KR20150138726A - Biocompatible Fluorescence Nanoparticles and Uses Thereof - Google Patents

Abstract

The present invention relates to a novel bipolar aryl vinyl compound, nano-probe particles including the same, a contrast agent using the same, and an imaging method thereof. The novel bipolar aryl vinyl compound can adjust the solid-state fluorescence (SSF) color according to donor-acceptor combinations and shows a hydrodynamic size less than 65 nm in an aqueous environment as well as exhibiting increased aggregation-enhanced fluorescence (AEF) and SSF. The nano-probe particles have a hydrodynamic size less than 25 nm, show increases SSF in a near-infrared (NIR) range of 650 nm and higher, and are accumulated in cells of a specific tissue such as the sentinel lymph node, brain, or tumors or on an in vivo level to detect the tissue specifically. The contrast agent and the imaging method can specifically contrast lymph nodes, tumors, or brain tissues to be applied effectively to the diagnosis of cancer, tumors, and brain diseases.

Description

≪ Desc / Clms Page number 1 > Biocompatible Fluorescence Nanoparticles and Uses Thereof &

The present invention relates to a novel bipolar aryl vinyl compound, a nanoprobe particle comprising the same, and a contrast agent composition and imaging method using the same.

Molecular fluorescence is high sensitivity, ease of access, the multi-variable led to the (multi-parametric) detection function and a variety of natural and big jump in the biological availability and biomedical research due to their synthetic fluorescent molecules ^1,2. Recently, organic nanoparticles composed of molecular-weight aggregates that emit solid-state fluorescence (SSF) or aggregation-enhanced fluorescence (AEF) there being great attention to the rising class, because they have a strong potential in bio-imaging applications requiring a powerful signal emitted (emission signal) ^{3 -12.} SSF is a unique phenomenon capable of maintaining fluorescence in concentrated or coagulated states to overcome the typical fluorescence quenching of common organic dyes. SSF provides dye-agglomerated molecule nanoprobes that allow for high-density dye loading inside the particle and increase the emission intensity in proportion to the loading density. Moreover, dye aggregation into nanoparticles facilitates the production of aqueous dispersions of dyes for biomedical applications without the need for chemical modification to water solubility. Nanoparticle formulation may provide additional benefits, including passive in vivo tumor targeting, and an active target surface engineering for by multiple bio-conjugation (bioconjugation) according to the increased cell permeability, enhanced permeability and retention (EPR) effect ^{13 , 14} .

Generally, SSF occurs in molecules of a particular class that show AEF. In most cases, the molecules have a non-planar structure with free torsional freedom which causes fluorescence quenching in a separate solution state, whereas when they block the torsional motion by solidification, ^{15 -20} . A wide variety of AEF-active dyes with broad spectrum emission ranges have been successfully applied in bio-imaging and sensing applications. However, most of them exhibit luminescence in a visible window of less than 650 nm; SSF in near-infrared (NIR) has been rarely reported until now ^{8 -12} . Low optical interference (absorption, scattering, and self-fluorescence), and a high signal-to-noise (signal-to-noise) NIR to for biomedical in vivo imaging since the NIR region have a most deep light transmission by biological tissue with a non- Spectrum tuning is required ²¹ . Thus, dyes that emit SSF at NIR, particularly above 650 nm, need to be further developed to provide advanced molecular nanoprobes for in vivo applications. In addition, in the development of the functional fluorescent dyes, chemical combination it is found to be a powerful methodology in order to reach the targeted goals, but ^{22 to 25,} this approach is hardly used to develop the SSF library dyes for application in vivo .

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to develop a functional fluorescent dye exhibiting excellent solid-state fluorescence (SSF) and a bio-imaging method using the functional fluorescent dye. As a result, we have synthesized / identified a bipolar aryl vinyl scaffold compound that emits increased SSF or AEF in the NIR region (especially in the wavelength region on 650 nm) that has high light transmission to biological tissue, The present invention relates to a method of forming nanoprobe particles of less than 20 nm in size self-assembled between biocompatible polymeric surfactants, wherein a composition comprising the nanoprobe particles or a method of using the nanoprobe particles is applied to a target (e.g., The surveillance lymph node, the brain or the tumor).

It is therefore an object of the present invention to provide a bipolar aryl vinyl compound.

It is another object of the present invention to provide fluorescent nanoprobe particles.

It is another object of the present invention to provide a contrast agent composition.

It is still another object of the present invention to provide a method of imaging a target tissue.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, the present invention provides a dipolar arylvinyl compound represented by the following general formula (I) or general formula (II): < EMI ID =

(I)

≪ RTI ID = 0.0 &

In the above formula (I) or (II), R ₁ is a cyano group; A is carbazole, aniline or derivatives thereof, B is a cyano group, a carbonyl group, a carboxyl group, a C ₂ -C ₁₀ ester, an aryl, or a C ₂ -C ₁₂ Linear or branched alkyl; C is a compound selected from 1,3-indandione or a 1,3-indanedione derivative substituted with one or more cyano groups.

According to another aspect of the present invention, the present invention provides a composition comprising (a) the above-described bipolar aryl vinyl compound; And (b) fluorescent nanoprobe particles (NPs) comprising a polymeric surfactant, wherein the bipolar aryl vinyl compound is embedded within the surfactant.

The present inventors have sought to develop functional fluorescent dyes exhibiting excellent SSF and a bio-imaging method using the same. As a result, we have synthesized / identified a bipolar aryl vinyl scaffold compound that emits increased SSF or AEF in the NIR region (particularly in the wavelength region above 650 nm) with high light transmission to biological tissues, and a biocompatible polymer self-between surfactant-stylized formulated in a nano-probe particles of the assembly is less than 20 nm in size, the target from a method using the composition or it containing the nano-probe particles cell or in vivo (e.g., the sentinel node, Brain or tumor) can be effectively imaged.

The compounds of the present invention represented by the general formula (I) or (II) exhibit a bipolar π-conjugation having a π-electron donor and a receptor at both ends. Based on this, a novel nanoprobe particle .

First, the present invention provides a bipolar aryl vinyl compound represented by the following formula (I) or (II):

(I)

≪ RTI ID = 0.0 &

In the formula (I) or (II), R ₁ is cyano group; A is carbazole, aniline or a derivative thereof, B is a cyano group, a carbonyl group, a carboxyl group, a C ₂ -C ₁₀ ester, an aryl, a C ₂ -C ₁₂ Linear or branched alkyl; C is selected from 1,3-indanedione or 1,3-indanedione derivatives substituted with one or more cyano groups.

In some embodiments of the present invention, the carbazole or derivative thereof is a compound represented by the following formula (III)

(III)

In the formula (III), R ₂ is C ₁ -C ₁₂ Straight or branched chain alkyl, C ₂ -C ₁₂ straight or branched alkyl alcohol, or C ₁ -C ₁₂ And R < ₃ > is a position at which a vinyl group is bonded in the formula (I) or (II).

In another embodiment of the present invention, the aniline or a derivative thereof is a compound represented by the following formula (IV)

(IV)

In Formula IV, R ₂ is independently H, C ₁ -C ₆ together Straight or branched chain alkyl, C ₂ -C ₆ straight or branched chain alkyl alcohol, or C ₁ -C ₆ And R < ₃ > is a position at which a vinyl group is bonded in the formula (I) or (II).

The term " aryl " as used herein refers to a substituted or unsubstituted monocyclic or polycyclic carbon ring which is totally or partially unsaturated, specifically monoaryl or biaryl. The monoaryl preferably has from 5 to 6 carbon atoms, and the biaryl preferably has from 9 to 10 carbon atoms. Specifically, the aryl includes, but is not limited to, substituted or unsubstituted phenyl, benzene, biphenyl, thiophene, 1,3-indanedione, and the like. Monoaryl, e.g., in the case where the substituted phenyl, substituted, but can be made by a variety of substituents at various positions, particularly, cyano, bromo, halo, hydroxy, nitro, C ₁ -C ₄ Substituted or unsubstituted straight or branched chain alkyl, C ₁ -C ₄ C ₃ -C ₆ cycloheteroalkyl, or a substituted or unsubstituted amino group, and more specifically, cyano or bromo. The aryl group includes, but is not limited to, a phenyl group, a substituted phenyl group, a naphthyl group, a substituted naphthyl group, and the above-mentioned aryl group substituent may specifically include a small number of alkyl or halogen . The term " heteroaryl ", as used herein, is a heterocyclic aromatic group that includes N, O, or S as a heteroatom. Specifically, the heteroaryl is a heterobialyl containing S as a heteroatom. The term " aryl group substituent " as used herein may include alkyl, cycloalkyl, cycloaryl, aryl, heteroaryl, and is optionally substituted with halo, haloalkyl, alkyl, arylalkyl, , Alkenyl having 1 to 2 double bonds, alkynyl having 1 to 3 triple bonds, hydroxy, polyhaloalkyl, and the like. Specifically, the aryl group substitution is selected from the group consisting of C ₁ -C ₅ Alkyl, C ₁ -C ₅ Alkoxy or halogen. The term " alkoxy ", as used herein, is an -O alkyl group, including, but not limited to, methoxy, ethoxy, propoxy, and the like.

As used herein, the term " alkyl " means a straight or branched unsubstituted or substituted saturated hydrocarbon group, for example, methyl, ethyl, propyl, isobutyl, pentyl, hexyl, heptyl, octyl, Undecyl, tridecyl, pentadecyl, heptadecyl and the like. In some embodiments of the present invention, the alkyl is C ₂ -C ₁₂ straight chain or branched chain alkyl, more specifically C ₁ -C ₆ Straight or branched chain alkyl, more particularly C ₂ -C ₄ Straight or branched chain alkyl. The term "C ₁ -C ₁₂ alkyl" means a saturated hydrocarbon group having from _{1 to 12} carbon atoms, and includes lower alkyl such as methyl, ethyl, n -propyl, isopropyl, isobutyl, n -butyl and t- .

The term " halogen " as used herein includes F, Cl, Br and I. In some embodiments of the present invention, said halogen is Br.

As used herein, the term " ester " means a functional group represented by -COOR (R is alkyl or aryl), specifically an ester having 2 to 10 carbon atoms. As used herein, the term " carboxyl group " means a functional group represented by -COOH, specifically a carboxyl group having 2-10 carbon atoms.

The term " alcohol " as used herein means a compound in which the hydroxyl group is bonded to the carbon atom of the alkyl or substituted alkyl group. Specifically, it is an alcohol having from 1 to 20 carbon atoms, more specifically, an alcohol having from 1 to 10 carbon atoms, and still more specifically, an alcohol having from 1 to 5 carbon atoms. As used herein, the term " alkyl alcohol " means a compound in which a hydroxyl group is bonded to a carbon atom of an alkyl or substituted alkenyl group, specifically, an alkenol having 1 to 20 carbon atoms, more specifically, Alkenols, more specifically alkenols having 1-5 carbon atoms.

