Patent Description:
The present invention relates to fluorescent water-soluble polycationic chitosan polymers and use thereof as imaging agents for imaging glycosamine-containing structures, e.g., vascular structures, <NUM>-dimensionally.

Vascular studies are important to understand the structure and function of arteries, capillaries, and veins. This information is useful to understand if there is a proper blood flow through different organs. Compromised vascular functioning is associated with many diseases, such as atherosclerosis, aneurysm, hypertension, chronic kidney diseases, etc. [<NUM>].

This task has been approached by various methods. Ultrasound (US) is mostly used in clinics. It can measure the amount of blood pumped with each heartbeat, which indicates the measure of vascular diameter. US can also detect abnormal blood flow, indicating blockage. However, this technique has very low spatial resolution, is sensitive towards artifacts (such as movement artifacts) and has a high cost of instrumentation [<NUM>,<NUM>].

Magnetic resonance imaging (MRI) is used to study vascular anatomy and blood flow to learn more about disease progression or is part of routine check-up. But it uses expensive instrumentation, along with expensive contrast agents. MRI provides low spatial resolution and sensitivity and is prone to artifacts, in addition its use is limited in patients with pacemakers or metallic implants [<NUM>,<NUM>].

Like MRI, computed tomography (CT) can be used to study blood vessels. It is used to diagnose acute coronary symptoms and aneurysms. CT has a higher spatial resolution than US and MRI. But it has serious problems with the use of ionising radiation and the expensive contrast agents. Furthermore, the contrast media can be nephrotoxic [<NUM>,<NUM>].

A substantial improvement is the use of optical imaging with fluorescence-based staining of intra- and extra-cellular biomolecules. Fluorescent markers such as Alcian Blue, indocyanine green dye, green fluorescent proteins and various synthetic chromophores have been used for the vascular visualisation [<NUM>]. Vascular biopsies combined with cellular staining is a common procedure in hospitals for diagnostic purposes. It is very efficient in site-specific study of tissue abnormalities, but is invasive, and can lead to infection, damage to nearby tissues, or excessive bleeding. Fluorescently labelled antibodies are another example for using fluorescence based vascular visualisation. For example, Alexa Fluor <NUM> labelled anti-CD31 is used for staining the endothelial cell lining of the vessels. This technique offers high specificity for the target but is very expensive. Moreover, the wavelength of these dyes is mainly in the blue and green region of the spectrum. This fact limits their application in visualising and imaging the inner organ structures because of the poor tissue penetration depth and the disturbance of the signal by autofluorescence of the tissue, compared to the red region of the spectrum [<NUM>].

Recently, many studies have focused on the use of fluorescent polymers instead of antibodies as a cheaper alternative to a diverse range of therapeutic and diagnostic biomedical applications, such as tools for gene delivery, immunotherapy, visualisation of tissue structures etc..

<CIT> demonstrates the use of one such fluorescent polymer for vascular staining. It describes the application of polyethyleneimine (PEI) conjugated to MHI148 dye for the visualisation and counting of glomeruli. MHI148-PEI uses branched PEI of molecular weight <NUM> kDa, conjugated to a cationic cyanine <NUM> dye and binds to glycosamine containing biological structures basing on an electrostatic interaction [<NUM>].

However, this compound has multiple drawbacks. MHI148 dye is well known for its chemical instability. It consists of a meso-positioned amino alkyl chain, which is sensitive to chemical and photoinduced decomposition [<NUM>]. Also, the use of PEI is limited in biological settings because of its toxicity. PEI has been reported to induce cellular toxicity and hence creates problems with the Regulatory Affairs [<NUM>].

, et al, (<NUM>) discloses cancer-targeting heptamethine dye MHI-<NUM> conjugated to a hydrophobically modified (5β-cholanic acid) glycol chitosan (forming nanomicelle).

There is therefore the need of new fluorescent markers for use in visualising glycosamine-containing structures, like blood vessels, that are chemically stable, non-toxic, and more efficient in imaging.

The object of the present invention is to provide a novel substance which can be used as an imaging agent to study vascular structures and that overcomes the disadvantages of the markers known in the prior art.

An object of the present invention is to provide new fluorescent markers that are chemically stable.

A further object of the present invention is to provide fluorescent markers that are non-toxic for their use in in vivo or ex vivo imaging of glycosamine-containing structures.

A further object of the present invention is to provide fluorescent markers that are more efficient in visualising glycosamine-containing structures than the known markers.

A further object of the present invention is to provide fluorescent markers that deliver reproducible results in visualising glycosamine-containing structures.

According to the invention, the above objects are achieved thanks to the matter specified in the ensuing claims, which are understood as forming an integral part of the present invention.

The current invention concerns new fluorescent polymers as a non-toxic, cheap, reproducible, and more efficient alternative than the known markers for imaging glycosamine-containing structures using <NUM>-dimensional imaging.

An embodiment of the present invention relates to a fluorescent water-soluble polycationic chitosan polymer of formula (I) and salts thereof:
<CHM>
wherein.

A further embodiment of the present invention relates to an in vitro or ex vivo method of imaging a glycosamine-containing structure in a biological sample using a fluorescent water-soluble polycationic chitosan polymer of formula (I).

