MEANS AND METHODS FOR VISUALIZATION OF TISSUE STRUCTURES

Abstract

The present invention relates to a chemical compound comprising (i) a polycationic polymer, coupled to (ii) a dye. The present invention further relates to a method for visualizing a glycosamine-containing structure in a biological sample comprising a) contacting an inner lumen of said biological sample with a dye-conjugated polycationic polymer, preferably with the chemical compound according to the present invention; b) tissue-clearing said biological sample; and, thereby, c) visualizing an internal glycosamine-containing structure in said biological sample. The present invention also relates to a method for determining the number and/or size of glomeruli in a kidney or a sample thereof making use of the method for visualizing a glycosamine-containing structure; and also relates to kits and uses related to said chemical compounds and said methods.

Claims

1. A method for visualizing a glycosamine-containing structure in a biological sample comprising: a) contacting an inner lumen of the biological sample with a dye-conjugated polycationic polymer, comprising (i) a polycationic polymer, coupled to (ii) a dye, wherein said polycationic polymer comprises at least one positive charge per 250 Da of molecular weight at pH=7 and wherein said dye is a polymethine dye; b) tissue-clearing the biological sample; and, thereby, c) visualizing an internal glycosamine-containing structure in the biological sample.

2. The method of claim 1, wherein the glycosamine-containing structure is (i) an endothelial matrix or a cartilage; and/or (ii) a glomerular filtration barrier matrix.

3. The method of claim 1, wherein said glycosamine-containing structure is an endothelial matrix.

4. The method of claim 1, wherein said glycosamine-containing structure is an endothelial matrix of a blood vessel.

5. The method of claim 1, wherein said glycosamine-containing structure is a basement membrane and/or a glycocalix of a blood vessel.

6. The method of claim 1, wherein said glycosamine-containing structure is a glomerular basement membrane.

7. The method of claim 1, wherein said dye is a fluorescent dye, preferably a fluorescent dye having an emission maximum at a wavelength of from 500 nm to 950 nm.

8. The method of claim 1, wherein said dye is Cy7, Cy5 or a fluorescent derivative thereof, preferably is Cy7 or a fluorescent derivative thereof.

9. The method of claim 1, wherein said polycationic polymer has an average molecular weight of from 20 kDa to 150 kDa.

10. The method of claim 1, wherein said polycationic polymer comprises at least one proton-accepting functional group with a pKs higher than 7 per 250 Da of molecular weight and/or comprises at least one functional group with a pH independent positive charge per 250 Da of molecular weight.

11. The method of claim 1, wherein said polycationic polymer comprises at least one positive charge per 100 Da at pH=7.

12. The method of claim 1, wherein said polycationic polymer is polyethyleneimine; a polycationic glycan; a poly-lysine; or ferritin.

13. The method of claim 1, wherein said polycationic polymer is polyethyleneimine.

14. The method of claim 1, wherein the sample is an organism, an organ, a tissue, or a section thereof.

15. The method of claim 1, wherein said sample is a liver, a muscle, a skin, a heart, a kidney, a brain, a lung, or a tumor, more preferably is a kidney.

16. The method of claim 1, wherein the sample is a sample of an experimental animal.

17. The method of claim 1, wherein said sample is a sample of a mouse, a rat, a guinea pig, a goat, a sheep, a cat, a dog, a rabbit, a donkey, a horse, or a cow.

18. A method for determining the number and/or size of glomeruli in a kidney or a sample thereof comprising: a) visualizing glomeruli according to the method for visualizing an internal glycosamine-comprising structure in a biological sample according to claim 1; b) determining the number of glomeruli and/or determining the size of glomeruli in at least part of the kidney or in the sample thereof; and, thereby, c) determining the number and/or size of glomeruli in a kidney or a sample thereof.

Description

FIGURE LEGENDS

[0058] FIG. 1: Chemical structure of the dye-conjugated polycationic polymer (compound 6); per polycationic polymer molecule, at least one R is R1 as depicted. X indicates possible coupling sites, i.e. potential sites for a covalent bond between the dye moiety and the polycationic polymer moiety; per dye moiety, at least one site X is covalently bound to the polycationic polymer moiety.

[0059] FIG. 2: Absorption and emission spectra of the dye-conjugated polycationic polymer (compound 6). Absorption maximum was at 654 nm, emission maximum was at 762 nm; x-axis: wavelength in nm; left y-axis: absorption; right y-axis: emission (arbitrary units).

[0060] FIG. 3: A) Fluorescence microscopic picture of a section of a mouse kidney stained and cleared according to Example 3; glomeruli can be unambiguously identified; B) same as in A), but only signal from dye-conjugated polycationic polymer shown.