In certain embodiments of the present invention, the aryl includes bromomethylbenzene, methylthiophene and methylbiphenyl. In some embodiments of the present invention, the ester is ethyl propionic acid. In some embodiments of the present invention, the 1,3-indandione may be substituted with one to four cyano groups when substituted, or may be substituted with a cyano group, for example, 3- (3-dicyanomethylidene) -1-indanone, and 1,3-bis (dicyanomethylidene) indane. It is not.

According to the present invention, A in the above formula (I) or (II) functions as a? -Electron donor and B or C functions as a? -Electron acceptor. The compounds of the present invention were synthesized by condensation reaction (e.g., Knoevenagel condensation reaction) between the building blocks (A and B or C). Since the above receptors composed of alpha, alpha -duplexed vinyl segments cause intracellular steric hindrance and cause severe distortion which is advantageous for generation of solid-state fluorescence (SSF), aryl vinyl compounds composed of the building blocks Can exhibit increased fluorescence in solids.

In some embodiments of the present invention, the arylvinyl compound of the present invention exhibits solid phase fluorescence or aggregation-enhanced fluorescence (AEF), and more particularly the SSF is an intramolecular charge transfer characteristics due to severe distortion due to steric hindrance.

The aryl vinyl compounds of the present invention exhibit increased fluorescence emission in weak fluorescence and aggregation in solution. In some embodiments of the present invention, the SSF color can be tunable through the combination of A and B or C, which are building blocks (see Figures 3 and Table 2).

In some embodiments of the present invention, the arylvinyl compound exhibits increased SSF over a broad spectral range, including near-infrared (NIR) regions, and more specifically near-infrared (NIR) Lt; RTI ID = 0.0 > SSF. ≪ / RTI >

In some embodiments of the present invention, the aryl vinyl compound represented by Formula (I) or (II) is a compound represented by the following Formula 1 to Formula 20:

[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

(7)

[Chemical Formula 8]

[Chemical Formula 9]

[Chemical formula 10]

(11)

[Chemical Formula 12]

[Chemical Formula 13]

[Chemical Formula 14]

[Chemical Formula 15]

[Chemical Formula 16]

[Chemical Formula 17]

[Chemical Formula 18]

[Chemical Formula 19]

[Chemical Formula 20]

Accordingly, the present invention relates to (a) the above-described bipolar aryl vinyl compound; And (b) fluorescent nanoprobe particles (NPs) comprising a polymeric surfactant. In the nanoprobe particle of the present invention, the bipolar aryl vinyl compound is embedded in the above-mentioned surfactant.

In some embodiments of the present invention, the polymeric surfactant of the present invention can be any substance that exhibits biocompatibility, and more specifically, polyoxyethylene-polyoxypropylene block copolymer, polyoxyethylene-polyoxypropylene block copolymer, Polyvinyl alcohol, and gelatin.

In some embodiments of the present invention, the polyoxyethylene-polyoxypropylene block copolymer (poloxamer) has the formula (PEO) _x - (PPO) _y - is a non-ionic high molecular material represented by (PEO) _z: Formula , PEO is ethylene oxide, PPO is propylene oxide, x, y and z are each independently an integer of 1-10,000. The biocompatible polymeric surfactant is an amphiphilic tri-block structure composed of hydrophobic centers of polypropylene oxide and hydrophilic polytags at both ends of polyethylene oxide to increase the water solubility of the hydrophobic oil material Can be used to increase the miscibility of the two materials having different properties and can be purchased as a variety of pluronics trade names (BASF Corporation) depending on the composition of x, y and z.

In some embodiments of the invention, the biocompatible polymeric surfactant is Pluronic 127.

The poloxamer is applied to industry, cosmetics or medical field. In particular, poloxamer can be used not only as a model system of a drug delivery system but also because of cell cushioning effects, (For example, a cell culture medium, bioimaging, etc.) because it puts the cell into a condition where the cells are in contact with each other.

The nanoprobe particle of the present invention is composed of the above-mentioned aryl vinyl compound (particularly, Formula 9, Formula 10, Formula 18, Formula 19 or Formula 20) and a biocompatible polymer surface active agent. In some embodiments of the invention, the hydrodynamic size of the nanoprobe particles of the present invention is less than 100 nm, more specifically less than 65 nm, more specifically less than 50 nm, and most specifically less than 20 nm to be.

Fluorescent nanoparticles for biological tissues should have high sensitivity as well as easy accessibility. The nanoprobe particles of the present invention not only can be easily prepared in water-dispersed formulations, but also exhibit increased SSF in the near-infrared (NIR) region above 650 nm (see FIG. 10).

In some embodiments of the present invention, the nanoprobe particles of the present invention exhibit a high signal-to-background (S / B) NIR-SSF release and are well suited for biomedical applications.

In some embodiments of the invention, the nanoprobe particles of the invention are colloidal in aqueous environment and do not cause a serum response in body fluids (see Figure 11).

Interestingly, the nanoprobe particles of the present invention specifically accumulate specific tissues (e.g., surveillance lymph nodes, tumors or brain) in cells or in vivo (see Figures 12-15). The ability of the nanoprobe particles of the present invention to accumulate specifically in the surveillance lymph nodes or tumor cells due to increased cell permeability, increased permeability, and retardation (EPR) effects and to be detected through fluorescence can be used / Can be applied. Furthermore, the nanoprobe particles of the present invention were accumulated specifically in brain tissue (see Fig. 15B).

The difficulty in diagnosing and treating diseases in the brain is due to the unique structure of the blood-brain barrier (BBB) present in the cerebral blood vessels, It tightly surrounds the blood vessels, allowing the nerve cells to survive and pass the toxins through to the brain. However, the blood-brain barrier is inhibiting the development of brain-related diagnostic and therapeutic technologies by preventing the passage of drugs when diseases such as tumors, Alzheimer's disease, and Parkinson's disease occur, as well as various imaging agents for diagnosis. Accordingly, a method of administering a drug or imaging agent after collapsing the blood-brain barrier through a temporary chemical attack using a drug such as OX26 (antibody), OX26-polyethylene glycol, mannitol, or transferrin has been used U. S. Patent No. 6,372, 250, U.S. Patent Application Serial No. 10 / 025,732). (See, for example, U.S. Patent Nos. 6,117,454 and 6,821,594, U.S. Patent Application No. 2004-0131692, European Patent Registration No. 1,071,408 number). However, the above methods are not easy to apply in that the blood-brain barrier is restored after a certain period of time and the additional delivery is difficult or the risk of causing shock when the blood-brain barrier is destroyed by the drug is difficult.

In some embodiments of the invention, the nanoprobe particles of the present invention are more specifically distributed in lymph nodes (especially surveillance lymph nodes), tumor cells, or brain, and the term "distribution" Implies the degree of localization of nanoprobe particles in vivo. Therefore, the nanoprobe particle of the present invention has the advantage of being able to pass through the blood-brain barrier without administering an additional drug in view of the application of the contrast agent / imaging agent, and also has a specific accumulation in the brain tissue, Can be usefully applied.

The term " diagnosing " as used herein includes determining whether a subject currently has a particular disease or disorder, or determining the prognosis of an object that has suffered a particular disease or disorder do.

In certain embodiments of the invention, the cancer / tumor that can be diagnosed / detected by the nanoprobe particle of the invention is selected from the group consisting of brain cancer, neuroendocrine carcinoma, myeloma, lymphoma, leukemia, lymphangioendotheliosarcoma, (Extraskeletal myxoid chondrosarcoma), stomach cancer, lung cancer, breast cancer, ovarian cancer, liver cancer, bronchial carcinoma, non-human carcinomatous carcinoma, papillary carcinoma, papillary carcinoma, Cancer of the prostate, prostate cancer, bone cancer, skin cancer, thyroid cancer, pituitary cancer, ureter cancer, cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, pancreatic cancer, pancreatic cancer, pancreatic cancer, Cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, renal cell carcinoma, hepatocellular carcinoma, biliary cancer, fibrosarcoma, mucinous carcinoma The present invention also relates to a method for treating a disease selected from the group consisting of carcinosarcoma, sarcoma, osteosarcoma, chondrosarcoma, angiosarcoma, endothelial cell sarcoma, lymphangiosarcoma, synovial sarcoma, mesothelioma, mesothelioma, leiomyosarcoma, rhabdomyosarcoma, rhabdomyosarcoma, But are not limited to, papillary adenocarcinoma, cystadenocarcinoma, soft tissue thyroid carcinoma, bronchial carcinoma, choriocarcinoma, testicular, embryonal carcinoma, Wilm's tumor, Kaposi sarcoma or retinoblastoma.

In addition, the brain localization characteristic of the nanoprobe particle of the present invention described above is a very interesting feature in comparison with conventional blood-brain-barrier-passing particles. In the case of conventional blood-brain barrier particles, But the nanoprobe particles of the present invention exhibit a higher distribution in the brain when injected into the body by itself alone (see Fig. 15B). Accordingly, the nanoprobe particles of the present invention can be used not only as a means for efficiently passing the blood-brain barrier but also as a carrier for transporting a specific substance (for example, a therapeutic agent) to the brain without using a conventional adjuvant (e.g., mannitol).

According to another aspect of the present invention, the present invention provides a contrast agent composition comprising nanoprobe particles as described above.

According to another aspect of the present invention, there is provided a method of preparing a nanoprobe comprising: (a) administering to a subject a nanoprobe particle as described above; And (b) measuring fluorescence in the tissue of interest of the subject.

Since the contrast agent composition and imaging method of the present invention include the above-described nanoprobe particles of the present invention as an active ingredient, the description common to both of them is omitted in order to avoid the excessive complexity of the present specification.

The nanoparticles for passing through the blood-brain barrier according to the present invention are very useful for contrasting the brain because they pass through the blood-brain barrier and are located in the brain.

The contrast agent composition of the present invention can be applied to various imaging techniques. The nanoprobe particles prepared according to the present invention can be used for optical imaging and spectroscopy (optical imaging and spectroscopy) or BL (bioluminescence) imaging according to emission fluorescence. The general contents of BL imaging are disclosed in U.S. Patent No. 5,650,135.

