PL219326B1 - Fluorescent marker for collagen-dye - Google Patents

Description

Description of the invention

The subject of the invention is a new fluorescent marker for staining collagen suitable for use in scientific research and medical diagnostics.

Collagens are components of the extracellular matrix [Aumailley and Gayraud, J. Mol. Med. 76 (3-4): 253-65,1998] and reconstituted tissue substitutes. Collagens influence the behavior of cells in tissues - adhesion, migration, proliferation and metabolism, and play a role in maintaining the mechanical resistance of tissues [Friedl et al. Microsc. Res. Tech. 43 (5): 369-78,1998; Friedl et al., Int. J. Dev. Biol. 48 (5-6): 441-9,2004].

Mutations in the genes encoding collagens of various types lead to disturbances in synthesis, post-translational modifications, polymerization and assembly of fibrils into fibers, and the structure of the fiber network. These disorders result in hereditary diseases such as neonatal pemphigus, Epidermolysis bullosa, Alport syndrome, Ehlers-Danlos syndrome and others [Baxter Cardiovasc. Pathol., 14 (4): 185-8, 2005]. During the aging process, collagen is denatured, which leads to the loss of the mechanical properties of the tissues.

Fluorescent staining of collagens is aimed at obtaining selective and specific (i.e. derived exclusively or almost exclusively from collagen or collagens) fluorescence of collagen or collagen-containing structures with the desired spectral and biophysical properties, including the desired color and intensity and stability during measurements ( i.e. low loss of luminescence intensity during irradiation and observation), both in living and fixed tissues, to create three-dimensional images of collagen-containing structures with high spatial resolution (fractions of a micrometer) and its changes or to measure, in living or fixed tissues and for examination by microscopic and cytometric methods such as fluorescence microscopy, confocal microscopy (CLSM), multiphoton microscopy (MP), fluorescence recovery after photo-fading (FRAP), fluorescence loss on photobleaching (FLIP), fluorescence resonance energy transfer (FRET) fluorescence spectroscope ia correlation (FCS), modulated illumination microscopy, laser scanning cytometry (LSC), flow cytometry and the like.

The microscopic identification of collagen fibers is used in studies of the extracellular matrix and reconstituted tissue substitutes.

Methods of observing collagen in gels are known [Brightman et al., Biopolymers. 54 (3): 222-34,2000; Wu et al. Microsc. Microanal. 9 (6): 574-80,2003; Wu et al., J. Microsc. 210 (Pt 2): 158-65, 2003], histochemical and immunofluorescence staining in fixed tissues and collagen contrasting in preparations for electron microscopy, however, there is no specific method of visualization and study of the three-dimensional structure of the collagen fiber network in living tissues.

Known methods of in situ testing of collagens are presented in the table below.

Method Tissues fix- ne Tissues alive Benefits Restrictions Equipment needed 1 2 3 4 5 6 Picrosyrius Red staining and silver staining ining ") Yes No Relatively specific Tissue fixation changes the structure of the collagen fiber network, only two-dimensional images are obtained from the selected tissue section, not suitable for the simultaneous study of other structures and functions, not suitable for the study of time relationships, laborious procedure Histochemical laboratory. Optical microscope Contrasting to electron microscopy ("freeze substitution") Yes No Relatively specific Tissue fixation changes the structure of the collagen fiber network, only two-dimensional images are obtained from the selected tissue section, not suitable for the simultaneous study of other structures and functions, not suitable for the study of time relationships, laborious procedure Microtome. electron microscope

In the table below

1 2 3 4 5 6 Reflected or diffused light No Only layouts model- in No specificity No specificity, only useful for in vitro testing of collagen gels; limited suitability for the study of living cells and tissues; in reflected light confocal microscopy strong interference and hot spot effects that must be removed by Fourier analysis methods Optical microscope with a dark field attachment; confocaine microscope with a reflection light detection system + special procedures for removing noise and interference stripes in the frequency domain in 4D space Autofluoresce naction Yes Yes No specificity Lack of specificity (other components of cells and extracellular matrix show similar autofluorescence) Fluorescence or confocal microscope SHG (Second Harmonies Generation) No Yes It allows, to a limited extent, to reconstruct oat the three-dimensional structure of the fiber network collagen, study other structures and functions of cells, study time dependencies Not very specific; other structures also give the signal of the second harmonic; cell photodamage, tissue heating during observation; requires expensive equipment (multi-photon microscope) and a special detector Multiphoton microscope, equipped with a special detector

The aim of the invention is to make it possible to observe collagens in vivo in intact tissues.

