New ferritin-based delivery system for bodipy molecules

Abstract

The present invention relates to the field of neurodegenerative diseases detection and diagnosis, in particular to a delivery system comprising bodipy markers, said delivery system consisting of a modified humanized ferritin from Archaeoglobus fulgidus (HumAfFt) and a bodipy fluorescent marker that selectively binds the TAU protein, compositions comprising said delivery system and methods ad uses thereof.

Description

[0001] NEW FERRITIN-BASED DELIVERY SYSTEM FOR BODIPY MOLECULES

[0002] The present invention relates to the field of neurodegenerative diseases detection and diagnosis, in particular to a delivery system comprising Bodipy markers, said delivery system consisting of a modified humanized ferritin from Archaeoglobus fulgidus (HumAfFt) and a bodipy fluorescent marker that selectively binds the TAU protein, compositions comprising said delivery system and methods ad uses thereof.

[0003] STATE OF THE ART

[0004] Alzheimer's disease (AD) is a neurodegenerative disease responsible for more than 80% of cases of senile dementia. The limited scientific evidence underlying the pathogenic mechanisms of AD has made it difficult to develop targeted and effective therapies and diagnoses and, to date, no solutions have been found that guarantee an improvement in the life of high-risk patients or effective prevention that allows to reduce health expenditure.

[0005] Despite numerous studies to elucidate the pathogenic mechanisms and efforts of the pharmaceutical industry, there is still no effective therapy to treat AD or significantly block the progression of symptoms.

[0006] Consequently, in order to develop a therapeutic strategy capable of preventing AD, research has focused on the study of accurate and specific diagnostic methods in order to monitor the progression of the disease. Numerous advances have been made thanks to the introduction of cerebrospinal fluid analysis (CSF) and diagnostic neuroimaging techniques. These techniques allow to study the presence of functional alterations in the brain and biomarkers associated with AD.

[0007] In particular, the coupled units of hyperphosphorylated protein (P-Tau) and the total- Tau monomers (t-Tau), the key brick of the neurofibrillary tangles (NFTs), appeared as the most reliable markers for AD. Recently, it has been shown that the presence and quantity of NFTs in patients with dementia can be related to the progression and clinical manifestations of the disease itself. In this framework, possible identification of early NFTs in the retinal tissue was considered as a most promising diagnostic tool. Therefore, NFTs have become the preferred biomarkers for the development of fluorescent probes that can be used in the clinical diagnosis of AD by simple fluorescence detection from the retina with an appropriate TAU specific fluorescent probe [Verwilst P. et al., Chem. Soc. Rev., 2018, 47, 2249-2265], In this context the construction of a computational model of the TAU oligomers (involved in the nucleation of the fibrillogenesis process) [Sci Rep. 2022 Mar 28;12(1):5257] allowed researchers towards the realization of early diagnostic methods through the rational design of highly specific Tau fluorophores. Along this line, WO2022 / 224151 discloses novel fluorescent probes (namely BT-1) for the identification of AD biomarkers have been patented recently and their specific binding to TAU tangles has been demonstrated in in vitro models. Although highly specific, BT-1 suffer from intrinsic low water solubility and it’s in vivo delivery to the retina may not be readily feasible.

[0008] Therefore, the need is strongly felt to identify a class of delivery cargoes capable of entering retinal cells and release the fluorescent molecule to the NFTs of the Tau protein in a highly selective way.

[0009] Ferritin proteins are natural nanocages capable of embedding small molecules and able to enter a number of human cells expressing the transferrin receptor on their surface. Ferritins are iron storage and transport proteins, found in most living organisms, characterized by a typical tetraeicosameric subunits assembly.1 Ferritin assembly results in the formation of a 8 nm diameter central cavity, which can be efficiently loaded with transition metals, drugs, fluorescent molecules or contrast agents.2-4 Thus, encapsulated molecules can be delivered through the ferritin nanocages, to target the transferrin receptor (TfR1 , or CD71), highly expressed in several cell types. These attributes make ferritins and their derivatives a powerful delivery system with potential application in nanomedicine.

[0010] Recombinant mammalian ferritins, however, are difficult to disassemble into subunits as they can be dissociated only under extreme conditions, thus rendering the encapsulation of many substrates impractical. Commonly employed cargo encapsulation techniques involve the disassembly and re-assembly of ferritins cage by pH jump, reaching drastic pH values (pH 2)9 which cause a partial and incomplete re-assembly.10

[0011] Recently, novel ferritins from lower eukariotes, bacteria and archea with different polymer association-dissociation thermodynamics and kinetics have emerged as possible alternative to human ferritin, once surface mutated in order to keep the receptor binding properties of their human counterpart and reduced immunogenicity. Among those, De Turris et. Al., Nanoscale. 2017 Jan 5;9(2):647-655 discloses a humanized ferritin from Archaeoglobus fulgidus (HumAfFt) capable of incorporating diverse substructures at neutral pH while being recognized and internalized in mammalian cells by human transferrin receptor TfR1 as human ferritin H homopolymer.