A further embodiment of the present invention relates to a fluorescent water-soluble polycationic chitosan polymer of formula (I) for use in an in vivo imaging method of a glycosamine-containing structure in a mammal.

The current invention concerns also a kit comprising at least one fluorescent water-soluble polycationic chitosan polymer of formula (I) for the application in a 3D imaging method of a glycosamine-containing structure.

The invention will now be described in detail, purely by way of illustrative and non-limiting example, with reference to the attached figures, wherein:.

In the following description, numerous specific details are given to provide a thorough understanding of the embodiments. The embodiments can be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment.

The invention relates to fluorescent water-soluble polycationic chitosan polymers and use thereof as markers for imaging glycosamine-containing structures in <NUM>-dimensions.

In one embodiment, the present invention concerns a fluorescent water-soluble polycationic chitosan polymer represented by the general formula (I) and salts thereof:
<CHM>
wherein.

Chitosan is a polysaccharide, composed of β-linked D-glucosamine and N-acetyl-D-glucosamine arranged randomly in a linear fashion. Chitosan is readily available by treating chitin with an alkaline compound. Furthermore, chitosan is inexpensive, U. Food and Drug Administration (FDA)-approved food additive and has shown no toxicity in biological systems till now [<NUM>].

It is well known that chitosan is insoluble in water due to its weak basicity. Therefore, chitosan was acetylated to create its water-soluble variants as reported in Sashiwa H. , Kawasaki N. , Nakayama A.

According to the present invention, to realize a water-soluble variant of chitosan, a partial substitution of the amine and hydroxyl groups of the chitosan with water soluble residues has been done, i.e., the substitutions took place only in some of the monomers of the chitosan polymer ('a' structure of formula (I)).

The present inventors finely modulated the degree of acetylation to find an optimal balance between the solubility of the polymer, the labelling of the polymer with the dye for its biological imaging, the amount of positive charge present for the biological tissue staining and selecting the appropriate fluorescent NIR tag to suit the instrumentational/application-based needs of the final user.

In an embodiment, R<NUM> and R<NUM> are CH<NUM>CO in at most <NUM> % of the O-positions of the chitosan polymer structure of formula (I).

In an embodiment, R<NUM> and R<NUM> are CH<NUM>CO in a percentage of O-positions of formula (I) between <NUM>% and <NUM>%, preferably between <NUM>% and <NUM>%.

In an embodiment, the fluorescent water-soluble polycationic chitosan polymer of formula (I) has a degree of substitution of R<NUM> and R<NUM> with CH<NUM>CO between <NUM> and <NUM>, preferably between <NUM> and <NUM>.

In an embodiment, R<NUM> is CH<NUM>CO in at most <NUM> % of the N-positions of formula (I).

In an embodiment, R<NUM> is CH<NUM>CO in a percentage of N-positions of formula (I) between <NUM>% and <NUM>%.

In an embodiment, R<NUM> is Dye in at most <NUM> N-positions of formula (I).

In an embodiment, R<NUM> is Dye in between <NUM> and <NUM> N-positions of formula (I).

In an embodiment, the fluorescent water-soluble polycationic chitosan polymer of formula (I) has a degree of substitution of R<NUM> with CH<NUM>CO between <NUM> and <NUM>, preferably between <NUM> and <NUM>.

Dyes belonging to the class of cyanine have already found extensive use in clinical diagnostics; in particular, Indocyanine Green has been used for kidney function tests and fluorescence angiography for more than <NUM> years [<NUM>]. Principally, Cyanine fluorophores that cover the near infrared (NIR) region (<NUM>-<NUM>) are especially suitable for in vivo imaging and therapy due to their improved penetration depth (<NUM>-<NUM>) as compared with fluorophores that absorb and emit below <NUM> and in the visible wavelength region. In addition, in vivo background (or autofluorescence) and scattering from water and chromophores are minimized in the NIR range.

Cyanine <NUM> and cyanine <NUM> dyes absorb and emit light in the near-infrared region (NIR) (<NUM>-<NUM>). These dyes are especially suitable for in vivo imaging and therapy, since biological tissues absorb poorly in the near-infrared spectral region, thereby allowing NIR dye light to penetrate deeply in such tissues. In addition, these dyes do not get affected from biological tissues' auto-fluorescence in the near-infrared region.

In the current invention, the water-soluble chitosan was conjugated to NIR cyanine dyes, specifically to positively, negatively, and neutrally charged cyanine <NUM> and cyanine <NUM> dyes. The Cyanine <NUM> and cyanine <NUM> dyes demonstrate higher chemical and photostability, reproducibility, signal-to-noise ratio, and fluorescent quantum yields as compared to MHI148-PEI. These improved properties also lead to lower dosage for in vivo use.

In an embodiment, the Dye residue has an excitation wavelength between <NUM> to <NUM>, preferably <NUM> to <NUM>.

In an embodiment, the polymer has an average molecular weight between <NUM> kDa and <NUM>,<NUM> kDa, preferably <NUM> kDa and <NUM> kDa.

In an embodiment, the polymer has an average molecular weight comprised between <NUM> and <NUM> kDa.

