10 min, followed by closure of the abdomen, utilizing surgical sutures. On the other hand, for mice in "Cor-AIE dots" group, the mouse abdomen was subsequently closed without white light irradiation. For "DSPE-AIE dots + L" group, DSPE-AIE dots (1 mg mL ^-1 based on TPP-TPA; 150 μL) were administered to the mice via the tail vein on day 0, followed by the same treatment as that for the mice in "Cor-AIE dots + L" group on day 1. For "Saline" group, saline was intravenously injected into the mice on day 0, followed by the same treatment as that for the mice in "Cor-AIE dots" group on day 1. Finally, for "Light" group, the mice were untreated on day 0, but on day 1, the mice were treated following the same procedure as that for "Cor-AIE dots + L" group. The tumor size and growth were monitored during a 9-day study duration through bioluminescence imaging with the Xenogen

Lumina II system post intraperitoneal injection of D-luciferin (150mg/kg) into the mice. The survival rates were also examined throughout the study.

Fig. 21A and Fig. 22 exhibit time-dependent bioluminescence imaging of the tumor-bearing mice in each group. It was apparent that tumors with intense bioluminescence signal exist in the abdomen of mice before different treatments on day 0. Dramatically, after receiving the treatment of “Cor-AIE dots + L” , the intraperitoneal tumor growth of mice was considerably suppressed, as evidenced by the similar average bioluminescence intensity of intraperitoneal tumors on day 9 to that on day 0 (Fig. 21B) . As controls, both the treatments of “Cor-AIE dots” and “Light (L) ” failed to slow down the growth of intraperitoneal tumors, compared with that of “Saline” (Fig. 22 and Fig. 21B) , revealing that the highly efficacious anticancer activity in “Cor-AIE dots + L” group roots in the PDT via Cor-AIE dots generating ROS in tumors. It is important to note that the PDT of DSPE-AIE dots did not have any inhibitory effects on tumor growth (Fig. 21A and 21B) . This result not only indicates the high malignancy of intraperitoneal tumors, but also implies that the ROS production ability of DSPE-AIE dots is not strong enough to work in this tumor-bearing animal model. The treatment of “Cor-AIE dots + L” lead to greatly prolonged lifetimes of mice and the median survival time for “Cor-AIE dots + L” group was far longer than that for “DSPE-AIE dots + L” group (Fig. 21C) . The sharp comparison in the antitumor efficacy between the PDT of Cor-AIE dots and DSPE-AIE dots reasonably highlights the necessity and importance of the Cor-AIE dots.

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 and near-infrared absorption, the compound having a backbone structural formula selected from the group consisting of:

wherein each R ₁, R ₁′, R ₁” and R ₁”’ is independently selected from the group consisting of

wherein each R ₂, R ₃, R ₄, R ₅, R ₆, and R ₇ is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, C _nH _2n+1, C ₁₀H ₇, C ₁₂H ₉, alkoxy, OC ₆H ₅, OC ₁₀H ₇ and OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nSH, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, N (C _nH _m) ₂, and SC _nH _m;

wherein n and m are each independently an integer from 0-10;

wherein A is a monovalent counter ion; and

wherein the monovalent counter ion is always present in the compound.
The compound according to claim 1, wherein the compound is:
The compound according to claim 1, wherein the compound is in nanoparticle form.
The compound according to claim 1, wherein the monovalent counter ion is selected from the group consisting of I ^-, Cl ^-, Br ^-, PF ₆ ^-, ClO ₄ ^-, BF ₄ ^-, BPh ₄ ^-, and CH ₃PhSO ₃ ^-.
A theranostic agent comprising the fluorescent compound of claim 1 in nanoparticle form and corannulene-modified polyethylene glycol in nanoparticle form, the corannulene-modified polyethylene glycol encapsulating the fluorescent compound.
A method of preparing the theranostic agent of claim 5, comprising combining the fluorescent compound with the corannulene-modified polyethylene glycol using nanoprecipitation under ultrasonic conditions.
A method of killing cancer cells, comprising:

contacting a target cancer cell with the theranostic agent according to claim 5;

imaging the target cancer cell while the theranostic agent contacts the target cancer cell using an imaging method selected from the group consisting of fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy; and

subjecting the target cancer cell to near infrared light irradiation while the theranostic agent is contacting the target cancer cell to kill the target cancer cell.
The method of claim 7, further comprising combining the theranostic agent with a buffer solution prior to contacting the target cancer cell.
A method of locating a tumor site in a patient, comprising:

administering the theranostic agent of claim 5 to the patient;

contacting the tumor site with the theranostic agent; and

locating the tumor site using an imaging method after the tumor site is contacted with the theranostic agent.
The method of claim 9, wherein the theranostic agent is administered by intravenous injection.
The method of claim 9, further comprising combining the theranostic agent with a buffer solution prior to administering the theranostic agent to the patient.
The method of claim 9, wherein the imaging method comprises at least one of fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy.
The method of claim 9, further comprising administering luciferin to the patient, wherein the imaging method comprises bioluminescence imaging.
A method of stopping or inhibiting tumor growth in a patient, comprising:

administering the theranostic agent of claim 5 to the patient;

contacting a tumor site with the theranostic agent;

locating the tumor site using an imaging method after the tumor site is contacted with the theranostic agent; and

subjecting the tumor site to near-infrared light irradiation while the theranostic agent is present at the tumor site to stop or inhibit the growth of the tumor.
The method of stopping or inhibiting tumor growth in a patient according to claim 14, wherein subjecting the tumor site to near-infrared light irradiation while the theranostic agent is present at the tumor site generates reactive oxygen species to stop or inhibit the growth of the tumor.
The method of claim 14, wherein the theranostic agent is administered by intravenous injection.
The method of claim 14, further comprising combining the theranostic agent with a buffer solution prior to administering the theranostic agent to the patient.
The method of claim 14, wherein the imaging method comprises at least one of fluorescence microscopy, bioluminescence imaging, and confocal laser scanning microscopy.
A fluorescent nanoparticle composition, comprising a fluorescent compound exhibiting aggregation induced emission properties and corannulene-modified polyethylene glycol, the corannulene-modified polyethylene glycol encapsulating the fluorescent compound, the fluorescent compound having a backbone structural formula selected from the group consisting of:

wherein each R ₁, R ₁′, R ₁” and R ₁”’ is independently selected from the group consisting of

wherein each R ₂, R ₃, R ₄, R ₅, R ₆, and R ₇ is independently selected from the group consisting of H, alkyl, unsaturated alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, C _nH _2n+1, C ₁₀H ₇, C ₁₂H ₉, alkoxy, OC ₆H ₅, OC ₁₀H ₇ and OC ₁₂H ₉, C _nH _2nCOOH, C _nH _2nNCS, C _nH _2nN ₃, C _nH _2nNH ₂, C _nH _2nSH, C _nH _2nCl, C _nH _2nBr, C _nH _2nI, N (C _nH _m) ₂, and SC _nH _m, ;

wherein n and m are each independently an integer from 0-10;

wherein A is a monovalent counter ion selected from the group consisting of I ^-, Cl ^-, Br ^-, PF ₆ ^-, ClO ₄ ^-, BF ₄ ^-, BPh ₄ ^-, and CH ₃PhSO ₃ ^-; and

wherein the monovalent counter ion is always present in the compound.
The fluorescent nanoparticle composition according to claim 19, wherein the compound is: