DE102011054396B4 - Use of broadband absorbing and emitting NIR fluorophores with large Stokes shift as dyes in core-shell nanoparticles for biomarker analysis and as components of FRET systems and method based thereon - Google Patents

Abstract

Use of a compound of the general formula (I) wherein B and B 'are independently selected from methyl, ethyl, phenyl, a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 Radical, a 5,6-R'-benzothiazole-2 radical and a 5,6-R'-indole-2 radical; X, X 'and X' 'are independently selected from methyl, ethyl, phenyl, a propanesulfonic acid radical and a butanesulfonic acid radical; D is selected from hydrogen, SO3, COO, methyl, ethyl, phenyl and trifluoromethyl; Z is nitrogen; R is carbon or hydrogen; K is H, Cl, Br, is; L is selected from thiol, amine, azide, aldehyde, carboxyl, ethylene and maleimide; n is 0 or a natural number; with the proviso that n is 0 when R is hydrogen; m is 0 or a natural number between 1 and 12; ...

Description

The present invention relates to near-infrared spectral range (NIR) fluorescent dyes and their labeled particles and to their applications in analytical and imaging processes.

In principle, the penetration depth of excitation radiation in the NIR allows detection of the localization of NIR fluorophores in deeper areas of the investigated material, without the surface of the investigated material having to be removed or damaged. With appropriate addressing of the fluorophores, specific events at the molecular level should be able to be detected and assessed non-invasively, for example under the skin, with the aid of an adapted optical system.

Particular advantages of using NIR fluorophores may result if the high sensitivity of the principles used extensively analytically in the UV / vis spectral range is achieved: Förster resonance energy transfer or fluorescence resonance energy transfer (FRET), fluorescence lifetime measurement and fluorescence quantum yield measurement for the near-infrared spectral range.

However, the previously known NIR dyes are predominantly characterized by a very small Stokes shift. This complicates the spectral separation of the emitted light from the scattered excitation light, reduces the validity of the findings and limits the resolution of fluorescence-optical images.

The use of known NIR dyes for highly sensitive analysis and detection methods is particularly complicated by the partial reabsorption of the emitted light, the so-called Crosstalk, when using the dyes in FRET pairs and by the comparatively low fluorescence quantum yields. In addition, the known NIR fluorescent dyes already tend to form aggregates in dilute solutions. These aggregates fluoresce mostly not or only minimally, for example due to self-extinction.

Against this background, the use of a NIR fluorescent compound as a marker or contrast agent in imaging and biomedical analysis, according to claim 11 a spectroscopic and / or imaging method is proposed. Further advantageous embodiments, details and features of the present invention will become apparent from the dependent claims, the description, the embodiments and the accompanying figures.

The present invention is first explained by way of example with reference to figures. Showing:

1 the structural skeleton of the here proposed NIR fluorophores according to formula (I);

2 the basic structure of fluorine-containing NIR dyes according to formula (II).

According to the present description, a family of compounds is proposed which absorb wideband in the vis spectral range and in the near infrared spectral range and emit in the NIR spectral range and are distinguished from known NIR chromophores by: a comparatively displeased Stokes shift; a comparatively small ambient dependence of the spectral position of the absorption and in particular the emission spectra; as well as by a comparatively high fluorescence quantum yield in polar solvents and in a polar environment, in particular in matrices, which can form hydrogen bonds.

1 shows the structural backbone (I) of the NIR-fluorophores proposed here, which in their entirety show surprisingly favorable spectroscopic properties which are advantageous in particular for the task described. These advantageous properties enable the application in the UV / vis spectral range of proven fluorescence optical measuring principles and detection methods for the vis / NIR spectral range.

The designated measurement principles and detection methods include the use of the proposed fluorescent dyes as labels, "markers" or "labels" for molecules such as peptides, proteins (including antibodies, antibody fragments, enzymes), nucleic acids (including single nucleotides or bases, oligonucleotides, Plasmids, DNA, RNA), for dendrimers or for nano- and microparticles (especially nanocrystals, core-shell nanoparticles, atom clusters and molecular complexes, such as for vesicles, liposomes, polymer particles) but also for membranes and films, as well as for cells and tissues , Like other fluorescence-optical measuring and detection methods, the applications made possible with the aid of the proposed dyes thus relate to different levels oforganization (for example: molecule-particle-cell-tissue-organ-organism) and specialist fields (for example: analytical chemistry, molecular biology, Biochemistry, cell biology, histology, diagnostics).

Thus, the use is proposed for the spectroscopic analysis and imaging of a family of compounds having a general basic structure according to formula (I). The use of substituents adapted to the particular use permits the use of the resulting compounds according to formula (I) for fluorescence-optical analyzes in polar media, materials and environments (matrices), in particular in an aqueous environment. The entirety of the compounds included is described in claim 1 and the dependent claims 2 to 10.

Particularly advantageous for the compounds described the usability of phenomena of radiationless energy transfer, which are used for example in FRET measurements or fluorescence quenching measurements for the ultrasensitive detection of molecular events or circumstances, in the NIR. Equally advantageous are measurements in the NIR which are based on influencing the fluorescence lifetime and the fluorescence quantum yield through the respective submicron environment, for example with the aid of potentiometric fluorescence samples (indicator dyes).

Such measuring principles can be used to detect a specific redox potential, an electrostatic (membrane) potential, to detect ion gradients or concentrations, to measure the pH, to detect the presence or absence of certain reactants and similar events at the molecular level (single molecule Spectroscopy), as well as on the level of individual cells (single cell spectroscopy), single or only a few molecules or particles (for example, various tracer techniques) are used.

The use of the proposed compounds as markers and / or molecular probes thus permits fluorescence-optical measurements and fluorescence-optical imaging in the near-infrared spectral range (NIR) under real conditions of use, ie. H. in aqueous environment, in physiological media and in polar matrices such. In tissues.

In order to adapt the compounds to the particular measuring task or situation, the basic skeleton shown in formula (I) can be modified specifically by incorporating certain substituents at specific positions.

Advantageously, compounds of the formula (I) can be provided, for example, with polar substituents in the X position, in particular with acid radicals of propanesulfonic acid or butanesulfonic acid. Likewise and independently of the particular substituent in the X-position or in addition to one of the specified substituents in the X position, the compounds may be substituted in the D-position. For example, hereby in position D sulfonated (-SO ₃ H) or carboxylated (-COOH) compounds according to formula (I) in aqueous medium hereby be included and named as NIR fluorescent dye.

According to one embodiment of the invention, in formula (I), B and B 'independently of one another are a methyl radical, an ethyl radical, a phenyl radical, a 5,6-R'-benzoxazole-2 radical, a 5,6- R'-benzimidazole-2 radical, a 5,6-R'-benzothiazole-2 radical or a 5,6-R'-indole-2 radical, where in each case R 'is a hydrogen radical, SO ₃ ^- or -SO ₃ H ^, respectively and / or COO ^- or -COOH. The use of these substituents offers particular advantages for the suitability of the NIR fluorophore in an aqueous environment.

Expressly excluded from the proposed family of compounds according to formula (I), the combination of substituents in which, for n = 0, the letter R (substituent) is a hydrogen radical, B is a benzoxazole or 5,6-R ' -Benzoxazol-2 radical with R 'as a hydrogen radical in which K corresponds to a hydrogen radical, Z corresponds to a nitrogen atom, X corresponds to an ethyl radical, X' and X '' both correspond to a phenyl radical and in which the substituents D and B 'respectively stand for a hydrogen radical or in which under otherwise identical occupation of the substituents and n = 0, the letter Z is a nitrogen atom, X = X "is a phenyl radical and X 'is an ethyl radical.

Thus, fluorophores are provided for applications in the NIR, preferably for measurements in the range 700 nm to 1400 nm, which are characterized by a pronounced Stokes shift. Also in high concentration above 5 mM, as well as in the bound state (up to 50 nmol per 1 mg of particles) a particle surface or a carrier molecule, the fluorophores described do not show the disadvantages of the known NIR fluorophores mentioned at the outset.

oder Untereinheiten dieses Dendrons, beispielsweise einer niedrigeren oder einer höheren Generation.According to a further embodiment, the abovementioned substituents in their respective R 'position carry a dendrimer or a dendron, for example the compound shown below

or subunits of this dendron, for example a lower or a higher generation.

modifiziert sein (PEGyliert sein).According to a further embodiment, the respective compound in R 'position with polyethylene glycol (PEG):

be modified (be PEGylated).

In this case, the number p may be any nonnegative natural number, for example 1, 2, 3.... According to exemplary embodiments, p is in the range of 2 to 150, for example between 20 and 50, in particular at p = 30.

For example, in other embodiments, the molecular weight of the PEG moieties of a substituent may be in the range of 110 to 7,000 daltons. According to another embodiment, it may be between 500 and 3000, in particular it may be 2 kD.

The decoration of the substituents with the shown lower-generation dendron or dendron, as well as the PEGylation described above, facilitates the formation of a hydrate shell and enhances the solubilization of the respective derivatives in polar solvents, for example in aqueous solution or in biological matrices. Advantageously, the stability of the fluorophores in the respective environment is increased, in particular in the bloodstream of living organisms. Dendrons can also be used to functionalize the surface of microparticles and nanoparticles by loading the particles with the fluorescent dye and using the hydroxyl groups of the dendrons to covalently anchor a target function. Examples of such particles are polymer particles. Other examples of nanoparticles are macromolecules, nanocrystals, and atomic clusters.

According to one embodiment, a polymer particle is first loaded with an NIR fluorophore, whose substituents in B and / or B'-position in the respective R'-position of the relevant substituent carry a dendron or which are PEGylated in R'-position. After the non-anchored portion of the NIR fluorophore molecules has been removed by suitable washing steps, ie the suspended particles no longer release a fluorophore, the particle is addressed. The addressing, or the equipment of the particle with the target function takes place, for example, by one or more of the hydroxyl groups of the dendrone or the PEG be used for covalent attachment of the target function. However, the provision of the particle with the target function can also be carried out prior to the anchoring of the NIR fluorophore and / or independently thereof.

According to a further embodiment, the respective compound, irrespective of the nature of the substituents at other positions, in the R'-position of the substituents at B and / or B'-position with a fluoroorganic compound, that is modified with an F-containing organic carbon compound, in particular with a trifluoromethyl radical in position 5 or 6 of the ring system.

The anchoring of fluorine-containing substituents offers the advantage of effecting an increase in photostability while maintaining the spectral absorption and fluorescence properties which are advantageous for the proposed family of compounds and the respective comparatively high fluorescence quantum yields.

A preferred fluorescence quantum yield of the NIR fluorophores specified according to claim 1 and according to claim 11 is at least 0.1 in aqueous solution at a pH of 7.4. According to further embodiments, the fluorescence quantum yield is ≥ 0.2 and preferably falls within the range of 0.2 to 0.5. The fluorophores described have a pronounced Stokes shift, d. H. a clear difference of the maxima of absorption and emission spectra in the NIR, which is, for example, above 150 nm, in particular range from 180 to 250 nm.

From the use of the compound designated by formula (I) and the substituents named as named at the designated positions, the applicability of the respective compounds for NIR fluorescence spectroscopy and for optical imaging results. In particular, the proposed fluorophores are suitable for FRET-based detection and analysis methods and suitable for detection and analysis methods based on the measurement of the fluorescence decay behavior (kinetics) or the fluorescence lifetime and / or the fluorescence quantum yield of the compound used as a molecular probe. This also allows the use of these NIR fluorophores as markers or as "labels" for the detection with different analytical readout methods.

Of particular importance is the recognition (qualifying) and assessment or measurement (quantification) of certain metabolic processes, molecular or cellular events or changes, infection states, differentiation, growth and / or tissue changes. This broad field of laboratory diagnostics is referred to herein as biomedical analysis.

A particular advantage of the proposed compound results from the use of the proposed compound as a molecular marker. In particular, various spectroscopic measurement methods can be combined with each other, since the compound with a suitable selection of the substituents according to the specification in claim 1 and according to the specification in claim 11 provides detectable signals with different spectroscopic measurement methods. For example, the combination of fluorescence NMR ( ¹⁹ F-NMR) or fluorescence XPS (detection of heteroatoms such as F, S or N) or fluorescence NMR-XPS offers particularly meaningful results. The element-specific energy spectra recorded here enable the quantitative detection of the compound.

Particular advantages result from the possibility of quantification of the dye, of the particles provided with the dye, or of analyte labeled with dye or labeled with dye-labeled particles (eg macromolecules, polymer particles, cells) by means of different spectroscopic methods. In particular, the average loading density of a particle or of a cell or of the surface of the particle with the NIR fluorophore can be determined if the particles have a largely monodisperse size distribution. A particular advantage of the formula (I) in 1 The proposed structure or the families of compounds described according to claim 1 and according to claim 11 represents the possibility of quantifying the loading density of micro- and nanoparticles with a compound which is specified in claim 1 or claim 11 and the associated subclaims.

In one embodiment, the use of compounds according to claim 1 as markers and / or contrast agents for imaging and / or for the quantitative and / or qualitative analysis of living cells, tissues, body fluids or other biological material (for example, smears, tissue sections or biopsies ) for the solution of medical diagnostic or biomedical or cell biological research questions. The questions addressed provide intermediate results which can be used for the diagnosis.

und
B = H, Methyl, Ethyl, Phenyl, 5,6-R'-Benzoxazol-2,5,6-R'-Benzimidazol-2, 5,6-R'-Benzthiazol-2, 5,6-R'-Indol-2;
B' = H, Methyl, Ethyl, Phenyl, 5,6-R'-Benzoxazol-2, 5,6-R'-Benzimidazol-2, 5,6-R'-Benzthiazol-2, 5,6-R'-Indol-2;
X = Methyl, Ethyl, Phenyl, Propanesulfonic acid, Butanesulfonic acid;
X' = Methyl, Ethyl, Phenyl, Propanesulfonic acid, Butanesulfonic acid;
X'' = Methyl, Ethyl, Phenyl, Propanesulfonic acid, Butanesulfonic acid;
D = SO₃ ^–, COO^–, Methyl, Ethyl, Phenyl, H, Trifluoromethyl;
R' = H, SO₃ ^–, COO^–, Dendrimere und PEG, 5-(trifluoromethyl), 6-trifluoromethyl;
Z = N;
R = H, C;
n = 0, 1, 2, ...;
A^– = Anionen wie z. B. ClO₄ ^–, Cl^–, Br^–, I^–, BF₄ ^–, Sulfat, Tosylat;
K = Cl, Br,

wobei m = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 (bevorzugt liegt m zwischen 1 und 5);
L = Thiol, Amine, Azide, Aldehyde, Carboxyl, Ethyne, Maleimide.In this case, the structure of the compound has the general formula (II):

and
B = H, methyl, ethyl, phenyl, 5,6-R'-benzoxazole-2,5,6-R'-benzimidazole-2, 5,6-R'-benzothiazole-2, 5,6-R'- indole-2;
B '= H, methyl, ethyl, phenyl, 5,6-R'-benzoxazole-2, 5,6-R'-benzimidazole-2, 5,6-R'-benzothiazole-2, 5,6-R'indole-2;
X = methyl, ethyl, phenyl, propanesulfonic acid, butanesulfonic acid;
X '= methyl, ethyl, phenyl, propanesulfonic acid, butanesulfonic acid;
X "= methyl, ethyl, phenyl, propanesulfonic acid, butanesulfonic acid;
D = SO ₃ ^- , COO ^- , methyl, ethyl, phenyl, H, trifluoromethyl;
R '= H, SO ₃ ^-, COO ^-, dendrimers and PEG, 5- (trifluoromethyl), 6-trifluoromethyl;
Z = N;
R = H, C;
n = 0, 1, 2, ...;
A ^- = anions such as. B. ClO ₄ ^-, Cl ^-, Br ^-, I ^-, BF ₄ ^-, sulfate, tosylate;
K = Cl, Br,

where m = 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 (preferably m is between 1 and 5);
L = thiol, amines, azides, aldehydes, carboxyl, ethyne, maleimides.

As shown, B and B 'are independent of each other z. B: a hydrogen radical, a methyl, an ethyl, a phenyl, a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical, a 5,6-R'-benzthiazole A radical or a 5,6-R'-indole-2 radical, where R 'can each be a hydrogen, SO ₃ , COO, a dendron, polyethylene glycol, 5- (trifluoromethyl) or 6- (trifluoromethyl) and X, X 'and X "independently of one another may be methyl, ethyl, phenyl, or an acid radical of propanesulfonic acid or butanesulfonic acid. The substituent D is selected from SO ₃ ^- , COO ^- , methyl, ethyl, phenyl or trifluoromethyl. Z represents a nitrogen or a carbon atom. R represents a hydrogen atom or a carbon atom. In the former case, when R is a hydrogen atom, n = 0. If R is a carbon atom, then n = 1.

wobei m jeweils eine beliebige natürliche Zahl 0, 1, 2 ... ist und L jeweils für ein Thiol, für Amin, für Azid, für Aldehyd, für Carboxyl, für Ethyn oder für Maleimid steht. Beispielsweise kann m hier einen Wert zwischen 1 und 12, bevorzugt zwischen 1 und 5 annehmen. Das mit A^– bezeichnete Anion kann ausgewählt sein unter Perchlorat, Chlorid, Bromid, Jodid, Tetrafluoroborat, Sulfat und Tosylat oder anderen Anionen mit ähnlichen Eigenschaften (z. B. Solvatationsverhalten, Wertigkeit).The substituent denoted by K is Cl, Br,

where m is any natural number 0, 1, 2... and L is in each case a thiol, an amine, an azide, an aldehyde, a carboxyl, an ethylene or a maleimide. For example, m here can assume a value between 1 and 12, preferably between 1 and 5. The anion designated A ^- may be selected from perchlorate, chloride, bromide, iodide, tetrafluoroborate, sulfate and tosylate or other anions having similar properties (eg, solvation behavior, valence).

According to a preferred embodiment, at least one substituent of the molecule has a fluorine atom. This means that at least one of the substituents B or B 'is a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical, a 5,6-R'-benzothiazole-2 radical or a 5,6-R'-indole-2 radical with R '= 5- (trifluoromethyl) or R' = 6- (trifluoromethyl), or at position D a trifluoromethyl is linked to the respective ring.

According to a further embodiment, the compound having the above formula (II) is used by detecting and / or analyzing a fluorescence signal, in particular an emission signal having a maximum in the wavelength range between 750 nm and 850 nm. In particular, the use of the compound according to formula (II) in imaging or in combined measuring methods is proposed, wherein in addition to the measurement and / or detection of a fluorescence emission signal, an element-specific energy spectrum is detected and / or analyzed and the element is selected from sulfur (S), nitrogen (N) and / or fluorine (F).

The incorporation of heteroatoms such as nitrogen atoms, fluorine atoms or sulfur atoms in the compound of formula (II) facilitates the quantification of the loading density of particles which are loaded with one or more different representatives of the described compounds of formula (II). This quantification is based on the fact that the average number of dye molecules per particle can be determined by means of different and independent analytical methods. Examples of this are optical-spectroscopic methods, such as. Example, the measurement of the absorption properties and the fluorescence properties of the loaded particles.

The absorption and fluorescence properties of the NIR fluorophore-labeled particles can be measured, for example, with an integrating sphere or with the aid of optical spectroscopic determination of the dye concentration after dissolution of the dye-labeled particles in solution.

Particular advantages for the particle-based analysis are opened when the at least one fluorine-containing substituent of a compound according to formula II is a compound of ¹⁹ F. This makes it possible to correlate the determined absorption and fluorescence properties of the NIR fluorophore-labeled particles with measurement results of ¹⁹ F-NMR, or to determine the dye via elemental analysis (TOC, eg S or N determination) ,

Using an adapted measurement method can be loaded with the dye according to formula (II) particles z. B. a number of chromophores, an absorption coefficient (per particle or per average chromophore loading density) and a quantum yield (per fluorophore) are assigned. The preferred average particle loading density for the particular practical application depends on the specific task. Typically, a preferred loading density is 1-10 nmoles per 1 mg of particles. Such a loading density is suitable, for example, for in vivo imaging applications using fluorescence-labeled particles.

There are no reliable methods for quantifying the loading density of microparticles and nanoparticles. At present, only a comparison of particle dye solutions according to the so-called MESF system, wherein MESF stands for Molecules of Equivalent Soluble Fluorophore. In principle, the novel approach to particle analysis described here can also be exploited to set up a new MESF-analogue quantification and feedback concept.

The quantification and jerk concept allows the use of the particle systems described and z. Example, the measurement of their absolute fluorescence quantum yields. Extinction coefficients and the dye concentration per particle the attachment (feedback) of many other fluorescence measurement techniques to absolute scales and spectroscopic measures and thus the cross-platform standardization. Thus, the derivatives of the compound of formula (I) described in claim 1 allow a novel approach to the standardization of fluorescence optical measured variables and the validation of measurement methods, equipment and laboratories (ring tests), which is not bound to the NIR spectral range. This recycling concept can also be applied to other dye systems.

The possibility of implementing the described quantification and recycling concepts is of particular importance for the field of application of the NIR fluorophores described according to claim 1 or according to formula (I) for applications in pharmaceutical and cell biological research, medical diagnostics and therapy Therapy monitoring as well as in practical laboratory diagnostics.

¹⁹ F-containing fluorescent systems such as optical probes (markers or labels), which are composed of a ¹⁹ F-labeled fluorophore (or a ¹⁹ F-labeled molecular system or loaded with the fluorophore particles) and a target-specific ligands or another analyte-specific Chromophore system included, then allow z. As well as the detection of different analytes by fluorescence and ¹⁹ F-NMR.

Regardless of or in addition to the above-described embodiments, the compound in the position of the substituent D carries a group selected from: SO ₃ ^- and -SO ₃ H, respectively; COO ^- or -COOH, methyl, ethyl, phenyl and trifluoromethyl. A sulpho or a carboxy group facilitates the solubility of the subject compound in a polar environment and has a beneficial effect on fluorescence quantum yields, extinction coefficients and maximum label density since dye aggregation is prevented.

In contrast, the trifluoromethyl-modified compound as indicated has advantageous properties for fluorophore applications in magnetic resonance imaging and / or magnetic resonance imaging (MRI). As an umbrella term for magnetic resonance imaging, the term MRI (magnetic resonance imaging) should be used here. Particular advantages result when the fluorine atom, for example a ¹⁹ F fluorine atom or a fluorine atom ¹⁸ F. Other isotopes are known and suitable. These isotopes are preferably used, for example, in nuclear magnetic resonance spectroscopy (NMR or ¹⁹ F-NMR) and MRI imaging methods based thereon. Different nuclear spin relaxation times in different biological tissues also form the basis for magnetic resonance imaging (magnetic resonance tomography) used in medicine as an imaging diagnostic method.

Likewise, other isotopes of hetero elements, for example, sulfur or nitrogen may be used in the described compound. Such dyes or nanoparticles or microparticles labeled with them can be used as contrast media (tracers), for example for the production of spatial images of a test object, for example cells, cell aggregates, tissues, organs or organisms by means of MRI techniques.

From the use of hetero elements and their isotopes, for example from the use of ¹⁹ F, there is the advantage of using two independent imaging methods with one and the same molecular probe or one and the same nanometer- or micrometer-scale particles containing a compound according to claim 1 are marked can be performed. This increases the safety of collected findings. For this purpose, the particles or other objects marked with the compound can be exposed or exposed to electromagnetic radiation of different wavelength ranges or spectral ranges (energies). Examples of useful radiation are UV radiation, UV / vis radiation, vis radiation, NIR radiation, X-radiation or gamma radiation.

According to a further embodiment, the substituent Z can independently of the other substituents B and B ', R', X, D, R, K, L, the size of n and independently of the anion A ^- in the formula (I) represent a nitrogen atom.

According to a further embodiment, the substituent R may independently of the other substituents B and B ', R', X, D, Z, K, L, the size of n, and independently of the anion A ^- in the formula image (I) a hydrogen or a carbon atom.

bedeuten, wobei m jeweils eine beliebige natürliche Zahl 0, 1, 2 ... bedeutet und L jeweils für ein Thiol, für Amin, für Azid, für Aldehyd, für Carboxyl, für Ethyn oder für Maleimid steht. Beispielsweise kann m hier einen Wert zwischen 1 und 12, bevorzugt zwischen 1 und 5 annehmen.In a further embodiment, the substituent K independently of the other substituents B and B ', R', X, D, Z, R, L and independently of the anion A ^- in the formula (I) Cl, Br,

where m is any natural number 0, 1, 2... and L is in each case a thiol, an amine, an azide, an aldehyde, a carboxyl, an ethyl or a maleimide. For example, m here can assume a value between 1 and 12, preferably between 1 and 5.

According to typical embodiments, the anion A ^- irrespective of substituents B and B ', R, R', R '', X, D, Z, K, L and the value of n and in the Formula image (I) is any anion in particular, it may be a perchlorate ion, a chloride anion, a bromide anion, a tetrafluoroborate anion, a sulfate anion, or a tosylate anion.

Particularly advantageous spectral characteristics and a markedly high photostability show the compound according to formula (II) (cf. 2 ).

The use for imaging and biomedical analysis proposed herein relates to compounds according to formulas (I) and (II), respectively, which are freely covalently bound to or associated with (for example through the formation of ion pairs or via ionic interaction) nano- and / or microparticles available. The compounds represent a broad band in the vis spectral range and in the NIR spectral range absorbing family of fluorescent dyes, which is characterized by a comparatively large for a NIR chromophore Stokes shift, by a relatively low environmental dependence of the spectral position of the absorption and in particular the emission spectra and characterized by a comparatively high fluorescence quantum yield, which is also present in polar matrices and in matrices which can form hydrogen bonds.

This results in the possibility of hedging collected findings largely method-independent and much better than was previously possible. Such a hedge is particularly for the application of the named measuring methods in analytics, laboratory diagnostics, clinical diagnostics, z. B. important for purposes of therapy control. The methods used for this must be subjected to a so-called validation procedure. The proposed group of compounds enables validation of the newly adapted and newly developed fluorescence spectroscopy and near-infrared spectral imaging (NIR) imaging techniques.

In 1 the basic structure of the compounds according to the invention according to formula (I) is shown.

Conventional fluorophores with charge transfer character (and therefore correspondingly large Stokes shift) emit either not at all in aqueous solvent mixtures, or there are only information on the fluorescence quantum yields in methanol, but not for aqueous solutions. Overall, the known fluorophores are characterized by narrow-band absorption and emission spectra with a vibronic fine structure. The difference between the maxima of absorption and emission spectra (Stokes shift) is low and is only a few 10 nm. This results in a significant overlap of absorption and emission and overall lower fluorescence quantum yield at higher loading levels z. B. by reabsorption or by aggregation.

By contrast, the compounds characterized by a basic structure described in claim 1 or claim 11 and its subclaims have a significantly larger Stokes shift and are therefore free of the disadvantages of the previously known dyes. This results in the applications described below by way of example:

a) Use as multichromophore fluorescence probe without and with target function:

According to an application example, the selected compound is incorporated into nanometer- to micrometer-sized polymer particles. In this context, polymer particles are understood as meaning more or less spherical particles which are produced by crosslinking the chains of a synthetic polymer. According to a first embodiment, the fluorescent probe is bound, for example, to the polymer chains prior to their crosslinking. The matrix of the polymer particle is then completely labeled with the NIR fluorophore.

According to a second embodiment, already present, for example commercially available, nanometer to micrometer-sized polymer particles can be incubated in a suitable solvent and swollen during this process. If the swollen polymer particles are brought into contact with a solution of the NIR fluorophore or swelled directly in the presence of the NIR fluorophore, the NIR fluorophore can penetrate into the interior of the particle by diffusion and be adsorptively, chemically or mechanically fixed or bound, when the solvent used for the swelling of the polymer particle is replaced by another solvent or a solvent mixture and the particle has shrunk again and assumed its dimensions typical for an aqueous medium. In this regard, it should be recalled that one aspect of the NIR fluorophore applications described herein is for use in polar media and matrices, such as aqueous solutions, physiological buffer or saline solutions, cell culture media and culture, and tissue culture the intracorporeal use is directed in the living organism or on the patient. After the unbound NIR fluorophore has been removed by washing, polymer particles are available whose matrix is marked with the NIR fluorophore where the NIR fluorophore could penetrate. With a suitable choice of polymer particles (polymer type, chain length, degree of crosslinking or porosity ...) and the solvent or a Solvent mixture can be obtained multiply labeled particles that release unbound dye, or show no leaking (leaking).

According to an exemplary embodiment, the respective fluorophore is dissolved in tetrahydrofuran (THF) or in dimethylformamide (DMF) at a dye concentration of 5 × 10 ^-3 mol / L to 5 × 10 ^-5 mol / L. For example, regardless of particle diameter, polystyrene particles are loaded with the respective fluorophore by mixing 100 μL of the dye solution with 600 μL of an aqueous dispersion of the particles (0.5 percent by weight (w%)). Particles of size <100 nm are then incubated for 30 minutes, larger particles for 1 h. Thereafter, another 800 μL of water are added and the occasionally shaken particle dispersion is centrifuged. This can be done, for example, with an Eppendorf 5415D centrifuge. 100 nm diameter polystyrene particles are separated at 15,000 g for 40 minutes, 1 μm diameter poystyrene particles at 5,000 g for 10 minutes, and 8 μm diameter polystyrene particles at 4,000 g for 10 minutes. For smaller particles, adapted rotors or other centrifuges may be used, for example a Beckman Coulter centrifuge Avanti J-20 XP. This proved to be suitable for polystyrene particles of a diameter of 50 nm 45000 g during 45 minutes of centrifugation. For polystyrene particles of 25 nm diameter, centrifugation was carried out at 75,000 g for 45 minutes. The separated particles are washed twice with distilled water and resuspended in double-distilled water in an ultrasonic bath. Depending on the substituents chosen or the swelling behavior of the respective polymer particles, THF or DMF for the incubation step can also be replaced by dichloromethane or trichloromethane. Likewise, other organic solvents or mixtures thereof can be used. If dichloromethane, trichloromethane or tetrachloromethane is used, the particles are first converted into isopropanol, washed twice with isopropanol to remove the water and each centrifuged. Other combinations of solvents in which the particle swells at least on its surface are possible. In particular, it is possible to use solvent mixtures which are adapted to the particular particle matrix, ie which are adapted to the polymer or polymers and / or their linkers from which the particle is composed.

In a further embodiment, the combination of the methods described above may be used to label particles with different fluorophores. For example, a particle may contain in its interior a compound according to formula (I) or (II) and be labeled on its surface with another fluorophore.

According to a further embodiment, a fluorophore according to the formulas (I) or (II) can be anchored exclusively or predominantly on the surface of a polymer particle. According to another embodiment, atom clusters, particles of paramagnetic compounds, for example iron oxides, or nanocrystals can also be labeled with the NIR fluorophore. This offers the advantage that the detection of the particles via the incorporated dye in the NIR can be combined with the detection of the specific interaction of the particle matrix with electromagnetic radiation of another energy spectrum. Examples are UV radiation, UV / vis radiation, vis radiation, NIR radiation, X-radiation or gamma radiation.

This results in advantages with regard to the development of standardized measuring methods. Furthermore, both different NIR fluorophores can be combined with each other as well as with other fluorophores or with other conventional detection reagents for other detection methods.

According to a further embodiment, the NIR flurophore can be incorporated into enveloped or multi-shell systems (core-shell systems). Multi-shell systems provide, for example, dendritic materials, i. H. different functionalized dendrimer systems of different generations. Other core-shell systems are encapsulated nanoparticles or atomic clusters in a polymer shell.

In another embodiment, the NIR fluorophores can be incorporated into liposome systems or vesicles or into micellar structures. The dye is present in the hydrophilic parts of the liposomes or micelles. This can be both the inside and the shell. Subsequently, the surface of the respective particle, the core / shell system, the vesicle, the liposome, the micelle, or the dendritic structure with the aid of suitable ligands, for example a peptide, a nucleic acid strand, a receptor, with so-called small molecules or with an antibody or antibody fragment, etc. or with an antigen or other methods customary in histochemistry, cytochemistry, cell biology or medical diagnostics. This modification determines the specificity of the fluorescent dye-equipped particle for the respective target (target function), ie, to bind or bind the ability of the particle at a specific target site or "address". The advantages of the individual listed markers or target function are known to the person skilled in the art. she arise from the respective task and can be, for example, in the field of therapy of a disease or its monitoring or in the field of cell biology or the field of medical and / or pharmaceutical research.

As a target, for example, a specific molecule, a specific molecular complex, a specific surface structure of a cell, a tissue of a microorganism or a virus come into consideration. In the context described, the surface structures named or corresponding to them can also be present, for example, in the form of a CD antigen or another tissue-specific or specific molecular structure for a particular disease process. The detection of such structures by means of the NIR fluorophores can give indications of a particular state of differentiation of cells or tissues, to a specific metabolic process, to an infection, to a tissue degeneration or to the effectiveness of a therapy, or to a specific course of disease. The detection of a target can be performed in situ or ex situ, or in vivo or ex vivo. Examples of the ex vivo application are the examination of samples of body fluids, for example of plasma samples, or biopsy samples (biopsies).

The application of the described highly fluorescent molecules or the particles or the probes produced with them can be carried out in different measuring formats or with the aid of different device platforms. The applications address specific questions and include applications for optical imaging and applications as biomarker-specific probes in various fluorescence assays. Examples are particle-based fluorescence assays that are read by microtiter plate reader, fluorescence microscope, flow cytometer or microarray scanner.

Depending on their size and surface functionalization, the particles loaded with a compound according to claim 1 or with a similar chromophore having the same advantageous spectral properties can be used for a variety of (bio) analytical and medical diagnostic issues and problem solutions. Of particular interest is the application of loaded with this dye or similar chromophores nanometer-sized particles, provided for. B. with different biomarker-specific ligands in the form of antibodies, antigen-binding (fab) antibody fragments, peptides, "small molecules" etc.

According to a further embodiment, the NIR chromophores according to claim 1 or the particles marked with them are used, for example in the form of particles smaller than 1 μm, by internalizing the particles in vivo or ex vivo by living cells, for example by macrophages or by stem cells by way of endocytosis. The uptake of the NIR-fluorophore-labeled particles can take place, for example, in cell culture.

It is likewise possible to bind the NIR fluorophore specified in claim 1 to a drug or an active substance or to active ingredient particles and to introduce the active substance labeled with the NIR fluorophore according to claim 1 into living cells, to infiltrate it elsewhere or to internalize it from living cells to let. Thereafter, the labeled cells for presentation, monitoring or monitoring of a therapy and the drug uptake and distribution (biodistribution of drugs) as target-specific probes in optical imaging (so-called in vivo imaging) for the highly sensitive and selective detection of pathological changes on molecular level in vivo.

According to further embodiments, the particles or cells labeled with the NIR fluorophore are used for preclinical pharmacokinetic studies to represent the distribution of therapeutic substances in the organism. For example, when the compound of claim 1 is introduced into macrophages, the cells so labeled can be used to display inflammatory changes in various organs (e.g., in the lung) or, ex vivo, in stem cells, to track the migration of stem cells in the organism in vivo become.

