JP2015017047A - Caged compound and composite - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a caged compound.SOLUTION: The compound is represented by formula (I) or (II) (where Z's are the same or different and each represent a biotinyl group or a maleimide group; Y represents a divalent linker group; Ar represents an optionally substituted arylene group or an optionally substituted heteroarylene group; and Ar1 represents an optionally substituted aryl group or an optionally substituted heteroaryl group).

Description

  The present invention relates to a caged compound and a complex in which a label is linked with the caged compound.

  Various and multifunctional nanoparticles have attracted attention in recent years due to their wide range of applications such as multiplex detection in vitro and in vivo.

  The combination of fluorescent and magnetic means in a single nanometer scale probe is useful for a variety of applications such as bioimaging, photothermal and photodynamic therapy, detection and isolation of various tumors.

  Quantum dots (QDs) are attracting attention in bioimaging due to their properties such as a wide absorption range, large molar extinction coefficient, narrow emission bandwidth, and high light stability.

Cheon, J .; Lee, J. H. Synergistically Integrated Nanoparticles as Multimodal Probes for Nanobiotechnology. Acc. Chem. Res. 2008, 41, 1630-1640.

  It is an object of the present invention to provide a labeling substance that is decomposed in vivo after imaging or treatment and is easily discharged outside the body.

The present invention provides the following compounds and complexes.
Item 1. Compound represented by the following formula (I) or (II):

(Wherein Z is the same or different and represents a biotinyl group or a maleimide group, Y represents a divalent linker group, Ar represents an optionally substituted arylene group or an optionally substituted heteroarylene group, Ar1 represents an optionally substituted aryl group or an optionally substituted heteroaryl group)
Item 2. A complex formed by binding a compound represented by formula (I) of Item 1, an avidinized labeling substance, and an avidinized functional substance.

  According to the present invention, the caged compound portion is decomposed by light, thereby reducing the size of the complex, and even when the complex is introduced into the living body, discharge to the outside of the body is promoted and safety is increased.

Synthesis and characterization of PUNP. (a) PCB, PCBB, and (b) PUNP synthesis scheme. (i) PBS, (ii) QD, (iii) Fe ₃ O ₄ NP and (iv) PUNP solution (c) optical image, (d) photoluminescence image, (e) T1-weighted MRI and (f) T2 Emphasized MRI. (g) FESEM images of PUNPs showing a uniform distribution of size and shape. Inset: Schematic representation of magnified and scanned FESEM images and PUNPs. Aggregation of PUNPs at 'g' is due to solvent drying during sample preparation for SEM imaging. Light uncaging. (a) SN1 photodecaylation reaction scheme under excitation at 397 nm, (b, c) (b) Photoactivation at 397 nm or (c) UV-Vis absorption spectrum of PCB in the dark Evolution. Inset: Kinetics of photodecaylation reaction, (d, e) MALDI-TOF mass spectrum of PCB before (d) and after (e) photodecaged, (f) light at 397 nm Temporal development of 1H NMR spectra of PCBs dissolved in DMSO-d6 upon activation, expanded 1H NMR spectra of (g, h) (g) allyl proton and (h) coumarinylmethyl proton, and (i) different bands Fluorescence image of uncaged product collected by pass filter. Intracellular delivery and cytotoxicity assays. (ac) Fluorescence and overlay images of B16 cells treated with PUNP-aratostatin complex and syto 21 and (df) H1650 cells treated with QD-EGF complex and syto 21. MTT assay histograms for (gi) (g) Fe ₃ O ₄ NP, (h) CdSe / ZnS QD and (i) PUNP. (J) Light transmission and (k) Fluorescence image of (i) labeled or (ii) unlabeled B16 cells using PUNP-aratostatin complex. (l) T1-weighted MRI and (m) T2-weighted MRI of (16) B16 cells labeled with Fe ₃ O ₄ NP, (ii) PBS, (iii) PUNP-aratostatin complex and (iv) unlabeled cells In vivo fluorescence imaging and MRI. (a) fluorescence image, and (b) T1-weighted MRI, (cf) T1-weighted MRI, (g, h) bright-field optical image and B6 mice injected subcutaneously with PBS, QD, Fe ₃ O ₄ NP, and PUNP (i, j) Fluorescence image of B6 mice intravenously injected with 10 nM PUNP solution (250 μL). UV-Vis absorption and PL spectra of PCB, PCBB, Fe ₃ O ₄ , QD and PUNP; Photocage of PCBB under irradiation of 340 nm light from a Xe lamp; SEM image of PUNP after photo-decaging; Fluorescence image of B16 cells treated with PUNP-aratostatin complex; PCB, PCBB and photoproduct MTT assays;

  The present invention provides a bifunctional labeling complex having two functions and a compound for obtaining such a labeling compound.

  The compounds used in the present invention are shown in the following formula (I) or formula (II).

(Wherein Z is the same or different and represents a biotinyl group or a maleimide group, Y represents a divalent linker group, Ar represents an optionally substituted arylene group or an optionally substituted heteroarylene group, Ar1 represents an optionally substituted aryl group or an optionally substituted heteroaryl group)

  The compound of the formula (II) is useful as a production intermediate for obtaining the compound of the formula (I).

