Modified cationic peptides for visualization of sulfated glycocalyx components on cells

Abstract

FIELD: bioorganic and biomedical chemistry; biochemistry; cell biology.SUBSTANCE: concerns modified cationic peptides that are used as probes for visualizing sulfated glycocalyx components on the surface of cells, primarily vascular endothelial cells. A modified peptide of the formula R-NH-(CH2)5-CO-Lys-(D-Lys)n-Arg- was proposed.βAla-βAla-NH2, where R = FITC, n=2 for compound (I), R = FITC, n=3 for compound (II), R = Bt, n=2 for compound (III) or R = Bt, n=3 for compound (IV), where FITC is fluorescein isothiocyanate and Bt is biotin, and the use of the peptide for visualization of endothelial glycocalyx.EFFECT: obtaining modified peptides that possess the proteolytic stability necessary for carrying out diagnostic procedures in vivo and the ability to interact with sulfated components of the carbohydrate portion of the glycocalyx on cell membranes.2 cl, 7 dwg, 1 tbl, 7 ex

Description

[0001] The invention relates to the field of bioorganic and biomedical chemistry, biochemistry, and cell biology and concerns new modified cationic peptides, their production and use for visualization of sulfated glycocalyx components on the surface of cells, primarily vascular endothelial cells.

[0002] The endothelial glycocalyx is a carbohydrate-rich layer lining the vascular endothelium, the main components of which are proteoglycans, glycoproteins, and glycosaminoglycans. The most important molecules in the glycocalyx are proteoglycans—complex, highly glycosylated proteins in which the carbohydrate moiety accounts for 90-95% of the total mass. Proteoglycans consist of a core protein covalently linked to one or more glycosaminoglycan chains, the main ones being heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronan (hyaluronic acid). These are linear polymers of disaccharides of variable length, modified by sulfation and / or acetylation to varying degrees.

[0003] The glycocalyx layer on the surface of the vascular endothelium plays a vital role in its numerous physiological functions, including regulation of vascular tone and lumen, hemostasis and cellular immune responses, maintenance of the barrier properties of the vascular wall, and angiogenesis. Glycocalyx damage during ischemia-reperfusion, chronic hypoxia, oxidative and metabolic stress, generalized infection, and aberrant hemodynamics leads to endothelial dysfunction and a wide range of pathophysiological changes in organs and tissues that are entirely dependent on the vascular system. The main diseases of modern humans—cardiovascular diseases and type 2 diabetes—are characterized by endothelial dysfunction, which, among other things, is caused by glycocalyx disruption. According to modern concepts, restoration of the endothelial glycocalyx is important for the correction of endothelial dysfunction.Therefore, the ability to non-invasively assess the state of the glycocalyx in human blood vessels during life is becoming a priority in medical diagnostics. Clinical studies indicate that the thickness of the glycocalyx in microvessels correlates with the severity of pathological processes, particularly septic conditions.

[0004] The main and actually the only clinically proven method for analyzing the glycocalyx of the vascular wall is the optical method - sidestream dark field imaging (SDF) and its variations - orthogonal polarization spectral imaging (OPS) and small-angle dark field imaging (IDF).

[0005] (Banerjee S., Mwangi JG, Stanley T.K., Mitra R., Ebong EE Regeneration and assessment of the endothelial glycocalyx to address cardiovascular disease. Ind. Eng. Chem. Res. 2021; 60, 17328-17347)

[0006] All of these methods visualize the glycocalyx as a transparent zone between the vessel wall and the red blood cells passing through it. They do not directly detect glycocalyx components and can only assess its presence and development in areas accessible to a camera, such as the sublingual region, retina, and nail beds. These methods are unable to visualize the glycocalyx in other vessels where atherosclerotic lesions are localized (aorta, femoral arteries), or those affected by diabetes (kidneys, lower extremities), arterial hypertension, and other pathologies.

[0007] At the same time, high-resolution methods (CT, MRI, fluorescence scanners) exist and are being successfully developed in the world, allowing for non-invasive visualization of structures in the human body in native or additional contrast.

[0008] However, the application of these imaging technologies requires probes that interact with the glycocalyx in vivo under blood flow conditions, which can be conjugated to reporter molecules used to contrast objects using one or another non-invasive imaging technology in the body.

[0009] Methods for identifying vascular glycocalyx components in vivo in animal model experiments are known; researchers mainly use lectins, which are proteins that bind certain disaccharide fragments of glycosaminoglycan chains, or antibodies to heparan sulfate, syndecan-1, or hyaluronan.

[0010] (Mulivor AW, Lipowsky N.N. Inflammation- and ischemia-induced shedding of venular glycocalyx. Am. J. Physiol. Heart Circ. Physiol., 2004, 286: H1672-H168)

[0011] Using fluorescently labeled probes on the glycocalyx, advanced microscopic methods can be used for its visualization. For example, FITC-labeled lectins have been used to visualize the glycocalyx of brain endothelium in vivo in mice [8]. The glycoprotein used by the authors—FITC-labeled wheat germ agglutinin (FITC-WGA), with a molecular weight of approximately 36 kDa—specifically bound heparan sulfate on the surface of endothelial cells.

[0012] (Yoon, J.-H., Lee, E.-S., & Jeong, Y. In vivo Imaging of the Cerebral Endothelial Glycocalyx in Mice. Journal of Vascular Research, 2017, 54(2), 59-67. doi:10.1159 / 000457799)

[0013] However, the above-mentioned macromolecules have not yet reached the clinical trial level for glycocalyx imaging in humans. Furthermore, the use of high-molecular-weight compounds, including proteins, for in vivo glycocalyx detection is associated with the risk of immune-mediated complications.

[0014] The objective of the present invention is to synthesize modified cationic peptides that can be used as small-sized molecular probes for visualizing sulfated components of the cellular, primarily endothelial, glycocalyx.

[0015] Detection of glycocalyx components can be carried out either by a direct method using fluorescently labeled peptides or by a two-step method using the "biotin - fluorescently labeled streptavidin" system based on the high affinity between biotin (Bt) and streptavidin. (The dissociation constant of the biotin-streptavidin complex Kd ~ 10 -14 mol / L). In a two-step detection method, biotin is attached to the target molecule (peptide), and then a fluorescently labeled streptavidin conjugate is used as a second-step reagent to detect this molecule using fluorescence microscopy.

[0016] The relevance of this task stems from the need to expand medical diagnostic capabilities to detect endothelial dysfunction, a key determinant of the development of atherosclerosis, arterial hypertension, and diabetic vasculopathies. Visualization and assessment of the vascular glycocalyx using small molecular probes will enable more effective monitoring and personalized therapy for these and other diseases.

[0017] The technical result consists in obtaining modified peptides of the general formula:

[0018] R-NH-(CH2)5-CO-Lys-(D-Lys) n -Arg-βAla-βAla-NH2, where

[0019] R = FITC, n = 2 for compound (I)

[0020] R = FITC, n = 3 for compound (II)

[0021] R = Bt, n = 2 for compound (III)

[0022] R=Bt, n=3 for compound (IV),

[0023] possessing the proteolytic stability necessary for carrying out diagnostic procedures in vivo and the ability to interact with the sulfated components of the carbohydrate part of the glycocalyx of the vascular wall.

[0024] The target module of the probe, which interacts with the sulfate groups of the glycocalyx components, is represented by a block of cationic amino acid residues consisting of three or four lysine residues and an arginine residue.

[0025] To protect the claimed compounds from the action of exopeptidases (amino- and carboxypeptidases), the N-terminal amino acid residue in them was acylated with 6-aminohexanoic acid, and the C-terminal was amidated. To protect the peptides from the action of endopeptidases, beta-alanine and D-lysine residues were introduced into their sequence. At the same time, the introduction of bulky modifications from the N-terminus and C-terminus of the target sequence of the probe prevents the possibility of its effective penetration through the plasma membrane into cells, which is typical for peptides with clusters of positively charged amino acid residues at the ends of the molecule (TAT peptide [9], PIK peptide

[10] , MLC 11-19

[11] , PIC7

[12] ). Fluorescein isothiocyanate was chosen as a fluorescent label when using both the direct and two-step methods for detecting sulfated glycocalyx components. FITC was attached to the cationic part of the peptide via a linker, 6-aminohexanoic acid, to ensure greater conformational freedom of the label. FITC has an excitation maximum of about 492-495 nm and an emission maximum of about 517-520 nm. Biotin was introduced into the peptide molecules using the same linker. The claimed peptides can also be modified with other fluorophores with emission ranges suitable for diagnostics, in particular, cyanine dyes (Cy3, Cy5, etc.), which are characterized by a high quantum yield and fluorescence stability, which can significantly increase the sensitivity of the method.

[0026] When applied to non-invasive visualization of glycocalyx in blood vessels using scintigraphy, CT, MRI technologies, other reporter compounds such as radioisotope-containing compounds, iodine-containing compounds, and paramagnetic substances containing gadolinium can be used as the R modifier, respectively.

[0027] The proposed small biocompatible molecular probes have no obvious analogues. Their use significantly reduces the risk of adverse effects, particularly those affecting the immune system. Due to their peptide nature, they exhibit low nonspecific binding to other molecular targets. Furthermore, standard protocols for the chemical synthesis and analysis of compounds in this class have been developed. Due to the small size of the developed probes compared to macromolecular probes such as antibodies and lectins, their in vivo use may offer advantages in terms of reduced interference with the test subject.

[0028] The claimed modified peptides (I-IV) were obtained by solid-phase synthesis using Fmoc-technology (Fmoc-9-fluorenylmethoxycarbonyl). Purification of the technical products was performed using preparative HPLC (high-performance liquid chromatography). The structure of compounds (I-IV) was confirmed by mass spectrometry, and their purity was confirmed by analytical HPLC. The technology for preparing the claimed compounds is described in Examples 1-4.

[0029] The ability of the claimed compounds to interact with sulfated glycocalyx components in vitro and in vivo / ex vivo is demonstrated in Examples 5 and 6.

[0030] The ability of the claimed compounds to detect glycocalyx on endothelial cells in vitro is demonstrated in Example 7.

[0031] To study the interaction of fluorescently labeled peptides FITC-NH-(CH2)5-CO-Lys-(D-Lys) n-Arg-β-Ala-βAla-NH2 with carbohydrate components of the glycocalyx was analyzed using affinity chromatography on a column with immobilized heparin in a sodium chloride concentration gradient. Heparin belongs to the glycosaminoglycan family. The heparin molecule is based on a chain of repeating disaccharides—α-D-glucosamine and uronic acid (L-iduronic or D-glucuronic), linked by 1–4 glycosidic bonds. Most α-D-glucosamine residues are sulfated at the amino and hydroxyl groups. Due to the high content of negatively charged sulfate and carboxyl groups, the heparin molecule is a strong polyanion of natural origin. Heparin is capable of interacting with many protein and synthetic compounds of a polycationic nature, carrying a net positive charge.Since a significant proportion of the carbohydrate composition of the glycocalyx is heparan sulfate, which has a high analogy with heparin, the study of the interaction of peptide probes with heparin immobilized on a sorbent is an adequate model of their interaction with sulfated components of the endothelial glycocalyx.

[0032] The invention is explained by drawings

[0033] Fig. 1 - Structural formula of the probe (I), its analytical HPLC profile on a column with a reversed-phase sorbent, confirming the individuality (purity) of the compound and the mass spectrum obtained using the electrospray ionization (ESI) method in the positive ion registration mode, confirming its authenticity.

[0034] Fig. 2 - Structural formula of the probe (II), its analytical HPLC profile on a column with a reversed-phase sorbent, confirming the individuality (purity) of the compound and the mass spectrum obtained using the electrospray ionization (ESI) method in the positive ion registration mode, confirming the authenticity.

[0035] Fig. 3. - Structural formula of the biotinylated probe (III), its analytical HPLC profile on a column with a reversed-phase sorbent, confirming the individuality (purity) of the compound and the mass spectrum obtained using the electrospray ionization (ESI) method in the positive ion registration mode, confirming the authenticity.

[0036] Fig. 4 - Structural formula of the biotinylated probe (IV), its analytical HPLC profile on a reversed-phase column confirming the individuality (purity) of the compound and the mass spectrum obtained using the electrospray ionization (ESI) method in the positive ion registration mode confirming its authenticity.

[0037] Fig. 5 - Elution profile of fluorescently labeled peptides (a) - FITC-NH-(CH2)5-CO-Lys-D-Lys-D-Lys-Arg-βAla-βAla-NH2(I) and (b) - FITC-NH-(CH2)5-CO-Lys-D-Lys-D-Lys-D-Lys-Arg-βAla-βAla-NH2(II) on a column with immobilized heparin Affi Gel® Heparin Bio-Rad in a sodium chloride concentration gradient at a wavelength of 495 nm.

