Fluorescent compounds capable of accelerating cancer diagnosis and treatment

Abstract

The invention discloses an aggregation-induced emission fluorescent compound with near infrared (NIR) luminescence. The fluorescent compound is wrapped by a polymer matrix to form a nano aggregate, which proves that the fluorescent compound shows excellent photothermal conversion efficiency while maintaining high fluorescence quantum yield. Therefore, compared with the mode of relying on high permeability and retention (EPR) effect, the nanometer aggregate accumulation guided by the photo-induced thermoacoustic effect (PTA) can further improve the photo-thermal treatment effect. In-vitro and in-vivo verification further confirms that the nano-aggregate shows excellent fluorescence-guided optical diagnosis and treatment capability in the aspect of tumor elimination.

Description

[0001] Cross - reference to related applications

[0002] This invention is a national phase application in China of International Application No. PCT / CN2023 / 122903, filed on September 28, 2023, which claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 417,322, filed on October 19, 2022. The entire content of each of the foregoing applications is incorporated herein by reference. Technical field

[0003] The present invention relates to near - infrared (NIR) luminescent compounds with aggregation - induced emission and their application in image - guided optical diagnosis and therapy. Background art

[0004] Cancer, as one of the most serious health problems globally, causes extremely high mortality. Although various intervention measures such as chemotherapy, radiotherapy, and surgery have been adopted to treat cancer, its severe side effects and unsatisfactory treatment outcomes are still far from satisfactory. Therefore, developing more effective cancer therapies is necessary but challenging. Fluorescence - guided optical diagnosis and therapy technology uses light irradiation to simultaneously achieve cancer diagnosis and treatment, and has become a promising tool due to its controllability, non - invasiveness, and simultaneous treatment process. However, robust phototherapy systems capable of both diagnosis and treatment are extremely scarce because it is very difficult to handle complex energy decay pathways during the excitation process.

[0005] Theranostics usually requires multifunctional reagents, which are difficult to achieve in a single molecule. Utilizing the energy of light irradiation, theranostic agents can ablate cancer cells under the guidance of single - or multimodal imaging, thus promoting cancer diagnosis and treatment simultaneously. Traditionally, several components with their respective functions, such as drugs and probes, need to be packaged in a nanoparticle as a multifunctional nanomedicine. However, the complexity and cost of this "one - size - fits - all" approach limit its development in clinical applications.

[0006] Another key issue limiting the effective utilization of theranostic agents is delivery efficiency. Although the enhanced permeability and retention (EPR) effect has been widely recognized for decades, only about 0.7% (median) of the administered dose reaches solid tumors. In addition, it takes 24 to 48 hours for the EPR to deliver the theranostic agent to the target tissue, and the targeting modification is usually chemical (click reaction) or biological (antibody, biomarker, etc.). Currently, the EPR-based delivery method is undoubtedly time-consuming, sensitive, complex, expensive, and inefficient. Most importantly, it has been proven that EPR only exists in small animals such as mice, and there is no EPR in the human body. Therefore, for cancer treatment, there is an urgent need to develop a simple and faster delivery method. Summary of the Invention

[0007] The present invention provides a simple physical strategy to promote the progress of drug development and reduce the toxic side effects of existing optoacoustic nanomedicine. Low-dose and milder laser irradiation can be achieved through the accumulation facilitated by the photoinduced thermoacoustic (PTA) effect. Therefore, by combining multifunctional near-infrared (NIR)-absorbing nanoclusters with a rapid delivery optoacoustic agent platform, the designed materials can be fully utilized for disease treatment.

[0008] In one embodiment, the present invention discloses a fluorescent compound. The fluorescent compound exhibits aggregation-induced emission characteristics, and the fluorescent compound has the following main structural formula:

[0009]

[0010] wherein R is selected from straight-chain, branched-chain, cyclic alkyl, alkylphenyl, alkylthienyl, and other alkyl aromatics containing 2 to 40 carbon atoms, and one or more non-adjacent carbon atoms may be selectively substituted by –O–, –S–, –C(O)–, –C(O–)–O–, –O–C(O)–, –O–C(O)–O–, or –C≡C–, and one or more hydrogen (H) atoms may be selectively replaced by fluorine (F), chlorine (Cl), bromine (Br), iodine (I), or cyanide (CN), or represents an aromatic, heteroaromatic, aryloxy, heteroaryloxy, aromatic carbonyl, heteroaromatic carbonyl, aromatic carbonyloxy, heteroaromatic carbonyloxy, aryloxycarbonyl, or heteroaryloxycarbonyl having a 4- to 30-atom ring, and these ring molecules are unsubstituted or substituted by one or more non-aromatic groups;

[0011] wherein each π is independently selected from the following combinations:

[0012]

[0013] and

[0014] wherein each X is independently selected from the following groups:

[0015]

[0016] In another embodiment, the present invention discloses a theranostic nanoaggregate, comprising:

[0017] nanoparticles comprising the aforementioned fluorescent compound; and

[0018] a polymeric nanoshell covering the aforementioned nanoparticles, wherein the polymeric nanoshell is formed by encapsulating the nanoparticles with a polymeric matrix.

[0019] In yet another embodiment, the present invention discloses a method for killing cancer cells, comprising:

[0020] contacting a target cancer cell with the aforementioned theranostic nanoaggregate;

[0021] when the aforementioned theranostic nanoaggregate contacts the target cancer cell, imaging the target cancer cell, using an imaging method selected from the following combination: fluorescence microscopy, bioluminescence imaging, confocal laser scanning microscopy, and photoacoustic microscopy; and

[0022] when the aforementioned theranostic nanoaggregate contacts the target cancer cell, irradiating the target cancer cell with light, converting the light into heat to kill the target cancer cell.

[0023] In still another embodiment, the present invention discloses a method for stopping, inhibiting, or eliminating tumor growth, comprising:

[0024] administering the aforementioned theranostic nanoaggregate to a mammal;

[0025] contacting the aforementioned theranostic nanoaggregate with a tumor site;

[0026] after the tumor site contacts the theranostic nanoaggregate, localizing the tumor site using an imaging method;

[0027] performing a preliminary pulsed light irradiation on the tumor site to enhance the vascular permeability near the tumor site, thereby promoting sufficient accumulation of the theranostic nanoaggregate at the tumor site; and

[0028] performing a subsequent continuous light irradiation on the tumor site, at which time there is sufficient theranostic nanoaggregate at the tumor site to convert light energy into heat energy, so as to raise the temperature of the tumor site to a desired temperature to stop or inhibit tumor growth, or eliminate the tumor.

[0029] The above description is only a summary of the technical solution of the present invention. The following provides a complete embodiment of the present invention in conjunction with the drawings, so that those of ordinary skill in the art can more easily understand the operation process, purpose, features, and advantages of the present invention. Description of the Drawings

[0030] Reading the following detailed description of the complete embodiment with reference to the drawings, the present invention can be fully understood, wherein:

[0031] Figure 1 Showing the synthetic routes of TBT-2(1P-DPA), TBT-2(2P-DPA), and TBT-2(TP-DPA).