A particular object of the invention is to provide a fluorescent dye capable of staining collagen.

The invention relates to the use of a compound of formula:

ie, eserolin-5'-N-cadaverine fluorescein carbamate or derivatives thereof, for the preparation of a fluorescent dye for staining collagen.

By derivatives, related or similar compounds of the fluorescent marker used are meant chemical compounds that have a related chemical structure, that is, they consist of a fluorophore (contain a chemical moiety that is immediately or after modification to emit luminescence) and the molecular structure attached to it, which together forms a molecule with a mass not exceeding 3000Da, showing affinity for one or more types of natural collagen, or for artificial collagens, in monomeric or polymerized form, in a biological, material or other sample.

PL 219 326 B1

Preferred eserolin-5'-N-cadaverine fluoresceincarbamate derivatives which can be used according to the invention are the compounds of formula I:

where R can be: H, -OH, -CH3.

Equally preferably, a derivative used according to the invention may be a compound selected from the group consisting of:

Eserolin-5'-N-methyl-cadaverine fluorescein carbamate:

Eserolin-5'-N-cadaverine methylfluoresceincarbamate and

Eserolin-5'-N-methanol-cadaverine fluorescein carbamate.

PL 219 326 B1

These derivatives can be obtained by known methods.

Another object of the invention is a fluorescent dye for staining collagen, containing a fluorescent label dissolved in a suitable solution, characterized in that it comprises eserolin-5'-N-cadaverine fluoresceincarbamate or a derivative thereof as the fluorescent label.

For the purposes of this description, a "fluorescent label" is understood to be a chemical compound having an affinity for a specific substance, in this case collagens, and fluorescent properties.

The applied fluorescent marker is a chemical compound composed of previously known elements (physostigmine, fluorescein, hydrocarbon chain). These molecules, not particularly linked to a single molecule, however have no affinity for collagen and are not suitable for dyeing monomers, fibrils or collagen fibers. These molecules, combined in one complex, show a strong affinity for collagen. The reaction (formation of chemical bonds between the fluorescein-x-physostigmine molecule and the collagen molecules) takes place within a few seconds. The tracer particles collected on the surface of collagen fibers emit green fluorescence when illuminated with radiation in the range of 300-500 nm, e.g. blue light, which enables easy detection of the location of collagen fibers in a tissue or other sample containing collagen.

On the other hand, the term "fluorescent dye" should be understood as a reagent suitable for the direct staining of the test preparation, usually in the form of a buffered aqueous solution, containing a fluorescent marker and appropriate additives, for example aqueous buffer solutions such as phosphate, carbonate or citrate buffers, suitable for use with stained substances. preparations and ensuring adequate stability of the reagent.

The label according to the invention can be used in the method of staining collagen for observation by optical methods using a fluorophore, in which the dye is administered to a laboratory animal, or samples of living tissue or a biomaterial sample are taken, the test sample containing collagen is contacted with the fluorescent marker according to the invention, thus prepared the sample is left for a period from a few seconds to several tens of minutes in order to generate, by non-covalent junctions, a chemical complex of a fluorescent marker-collagen, then the test sample is illuminated with light with a wavelength of 300 nm to 500 nm and observed or examined by known optical methods , microscopic or other using fluorescence, whereby, if necessary, the test sample may be fixed with formaldehyde or other fixing agents prior to observation.

Polymerized collagen forms fibers in animal tissues that are responsible for the mechanical integrity of the tissues and play a role in embryonic development, wound healing, aging and other important biological processes. A variety of acquired or genetic diseases are known to have causes or effects related to collagens. Possibility of detection and observation of collagen fibers with fluorescent methods, e.g. by fluorescence and confocal microscopy, it can be used in scientific research and medical diagnostics. The invention allows the observation of collagens in vivo in intact or fixed tissues or materials containing collagen. The non-covalent bonds of the fluorescein-x-physostigmine-collagen chemical complex prevent loss of fluorescence in stained collagen.

The attached figures allow the essence of the invention to be explained.

Figure 1 shows the affinity of the fluorescent tag for collagen fibers. Single in vitro polymerized collagen fibril - reflected light image (A) and fluorescence image, after staining with a fluorescent marker (B). The thickness of the confocal layer in the reflected light observation is smaller than in the fluorescence image, hence image A shows a shorter fragment of a slightly bent fiber than image B.

Figure 2 shows the specific binding of a fluorescent marker to collagen fibers in living tissues. Collagen fibers on the surface of mouse skeletal muscle (left) and in blood vessels (right). 160 x 160 μm field of view.

Figure 3 shows the structure of a collagen-containing biomaterial used in the treatment of burn wounds.