In addition, in the synthesis of the dipolar aryl vinyl compound of the present invention, when a radioisotope or a positron emitting isotope is bound to synthesize a bipolar aryl vinyl compound, the nanoprobe particles obtained from the resultant compound are analyzed by single photon emission computed tomography (SPECT, Single Photon Emission Computed Tomography), or Positron Emission Tomography (PET). When PET or SPECT images are obtained using the contrast agent composition of the present invention, examples of positron emission isotopes are ¹⁰ C, ¹¹ C, ¹³ O, ¹⁴ O, ¹⁵ O, ¹² N, ¹³ N, ¹⁵ F, ¹⁷ F, ^{18 F, 32 Cl, 33 Cl} , 34 Cl, 43 Sc, 44 Sc, 45 Ti, 51 Mn, 52 Mn, 52 Fe, 53 Fe, 55 Co, 56 Co, 58 Co, 61 Cu, 62 Cu, 62 Zn , ⁶³ Zn, ⁶⁴ Cu, ⁶⁵ Zn, ⁶⁶ Ga, ⁶⁶ Ge, ⁶⁷ Ge, ⁶⁸ Ga, ⁶⁹ Ge, ⁶⁹ As, ⁷⁰ As, ⁷⁰ Se, ⁷¹ Se, ⁷¹ As, ⁷² As ⁷³ Se, ⁷⁴ Kr, ⁷⁴ Br , ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ⁷⁷ Kr, ⁷⁸ Br, ⁷⁸ Rb, ⁷⁹ Rb, ⁷⁹ Kr, ⁸¹ Rb, ⁸² Rb, ⁸⁴ Rb, ⁸⁴ Zr, ⁸⁵ Y, ⁸⁶ Y, ⁸⁷ Y, ⁸⁷ Zr, ^{88 Y, 89 Zr, 92 Tc,} 93 Tc, 94 Tc, 95 Tc, 95 Ru, 95 Rh, 96 Rh, 97 Rh, 98 Rh, 99 Rh, 100 Rh, 101 Ag, 102 Ag, 102 Rh, 103 Ag, ^{104 Ag, 105 Ag, 106 Ag} , 108 In, 109 In, 110 In, 115 Sb, 116 Sb, 117 Sb, 115 Te, 116 Te, 117 Te, 117 I, 118 I, 118 Xe, 119 Xe, 119 I ^{, 119 Te, 120 I, 120} Xe, 121 Xe, 121 I, 122 I, 123 Xe, 124 I, 126 I, 128 I, 129 La, 130 La, 131 La, 132 La, 133 La, 135 La, 136 La, ¹⁴⁰ Sm, ¹⁴¹ Sm, ¹⁴² Sm, ¹⁴⁴ Gd, ¹⁴⁵ Gd, ¹⁴⁵ Eu, ¹⁴⁶ Gd, ¹⁴⁶ Eu, ¹⁴⁷ Eu, ¹⁴⁷ Gd, ^{1 48 Eu, 150 Eu, 190 Au} , 191 Au, 192 Au, 193 Au, 193 Tl, 194 Tl, 194 Au, 195 Tl, 196 Tl, 197 Tl, 198 Tl, 200 Tl, 200 Bi, 202 Bi, 203 Bi , ²⁰⁵ Bi, ²⁰⁶ Bi, or derivatives thereof. The PET imaging method and apparatus are described in U.S. Patent Nos. 6,151,377, 6,072,177, 5,900,636, 5,608,221, 5,532,489, 5,272,343, and 5,103,098, the disclosures of which are incorporated herein by reference . SPECT imaging methods and apparatus are disclosed in U.S. Patent Nos. 6,115,446, 6,072,177, 5,608,221, 5,600,145, 5,210,421, and 5,103,098, the disclosures of which are incorporated herein by reference.

As described above, the contrast agent composition of the present invention is very useful for imaging a specific region of a human body, particularly, blood vessels of the brain. The imaging process is performed by administering a diagnostically effective amount of contrast agent to a human, and then scanning the human body by imaging to obtain a visible image of the brain.

The contrast agent of the present invention can be administered together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers commonly used in the field of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, poly But are not limited to, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. Suitable pharmaceutically acceptable carriers and formulations include, but are not limited to, Remington's Pharmaceutical Sciences (19th ed., 1995).

The contrast agent of the present invention is preferably administered parenterally. For parenteral administration, it can be administered by intravenous infusion, intracranial infusion, intradermal infusion, intralesional infusion, intramuscular injection, and the like. A suitable dosage of the contrast agent of the present invention may be variously prescribed by factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient have. The term " diagnostically effective amount " as used herein means an amount sufficient to obtain an image of the human body, generally 0.0001-100 mg / kg.

In some embodiments of the invention, the compositions and methods of the present invention are used to treat or prevent a mammal, more particularly a human, a mouse, a rat, a guinea pig, a rabbit, a monkey, a pig, a horse, But are not limited to,

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a novel bipolar aryl vinyl compound, a nanoprobe particle comprising the same, and a contrast agent composition and imaging method using the same.

(b) The bipolar arylvinyl compound of the present invention forms solid-phase nano-aggregates under physiological conditions to exhibit increased SSF and AEF as well as the ability to adjust the SSF color according to the donor-receptor combination, Exhibit a hydrodynamic size of less than about 65 nm.

(c) Nanoprobe particles of the present invention have hydrodynamic size of less than about 25 nm and exhibit increased SSF in the near-infrared (NIR) region of greater than or equal to 650 nm, Brain) in a cell or an in vivo level.

(d) Therefore, the contrast agent composition and imaging method comprising the nanoprobe particle of the present invention can be efficiently applied for the diagnosis of cancer / tumor or brain disease in that lymph node, tumor or brain tissue can be specifically expressed .

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing the structure and synthesis path of a solid-phase fluorescent bipolar aryl vinyl (ArV).
Figure 2 is an optimized geometry result of ArV overlaid with HOMO and LUMO contour diagram (HF / PM3).
Figure 3 shows the fluorescence of ArV powder following 365 nm irradiation. NIR fluorescence images of CbV9-10 and AnV9-10 were obtained with a Kodak imaging system with an excitation / emission filter set for Cy5.5.
Figure 4a shows the hydrodynamic size of self-aggregated nanoparticles of CbV and AnV in THF (tetrahydrofuran) / H ₂ O (= 1/9 by volume) as measured by DLS. Label: size, d.nm (diameter, nm); And number,%.
Figure 4b shows the normalized UV and PL spectra of representative ArV dyes: (a) THF solution, (b) self-aggregates and (c) absorbency at FArV NPs; (d) THF solution, (e) self-aggregates, and (f) FArV NPs. (1) CbV5, (2) AnV1, (3) CbV7, (4) AnV7, (5) CbV8, (6) AnV8, (7) CbV9 and (8) CbV10. Label: absorbance, absorbance; PL intensity (au), photoluminescence intensity (au); And wavelength, wavelength (nm).
Figure 5 shows the results of a solution of 5 μM ArV [(a) CbV5, (b) AnV1, (c) in solution (THF, dotted line) and dispersion of self-agglomerated nanoparticles (THF / deionized water = 1/9 v / v, ) CbV7, (d) AnV7, (e) AnV8, and (f) CbV9. (Insertion figure) Fluorescence emission according to 365 nm irradiation. The NIR fluorescence image of CbV9 of (f) was obtained with a Kodak imaging system with an excitation / emission filter set for Cy5.5.
(A) CbV8 (6.3 ± 1.6 nm), (b) AnV8 (7.6 ± 2.2 nm), (c) CbV9 (7.2 ± 1.4 nm), and (d) CbV10 (6.1 +/- 1.5 nm). Scale bar: 40 nm.
Figure 6b shows the hydrodynamic magnitudes of FArV NPs measured by DLS. Label: size, d.nm (diameter, nm); And number,%.
Figure 7 shows the fluorescence emission of representative FArV NPs under 365 nm illumination: (a) CbV5, (b) AnV1, (c) CbV7, (d) AnV7, (e) CbV8, ) CbV10 and (h) CbV9. The corresponding chemical structures of ArV are shown below. The NIR fluorescence image of CbV9 (h) was obtained with a Kodak imaging system with an excitation / emission filter set for Cy5.5.
Figure 8 shows the results of optical, fluorescent and merged images in HeLa cells treated with FArV NPs: (a) CbV5, (b) AnV1, (c) CbV7, (d) AnV7, AnV8, and (f) CbV9. DAPI stained nuclei are shown in blue. (Insertion figure) Local fluorescence spectrum of cytoplasmic region. Label: PL intensity (au), photoluminescence intensity (au); And wavelength, wavelength (nm).
Figure 9 shows the results of in vitro MTT assay showing the cytotoxicity of FArV NPs at concentrations of (a) 40 mg / mL and (b) 20 mg / mL on HeLa cells. (1) CbV5, (2) AnV1, (3) CbV7, (4) AnV7, (5) CbV8, (6) AnV8, (7) CbV10, (8) CbV9 and (9)
Figure 10 shows the fluorescence signal (S) of FArV NPs and the quantification of the organizer fluorescence background (B) and the S / B ratio thereof. Photoluminescence was imaged using biological excitation (Ex.) And emissive (Em.) Filters excited from biological tissue (3.5 mm thick pig ham, n = 3) with the wavelengths given in the images. The signaling (S) area is indicated by a white circle.
Figure 11 shows the results of a stability test of FCbVlO NPs under biological conditions (90% FBS, 37 ° C) monitored by fluorescence and transmission (scattering) changes. Label: PL intensity (% of initia), photoluminescence intensity (% relative to the starting value); Transmittance (% of initia), permeability (% relative to initial value); And time (H).
12A is a graph showing NIRF images of mice (n = 5) injected intracutaneously into the left front foot of FCbV10 NPs. Imaging times after injection are shown. The yellow arrow and the red arrow indicate the injection site and the sentinel lymph node, respectively. Label: S, Signal; And B, background.
12B shows a result of showing a pseudo-color x - ray image. Excised lymph nodes (shown as red and green, respectively) from the injected site (left) and the other site (right) were not spectrally mixed according to their spectral profiles (right graph) presented in the same color.
12C shows the results of in-bobe and x- bobe intensities of FCbV10 NPs in the region of the signal S and the background B indicated in Figs. 12A and 12B.
Figure 13a shows the results of NIRF images of mice (n = 5) in which FCbVlO NPs were injected into the tail vein. Imaging times after injection are shown. Subcutaneous SCC7 tumors not seen in the initial growth phase (one week after inoculation) are indicated by red arrows. Label: S, Signal; And B, background.
Figure 13b is a binary pseudo read after inoculation 12 hours shows the in vivo image (left panel) color. The organizer was not spectrally mixed according to the spectral profiles of the fluorescence (green) and the signal (red) of FCbV10 NPs presented in the same color (right panel). Label: Tissue, tissue; And Tumor, Tumor.
Fig. 13C is a result showing the in-vivo strength of FCbV10 NPs in the region of the signal S and the background (B) indicated in Fig. 13A.
Figure 14 shows the results of a false-color ex vivo image of tumors and other major organs that were excised from SCC7 tumor-inoculated mice (observed after 2 weeks of tumor cell injection). Images were obtained 5 hours after the tail vein injection of FCbV10 NPs. The autologous fluorescence (a) from the FCbVlO NP signal from the tumor (red) and other organs (green) was not spectrally mixed according to their spectral profiles (b) presented in the same color. (a) My insert shows the intensity of the ex vivo activity of FCbV10 NPs in tumor and background (BG). Label: Liver, liver; Heart, heart; Kidney, kidney; Spleen, spleen; And Tumor, Tumor.
15A and 15B are brain imaging results using the control group (Cy.5) and the polymeric NPs of the present invention, respectively. Label: Liver, liver; Heart, heart; Kidney, kidney; Spleen, spleen; Brain, brain; And Ctr1, control group.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Materials and machines. All chemical reagents were purchased from Aldrich and TCI and used as obtained. The chemical structures were identified by elemental analysis carried out on FT-IR (Thermo Mattson Model Infinity Gold FT-IR), ¹ H NMR (Varian unity plus 300) and CHNS analyzer (EA 1180, FISONS Instruments). The particle size distribution of the nanoparticle dispersion in deionized water was determined at 25 ° C using zeta-sizer (Nano-ZS, Malvern). Transmission electron microscopy (TEM) images were obtained with a CM30 electron microscope (FEI / Philips) operated at 200 kV. For TEM sample preparation, one drop of the particle dispersion was dried on a 300-mesh copper grid coated with carbon and negatively stained with a 2 wt% uranyl acetate solution. From the TEM images, 60 particles were counted for size analysis of each sample. Absorption and emission spectra were obtained using a UV-visible spectrophotometer (Agilent 8453) and a fluorescence spectrophotometer (Hitachi F-7000, wavelength calibrated for excitation and emission), respectively. The relative fluorescence quantum yield (? F) was determined using an ethanol solution containing rhodamine B as a reference.