[0032] Figure 2 Showing the chemical structure of TBT-2(TP-DPA).

[0033] Figure 3a The molecular design principles and chemical structures of TBT-2(1P-DPA), TBT-2(2P-DPA), and TBT-2(TP-DPA). Figure 3b Diagrams of the S0 optimized geometric structures and frontier molecular orbitals (LUMOs and HOMOs) obtained from theoretical calculations using B3LYP / 6-31G*. Figure 3c For the normalized absorption spectra of the compound in THF solution (10 μM), and Figure 3d For the normalized photoluminescence (PL) spectra. Figure 3e A graph of the maximum absorption wavelength peak (hollow circle) and the maximum emission wavelength peak (solid circle) in THF, as well as the corresponding Stokes shifts of the three compounds.

[0034] Figure 4a Schematic diagram for the preparation of PTA nanoaggregates. Figure 4b Dynamic light scattering (DLS) distribution map of PTA nanoaggregates in water. Inset: Transmission electron microscope (TEM) image of the nanoaggregates. Figure 4c Normalized absorption and photoluminescence (PL) spectra of PTA nanoaggregates. Figure 4d Photothermal conversion curve of PTA nanoaggregates. Figure 4e Photoacoustic spectrum of PTA nanoaggregates in aqueous solution. Inset: Photoacoustic imaging maps of PTA nanoaggregates at different concentrations (0, 50, 100, 200, and 500 μg / mL).

[0035] Figure 5Calculation of the photothermal conversion efficiency of TBT-2(TP-DPA) nanoclusters. The PTA nanoclusters (1 mg / mL) were irradiated with 808 nm light (1 W / cm2) for 5 minutes and then the laser was turned off. The heat transfer time constant of the PTA nanoclusters was calculated to be τS = 186.1 s.

[0036] Figure 6 Photacoustic spectrum of TBT-2(TP-DPA) nanoclusters in aqueous solution.

[0037] Figure 7a Dual-wavelength (532 nm and 840 nm) ORPAM imaging of a representative mouse tumor that was intravenously injected with TBT-2(TP-DPA) PTA nanoclusters (1 mg / mL, 200 μL), followed by a series of consecutive scans. Scale bar: 1 mm. Figure 7b Quantitative analysis of the accumulation of nanoclusters in the flank tumor by the photoacoustic intensity in Fig. a. Figure 7c NIR-II fluorescence imaging of the EPR group after intravenous injection of PTA nanoclusters (1 mg / mL, 200 μL). Figure 7d Quantitative analysis of the time-dependent accumulation of nanoclusters in the flank tumor in the EPR group by the fluorescence intensity in Fig. c. Figure 7e NIR-II fluorescence imaging of bilateral tumors after intravenous injection (i.v.) of the prepared nanoclusters (1 mg / mL, 200 μL). The right tumor received 808 nm pulsed laser (ORPAM and ORPAM+PAT groups) or CW laser treatment, while the left tumor received no treatment. Figure 7f Quantitative analysis of the accumulation of nanoclusters in the flank tumor by the fluorescence intensity in Figs. c and e.

[0038] In vitro photothermal properties of PTA nanoclusters. Figure 8a Temperature changes of PTA nanoclusters under 808 nm laser irradiation at different power densities (from low to high: 0.2, 0.4, 0.6, 0.8, and 1 W / cm 2 ) for 5 minutes. Figure 8b Temperature changes of nanoclusters at different concentrations (from low to high: 0, 0.03125, 0.0625, 0.125, 0.25, 0.1, and 1 mg / mL) under 808 nm laser irradiation (1 W / cm 2 ) for 5 minutes. Figure 8c Photothermal conversion curve of five near-infrared laser on / off cycles (808 nm). Figure 8dFlow cytometry analysis of 4T1 cells after various treatments, including untreated cells (control), cells treated with PTA nanoclusters (PTA nanoclusters), cells treated with 808 nm CW laser (808 nm CW laser), and cells treated with both PTA nanoclusters and 808 nm CW laser (PTA nanoclusters + 808 nm CW laser). Figure 8e Live / dead detection results of 4T1 cells after different treatments shown in d. Laser power: 1 W / cm 2 . Scale bar: 50 μm.

[0039] Figure 9 In vitro photothermal properties of PTA nanoclusters. Infrared thermal imaging of TBT-2 (TP-DPA) nanoclusters at different concentrations (0.03125 - 1 mg / mL) irradiated with 808 nm laser (1 W / cm2) for 5 minutes.

[0040] Figure 10 Survival rate of 4T1 cells after treatment with different concentrations of TBT-2 (TP-DPA) nanoclusters (0, 10, 25, 50, 100, and 250 μg / mL) for 24 hours.

[0041] Efficacy of photothermal therapy (PTT) of PTA nanoclusters against subcutaneous 4T1 tumors (in vivo). Figure 11a For: Infrared thermal imaging of 4T1 tumor-bearing mice after 808 nm (1 W / cm 2 ) laser irradiation for 10 minutes after various treatments. Figure 11b For: Corresponding temperature change curves at the tumor sites of 4T1 tumor-bearing mice in each group. Figure 11c For: Tumor growth curves of mice in different groups. Figure 11d For: Body weight change curves of mice in different groups. Figure 11e For: Representative digital images and corresponding H&E staining results of tumor sections of mice in each experimental group at different time points. Data are presented as mean ± standard deviation (n = 3 - 5 mice in all groups); 0.01 < *P < 0.05, 0.001 < **P < 0.01, ***P < 0.001. Scale bar: 50 μm.

[0042] Figure 12 Digital images of tumors on the 14th day after various treatments in photothermal therapy. Note: (a) control group, (b) 840 nm pulsed laser + 808 nm continuous wave laser, (c) optical diagnostic agent (PTA), (d) EPR 24 hours + photothermal therapy (PTT), (e) optical diagnostic agent (PTA) + photothermal therapy (PTT).

[0043] Figure 13For the in vivo biosafety analysis of PTA nanoaggregates. Blood routine analysis data, including the values of white blood cells (WBC), red blood cells (RBC), platelets (PLT), lymphocytes (Lym), hemoglobin (HGB), mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), and mean corpuscular hemoglobin concentration (MCHC) of the control group and mice treated with PTA nanoaggregates.

[0044] Figure 14 For the in vivo biosafety analysis of PTA nanoaggregates. Serum biochemical data of the control group and mice treated with PTA nanoaggregates were measured, including ALT, AST, total protein, albumin (ALB), creatinine (CREA), total cholesterol, blood urea nitrogen (BUN), and triglyceride levels.

[0045] Figure 15 H&E staining images of the major organs (heart, liver, spleen, lung, and kidney) of control group mice and mice treated with PTA nanoaggregates collected on the 7th and 14th days after intravenous injection of PTA nanoaggregates. Scale bar: 100 μm. Detailed implementation manners

[0046] Definition

[0047] In the present application, when an element or molecular group is said to be included in and / or selected from the list of the recited elements or molecular groups, it should be understood that the element or molecular group can be any one of the recited elements or molecular groups, or the element or molecular group can be selected from a group consisting of two or more of the recited elements or molecular groups. In addition, it should be understood that, without departing from the spirit and scope of the present guidance, the elements, compositions, devices, or methods described herein can be combined in various ways, whether explicitly or implicitly in this document.

