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

1-15. (canceled)

16. A chemical compound comprising: (i) a polycationic polymer, coupled to (ii) a dye, wherein the polycationic polymer comprises at least one positive charge per 250 Da of molecular weight at pH=7, wherein the dye is a polymethine dye, and wherein the polycationic polymer comprises polyethyleneimine.

17. The chemical compound of claim 16, wherein the dye is a fluorescent dye.

18. The chemical compound of claim 16, wherein the dye is Cy7, Cy5, or a fluorescent derivative thereof.

19. The chemical compound of claim 16, wherein the polycationic polymer has an average molecular weight of from 0.5 kDa to 1000 kDa.

20. The chemical compound of claim 16, wherein the polycationic polymer comprises at least one proton-accepting functional group with a pKs higher than 7 per 250 Da of molecular weight.

21. The chemical compound of claim 16, wherein the polycationic polymer comprises at least one positive charge per 100 Da, at pH=7.

22. The chemical compound of claim 16, wherein the polycationic polymer is polyethyleneimine.

23. 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; b) tissue-clearing the biological sample; and, thereby, c) visualizing an internal glycosamine-containing structure in the biological sample.

24. The method of claim 23, wherein the glycosamine-containing structure is (i) an endothelial matrix or a cartilage; and/or (ii) a glomerular filtration barrier matrix.

25. The method of claim 23, wherein the sample is an organism, an organ, a tissue, or a section thereof.

26. The method of claim 23, wherein the sample is a sample of an experimental animal.

27. 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 23; 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.

28. A kit comprising the chemical compound of claim 16 and a means for application thereof, wherein the kit further comprises a tissue clearing agent.

29. The chemical compound of claim 16, wherein the polymethine dye comprises 2, 3, 4, 5, 6, 7, or 8 methine units.

30. The chemical compound of claim 17, wherein the fluorescent dye has an emission maximum at a wavelength of from 500 nm to 950 nm.

31. The method of claim 23, wherein the dye-conjugated polycationic polymer is a chemical compound comprising: (i) a polycationic polymer, coupled to (ii) a dye, wherein the polycationic polymer comprises at least one positive charge per 250 Da of molecular weight at pH=7, wherein the dye is a polymethine dye, and wherein the polycationic polymer comprises polyethyleneimine.

Description

[0052] In view of the above, the following embodiments are preferred:

[0053] 1. A chemical compound comprising [0054] (i) a polycationic polymer, coupled to, preferably covalently coupled to, [0055] (ii) a dye.

[0056] 2. The chemical compound of embodiment 1, wherein said dye is a fluorescent dye.

[0057] 3. The chemical compound of embodiment 1 or 2, wherein said dye is a polymethine dye.

[0058] 4. The chemical compound of any one of embodiments 1 to 3, wherein said dye is a cyanine dye, preferably comprising 2, 3, 4, 5, 6, 7, or 8 methine units.

[0059] 5. The chemical compound of any one of embodiments 1 to 4, wherein said dye is Cy5, Cy7 or a fluorescent derivative thereof, preferably is Cy7 or a fluorescent derivative thereof.

[0060] 6. The chemical compound of any one of embodiments 1 to 5, wherein said dye has an absorption maximum at a wavelength of from 400 nm to 900 nm.

[0061] 7. The chemical compound of any one of embodiments 1 to 6, wherein said dye has an emission maximum at a wavelength of from 500 nm to 950 nm.

[0062] 8. The chemical compound of any one of embodiments 1 to 7, wherein said polycationic polymer has an average molecular weight of from 0.5 kDa to 1000 kDa, preferably of from 1 kDa to 500 kDa, more preferably of from 1 kDa to 300 kDa, more preferably of from 20 kDa to 150 kDa.

[0063] 9. The chemical compound of any one of embodiments 1 to 8, wherein said polycationic polymer is ferritin; a polycationic glycan, preferably a polycationic glucan, more preferably diethylaminoethyl-dextran or trialkyl-aminoalkyl-dextran; a poly-lysine; or polyethyleneimine.