Figure 4 shows a scheme for the synthesis of a fluorescent marker that consists of a physostigmine-conjugate fluorescein.

Exemplary embodiments of the above-defined invention are presented below.

PL 219 326 B1

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Obtaining fluorescent markers.

The original method for synthesizing the fluorescein tag, conjugated with physostigmine, consisted of the five reactions shown in Figure 4. Modifications to the methods described by Yu et al. Were used to construct the sequence of reactions leading to the synthesis of the tag. (Yu et al., Heterocycles 1987, 26 (5): 1271-1275) and Atack et al (Atack et al., J. Pharmacol. Exp. Ther. 1989, 249 (1): 194-202). Carbamate synthesis by direct attachment of 5 - ((5-aminopentyl) thiourea) fluorescein to eserolin, using several carbamate formation methods, failed to obtain the desired structure.

Isocyanate (2) is prepared according to a modified procedure described by Nowick et al. (Nowick et al., J. Org. Chem. 1992, 57: 7364-7366; Nowick et al., J. Org. Chem. 1996, 61 : 3929-3934; Nowick et al., J. Org Chem. 1998, 63: 9144). A solution of mono-N-t-BOC-cadaverine (1) in dichloromethane is cooled to 0 ° C and saturated with calcium bicarbonate. Triphosgene is added and the two-phase system is mixed. 5-N- (t-Butyloxycarbonyl) -1-N'-isocyanatopentane (2) is formed.

The reaction of the isocyanate (2) with eseroline (3) is performed according to the method of Yu et al. (Yu et al., J. Med. Chem. 1999, 42: 1855-1861) and Atack et al. (Atack et al., J. Pharmacol. Exp. Ther. 1989, 249: 194-202). Esperoline fumarate is extracted into the diethyl ester and evaporated. N-t-BOC-cadaverine isocyanate (2) is added as an emulsion in a diethyleter. 5'-N-t-BOC-cadaverine-eseroline carbamate is formed (4).

Trifluoroacetic acid (TFA) is added to a solution of N-t-BOC-cadaverineeseroline carbamate (4) at 0 ° C to remove the protecting group (t-BOC). The trifluoroacetate salt of eserolin-5'-N-cadaverine carbamate is formed (5).

Compound (5) is dissolved in diisopropylethylamine (DEA), and then fluorescein 5-isothiocyanate (FITC) is added to the solution. The desired reaction product is formed - fluorescein thiourea adduct to eserolin-5'-N-cadaverine carbamate (6).

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Dyeing of collagen fibers

Dye storage

The fluorescent marker according to the invention, in the form of a powder, dissolved in DMSO to a concentration of mM and divided into 5 µl aliquots. Store Fluorescent Tracer Solution at -18 ° C. Protect from light.

Preparation of the staining solution

Thaw an aliquot of the fluorescent tracer solution immediately prior to staining. Mix using a shaker (Vortex). Protect the solution from light.

Preparation of tissues for staining

Leave the isolated piece of tissue at room temperature, do not cool it. Place the piece of tissue on a petri dish with a glass bottom 0.17 mm thick (e.g. Mattek or similar trays). Flood the tissue with a sufficient amount of air-buffered culture medium at 37 ° C (i.e., medium that maintains an acidity of pH 7.2-7.4 in equilibrium with air), without the addition of phenol red. The medium should contain 1% of serum.

Coloring

Add such a volume of the inventive fluorescent tracer stock solution to the medium to obtain a final concentration of 10 μΜ. Remove the normal medium from the Petri dish containing the tissue and replace it with the medium containing the fluorescent marker. Incubate for 5 to 60 minutes, depending on tissue type and thickness.

If it is necessary to simultaneously stain other structures in the test tissue, the necessary dyes can be added to the medium containing the fluorescent label or directly to the tissue stained with the fluorescent label.

Non-specific staining

Staining with a fluorescent label may lead to the appearance of a non-specific fluorescence signal. The reasons and how to minimize this problem are given below:

a. Nonspecific staining of damaged cells occurs when a fluorescent label accesses the interior of cells that have a damaged plasma membrane. This phenomenon occurs when staining damaged tissue or tissue that has been isolated many hours before staining. Under these circumstances, the outer membrane loses over time

The ability to separate the external and internal environment of the cell. The dye penetration of the fluorescent marker allows for the production of weak bonds with cell components and weak staining of internal structures. The entire interior of the cell then takes on a weak green color. The nature of this staining pattern is relatively easy to recognize and distinguish from stained collagen fibers. The problem can be avoided by staining undamaged tissues immediately after isolation.