[0048] Unless otherwise explicitly stated, the use of the terms "comprising", "including", "having", or "possessing" is generally understood to be open-ended and non-restrictive.

[0049] The use of the singular here includes the plural (and vice versa) unless otherwise explicitly stated. In addition, under the description of using the term "about" before a quantitative value, the present patent also includes the specific quantitative value itself unless otherwise explicitly stated. When the term "about" is used herein, it means within ±10% of the standard value unless otherwise stated or inferred.

[0050] There is no corresponding critical relationship between the order of steps and the execution of an operating procedure as long as the guiding rules in the patent are still valid. In addition, two or more actions can be performed simultaneously.

[0051] “λ ex ” herein refers to the excitation wavelength.

[0052] The term “aggregation-caused quenching” or “ACQ” herein refers to a significant decrease in the fluorescence intensity of a fluorophore after the aggregation of π-conjugated fluorophores. The formation of aggregates is considered to “quench” the luminescence of the fluorophore.

[0053] The term “aggregation-induced emission” or “AIE” herein refers to a phenomenon in which a compound exhibits a significant enhancement in luminescence when aggregated in an amorphous or crystalline (solid) state, while showing weak or almost no luminescence in dilute solutions.

[0054] 「Emission intensity」 herein refers to the magnitude of the fluorescence / phosphorescence amount usually obtained from fluorescence spectrometer or fluorescence microscope measurements; 「fluorophore」 or 「fluorescent source」 herein refers to a molecule that exhibits fluorescence emission; 「luminescent source」 or 「lumophore」 herein refers to a molecule that exhibits luminescence; and 「AIEgen」 herein refers to a molecular group that exhibits AIE properties.

[0055] The 「donor」 material used herein refers to an organic material, such as an organic nanoparticle material, in which holes are the main current or valence charge carriers.

[0056] The 「acceptor」 material used herein refers to an organic material, such as an organic nanoparticle material, in which electrons are the main current or valence charge carriers.

[0057] The 「theranostic agent」 used herein refers to an organic material, such as an organic nanoparticle material, that has both diagnostic and therapeutic capabilities.

[0058] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0059] Fluorescent compounds and fluorescent nanoclusters

[0060] Nanoscale aggregates, including the aggregation of molecules and nanoparticles, have attracted extensive research interest due to their different properties from molecular-level materials. For example, nanocrystals of drugs can solve the problems of insolubility and bioavailability, and have better clinical effects than molecules. Molecular nanoscale aggregates with aggregation-induced emission (AIE) properties can emit higher-intensity luminescence compared to their dispersed molecules, which has advanced the development of the fields of imaging, diagnosis, treatment, and optoelectronic devices in the past 20 years.

[0061] However, aggregate science remains largely unexplored because, although aggregates may be the smallest entities in practical operation, they have received far too little attention, especially in the application of multifunctional materials. Aggregates can be homogeneous or heterogeneous. Homogeneous aggregates of a single component can exhibit properties that molecules do not have by packing them closely together. Most AIE materials are homogeneous aggregates, in which different parts of the nanoaggregates can perform various functions. The research results of J-aggregates also show that the packing pattern can affect the behavior of aggregates. Heterogeneous aggregates offer more possibilities due to the interactions between components, but the results are difficult to predict due to the complexity of the system. Therefore, controlling multifunctionality without changing the original chemical formula and synthesis of nanoaggregates is worth considering.

[0062] Homogeneous aggregates from a single component are thus highly regarded, and their properties and functions are controllable and predictable. Aggregation-induced emission luminogens (AIEgens) are ideal design templates for constructing "all-in-one" optical diagnostic agents, perfectly solving the aggregation-caused quenching (ACQ) challenge and showing low in vivo side toxicity. AIEgens usually do not emit light in solution because the excited-state energy is consumed by non-radiative relaxation of intramolecular motion. This unusual property makes AIEgens very suitable for fabricating fluorescent nanoaggregates with ultra-high brightness and photobleaching thresholds.

[0063] The present invention provides a fluorescent compound having aggregation-induced emission (AIE) properties and showing near-infrared emission. Near-infrared (NIR) fluorescence imaging has attracted extensive attention because it has lower tissue autofluorescence interference and deeper tissue penetration depth compared with visible light. The fluorescent compound can exist in the form of nanoparticles.

[0064] The present invention also provides a fluorescent nanoaggregate, which comprises:

[0065] nanoparticles containing a fluorescent compound; and

[0066] a polymer nanoshell, wherein the polymer nanoshell covers the nanoparticles and is formed by using a polymer matrix to wrap the nanoparticles. Hereinafter, the fluorescent nanoaggregate can also be referred to as a "theranostic nanoaggregate". Wrapping the fluorescent compound in a polymer matrix can improve the microenvironment inside the particles, thereby enhancing fluorescence.

[0067] In one embodiment, the present invention provides a fluorescent compound. The fluorescent compound exhibits aggregation-induced emission properties, and the fluorescent compound has the following main structural formula:

[0068]

[0069] wherein R is selected from linear, branched, cyclic alkyl, alkylphenyl, alkylthienyl and other alkyl aromatics containing 2 to 40 carbon atoms, and one or more non-adjacent carbon atoms may optionally be substituted by –O–, –S–, –C(O)–, –C(O–)–O–, –O–C(O)–, –O–C(O)–O– or –C≡C–, and one or more hydrogen (H) atoms may optionally be replaced by fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or cyanide (CN), or represents an aromatic, heteroaromatic, aryloxy, heteroaryloxy, aromatic carbonyl, heteroaromatic carbonyl, aromatic carbonyloxy, heteroaromatic carbonyloxy, aryloxycarbonyl or heteroaryloxycarbonyl having a 4- to 30-atom ring, and these ring molecules are unsubstituted or substituted by one or more non-aromatic groups;

[0070] wherein each π is independently selected from the following combinations:

[0071]

[0072] and wherein each X is independently selected from the following groups:

[0073]

[0074] Preferably, the fluorescent compound includes:

[0075]

[0076] In another embodiment, the present invention provides a theranostic nanoaggregate, which includes:

[0077] nanoparticles containing a fluorescent compound; and

[0078] a polymer nanoshell covering the aforementioned nanoparticles, and the polymer nanoshell is formed by using a polymer matrix to wrap the nanoparticles. The fluorescent compound in this embodiment has the following main structural formula:

[0079]

[0080] Wherein R is selected from linear, branched, cyclic alkyl, alkylphenyl, alkylthienyl and other alkyl aromatics containing 2 to 40 carbon atoms, and one or more non-adjacent carbon atoms may optionally be replaced by –O–, –S–, –C(O)–, –C(O–)–O–, –O–C(O)–, –O–C(O)–O– or –C≡C–, and one or more hydrogen (H) atoms may optionally be replaced by fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or cyanide (CN), or represents an aromatic, heteroaromatic, aryloxy, heteroaryloxy, aromatic carbonyl, heteroaromatic carbonyl, aromatic carbonyloxy, heteroaromatic carbonyloxy, aryloxycarbonyl or heteroaryloxycarbonyl having a 4- to 30-atom ring, and these ring molecules are unsubstituted or substituted by one or more non-aromatic groups;

[0081] Wherein each π is independently selected from the following combinations:

[0082]

[0083] And wherein each X is independently selected from the following groups:

[0084]

[0085] Preferably, the fluorescent compound in the theranostic nanoaggregates is selected from the following combinations:

[0086]

[0087] Figure 1 Shows exemplary reaction schemes for preparing the TBT-2(1P-DPA), TBT-2(2P-DPA) and TBT-2(TP-DPA) compounds.