[0064] 10. The chemical compound of any one of embodiments 1 to 9, wherein said polycationic polymer comprises at least one proton-accepting functional group with a pKs higher than 7 per 250 Da of molecular weight, preferably per 100 Da, more preferably per 50 Da and/or comprises at least one functional group with a pH independent positive charge per 250 Da of molecular weight, preferably per 100 Da, more preferably per 50 Da

[0065] 11. The chemical compound embodiment 10, wherein said proton-accepting functional group with a pKs higher than 7 is selected from a primary amino group or a secondary amino group, and/or and/or wherein said functional group with a pH independent positive charge is a tertiary amino group.

[0066] 12. The chemical compound of any one of embodiments 1 to 11, wherein said polycationic polymer comprises at least one positive charge per 250 Da of molecular weight, preferably per 100 Da, more preferably per 50 Da, at pH=7.

[0067] 13. The chemical compound of any one of embodiments 1 to 12, wherein said polycationic polymer comprises ethyleneimine units.

[0068] 14. The chemical compound of any one of embodiments 1 to 13, wherein said polycationic polymer consists of ethyleneimine units.

[0069] 15. The chemical compound of any one of embodiments 1 to 14, wherein said polycationic polymer is polyethyleneimine, preferably is branched polyethyleneimine.

[0070] 16. The chemical compound of any one of embodiments 1 to 15, wherein said chemical compound consists of said polycationic polymer and said dye.

[0071] 17. A method for visualizing a glycosamine-containing structure, preferably in a biological sample, comprising

a) contacting an inner lumen of said biological sample with a dye-conjugated polycationic polymer;
b) preferably, tissue-clearing said biological sample; and, thereby,
c) visualizing an internal glycosamine-containing structure in said biological sample.

[0072] 18. The method of embodiment 17, wherein said glycosamine-containing structure is (i) an endothelial matrix or a cartilage, preferably an endothelial matrix, more preferably an endothelial matrix of a blood vessel, more preferably a basement membrane and/or a glycocalix of a blood vessel; and/or (ii) a glomerular filtration barrier matrix, preferably a glomerular basement membrane.

[0073] 19. The method of embodiment 17 or 18, wherein said method comprises a further step a0) of flushing said inner lumen, preferably with phosphate-buffered saline (PBS) before step a).

[0074] 20. The method of any one of embodiments 17 to 19, wherein said method comprises a further step a1) of flushing said inner lumen, preferably with phosphate-buffered saline (PBS) after step a).

[0075] 21. The method of any one of embodiments 17 to 20, wherein said method comprises a further step a2) of fixing biological structures in said biological sample by flushing said inner lumen with a fixing agent, preferably with a solution comprising formaldehyde, before step b) and, preferably, following step a1).

[0076] 22. The method of embodiment 21, wherein said step a2) comprises incubating said biological sample with said fixing agent for at least 10 min, preferably for at least 30 min, more preferably for at least 1 h.

[0077] 23. The method of any one of embodiments 17 to 22, wherein said method comprises the further step a3) of flushing said inner lumen, preferably with phosphate-buffered saline (PBS) after step a2)

[0078] 24. The method of any one of embodiments 17 to 23, wherein said method comprises the further step of b1) analyzing at least part of said biological sample by optical means, preferably by microscopy, more preferably by fluorescent light microscopy, more preferably confocal microscopy and/or by lightsheet fluorescence microscopy.

[0079] 25. The method of any one of embodiments 17 to wherein said dye-conjugated polycationic polymer is a chemical compound according to any one of embodiments 1 to 16.

[0080] 26. The method of any one of embodiments 17 to 25, wherein said sample is an organism, an organ, a tissue, or a section thereof, preferably is a liver, a muscle, a skin, a heart, a kidney, a brain, a lung or a tumor, more preferably is a kidney.

[0081] 27. 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 any one of embodiments 17 to 26;
b) determining the number of glomeruli and/or determining the size of glomeruli in at least part of said kidney or in said sample thereof; and, thereby,
c) determining the number and/or size of glomeruli in a kidney or a sample thereof.

[0082] 28. A method for providing an indication for cancer, comprising

a) visualizing blood vessels according to the method for visualizing an internal glycosamine-comprising structure in a biological sample according to any one of embodiments 17 to 26;
b) determining vascularization in said sample,
c) comparing the vascularization determined in step b) to a reference; and,
d) based on the result of comparing step c), providing an indication for cancer.