[0038] These profiles demonstrate that polycationic fluorescent probes (I) and (II) bind to immobilized heparin, and the resulting complexes are stable under physiological conditions at pH 7.4 in 0.15 M PBS. However, probe (I) exhibits lower retention on the sorbent with immobilized heparin in a sodium chloride concentration gradient than probe (II).

[0039] Fig. 6 - Ex vivo desorption of FITC-labeled peptide (I) from the luminal surface of rat thoracic aorta coated with developed glycocalyx after adsorption of peptide (I) to the aortic wall under in vivo blood flow conditions (direct detection method). The graph shows a more efficient desorption of peptide (I) from the aortic surface in the presence of glycocalyx-degrading enzymes heparinase III and hyaluronidase compared to the control (incubation in saline) and less residual dissociation of peptide (I) under these conditions in the presence of a high concentration of NaCl, sufficient to disrupt the electrostatic interactions of peptide (I) with the sulfated components of the glycocalyx.

[0040] Fig. 7 - Detection of sulfated glycocalyx components on the surface of fixed non-permeabilized human umbilical vein endothelial cells (HUVEC) grown in a flowing culture medium at a shear stress of 5 dynes / cm 2for 7 days, using biotinylated peptide (III) and FITC-streptavidin (two-step detection method).

[0041] These micrographs show that the peptide (III) exhibits punctate structures on the cell surface, sometimes forming dotted linear structures, and short filaments, which appear as thin fibers located mainly vertically to the cell surface in 3D reconstruction of fluorescence images.

[0042] The invention is described in seven examples

[0043] List of abbreviations:

[0044] AA - amino acid;

[0045] Boc - tert-butyl;

[0046] FITC - fluorescein isothiocyanate;

[0047] Fmoc - 9-fluorenylmethoxycarbonyl;

[0048] TBTU - N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium tetrafluoroborate

[0049] DCM - dichloromethane;

[0050] DMB - 1,3-Dimethoxybenzene;

[0051] DMF - N,N-dimethylformamide;

[0052] ESI +- ionization by spraying in an electric field;

[0053] NMM - N-methylmorpholine;

[0054] Pbf - 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl;

[0055] TFA - trifluoroacetic acid

[0056] DAPI - 4',6-diamidino-2-phenylindole

[0057] EGM - Endothelial growth medium, endothelial culture medium

[0058] HUVEC - Human umbilical vein endothelial cells

[0059] Example 1. Synthesis of FITC-NH-(CH2)5-CO-Lys-D-Lys-D-Lys-Arg-βAla-βAla-NH2- probe (I)

[0060] The following compounds were used in the work: amino acid derivatives and TBTU (NovaBiochem, Switzerland), NMM, HOBt, TIBS (Fluka, Switzerland), pyrrolidine (ReaChemLab, Russia), FITC (isomer I) from Sigma (USA). DMF, dichloromethane and TFA (Fluka, Switzerland) were used for synthesis, and acetonitrile (Panreac, Spain) was used for chromatography. Analytical HPLC was performed on a chromatograph (Gilson, France). HPLC conditions: Diaspher 110-C18 column (BioChemMac, Russia) (4.0x250 mm), sorbent particle size 5 μm. Mobile phase: buffer A - 0.1% aqueous TFA solution, buffer B - 80% acetonitrile in buffer A, elution rate 0.75 ml / min with a concentration gradient of buffer B in buffer A from 10 to 70% in 30 min, detection at 220 nm.

[0061] Preparative HPLC was performed on a Knauer instrument (Germany), Eurosphere-C18 column (USA) (20 x 250 mm) with a particle size of 10 μm. Buffer A - 0.1% aqueous TFA solution, buffer B - 80% acetonitrile in buffer A were used as eluents. Elution was carried out from 100% buffer A in a concentration gradient of 0.5% / min buffer B at a rate of 10 ml / min. Mass spectra of peptides were recorded on an Amazon Bruker instrument (Germany) by electrospray ionization (ESI) in the positive ion registration mode (capillary voltage - 3500 V). Mass scanning range - m / z - 70 - 2200 D. Syringe injection of a sample dissolved in a mixture of water and acetonitrile was used. Nebulizer gas: nitrogen, interface temperature -100°C.