From the point of view of avoiding artifacts, the effective labeling of living cells with a compound as described in claim 1 is particularly advantageous, since a strong fluorescent signal is delivered even from the lowest absolute amounts.

The formation of emissive dimers allows for the proposed chromophores, the production of highly highly fluorescent particles with pronounced NIR emission, which is also largely independent of the degree of loading. In contrast, common NIR dyes that adhere to particles are bound or incorporated into particles, above 1 nmol per 1 mg of particles, by the formation of non-emissive or only very low fluorescent molecular aggregates rapidly into areas of self-quenching.

As explained above, even at lower loading concentrations above 1 nmol per 1 mg of particles, other dyes already show the formation of non-emissive or only very low-fluorescent aggregates. In addition, the significantly lower Stokes shift makes the separation of excitation and emission light more difficult. This results in disadvantages in almost all applications of a fluorescent dye.

According to another embodiment of the described NIR fluorophores biomarkers z. B. be detected in body fluids from experimental animals. An exemplary application in oncology concerns z. B. the safe detection of even a very small number of cancer cells in the blood, in ascites or in pleural fluid, in urine, in biopsies or in smear preparations. Here, the cancer cells are specifically detected by fluorescence microscopy using a microtiter plate-based fluorescence assay using loaded with these fluorophores and functionalized with biomarker-specific ligands particles. Likewise, the relevant cancer cells can be specifically labeled and then detected individually (single-cell analysis).

Alternatively, reading such an assay is also possible by fluorescence microscopy or the use of biomarker-functionalized particles loaded with these fluorophores and magnetic materials to separate the analytes from the sample by an external magnetic field and then fluorescence detection.

b) Use as a component in a bi or multichromophore FRET system:

According to an associated embodiment, the NIR fluorophore can also fulfill the function of a sensor. The optical properties of the FRET system can be designed switchable or non-switchable. A switchable embodiment of the FRET system in this context means that a FRET signal is generated in response to a binding event on a specific ligand or in response to a specific environment or in response to another external trigger. Similarly, the NIR fluorophore can be used as a component of one or more FRET systems or used in combination with magnetic, paramagnetic and superparamagnetic materials.

The NIR fluorophores can either be used directly or they are, as already explained, incorporated or as superficial marking. Examples of the presence of the dyes in the installed state are, for. For example, the presence of the NIR fluorophores in polymer particles such as polystyrene particles with or without functional surface groups (such as -COOH, -NH ₂ ); with covalently or non-covalently bound polymers - such. B. PEG, with sugars - such. As sorbitol or dextran. Other examples are the binding to or association with various lipids or dendrons, with dendritic carrier systems, as core-shell systems, as layer-by-layer polyelectrolyte shell systems (cf. LbL technique). Likewise, the NIR fluorophores may be entrapped in liposomes and micelles or bound in and to block co-polymer particles.

Examples of such polymer particles are hydrophilic co-polymers z. Based on N- (2-hydroxypropyl) -methacrylamide (HPMA) or poly (lactic-co-glycolic acid) (PLGA) or other synthetic polymers. Likewise, it is possible to use particles based on biological components, for example human serum albumin (HSA) or chitosan. Likewise, hydrophobic polymer particles can be marked.

In another embodiment, the listed components may function as film forming components in a FRET system. It is also possible to use polymers such as PMMA with different surface functionalizations.

Of particular interest for the use of the described NIR fluorophores in FRET systems is the application of the NIR fluorophores as a broadband emitting NIR donor. Application examples are in optical imaging. Related applications include target-specific probes with sensor function based on an analyte switchable FRET signal.

For example, analyte switchability may be based on a molecular linker that is cleaved in the presence of the analyte, which cleavage either triggers energy transfer (FRET signal) or causes it to go out. Examples of the cleavability of the linker include cleavage at or from a certain pH, enzymatically, for example in the presence of a protease, through a reductive environment, under the influence of light of a certain wavelength (photocleavable linkers) or other principles known to those skilled in the art (temperature switchability, eg in combination with Schiff's bases).

Advantageous compared to conventionally used NIR chromophores here is due to the large Stokes shift only very small pronounced spectral crosstalk between donor and acceptor. This allows excitation of the donor without simultaneous excitation of the acceptor. This is not possible with commonly used NIR-FRET pairs, since conventional NIR-FRET pairs are composed of narrow-band absorbing and emitting NIR dyes with a low Stokes shift. In this case, an unintentional acceptor direct excitation can not be avoided. This, in turn, adversely affects the specificity and / or the signal-to-noise ratio of the particular assay.

Usually, corrections are made to take into account in the signal evaluation, the proportion of direct excitation of the acceptor at the conclusion of the signal. These corrections complicate the implementation of FRET experiments in the NIR anyway, but are hardly feasible, especially in the in-vivo FRET, since no blank measurement and no measurement of individual components is possible.

As FRET acceptors, the z. B. can be bound via analyte-specific cleavable linker or show the self-analyte-specific changes in their optical properties, come NIR dyes into consideration that either absorb only or absorb and emit. Particularly advantageous examples of suitable FRET acceptors are the dyes DY-78x (DY-78D, DY-781, DY-782) and ICG-analogous dyes (also Cypate).

The described NIR fluorophores according to claim 1 act as FRET donors. It is advantageous in this case that the dye ICG is approved as NIR fluorophore for applications in medical diagnostics.

In another embodiment, the absorption and fluorescence properties of the named FRET acceptors are controlled by aggregation. For example, the fluorescence intensity of the proposed according to claim 1 NIR fluorophores can be switched. Also for this application, for example, surface-functionalized particles or core-shell systems such as dendrimers with nonpolar cores containing in the non-polar part proposed according to claim 1 NIR dyes and outside at the periphery carry the corresponding, non-aggregated or aggregated FRET acceptors. In the aggregated state, the fluorescence is quenched, with de-aggregation, a "turn on" the fluorescence, d. H. the acceptor becomes emissive.

According to further embodiments, the proposed according to claim 1 NIR fluorophores can also act as a FRET acceptor. An example of the function of the proposed NIR fluorophores is the combination with the polarity probe Nile Red. The comparatively low spectral crosstalk is also advantageous here.

c) Combination with (super) paramagnetic and strongly UV absorbing systems in the UV / vis spectral range:

Because of the comparatively high fluorescence quantum yield, the long-wave absorption and the long-wave emission, the NIR fluorophores proposed according to claim 1 also exhibit combinations in (super) paramagnetic and in the UV / vis spectral range strongly absorbing systems, for example with iron oxide particles or other contrast media such as iodine, Mn (II) systems or Gd-containing compounds, nor an intense fluorescence, even if encapsulated as described d. H. in the installed state. The majority of vis-emitter compounds in the visual spectrum can not fulfill this criterion.

According to a further embodiment, the compounds proposed according to claim 1 are used in combination with contrast agents for positron emission tomography (PET).

The compounds described herein can be used in combined methods of hybrid imaging (multimodal imaging). For example, the visualization of an NIR fluorescence signal can be overlaid with the visualization of an XPS signal of the connection. Likewise, combinations with contrast agents are conceivable for other detection techniques and imaging methods.

d) Use as a Fluorescence Marker for Lifetime Multiplexing:

The NIR fluorophores encapsulated as described above can be used as biospecific markers for fluorescence lifetime measurements. This offers the advantage of being able to build extremely sensitive detection systems, since the NIR fluorophores can be encapsulated in differently functionalizable support materials as shown. Fluorescence lifetime measurement requires chromophores that can be excited and detected at one and the same wavelength, but that differ significantly in their fluorescence decay behavior. This is very difficult to achieve in the NIR spectral range because the fluorescence lifetime of most dyes is quite similar. Using the proposed fluorophores, a varying particle fraction or differently charged particles can be distinguished by the resulting different decay curves of a sample, thus allowing for differential labeling using otherwise identical fluorophores.

Advantageously, the PS-NPs can be used for fluorescence lifetime multiplexing, both in free solution and in living tissue or in cells. The respective different surface functionalization such lifetime label, z. As the connection to different ligands and thus the interaction with different biomarkers or the incorporation into particles of different sizes or different surface chemistry, allows the detection of different analytes. The respective surface chemistry of the particles can be used to their specific enrichment in different areas z. B. in cells or in the tissue pretend or prove.

e) Use as external or integrated spectral fluorescence standard and as fluorescence intensity standard:

According to a further embodiment, the use of the fluorophore according to claim 1 as integrated fluorescence standard and as fluorescence intensity standard for various fluorescence methods such as fluorescence imaging method is proposed. Applications include fluorescence tomography, fluorescence reflectance imaging, fluorescence microscopy and flow cytometry.