[0012] SUMMARY OF THE INVENTION The authors of the present invention had previously designed the Bodipy-based markers capable to bind with high affinity to the R3 region of the microtubule-binding tau protein, the region responsible of the propensity of the protein itself to assemble into fibrils. Said markers are disclosed in WO2022 / 224151 , herein enclosed by reference in its entirety, the preferred marker being identified as BT 1 and reported in Formula I below.

[0013] BT1 Formula II

[0014] Said markers have been shown to possess high binding affinity to TAU protein oligomers in the retinal tissue in vitro experiments. However, the markers disclosed in WO2022 / 224151 (hereinafter BT markers) have low water solubility which renders their delivery in vivo difficult as also shown in Figure 4D. Due to their low water solubility, the BT markers show no obvious possibility for in vivo targeted delivery.

[0015] In the present application, the authors have discovered that, ferritin nanocages known in the art were not able to efficiently upload the BT markers. The authors have identified a surprisingly efficient modified ferritin nanocage system, obtained by engineering a ferritin molecule and have found that said modified ferritin efficiently uploads BT markers, shows an efficient uptake by retinal cells in vivo and releases the uploaded BT molecule to the intracellular TAU oligomers.

[0016] At present, most of the conventional methods to detect tau neurofibrillary tangles are based on the post-mortem analysis of brain tissues. One major challenge to early diagnosis of tauopathies is represented by the complexity of conventional diagnostic methods as these generally involve not only the immunohistochemical demonstration of abnormal tau deposition in the brain, but also the detection of the presence or absence and amount of other non-tau proteins in the brain, as well as the study of the morphological characteristics of the tau in different regions of the brain.

[0017] Advantageously, thanks to their high affinity and specificity for the neurofibrillary tangles of the Tau protein, and thanks to the new cargo system provided in the present application, the fluorescent BT markers disclosed in WO2022 / 224151 the resulting delivery system of the invention can be used to provide new imaging methods providing images that can be subsequently analysed by a specialist in order to obtain information allowing an effective, non-invasive, early diagnosis of tauopathies or that can be used in the assessment of the effectiveness of a medical treatment of a tauopathy and / or of the progression of a tauopathy.

[0018] Therefore, the invention refers to the combination of one or more BT compound with an engineered recombinant ferritin cargo of specified sequence.

[0019] Therefore, object of the present invention is:

[0020] - A delivery system consisting of a nanocage of humanised ferritin from Archaelogus fulgidus (HumAfFt) of SEQ ID NO 1 , and the fluorescent marker of formula I: wherein X and Y are carbon atoms linked either by a double bond with E or Z configuration or by an aromatic or heteroaromatic para-substituted ring, or by an aromatic or heteroaromatic 1,4 disubstituted ring; R is hydrogen, halogen, NH(RA), N( A)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)s, or C5-7 aromatic or aliphatic heterocycle; and in each substituent, RA is: hydrogen; halogen, hydroxyl; CF3, a C1-7 saturated or unsaturated chain, linear or branched containing up to three independent heteroatoms selected among nitrogen oxygen and sulphur; C5-7 cycloalkyl; phenyl; C5-7 heterocycle or n-etilenglycol, wherein said HumAfFt is covalently bound to moiety comprising a thiol-reactive group.

[0021] -A composition comprising the delivery system according to any of the embodiments of the invention and one or more additional excipients and / or carriers.

[0022] -The use of the delivery system or of the pharmaceutical composition of the invention for the detection of neurofibrillary tangles of the Tau protein.

[0023] -A method for the detection of neurofibrillary tangles of the Tau protein comprising the steps of:

[0024] - contacting the delivery system or the composition according to the invention with a biological sample under conditions wherein said fluorescent marker binds to the neurofibrillary tangles of the tau protein;

[0025] - detecting said fluorescent marker bound to the biological sample.

[0026] -An imaging method comprising the steps of:

[0027] - administering the delivery system or the composition according to the invention to a subject,

[0028] - carrying out a non-invasive fluorescence imaging of the retina of said subject, wherein the detection of fluorescence from said fluorescent marker indicates the binding of said marker to the retina.

[0029] -A method for determining a fluorescence graph comprising the steps of:

[0030] - administering to a subject the delivery system or the composition according to the invention and carrying out a non-invasive quantitative fluorescence imaging on the retina of said subject, at a plurality of successive time instants ti, where i is 0 to n, thus obtaining corresponding fluorescence values;

[0031] - using the obtained fluorescence values for determining a graph of the fluorescence as a function of time.

[0032] - Use of the delivery system or the composition according to the invention in the diagnosis of a tauopathy. - A method for the diagnosis of neurodegenerative diseases characterised by tau tangles comprising the steps of:

[0033] - administering to a subject the delivery system or the composition according to the invention ; carrying out a non-invasive quantitative fluorescence imaging on the retina of said subject, wherein the presence of tangles indicates the presence of a neurodegenerative disease.