  “Aryl group” means a monocyclic or polycyclic group consisting of a 5- or 6-membered aromatic hydrocarbon ring, and specific examples thereof include phenyl, naphthyl, fluorenyl, anthryl, biphenylyl, tetrahydronaphthyl, chromanyl, 2,3-dihydro-1,4-dioxanaphthalenyl, indanyl and phenanthryl are mentioned.

  The “arylene group” means a monocyclic or polycyclic divalent group composed of a 5- or 6-membered aromatic hydrocarbon ring. Specific examples thereof include phenylene, naphthylene, fluorenylene, anthrylene, biphenylylene, tetrahydronaphthylene. Len, chromanilene, 2,3-dihydro-1,4-dioxanaphthalenylene, indanylene and phenanthrylene.

  “Heteroaryl group” means a monocyclic or polycyclic group consisting of a 5- or 6-membered aromatic ring containing 1 to 3 heteroatoms selected from N, O and S. In this case, at least one ring may be an aromatic ring. Specific examples include furyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl, indolyl, quinolyl, isoquinolyl, benzo [b] thienyl and benzimidazolyl, coumaryl, benzopyranyl, Benzofuranyl is mentioned.

  “Heteroarylene group” means a monocyclic or polycyclic divalent group consisting of a 5- or 6-membered aromatic ring containing 1 to 3 heteroatoms selected from N, O and S; In the case of a polycyclic system, at least one ring may be an aromatic ring. Specific examples include furylene, thienylene, pyrrolylene, imidazolylene, pyrazolylene, oxazolylene, thiazolylene, isoxazolylene, isothiazolylene, pyridylene, pyrazinylene, pyrimidinylene, pyridazinylene, indolylene, quinolylene, isoquinolylene, and benzo [b] thienylene and benzidylene. Coumarinene, benzopyranylene, and benzofuranylene.

  When Z is a biotinyl group, Z—OH is biotin.

  The biotinyl group can be bonded to the avidin material, and the maleimide group can be bonded to the SH group.

  Examples of the divalent linker group represented by Y include an alkylene group having 1 or more carbon atoms, a cycloalkylene group, -CO-,-(arylene)-(alkylene having 1 or more carbon atoms)-group, and an arylene group. .

  Examples of the alkylene group having 1 or more carbon atoms include linear or branched alkylene groups such as methylene, ethylene, propylene, butylene, pentylene, hexylene, -CH2CH (CH3)-, -CH (CH3) CH2-. .

  Examples of the cycloalkylene group include cycloalkylene groups having 3 to 8 carbon atoms such as a cyclopentylene group and a cyclohexylene group.

  As the-(arylene)-(alkylene having 1 or more carbon atoms)-group, the above arylene group and an alkylene group having 1 or more carbon atoms (preferably an alkylene group having 1 to 4 carbon atoms, more preferably 1 to 2 carbon atoms). Group of the above-mentioned alkylene group).

Examples of the substituent for the arylene group, heteroarylene group, aryl group and heteroaryl group include halogen, hydroxy, C _1-6 alkyl, C _1-6 alkoxy, trifluoromethyl, trifluoromethoxy, trifluoroethoxy, cyano, nitro , Amino, mono C _1-6 alkyl substituted amino, di C _1-6 alkyl substituted amino, carbamoyl, sulfamoyl, methylenedioxy, and the number of substituents is 1 to 3, preferably 1 to 2. is there.

“C _1-6 alkyl” may be either linear or branched. Examples thereof include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl and hexyl.

  Examples of halogen include chlorine, bromine, fluorine, and iodine.

Specific examples of C _1-6 alkoxy include methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, tert-butoxy, pentyloxy, isopentyloxy and hexyloxy.

Mono C _1-6 alkyl substituted amino includes methylamino, ethylamino, n-propylamino, isopropylamino, n-butylamino, isobutylamino, tert-butylamino, n-pentylamino, isopentylamino, hexylamino Can be mentioned.

Di-C _1-6 alkyl-substituted amino groups include dimethylamino, diethylamino, di-n-propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, ditert-butylamino, di-n-pentylamino, diisopentyl Examples include amino and dihexylamino.

  Examples of labeling substances include quantum dots (QD), fluorescent substances, fluorescent proteins (eg, GFP, CFP, YFP, etc.), luminescent enzymes (eg, luciferase, etc.), enzymes, gold colloids, europium complexes, terbium complexes, and the like. .

  Examples of fluorescent substances include cy5, cy7, cy3B, cy3.5, Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alex Fluor 594, Alex Fluor 594, Alex Fluor 594, Alex Fluor 564 Alexa Fluor750, fluorescein-4-isothiocyanate (FITC), etc. are mentioned.

  Examples of the enzyme include β-galactosidase, horseradish peroxidase, alkaline phosphatase and the like.

  As the europium complex and terbium complex, a complex in which a known ligand is coordinated can be used without limitation. Examples of ligands for europium ions and terbium ions include ligands having a β-diketone structure, ligands having a β-disulfonyl structure, ligands having a carbonylimide structure, and coordination having a sulfonimide structure. These ligands are preferable in that the luminous efficiency of the complex formed is good.

  These labeling substances are combined with avidin substances to form avidinized labeling substances. Examples of the avidin substance bound to the labeling substance include avidin, streptavidin, neutravidin, and the like. The labeling substance (avidinated labeling substance) bound to the avidin substance can be a commercially available product or can be produced according to a conventional method. The avidin substance and the labeling substance may be bonded directly or via an appropriate linker.