It is advantageous here that the connection can be used simultaneously as optical probe and as standard for device calibration and characterization. In particular, the compounds according to claim 1 are used for simultaneous use as an optical probe or sensor system and as an internal or integrated NIR emission standard or NIR intensity standard. This opens up the benefits of internal calibration. It is particularly advantageous that the sample used for calibration is located exactly at the same measuring location. When calibrating the measurement signal with the control signal, the reliability of the measurement signal and the statements derived from it can be increased without extensive technical effort.

f) Use as reference dye for ratiometric measurements:

According to further embodiments, a compound according to claim 1 is used as a reference dye in ratiometric analyte-specific molecular probes and sensors. For example, by combining the NIR fluorophore with an oxygen-sensitive dye, e.g. As a ruthenium complex such as tris (4,7-diphenyl-1,10-phenanthroline) ruthenium (II) dichloride (Ru (dpp) ₃ ), an oxygen concentration-dependent photoluminescence quantum yield and / or life can be measured. Ru (dpp) ₃ can be excited at many relevant laser (diode) wavelengths and LED wavelengths, and is therefore of particular practical importance for use with existing device technology.

According to a further embodiment, the NIR fluorophore according to claim 1 is combined with a pH-sensitive dye. For example, sensor molecules are used which can be excited at a wavelength of 488 nm and whose analyte-specific emission is then determined based on the observed under these conditions emission intensity of the NIR fluorophore according to claim 1. The prerequisite for this are broadband and long-wave absorbing dyes, such as those described in claim 1 and their characteristic large Stokes shift by typically more than 180 nm in the NIR. These properties are extremely rare in previously known NIR chromophores.

g) Water-soluble labels and probes based on the dye systems described above in accordance with the applications a) -e):

Based on the applications described under a) -e) above, NIR fluorophores can be used as water-soluble labels and molecular fluorescence probes without and with target and sensor function, as a label for fluorescence lifetime multiplexing and as a fluorescence standard and as a reference dye.

In a typical embodiment, the NIR fluorophore of claim 1 may be coupled to a dendron via at least one ligand B or B '. An example of a candidate dendron is shown above. Dendrons which are directly bonded to the compound of the formula (I) or bonded via the substituents B or B 'can envelop the dye molecule and in particular contribute to the formation of an advantageous hydrate coat.

According to a further embodiment, functional groups of the dendron, for example hydroxyl groups, amino groups or carboxyl groups, are used for surface functionalization and thus for addressing the NIR fluorophore.

The water solubility and / or biocompatibility of the NIR fluorophore according to formula (I) can be improved if the structure is modified with one or more dendrons without the spectral or other fluorescence properties changing as a result of this modification. Preferred water solubilities of the NIR fluorophores are at 37 ° C. in the range between 10 and 100 μmol / L or above. The solubility and / or biocompatibility of a solution which has both a modified with at least one dendritic structure NIR fluorophore and a NIR fluorophore without dendron, can - based on a solution that has no dendrene-modified NIR fluorophores - increased or be improved. Equally advantageous is the PEGylation of the NIR fluorophore according to claims 1, 7, 11 or 2 ,

h) incorporation of fluorine (F) -containing substituents:

To increase the photostability of the NIR fluorophores, while retaining the advantageous spectral absorption and fluorescence properties and the comparatively high fluorescence quantum yield, fluorine-containing substituents can be introduced into the structure of the formula (I). Advantageously, F-containing organic carbon compounds such as 5- (trifluoromethyl) - or 6- (trifluoromethyl) radicals are attached in the R 'position of the substituents at B and / or B' position. Examples of substituents in the B and / or B 'position of the formula (I) which are provided with fluorine-containing organic radicals R' include: 5,6-R'-benzoxazole-2 radicals, 5,6-R'-benzimidazole-2 radicals, 5,6-R'-benzothiazole-2 radicals and 5,6-R'-indole-2 radicals.

Particular advantages thus provide a compound according to the in 2 constructed formula (II) is constructed.

According to one or more embodiments, using the fluorine-containing compound of the formula (II), various spectroscopic measurement methods can be combined with each other. In particular, NIR fluorescence measurements can be combined with ¹⁹ F NMR. Likewise, at least the measurement of the fluorescence emission of the compound in the NIR can be combined with detection of element-specific signals of fluorine, sulfur or nitrogen by means of XPS. Likewise, fluorescence measurements can also be combined with nuclear magnetic resonance spectroscopy or tomography and X-ray photoelectron spectroscopy (XPS). In this case, the analysis and billing of the measured data both for two-dimensional representation of the examined object, for example by means of microscopy, or for displaying spatial images of the test object or objects by means of tomographic imaging is possible.

A particular advantage is the collection of information on the specific loading density of particles, such as polymer particles, nanocrystals or cells with the dye of formula (II). With the above-described substituted compounds of the formulas (I) or (II) can in particular Nanoparticles, submicron particles and microparticles are advantageously labeled, since these particles fluorescence optically when using a suitable anchorage of the dye in or on the particle even when viewed in a sectional plane whose depth (z-direction) is below 100 nm (nanoscale) (NIR) still provides evaluable measuring signals. In particular, objects such. For example, living cells that have nanoscale dimensions in at least one spatial extension direction can be displayed, counted or measured in high resolution using the compounds described or using particles labeled with the described compound, without compromising the viability of the cells ,

Likewise, other nanoscale or microscale particles can be labeled with the dye according to the formulas (I) or (II) or according to claims 1 and / or 11.

According to another embodiment, molecules labeled with a dye of formulas (I) or (II), such as DNA or RNA strands, oligonucleotides, peptides, proteins, receptor-ligand pairs; Nanocrystals; Atom clusters; vesicles; liposomes; Microorganisms or individual cells or tissue are either followed by fluorescence optics, imaged, measured and / or counted.

According to a further embodiment, spherical polymer particles labeled with a dye according to the formulas (I) or (II) are either traced, measured and / or counted or imaged by fluorescence optics.

According to a further embodiment, cells and tissues which at least partially comprise, bear or contain spherical polymer particles labeled with a dye according to formulas (I) or (II) and are present in a physiological medium or are present in matrices which can form hydrogen bonds are fluorescence optically mapped, assessed, measured and / or counted.

• – die für bekannte NIR-Fluorophoren ungewöhnlich breiten und unstrukturierten Absorptions- und Emissionsspektren der im Anspruch 1 beschriebenen Verbindungen in Lösungsmitteln und festen Matrices verschiedener Polarität (auch in Matrices relativ geringer Polarität wie z. B. in Polystyren (PS) oder Polymethylmethacrylat (PMMA)),
• – die geringe Polaritätssensitivität der optischen Eigenschaften des Farbstoffes, insbesondere bezüglich der Form und spektralen Lage der Emissionsbande,
• – der für einen NIR-Fluorophor relativ große Stokes Shift und
• – die vergleichsweise hohe Fluoreszenzquantenausbeute in polaren Lösungsmittel wie Ethanol, in Lösungsmittelgemischen aus einem organischen Lösungsmittel und Wasser und in festen Matrices,
• – die Bildung von emissiven Aggregaten sowie
• – die vergleichsweise hohe Stabilität der beschriebenen NIR-Fluorophore in luftgesättigter Lösung und in festen Matrices.

The basis for the applications described under a) to h) form:

• The unusually broad and unstructured absorption and emission spectra of the known NIR fluorophores of the compounds described in claim 1 in solvents and solid matrices of different polarity (also in matrices of relatively low polarity such as in polystyrene (PS) or polymethyl methacrylate (PMMA) )
• The low polarity sensitivity of the optical properties of the dye, in particular with regard to the shape and spectral position of the emission band,
• - The relatively large Stokes shift for a NIR fluorophore and
• The comparatively high fluorescence quantum yield in polar solvents such as ethanol, in solvent mixtures of an organic solvent and water and in solid matrices,
• - the formation of emissive aggregates as well
• The comparatively high stability of the described NIR fluorophores in air-saturated solution and in solid matrices.

Particular advantages of the use according to claim 1, of the method according to claim 11 and the particles result overall from the typical for the proposed class of compounds, however, for a conventional NIR fluorophore relatively unusual spectral properties. The clear separation of the maximum of the absorption band from the maximum of the emission band and the only slight spectral overlap - overall the large Stokes shift - allow the excitation of the dye and of particles loaded with this dye with various conventional laser diodes (eg 570 nm, 633 and 635 nm, 670 nm), which are routinely used as excitation light sources in many measuring instruments in medical diagnostics and bioanalytics.

The large Stokes shift allows the easy separation of excitation light and emission light and, together with the relatively high fluorescence quantum yield for an NIR chromophore, enables a low detection limit.

The achievable as described above homogeneous loading z. B. of polystyrene (PS) particles of different surface functionalization allows the production of NIR fluorescent particles of various size and surface chemistry, which differ in their absorption and in particular their emission properties only comparatively little from the chromophore in free solution. Depending on the loading density, the fluorescent particles loaded with the dye presented here show z. Example, at an excitation wavelength of 633 nm, a 5- to 10-fold increased brilliance compared to commercial NIR systems. An example of a typical commercial NIR system is the dye Alexa 750. It even shows a 30- to 60-fold increased brilliance at an excitation wavelength of 543 nm.