[0034] - A method for the preparation of a delivery system according to the invention, comprising the following steps: i) reacting a solution comprising a moiety comprising a thiol reacting group? with cysteine 54 of the HumAfFt of SEQ ID NO 1 thereby obtaining a moiety comprising a thiol reacting group -HumAfFt with a hydrophobic surface within the protein nanocage; ii) adding a solution of a fluorescent marker of formula I to the solution obtained in step i) thereby obtain a moiety comprising a thiol reacting group- HumAfFt conjugated with a fluorescent marker of formula I; iii) at the end of the reaction as determined by MALDI mass spectroscopy, adding a divalent cation salt in order to close the HumanAfFt as 24-mer, thereby creating an internal cavity.

[0035] DETAILED DESCRIPTION OF THE FIGURES

[0036] Figure 1 Molecular structure of the lead bodipy compound designed as BT1 and capable of high affinity binding to the Tau 1 protein.

[0037] Figure 2 Bioconjugation of NPM to HumAfFt. of SEQ ID NO 1. Mass Spectrometry (MALDI) analysis of 2A) HumAfFt control with a peak at 20254 Da and 20386 Da corresponding to the molecular mass of HumAfFt without and with the first methionine, respectively. 2B) NPM-labelled HumAfFt with peak without (20565 Da) and with (20700 Da) the first methionine. The peak at 20268 Da is related to HumAfFt unreacted.

[0038] Figure 3: UV-Vis spectra of BT1 in DMSO (A) and in Hepes aqueous solution (B). In panel A), 14 pM BT1 in 100% DMSO is shown and in panel B), 150 pM BT1 in buffer 20 mM Hepes is reported. All the spectra were measured by using a Jasco V-750 Spectophotometer, in the range 700 - 240 nm (Data interval 0.2 nm, Bandwidth 1.0 nm Response 0.24 sec Scan speed 400 nm / min). All spectra have been measured at 25°C in a 1 cm pathlength quartz cuvette.

[0039] Two major broad peaks are observed around 360 and 590 nm, whose relative intensities are different in DMSO or Hepes aqueous solution. In particular, the peak at 590 nm decreases when changing solvent from DMSO to water. The feature is ascribed to aggregation induced phenomenon in aqueous solvents, due to the poor solubility of BT 1 in water.

[0040] Figure 4: Fluorescence emission spectra of free (A and B) and ferritin embedded (C and D) BT1 compound. In panel A), BT1 at a final concentration 1 pM in 100% DMSO, is shown. In panel B), BT1 in 20 mM Hepes pH 7.5 at a final concentration 150 pM,is reported. In panel C), BT1 embedded in NPM-HumAfFt at a concentration of 1 pM is shown and in panel D), BT1 embedded in HumAfFt at a final concentration 2 pM in 20 mM Hepes, pH 7.4, is reported. All the spectra were measured by using a Shimadzu 6000 Fluorimeter in the range 540 - 700 nm (Aexc =520 nm, Bandwidth 5.0 nm, Scan speed 400 nm / min).

[0041] In the emission spectra (panel A, in DMSO, panel 4 B in Hepes aqueous solution), a dramatic decrease of the intensity of fluorescence emission is observed in aqueous solution with respect to DMSO. In agreement with the absorption spectra, the phenomenon is attributed to aggregation induced quenching of BT 1 in aqueous solution. Most interestingly, when BT1 is incorporated into HumAfFt (in aqueous solution, panel C), a significant aggregation induced quenching is observed, whereas, once BT 1 is embedded into the hydrophobic environment of the NPM-HumAfFt, even in aqueous solution, a high fluorescence intensity peak is observed (within the same scale as of free BT1 in DMSO). These data demonstrate that, when incorporated into NPM-HumAfFt, BT1 is not aggregated and displays full fluorescent properties. Panel D shows that the unmodified HumAfFt is not sufficient to efficiently incorporate BT 1 in acquoeus solution.

[0042] Figure 5. Fluorescence analysis of BT1-loaded NPM-HumAfFt and K18 Tau protein. Representative plot showing the increasing of BT1 fluorescence at 565 nm as a function of time (Aex = 520 nm) in the presence of unfibrillated K18 tau protein (70 pM) (CTRL) and fibrillated K18 upon induction with heparin (70 pM) after 1 day, 3 days and 7 days at 37 °C.

[0043] Figure 5 shows the ability of BT1-loaded in NPM-HumAfFt to bind to Tau fibrils in time-course experiments by fluorescence emission spectroscopy. Unfibrillated K18 domain (residues from 244 to 376) of human Tau protein containing the 4 microtubule-binding repeats and fibrillated K18 upon the addition of the aggregation promoter heparin (ratio 1:1) were analysed after the addition of BT1-loaded NPM-HumAfFT (5 pM in protein concentration). The fluorescence of the BT1-embedded in NPM-HumAfFt increases with the formation of K18 fibrils as shown in Fig. 5 where the maximum fluorescence (at Aem = 565 nm) is plotted as a function of time of fibrillogenesis. These data, demonstrated that BT1, when loaded into NPM-HumAfFt, is slowly transferred to the nascent Tau fibrils where its fluorescent properties are further magnified.