[0062] For solid-phase synthesis, a copolymer of styrene with 1% divinylbenzene with a 4-(2,4-dimethoxyphenyl)-Fmoc-aminomethylphenoxy anchor group (Rink-amide polymer) from Nova BioChem (Switzerland) was used. It is intended for obtaining peptide amides and contains 0.8 mmol / g of amino groups. The synthesis of the claimed compounds was carried out in automatic mode on a Tribute UV peptide synthesizer (Protein Technologies Inc., USA) from the C-terminus, stepwise (adding one amino acid at a time), starting from 0.25 g (0.2 mmol) of Rink-amide polymer according to a standard program for a single condensation using a 4-fold excess of Fmoc-amino acids. To block the guanidine groups of Arg residues, Pbf protection was used, and for the 6-amino groups of Lys, Boc protection was used. To create the amide bond, the uronium salt method (TBTU / NMM) was used

[13] . The solid-phase synthesis cycle included the following steps: 1) elimination of N α-Fmoc-3aniHTbi with 20% pyrrolidine solution in DMF (2 x 5 ml x 3 min)

[14] ; 2) DMF washes (4 x 5 ml x 1 min); 3) condensation (0.8 mmol Fmoc-AA-OH + 0.8 mmol TBTU + 2 mmol NMM in 5 ml DMF for 90 min); 4) DMF washes (4 x 5 ml x 1 min); 5) ninhydrin test for residual amino groups. Upon completion of the synthesis of N α -amino-free peptidyl polymer NH2-(CH2)5-CO-Lys(Boc)-D-Lys(Boc)-D-Lys(Boc)-Arg(Pbf)-βAla-βAla-NH- P was transferred to a filter, washed with DMF 2x10 ml, DCM 2x10 ml, and dried to constant weight.

[0063] FITC labeling was performed on the solid phase in a dark reaction vessel for 20 h. A solution containing 40 mg (2 equivalents) of FITC and 0.05 ml (6 equivalents) of DIPEA in 0.5 ml of DMF was added to a suspension of 100 mg of peptidyl polymer (0.05 mmol of amino groups) in 1 ml of DMF. The reaction mixture was kept for 20 h with stirring at room temperature. The completeness of labeling was monitored using a ninhydrin test for unsubstituted amino groups.

[0064] The final cleavage of the peptide from the polymer with simultaneous deprotection of the side functional groups was carried out by treating the FITC-peptidyl polymer with a mixture of 91% trifluoroacetic acid with the addition of 5% deionized water, 2% DMB, 2% TIBS for 1.5 h. Then the polymer was filtered off, washed 2×2 ml with the deprotecting mixture, the filtrate was evaporated, and the product was precipitated with dry cold diethyl ether. The precipitate was filtered off, washed on the filter with DCM (2×5 ml), diethyl ether (3×5 ml), and dried in a vacuum desiccator. The crude product was purified by preparative HPLC and lyophilized. 45 mg of probe I was obtained (65%> based on the starting amino acid attached to the polymer). The structure of the claimed peptide was confirmed by mass spectrometry, and its purity was confirmed by analytical HPLC. The data are presented in the table.

[0065] Example 2. Synthesis of FITC-NH-(CH2)5-CO-Lys-D-Lys-D-Lys-D-Lys-Arg-βAla-βAla-NH2- probe (II)

[0066] The synthesis of peptide (II) was carried out similarly to the synthesis of peptide (I) described in Example 1. An additional cycle of addition of the Fmoc-D-Lys(Boc)-OH residue was added to the synthetic protocol. After purification, carried out as described in Example 1, 30 mg of compound (II) was obtained; the corresponding characteristics are listed in the table.

[0067] Example 3. Bt-NH-(CH2)5-CO-Lys-D-Lys-D-Lys-Arg-βAla-βAla-NH2K ​​suspension 200 mg (0.1 mmol) N α-amino-free peptidyl polymer NH2-(CH2)5-CO-Lys(Boc)-D-Lys(Boc)-D-Lys(Boc)-Arg(Pbf)-βAla-βAla-NH- P obtained as described in Example 1, 37 mg (0.152 mmol) of biotin, 23 mg (0.152 mmol) of HOBt and 35 μl (0.200) of DIC were added to 1.5 ml of DMF and gently stirred for 4 h. The completeness of the reaction was monitored using a ninhydrin test for the unsubstituted amino group. Upon completion of the reaction, the peptidyl polymer was washed with DMF 2x10 ml, DCM 2x10 ml, and dried to constant weight. The final deprotection of the modified peptide and purification of the technical product were carried out as described in Example 1. 62.0 mg (59%) of compound (III) were obtained. The characteristics of the synthesized compounds are given in the table; HPLC, mass spectrum and structural formula are shown in Fig. 3