Uses according to claim 1 and 11 or particles proved to be suitable in leaking experiments under application-relevant conditions and in cytotoxicity studies in vitro (cell culture - 3T3 mouse fibroblasts) and in vivo (mouse model) and showed application-relevant doses or particle concentrations excellent colloidal and photostability ,

Thus, loaded with the proposed dye polystyrene particles (PS) particles (with different surface chemistry e.g. unfunctionalized particles, PS-COOH, PS-NH _2;. PEGylated systems PEGylated and a biomarker-specific antibodies such as trastuzumab (Herceptin ^®) provided systems) under in vitro and in vivo conditions for evaluating and assessing the particular molecular situation.

In addition, these particles and all other objects loaded or doped with this chromophore or similar chromophore systems can be used to determine the emission correction for the spectral range from about 650 nm to approx. 950 nm and to determine the excitation correction for the spectral range from about 450 nm to about 800 nm. Further advantages result from the fact that the compounds used in claim 1 and 11 and the respective subclaims as quantum yield standards and as internal standards z. B. can be used for sensor systems.

Particularly advantageous is the applicability of the representatives of the proposed family of NIR fluorescence dyes for ratiometric measurements (ratiometric sensing) with particles that are loaded inside with this dye as a reference and carry on their outer side another analyte-sensitive chromophore or together z. B. form a FRET pair.

Particular advantages of the compounds described above and their applications thus result from named applications from the suitability of these dyes alone as a molecular optical probe and / or in combination with other dyes in complex sensor systems.

Another advantage is the suitability of the described dyes for simultaneous use as NIR emission standard and NIR intensity standard in aqueous medium or in living cells.

• – den hochsensitiven Nachweis von Analyten wie von krankheitsrelevanten Biomarkern in der klinischen Anwendung zu diagnostischen Zwecken in Körperflüssigkeiten wie beispielsweise in Blut, Urin, Liquor, in Aszites oder Pleuraerguß, in Biopsaten und Knochenmark oder Abstrichen von Zellen (Zervix, Blutausstrichen);
• – den hochsensitiven Nachweis von Zellen im Blut beispielsweise Stammzellen ex-vivo, in der klinischen Anwendung beispielsweise zum Nachweis des Anteils von Zellanteilen im Blut von Patienten;
• – den hochsensitiven Nachweis von beispielsweise Tumorzellen in vivo, in der klinischen Anwendung beispielsweise in Verbindung mit endoskopischen Systemen oder während chirurgischer Eingriffe, beispielsweise in der Onkologie zur frühen Diagnose und zum Nachweis der Ausbreitung der Tumorerkrankung im Organismus.

Further advantages of the compounds described above and their special spectral characteristics arise for clinical applications, for example for:

• - the highly sensitive detection of analytes such as disease-relevant biomarkers in clinical use for diagnostic purposes in body fluids such as in blood, urine, cerebrospinal fluid, in ascites or pleural effusion, in biopsies and bone marrow or smears of cells (cervix, blood smears);
• - The highly sensitive detection of cells in the blood, for example, stem cells ex-vivo, in clinical use, for example, to detect the proportion of cell portions in the blood of patients;
• - The highly sensitive detection of, for example, tumor cells in vivo, in clinical use, for example, in connection with endoscopic systems or during surgical procedures, for example in oncology for early diagnosis and to detect the spread of the tumor disease in the organism.

• – den hochsensitiven Nachweis der Expression und der Lokalisation von Analyten wie von krankheitsassoziierten Biomarkern in Zellen in Abhängigkeit bestimmter Versuchsbedingungen und über die Zeit in Zell-basierten Assays;
• – den hochsensitiven Nachweis und die Verfolgung von NIR-fluoreszenzmarkierten Zellen wie beispielsweise Makrophagen in Zellsystemen in Abhängigkeit von der Zeit.

Typical advantages for the use of the above-described compounds according to claim 1 also arise for applications in cell-based systems, for example for:

• The highly sensitive detection of the expression and localization of analytes such as disease-associated biomarkers in cells as a function of specific experimental conditions and over time in cell-based assays;
• The highly sensitive detection and tracking of NIR-fluorescently labeled cells such as macrophages in cell systems as a function of time.

• – den hochsensitiven Nachweis von Analyten wie von krankheitsrelevanten Biomarkern in der präklinischen Forschung in in vivo Modellen über die Zeit, beispielsweise in verschiedenen krankheitsrelevanten Tiermodellen, wie beispielsweise in der Onkologie in Tumor-Tiermodellen;
• – die hochsensitive Darstellung von mit NIR-Fluoreszenzfarbstoffen beladenen Zellen, beispielsweise von Stammzellen oder Makrophagen (ex vivo beladen) in Tiermodellen zur Darstellung der Wanderung und Lokalisationen dieser Zellen in vivo und nicht-invasiv über die Zeit;
• – die Entwicklung von Strategien zum Therapie-Monitoring und zur Bestimmung der Therapieeffizienz in der präklinischen Evaluation in krankheitsrelevanten Tiermodellen.

Typical advantages for the use of the compounds described above according to claim 1 also result for preclinical applications, for example for:

• The highly sensitive detection of analytes as well as of disease-relevant biomarkers in preclinical research in in vivo models over time, for example in various disease-relevant animal models, such as in oncology in tumor animal models;
• The highly sensitive imaging of NIR fluorescent dye-loaded cells, for example, stem cells or macrophages (ex vivo loaded) in animal models to represent the migration and localization of these cells in vivo and non-invasively over time;
• - the development of therapy monitoring strategies and the determination of therapeutic efficacy in preclinical evaluation in disease-relevant animal models.

The applications described above require NIR emissive probes and sensor molecules that are as highly fluorescent as possible, that specifically recognize their target, that are multiplexable for the parallel detection of various analytes that are easily stimulated and detected with existing device platforms that exhibit low cytotoxicity Therefore, they are suitable for in vivo applications and are suitable for the implementation of concepts for traceable calibration. As stated, these requirements are met by the proposed compounds.

The present invention has been explained with reference to exemplary embodiments. These embodiments should by no means be construed as limiting the present invention. The following claims are a first, non-binding attempt to broadly define the invention.

References:

• [1] Peng, X .; Song, F .; Lu, E .; Wang, Y .; Zhou, W .; Fan, J .; Gao, Y., J. Am. Chem. Soc. 2005, 127, 4170-4171.
• [2] Yu, Y. H .; Descalzo, A.B .; Shen, l .; Tube, H .; Liu, Q .; Wang, Y. W .; Game, M .; Li, Y. Z .; Rurack, K .; You, X. Z., Chemistry-an Asian Journal 2006, 1, 176-187.
• [3] Lammers T, Ulbrich K, Adv Drug Deliv Rev. 2010 Feb 17; 62 (2): 119-21. (HPMA copolymer: 30 years of advances.)
• [4] Song X, Zhao X, Zhou V, Li S, Ma Q., Curr Drug Metab. 2010 Dec; 11 (10): 859-69. (Pharmacokinetics and disposition of various drug loaded biodegradable poly (lactide-co-glycolide) (PLGA) nanoparticles).
• [5] Steinhauser, L, Spankuch, B., Strebhardt, K. & Langer, K., Biomaterials 27, 4975-4983 (2006) (Trastuzumab-modified nanoparticles: optimization of preparation and uptake in cancer cells).
• [6] Calderon, M .; Quadir, M.A .; Strumia, M .; Haag, R., Biochimie 2010, 92, 1242-1251.
• [7] Xu, S.J .; Luo, V .; Graeser, R .; Warnecke, A .; Kratz, F .; Hauff, P .; Licha, K .; Haag, R. Bioorganic & Medicinal Chemistry Letters 2009, 19, 1030-1034; Christie, R. J .; Anderson, D.J .; Grainger, D.W., Bioconjugate Chemistry 2010, 21, 1779-1787.
• [8] Christie, R. J .; Anderson, D.J .; Grainger, D.W., Bioconjugate Chemistry 2010, 21, 1779-1787; White Leather, R .; Tung, C.H .; Mahmood, U .; Bogdanov, A., Nature Biotechnology 1999, 17, 375-378.
• [9] Burakowska, E .; Zimmerman, S. C .; Haag, R., Small 2009, S, 2199-2204.
• [10] Steinhilber, D .; Sisson, A.I .; Mangoldt, D .; Welker, P .; Licha, K .; Haag, R. Advanced Functional Materials 2010, 20, 4133-4138.
• [11] Grabolle, M .; Kapusta, P .; Nann, T .; Shu, X .; Ziegler, J .; Resch-Genger, U. Analytical Chemistry 2009, 81, 7807-7813.
• [12] Resch-Genger, U .; Grabolle, M .; Cavaliere-Jaricot, S .; Nitschke, R .; Nann, T., Nature Methods 2008, 5, 763-775.
• [13] EP2145925 ; DE 10 2008 040 513 ,
• [14] Heek, T .; Fasting, C .; Rest, C .; Zhang, X .; Wurthner, F .; Haag, R., Chemical Communications 2010, 46, 1884-1886.
• [15] Emmerling, F .; Orgzall, l .; Dietzel, B .; Schulz, B.W .; Reck, G .; Schulz, B., Journal of Molecular Structure 2007, 832, 124-131.
• [16] Renikuntla, B.R .; Rose, H. C .; Eldo, J .; Wagoner, A. S .; Armitage, B.A. Organic Letters 2004, 6, 909-912.
• [17] Rurack, K .; Spieles, M., Analytical Chemistry 2011, 83, 1232-1242.
• [18] Cooper et al, patent US 7,767,829 B2 , Aug. 2, 2010.
• [19] Schwartz, A .; Gaigalas, A.K .; Wang, L .; Marti, G. E .; Vogt, R. F .; Fernandez-Repollet, E., Cytometry 2004, 578, 1-6.
• [20] Marti, G. E .; Vogt, R. F .; Gaigalas, A.K .; Hixson, C.S. Hoffman, R. A .; Lenkei, R .; Magruder, L.E .; Purvis, N.B .; Schwartz, A .; Shapira, H.M .; A., W. NCCLS, I / LA24-A 2004, 24.