General procedure for ArV synthesis . All ArV compounds were synthesized by the Knoevenagel condensation illustrated in FIG. Typically, the piperidine is treated portion wise with a solution of the aromatic aldehyde donor (Ar-CHO, 1.0 mmol) and active methylene acceptor (AM, 1.0 mmol) dissolved in 100% EtOH (10 mL) And stirred at 60 [deg.] C for 2-3 hours. After cooling to 0 < 0 > C, the precipitate was filtered and washed with cold EtOH. The dried product was purified by silica gel column chromatography using chloroform as an eluent to obtain a pure solid. Characteristic data for all compounds are provided as follows:

2- (4-bromophenyl) -3- (9-ethyl-9H-carbazol-3-yl) acrylonitrile (CbV1);

Light-yellow solid. Yield: 71%. FT-IR cm- ¹ (KBr), 1622 (aromatic C = C), 2208 (CN), 2968 (CH of aliphatic) and 3046 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 1.46 (3H, t, -CH3, J = 7.2), 4.4 (2H, q, -CH2, J = 7.2), 7.30 (1H, t, CbPhH, J = 7.2), 7.42-7.56 (8H, m, CbPhH & COPhH), 7.68 (1H, s, CH), 8.15 (1H, d, CbPhH, J = 7.5), 8.63 (1H, s, CbPhH) . ¹³ C NMR (150 MHz, CDCl ₃ ) 隆 143.92, 141.37, 140.66, 134.39, 132.20, 127.28,127.13,126.66,124.63,123.49,122.96,122.86,122.58,120.91,12.03,118.89,109.10,108.99,106.22,37.95 , 13.99 ppm. Elemental analysis as determined by C ₂₃ H ₁₇ BrN ₂ (C, 68.84; H, 4.27; N, 6.98): Found, C, 68.63; H, 4.16; N, 6.96%. MS (MALDI) measured for C ₂₃ H ₁₇ BrN ₂ ( m / z = 402.06): measured, m / z = 402.05.

3- (9-ethyl-9H-carbazol-3-yl) -2- (thiophen-2-yl) acrylonitrile (CbV2);

Yellow solid. Yield: 75%. FT-IR cm- ¹ (KBr), 1628 (C = C of aromatic), 2212 (CN), 2977 (CH of aliphatic) and 3096 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 1.47 (3H, t, J = 7.2 Hz), 4.40 (2H, q, J = 7.2 Hz), 7.08 (1H, t, J = 7.5 Hz ), 7.27-7. 32 (2H, m), 7.37 (1H, d, J = 6.9Hz), 7.43-7. D, J = 8.7 Hz), 8.15 (1H, d, J = 7.8 Hz), 8.61 (1H, s). ¹³ C NMR (150 MHz, CDCl ₃ ) δ 141.33, 141.21, 140.64, 140.28, 128.13, 126.98, 126.59, 126.10, 125.26, 124.55, 123.48, 122.97, 122.58, 120.92, 119.97, 118.05, 109.06, 109.01, 102.21, , 13.99 ppm. C ₂₁ H ₁₆ N ₂ S elemental analysis measured in (C, 76.80; H, 4.91 ;; N, 8.53 S, 9.76): measurement value, C, 76.44; H, 4.89; N, 8.39; S, 9.60%. _{C 21 H 16 N 2 S (} m / z = 328.10) the MS (MALDI) measured by: measured, m / z = 328.11.

2- (biphenyl-4-yl) -3- (9-ethyl-9H-carbazol-3-yl) acrylonitrile (CbV3);

Light-yellow solid. Yield: 62%. FT-IR cm ^-1 (KBr), 1612 (C = C of aromatic), 2213 (CN), 2908 (CH of aliphatic) and 3038 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 1.48 (3H, t, -CH3, J = 7.2), 4.41 (2H, q, -CH2, J = 7.2), 7.30 (1H, t, CbPhH, J = 7.5), 7.38 (1H, t, BiPhH, J = 7.2), 7.44-7.55 (6H, m, BiPhH & CbPhH), 7.63-7.68 (3H, m, CbPhH), 7.71 (1H, s, CH), 7.79 (2H, d, CbPhH, J = 8.4), 8.17 (2H, d, BiPhH, J = 8.4), 8.67 (1H, s, CbPhH). ¹³ C NMR (150 MHz, CDCl ₃ ) 隆 143.29, 141.31, 141.25, 140.65, 140.27, 134.32,129.04,127.81,127.70,127.31,127.13,126.58,126.20,124.97,124.48,123.02,122.75,201.94,119.97,119.25 , 109.07, 108.96, 107.04, 37.93, 14.0. Elemental analysis as determined by C ₂₉ H ₂₂ N ₂ (C, 87.41; H, 5.56; N, 7.03): found, C, 86.27; H, 5.51; N, 6.94%. MS (MALDI) measured with C ₂₉ H ₂₂ N ₂ ( m / z = 398.18): measured, m / z = 398.17.

2-Cyano-3- (9-ethylcarbazol-3-yl) acrylic acid (CbV4);

Yellow-green solid, 57% yield; FT-IR cm- ¹ (KBr), 1624 (C = C of aromatic), 1694 (C = O of carboxyl group), 2224 (CN), 2980 (CH of aromatic), 3440 (OH, carboxylic acid). ^{1 H NMR (300 MHz, DMSO} , TMS, ppm): δ 1.35 (3H, t, CH3, J = 6.9), 4.51 (2H, q, CH2, J = 6.9), 7.32 (1H, t, J = 7.2 ), 7.55 (1H, t, J = 7.5), 7.72 (1H, d, J = 8.4), 7.83 (1H, d, J = 8.7), 8.15 (1H, d, J = 7.5) 8.29 (1H, d , J = 8.4), 8.46 (1H, s), 8.86 (1H, s), 13.64 (1H, s, ¹³ C NMR (150 MHz, DMSO) δ 164.78, 155.79, 142.73, 140.78, 128.33, 127.42, 126.07, 123.13, 122.85, 122.62, 121.09, 120.86, 117.88, 110.51, 98.34, 62.74, 38.03, 14.0. _{C 18 H 14 N 2 O 2} (C, 74.47; H, 4.86; N, 9.65; O, 11.02) The elemental analysis determined by: measurement value, C, 74.39%; H, 4.76%; N, 9.52%. _{C 18 H 14 N 2 O 2} (m / z = 290.11) The MS measurement in (MALDI): measurement value, m / z = 290.26.

Ethyl 2-cyano-3- (9-ethyl-9H-carbazol-3-yl) acrylate (CbV5);

Light-yellow solid, yield: 58%. FT-IR cm ^-1 (KBr), 1628 (C = C of aromatic), 1722 (C = O, ester), 2218 (CN), 2974 (CH of aliphatic) and 3068 (CH of aromatic). ¹ H NMR (300 MHz, CDCl ₃ , TMS, ppm):? 1.39-1.50 (6H, m, -CH3), 4.36-4.44 (4H, m, -CH2), 7.32 J = 7.2), 7.46 (1H , t, Cb-PhH, J = 6.9), 7.52-7.57 (2H, m, Cb-PhH), 8.16 (1H, d, Cb-PhH, J = 8.4), 8.22 ( (1H, d, Cb-PhH, J = 8.7), 8.42 (1H, s, CH), 8.75 (1H, s, Cb-PhH). ^{13 C NMR (150 MHz, CDCl} 3) δ 163.76, 156.01, 142.89, 140.72, 129.16, 127.05, 125.48, 123.67, 122.72, 121.06, 120.66, 117.08, 109.33, 109.19, 103.3, 97.76, 62.39, 38.05, 14.56, 14.2 ppm. _{C 20 H 18 N 2 O 2} (C, 75.45; H, 5.70; N, 8.80) The elemental analysis determined by: measurement value, C, 74.94%; H, 5.61%; N, 8.66%. _{C 20 H 18 N 2 O 2} (m / z = 318.14) The MS measurement in (MALDI): measurement value, m / z = 318.50.