b. Nonspecific staining of the nuclei of some cell types may occur as a result of penetration of free fluorescein through the functional cell membrane. Free fluorescein may be present in the fluorescent tracer solution - this is an impurity left over from dye synthesis. The concentration of free fluorescein depends on the efficiency of the purification process of the synthesized compound.

c. staining of structures containing collagen-like molecules. Some types of cells and tissues may have molecules that contain a subunit that is very similar to collagen. This group of molecules includes a form of acetylcholinesterase anchored in cell membranes. It has collagen chains in its structure, the so-called collagen Q. Another representative is lectins, which contain collagen molecules (collectins). The tissue concentration of these compounds is usually many times lower than that of collagen, so the fluorescence signal from molecules containing collagen-like polypeptide chains is below the detection limit.

d. Acetylcholinesterase staining by binding a fluorescent label to the active site of the enzyme. The component of the fluorescent marker molecule is physostigmine - a molecule showing affinity to the active site of cholinesterases (acetyl- and butyrylcholinesterase). It was established that the binding between the fluorescent marker and the isolated cholinesterases takes place, however, it is weak and unstable. The affinity of the fluorescent marker for cholinesterases does not lead to clear staining of the cholinergic nerves.

Fading and dynamic dye exchange (insulated fibers, intact tissue)

The light exciting the fluorescence of the fluorescent marker leads to damage of the fluorophore and loss of the ability to emit fluorescence. The effect of fading is to reduce the fluorescence intensity of the fluorescent marker in the test area of the sample. The decrease in fluorescence intensity, however, is much slower than it would appear from the fading rate of the fluorophore of the fluorescent tracer used. The reason for the unexpected signal stability is the dynamic exchange between the pool of fluorescent marker associated with collagen fibers and the pool present in solution. This leads to a constant reproduction of the population of undyed fluorescent marker molecules associated with the collagen fibers. This was demonstrated by the FRAP method.

Collision blanking

When the concentration of the fluorescent marker is used in excess of 10 μ takiej, the amount of dye particles may accumulate on the surface of collagen fibers that the collision quenching phenomenon occurs. This leads to a decrease in the intensity of the fluorescence signal, despite the increase in the concentration of the dye in the sample. To obtain optimal staining conditions, titration is required. In most cases, the optimal concentration of the fluorescent marker in the solution is approx. 10 µΜ.

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Simultaneous staining of collagen fibers, cell nuclei and active mitochondria

Add the TMRE dye to the medium containing the fluorescent label. Final TMRE concentration should be 1 µΜ. Pour the tissue with the prepared solution. Then add the Draq5 dye taken directly from the solution provided by the manufacturer (Biostatus, UK). The final concentration of Draq5 should be 1 µΜ. Incubate the tissue with the three dye solution for 5-60 minutes.

Recording of microscopic images

Fluorescence images of collagen fibers stained with a fluorescence marker can be viewed, recorded and examined using a standard fluorescence microscope (the so-called wide field) or a confocal microscope, equipped with a blue light source and typical optical filters for the detection of fluorescein or other research device using fluorescence and one or more with several methods, such as MP, FRAP, FLIP, FRET, FCS, 4Pi, STED, microscopy using modulated illumination.

PL 219 326 B1

Measurement conditions for sample stained with fluorescent marker, Draq5 and TMRE, Bio-Rad MRC1024 microscope. Fluorescence excitation wavelength - 488 nm,

568 nm, KrAr laser, T1 first dichroic mirror, T2A second dichroic mirror, emission filters

540/30, 580LP, 630LP.

Fixation of colored preparations

Slides stained with a fluorescent marker can be fixed with formaldehyde and remain stable for at least a few days, but the image quality is inferior to that of slides containing viable tissues.

In the case of the staining described in the example above, staining with the fluorescent marker and Draq5 remains visible after fixation of the tissue with formaldehyde, while the staining of the mitochondria disappears once the mitochondrial potential is lost.

Claims (2)

1. Use of a compound of formula: where R is: H, -OH or -CH3, for the production of a fluorescent dye for staining collagen, the compound used being especially selected from the group consisting of: eserolin-5'-N-cadaverine fluoresceincarbamate, eserolin-5'-N-methylcadaverine fluoresceincarbamate , eserolin-5'-N-cadaverine methylfluoresceincarbamate and eserolin-5'-N-methanolcadaverine fluoresceincarbamate. 2. A fluorescent dye for staining collagen comprising a fluorescent label dissolved in a suitable solution, characterized in that the fluorescent label is a compound according to claim 1 or 2. 1.