EXAMPLE 1: DYE SYNTHESIS

[0061] 1.1 Synthesis of Compound 2

##STR00003##

[0062] To a 50 mL round-bottom flask, glacial acetic acid (30 mL) was added to a mixture of phenylhydrazine (compound 1, 2.163 g, 20 mmol), methyl isopropyl ketone (2.584 g, 30 mmol) and sodium acetate (3.2 g, 38 mmol). The suspension was refluxed under 110° C. for 24 h, the hot solution was cooled to room temperature and concentrated, the residues were purified by silica gel column chromatography with ethyl acetate/petroleum ether=1/2. A brown oil acetated salt (compound 2, 3.26 g, yield 75%) was obtained. .sup.1H NMR (300 MHz, DMSO-d.sub.6): δ 1.3 (s, 6H), 2.08 (s, 3H), 2.30 (s, 3H), 7.22 (d, J=9.0 Hz, 1H), 7.32 (m, 2H), 7.64 (d, J=6.0 Hz, 1H), 10.34 (s, 1H). .sup.13C NMR (75 MHz, DMSO-d.sub.6): δ 14.99, 23.53, 53.57, 121.33, 127.69, 145.30, 152.58, 175.62, 188.64. LRMS (m/z): calcd: 159.10, found: 159.14.

[0063] 1.2 Synthesis of Compound 3:

##STR00004##

[0064] A mixture of 2,3,3-trimethyl-3H-indole (compound 2, 1.59 g, 10 mmol) and 6-bromohexanoic acid (1.95 g, 10 mmol) in toluene (20 mL) was heated at 130° C. for 48 hours under argon. The mixture was cooled to room temperature and concentrated under reduced pressure. The product was extensively washed with Hexane to obtain a pink solid (compound 3, 1.94 g, 55%), which was used in the next step without further purification.

[0065] 1.3 Synthesis of Compound 4:

##STR00005##

[0066] A mixture of sodium salt of compound 3 (800 g, 2.26 mmol), Vilsmeier-Haack reagent (0.36 g, 1 mmol) and anhydrous sodium acetate (0.252 g, 3 mmol) in 20 ml of absolute ethanol was refluxed for 8 h under argon. The reaction mixture was cooled to room temperature, and then concentrated under reduced pressure to yield a brownish green residue. The crude product was washed with ethyl acetate/petroleum ether=1/2. The residues were purified by silica gel column chromatography with methanol/ethyl acetate=1/2, a green solid (compound 4, 0.174 g, yield 25%) was yield. LRMS (m/z): calcd: 683.36, found: 683.49.

[0067] 1.4 Synthesis of Compound 5:

##STR00006##

[0068] A mixture of compound 4 (69 mg, 0.1 mmol) and 4-aminobutanoic acid (62 mg, 0.6 mmol) in DMSO was heated at 65° C. overnight. The reaction mixture was cooled to room temperature and precipitated in ethyl acetate and washed with dichloromethane. The residues were purified by silica gel column chromatography and washed with methanol/dichloromethane=1/2. A blue solid was obtained (compound 5, 53.3 mg, yield 71%). LRMS (m/z): calcd: 750.45, found: 750.60.

##STR00007##

EXAMPLE 2: SYNTHESIS OF DYE-CONJUGATED POLYCATIONIC POLYMER (COMPOUND 6)

[0069] A mixture of dye compound 5 (cf 1. above; 35 mg, 0.046 mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (44.7 mg, 0.23 mmol), 4-dimethylaminopyridine (10 mg, 0.08 mmol) and branched polyethylenimine (1000 mg) and DMSO (20 mL) was stirred at room temperature under argon gas protection and exclusion of light. After 12 hours the reaction mixture was then dialysis in membrane against PBS for 48 h. The product was freeze dried to yield a blue solid (Compound 6, 350 mg). Absorption and emission spectrum of the dye-conjugated polycationic polymer (compound 6) obtained is shown in FIG. 2. All spectroscopic measurements were conducted in phosphate buffered saline (PBS). UV-vis and fluorescence spectra were acquired using a microplate reader (Tecan Infinite M200).

EXAMPLE 3: VISUALIZATION OF MOUSE GLOMERULI

[0070] Mice were sacrificed and kidneys were immediately withdrawn. Kidneys were perfused and stained by retrograde perfusion via the renal vein (V. renalis) according to the scheme of Table 1, followed by tissue clearing according to the scheme of Table 2 (PFA: paraformaldehyde, PBS: phosphate buffered saline, EDTA: ethylenediaminetetraacetic acid, ECi: Ethyl cinnamate, EtOH: Ethanol). After tissue clearing, glomeruli were visualized by fluorescence microscopy. Exemplary results obtained are shown in FIG. 3.

TABLE-US-00001 TABLE 1 Mouse perfusion Substances Time (min) Pressure (mBar) Saline and EDTA/Heparin 6 200-300 Compound 6 in PBS 10 200-300 Saline 1 200-300 PFA/PBS 6 200-300

TABLE-US-00002 TABLE 2 Tissue clearing Procedure Temperature Time Sum time Post-fixation Room temperature 60-120 min 60-120 min in 4% PFA/PBS Dehydration Room temperature 30-45 min 120-180 min in 50% EtOH Dehydration Room temperature 30-45 min in 80% EtOH Dehydration Room temperature 30-45 min in 99% EtOH Dehydration Room temperature 30-45 min in 99% EtOH ECi clearing Room temperature 2-16 hours 2-16 hours or days or days