2 - ((9-ethyl-9H-carbazol-3-yl) methylene) malononitrile (CbV6);

Yellow solid. Yield 60%. FT-IR cm- ¹ (KBr), 1626 (C = C of aromatic), 2228 (CN), 2980 (CH of aliphatic) and 3056 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 1.48 (3H, t, CH 3, J = 7.2 Hz,), 4.41 (2H, q, CH 2, J = 8.0 Hz), 7.36 (1 H, t, PhH, J = 7.2 Hz), 7.47 (2H, d, PhH, J = 9.3 Hz), 7.57 (1H, t, PhH, J = 7.2 Hz), 7.86 (1H, s, CH), 8.07 -8.15 (2H, m, PhH), 8.62 (1H, s, PhH) ppm. ¹³ C NMR (150 MHz, CDCl 3)? 160.0, 143.30, 140.65, 128.52, 127.33, 124.98, 123.69, 122.56, 122.26, 120.93, 115.02, 114.08, 109.40, 109.30, 76.36, 38.02, 13.77 ppm. _{C 18 H 13 N 3 (C} , 79.68; H, 4.83; N, 15.49%) elemental analysis determined by: measurement value, C, 79.62%; H, 4.72%; N, 15.41%. _{C 18 H 13 N 3 (m} / z = 271.11) The MS measurement in (MALDI): measurement value, m / z = 271.12.

2- Benzoyl -3- (9-ethyl-9H- Carbazole -3 days) Acrylonitrile ( CbV7 );

Yellow-green solid, yield: 68%. FT-IR cm ^-1 (KBr), 1628 (C = C of aromatic), 1665 (C = O), 2210 (CN), 2978 (CH of aliphatic) and 3055 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 1.48 (3H, t, -CH3, J = 7.2), 4.41 (2H, q, -CH2, J = 7.2), 7.33 (1H, t, CbPhH, J = 7.2), 7.45-7.57 (6H, m, COPhH & CbPhH), 7.63 (1H, t, COPhH, J = 7.2), 7.91 (2H, d, COPhH, J = 7.8), 8.15 (1H, d, CbPhH, J = 7.5), 8.30 (1H, s, CH), 8.78 (1H, s, CbPhH). ¹³ C NMR (150 MHz, CDCl ₃ ) δ 189.34, 157.17, 143.14, 140.79, 136.91, 132.93, 129.41, 129.28, 128.66, 127.15, 125.87, 123.86, 123.07, 122.97, 121.12, 120.79, 118.56, 109.41, 109.35, 105.38 , 38.15, 14.0 ppm. Elemental analysis as determined by C ₂₄ H ₁₈ N _{2 O} (C, 82.26; H, 5.18; N, 7.99; O, 4.57%): C, 81.96%; H, 5.06%; N, 7.98%. MS (MALDI) measured with C ₂₄ H ₁₈ N _{2 O} ( m / z = 350.14): measured, m / z = 350.13.

2 - ((9-ethyl-9H- Carbazole Yl) methylene) -1H- Inden -1,3 (2H) - Dion ( CbV8 );

Orange solid. Yield: 81%. FT-IR cm ^-1 (KBr), 1625 (C = C of aromatic), 1665 (C = O), 2976 (CH of aliphatic) and 3050 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 1.47 (3H, t, -CH3, J = 7.2), 4.39 (2H, q, -CH2, J = 7.2), 7.33 (1H, t, CbPhH, J = 7.5), 7.41-7.54 (3H, m, CbPhH), 7.73-7.80 (2H, m, InPhH), 7.95-8.03 (2H, m, InPhH), 8.07 (1H, s, CbPhH), 8.24 (1H, d, CbPhH, J = 8.4), 8.66 (1H, d, CbPhH, J = 8.7), 9.44 (1H, s, CH). ¹³ C NMR (150 MHz, CDCl ₃ ) δ 191.29, 143.32, 142.50, 140.71, 140.02, 134.92, 134.66, 133.48, 128.88, 126.79, 125.02, 123.79, 123.41, 122.96, 121.19, 120.69, 109.25, 108.78, ppm. Elemental analysis as measured by C ₂₄ H ₁₇ NO ₂ (C, 82.03; H, 4.88; N, 3.99%): measured, C, 81.97%; H, 4.79%; N, 3.99%. MS (MALDI) as measured by C ₂₄ H ₁₇ NO ₂ ( m / z = 351.13): found, m / z = 351.12.

2- (2 - ((9-ethyl-9H- Carbazole Yl) methylene) -3-oxo-2,3- Dihydro -1H- Inden -One- Iriden ) Malononitrile ( CbV9 )

Dark red solid. Yield: 67%. FT-IR cm- ¹ (KBr), 1628 (C = C of aromatic), 1704 (C = O), 2220 (CN), 2974 (CH of aliphatic) and 3050 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 1.50 (3H, t, -CH3, J = 7.2), 4.42 (2H, q, -CH2, J = 7.2), 7.36 (1H, t, CbPhH, J = 7.8), 7.46-7.57 (3H, m, CbPhH), 7.75-7.81 (2H, m, onmalPhH), 7.95 (1H, d, CbPhH, J = 7.5), 8.23 (1H, d, onmalPhH, J = 7.8), 8.50 (1H, d, OnMalPhH, J = 8.7), 8.71 (1H, d, CbPhH, J = 7.8), 8.84 (1H, s, CbPhH), 9.25 (1H, s, CH). ¹³ C NMR (150 MHz, CDCl ₃ ) δ 188.32, 163.17, 149.76, 143.83, 140.56, 137.54, 135.09, 134.58, 134.07, 129.6, 127.08, 125.18, 124.53, 123.84, 123.58, 123.4, 123.3, 121.30, 121.11, 119.50 , 115.29, 114.82, 109.46, 108.80, 69.88, 38.21, 14.01 ppm. _{C 27 H 17 N 3O (C} , 81.19; H, 4.29; N, 10.52%) elemental analysis determined by: measurement value, C, 81.08%; H, 4.37%; N, 10.68%. _{C 27 H 17 N 3O (m} / z = 399.14) the MS (MALDI) measured by: measured, m / z = 399.13.

2,2 '- (2 - ((9-ethyl-9H- Carbazole Yl) methylene) -1H- Inden -1,3 (2H) - Diiridene ) Dimalononitrile ( CbV10 );

Dark blue solid. Yield: 85%. FT-IR cm- ¹ (KBr), 1630 (C = C of aromatic), 2208 (CN), 2956 (CH of aliphatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 1.51 (3H, t, -CH3, J = 7.2), 4.43 (2H, q, -CH2, J = 7.2), 7.38 (1H, t, CbPhH, J = 7.5), 7.48-7.58 (3H, m, CbPhH), 7.74-7.82 (2H, m), 7.92 (1H, d, J = 7.5), 7.96-8.1 (2H, m), 8.72 (1H , d, J = 7.8), 8.85 (1H, s), 9.3 (1H, s, CH). ¹³ C NMR (150 MHz, CDCl ₃ ) δ 190.1, 158.74, 148.54, 143.64, 140.54, 138.42, 135.02, 130.69, 129.2, 127.06, 125.2, 124.33, 123.54, 122.14, 118.51, 118.42, 109.26, 108.60, 103.3, , 39.6, 14.05 ppm. Elemental analysis as determined by C ₃₀ H ₁₇ N ₅ (C, 80.52; H, 3.83; N, 15.65%): measured, C, 80.26; H, 3.57; N, 15.37%. _{C 30 H 17 N 5 (m} / z = 447.15) The MS measurement in (MALDI): measurement value, m / z = 447.28.

2- (4- Bromophenyl ) -3- (4- (dimethylamino) Phenyl ) Acrylonitrile ( AnV1 );

Yellow solid, yield: 86%. FT-IR cm- ¹ (KBr), 1612 (C = C of aromatic), 2206 (CN), 2908 (CH of aliphatic) and 3037 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 3.1 (6H, s, (NCH 3) 2), 6.70 (2H, d, ArH, J = 9 Hz), 7.37 (s, 1H, CH ), 7.46-7.53 (m, 4H, PhBr), 7.84 (2H, d, ArH, J = 9 Hz). ^{13 C NMR (150 MHz, CDCl} 3) δ 151.95, 142.95, 134.74, 132.08, 131.56, 127.02, 121.94, 121.38, 119.24, 111.71, 103.25, 40.14. Elemental analysis as measured by C ₁₇ H ₁₅ BrN ₂ (C, 62.40; H, 4.62; Br, 24.42; N, 8.56%): measured, C, 62.56%; H, 4.61%; N, 8.65%. MS (MALDI) measured with C ₁₇ H ₁₅ BrN ₂ ( m / z = 326.04): measured, m / z = 326.04.

2- (4- (dimethylamino) Phenyl ) -3- (thiophen-2-yl) Acrylonitrile ( AnV2 );

Green solid. Yield: 80%. FT-IR cm ^-1 (KBr), 1612 (C = C of aromatic), 2208 (CN), 2908 (CH of aliphatic) and 3012 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 3.61 (6H, s, N (CH 3) 2), 6.71 (2H, d, ArH, J = 9 Hz), 7.02-7.05 (1H, m, TpH), 7.20 (1H , d, TpH, J = 6 Hz), 7.25 (1H, s, CH), 7.27 (1H, d, TpH, J = 6 Hz), 7.80 (2H, d, ArH, J = 9 Hz). ^{13 C NMR (150 MHz, CDCl} 3) δ 151.75, 140.64, 131.21, 128.03, 128.01, 125.25, 124.59, 121.30, 118.38, 111.77, 99.48, 40.16. C ₁₅ H ₁₄ N ₂ S elemental analysis measured in (C, 70.83; H, 5.55 ;; N, 11.01 S, 12.61%): measurement value, C, 70.67%; H, 5.54%; N, 10.85; S, 12.43%. _{C 15 H 14 N 2 S (} m / z = 254.09) the MS (MALDI) measured by: measured, m / z = 254.08.

2- (biphenyl-4-yl) -3- (4- (dimethylamino) Phenyl ) Acrylonitrile ( AnV3 );

Yellow solid. Yield: 85%. FT-IR cm ^-1 (KBr), 1612 (C = C of aromatic), 2210 (CN), 2908 (CH of aliphatic) and 3038 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 3.07 (6H, s, N (CH 3) 2), 6.74 (2H, d, ArH, J = 9 Hz), 7.46 (1H, s, CH), 7.40-7.51 (5H, m, PhH), 7.68-7.61 (4H, m, PhH), 7.88 (2H, d, ArH, J = 9 Hz). ^{13 C NMR (150 MHz, CDCl} 3) δ 151.82, 142.36, 141.24, 140.34, 134.66, 131.47, 128.95, 127.96, 127.20, 125.93, 121.76, 119.56, 111.76, 104.20, 40.18, 23.45. Elemental analysis as determined by C ₂₃ H ₂ O ₂ (C, 85.15; H, 6.21; N, 8.63%): measured, C, 85.55%; H, 6.12%; N, 8.48%. _{C 23 H 2 0N 2 (m} / z = 324.16) The MS measurement in (MALDI): measurement value, m / z = 324.16.