  The avidinized functional substance is a material in which the avidin substance and the functional substance are bonded directly or via an appropriate linker. Examples of functional substances include magnetic materials, antibodies, drugs, hormones, cytokines, aptamers, folic acid, ligand molecules of receptors such as RGD peptides, and the like.

  The magnetic material is useful as a contrast agent for MRI examination. For example, a magnetic material such as a gadolinium complex, iron oxide, manganese oxide, cobalt complex, or niobium complex can be used as a contrast agent for MRI examination. The complex of the present invention containing these magnetic materials and a label can be used as a contrast agent by intravenous injection or injection into an organ or tissue to be observed.

  In a preferred embodiment, the present invention provides a complex consisting of photouncaged and bifunctional labeled nanoparticles. “Bifunctional” means that a functional substance (magnetic material) and a labeling substance are included, and a preferred complex of the present invention is a combination of a magnetic material and a labeling substance with a compound of formula (I) or (II). To do. The labeling substance is removed after imaging or treatment to facilitate excretion outside the body.

  In the present specification, a complex in which a labeling substance and a functional substance are caged by a photodecaged ligand may be referred to as (PUNP). The complex of the present invention can be easily decomposed by applying light without using a chemical reagent. The conjugates of the present invention have found interesting uses in different aspects of biochemistry such as spatially and temporally controlled drug and biomolecule delivery. Among the various photodecaged molecules known to date, coumarinyl methyl ester has been studied in detail for its effective photodegradability. Biotin derivatives or maleimide derivatives prepared using coumarinyl methyl ester allowed the binding of multiple fluorophores to the surface of the magnetic NP and to obtain a complex with high fluorescence and magnetism.

  Hereinafter, the present invention is based on quantum dots as fluorescent substances, magnetic nanoparticles as functional substances, photodecaged biotin derivatives or photodecaged maleimide derivatives as coumarinylmethylbiotin ester (PCB, 3) and coumarinylmethylbis-biotin. An example using an ester (PCBB, 5) will be mainly described. However, the present invention is not limited to these, and includes all embodiments of the claims.

The present inventors synthesized two novel photodecaged biotin derivatives, namely, coumarinylmethyl biotin ester (PCB, compound 3) and coumarinylmethyl bis-biotin ester (PCBB, compound 5), and using CCB, CdSe A PUNP composed of / ZnS QD and Fe ₃ O ₄ NP is prepared and characterized by FESEM imaging. In addition, PUNP was complexed with the peptide hormone allatostatin I, allowing intracellular delivery of PUNP. PCB, PCBB and PUNP decaging were studied in detail using time-dependent UV-Vis absorption spectra, NMR spectra, MALDI-TOF mass spectra and SEM. The formation of coumarinylmethyl alcohol derivatives upon the photoactivation of PCB or PCBB strongly supports photodecaylation. Cytotoxicity of PCB, PCBB, peptide-coated PUNP, and photodecaged products in human lung epithelial adenocarcinoma cells (H1650) and mouse melanoma cells (B16) using MTT assay is over 90% Shows cell viability, suggesting the non-toxic nature of PUNP and its components. Finally, we used peptide-complexed PUNP as an effective contrast agent for MRI and fluorescence imaging of B16 cells and C57BL / 6 (B6) mice. The enhanced magnetic and fluorescence contrast collected from cell samples treated with peptide-complexed PUNP or mice injected with peptide-complexed PUNP confirmed its bimodal character.

Figures 1a and 1b show the synthesis steps of PCB, PCBB, and PUNP. Details of organic chemical reactions and their characterization for the preparation of PCB and PCBB are given in the examples. During the preparation of PUNP, the concentration of Fe ₃ O ₄ NP and PCBB is a ratio of 1: 100, so that the streptavidin pocket on Fe ₃ O ₄ NP is completely occupied by the PCBB molecule, and the photodecaged biotin functional Provide Fe ₃ O ₄ NP. By adding excessive Fe ₃ O ₄ NP to PCBB, PCBB-mediated aggregation of Fe ₃ O ₄ NP can be prevented. This can be confirmed by SEM imaging. Free PCBB molecules are continuously removed by dialysis against a 2 KDa membrane. Biotinylated Fe ₃ O ₄ NP is then added to the streptavidin-functionalized QD in a 1:10 ratio at a 1:10 ratio, and about 10 QD is ligated onto the Fe ₃ O ₄ NP by the photodecaged biotin ligand. Repeated magnetic separation using a bar magnet was performed overnight to remove any free QD in the reaction mixture. Details of PUNP preparation are given in the experimental section. Brightfield light image (c), fluorescence image (d) for phosphate buffered saline (PBS) solution (i), CdSe / ZnS QD (ii), Fe ₃ O ₄ NP (iii) and PUNP (iv) T1 weighted MRI (e) and T2 (f) weighted MRI are shown in FIGS. 1c-f. In addition to the overall characteristics of QD, PCBB and Fe ₃ O ₄ NP in the light absorption and emission spectra (FIG. 5), the strong magnetic and fluorescence contrast from PUNP suggests the bifunctional nature of PUNP. MR and fluorescence contrast are not due to the presence of free Fe ₃ O ₄ and QD, which is confirmed by obtaining the form of PUNP using SEM. The size distribution and shape of PUNP is shown in the SEM image (Figure 1g). To further understand the morphology of PUNP, we obtained a magnified and scanned FESEM image (FIG. 1g), suggesting the binding of multiple QDs on Fe ₃ O ₄ NP. On the other hand, the present inventors could not observe the characteristic morphology of Fe ₃ O ₄ , QD or a mixture thereof in the sample. Therefore, nonspecific aggregation of QD and Fe ₃ O ₄ NP was excluded. On the other hand, the aggregation of PUNP shown in FIG. 1g is due to evaporation of the solvent under reduced pressure during sample preparation for SEM imaging.