[0088] During photoexcitation, a typical radiative pathway for a molecule to process the energy received after excitation is fluorescence (FL). Another pathway is non-radiative dissipation to provide a photothermal effect, which can be used for photothermal therapy (PTT).

[0089] In a preferred embodiment of the present invention, an integrated AIE system is provided for image-guided optical diagnosis and treatment, which has excellent photothermal conversion efficiency while maintaining an appropriate fluorescence quantum yield. By developing a series of D-(π)-A-(π)-D organic small molecules with carefully designed π-bridges, named TBT-2(1P-DPA), TBT-2(2P-DPA) and TBT-2(TP-DPA), as candidates for integrated optical diagnosis and treatment agents. The results show that TBT-2(TP-DPA) containing a thiophene bridge exhibits the most balanced near-infrared imaging and treatment potential. Computational results indicate that TBT-2(TP-DPA) shows the strongest D-A interaction and favorable intramolecular charge transfer effect, which contributes to extending the emission wavelength to about 1500 nm. After simple coating with DSPE-PEG2000, the nanogels of TBT-2(TP-DPA) exhibit a photothermal conversion efficiency as high as 51%, while maintaining an acceptable photoluminescence quantum yield in the near-infrared region.

[0090] The fluorescent compound TBT-2(TP-DPA) shows NIR-I excitation and NIR-II emission. The significant progress achieved in the second near-infrared region (NIR-II, 1000 - 1600 nm) has significantly promoted the development of biomedical imaging. NIR-II fluorescence imaging has several advantages over traditional and NIR-I (650 - 950 nm) imaging modes in basic research applications, such as reduced photon scattering, autofluorescence, and improved penetration depth.

[0091] Cancer diagnosis and / or cancer treatment

[0092] The theranostic nanogels described in the present invention have potential benefits in cancer diagnosis and optical diagnosis and treatment applications, especially in near-infrared (NIR) image-guided cancer surgery, with excellent photothermal conversion efficiency while maintaining an appropriate fluorescence quantum yield. Using NIR fluorescence for image-guided cancer surgery has been proven feasible in clinical cancer surgery and has great potential for successful outcomes of cancer surgery. The theranostic nanogels described herein can serve as efficient NIR fluorescence probes to meet the necessary requirements for image-guided cancer surgery.

[0093] As described in the present invention, the absorption and emission spectra of TBT-2(1P-DPA), TBT-2(2P-DPA) and TBT-2(TP-DPA) in various polar solvents were measured. The results show that TBT-2(1P-DPA) and TBT-2(2P-DPA) have absorption peaks at 580 and 660 nm, respectively, in tetrahydrofuran (THF) solution, and their absorption extends to 800 nm (as Figure 3c)。However, the absorption peak of TBT-2(TP-DPA) is around 800 nm, and the entire absorption wavelength can extend to the NIR-I region (such as Figure 3c )。As expected, the emission spectra of TBT-2(1P-DPA) and TBT-2(2P-DPA) are both below 1000 nm, while the emission maximum of TBT-2(TP-DPA) under 808 nm excitation is 1046 nm (such as Figure 3d )。Therefore, TBT-2(TP-DPA) is an ideal candidate as a theranostic agent template in the NIR-II region.

[0094] Under 808 nm excitation, the absolute quantum yield of TBT-2(TP-DPA) in the near-infrared region was determined to be 10.4% in toluene. The quantum yield (QY) values of this molecule in other solvents and its solid state all show its superior fluorescence emission ability in the near-infrared region, making it a preferred probe for in vivo imaging.

[0095] The theranostic nanoaggregates of TBT-2(TP-DPA) were prepared by simply coating TBT-2(TP-DPA) with DSPE-PEG2000 (such as Figure 4a )。The average size of the TBT-2(TP-DPA) nanoaggregates is about 32 nm, and they show a uniform morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM) (such as Figure 4b )。The absorption and emission spectra of the nanoaggregates were recorded ( Figure 4c ), which are similar to the spectra of the pure organic molecule.

[0096] According to one embodiment, the present invention provides a method for killing cancer cells, which may include contacting the theranostic nanoaggregates with target cancer cells, imaging the target cancer cells while the theranostic nanoaggregates are in contact with the target cancer cells, and irradiating the target cancer cells with light to kill the target cancer cells while the theranostic nanoaggregates are in contact with the target cancer cells. The imaging method can be selected from fluorescence microscopy, bioluminescence imaging, confocal laser scanning microscopy, and photoacoustic microscopy. The theranostic nanoaggregates can be combined with a buffer solution before contacting the target cancer cells. The target cancer cells can be present in a living mammal.

[0097] The light irradiation can be near-infrared light irradiation or laser irradiation.

[0098] Currently, the EPR-based drug delivery method is undoubtedly time-consuming, sensitive, complex, expensive, and ineffective. Most importantly, it has been proven that EPR only exists in small animals such as mice, and does not exist in the human body. Therefore, there is a great clinical need for a simple physical drug delivery method.

[0099] Surprisingly, based on photoinduced thermoacoustic processes, it has been found that the photoacoustic effect can not only be used as an imaging technique, but also become a driving force for rapid delivery in vivo and in vitro. The mechanism may involve two aspects. First, blood vessels are not a seamless structure, and their permeability can be enhanced or even damaged due to the heat generated by the photothermal process. There have been some previous studies discussing photothermal agents for enhancing vascular permeability, however, potential risks must be considered because overheating by continuous wave lasers may directly damage blood vessels and cause harm to biological tissues.

[0100] However, if we use pulsed lasers, their photothermal effects can be negligible because the duration of the photoinduced stimulation is only in the nanosecond range, while the interval between two pulses is in the microsecond range, leaving enough time for the instantaneous heat to dissipate into the surrounding medium. Therefore, the photoinduced thermoacoustic process will generate a PTA field or PTA force (or photoacoustic radiation force, PAF as called by researchers in the photoacoustic field) to enhance vascular permeability in a non-invasive, specific and instantaneous manner. This can prompt sufficient reagents to accumulate in tumor tissues at high efficiency (within one hour), solving the time-consuming and inefficient problems in traditional EPR methods.