2- Cyano -3- (4- (dimethylamino) Phenyl ) Acrylic acid ( AnV4 );

Yellow solid. Yield 63%. FT-IR cm- ¹ (KBr), 1615 (C = C of aromatic), 1668 (C = O of carboxyl group), 2219 (CN), 2972 (CH of aromatic), 3436 (OH, carboxylic acid). ^{1 H NMR (300 MHz, DMSO} , TMS, ppm): δ 3.07 (6H, s, NCH 3), 6.82 (2H, d, ArH, J = 9), 7.93 (2H, d, ArH, J = 9) , 8.07 (1H, s, CH), 13.25 (1H, s, OH). ¹³ C NMR (150 MHz, DMSO) δ 165.40, 154.36, 154.02, 134.06, 118.97, 118.49, 112.17, 94.02, 39.64 ppm. Elemental analysis as determined by C ₁₂ H ₁₂ N ₂ O ₂ (C, 66.65; H, 5.59; N, 12.96%): found, C, 66.69; H, 5.66; N, 12.97%. MS (MALDI) measured with C ₁₂ H ₁₂ N ₂ O ₂ ( m / z = 216.09): measured, m / z = 216.10.

Ethyl 2- Cyano -3- (4- (dimethylamino) Phenyl ) Acrylate ( AnV5 );

Orange solid. Yield: 72%. FT-IR cm ^-1 (KBr), 1612 (C = C of aromatic), 1706 (C = O, ester), 2214 (CN), 2935 (CH of aliphatic) and 2990 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 8.06 (1H, s, CH), 7.93 (2H, d, J = 9.3 Hz, ArH), 6.69 (2H, d, J = 9 Hz, (2H, q, J = 7.1 Hz, CH3), 3.10 (s, 6H, NCH3), 1.37 (3H, t, J = 7.2 Hz, CH3). ^{13 C NMR (150 MHz, CDCl} 3) δ 164.39, 154.63, 153.68, 134.15, 119.46, 117.70, 111.59, 86.16, 61.8, 40.26, 14.26 ppm. _{C 14 H 16 N 2 O 2} (C, 68.83; H, 6.60; N, 11.47%) elemental analysis determined by: measurement value, C, 68.61%; H, 6.66%; N, 11.40%. _{C 14 H 16 N 2 O 2} (m / z = 244.12) The MS measurement in (MALDI): measurement value, m / z = 244.12.

2- (4- (dimethylamino) Benzylidene ) Malononitrile ( AnV6 );

Yellow solid. Yield: 67%. FT-IR cm- ¹ (KBr), 1618 (C = C of aromatic), 2216 (CN), 2925 (CH of aliphatic) and 3060 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 7.81 (2H, d, ArH, J = 9.0 Hz), 7.45 (1 H, s, CH), 6.69 (2 H, d, ArH, J = 9.0 Hz), 3.14 (6 H, s, (NCH 3) 2). ^{13 C NMR (150 MHz, CDCl} 3) δ 158.20, 154.37, 133.92, 119.40, 116.13, 115.05, 111.73, 39.76. Elemental analysis as determined by C ₁₂ H ₁₁ N ₃ (C, 73.07; H, 5.62; N, 21.30%): found, C, 72.89; H, 5.58; N, 20.99%. MS (MALDI) measured with C ₁₂ H ₁₁ N ₃ ( m / z = 197.10): found, m / z = 197.09.

2- Benzoyl -3- (4- (dimethylamino) Phenyl ) Acrylonitrile ( AnV7 );

Orange solid. Yield: 69%. FT-IR cm ^-1 (KBr), 1618 (C = C of aromatic), 1654 (C = O), 2204 (CN), 2948 (CH of aliphatic) and 3054 (CH of aliphatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 3.13 (6Н, s, N (CH 3) 2); 6.72 (2H, d, ArH, J = 9 Hz); 7.46-7.55 (2 H, m, COPh, J = 7.2); 7.57 (1H, t, COPh, J = 4.8 Hz); 7.84 (2H, d, COPh, J = 7.2); 7.89 (1H, s, CH), 8.0 (2H, d, ArH, J = 9 Hz). ^{13 C NMR (150 MHz, CDCl} 3) δ 188.74, 155.87, 153.98, 137.46, 134.65, 132.46, 129.05, 128.49, 119.84, 119.33, 111.73, 101.75, 40.26 ppm. C ₁₈ H ₁₆ N ₂ O as measured by elemental analysis (C, 78.24;; H, 5.84 N, 10.14%): measurement value, C, 77.94; H, 5.80; N, 10.03%. _{C 18 H 16 N 2 O (} m / z = 276.13) The MS measurement in (MALDI): measurement value, m / z = 276.12.

2- (4- (dimethylamino) Benzylidene ) -1H- Inden -1,3 (2H) - Dion ( AnV8 );

Red solid. Yield: 80%. FT-IR cm ^-1 (KBr): 1608 (vC = C of aromatic), 1662 & 1708 (vC = O), 2948 (vC-H of aliphatic) and 3000 (vC-H of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 3.15 (6H, s, N (CH 3) 2), 6.75 (2H, d, ArH, J = 9 Hz), 7.71-7.75 (2H, m, COPh), 7.83 (1H, s, CH), 7.91-7.95 (2H, m, COPh), 8.55 (2H, d, ArH, J = 9 Hz) ppm. ^{13 C NMR (150 MHz, CDCl} 3) δ 190.1, 154.06, 147.62, 142.35, 139.99, 138.09, 134.49, 134.21, 122.59, 111.51, 40.18 ppm. C ₁₈ H ₁₅ NO ₂ measured by elemental analysis (C, 77.96;; H, 5.45 N, 5.05%): measurement value, C, 77.38; H, 5.45; N, 5.03%. _{C 18 H 15 NO 2 (m} / z = 277.11) The MS measurement in (MALDI): measurement value, m / z = 277.10.

2- (2- (4- (dimethylamino) Benzylidene ) -3-oxo-2,3- Dihydro -1H- Inden -One- Iriden ) Malononitrile ( AnV9 );

Dark red solid. Yield 60%. FT-IR cm- ¹ (KBr), 1608 (C = C of aromatic), 1674 (C = O), 2214 (CN), 2948 (CH of aliphatic) and 2994 (CH of aromatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 3.07 (6H, s, N (CH 3) 2), 6.68 (2H, d, J = 8.7 Hz), 7.20-7.36 (2H, m) , 7.70 (1H, t, J = 8.1 Hz), 7.85 (1H, t, J = 8.4 Hz), 8.37 (2H, d, ArH, J = 9.3 Hz), 8.50 (1H, s, ¹³ C NMR (150 MHz, CDCl ₃ ) δ 198.93, 189.55, 159.17, 149.81, 139.87, 135.86, 134.08, 131.65, 129.51, 127.41, 125.74, 123.54, 120.70, 115.39, 111.64, 61.08, 40.33 ppm. Elemental analysis as determined by C ₂₁ H ₁₅ N ₃ O (C, 77.52; H, 4.65; N, 12.91%): found, C, 76.98; H, 4.80; N, 13.39%. _{C 21 H 15 N 3 O (} m / z = 325.12) The MS measurement in (MALDI): measurement value, m / z = 325.74.

2,2 ' - (2- (4- (dimethylamino) Benzylidene ) -1H- Inden -1,3 (2H) - Diiridene ) Dimalononitrile ( AnV10 );

Dark blue solid. Yield 77%. FT-IR cm- ¹ (KBr), 1632 (C = C of aromatic), 2200 (CN), 2950 (CH of aliphatic). ^{1 H NMR (300 MHz, CDCl} 3, TMS, ppm): δ 3.08 (6H, s, N (CH 3) 2), 5.66 (1H, s, CH), 6.59 (2H, d, ArH, J = 9.3 Hz), 7.21-7.26 (2H, m, PhH), 7.48 (2H, d, ArH, J = 9.3 Hz), 7.92-7.96 (2H, m, PhH). ^{13 C NMR (150 MHz, CDCl} 3) δ 190.39, 164.78, 158.72, 138.42, 136.49, 132.1, 122.13, 118.51, 118.41, 111.61, 103.32, 59.84, 39.96 ppm. Elemental analysis as determined by C ₂₄ H ₁₅ N ₅ (C, 77.20; H, 4.05; N, 18.76%): measured, C, 77.29; H, 4.37; N, 18.27%. MS (MALDI) measured with C ₂₄ H ₁₅ N ₅ ( m / z = 373.13): measured, m / z = 373.56.

Preparation of - (Aggregates Self) - ArV self-aggregates. Aqueous dispersions of ArV nano-aggregates were prepared without surfactant for the study of AEF behavior. Briefly, a THF stock solution of each ArV compound (50 μM, 1 mL) was added to 9 mL of deionized water at one time under ultrasound to obtain an ArV solution at a final concentration of 5 μM. For comparative studies, the same concentration of THF solution was prepared by mixing the THF stock solution with 9 mL of THF.

FArV Manufacture of NPs . For all ArV compounds, polymer-captured nanoparticles of dye aggregates were prepared with a Pluronic F-127 surfactant for in vitro and in vivo imaging. Typically, a mixture of F-127 (20 mg) and ArV (0.3 mg) was dissolved in 1 mL of THF. After blowing air to evaporate the solvent, the dried mixture is mixed with 2 mL of deionized water and stirred under ultrasonic treatment for several seconds to obtain a transparent aqueous dispersion of hydrophobic dye aggregates having an ArV concentration of 150 μg / mL. In the case of in vivo brain imaging, a mixture of F-127 (20 mg) and ArV (0.5 mg) was dissolved in 200 μL of MC (methyl cellulose) to obtain a water-soluble dispersion. 200 μL The dispersion was injected intravenously.

In vitro cell labeling and imaging . HeLa cells were maintained in DMEM (Dulbecco's modified Eagle medium) supplemented with 10% FBS, 5 mM L-glutamine, and 5 μg / mL gentamycin in a humidified 5% CO ₂ , 37 ° C incubator. Cells were seeded in dishes under 35 mm cover glass and incubated until reaching 70% confluence. Prior to the experiment, cells were washed twice with PBS (pH 7.4) to remove residual growth medium and then incubated in serum-free medium (1.8 mL) containing FArV NPs (200 μL) for 30 min. For photoluminescence imaging, the labeled cells were washed twice with PBS (pH 7.4) and then directly imaged using LEICA DMI 3000B (CRI, USA) equipped with a nuance FX multispectral imaging system.

In Vivo Surveillance lymph nodes and tumor imaging . Animal studies were approved by the Animal Experimental Ethics Committee of KIST and the treatment of mice was carried out in accordance with the regulations of the Commission. For the production of tumor models, SCC7 cell lines were injected into BALB / c mice (5-week old male; Orient Bio Inc., Korea). The mice were anesthetized with an intraperitoneal injection of 0.5% pentobarbital sodium (0.01 mL / g) before the experiment. For surveillance lymph node imaging, 20 μL of FCbV10 NPs were injected into the forefoot of normal mice, and for tumor imaging, 200 μL of FCbV10 NPs were injected intravenously via the tail vein of tumor-bearing mice. In vivo imaging was performed with an IVIS spectral imaging system (Caliper, USA).