[0044] Figure 6. Retinal culture differentiation from human iPSCs express the transferrin receptor CD71. 6A) Temporal gene expression of retinal markers over a 30-day time course in human iPSC. Gene expression data are reported as relative expression (2-AACT) normalized to internal control (GAPDH) and compared to control sample (day 0, undifferentiated iPSCs). Error bars represent mean ± SEM of 3 biological replicates (3 batches). Day 0 was set at 1. Cell type markers: Stem cells-NANOG, Transferrin receptor-CD71 , Retinal ganglion cells-BRN3a; Photoreceptors-CRX; Muller glia-RLBP1. 6B) Representative confocal images of BRN3a, TLIJ1 and MAP2 staining in differentiated hiPSC-derived retinal neurons at day 30 of differentiation. Scale bar: 50 pm. Nuclei were stained with DAPI. 6C) Representative confocal images of CD71, at day 30 of hiPSC-derived retinal cultures. The cytoskeleton marker MAP2 was used to identify cells. Scale bar: 50 pm. Images show a maximum intensity projection of a region of interest (20 stacks, z=0.5 pm). 6D) Bar chart reporting the quantification of CD71 expression at day 6 and day 30 of retinal differentiation.

[0045] Figure 7. Internalization of the NPM-HumAfFT complex in human iPSC derived retinal neurons. 7A) Representative fluorescent confocal images of live hiPSC-derived retinal neurons (day 30) exposed to 150 nM rhodamine-ferritin conjugate to identify the upload and the localization of ferritin. The internalization was assessed for different hours of exposure (6-8-16-24-48h). Living cells are stained with Fluorescein Diacetate (FDA) and nuclei are stained with DAPI. Scale bar: 50 pm. Images show a maximum intensity projection of a region of interest (20 stacks, z=0.2 pm). 7B) Bar charts show the quantification as integrated density of rhodamine-ferritin conjugate (150 nM) internalized within retinal neurons (day 30). The fluorescence of rhodamine increases over time. All experiments were performed in triplicate (3 different batches) for different hours of exposure (6-8-16- 24-48h). Significant differences are reported compared to 6 hours (one-way ANOVA, ****p<0.0001, ***p<0.001). 7C) Representative fluorescent confocal images of NPM-HumAfFT -BT1 complex within live hiPSC-derived retinal neurons (day 30). Retinal neurons were incubated with 150 nM of NPM-HumAfFT -BT1 complex for 24 hours. Nuclei were stained with DAPI. Scale bar: 50 pm. Images show a maximum intensity projection of a region of interest (16 stacks, z=0.2 pm).

[0046] Figure 8. Evaluation of NPM-HumAfFT BT1 neurotoxicity in retinal neurons. 8A) Representative fluorescent confocal images of live hiPSC-derived retinal neurons (day 30) exposed to 150 nM of NPM-HumAfFT -BT1 complex for 24 hours to evaluate BT1 cytotoxicity. Living cells are stained with Fluorescein Diacetate (FDA). Scale bar: 50 pm. Images show a maximum intensity projection of a region of interest (20 stacks, z=0.2 pm). 8B) Bar charts showing the dose-response effect on cell survival following 24-hour exposure to NPM-HumAfFT -BT1 (dotted gray bars). Cells treated with 10% triton for 4 minutes are used as negative control (black bar), while cells untreated are used as positive control (white bar). Cells incubated with human NPM-HumAfFT (vehicle, light gray bars) are used as control to evaluate BT1 cytotoxicity. All experiments were performed in triplicate (3 different batches). Significant differences are reported compared to the positive control (ANOVA, p<0.05). Note that the cell viability was left unaltered by 24 hours incubation with 150 nM NPM-HumAfFT -BT1 complex.

[0047] Figure 9. BT1 detects hyperphosphorylated and oligomeric tau in frontotemporal dementia retinal neurons. Quantification of BT1 staining (obtained after administration of NPM-HumAfFT -BT1 complex for 24 hours to living neurons) and immunostaining performed after cell fixation, with specific antibodies against different forms of tau. Specifically, in (9A) we used anti- total TAU (HT7), in (9B) anti PHF-tau Ser202 / Thr205 (AT8) and in (9C) anti- oligomeric TAU (T22). Experiments were performed in retinal neurons (at day 30) derived from healtly control iPSC (iPS28) in control condition (WT CTRL), after treatment with okadaic acid (WT OA TREATED), and in retinal neurons derived from isogenic iPSC harboring the 10-16 FTD related intronic mutation (iVS10-16) (MUT).