In order to evaluate the photo-decaging of PCBs, PCBBs and PUNPs, we used photo-cage using time-dependent UV-Vis absorption spectroscopy, 1H NMR spectroscopy and MALDI-TOF mass spectrometry. We studied the kinetics of crystallization. First, irradiate a 1 μM PCB solution in a DMF: water (10: 1) mixture with 397 nm light from a Xe lamp or 400 nm laser light (7 mW), and keep the UV-Vis absorption spectrum of the solution constant. Recorded at time intervals. As shown in FIG. 2b, the continuous decrease in absorbance at about 410 nm and the generation of a new band at about 275 nm due to the isosbestic point suggests a photodecaging reaction. Conversely, the absorption spectrum of the control sample in the dark was essentially unchanged (FIG. 2c). A plot of absorbance vs. time during the course of photodecaging at 410 nm and 275 nm is shown in FIG. 2b, which follows the first order rate law. Using the number of reacting molecules calculated from the molar extinction coefficient and the difference absorption spectrum and the number of absorbed photons calculated from the excitation laser intensity and photon energy, the inventor achieved a quantum efficiency of the photodecaylation reaction of about 4.8%. It was evaluated. In order to further evaluate the photodecaging reaction, we recorded and evaluated MALDI-TOF mass spectra of PCB samples loaded on a standard α-cyano-4-hydroxycinnamic acid matrix. The MALDI-TOF mass spectra of the PCB before and after photoactivation are shown in FIGS. 2d and 2e. As expected, the molecular peak of PCB (m / z = 453) in the photoactivated sample disappeared completely, which is associated with the formation of the photodecaged product shown in Figure 2a. The change is in good agreement with the photodecaking process seen in the absorption spectrum (Figure 2b). The photodecaylation properties of allyl esters and the formation of primary alcohols allow further evaluation of photodecaylation reactions by time-dependent 1H NMR spectra of PCBs dissolved in deuterated DMSO and irradiated with 397 nm light (Fig. 2f). The broad field downfield shift of coumarinylmethyl protons (Fig. 2g) and the appearance of triplets due to the spatial coupling of allyl protons with newly formed hydroxymethyl groups (Fig. 2h) confirm the photodecaylation reaction. . More interestingly, the photouncaged sample irradiated with UV light in an NMR tube shows white light emission, which can be resolved into individual colors using a bandpass filter as shown in FIG. 2i. Compared with PCB, white light emission is attributed to the formation of H-type aggregates and excimers of hydroxymethylcoumarin promoted by its simple molecular structure. Similarly, the photo-decaging of PCBB can be characterized using a time-dependent UV-Vis absorption spectrum (FIG. 6). However, the kinetics of PCBB photodecaylation appears to be slower than that of PCB, due to differences in the stability of the carbocation formed during the photoSN1 reaction (FIG. 2a). In other words, the initial PCB photodecaylation causes the formation of a 7-hydroxy-coumarinylmethyl carbocation, which is more stable than the carbocation formed during the photodecasement of PCBB. In order to evaluate the photo-uncaging of PUNP, we recorded and analyzed the SEM image of PUNP after 340nm light irradiation from Xe lamp. Interestingly, we observed fragmented nanoparticles rather than large spheroid nanostructures in most SEM samples (FIG. 7). The formation of small nanostructures in the SEM image of the light-capped PUNP sample and PCB and PCBB suggests the photo-uncaging of PUNP. The combination of MR and fluorescent contrast agent makes it possible to obtain MRI and fluorescent images in vivo and in vitro. First, the inventors tested the bimodal properties and cytotoxic effects of PUNP in cultured human and mouse cells prior to application in vivo. The inventor has used allatostatin I (a peptide hormone present in insects) and crustacean as the endocytosis mechanism. Allatostatin was biotinylated using 3-sulfo-N-hydroxysuccinimide ester of biotin and then complexed with the streptavidin moiety of PUNP at a 1: 5 molar ratio (PUNP: aratostatin). Detailed procedures for biotinylation of allatostatin and bioconjugation of PUNP are shown in the examples. Mouse melanoma cells cultured to 60% confluence (B16) were incubated for 1 hour at 4 ° C in a solution containing 5nM PUNP-aratostatin complex in Dulbecco's modified Eagle's medium (DMEM) without phenol red or fetal bovine serum (FBS) , Washed 3 times with PBS and treated with a solution of 5 nM nuclear staining dye syto 21 for 10 minutes. After treatment, the cells were washed 3 times with PBS and the medium was replaced with DMEM supplemented with 10% FBS. Intracellular delivery of PUNP in B16 cells was studied in detail using fluorescence microscopy. The excitation light source used for imaging was a 400 nm fs laser for syto 21 and QD, and a 532 nm cw laser for QD alone. FIG. 3a shows the fluorescence image of B16 cells obtained by excitation at 400 nm and the fluorescence signal collected with a 580 nm long pass filter. The cell nucleus appears orange due to the fluorescent tailing of the green fluorescent syto 21, and the cytoplasmic red fluorescence is due to intracellular PUNP. FIG. 3b shows an overlay image of the images obtained by exciting at 400 nm and collecting the fluorescence signal with a 510-550 nm optical bandpass filter for syto21 and a 580 nm longpass filter for PUNP. FIG. 3c shows an overlay of light transmission and fluorescence images, clearly showing PUNP endocytosis in B16 cells. Fluorescence images of large areas of cells are shown in FIGS. 8a-c. The inventor further compared the efficiency of allostatin-mediated endocytosis of PUNP and EGF-mediated endocytosis of QD. FIG. 3d shows an overlay fluorescence image of human lung epithelial adenocarcinoma cells (H1650) treated with QD-EGF complex. In FIG. 3d, the nucleus appears green due to syto21 (FIG. 3e) and the cytoplasmic red fluorescence is due to intracellular QD. FIG. 3f shows an overlay of light transmission and fluorescence images, indicating effective intracellular delivery in H1650 cells. On the other hand, the EGF complex of QD or PUNP is not effectively delivered due to the low expression of EGFR receptor in B16 cells. The toxicity of nanomaterials is critical for biological applications. Although QD-based fluorescent markers are widely used in bioimaging, QD cytotoxicity has not been shown to be a problem. Recent studies have shown air oxidation of unprotected CdSe QDs and leaching of cadmium ions when exposed to light, which has been shown to be suppressed by surface protection using ZnS and polymer shells. Polyethylene glycol (PEG)-and ZnS-coated CdSe QD and PEG-coated Fe ₃ O ₄ NP are included in PUNP, but we measured the inhibition of cell proliferation using MTT assay. The cytotoxicity of QD, Fe ₃ O ₄ NP, PUNP and photodecaged products was further evaluated.