In an embodiment, the Dye residue is selected from formulas (III) and (IV)
<CHM>
wherein residues R<NUM>-R<NUM> and Q have the meanings set forth above.

In an embodiment, R<NUM> and R<NUM> are independently selected from (CH<NUM>)<NUM>SO<NUM>-, (CH<NUM>)<NUM>N+(CH<NUM>)<NUM> and salts thereof.

In an embodiment, D is selected from the group consisting of
<CHM>.

In an embodiment, the Dye residue is a compound of formula (V):
<CHM>
wherein WSC is the water-soluble chitosan polymer that is conjugated to the dye molecule through an amide bond formed in the coupling reaction of carboxyl group of Dye of D
<CHM>
corresponding to the carboxyl group residue having the structure with the NH group present in the N-position of the chitosan polymer of formula (I).

In an embodiment, the Dye residue is a compound of formula (VI)
<CHM>
wherein WSC is the water-soluble chitosan polymer that is conjugated to the Dye through an amide bond formed in the coupling reaction of carboxyl group of the Dye corresponding to the carboxyl group of D residue having the structure
<CHM>
with the NH group present in the N-position of the chitosan polymer of formula (I).

In an embodiment, the Dye residue is a compound of formula (VII)
<CHM>
wherein WSC is the water-soluble chitosan polymer that is conjugated to the Dye through an amide bond formed in the coupling reaction of carboxyl group of the Dye corresponding to the carboxyl group of D residue having the structure
<CHM>
with the NH group present in the N-position of the chitosan polymer of formula (I).

In an embodiment, the present invention relates to a fluorescent water-soluble polycationic chitosan polymer of formula (I) for use in an in vivo imaging method of a glycosamine-containing structure in a mammal.

In an embodiment, the present invention concerns a fluorescent water-soluble polycationic chitosan polymer of formula (I) for use in a method of imaging a glycosamine-containing structure in a mammal comprising the steps of:.

The in vivo imaging method allows for visualizing the glycosamine-containing structure for diagnosing a vascular pathology or condition, like for example kidney or lung diseases.

The in vivo imaging method allows for assessing the effectiveness of a therapeutic treatment of the glycosamine-containing structure, like for example in case of haemangiomas treatment.

In fact, the imaging method allows visualizing and/or quantifying internal glycosamine-containing structures, preferably inner lumen of blood vessels and/or capillaries, i.e. the vascularization. The term vascularization refers to the density of blood vessels in a tissue or organ and, as will be understood by the skilled person, an increase or a decrease of the vascularization is often a diagnostic finding associated to a pathological state, thereby the disclosed method provides a diagnostic method for determining vascular changes occurring in diseased subjects in comparison to healthy subjects. The vascular staining comprises staining of vessels, peritubular capillaries, glomeruli and pre- or post-glomerular capillaries for providing an indication of pathological conditions, wherein the fluorescent water-soluble polycationic chitosan polymer of formula (I) does not enter the cell but stays in the lumen.

The imaging method herein described also allows for studying the vascularization of organs, preferably kidney.

In an embodiment, the glycosamine-containing structure is selected from blood vessels including vascular networks of capillaries of a mammal, preferably kidney vasculature.

In an embodiment, the glycosamine-containing structure is selected from:.

In an embodiment, the polymer of formula (I) is suitable for administration to the mammal and the fluorescent signal emitted from the fluorescent polymer of formula (I) is detected and visualised <NUM>-dimensionally by optical imaging.

In an embodiment, the fluorescent water-soluble polycationic chitosan polymer of formula (I) is suitable for administration to the mammal intravenously or locally.

The imaging method is accomplished in a period that is such to allow the complete binding of the fluorescent water-soluble polycationic chitosan polymer of formula (I) to glycosamine-containing structures in the inner lumen of biological structures. The skilled person understands that the timing depends on specific parameters of the samples, e.g., size, volume and the like.

In an embodiment, the in vivo detection of the fluorescence emitted signal from the fluorescent water-soluble polycationic chitosan polymer of formula (I) is performed by means of sensors for fluorescent light, i.e., cameras or little sensors for transcutaneous measurement or intravascular applications.

In an embodiment, the mammal is a laboratory animal selected from a mouse, a rat, a guinea pig, a cat, a dog, a sheep, a goat, a pig, a cow, a horse, and a primate (not a human).

In an embodiment, the mammal is a laboratory mammalian model of kidney disease linked to cardiovascular defects, wherein the disease can be created by causing an appropriate direct or indirect injury and/or by means of a surgical technique, a controlled administration of toxic agents or by means of a transgenesis, wherein transgenesis can be induced by pronuclear injection (additive transgenesis), homologous recombination (targeted transgenesis) or by gene specific silencing of gene function (knock-down) that can be determined by RNA interference (RNAi) or CRISPR/Cas9-based technology [<NUM>], [<NUM>].

In a further embodiment, the present invention concerns an in vitro or ex vivo imaging method of glycosamine-containing structures in a biological sample comprising contacting the biological sample with at least one fluorescent water-soluble polycationic chitosan polymer of formula (I) and visualizing the glycosamine-containing structure by means of fluorescence microscopy.