[0048] 9A) Left: bar chart showing the % of the retinal cell area stained by total tau (HT7) in control cells, after treatment with Okadaic acid, and in mutant cells. Right: bar chart reporting the overlap between BT 1 staining and total tau (HT7) staining in the three conditions, estimated as the Manders’ Overlap Coefficient. Note that the amount of total tau and the BT1 / HT7 overlap are similar in the three conditions as expected.

[0049] 9B) Left: bar chart showing the % of the retinal cell area stained by phospho- tau (AT8) in control cells, after treatment with Okadaic acid, and in mutant cells. Right: bar chart reporting the overlap between BT 1 staining and phosphorylated tau (AT8) staining in the three conditions, estimated as the Manders’ Overlap Coefficient. Note that the amount of phospho-tau is higher in FTD mutant cells, and that the BT1 / AT8 overlap is increases when tau phosphorylation is higher.

[0050] 9C) Left: bar chart showing the % of the retinal cell area stained by oligomeric tau (T22) in control cells, after treatment with Okadaic acid, and in mutant cells. Right: bar chart reporting the overlap between BT 1 staining and tau oligomers (T22) staining in the three conditions, estimated as the Manders’ Overlap Coefficient. Note that oligomeric tau is higher in FTD mutant cells, and that the BT1 / T22 overlap is increases when tau phosphorylation is higher.

[0051] GLOSSARY IN THE MEANING OF THE INVENTION:

[0052] Bodipy-based markers or bodipy compounds or BT in the present description refers to the markers, in particular to markers BT 1-8 disclosed in WO2022 / 224151 and dpeicted in scheme 1 of the present description.

[0053] BT 1 in the present description and drawings refers to marker 3-((E)-4-((E)-4- (dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',T- f][1,3,2]diazaborinin-4-ium-5-uide also depicted in scheme 1 of the present description.

[0054] HumAfFt in the present description and drawings indicates the humanized ferritin from Archaeoglobus fulgidus of SEQ ID NO 1 disclosed in De Turris et. Al., Nanoscale. 2017 Jan 5;9(2):647-655.

[0055] X-HumAfFt in the present description refers to the HumAfFt of SEQ ID NO 1 as defined above, covalently bound to a moiety comprising a thiol-reactive group, X representing the moiety comprising a thiol-reactive group.

[0056] X-HumAfFt-BT in the present description refers to a delivery system consisting of a nanocage of HumAfFt of SEQ ID NO 1, covalently linked to a moiety comprising a thiol-reactive group “X” according to any of the embodiment disclosed in the present description and claims, and a fluorescent marker selected from BT1- 8.

[0057] Thiol-reacting group according to the present description refers to a chemical moiety or functional group that is capable of forming covalent bonds or reacting specifically with thiol (-SH) groups.

[0058] NFM-HumAfFt-BT in the present description refers to a delivery system consisting of a nanocage of HumAfFt of SEQ ID NO 1, covalently linked to N-substituted maleimide is N-(1-Pyrenil)maleimide (NPM) and a fluorescent marker selected from BT1-8.

[0059] NFM-HumAfFt-BT1 in the present description refers to a delivery system consisting of a nanocage of HumAfFt of SEQ ID NO 1, covalently linked to N-substituted maleimide is N-(1-Pyrenil)maleimide (NPM) and the fluorescent marker 3-((E)-4- ((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',T- f][1 ,3,2]diazaborinin-4-ium-5-uide (BT1).

[0060] Conjugated HumAfFt in the present description refers to a delivery system consisting of a nanocage of HumAfFt of SEQ ID NO 1, covalently linked to a moiety according to any of the embodiment disclosed in the present description and claims, and a fluorescent marker selected from BT1-8.

[0061] Non-conjugated HumAfFt in the present description refers to a HumAfFt which is not covalently linked, and corresponds to the humanized ferritin from Archaeoglobus fulgidus of SEQ ID NO 1 disclosed in De Turris et. Al., Nanoscale. 2017 Jan 5;9(2):647-655.

[0062] BODIPY in the present invention has the meaning commonly intended in the art, i.e. is the technical common name of a chemical compound with formula C9H7BN2F2, whose molecule consists of a boron difluoride group BF2 joined to a dipyrromethene group C9H7N2; specifically, the compound 4,4-difluoro-4-bora-3a,4a-diaza-s- indacene in the IIIPAC nomenclature. The common name is an abbreviation for "boron-dipyrromethene".

[0063] The terms aromatic and not aromatic are as commonly understood in the state of the art and can therefore indicate, respectively, any aromatic heterocycle and any non-aromatic heterocycle.

[0064] As used herein, the term “ocular tissue” can include any tissue of an eye and / or the optic nerve of a mammal, such as a retina or substructures within the retina perimeter such as macula lutea and fovea. The retina can include one or more of: an inner nuclear layer of the retina and a retinal ganglion cell of the retina.