In the MTT assay, yellow tetrazolium salts taken up by cells are converted to purple formazan as a result of reduction by NADP / H-dependent oxidoreductase in the cytoplasm of metabolically viable cells. Thus, the optical density of intracellular formazan directly measures cell proliferation or cell viability. Detailed procedures for the MTT assay are described in the examples. Here, the cytotoxicity of Fe ₃ O ₄ NP (Figure 3g), CdSe / ZnS QD (Figure 3h), PUNP (Figure 3i), PCB (Figure 9a), PCBB (Figure 9b) and photoproduct (Figure 9c) The assay shows greater than 90% cell viability in the nanomolar to micromolar concentration range, suggesting the non-toxic nature of PUNP and its components and photoproducts. To evaluate the potential of the bimodal imaging probe PUNP, we recorded MRI and fluorescence images of cells treated with the PUNP-aratostatin complex. 90% confluent B16 cells are incubated with 5 nM complex solution in DMEM without phenol red or FBS for 1 hour at 4 ° C. The cells were washed 3 times with PBS, harvested with trypsin, washed 3 times with DMEM medium supplemented with FEB, and then centrifuged. Figures 3j and 3k show bright field and fluorescence images of (i) unlabeled and (ii) labeled B16 cell pellets using PUNP-aratostatin conjugates.

Fluorescence images of individual cells (Figure 3a) and cell pellets (Figure 3k) show that the fluorescence means provided by PUNP is effective. Furthermore, Figure 3l and 3m show the _{Fe 3 O 4 NP (i)} , PBS (ii), has been (iii) and unlabeled labeled with PUNP (iv) T1 and T ₂ enhancement MRI. The dark contrast of labeled cells in T2-weighted MRI is due to PUNP contrast enhancement at longer echo times (TE) and repetition times (TR). The MRI contrast provided by PUNP shown in FIGS. 1f and 3m distinguishes labeled cells from lipid / water / adipose molecules that appear shining with T2-weighted MRI.

In order to further evaluate the bimodal properties of PUNP, we obtained in vivo MRI and fluorescence images of C57BL / 6 (B6) mice that received PUNP. Subcutaneously or intravenously. First, anesthesia was performed by intraperitoneally administering sodium pentobarbital (40 mg kg-1). Following anesthesia, a 10 nM solution of QD, Fe ₃ O ₄ NP, PUNP and PBS was administered subcutaneously at different locations in B6 mice as shown in FIGS. 4a-b. In vivo fluorescence images were collected immediately after injection by excitation with bandpass filter light (yellow-green light from Xe lamp). Pictures of mice collected with a 580 nm long pass filter show a fluorescent signal with a high S / N ratio from where QD (control) or PUNP was injected (FIG. 4a). Similarly, T1-weighted MRI of mice shows enhanced subcutaneous contrast in the area injected with Fe ₃ O ₄ NP (control) or PUNP (FIG. 4b). Furthermore, in order to test the possibility of PUNP fluorescence and MRI contrast detection in deep organs such as the liver, the present inventors injected PUNP (10 nM, 250 μL) into the vein of C57BL / 6 (B6) mice different from the above. MRI and fluorescence images were obtained at various times after injection. As shown in FIGS. 4c-f, T1-weighted MRI of mice obtained from t = 0 to t = 24h shows contrast enhancement in the liver. Interestingly, PUNP accumulates in the liver within 30 minutes after injection. The fluorescence image of the mouse collected by a 580 nm long pass filter 30 minutes after injection showed a faint red fluorescence from the liver region (FIG. 4i), which is due to the fluorescence imaging method of PUNP provided by QD. Interestingly, as shown in FIGS. 4d-f, PUNP gradually accumulates clearly in the liver and disappears completely within 24 hours. In addition, the intensity of red fluorescence from the liver region decreases with time, and disappears completely within 24 hours as shown in FIG. 4j. This is consistent with the decrease and disappearance of MRI contrast shown in FIGS. 4e and 4f.