Claims (19)

Use of a compound according to the general formula (I)

where B and B 'are independently selected from methyl, ethyl, phenyl, a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical, a 5,6-R' radical Benzothiazole-2 radical and a 5,6-R'-indole-2 radical; X, X 'and X''are independently selected from methyl, ethyl, phenyl, a propanesulfonic acid radical and a butanesulfonic acid radical; D is selected from hydrogen, SO ₃ ^- , COO ^- , methyl, ethyl, phenyl and trifluoromethyl; Z is nitrogen; R is carbon or hydrogen; K is H, Cl, Br,

is; L is selected from thiol, amine, azide, aldehyde, carboxyl, ethylene and maleimide; n is 0 or a natural number; with the proviso that n is 0 when R is hydrogen; m is 0 or a natural number between 1 and 12; R 'is selected from H, SO ₃ ^- , COO ^- , a dendron according to the following structure

or its subunits, a lower generation dendron or subunits of a lower generation dendron or polyethylene glycol (PEG) or 5- (trifluoromethyl) or 6- (trifluoromethyl); and the anion A ^{- is} selected from ClO ₄ ^- , Cl ^- , Br ^- , I ^- , BF ₄ ^- , a sulfate, and a tosylate anion, as markers and / or contrast agents in methods for imaging and / or in biomedical analysis, wherein a fluorescence signal of the compound is detected in the NIR, wherein a fluorescence signal of the compound excites another organic compound to fluoresce and wherein an emission signal of the other organic compound is optically detected and / or analyzed. Use according to claim 1, wherein B and B 'are different from each other and in particular are selected from a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical and a 5,6-R '-Benzothiazole-2 remainder. Use according to claim 1, wherein B and B 'are the same and in particular are selected from a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical or a 5,6-R' -Indol-2 rest. Use according to any one of the preceding claims wherein X, X 'and X "are each independently methyl, ethyl, a propanesulfonic acid residue or a butanesulfonic acid residue. Use according to any one of claims 2 to 4, wherein R 'is 5- (trifluoromethyl) or 6- (trifluoromethyl), the compound of general formula (I) is used as a molecular marker, and magnetic resonance imaging (MRI) or tomographic imaging Includes method. Use according to any of the preceding claims, wherein D is trifluoromethyl. Use according to any of the preceding claims, wherein K is Cl, Br,

is; L is thiol, amine, carboxyl, or maleimide; R 'is selected from polyethylene glycol (PEG), 5- (trifluoromethyl) or 6- (trifluoromethyl), and A ^- is ClO ₄ ^- . Use of a compound according to any one of the preceding claims, wherein the compound is bonded to at least one surface of a particle and wherein the particle has a diameter in the range of 2 nm to 500 μm. Use of a compound according to claim 8, wherein the particle has a size below 100 nm at least in a spatial direction of extension. Use of a compound according to one of the preceding claims, wherein different energy spectra and / or spectral regions are detected synchronously and image information derived therefrom is used for multimodal imaging. A spectroscopic and / or imaging method comprising the steps of: providing a compound according to the general formula (II):

in which B and B 'are independently selected from H, methyl, ethyl, phenyl, or a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical, a 5,6-R 'Benzothiazole-2 radical or a 5,6-R'-indole-2 radical; wherein R 'is selected from H, SO ₃ ^- , COO ^- , a dendrimer, PEG, 5- (trifluoromethyl) and 6- (trifluoromethyl); X and X 'and X''are independently selected from methyl, ethyl, phenyl, an acid radical of propanesulfonic acid and an acid radical of butanesulfonic acid; D is selected from H, SO ₃ ^- , COO ^- , methyl, ethyl, phenyl and trifluoromethyl; Z is nitrogen; K is equal to Cl, Br,

is; L is selected from thiol, amine, azide, aldehyde, carboxyl, ethylene and maleimide; m is a natural number between 1 and 12; R is hydrogen and n = 0 or R is carbon and n = 1, where the compound has at least one trifluoromethyl group and the anion A ^{- is} selected from ClO ₄ ^- , Cl ^- , Br ^- , I ^- , BF ₄ ^- , a sulfate, and a tosylate anion; Providing a suspension of particles labeled with said compound selected from polymer nanoparticles, macromolecules, dendrons, dendrites, core-shell nanoparticles, layer-by-layer polyelectrolyte systems, micelles, vesicles, liposomes, atomic clusters, iron oxide particles, polymer microparticles; - contacting the provided suspension with sample material; - exposing the sample material to electromagnetic radiation; - detecting and / or measuring an electromagnetic spectrum of the compound in the NIR and - measuring on particles in which the compound of formula (II) forms a FRET pair with another chromophore. A process according to claim 11, wherein in the step of providing B and B 'are independently selected from a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical, a 5,6- R'-benzothiazole-2 radical or a 5,6-R'-indole-2 radical; wherein each R 'is selected from H, SO ₃ ^-, COO ^-, dendrimers, polyethylene glycol, 5- (trifluoromethyl) and 6- (trifluoromethyl); X and X 'are independently selected from methyl, ethyl, phenyl, an acid residue of propanesulfonic acid and an acid residue of butanesulfonic acid; D is selected from SO ₃ ^- , COO ^- , methyl, ethyl, phenyl and trifluoromethyl; Z is nitrogen; R is hydrogen and n = 0 or R is carbon and n = 1, wherein the compound carries at least one trifluoromethyl group. A process according to claim 11, wherein in the step of providing B and B 'are independently selected from a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical, a 5,6- R'-benzothiazole-2 radical or a 5,6-R'-indole-2 radical; wherein R 'is each selected from 5- (trifluoromethyl) and 6- (trifluoromethyl); X and X 'are independently selected from methyl, ethyl, phenyl, an acid residue of propanesulfonic acid and an acid residue of butanesulfonic acid; D is selected from SO ₃ ^- , COO ^- , methyl, ethyl, phenyl and trifluoromethyl; Z is nitrogen; R is hydrogen and n = 0 or R is carbon and n = 1, wherein the compound carries at least one trifluoromethyl group. A process according to claim 11, wherein in the step of providing B and B 'are independently selected from a 5,6-R'-benzoxazole-2 radical, a 5,6-R'-benzimidazole-2 radical, a 5,6- R'-benzothiazole-2 radical or a 5,6-R'-indole-2 radical; wherein R 'is each selected from 5- (trifluoromethyl) and 6- (trifluoromethyl); X and X 'are independently selected from methyl, ethyl, phenyl, an acid residue of propanesulfonic acid and an acid residue of butanesulfonic acid; D is selected from -SO ₃ H and trifluoromethyl; Z is nitrogen; R is hydrogen and n = 0 or R is carbon and n = 1, wherein the compound carries at least one trifluoromethyl group. A method according to any one of claims 11 to 14, wherein the step of detecting and / or measuring a fluorescence emission signal is detected with a maximum in the wavelength range between 750 nm and 850 nm. A method according to any one of claims 11 to 15, further comprising - Detecting and / or measuring an element-specific energy spectrum, wherein the element is selected from sulfur, nitrogen and / or fluorine. The method of any of claims 11 to 16, further comprising Detecting and / or measuring a fluorescence quantum yield and / or a fluorescence decay behavior and / or a fluorescence lifetime of the compound in the NIR. Method according to one of claims 11 to 17, further comprising at least one of the steps: Ratiometric measurement on particles which are internally charged with the compound of the formula (II) and which carry on their surface another analyte-sensitive chromophore, or - Measuring fluorescence quenching. Method according to claim 11 to 18, wherein the electromagnetic radiation is selected from radiation in the UV spectral range, in the UV / vis spectral range, in the vis spectral range, in the NIR spectral range, in the X-ray range, and / or gamma radiation.

Images