[0101] According to one embodiment, the present invention provides a method for stopping, inhibiting or eliminating tumors in mammals, the method may include administering a theranostic nanoaggregate to a mammal; contacting the theranostic nanoaggregate with a tumor site; after contacting the tumor site with the theranostic nanoaggregate, using an imaging method to locate the tumor site; performing an initial pulsed light irradiation on the tumor site to enhance the vascular permeability near the tumor site, so as to prompt sufficient theranostic nanoaggregates to accumulate at the tumor site, and then performing continuous light irradiation on the tumor site, using the theranostic nanoaggregates accumulated at the tumor site to convert light energy into heat energy to raise the temperature of the tumor site to a desired temperature to stop or inhibit the growth of the tumor, or eliminate the tumor. The desired temperature is preferably higher than 55 degrees Celsius. The theranostic agent can be administered by intravenous injection. The imaging method includes at least one of the following: fluorescence microscopy, bioluminescence imaging, confocal laser scanning microscopy and photoacoustic microscopy.

[0102] The light irradiation can be near-infrared irradiation or laser irradiation.

[0103] The initial pulsed light irradiation includes more than 3 light pulse sequences. The peak irradiation intensity is higher than 0.5 W / cm 2 and lower than 100 W / cm 2 . The effective pulse duration is between 5 microseconds and 5 seconds.

[0104] The off - time between every two adjacent optical pulses is sufficient to allow the transient heat of the blood vessels near the tumor site to dissipate into the surrounding medium, so as to avoid overheating and damaging the blood vessels near the tumor site.

[0105] The total duration of the initial pulsed light irradiation and subsequent continuous light irradiation is less than one hour.

[0106] It is worth mentioning that in non - radiative decay, the thermally generated acoustic waves can not only assist photoacoustic imaging (PAI), but also serve as a guiding force for in - vivo and in - vitro particle delivery. In addition, it has been confirmed that many types of nanoparticles or nano - aggregates have photoacoustic responses. Whether it is ICG, small molecules, polymers or gold nanoparticles, they can be triggered by this photoacoustic radiation force.

[0107] According to one embodiment, the present invention provides a method for stopping, inhibiting or eliminating tumor growth in a mammal. The method may include administering a theranostic agent to the mammal; bringing the theranostic agent into contact with the tumor site; after the tumor site comes into contact with the theranostic agent, using an imaging method to locate the tumor site; performing an initial pulsed light irradiation on the tumor site to enhance the vascular permeability near the tumor site, so as to promote sufficient accumulation of the theranostic agent at the tumor site, and then performing continuous light irradiation on the tumor site, converting the light energy into heat energy by using the theranostic agent accumulated at the tumor site to raise the temperature of the tumor site to a desired temperature to stop or inhibit tumor growth, or eliminate the tumor. The desired temperature is preferably higher than 55 degrees Celsius. The theranostic agent can be administered by intravenous injection. The imaging method includes at least one of the following: fluorescence microscopy, bioluminescence imaging, confocal laser scanning microscopy, and photoacoustic microscopy.

[0108] The aforementioned theranostic agent includes nanoparticles or nano - aggregates having a photoacoustic response. The theranostic agent may include ICG, small molecules, polymers, gold nanoparticles or aggregation - induced emission materials (AIEgens).

[0109] The light irradiation can be near - infrared irradiation or laser irradiation.

[0110] The initial pulsed light irradiation includes a sequence of more than 3 light pulses. The peak irradiation intensity is higher than 0.5 W / cm 2 and lower than 100 W / cm 2 . The effective pulse length is between 5 microseconds and 5 seconds.

[0111] The off - time between every two adjacent optical pulses is sufficient to allow the transient heat of the blood vessels near the tumor site to dissipate into the surrounding medium, so as to avoid overheating and damaging the blood vessels near the tumor site.

[0112] The total duration of the initial pulsed light irradiation and subsequent continuous light irradiation is less than one hour.

[0113] The following examples provide further illustration and facilitate understanding of the present invention, and do not limit the present invention in any way.

[0114] Examples

[0115] Example 1 - Synthesis and characterization

[0116] The synthetic routes of TBT-2(1P-DPA), TBT-2(2P-DPA) and TBT-2(TP-DPA) are as Figure 1 shown.

[0117] To construct D-π-A-π-D NIR-II emissive molecules, the benzobisthiadiazole (BBT) unit is the most commonly used acceptor moiety. Although it exhibits strong electron-withdrawing ability and quinoid characteristics that are favorable for red-shifted absorption and emission, it usually results in low fluorescence quantum yields. Therefore, it is of great significance to develop alternative NIR-II acceptor cores with bright NIR-II fluorescence. Herein, we applied another strong electron-withdrawing unit, named thiadiazolotriazole (TBT), as the acceptor core to design a series of NIR emissive molecules. Using diphenylamine (DPA) as the electron donor unit, we employed three different aromatic moieties, namely phenyl, biphenyl, and phenylthienyl as the π units to bridge the TBT acceptor and DPA donor. Although the structures of these π units are simple, they are expected to endow the molecules with various electronic effects and aggregation behaviors when incorporated into the D-π-A-π-D molecules. The three final products TBT-2(1P-DPA), TBT-2(2P-DPA) and TBT-2(TP-DPA) can be synthesized via the face-to-face Stille coupling reaction in high yields and with easy purification, as Figure 3a shown.

[0118] Density functional theory (DFT) calculations were performed using the B3LYP functional and the 6-31G* basis set to obtain the optimized geometries and molecular orbitals of TBT-2(1P-DPA), TBT-2(2P-DPA) and TBT-2(TP-DPA) Figure 3b)。In terms of optimizing the geometric structure, the benzene rings of TBT-2(1P-DPA) and TBT-2(2P-DPA) are directly connected to the TBT unit, showing a twisted molecular backbone, where the dihedral angle between the TBT unit and the benzene ring is 30-35°. In addition, the two benzene rings of the π-bridge in TBT-2(2P-DPA) also form a torsional angle of about 30° due to steric hindrance. In contrast, the thiophene ring of TBT-2(TP-DPA) generates smaller dihedral angles of about 2° and about 16° with the adjacent TBT unit and benzene ring respectively, resulting in a coplanar central part and a twisted wing part of the molecule. Therefore, the different planarity of these molecules significantly affects the conjugation and D-A interaction. It can be observed from the HOMO / LUMO distribution that TBT-2(1P-DPA) shows typical D-A characteristics and can be regarded as a reference. The additional benzene ring of TBT-2(2P-DPA) hinders the conjugation between the DPA and TBT units, resulting in no LUMO on the N atom of the DPA unit. On the contrary, the thiophene ring of TBT-2(TP-DPA) not only extends the conjugation length due to the smaller torsional angle but also enhances the intramolecular charge transfer (ICT) due to its richer electronic properties. Therefore, the HOMO-LUMO energy gaps of these molecules are TBT-2(2P-DPA), TBT-2(1P-DPA), and TBT-2(TP-DPA) in turn, specifically 1.70, 1.68, and 1.37 eV. The calculation results show that different π-bridging can change the electron cloud distribution of the molecule and thus regulate the optical properties of the material.