[0065] TALI1 probe is the structure described in the manuscript by Verwilst P, et. al, "Rational Design of in Vivo Tau Tangle-Selective Near-Infrared Fluorophores: Expanding the BODIPY Universe." J Am Chem Soc. 2017 Sep 27; 139(38) : 13393- 13403.

[0066] As used herein, the term “tauopathy” encompasses the class of neurodegenerative diseases involving the aggregation of tau proteins into neurofibrillary or gliofibrillary tangles (NFTs), such as Alzheimer’s disease, Down syndrome, amyotrophic lateral sclerosis, Pick’s disease, Parkinson’s disease, primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CDB), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), lytico-bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), as well as lead encephalopathy, tuberous sclerosis, Pantothenate kinase-associated neurodegeneration, and lipofuscinosis.

[0067] As used herein, the term “polyaromatic maleimide derivative” means a chemical compound containing the maleimide group with the formula H2C2(CO)2NH where the H atom in the NH group is substituted with one or more than one fused benzene rings, resulting in a flat aromatic system.

[0068] As used herein, the expression “at the end of the reaction as determined by MALDI mass spectroscopy” means a matrix-assisted laser desorption / ionization (MALDI) technique that uses a laser energy-absorbing matrix to create ions from large molecules with minimal fragmentation and is used to determine their molar mass.

[0069] As used herein, the term “divalent cations” means ions with a double positive charge including the elements of the second group of the periodic table (such as beryllium, calcium, Magnesium, barium, radio, strontium, cesium) or the transition metals including iron, cobalt, copper, zinc, manganese, nickel.

[0070] DETAILDE DESCRIPTION OF THE SEQUENCES

[0071] SEQ ID NO 1 : HumAfFt: humanized ferritin from Archaeoglobus fulgidus disclosed in De Turris et. Al., Nanoscale. 2017 Jan 5;9(2):647-655

[0072] MASISEKMVEALNRQINAEIYSAYLYLSMASYFDSIGLKGFSNWMRVQWQ EELCHANKMFDFVSERGGIRIFLQDIKKPDSEWESPLAAFEHVYEHEVNVTKRIHEL VEMAMQEKDFATYNFLQWYVAEQVEEEASALDIVEKLRLIGEDKRALLFLDKELSL RQFTPPAEEEK

[0073] SEQ ID NO 2: Fwd primer for GADPH

[0074] GGC CAT CCA CAG TCT TCT G

[0075] SEQ ID NO 3: Rev primer for GADPH

[0076] TCA TCA GCA ATG CCT CCT G

[0077] SEQ ID NO 4: Fwd primer for RECOVERIN

[0078] TTCAAGGAGTACGTCATCGCC

[0079] SEQ ID NO 5: Rev primer for RECOVERIN

[0080] GATGGTCCCGTTACCGTCC SEQ ID NO 6: Fwd primer for RLPB1

[0081] GGCAGGGAACAACCAAGACT

[0082] SEQ ID NO 7: Rev primer for RLPB1

[0083] AGTCAGGGCCAAGTTGTGAC

[0084] SEQ ID NO 8: Fwd primer for CD71

[0085] CGTGAGGCTGGATCTCAAA

[0086] SEQ ID NO 9: Rev primer for CD71

[0087] CCAGGATTCTCCACCAGGTA

[0088] SEQ ID NO 10: Fwd primer for NANOG

[0089] GAAATACCTCAGCCTCCAGC

[0090] SEQ ID NO 11 : Rev primer for NANOG

[0091] GCGTCACACCATTGCTATTC

[0092] DETAILED DESCRIPTION OF THE INVENTION

[0093] The present invention relates to a novel delivery system consisting of humanised ferritin from Archaelogus fulgidus (HumAfFt) of SEQ ID NO 1, and the fluorescent marker fluorescent marker of formula I: Formula I wherein X and Y are carbon atoms linked either by a double bond with E or Z configuration or by an aromatic or heteroaromatic para-substituted ring, or by an aromatic or heteroaromatic 1,4 disubstituted ring; R is hydrogen, halogen, NH(RA), N( A)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)s, or C5-7 aromatic or aliphatic heterocycle; and in each substituent, RA is: hydrogen; halogen, hydroxyl; CF3, a C1-7 saturated or unsaturated chain, linear or branched containing up to three independent heteroatoms selected among nitrogen oxygen and sulphur; C5-7 cycloalkyl; phenyl; C5-7 heterocycle or n-etilenglycol, wherein said HumAfFt is covalently bound to moiety comprising a thiol-reactive group, to compositions comprising it, to the method for the preparation of said delivery system and to methods and uses of said delivery system or of said compositions.

[0094] The delivery system of the invention allows an efficient solubilisation and delivery in vivo of BODIPY markers, such as the markers disclosed in fluorescent BT1-8 markers disclosed in WO2022 / 224151 that specifically bind the TAU protein, that would otherwise be poorly soluble in aqueous environments.