The low fluorescence intensity of PUNP from the liver region compared to the bright fluorescence from the subcutaneous region (FIG. 4a) is due to the low transmittance of excitation light to deep organs such as the liver. The large absorption cross-section and strong fluorescence of QD and the presence of Fe ₃ O ₄ nanoparticles in PUNP allow detection of both fluorescence and MRI contrast from the liver. Nevertheless, the low transmittance of externally applied excitation light into the internal organs should be noted.

  In summary, the first successful photodecaged fluorescent-magnetic bimodal nanoparticles prepared with a novel photodecaged ligand were reported. Here, we have introduced a simple method using light as an innovative tool for fragmentation of ligands that bind to both the magnetic and fluorescent components of the particles. The photo-decaging process is revealed by observing regular changes in absorption spectrum, mass spectrum and NMR spectrum of the photo-deccaged ligand and SEM images of the particles. Nanoparticles prepared by conjugation of peptide hormones to bimodal nanoparticles or its components or photoproducts did not show toxicity by MTT assay. Photocaged nanoparticles can be applied as bimodal contrast agents combining MRI and fluorescence imaging of melanoma cells and B6 mice. In vivo fluorescence imaging and MRI of B6 mice injected with subcutaneously or intravenously photocaged nanoparticles showed enhanced fluorescence and magnetic contrast in the subcutaneous and liver. Photodecaged nanoparticles are interesting and worth evaluating on-demand clearance of nanomaterials left in biological systems after bioimaging or treatment as well as various bioimaging probes.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, it cannot be overemphasized that this invention is not limited to these Examples.

Materials and methods:
All reagents and solvents were analytical grade and used without further purification. CdSe / ZnS QD (PL maximum about 605 nm) was purchased from Invitrogen. Fe ₃ O ₄ nanoparticles were purchased from NANOCS. 4-chlororesorcinol, ethyl-4-chloroacetoacetate, methanesulfonic acid, biotin and 1,8-diazabicyclo [5.4.0] -7-undecene (DBU) were purchased from Sigma Aldrich. K2CO3 and 1,3-dibromopropane were purchased from Tokyo Chemical Industry.

  1H and 13C NMR measurements were performed on a JEOL 400 MHz spectrometer. MALDI / LDI-TOF mass measurement was performed by BRUKER Microflex using α-cyano-4-hydroxycinnamic acid as a matrix. Fluorescent images of labeled cells include 40X objective lens (Olympus-LUCPlanFl), a2.5X telephoto lens, bandpass / longpass filters for PUNP and syto21 dye, and electron multiplying CCD (EMCCD, Andor Technology) or CCD camera (Olympus ) With an inverted optical microscope (Olympus IX70). The excitation light source used for fluorescence imaging was a 400 or 532 nm laser. FESEM images were obtained using a JSM-6700FZ (JEOL) microscope operating at 10 μA and 15 kV. MRI data was obtained using a small animal imaging MRI machine (MR Technology, Inc., Tsukuba, Japan). Fluorescence images of B16 cell pellets and B6 mice were recorded using a Maestero small animal imaging system (Perkin Elmer). Cell pellets and mouse MRI and fluorescence images were analyzed using Maestro software (Cambridge Research & Instrumentation, Inc.) and Image-Pro Plus software (Roper Industries, Inc., Sarasota, FL, USA).

Synthesis of compound 1:
A mixture of 4-chlororesorcinol (14.4 g, 100 mmol) and ethyl-4-chloroacetoacetate (19.9 g, 120 mmol) dissolved in methanesulfonic acid (160 mL) was stirred at room temperature for 4 h, then poured into ice-cold water to make a constant amount. Stirring was performed for 1 h. The precipitate was filtered, washed with ice cold water and dried under reduced pressure to give the desired product (22 g, 90%). 1H NMR (400MHz, DMSO-d6) δ = 4.99 (s, 2H), 6.49 (s, 1H), 6.96 (s, 1H), 7.8 (s, 1H), 11.5 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ = 59.70, 104.30, 108, 111.90, 112.30, 117.30, 126, 153.89, 156.84, 160.63; FT-IR, (νmax) = 3297, 1702, 1611, 1553, 1504, 1430, 1399, 1342, 1291, 1170, 1138, 1068, 1036, 941, 891, 738, 643, 534 cm-1; MALDI-TOF (C10H6Cl2O3), m / z = 245. 15