[0119] Example 2 - Photophysical, photodynamic and photothermal properties

[0120] To confirm the demonstration of the molecular simulation results, the absorption and emission spectra of TBT-2(1P-DPA), TBT-2(2P-DPA), and TBT-2(TP-DPA) in various polar solvents were then measured. The results show that TBT-2(1P-DPA) and TBT-2(2P-DPA) have absorption peaks at 580 and 660 nm respectively in the THF solution, and their absorption extends to 800 nm ( Figure 3c ). However, the absorption peak of TBT-2(TP-DPA) is about 800 nm, and the overall absorption wavelength can extend to the NIR-I region ( Figure 3c ), showing the potential as a NIR-II imaging agent. As expected, the emission spectra of TBT-2(1P-DPA) and TBT-2(2P-DPA) are both below 1000 nm, while the emission maximum of TBT-2(TP-DPA) under 808 nm excitation is 1046 nm ( Figure 3d)。The results showed that as the degree of twisting increased, the absorption and emission spectra of TBT-2(1P-DPA), TBT-2(2P-DPA), and TBT-2(TP-DPA) showed an increasing trend in wavelength ( Figure 3c and Figure 3d ), where TBT-2(TP-DPA) showed the largest Stokes shift ( Figure 3e ). The experimental characteristics were consistent with the theoretical hypothesis, making TBT-2(TP-DPA) an ideal candidate for the template of theranostic agents in the NIR-II region.

[0121] Under 808 nm excitation, the absolute quantum yield of TBT-2(TP-DPA) in the near-infrared region was determined to be 10.4% in toluene. The quantum yield values of this molecule in other solvents and its solid state all showed its superior fluorescence emission ability in the near-infrared region, making it the first qualified in vivo imaging probe.

[0122] Example 3 - Construction of nanoclusters and their multifunctional potential

[0123] According to the Jablonski diagram, obtaining near-infrared fluorescent dyes has always been a win-win optical theranostic strategy. As the red-shifted emission in the near-infrared region is quenched, we can make full use of non-radiative decay for multimodal theranostics, such as photothermal or photoacoustic imaging and related treatments. In biological applications, polymer nanoprecipitation is usually used to introduce hydrophobic molecules into the biological medium. Among them, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (abbreviated as DSPE-PEG) is widely used for drug delivery. Its biocompatibility, biodegradability, and amphiphilicity make it an ideal nanoparticle shell for contrast agents including AIEgens.

[0124] This coating can further trigger the restriction of intermolecular motion, making AIEgens have stronger fluorescence emission when aggregated. In addition, thanks to the flexible surfactant, the internal part of the nanoparticle aggregates is closely packed, while the molecules in the outer part near the nanoparticle surface are relatively loose, which enables the aggregates to achieve both radiative and non-radiative decay. Inspired by the previous design, we simply coated TBT-2(TP-DPA) with DSPE-PEG2000 ( Figure 4a ). The average size of the nanoparticle aggregates of TBT-2(TP-DPA) was about 32 nm, and showed a uniform morphology by dynamic light scattering (DLS) and transmission electron microscopy (TEM) ( Figure 4b ). The absorption and emission spectra of the nanoparticle aggregates were recorded ( Figure 4c), similar to the spectra of pure organic molecules.

[0125] To test the multifunctional potential of the nanoaggregates, their heat generation ability was first evaluated by calculating their photothermal conversion efficiency (PCE). As expected, the TBT-2(TP-DPA) nanoaggregates exhibited good photothermal conversion ability. When suspended in water at a concentration of 1 mg / mL and irradiated with an 808 nm continuous laser for 5 minutes, the temperature rose above 60 degrees Celsius ( Figure 4d ). The PCE value of the above nanoaggregates was calculated to be 51% ( Figure 5 ). When applying pulsed laser, due to the photoinduced thermoacoustic process, a photoacoustic response was triggered. The photoacoustic spectrum of the nanoaggregates in water was consistent with their absorption characteristics, ranging from 650 - 1000 nm, and having a strong photoacoustic signal at 808 nm ( Figure 6 ). In particular, as the concentration of the nanoaggregates increased, the photoacoustic brightness gradually enhanced ( Figure 4e , inset). The linear relationship between the photoacoustic signal and the nanoaggregate concentration can ensure the rationality of quantifying the nanoaggregate accumulation by PAI ( Figure 4e ). These characteristics can initially meet the requirements of photothermal imaging, photoacoustic imaging, and photothermal therapy. Example 4 - Fast delivery via photoinduced thermoacoustic process

[0126] It is worth mentioning that in non-radiative decay, the thermally generated acoustic waves can not only assist photoacoustic imaging (PAI), but also become the guiding force for particle delivery in vivo and in vitro. In addition, it has been confirmed that many nanoparticles with photoacoustic responses, whether ICG, small molecules, polymers, or gold nanoparticles, can be triggered by this photoacoustic radiation force. Therefore, we evaluated the possibility and efficiency of the TBT-2(TP-DPA) nanoaggregates to achieve delivery during the in vivo photoinduced thermoacoustic (PTA) process. A bilateral xenograft 4T1 tumor mouse was used as a solid tumor model. A dual-wavelength optical resolution photoacoustic microscopy (ORPAM) system was established to assist the delivery of the nanoaggregates to the tumor (808 nm pulsed laser), and simultaneously visualize the tumor site and its surrounding vascular structure (532 nm) and the accumulation of the nanoaggregates (808 nm).

[0127] After intravenous injection of the nanoaggregates, two tumors on the same mouse were used as the PTA experimental group and the EPR control group, respectively. On one side of the PTA tumor, we used an 808 nm pulsed laser for continuous laser scanning for 30 minutes (20 seconds per cycle) to deliver the nanoaggregates through the PTA process. As Figure 7aAs shown, the nanoaggregates accumulate in solid tumors in increasing amounts over time and are almost distributed throughout the area at 30 minutes of scanning, indicating that this method has significant effects. The quantitative results based on photoacoustic signals show the accumulation of nanoaggregates over time within half an hour (see Figure 7b ).

[0128] To study this PTA-driven delivery precision, we also imaged a larger field of view using the NIR-II fluorescence emitted by the nanoaggregates. In the EPR group, after the same 30-minute interval, almost no detectable fluorescence signal was observed in the tumors. However, in the PTA group, the fluorescence could clearly distinguish the tumor and its surrounding area, and after a further 10-minute laser scan using photoacoustic tomography (PAT), more signals appeared, showing that this method can promote delivery in deeper tissues ( Figure 7e ). Since these nanoaggregates are only coated with DSPE-PEG and not otherwise targeted modified, these results verify the feasibility of PTA-driven delivery of TBT-2(TP-DPA) nanoaggregates because the PTA process can enhance the vascular permeability of tumor tissues.