In an embodiment, the detection of the fluorescence emitted signal from the fluorescent water-soluble polycationic chitosan polymer of formula (I) is performed by means of fluorescence microscopy, preferably confocal or light-sheet microscopy, on harvested biological samples, wherein the sample is an isolated organ or a tissue section, preferably a liver, a muscle, a skin, a heart, a kidney, a brain, a lung, more preferably a kidney.

In an embodiment, the imaging method comprises the following steps, preferably in the indicated sequence:.

In a preferred embodiment, the imaging method comprises the following steps:.

In an embodiment, the kit contains a washing agent, preferably an isosmotic solution and/or a physiological buffer. Preferably, the washing agent is selected from phosphate-buffered saline (PBS) or Saline/EDTA/Heparin.

In an embodiment, the kit includes a fixing agent for preserving the tissue structures, more preferably a solution of formaldehyde in an isosmotic solution and/or a physiological buffer within a range of from <NUM>% (w/v) to <NUM>% (w/v), preferably <NUM>% to <NUM>% (w/v).

In an embodiment, the kit comprises a clearing agent that does not interfere with the effectiveness of the staining and/or with the detection of the fluorescence of the fluorescent water-soluble polycationic chitosan polymer formula (I), preferably ethyl-cinnamate (ECi).

The following examples are intended to illustrate aspects of the present invention and should not be construed as limiting the scope thereof as defined by the claims. They provide a detailed description of the synthesis of the fluorescent water-soluble polycationic chitosan polymer of formula (I), along with the demonstration of its photostability, safety to use in biological testing and biological application as imaging agents (Example <NUM>; <FIG>).

A mixture of <NUM>,<NUM>,<NUM>-trimethyl-<NUM>-indole-<NUM>-sulfonic acid (<NUM>, <NUM> mmol) and (<NUM>-bromopropyl) trimethyl ammonium bromide (<NUM>, <NUM> mmol) in <NUM>,<NUM>-dichlorobenzene (<NUM>) was heated at <NUM> for <NUM> hours under argon. The mixture was cooled to room temperature and the solvent was decanted. The crude product was washed with dichloromethane, dissolved in acetone and re-precipitated into a large volume of ethyl acetate to obtain a red solid (Compound <NUM>, <NUM>, yield <NUM>%), which was used in the next step without further purification.

A mixture of <NUM>-ethyl-<NUM>,<NUM>,<NUM>-trimethylindolinium iodide (<NUM>, <NUM> mmol), Vilsmeier-Haack reagent (<NUM>, <NUM> mmol) and anhydrous sodium acetate (<NUM>, <NUM> mmol) in <NUM> of absolute ethanol was refluxed for <NUM> hours under argon. The reaction mixture was cooled to room temperature, and then concentrated under reduced pressure to yield a brownish green residue. The residue was suspended in dichloromethane, filtered, and dried in vacuum. The crude product was purified by silica gel column chromatography in methanol/dichloromethane = <NUM>/<NUM> to yield a golden-green solid (Compound <NUM>, yield <NUM>%). <NUM>H NMR (<NUM>, CDCl<NUM>): δ <NUM> (t, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (t, <NUM>), <NUM> (q, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM>(m, <NUM>), <NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>).

A mixture of bromide salt of compound <NUM> (<NUM>, <NUM> mmol), Vilsmeier-Haack reagent (<NUM>, <NUM> mmol) and anhydrous sodium acetate (<NUM>, <NUM> mmol) in <NUM> of absolute ethanol was refluxed for <NUM> hours 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 dichloromethane. The residue was suspended in methanol/dichloromethane = <NUM>/<NUM>, filtered, and dried in vacuum to yield a golden-green solid (Compound <NUM>, yield <NUM>%). <NUM>H NMR (<NUM>, D<NUM>O): δ <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>) m <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>) <NUM> (s, <NUM>), <NUM> (d, J=<NUM>, <NUM>). LRMS (m/z): calcd: <NUM>, found: <NUM>.

In a <NUM> round-bottom flask, sodium pellets (<NUM>, <NUM> mmol) were dissolved completely in methanol, and then <NUM>-((<NUM>-Hydroxyphenyl)amino)-<NUM>-oxobutanoic acid (<NUM>, <NUM> mmol) was added and stirred altogether at room temperature for <NUM> hours. The reaction was stopped and precipitated in diethyl ether. The precipitate was filtered and dried to obtain a white solid, which was directly used for the next step (Compound <NUM>, <NUM>% yield).

A mixture of compound <NUM> (<NUM>, <NUM> mmol) and <NUM>-aminobutanoic acid (<NUM>, <NUM> mmol) in DMSO was heated at <NUM> for <NUM> hours. The reaction mixture was cooled to room temperature and dissolved in dichloromethane. It was extracted twice in water and once in brine. The organic layer was collected, and dried using sodium sulphate, filtered, and dried overnight. The crude product was purified by silica gel column chromatography in methanol/dichloromethane = <NUM>/<NUM> to yield a golden-blue solid (SV620C-<NUM>, yield <NUM>%). <NUM>H NMR (<NUM>, DMSO) : δ <NUM> (t, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (t, <NUM>), <NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (m, <NUM>), <NUM> (t, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>).