[0068] Example 4. Bt-NH-(CH2)5-CO-Lys-D-Lys-D-Lys-D-Lys-Arg-βAla-βAla-NH2(IV) The synthesis of peptide (IV) was carried out similarly to the synthesis of peptide (III) described in Example 3 by biotinylation of the peptidyl polymer NH2-(CH2)5-CO-Lys(Boc)-D-Lys(Boc)-D-Lys(Boc)-D-Lys(Boc)-Arg(Pbf)-βAla-βAla-NH- P After purification, 57 mg (49%) of compound (IV) was obtained, the corresponding characteristics are given in the table.

[0069]

[0070]

[0071] Example 5. Interaction of fluorescent probes with sulfated glycocalyx components

[0072] A) A solution of 1.5 mg of peptide (I) in 1 ml of 0.075 M phosphate buffered saline (PBS) with pH 7.4 was applied to a column (1.5 x 10 cm) with Affi Gel® Heparin Bio-Rad (USA) sorbent containing 1 (±0.2) mg of heparin per 1 ml of gel, equilibrated with the same buffer, washed with 10 ml of starting buffer and the peptide was eluted with a linear concentration gradient of sodium chloride from 75 to 750 mM in the initial PBS with pH 7.4 (60+60 ml), collecting 4.5 ml fractions. Fig. 3 (a) shows the elution profile of fluorescently labeled peptide (I) at a wavelength of 495 nm.

[0073] B) A solution of 1.0 mg of peptide (II) in 1 ml of 0.075 M phosphate-buffered saline (PBS) with pH 7.4 was applied to a column (1.5 x 10 cm) with Affi Gel® Heparin Bio-Rad (USA) sorbent containing 1 (±0.2) mg of heparin per 1 ml of gel, equilibrated with the same buffer, washed with 10 ml of starting buffer and the peptide was eluted with a linear concentration gradient of sodium chloride from 75 to 750 mM in the initial PBS with pH 7.4, collecting 4.0 ml fractions. Fig. 3 (b) shows the elution profile of fluorescently labeled peptide (II) at a wavelength of 495 nm.

[0074] Example 6. Interaction of FITC-labeled peptide (I) with rat aortic glycocalyx in vivo / ex vivo (direct detection method).

[0075] FITC-labeled peptide (I) was dissolved in sterile saline to a final concentration of 3 mM, and 0.5 ml of the resulting solution was injected into the jugular or tail vein of 300 g Wistar rats previously anesthetized by intraperitoneal administration of a mixture of xylazine (5 mg / kg) and zoletil (30 mg / kg). After 5 min, the rats were euthanized, a fragment of the thoracic aorta was removed, washed, and connective tissue was removed. An aortic fragment approximately 1 cm in length was transferred to a plastic tube containing 300 μl of phosphate-buffered saline and incubated for 30 min at 37°C in the presence of 30 mU / ml heparinase III (Sigma, USA) and 15 mU / ml hyaluronidase (Liporase, Daehan New Pharm Co., Ltd., South Korea). Control aortic fragments were incubated without the addition of enzymes. After incubation, the aortic fragments were transferred to clean tubes and washed once with 300 μl of phosphate-buffered saline.The aortic fragments were then transferred to clean tubes, and fluorescent peptides were extracted for 30 minutes at room temperature using 300 µl of phosphate-buffered saline supplemented with 500 mM NaCl. FITC fluorescence was measured in each extract using a Victor X3 fluorescence plate reader (Perkin Elmer, USA). All measurements were performed in duplicate.

[0076] As shown in Fig. 6, peptide (I) extraction was more efficient in the presence of glycocalyx-degrading enzymes. These data indicate that tissue treatment with heparinase III and hyaluronidase released glycocalyx components and the bound peptide (I). Upon subsequent extraction of peptide (I) with a high ionic strength solution, the fluorescent signal was lower in the extracts previously treated with enzymes than in tissues not exposed to glycocalyx-degrading enzymes. Repeated treatment of aortic fragments with a high-salt solution did not result in additional peptide (I) extraction (not shown in Fig. 6).Thus, all remaining peptide (I) reversibly bound to the luminal surface of rat aorta in vivo was dissociated by phosphate-buffered saline additionally containing 500 mM NaCl, which indicates the electrostatic nature of the binding of peptide (I) to structures on the aortic surface and is consistent with the interaction of the polycationic peptide (I) with negatively charged sulfate groups exposed on heparan sulfate and other sulfated components of the vascular wall glycocalyx.