[0129] Next, to compare the accumulation effects of nanoaggregates in the EPR effect and the PTA effect, we injected the same dose of nanoaggregates via the tail vein and observed the fluorescence signals of another group of mice over time. In the case of almost only the EPR effect, the fluorescence emission intensity in the tumors reached the maximum at 24 hours after injection ( Figure 7c and Figure 7d ), but its signal intensity was still much lower than the level of the PTA group within one hour ( Figure 7e and Figure 7f ). The quantitative results show that the signal intensities of the ORPAM and ORPAM+PAT groups are almost 3 to 4 times that of the EPR group. Even when the EPR group was given 24 hours of accumulation time, the fluorescence signal of the ORPAM+PAT group was still twice that of it ( Figure 7f ), demonstrating the high efficiency of this PTA-driven delivery method. It is worth noting that in the present invention, we first evaluated the possibility of delivering continuous laser energy to solid tumors by irradiating the tumors of mice with 808nm CW laser for 30 minutes. The fluorescence signals of the CW group indicate that the continuous wave does provide more energy to drive the particles forward. However, the results only show slight differences from those of the EPR group and are much lower than the delivery efficiency of pulsed lasers. This indicates that the transient enhancement of vascular permeability can only be achieved by the photoinduced thermoacoustic process triggered by pulsed lasers, without interference from heat generation.

[0130] Example 5 - In vitro evaluation of photothermal effect

[0131] Based on the regulation of the TICT effect in molecular design, near-infrared fluorophores are more likely to generate heat. Since TICT can weaken fluorescence intensity but enhance its photothermal effect, balancing the radiative decay and non-radiative decay of a single molecule makes it a multifunctional theranostic agent. The photothermal effect of the TBT-2(TP-DPA) nanoclusters depends on the power density ( Figure 8a ) and concentration ( Figure 8b and Figure 9 ), which provides a basis for the subsequent optimization of in vitro experiments. It is worth noting that even after undergoing 5 consecutive heating / cooling cycles, the nanoclusters still exhibit excellent photostability ( Figure 8c ).

[0132] As a qualified theranostic agent, the material should have no cytotoxicity when the laser is not activated. Therefore, we evaluated the dark toxicity of the nanoclusters on 4T1 cells through CCK-8 assays. The results showed that even at a concentration of 250 μg / mL, after co-incubating the TBT-2(TP-DPA) nanoclusters with 4T1 cells for 24 hours, the cell viability could still remain above 80%, indicating good biocompatibility ( Figure 10 ). However, after subsequent continuous laser irradiation at 808 nm for 10 minutes, the cells incubated with the nanoclusters showed apoptotic phenomena. Flow cytometry results showed that approximately 42.1% of the cells were in the late apoptotic stage, while 50.9% of the cells were in the early apoptotic stage after the above treatment ( Figure 8d ). Meanwhile, the percentage of apoptotic cells in the control group, the group only receiving 808 nm laser irradiation, and the group only exposed to the nanoclusters without irradiation was all less than 15%. The significant cell killing effect through photothermal therapy can effectively ablate cancer cells, providing an efficacy guarantee for subsequent in vivo experiments.

[0133] We also used Calcein AM and propidium iodide (PI) as live / dead cell detection to visually observe the cell morphology under PTT treatment. Confocal imaging showed that dead cells in the TBT-2(TP-DPA) nanoclusters + laser group emitted strong red fluorescence, while other groups including the control group only emitted green fluorescence representing live cells ( Figure 8e ), indicating the consistency and reliability of our in vitro PTT results.

[0134] Example 6 - In vivo evaluation of PTA-enhanced photothermal therapy

[0135] After confirming the accumulation of the nanoclusters facilitated by the photoinduced thermoacoustic process and its photothermal effect on cancer cells, we have sufficient reason to study its therapeutic effect in vivo. Multiple 4T1 tumor-bearing mice were randomly divided into 5 groups.

[0136] 1) TBT-2(TP-DPA) nanoaggregates + 808 nm pulsed laser + 808 nm continuous wave laser (PTA + PTT);

[0137] 2) TBT-2(TP-DPA) nanoaggregates + 808 nm continuous wave laser (EPR for 24 h + PTT); 3) TBT-2(TP-DPA) nanoaggregates + 808 nm pulsed laser (PTA); 4) 808 nm pulsed laser + 808 nm continuous wave laser; 5) PBS (control).

[0138] To observe the actual effect of photothermal effect in vivo in real time, an infrared thermal imaging camera was set up to monitor the infrared imaging of all mice in groups 1), 2), 4), and 5) Figure 11a ). As Figure 11b quantified, the tumor temperature in the PTA + PTT group increased to 53 °C only 2 minutes after receiving 808 nm laser irradiation and further increased to nearly 60 °C within the next 8 minutes. In contrast, although we gave the EPR group 24 hours to accumulate sufficient nanoaggregates in the tumor, under the same laser treatment, they only rose to 51 °C within 10 minutes Figure 11b ). On the contrary, whether treated with only 808 nm CW laser (group 5, control group) or plus 808 nm pulsed laser (group 4), without injecting nanoaggregates, from the initial body temperature of the mice, the laser itself had a negligible effect on the temperature change of the tumor, and the final temperature was below 40 °C, which did not affect the morphology of the tumor tissue Figure 11b ).

[0139] During the next 14 days, the digital images, tumor volume, and body weight of all 5 groups of mice were recorded and analyzed. As Figure 11c , Figure 11e and Figure 12 shown, the solid tumors of the mice in the PTA + PTT group gradually shrank and disappeared significantly without any recurrence. However, the groups that received only 808 nm CW or CW plus pulsed laser irradiation but did not administer nanoaggregates (groups 4 and 5 as controls) had little therapeutic effect. In contrast, although the EPR + PTT group showed an inhibitory effect in the first 4 days, the tumor would still regenerate thereafter, which could be attributed to the incomplete killing of cancer cells in EPR.

[0140] The body weights of the mice in each group remained within the normal range during the observation period Figure 11d ). In addition, according to hematoxylin-eosin (H&E) staining, more significant signs of apoptosis were observed in the PTA + PTT group Figure 11e)。Meanwhile, the treatment process of PTA+PTT only requires 40 minutes of 808nm pulsed laser scanning, while traditional EPR cannot achieve the same tumor killing effect as PTA-guided drug delivery even with up to 24 hours of accumulation before PTT. We also investigated and analyzed the biosafety of the designed TBT-2(TP-DPA) nanoclusters in mice through blood tests and H&E staining (such as Figures 13 - 15 ). All blood routine and serum biochemical results showed that there were no significant differences between the nanocluster administration group and the control group 14 days after injection. The major organs collected from the sacrificed mice on the 14th day after injection, including the heart, liver, spleen, lung, and kidney, did not show obvious lesions or injuries.