A mixture of compound <NUM> (<NUM>, <NUM> mmol) and compound <NUM> (<NUM>, <NUM> mmol) in DMSO and water (<NUM>:<NUM>) was heated at <NUM> for <NUM> hours. The reaction mixture was cooled to room temperature and precipitated slowly in ethyl acetate and absolute ethanol (<NUM>:<NUM>) with <NUM>% formic acid. The precipitate was filtered and washed with excess ethanol. The crude product was purified by RP C18 chromatography to yield a golden-green solid compound (SV770Z-<NUM>, yield <NUM>%). <NUM>H NMR (<NUM>, D<NUM>O): δ <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (t, <NUM>), <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, J = <NUM>, <NUM>).

<NUM>-amino-<NUM>,<NUM>-naphthalenedisulfonic acid disodium salt (<NUM>, <NUM> mmol, <NUM> eq) was added to a <NUM> jacketed flask connected to a cryostat at -<NUM>. Concentrated hydrochloric acid (<NUM>) was added very slowly with continuous stirring. Once fully dissolved, a solution of sodium nitrate (<NUM>, <NUM> mmol, <NUM> eq) in <NUM> water was added very slowly using a dropping funnel over <NUM> minutes. The reaction was run for <NUM> mins at <NUM>. Temperature was again brought down to -<NUM> and a solution of tin (II) chloride dihydrate (<NUM>, <NUM> mmol, <NUM> eq) in concentrated hydrochloric acid (<NUM>) was added slowly using a dropping funnel over <NUM> hours. The reaction was allowed to run at <NUM> for <NUM> hour. The reaction mixture was filtered and washed with excess absolute ethanol. The precipitate was dried under vacuum for <NUM> days to give compound <NUM> (yield <NUM> %). <NUM> NMR (<NUM>, DMSO-d6): δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>).

Compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq) was taken in a round bottom flask with <NUM>-methyl-<NUM>-butanone (<NUM>, <NUM> mmol, <NUM> eq) and <NUM> of acetic acid. The reaction mixture was refluxed at <NUM> for <NUM> hours with continuous stirring. The reaction was then cooled down and precipitated into ethyl acetate. The precipitate was filtered over gooch funnel and dried under vacuum to give compound <NUM> (yield <NUM>%). <NUM>H NMR (<NUM>, D2O) : δ <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> (s, <NUM>).

Compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq) was taken in a round-bottom flask along with <NUM>,<NUM>-propanesultone (<NUM>, <NUM> mmol, <NUM> eq) in <NUM>,<NUM>-dichlorobenzene (<NUM>) and refluxed at <NUM> for <NUM> hours. The reaction was then cooled down and the solvent was decanted. The precipitate was triturated with <NUM> of ethyl acetate and filtered over gooch funnel. The solid precipitate was further triturated with ethyl acetate (<NUM> X <NUM>) after which it was re-dissolved in hot methanol (<NUM>) and precipitated in isopropanol. The precipitate was filtered and washed with isopropanol, diethyl ether and ethyl acetate. The solid was dried under vacuum to give compound <NUM> (yield <NUM>%). Compound <NUM> was directly used for the next step.

In a <NUM> round-bottom flask, Compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq) was stirred along with triethylamine (<NUM>, <NUM> mmol, <NUM> eq) in absolute ethanol (<NUM>) at room temperature for <NUM> mins till it fully dissolved. N-Phenyl-<NUM>-bromo-<NUM>-(phenylimino)-<NUM>-propene-<NUM>-amine (<NUM>, <NUM> mmol, <NUM> eq) and acetic anhydride (<NUM>) was added to the flask and the reaction was stirred at <NUM> for <NUM> hr. The reaction was cooled and precipitated in excess acetone. The precipitate was filtered over gooch funnel and was dried under vacuum. The crude was purified using reverse-phase C18 column chromatography to yield a blue powder (Compound <NUM>, <NUM>, yield <NUM> %). <NUM>-NMR (<NUM>, D<NUM>O): δ <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (dd, J = <NUM>, <NUM>, <NUM>), <NUM> (d, <NUM>), <NUM> (d, J = <NUM>, <NUM>). ε: <NUM>,<NUM>-<NUM>-<NUM>. λab: <NUM>, λem: <NUM> [<NUM>].

In a <NUM> round-bottom flask under argon atmosphere, Compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq) was taken along <NUM>-(<NUM>-carboxyethyl) benzene boronic acid (<NUM>, <NUM> mmol, <NUM> eq) and cesium carbonate (<NUM>, <NUM> mmol, <NUM> eq) in absolute ethanol and water (<NUM>/<NUM>) and stirred at room temperature for <NUM> mins, till it was fully dissolved. Tetrakis(triphenylphosphine)palladium (<NUM>) (<NUM>, <NUM> mmol, <NUM>% by weight) was added to the flask and the temperature was raised to <NUM>. The reaction was stirred for <NUM> hr and then extracted in ethyl acetate and water. The crude was purified using reverse-phase C18 column chromatography to yield a golden-blue solid (SV680A-<NUM>, <NUM>, yield <NUM> %). <NUM>-NMR (<NUM>, D<NUM>O): δ <NUM> (s, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM>-<NUM> (m, J = <NUM>, <NUM>), <NUM> (t, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (d, J = <NUM>, <NUM>), <NUM> (t, J = <NUM>, <NUM>), <NUM>-<NUM> (m, <NUM>), <NUM>-<NUM> (m, <NUM>). ε: <NUM>,<NUM>-<NUM>-<NUM>. λab: <NUM>, λem: <NUM> [<NUM>].