[0077] Example 7. Detection of sulfated glycocalyx components on the surface of human umbilical vein endothelial cells cultured under laminar flow conditions of growth medium using biotinylated peptide (III) (two-step detection method).

[0078] Human umbilical vein endothelial cells (HUVEC) were obtained from five healthy donors and pooled in equal proportions. HUVEC were cultured in EGM medium (Cell Applications) at 37°C under 5% CO2 in culture dishes pre-coated with 0.2% gelatin. Cells from passage 3 were seeded in flow chambers - μ-slide I 0.4 (ibidi.de, Germany) and incubated at 37°C in a 5% CO2 atmosphere until a monolayer was achieved. The chambers were then connected to the ibidi Pump System (ibidi.de, Germany), which created a laminar flow of the culture medium, simulating blood flow in vessels with an estimated shear stress of 5 dynes / cm. 2After 7 days, the cells were fixed in 3.7% formalin solution for 2 min at room temperature, washed with five volumes of phosphate-buffered saline and incubated for 30 min with biotinylated peptide (III). The final concentration of peptide (III) was 150 μM. The chamber was washed with five volumes of phosphate-buffered saline and incubated for 30 min at room temperature with FITC-labeled streptavidin (Imtek, Russia) at a final concentration of 50 ng / ml for visualization of peptide (III) and 4',6-diamidino-2-phenylindole (DAPI) for staining cell nuclei. The chambers were then washed with five volumes of phosphate-buffered saline, and the cells were visualized using a Stellaris 5 laser confocal microscope (Leica, Germany) at 10x or 63x magnification and identical imaging settings (laser intensity, exposure time, etc.). Control cells were prepared in parallel; the staining process for these cells did not include the incubation step with peptide (III).Images were processed using ImageJ 1.54f software (NIH, USA).

[0079] As can be seen from Fig. 7, in the absence of cell treatment with peptide (III), non-specific binding of FITC-labeled streptavidin to cells is weak and the fluorescent signal is virtually undetectable (A), whereas after incubation of cells with biotinylated peptide (III), the FITC fluorescent signal increases significantly and the cell outlines become clearly visible (B). Punctate structures, sometimes forming dotted linear structures, and short filaments are visible on the cell surface. Panel B shows a 3D reconstruction of confocal images of HUVEC cells obtained at high magnification after incubation of cells with peptide (III) and FITC-streptavidin. The 3D reconstruction reveals thin fibers located mainly vertically to the cell surface.

[0080] Thus, the claimed fluorescently labeled probes based on modified peptides of the general formula R- NH-(CH2)5-CO-Lys-(Z)-Lys) n -Arg-βAla-βAla-NH2, where

[0081] R = FITC, n = 2 for compound (I)

[0082] R = FITC, n = 3 for compound (II)

[0083] R = Bt, n = 2 for compound (III)

[0084] R=Bt, n=3 for compound (IV),

[0085] possesses the proteolytic stability necessary for in vivo diagnostic procedures, which is due to their structural modification and ability to interact with sulfated components of the carbohydrate portion of the glycocalyx on cells, as demonstrated in the examples. Detection / visualization of sulfated glycocalyx components using fluorescence microscopy can be accomplished either directly using fluorescently labeled peptides or via a two-step method using a biotin-fluorescently labeled streptavidin system. The two-step method involves the use of biotinylated peptides and fluorescently labeled streptavidin.

[0086] Literature

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Claims

1. Modified peptide of formula R-NH-(CH2)5-CO-Lys-(D-Lys) n -Arg-βAla-βAla-NH2, where R = FITC, n=2 for compound (I), R = FITC, n=3 for compound (II), R = Bt, n=2 for compound (III) or R = Bt, n=3 for compound (IV), where FITC is fluorescein isothiocyanate and Bt is biotin, which is capable of binding to sulfated components of the glycocalyx on cells.

2. Use of a peptide of the formula R-NH-(CH2)5-CO-Lys-(D-Lys) n -Arg-βAla-βAla-NH2 according to claim 1 for visualization of endothelial glycocalyx.

Images