Synthesis of compound 2:
To a suspension of compound 1 (2.4 g, 9.8 mmol) prepared while sonicating in 1,3-dibromopropane (10 mL, 98.95 mmol) was added DBU (1.5 mL, 10.05 mmol) at 150 ° C. Heated for 30 minutes. The product was extracted with dichloromethane (DCM) and the extract was washed with dilute HCl to remove DBU. The crude product was purified by silica gel (200-400mesh) column chromatography using DCM as an eluent to obtain Compound 2 in a yield of 65%. 1H NMR (400 MHz, CDCl3) δ = 2.40 (m, 2H), 3.62 (t, 2H), 4.20 (t, 2H), 4.56 (s, 2H), 6.40 (s, 1H), 6.90 (s, 1H ), 7.6 (s, 1H); 13C NMR (125 MHz, CDCl3) δ = 29.80, 31.90, 41.10, 67.50, 102.30, 111.34, 113.68, 120, 125, 149.00, 154, 157, 160; FT-IR, ( νmax) = 1731, 1607, 1552, 1505, 1464, 1416, 1369, 1288, 1208, 1152, 1206, 1152, 1036, 909, 871, 771, 724, 602, 534 cm-1; MALDI-TOF (C13H11BrCl2O3) , m / z = 365.

Synthesis of compound 3:
To an ethanol suspension of compound 1 (1.5 g, 6.25 mmol) was added a mixture of biotin (1 g, 4.08 mmol) and DBU (700 μL, 4.69 mmol) in dry ethanol, and the reaction mixture was heated and stirred for 4 h. Hexane was added to precipitate compound 3. The crude product was purified by silica gel column chromatography (200-400mesh) using 2% methanol in ethyl acetate as eluent to obtain compound 3 in a yield of 50%. 1H NMR (400 MHz, DMSO-d6) δ = 1.36 (m, 2H), 1.60 (m, 4H), 2.46 (t, 2H), 2.80 (d, 1H), 2.84 (d, 1H), 3.08 (m , 1H), 4.13 (m, 1H), 4.29 (m, 1H), 5.36 (s, 2H), 6.23 (s, 1H), 6.36 (s, 1H), 6.42 (s, 1H), 6.93 (s, 1H), 7.75 (s, 1H); 13C NMR (125 MHz, DMSO-d6) δ = 25, 28.54, 33.45, 43.33, 55.20, 59.42, 62.22, 74.20, 92.88, 104.32, 110.60, 117.66, 125.38, 150.64, 154.15, 157.66, 161.87, 163.98, 173.80; FT-IR, (νmax) = 3230, 3059, 2932, 1714, 1600, 1405, 1316, 1260, 1162, 1032, 952, 876, 838, 700, 554 cm-1 ; MALDI-TOF (C20H21ClN2O6S), m / z = 453.

Synthesis of compound 4:
A mixture of biotin (1 g, 4.08 mmol), 1,3-dibromopropane (1.3 mL, 12.86 mmol) and DBU (1.85 mL, 12.40 mmol) in acetonitrile (100 mL) was heated and stirred for 3 hours. Compound 4 was precipitated by adding hexane. The crude product was purified by silica gel (200-400 mesh) column chromatography using DCM as an eluent to obtain compound 4 in a yield of 80%. 1H NMR (400 MHz, CDCl3) δ = 1.46 (m, 2H), 1.70 (m, 4H), 2.18 (m, 2H), 2.35 (t, 2H), 2.74 (m, 1H), 2.94 (m , 1H), 3.16 (m, 1H), 3.47 (t, 2H), 4.21 (t, 2H), 4.34 (m, 1H), 4.56 (m, 1H), 5.16 (s, 1H), 5.56 (s, 13C NMR (125 MHz, CDCl3) δ = 29, 32.5, 34, 36, 38, 45, 53, 59, 64.5, 66.5, 101, 167, 178; FT-IR, (νmax) = 3241, 2941 , 2843, 1736, 1474, 1418, 1362, 1324, 1262, 1223, 1195, 1115, 994, 859, 752, 728, 662, 624, 569, 544 cm-1; MALDI-TOF (C13H21BrN2O3S), m / z = 365. 16

Synthesis of Compound 5 from Compound 2:
A mixture of compound 2 (1 g, 2.75 mmol), biotin (2 g, 8.2 mmol) and K2CO3 (1.2 g, 828 mmol) was suspended in DMF and refluxed for 1 hour. Unreacted biotin and K2CO3 were precipitated and removed by adding chloroform to the reaction mixture. Compound 5 was then precipitated by adding ice cold water. The crude product was washed thoroughly with water and dried in a desiccator under reduced pressure to give the desired product in about 30% yield.