Compound <NUM> was synthesised using the same procedure as reported in scheme <NUM>, <NUM> and <NUM>, with the only difference of using <NUM>-Amino-<NUM>-naphthalenesulfonic acid as the starting material in Scheme <NUM>.

Compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq) was taken in a round-bottom flask and dissolved completely in sulfolane (<NUM>), to which <NUM>,<NUM>-propanesultone (<NUM>, <NUM> mmol, <NUM> eq) was added. Altogether they were refluxed at <NUM> for <NUM> hours. The reaction was then cooled down and the solvent was decanted. <NUM> of methanol was added to quench the reaction and dissolve the solid block, which was then precipitated in acetone. The precipitate was filtered over gooch funnel and washed with acetone, ethanol, and diethyl ether. The solid was dried under vacuum to give compound <NUM> (yield <NUM>%). Compound <NUM> was used for synthesising SV680A-<NUM> using the same procedure as used for SV680A-<NUM>.

In a <NUM>-necked round-bottom flask, SV620C-<NUM> (<NUM>, <NUM> mmol, <NUM> eq) was dissolved in dichloromethane (<NUM>) in an ice bath under argon atmosphere. Acetyl chloride (<NUM>µL, <NUM> mmol,<NUM> eq) and N, N-diisopropylethylamine (<NUM>µL, <NUM> mmol, <NUM> eq) were injected into the reaction flask and stirred for <NUM> mins. The reaction was quenched in <NUM> N hydrochloric acid solution (<NUM>) and then dried off over rotary evaporator. The crude product was purified by silica gel column chromatography in methanol/dichloromethane = <NUM>/<NUM> to yield a golden-green solid (SV770C-<NUM>, yield <NUM>%). <NUM>H NMR (<NUM>, DMSO): δ <NUM> (t, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (t, <NUM>), <NUM> (m, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (m, <NUM>), <NUM> (t, <NUM>), <NUM> (d, J=<NUM>, <NUM>), <NUM> (d, J=<NUM>, <NUM>).

A mixture of compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq) and compound <NUM> (<NUM>, <NUM> mmol, <NUM> eq) in DMSO and water (<NUM>:<NUM>) was heated at <NUM> for <NUM> hours. The reaction mixture was cooled to room temperature and acidified with <NUM>% formic acid. The reaction was extracted in dichloromethane and water, dried over sodium sulfate and then put on rotary evaporator overnight to give solid crude. The crude product was purified by silica gel chromatography to yield a golden-green solid compound (SV770C-<NUM>, yield <NUM>%). <NUM>H NMR (<NUM>, D<NUM>O): δ <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (m, <NUM>), <NUM> (t, <NUM>), <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (m, <NUM>), <NUM> (s, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (d, J= <NUM>, <NUM>), <NUM> (s, <NUM>), <NUM> - <NUM> (m, J = <NUM>, <NUM>).

In a <NUM> round-bottom flask, Chitosan (<NUM>, <NUM> mmol) was stirred in <NUM> of methane sulfonic acid. After <NUM> hours, acetyl chloride was added to this flask, and was stirred altogether for <NUM> hours. The reaction was stopped by adding <NUM> ice to the flask and the reaction mixture was transferred to a dialysis bag (Slide-A-Lyzer™ Dialysis Flask, <NUM> MWCO, <NUM>) and dialysed against Milli-Q water. After <NUM> hours, the content of the dialysis bag was neutralised using sodium bicarbonate solution and continued to dialyse for <NUM> days. The dialysing medium was changed every <NUM> hours. After <NUM> days, the content of the dialysis bag was freeze-dried to give a white solid (WS Chitosan, yield <NUM>%).

In a <NUM> round-bottom flask, a mixture of SV680A-<NUM> (<NUM>, <NUM> mmol), N-Hydroxysuccinimide (<NUM>, <NUM> mmol) and <NUM>-Ethyl-<NUM>-(<NUM>-dimethylaminopropyl)carbodiimide (<NUM>, <NUM> mmol) was stirred in <NUM> anhydrous DMSO at room temperature under Argon for <NUM> mins. To this reaction mixture, a solution of WS Chitosan (<NUM>, <NUM> mmol) in <NUM> anhydrous DMSO was added and stirred altogether for <NUM> hours at room temperature. The reaction was stopped, and the content of the flask was transferred to a dialysis bag (Pur-A-Lyzer™ Mega Dialysis Kit, <NUM> MWCO, <NUM>-<NUM>). The reaction mix was dialysed against 1X PBS buffer (pH <NUM>) for <NUM> hours with change of the dialysis medium every <NUM> hours, and later against Milli-Q water for another <NUM> hours. The resulting solution inside the dialysis bag was lyophilised to obtain a blue powder (SV680A-<NUM>-WS Chitosan, <NUM>% yield). The chemical structure of the compound is shown in <FIG>.