[0083] 29. The method of embodiment 28, wherein an indication for cancer is found if vascularization is increased compared to a healthy reference; and/or wherein an indication for cancer is found if vascularization is similar or increased compared to a reference cancer sample is detected.

[0084] 30. The methods of any one of embodiments 17 to 29, wherein said methods are in vitro methods.

[0085] 31. The method of any one of embodiments 17 to 30, wherein said glycosamine-comprising structure is a structure comprised in a tissue lining an inner lumen of a body.

[0086] 32. The method of any one of embodiments 17 to 31, wherein said glycosamine-comprising structure is (i) an endothelial matrix or cartilage, preferably an endothelial matrix, more preferably an endothelial matrix of a blood vessel, more preferably a basement membrane of a blood vessel; and/or (ii) a glomerular filtration barrier matrix, preferably a glomerular basement membrane.

[0087] 33. A kit comprising the chemical compound according to any one of embodiments 1 to 16 and a means for application thereof.

[0088] 34. The kit of embodiment 33, wherein said kit further comprises a washing agent.

[0089] 35. The kit of embodiment 33 or 34, wherein said kit further comprises a fixing agent.

[0090] 36. The kit of any one of embodiments 33 to 35, wherein said kit further comprises a tissue clearing agent, preferably further comprises ethyl-3-phenylprop-2-enoate (ethyl cinnamate).

[0091] 37. Use of a dye-conjugated polycationic polymer, preferably according to any one of embodiments 1 to 16, for visualizing an internal glycosamine-containing structure, for determining the number and/or size of glomeruli in a kidney or a sample thereof, for visualizing vessels, and/or for providing an indication for cancer.

[0092] 38. Use of a dye-conjugated polycationic polymer, preferably according to any one of embodiments 1 to 16, for manufacturing a diagnostic composition for diagnosing increased vascularization, preferably for providing an indication for cancer.

[0093] 39. A method for detecting a glycosamine-comprising structure in a biological sample, wherein said method comprises contacting said biological sample with a dye-conjugated polycationic polymer.

[0094] 40. The method of embodiment 39, wherein said glycosamine-comprising structure is a structure comprised in a tissue lining an inner lumen of a body.

[0095] 41. The method of embodiment 39 or 40, wherein said glycosamine-comprising structure is (i) an endothelial matrix or a cartilage, preferably an endothelial matrix, more preferably an endothelial matrix of a blood vessel, more preferably a basement membrane of a blood vessel; and/or (ii) a glomerular filtration barrier matrix, preferably a glomerular basement membrane.

[0096] 42. The methods of any one of embodiments 17 to 31 or 39 to 41, wherein said method further comprises contacting an inner lumen of said sample with a further dye-conjugated polymer and/or with an agent specifically binding to a structure comprised or suspected to be comprised in said sample.

[0097] 43. The method of embodiment 42, wherein said further contacting an inner lumen with a further dye-conjugated polymer and/or with an agent specifically binding to a structure is performed sequentially to contacting said inner lumen of said sample with a dye-conjugated polycationic polymer, preferably is performed after contacting said inner lumen of said sample with a dye-conjugated polycationic polymer.

[0098] All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.

[0099] The following Examples shall merely illustrate the invention. They shall not be construed, whatsoever, to limit the scope of the invention.

FIGURE LEGENDS

[0100] 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.

[0101] 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).

[0102] 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

1.1 Synthesis of Compound 2

[0103] ##STR00003##

[0104] 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): ? 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): ? 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.

1.2 Synthesis of Compound 3:

[0105] ##STR00004##

[0106] 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.

1.3 Synthesis of Compound 4:

[0107] ##STR00005##

[0108] 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.

1.4 Synthesis of Compound 5:

[0109] ##STR00006##

[0110] 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)

[0111] 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

[0112] 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 in Room temperature 30-45 min 120-180 min 50% EtOH Dehydration in Room temperature 30-45 min 80% EtOH Dehydration in Room temperature 30-45 min 99% EtOH Dehydration in Room temperature 30-45 min 99% EtOH ECi clearing Room temperature 2-16 hours 2-16 hours or days or days