[0141] These results together confirmed the biosafety of TBT-2(TP-DPA) nanoclusters in potential biological applications. In summary, the present invention developed new near-infrared absorbing nanoclusters based on small molecules, which not only showed good effects in multifunctional optical diagnosis and treatment (FLI, PAI, PTI, and PTT), but also were applicable to drug delivery methods and platforms realized by the photoacoustic process induced by light. Based on this strategy, it is reasonable to believe that other near-infrared absorbing small molecule nanoclusters also have potential in this PTA-guided rapid delivery and efficient cancer treatment, can make up for the deficiencies of EPR, and save considerable time for tumor targeting design.

[0142] The above embodiments are only used to illustrate the principle of the present invention and should not be construed as any limitation to the invention of this patent. The above embodiments can be adjusted by those of ordinary skill in the art without departing from the scope of the invention of this patent as defined by the following appended claims.

Claims

1. A fluorescent compound showing aggregation-induced emission properties, the fluorescent compound having the following main structural formula: wherein R is selected from linear, branched, cyclic alkyl, alkylphenyl, alkylthienyl and other alkyl aromatics containing 2 to 40 carbon atoms, and one or more non-adjacent carbon atoms may optionally be replaced by – O–, –S–, –C(O)–, –C(O–)–O–, –O–C(O)–, –O–C(O)–O– or –C≡C–, and one or more hydrogen (H) atoms may optionally be replaced by fluorine (F), chlorine (Cl), bromine (Br), iodine (I) or cyanide (CN), or represents an aromatic, heteroaromatic, aryloxy, heteroaryloxy, aromatic carbonyl, heteroaromatic carbonyl, aromatic carbonyloxy, heteroaromatic carbonyloxy, aryloxycarbonyl or heteroaryloxycarbonyl having a 4- to 30-atom ring, and these ring molecules are unsubstituted or substituted by one or more non-aromatic groups; wherein each π is independently selected from the following combinations: wherein each X is independently selected from the following groups:

2. The fluorescent compound according to claim 1, characterized in that, The fluorescent compound shows near-infrared (NIR) emission.

3. The fluorescent compound according to claim 1, wherein, The fluorescent compound comprises:

4. The fluorescent compound according to claim 1, wherein The fluorescent compound exists in the form of nanoparticles.

5. A theranostic nanoaggregate, comprising: nanoparticles comprising the fluorescent compound as claimed in claim 1; and a polymer nanoshell covering the nanoparticles, the polymer nanoshell being formed by wrapping the nanoparticles with a polymer matrix.

6. A method of killing cancer cells, comprising: contacting target cancer cells with the theranostic nanoaggregate as claimed in claim 5; when the theranostic nanoaggregate contacts the target cancer cells, imaging the target cancer cells, using an imaging method selected from the following combinations: fluorescence microscopy, bioluminescence imaging, confocal laser scanning microscopy and photoacoustic microscopy; and when the theranostic nanoaggregate contacts the target cancer cells, irradiating the target cancer cells with light, converting the light into heat to kill the target cancer cells.

7. The method according to claim 6, characterized in that, The light irradiation is near-infrared light irradiation.

8. The method according to claim 6, wherein The light irradiation is laser irradiation.

9. A method of arresting, inhibiting or eliminating a tumor in a mammal, comprising: administering the theranostic nanoaggregate as claimed in claim 5 to a mammal; contacting the theranostic nanoaggregate with the tumor site; when the tumor site contacts the theranostic nanoaggregate, localizing the tumor site using an imaging method; performing a preliminary pulsed light irradiation on the tumor site to enhance the vascular permeability near the tumor site, thereby promoting sufficient accumulation of the theranostic nanoaggregate at the tumor site; and performing a subsequent continuous light irradiation on the tumor site, at which time there is sufficient theranostic nanoaggregate at the tumor site to convert the light energy into heat energy to raise the temperature of the tumor site to a desired temperature so as to arrest or inhibit the growth of the tumor, or eliminate the tumor.

10. The method according to claim 9, wherein The theranostic agent is administered by intravenous injection.

11. The method according to claim 9, wherein The imaging method comprises at least one of the following: fluorescence microscopy, bioluminescence imaging, confocal laser scanning microscopy and photoacoustic microscopy.

12. The method according to claim 9, wherein The light irradiation is near-infrared light irradiation.

13. The method according to claim 9, wherein The light irradiation is laser irradiation.

14. The method according to claim 9, wherein The required temperature is higher than 55 degrees Celsius.

15. The method according to claim 9, wherein The total duration of the initial pulsed light irradiation and the subsequent continuous light irradiation is less than one hour.

16. The method according to claim 9, wherein The initial pulsed light irradiation includes a sequence of light pulses more than 3 times.

17. The method according to claim 16, wherein The peak irradiation intensity is higher than 0.5 W / cm 2 and lower than 100 W / cm 2 .

18. The method according to claim 16, wherein The effective pulse length is between 5 microseconds and 5 seconds.

19. The method according to claim 16, wherein The off-time between every two adjacent light pulses is sufficient for the transient heat of the blood vessels near the tumor site to dissipate into the surrounding medium to avoid overheating and damaging the blood vessels near the tumor site.

20. A method for stopping, inhibiting or eliminating tumor growth in a mammal, comprising: administering a theranostic agent to a mammal; bringing the theranostic agent into contact with the tumor site; localizing the tumor site using an imaging method after the tumor site is in contact with the theranostic agent; performing an initial pulsed light irradiation on the tumor site to enhance the vascular permeability near the tumor site, thereby promoting sufficient accumulation of the theranostic agent at the tumor site; and performing a subsequent continuous light irradiation on the tumor site, at which time sufficient theranostic agent is used at the tumor site to convert light energy into heat energy to raise the temperature of the tumor site to the required temperature to stop or inhibit tumor growth, or eliminate the tumor.

21. The method according to claim 20, wherein The theranostic agent is administered by intravenous injection.

22. The method according to claim 20, wherein The theranostic agent includes nanoparticles or nanoaggregates having a photoacoustic response.

23. The method according to claim 20, wherein The theranostic agent includes ICG, small molecules, polymers, gold nanoparticles or AIEgens.

24. The method according to claim 20, wherein The imaging method includes at least one of the following: fluorescence microscopy, bioluminescence imaging, confocal laser scanning microscopy and photoacoustic microscopy.

25. The method according to claim 20, wherein The light irradiation is near-infrared light irradiation.

26. The method according to claim 20, characterized in that, The light irradiation is laser irradiation.

27. The method according to claim 20, wherein The required temperature is higher than 55 degrees Celsius.

28. The method according to claim 20, wherein The total duration of the initial pulsed light irradiation and the subsequent continuous light irradiation is less than one hour.

29. The method according to claim 20, wherein The initial pulsed light irradiation includes a sequence of light pulses more than 3 times.

30. The method according to claim 29, wherein The peak irradiation intensity is higher than 0.5 W / cm 2 and lower than 100 W / cm 2 .

31. The method according to claim 29, characterized in that, The effective pulse length is between 5 microseconds and 5 seconds.

32. The method according to claim 29, wherein The off-time between every two adjacent light pulses is sufficient for the transient heat of the blood vessels near the tumor site to dissipate into the surrounding medium to avoid overheating and damaging the blood vessels near the tumor site.

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