Experimental results and further discussion

The structure and synthesis route of ArV is shown in Fig. Dipolar acrylic plastic scaffolds is due can be readily prepared via a modular synthesis of the combination format and jyeotgi found to exhibit a ²⁵ part derivative are effective SSF is ^{26 - 28,} wherein the scaffold was chosen to construct the compound library. The ArV structure is characterized by bipolar π-conjugation with a π-electron donor and an acceptor at both ends. The compounds were synthesized by single-step Knoevenagel condensation between aromatic aldehyde donors (Ar-CHO) and active methylene (AM) receptor building blocks. The aldehydes of aniline (An-CHO) and carbazole (Cb-CHO) were selected as donor building blocks with different? -Electron donating strengths. Ten receptor building blocks were selected from active methylene compounds containing one or two electron-accepting cyano or carbonyl substituents. The condensation reaction was carried out in ethanol in the presence of piperidine, and the precipitated products were purified by column chromatography. The hydrophobic products are soluble in common organic solvents, but are insoluble in alcohol and water, which facilitates the formation of solid-phase nano-aggregates under physiological conditions.

Semiempirical molecular orbital (MO) calculations show that all of the bipolar ArV compounds exhibit the highest occupied molecular orbital (HOMO) and the lowest intrinsic molecular orbital (LUMO) diagram, transfer properties: the π-electrons are distributed around the donor in the HOMO (reflecting the electronic ground state), but redistributed from the LUMO towards the receptor (reflecting the first excited state; Figure 2 ). The optimized geometry showed that the bulk receptors composed of alpha, alpha -duplex substituted vinyl segments add intracellular steric hindrance and cause severe distortion that is appropriate for the generation of SSF. Indeed, all of the ArV compounds obtained exhibited fluorescence in solids, which means that our molecular design strategy for obtaining SSF is valid (FIG. 3).

The visual properties of ArV according to molecular states were studied in comparison with a suitable solvent (THF) and a poorly water soluble solvent (THF / H ₂ O = 1/9 on volume). In the latter aqueous environment, hydrophobic ArV molecules were precipitated with self-aggregated nanoparticles having a hydrodynamic size of about 50 nm (Table 1 and Fig. 4A). The above results are summarized in Table 2 below (some are for representative spectra, Figure 4b).

Hydrodynamic size (nm) determined by DLS. ArV Self-aggregate FArV ArV Self-aggregate FArV CbV1 56.37 15.02 AnV1 40.37 14.03 CbV2 32.65 13.83 AnV2 33.29 11.53 CbV3 49.34 16.53 AnV3 62.08 14.62 CbV4 29.56 12.58 AnV4 29.74 15.44 CbV5 43.44 14.93 AnV5 52.18 14.96 CbV6 34.96 17.97 AnV6 50.71 17.34 CbV7 43.76 12.92 AnV7 34.16 16.50 CbV8 47.34 13.95 AnV8 60.01 15.65 CbV9 30.48 12.89 AnV9 59.47 22.42 CbV10 28.62 8.78 AnV10 31.75 11.35

As shown in FIG. 5, most of the ArV compounds exhibited AEF behavior, i.e., weak fluorescence in solution and increased release in nanoaggregation. As shown by the calculation results (Fig. 2), the twisted pi-conjugation of AvrV can cause free distorted brass in a separate solution state, which results in a faster nonradiative decay and a lower fluorescence quantum It leads to yield ^7. Thus, the limited quenching activity by agglomeration may be a valid reason for the AEF behavior. Bathochromic alterations in the fluorescence spectrum were observed under agglutination, suggesting that the coagulation-induced planarization of the twisted geometry prolongs the effective pi-conjugation length to narrow the visual bandgap it means. This suggests that different molecular stacking modes of each ArV can affect the resulting SSF color of the nano-aggregates formed.

Bipolar ArV libraries with various donor-receptor combinations enabled easy tunability of SSF colors, as summarized in Table 2 below.

Spectral characteristics of ArV in the form of solutions, self-aggregates and polymer-trapped nanoparticles (FArV NPs) ^a .

A rV solution( THF ) Self-aggregate FArV NPs Abs
( nm ) Em
( nm ) QY
(%) Abs
( nm ) Em
( nm ) QY
(%) Abs
( nm ) Em
( nm ) QY
(%) CbV1 369 452 0.23 387 500 2.23 380 460 3.5 CbV2 370
447
548 0.26
397
530
2.94 383
465
3.6 CbV3 386 465 0.41 394 519 6.5 383 470 6.3 CbV4 388 458 0.49 370 506 2.2 372 482 2.6 CbV5 394 460 0.37 432 510 22.9 398 470 12.3 CbV6 405 472 0.29 421 513 12.1 466 536 40.3 CbV7 406 486 0.32 437 546 8.7 424 548 7.5 CbV8 448 510 0.23 460 630 15.2 440 624 14.7 CbV9 513 574 0.61 562 740 19.8 550 732 18.2 CbV10 580
630 654
705 12.6
584
624 661
709 15.8
582
633 658
708 18.7
AnV1 392 485 0.003 442 503 7.6 398 503 27.1 AnV2 386
455
546 0.18
416
534
5.5
397
524
10.6
AnV3 386 476 0.16 419 550 7.9 399 564 15 AnV4 396 462 0.30 410 486 3.2 402 487 2.9 AnV5 414 467 0.21 437 572 4.7 432 566 5.4 AnV6 426 476 0.71 440 564 44 438 569 20 AnV7 432 512 0.03 465 601 36 462 606 15.3 AnV8 474 534 0.04 502 670 9.3 494 720 5.2 AnV9 547 638 1.2 566 715 12.3 552 656 8.5 AnV10 582
630 643
704 5.4
586
632 657
715 6.2
584
634 658
710 10.2

^a Abs, absorption; Em, emission; QY, quantum yield; Standard for QY, rhodamine dissolved in ethanol B. ArV concentration for all samples is 5 μM. Self-aggregates and FArV NPs were dispersed in a THF / deionized water mixture (THF / H ₂ O = 1/9 v / v) and pure deionized water, respectively.

Significantly, the emission color includes a broad spectral range up to the NIR region (FIG. 5). In the same receptor units, aniline derivatives (AnV) showed red-shifted absorption and emission relative to the corresponding carbazole counterpart (CbV) Which is a more powerful π-charge donor. Compared to the loan pair electrons in the nitrogen of aniline, the lambda pair electrons of carbazole preferentially delocalize in the carbazole ring due to the stabilization of the further aromatic, interfere with the directed charge transfer to lower the degree of widening the visual ICT bandgap ^29. Similarly, variations in receptor structures with different numbers of receptors (cyano >ketone> esters) with different acceptance intensities showed significant effects on the SSF color of the nano-aggregates. CbV1-3 and AnV1-3 with one cyano group attached to the vinyl end and a geminal aromatic ring showed a large bandgap emitting blue-to-green SSF. The presence of two receptors (CbV4-7 and AnV4-7) at the same vinyl terminus adjusted the SSF color to a green-to-orange window. The SSF color is further coupled with an additional phenyl ring by cyclic bridging of two identical receptors, i.e., 1,3-indanedione-based receptors (CbV8-10 and AnV8-10) Could be moved. The phenyl bridge was found to play a key role in the red-shift of the spectrum by locking the two receptors into an in-plane geometry and extending effective pi-conjugation with additional orientation presumably ^30. This inference is supported by the computational results for CbV8-10 and AnV8-10, which demonstrated HOMO-to-LUMOπ-electron redistribution to the plane of the ring-like receptors and to the end of the phenyl bridge by ICT ). With increasing dicyanomethylidene substitution of the 1,3-indanedione unit, the resulting SSF color approaches the correct NIR (greater than 700 nm), which is a combination approach of our inventors to adjust SSF to NIR Is valid. Overall, our ArV library provides SSF colors in the blue-to-NIR region useful for a wide range of biological applications.

To apply the resulting SSF release to bioimaging, ArV compounds were captured with Pluronic F-127, a biocompatible polymer surfactant, to produce dye-agglomerated molecular nanoprobe (FArV NPs). FArV NPs were prepared by mixing ArV with Fluoronic F-127 in solution and adding water to the dried mixture. During the latter step, uniformly dispersed colloids were spontaneously formed in water without a clear precipitate observed in the dispersion. Furthermore, the hydrodynamic size of FArV NPs (less than 20 nm) was measured to be much smaller than the size of the ArV self-aggregates (Table 1), suggesting that the water-insoluble ArV dyes are less than the self- Implies that it is completely embedded within the hydrophobic structure of the structure.

Transmission electron microscopy (TEM) images of representative NIR-emitting FArV NPs clearly showed that the colloid obtained was spherical nanoparticles having an average diameter of less than 10 nm (Figure 6a, DLS size of all compounds; Figure 6b) . The size of the colloid obtained is small enough to facilitate circulation in the bloodstream and is suitable for in vivo application of nanomaterials ^{13 , 14} . Figures 3 and 4b confirm the blue-to-NIR fluorescence modulation of the resulting ArV-based molecular nanoprobe. Significantly, FArV NPs emitted red-shifted and increased fluorescence compared to corresponding solutions (Table 2). As observed in the self-aggregates, this means that the ArV dyes phase-separate and aggregate within the pluronic nanoparticles to exhibit SSF release.

The utility of FArV NPs for biomedical applications has been demonstrated by conducting fluorescence imaging experiments on cells and in vivo . For live cancer cell imaging, HeLa (human cervical epitheloid carcinoma) cells were reacted with FArV NPs. After 30 minutes of staining, the cytoplasmic site exhibited clear multiple-color fluorescence in the blue-to-NIR window with the emission spectrum of each nanoprobe well maintained in the intracellular environment (FIG. 8). Efficient intracellular uptake of FArV NPs is due to surface features of pluronic nanoparticles known to promote various non-destructive endocytosis pathways ^{31 , 32} . Moreover, the pluronic-surfaced nanoparticles exhibited minimal cytotoxicity under imaging conditions, due to their biocompatible surface properties and small colloidal size (FIG. 9).