[0095] Although highly specific, BT1-8 molecules, suffer from intrinsic low water solubility therefore, their in vivo delivery to the retina may not be readily feasible. In fact, solubility of BT1 can be quantified in the millimolar range only in aqueous solutions containing at least 50% DMSO. At such concentrations, acute toxicity is observed. Thus, it is necessary to find a delivery, or cargo system for BT1 that can assure bioavailability of the same in aqueous solution.

[0096] It was therefore important to identify a class of delivery cargoes capable of entering retinal cells and release the fluorescent molecule to the NFTs (neurofibrillary tangles) of the Tau protein in a highly selective way.

[0097] The preferred cargo was identified among ferritin proteins, natural nanocages capable of embedding small molecules and able to enter a number of human cells expressing the transferrin receptor on their surface. Ferritins are iron storage and transport proteins, found in most living organisms, characterized by a typical tetraeicosameric subunits assembly. Ferritin assembly results in the formation of an 8 nm diameter central cavity, which can be efficiently loaded with transition metals, drugs, fluorescent molecules or contrast agents.2-4 Thus, encapsulated molecules can be delivered through the ferritin nano-cages, to target the transferrin receptor (TfR1 , or CD71), highly expressed in several cell types. These attributes make ferritins and their derivatives a powerful delivery system with potential application in nanomedicine.

[0098] Recombinant mammalian ferritins, however, are difficult to disassemble into subunits as they can be dissociated only under extreme conditions, thus rendering the encapsulation of many substrates impractical. Commonly employed cargo encapsulation techniques involve the disassembly and re-assembly of ferritins cage by pH jump, reaching drastic pH values (pH 2-9) which cause a partial and incomplete re-assembly. The inventors identified as a suitable ferritin, the humanized ferritin from Archaeoglobus fulgidus (HumAfFt)(De Turris et. Al., 2017), which surprisingly proved, however, to be incapable of properly uploading the BT molecules (cfr Figure 4D).

[0099] The inventors found that a specific modification of said ferritin provided a cargo capable, not only of uploading the BT molecules efficiently as shown in figure 4C, but also of releasing them to the TAU protein aggregates in solution as shown in figure 5.

[0100] The proof of concept of the novel delivery system of the invention is given in the examples (and related figures) below. In particular it has been shown that a delivery system according to the invention is capable of entering target cells (Retinal ganglion cells--BRN3a) using the CD71 receptor, highly expressed in these cells.

[0101] Details are given as follows:

[0102] A) The differentiated human iPSC and Retinal ganglion cells-BRN3a were first demonstrated to be able to express CD71 receptor within the cell surface, ad shown in Fig. 6. In particular, retinal markers for differentiation were verified together with CD71 each one with appropriate antibody staining. Bar graphs in fig 6A refers to the presence of appropriate markers as a function of days of differentiation.

[0103] B) Fluorescent confocal images of live hiPSC-derived retinal neurons (day 30) were assessed for HumAfFt internalization at different treatment times (6-8- 16-24-48h) as shown in Fig. 7. As depicted in the bar charts of Fig. 7, rhodamine- HumAfFt conjugate were readily internalized within retinal neurons (day 30). Finally, representative fluorescent confocal images of NPM-HumAfFt-BT1 complex within live hiPSC-derived retinal neurons (day 30) demonstrated uptake of the ferritin-fluorophore complex.

[0104] C) After demonstration of the claimed delivery system internalization, it was demonstrated that the Bodipy molecule is indeed released in the cytosol and binds specifically to TAU oligomers when present. In order to prove the specific staining of BT1, representative confocal images of BT1 fluorescence and immunostaining for total TAU were carried out in control and OKA-treated iPSC- derived retinal neurons. Cells were first incubated with 150 nM NPM-HumAfFt-BT1 complex for 24 hours and then fixed and processed for immunostaining. Zoomed images reveals the cytoplasmic co-localization of BT1 with HT7 co-localization in retinal neurons. Further proofs were given in cells overexpressing the TAU mutant Ser202 / Thr205 (AT8) Of note, the increased colocalization of BT1 with total, phosphorylated and oligometic tau in OKA treated cultures.

[0105] According to the invention and to the state of the art, the compound of formula I is a fluorescent molecule with a high affinity for Tau protein neurofibrillary tangles (NFTs).

[0106] According to one embodiment of the invention, in the fluorescent marker of formula I, X and Y are carbon atoms linked by an aromatic para-substituted ring or by an aromatic 1,4 disubstituted ring;

[0107] R is H, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)s, or C5-7 aromatic or aliphatic heterocycle; and in each substituent, RA is: hydrogen; halogen; hydroxyl; CF3, a C1-7 saturated or unsaturated chain, linear or branched containing up to three independent heteroatoms selected among nitrogen oxygen and sulphur; C5-7 cycloalkyl; phenyl; C5-7 heterocycle or n-etilenglycol.

[0108] According to the present invention, preferred aromatic heterocycles are selected among pyrrole, imidazole and piperidine.

[0109] According to the present invention, preferred non-aromatic heterocycles are selected among morpholine, piperazole and pyrrolidine.