In a <NUM> round-bottom flask, a mixture of SV680A-<NUM> (<NUM>, <NUM> mmol), N-Hydroxysuccinimide (<NUM>, <NUM> mmol) and <NUM>-Ethyl-<NUM>-(<NUM>-dimethylaminopropyl)carbodiimide (<NUM>, <NUM> mmol) was stirred in <NUM> anhydrous DMSO at room temperature under Argon for <NUM> mins. To this reaction mixture, a solution of WS Chitosan (<NUM>, <NUM>\ mmol) in <NUM> anhydrous DMSO was added and stirred altogether for <NUM> hours at room temperature. The reaction was stopped, and the content of the flask was transferred to a dialysis bag (Pur-A-Lyzer™ Mega Dialysis Kit, <NUM> MWCO, <NUM>-<NUM>). The reaction mix was dialysed against 1X PBS buffer (pH <NUM>) for <NUM> hours with change of the dialysis medium every <NUM> hours, and later against Milli-Q water for another <NUM> hours. The resulting solution inside the dialysis bag was lyophilised to obtain a blue powder (SV680A-<NUM>-WS Chitosan, <NUM>% yield). The chemical structure of the compound is shown in <FIG>.

In a <NUM> round-bottom flask, a mixture of SV620C-<NUM> (<NUM>, <NUM> mmol), N-Hydroxysuccinimide (<NUM>, <NUM> mmol) and <NUM>-Ethyl-<NUM>-(<NUM>-dimethylaminopropyl)carbodiimide (<NUM>, <NUM> mmol) was stirred in <NUM> anhydrous DMSO at room temperature under Argon for <NUM> mins. To this reaction mixture, a solution of WS Chitosan (<NUM>, <NUM> mmol) in <NUM> anhydrous DMSO was added and stirred altogether for <NUM> hours at room temperature. The reaction was stopped, and the content of the flask was transferred to a dialysis bag (Pur-A-Lyzer™ Mega Dialysis Kit, <NUM> MWCO, <NUM>-<NUM>). The reaction mix was dialysed against 1X PBS buffer (pH <NUM>) for <NUM> hours with change of the dialysis medium every <NUM> hours, and later against Milli-Q water for another <NUM> hours. The resulting solution inside the dialysis bag was lyophilised to obtain a blue powder (SV620C-<NUM>-WS Chitosan, <NUM>% yield). The chemical structure of the compound is shown in <FIG>.

In a <NUM> round-bottom flask, a mixture of SV770Z-<NUM> (<NUM>, <NUM> mmol), N-Hydroxysuccinimide (<NUM>, <NUM> mmol) and <NUM>-Ethyl-<NUM>-(<NUM>-dimethylaminopropyl)carbodiimide (<NUM>, <NUM> mmol) was stirred in <NUM> anhydrous DMSO at room temperature under Argon for <NUM> mins. To this reaction mixture, a solution of WS Chitosan (<NUM>, <NUM> mmol) in <NUM> anhydrous DMSO was added and stirred altogether for <NUM> hours at room temperature. The reaction was stopped, and the content of the flask was transferred to a dialysis bag (Pur-A-Lyzer™ Mega Dialysis Kit, <NUM> MWCO, <NUM>-<NUM>). The reaction mix was dialysed against 1X PBS buffer (pH <NUM>) for <NUM> hours with change of the dialysis medium every <NUM> hours, and later against Milli-Q water for another <NUM> hours. The resulting solution inside the dialysis bag was lyophilised to obtain a blue powder (SV770Z-<NUM>-WS Chitosan, <NUM>% yield). The chemical structure of the compound is shown in <FIG>.

In a <NUM> round-bottom flask, a mixture of SV770C-<NUM> (<NUM>, <NUM> mmol), N-Hydroxysuccinimide (<NUM>, <NUM> mmol) and <NUM>-Ethyl-<NUM>-(<NUM>-dimethylaminopropyl)carbodiimide (<NUM>, <NUM> mmol) was stirred in <NUM> anhydrous DMSO at room temperature under Argon for <NUM> mins. To this reaction mixture, a solution of WS Chitosan (<NUM>, <NUM> mmol) in <NUM> anhydrous DMSO was added and stirred altogether for <NUM> hours at room temperature. The reaction was stopped, and the content of the flask was transferred to a dialysis bag (Pur-A-Lyzer™ Mega Dialysis Kit, <NUM> MWCO, <NUM>-<NUM>). The reaction mix was dialysed against 1X PBS buffer (pH <NUM>) for <NUM> hours with change of the dialysis medium every <NUM> hours, and later against Milli-Q water for another <NUM> hours. The resulting solution inside the dialysis bag was lyophilised to obtain a blue powder (SV770C-<NUM>-WS Chitosan, <NUM>% yield). The chemical structure of the compound is shown in <FIG>.