Synthesis of compound 5 from compounds 3 and 4:
A mixture of compound 3 (1 g, 2.20 mmol), compound 4 (0.9 g, 2.47 mmol) and K2CO3 (1 g, 7.7 mmol) was suspended in DMF and refluxed for 6 hours. The solid was removed by filtration and the product was precipitated by adding ice cold water to the filtrate. The crude product was washed thoroughly with water and dried in a desiccator under reduced pressure to give the desired product in about 50% yield. 1H NMR (400 MHz, DMSO-d6) δ = 1.36 (m, 4H), 1.63, (m, 8H), 2.11 (m, 2H), 2.31 (t, 2H), 2.46 (m, 2H), 2.59 ( m, 2H), 2.81 (m, 2H), 3.07 (m, 2H), 3.30 (t, 2H), 4.13 (m, 2H), 4.23 (t, 2H), 4.3 (m, 2H), 5.36 (s , 2H), 6.32 (s, 1H), 6.35 (s, 1H), 6.40 (s, 1H), 6.43 (s, 1H), 7.24 (s, 1H), 7.82 (s, 1H), 8.23 (s, 13C NMR (125 MHz, DMSO-d6) δ = 25, 28, 33.6, 56, 60, 61.6, 67, 103, 118.7, 126, 144, 150.1, 154, 157, 160, 163, 173, 177 ; FT-IR, (νmax) = 3248, 3091, 2920, 2848, 2365, 3091, 1737, 1694, 1612, 1550, 1507, 1464, 1414, 1386, 1336, 1279, 1205, 1165, 1065, 887, 772 , 734, 689, 602, 545; MALDI-TOF (C33H41ClN4O9S2), m / z = 737.

Production of PUNP:
The production of PUNP was performed in two steps. In the first step, a 200 nM aqueous solution (50 μL) of streptavidin-complexed Fe ₃ O ₄ NP and a 20 μM DMSO solution (50 μL) of PCBB are mixed to strep the biotin moiety present at the end of the PCBB. Complexed with avidin-functionalized Fe ₃ O ₄ NP. This mixture was incubated at 37 ° C. for 2 hours, and unreacted PCBB was removed by dialysis using a 2 KDa dialysis membrane. In the second step, 50 μL of Fe ₃ O ₄ NP PCBB derivative was added to the QD solution (50 μL, 100 nM) in 5 portions, and then incubated at 37 ° C. for 2 hours to prepare in the first step. The biotin derivative of Fe ₃ O ₄ NP was complexed with CdSe / ZnS QD coated with streptavidin. This conjugation resulted in PUNPs that were cross-linked by photodecaged PCBB molecules.

Preparation of PUNP-aratostatin conjugate:
Lyophilized allatostatin (Genscript Corporation) was reconstituted in a 0.75 mM solution in sterile water and biotinylated with a 15 mM aqueous solution of biotin-3-sulfo-NHS ester at 25 ° C. for 1 hour. Biotinylation Allatostatin is purified by gel filtration on a sephadex G-25 column (Sigma), diluted to 250 nM (100 mL) solution with sterile filtered water, and 30 nP PUNP in 50 nM (100 μL) at 25 ° C. The reaction was allowed to proceed for a minute to obtain a PUNP-aratostatin complex.

Cytotoxicity assay:
Cytotoxicity of CdSe / ZnS QD, Fe ₃ O ₄ NP, PCB, PCBB, PUNP and photodecaged products is 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium chloride (MTT) Evaluated by MTT assay using cell proliferation kit (Roche Diagnostics). Here, about 1 × 10 6 human epithelial lung adenocarcinoma cells (H1650) were inoculated into a 96-well tissue culture plate (FALCON) containing DMEM supplemented with 10% FBS and cultured at 37 ° C. for 48 hours. The cells are then washed thoroughly with PBS and the medium is mixed with various concentrations of coumarinylmethyl alcohol (0.1 μM to 10 μM), PCB (0.01 μM to 1 μM), PCBB (0.01 μM to 1 μM), Fe ₃ O ₄ Replaced with DMEM containing NP (1 nM to 100 nM), CdSe / ZnS QD (0.1 nM to 10 nM), or PUNP (0.1 nM to 10 nM). After 24 hours of culture, the cells were washed 3 times with PBS and treated with MTT solution (10 μL, 5 mg / mL) for 4 hours. Treated cells were lysed by adding 10% SDS in 0.01 M HCl (100 μL / well) and incubated overnight. The absorbance of formazan formed from MTT was measured at 550 nm with a microplate reader, and cell viability was determined by comparing the absorbance of formazan produced in treated cells with the absorbance of control cells.

  The complex of the present invention is useful as a contrast agent for PET, CT, MRI and the like. This contrast agent is applied to a living body as an antitumor antibody, a disease marker, etc., and then removed by photocleavage.

  Complexing of imaging probe and drugs combined with cancer specific antibodies such as anti-EGFR antibody, anti-PSMA antibody, trastuzumab, rituximab etc. is useful for cancer treatment such as lymphoma, breast cancer, prostate cancer . Release of the drug and removal of the imaging probe are possible after application of such an imaging probe-drug complex.

  The drug can be bound to nanoparticles such as gold and polymer liposomes via the compound of the present invention. The nanoparticles can be concentrated locally and can be released locally by photocleavage.

  Aptamers, Bortezomib, RGD peptides, folic acid and the like can be bound to the complex via the compounds of the present invention and released site-specifically.

  Photodynamic and photothermal therapeutic agents are complexed and cleaved and removed by different excitation light (400/800 nm).

Claims (2)

Compound represented by the following formula (I) or (II):
(Wherein Z is the same or different and represents a biotinyl group or a maleimide group, Y represents a divalent linker group, Ar represents an optionally substituted arylene group or an optionally substituted heteroarylene group, Ar1 represents an optionally substituted aryl group or an optionally substituted heteroaryl group) A complex formed by binding the compound represented by the formula (I) of claim 1 with an avidinylated labeling substance and an avidinized functional substance.