Prior to in vivo imaging, the advantage of using NIR-SSF nanoparticles was evaluated in vivo -mimicking conditions. The bright NIR-SSF emission (720-740 nm) from FANV10 NP and FCbV10 NPs can be obtained by far-red (NIR) excitation at 640 nm and the emission has better tissue permeability Lt; RTI ID = 0.0 > fluorescence < / RTI > background derived from biological tissues. This advantage of the NIR-excited NIR-SSF is that the signal-to-background (S / B) ratio in a thick biological specimen (3.5 mm thick pork ham) . For comparison, FArV NPs with different excitation / emission wavelengths were used and imaged with different excitation / emission filters: FCbV8 (430/620 nm; visible light / visible light), FAnV8 (430/700 nm; visible light / NIR) and FCbV10 (640/720 nm; NIR / NIR). As seen in Figure 10, the NIR / NIR combination at FCbVlO showed the highest S / B ratio, as compared to others, because it has higher tissue permeability in both excitation and emission light. These observations are both - proves the potential (potential) of the nanoparticles as a useful light emission SSF- NIRF probes for in vivo medium (media) rich-limited and the background. Moreover, the visual characteristics of the colloidal dispersion and FCbVlO NPs were shown to be well maintained over time in 90% serum at 37 < 0 > C. No significant changes in fluorescence intensity and scattering were observed during the 12 hour serum reaction, indicating chemical stability and colloidal stability in body fluids (FIG. 11).

In order to verify the in vivo imaging performance, FCbV10 NPs were injected intradermally into the forefoot of the mouse paw and applied to clinically important sentinel lymph node (SLN) mapping in the cancer stage and in surgery ³³ . It was clearly imaged that some administered nanoprobes had already accumulated in the lymph nodes at 5 minutes after injection (Fig. 12A). In the X-vivo images, the typical NIR spectral characteristics of FAnV10 NPs were observed in only two sections (armpits and arms) that were excised from the injection surface (Fig. 12b), suggesting that the nanoprobes efflux associated with the lymph were effectively trapped in the SLN (trapped). Rapid SLN mapping by these nanoprobes is due to colloidal size (<20 nm) small enough to allow efficient lymphatic efflux and migration ³⁴ . Significantly, the FAnV10 NPs showed a very good in vivo image contrast capability (S / B ratio = 8.2), such that only a half reduction compared to the autofluorescence-free ex vivo value (S / B ratio = 16.5) 12c), which means that the bright NIR-SSF nanoprobes can describe the tissue distribution with high accuracy.

The possible utility of bright NIR-SSF release for disease imaging has been demonstrated by noninvasive body-whole visualization of mice with subcutaneous small tumors that are not visually detectable by systemic administration. FCbV10 NPs were injected into the tail vein of BALB / c nude mice one week after SCC7 cell inoculation and monitored under NIR excitation / detection settings. As can be seen in FIG. 13A, strong NIRF signals successfully represent transient tissue distribution of blood-circulating nanoprobes. Significantly, clear image contrasts were found within 10 minutes at the tumor inoculation site and increased to 8 hours post-injection to easily locate tumors that were not visually detectable. These spectral characteristics reliably evidenced that the strong tumor signal is fluorescence from FCbVlO NPs, which means their accumulation in the tumor (Figure 13b). This behavior indicates that the prominent tumor accumulation of FCbVlO NPs is due to the small colloidal size (< 20 nm) and polyethylene glycol (PEG) -rich surface properties of the pluronic nanoparticles. The above-described properties can promote passive tumor targeting through size-induced EPR effects by reducing the removal of nanomaterials by the reticulo-endothelial system (RES), prolonging circulation in the bloodstream ^{13 , 14} . The x-vivo image of the resected organs shows that the nanoprobe is primarily accumulated in tumors with a stronger fluorescence signal than fluorescence in other organs (Fig. 14). The in vivo image contrast ability was confirmed to be very high (S / B ratio = 7.9) as in the case of SLN imaging (Fig. 13C).

In addition, CbV10-F127 NPs dissolved in MC were injected into the tail vein of BALB / c nude mice and monitored for brain imaging efficacy under NIR excitation / detection settings. As can be seen in FIG. 15A, strong NIRF signals began to appear from 30 minutes after inoculation, indicating a strong signal in the brain at 2 hours after injection, which persisted for up to 5 hours (left panel in FIG. 15A). Interestingly, the tissue was excised from the mouse, and accumulation of nanoparticles was observed for each tissue. As a result, it was confirmed that the accumulation was specifically accumulated in the brain (right panel in FIG. 15A). Imaging for Cy.5-F127 NPs as controls also proceeded in the same manner, the results of which are illustrated in FIG. 15b. The Cy5-F127 NPs tended to be distributed evenly throughout the mouse body, especially at high concentrations in the liver and kidney, and no fluorescence signal could be detected in the brain tissue.

Overall, the imaging results described above have shown that NIR-SSF nanoprobes for detecting and diagnosing tumors at a very early stage of tumorigenesis due to their high image contrast capability as well as their ability to effectively target tumors with a robust fluorescent signal in the tissue- Proving their potential and accumulating specifically in the brain, making them particularly efficient for brain imaging.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims. It will be obvious. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

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Claims (36)

A dipolar arylvinyl compound represented by the following formula (I) or (II):
(I)

&Lt; RTI ID = 0.0 &

In the above formulas (I) and (II), R ₁ is a cyano group; A is carbazole, aniline or derivatives thereof, B is a cyano group, a carbonyl group, a carboxyl group, a C ₂ -C ₁₀ ester, an aryl, or a C ₂ -C ₁₂ linear or branched alkyl ; C is a compound selected from 1,3-indanedione or a 1,3-indanedione derivative substituted with one or more cyano groups. 2. The aryl vinyl compound according to claim 1, wherein A functions as an electron donor. The carbazole or derivative thereof according to claim 1, wherein the carbazole or a derivative thereof is a compound represented by the following formula (III)
(III)

In the formula (III), R ₂ is C ₁ -C ₁₂ Straight or branched chain alkyl, C ₁ -C ₁₂ straight or branched chain alkyl alcohol, or C ₁ -C ₁₂ Alkoxy, and R &lt; ₃ &gt; is a position at which a vinyl group is bonded in the above formula (I) or (II). 2. The compound according to claim 1, wherein the aniline or a derivative thereof is a compound represented by the following formula (IV)
(IV)

In Formula IV, R ₂ is independently H, C ₁ -C ₆ together Linear or branched alkyl, C ₁ -C ₆ straight or branched chain alkyl alcohol, or C ₁ -C ₆ Alkoxy, and R &lt; ₃ &gt; is a position at which a vinyl group is bonded in the above formula (I) or (II). The aryl vinyl compound according to claim 1, wherein the aryl is bromomethylbenzene, methylthiophene, or methylbiphenyl. The aryl vinyl compound according to claim 1, wherein the ester is ethyl propionic acid. The aryl vinyl compound according to claim 1, wherein the 1,3-indanedione derivative is a compound substituted with one to four cyano groups. The aryl vinyl compound according to claim 7, wherein the 1,3-indandione derivative is 3-dicyanomethylidene-1-indanone or 1,3-bis (dicyanomethylidene) indane. The aryl vinyl compound according to claim 1, wherein the aryl vinyl compound is represented by any of the following formulas (1) to (20).
[Chemical Formula 1]

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

[Chemical Formula 6]

(7)

[Chemical Formula 8]

[Chemical Formula 9]

[Chemical formula 10]

(11)

[Chemical Formula 12]

[Chemical Formula 13]

[Chemical Formula 14]

[Chemical Formula 15]

[Chemical Formula 16]

[Chemical Formula 17]

[Chemical Formula 18]

[Chemical Formula 19]

[Chemical Formula 20]

The aryl vinyl compound of claim 1, wherein the aryl vinyl compound exhibits solid-state fluorescence (SSF) or aggregation-enhanced fluorescence (AEF). 11. The aryl vinyl compound according to claim 10, wherein the arylvinyl compound exhibits increased SSF in a spectral range including a near-infrared (NIR) region. 12. The aryl vinyl compound according to claim 11, wherein the near infrared region is 650 nm or more. 11. The aryl vinyl compound according to claim 10, wherein the SSF occurs through severe distortion due to steric hindrance due to ICT (intramolecular charge transfer) characteristics of the aryl vinyl compound. 11. The aryl vinyl compound according to claim 10, wherein the SSF color is tunable through the combination of A and B. (a) the bipolar aryl vinyl compound of any one of claims 1 to 14; And (b) nanoprobe particles (NPs) comprising a polymeric surfactant, wherein the bipolar aryl vinyl compound is embedded within the surfactant. 16. The nanoprobe particle of claim 15, wherein the polymeric surfactant is a biocompatible polymeric surfactant. 17. The nanoprobe particle of claim 16, wherein said biocompatible polymer surfactant is a polyoxyethylene-polyoxypropylene block copolymer, polyvinyl alcohol, or gelatin. The nanoprobe particle according to claim 17, wherein the polyoxyethylene-polyoxypropylene block copolymer is pluronic 127. 16. The nanoprobe particle of claim 15, wherein the nanoprobe particle has a size of less than 65 nm. 16. The nanoprobe particle of claim 15, wherein the nanoprobe particles are in colloidal form in an aqueous environment. 16. The nanoprobe particle of claim 15, wherein the nanoprobe particle exhibits a high signal-to-background (S / B) NIR-SSF emission. 16. The nanoprobe particle of claim 15, wherein the nanoprobe particle does not cause a serum reaction in body fluids. 16. The nanoprobe particle of claim 15, wherein the nanoprobe particle targets a sentinel lymph node (SLN), brain or tumor cell. A contrast agent composition comprising nanoprobe particles according to any one of claims 15 to 23. 25. The contrast agent composition of claim 24, wherein said composition is administered parenterally to a subject. 27. The contrast agent composition of claim 24, wherein the composition accumulates within 10 minutes to a tissue of interest and continuously displays fluorescence for up to 18 hours. 27. The contrast agent composition of claim 26, wherein the target tissue is a surveillance lymph node, brain or tumor cell. 27. The contrast agent composition of claim 26, wherein the fluorescence is observed in a near infrared region of 650 nm or more. (a) administering to the subject a nanoprobe particle according to any one of claims 15 to 23; And (b) measuring fluorescence in a tissue of interest of the subject. 30. The method according to claim 29, wherein the administration in step (a) is parenteral administration. 31. The method of claim 30, wherein the parenteral administration is intravenous administration. 30. The method of claim 29, wherein the subject is a mammal. 33. The method of claim 32, wherein the mammal is a human. 30. The method of claim 29, wherein the target tissue is a surveillance lymph node, brain or tumor tissue. 30. The method according to claim 29, wherein the fluorescence of step (b) is observed in a near infrared region of 650 nm or more. 30. The imaging method of claim 29, wherein the imaging method is performed in a cell or in vivo .

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