Time dependence photostability evaluation of fluorescent water-soluble chitosan polymers of formula (I) (SV680A-<NUM>-WS chitosan, SV680A-<NUM>-WS chitosan, SV770Z-<NUM>-WS chitosan, SV620C-<NUM>-WS chitosan) and the dye SV620C-<NUM> conjugated to PEI were performed under continuous illumination in PBS at <NUM>, using Cary <NUM> UV-vis spectrophotometer (Agilent) and FS5 spectrofluorometer (Edinburgh Instruments) at University of Parma. Mean emission intensity of the markers was calculated by averaging the experimental values collected from <NUM> replicative studies. The values were collected at time <NUM> mins, and after <NUM> minutes of continuous illumination.

SV620C-<NUM>-PEI was compared with SV620C-<NUM>-WS chitosan to evaluate the efficiency of WS chitosan as a substitute to PEI, which has been used in the prior art. SV620C-<NUM>-WS chitosan was found to be more stable than SV620C-<NUM>-PEI, with <NUM>% degradation of the WS chitosan conjugate as compared to <NUM>% degradation of the PEI conjugate. This indicates that WS chitosan offers higher stability to the dye, as compared to PEI, and therefore higher stability in comparison to MHI-<NUM>-PEI.

Among the remaining WS chitosan conjugates, SV770Z-<NUM>-WS chitosan and SV680A-<NUM>-WS chitosan show the highest stability, as seen in <FIG>.

Cytotoxicity assays were performed on the fluorescent water-soluble chitosan polymers of formula (I) (SV680A-<NUM>-WS chitosan and SV770Z-<NUM>-WS chitosan) and the dye SV620C-<NUM> conjugated to PEI at various concentrations:.

The cells were left to incubate with the markers at various concentrations for <NUM>, after which, they were treated with trypsin and the supernatants and cells were collected.

The samples thus obtained were labelled and analysed following the experimental laboratory procedures, for the evaluation of apoptosis. The test was performed in <NUM>-well plates with seeded <NUM>,<NUM> cells of the line 3T3-L1 (Merck) per well, in a volume of <NUM> of complete DMEM medium (<NUM>% FBS, <NUM>% Penicillin / Streptomycin / L-Glutamine). The cells were allowed to adhere to the surface overnight. The next day the fluorescent markers were dissolved in complete DMEM medium, and the treatment was carried out with the cells in <NUM> of DMEAM medium with different concentrations of compounds. The cells were left to incubate for <NUM>. After <NUM> hours, the supernatants and cells were collected after trypsinization. The samples thus obtained were labelled and analysed following the procedures laboratory tests, for the evaluation of apoptosis. Apoptosis was assessed by flow cytometric analysis.

The percentage of apoptotic cells in the different phases of the apoptotic process, was calculated using the formula: ((test% - control%) * <NUM> / (<NUM> - control%)).

From the tests carried out it is concluded that SV620C-<NUM>-PEI induces <NUM>% cell death at all concentrations tested. The remaining batches, according to our knowledge, do not induce significant apoptosis, although the values indicate a slight increase in specific apoptosis.

The fluorescent polymers of formula (I) were tested in mice. Organs were harvested and optically cleared following retrograde perfusion.

All animal experiments were conducted in accordance with the German Animal Protection Law and approved by the local authority (Regierungspräsidium Nordbaden, Karlsruhe Germany in agreement with EU guideline <NUM>/<NUM>/EU).

Procedure: Mice retrograde perfusion, vascular staining and optical tissue clearing were performed according to the protocol described in Huang et al [<NUM>]. Among the collected organs, mouse kidney sections were imaged by confocal microscopy for the <NUM>-dimensional visualization of the vascular structures.

Renal vessels, peritubular capillaries and glomeruli are shown in <FIG> (SV770Z-<NUM>-WS Chitosan staining) and <FIG> (SV680A-<NUM>-WS Chitosan staining).

A comparison between the new fluorescent chitosan polymers and the previously disclosed MHI148-PEI is shown in <FIG>.

Claim 1:
A fluorescent water-soluble polycationic chitosan polymer having formula (I) and salts thereof:
<CHM>
wherein
a is an integer between <NUM> and <NUM>,<NUM>;
R<NUM> and R<NUM> are independently selected from the group consisting of CH<NUM>CO and H, provided that at least <NUM>% of the O-positions of the chitosan polymer structure is substituted with CH<NUM>CO;
R<NUM> is selected from the group consisting of CH<NUM>CO, H and Dye, provided that (i) at least one Dye residue is present in the chitosan polymer structure, (ii) and at least <NUM>% of the N-positions of the chitosan polymer structure is substituted with CH<NUM>CO;
Dye is a polymethine dye, having a general formula (II) :
<CHM>
wherein
W represents <NUM>-<NUM> carbon atoms forming a benzocondensed or a naphto-condensed ring;
R<NUM> and R<NUM> are independently selected from the group consisting of CH<NUM>CH<NUM>, (CH<NUM>)<NUM>SO<NUM>-, (CH<NUM>)<NUM>N+(CH<NUM>)<NUM>;
R<NUM> and R<NUM> are independently selected from the group consisting of H, SO<NUM>-;
Q is selected from the group consisting of
<CHM>
wherein D is selected from the group consisting of
<CHM>