[0110] In one preferred embodiment, R is selected from NH2, NH(CH3), N(CHs)2, N(Ph)2, imidazole, morpholine or piperazine.

[0111] Non-limiting examples of fluorescent markers of formula I include:

[0112]

[0113] Scheme 1 - Structures of the compounds BT1-BT8

[0114] Therefore, the fluorescent marker in the delivery system according to the present invention may be selected from 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H- dipyrrolo[1,2-c:2',T-f][1,3,2]diazaborinin-4-ium-5-uide (also referred as BT1 in the present description, in the figures and in the schemes); 3-((E)-4-((E)-4-(diphenylamino)styryl)styryl)-5,5-difluoro-1 -methyl-5H-dipyrrolo[1 ,2- c:2',T-f][1,3,2]diazaborinin-4-ium-5-uide (also referred as BT2 in the present description and in the schemes);

[0115] 3-((E)-4-((E)-4-aminostyryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinin-4-ium-5-uide (also referred as BT3 in the present description and in the schemes);

[0116] 5.5-difluoro-1-methyl-3-((E)-4-((E)-4-(methylamino)styryl)styryl)-5H-dipyrrolo[1 ,2- c:2',T-f][1,3,2]diazaborinin-4-ium-5-uide (also referred as BT4 in the present description and in the schemes);

[0117] 5.5-difluoro-1-methyl-3-((1E,3E,5E)-6-(pyrrolidin-1-yl)hexa-1,3,5-trien-1-yl)-5 / 7- dipyrrolo[1,2-c:2',1'- / ][1,3,2]diazaborinin-4-ium-5-uide (also referred as BT5 in the present description and in the schemes);

[0118] 5.5-difluoro-1-methyl-3-((E)-4-((E)-4-morpholinostyryl)styryl)-5H-dipyrrolo[1 ,2-c:2',1'- f][1,3,2]diazaborinin-4-ium-5-uide (also referred as BT6 in the present description and in the schemes);

[0119] 5.5-difluoro-1-methyl-3-((E)-4-((E)-4-(piperazin-1-yl)styryl)styryl)-5H-dipyrrolo[1 ,2- c:2',T-f][1,3,2]diazaborinin-4-ium-5-uide (also referred as BT7 in the present description and in the schemes); and 3-((E)-4-((E)-4-(1 H-imidazol-1-yl)styryl)styryl)-5,5-difluoro-1-methyl-5H- dipyrrolo[1,2-c:2',1'-f][1,3,2]diazaborinin-4-ium-5-uide (also referred as BT8 in the present description and in the schemes).

[0120] In a preferred embodiment the fluorescent marker is: 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1 ,2- c:2',T-f][1,3,2]diazaborinin-4-ium-5-uide depicted in formula II below Formula II Said markers can be prepared using the method disclosed in pages x-y of WO2022 / 224151, in particular in example 2.

[0121] In one embodiment of the present invention, the moiety comprising the thiol-reactive group to which humanised ferritin from Archaelogus fulgidus of SEQ ID NO 1 is covalently bound, is selected from N-substituted maleimide, N-substituted halobenzene sulphonamide, and haloacetoamide.

[0122] In preferred embodiments said halo group is selected from iodine, bromine and fluorine.

[0123] In further preferred embodiments said N-substituted maleimide hydrophobic moieties are selected from maleimide where the substituted compound may be composed of alkyl groups composed of 1 to 12 carbon atoms, and of aliphatic rings, such as N-cyclohexyl maleimide and of fused rings of various sizes, including some that are not aromatic, such as benzyl, biphenyl, pyrenyl and perylenyl maleimide; and said N-substituted halobenzene sulphonamide hydrophobic moieties are selected from the group consisting substituted or unsubstituted phenyl and pyrenyl moieties halobenzene sulphonamide, where hydrogens atoms may be substituted with 1 to 5 halogens such as fluorine, chlorine, bromine and the N-substituted compound may be composed of alkyl groups from 1 to 12 carbon atoms, aliphatic rings such as N-cyclohexyl and of fused rings of various size including some that are not aromatic, such as benzyl, biphenyl, pyrenyl and perylenyl group..

[0124] In a particularly preferred embodiment said N-substituted maleimide is N-(1- Pyrenil)maleimide (NPM).

[0125] According to a preferred embodiment of the invention, the delivery system hence consists of NFM-HumAfFt-BT1, i.e., of a nanocage of HumAfFt of SEQ ID NO 1 , covalently linked to N-substituted maleimide is N-(1-Pyrenil)maleimide (NPM) and the fluorescent marker 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1- methyl-5H-dipyrrolo[1 , 2-c: 2' , 1 -f] [ 1 ,3,2]diazaborinin-4-ium-5-uide (BT1).

[0126] The invention also relates to a composition comprising the delivery system according to anyone the embodiments described herein, and one or more additional excipients and / or carriers.

[0127] In an embodiment, the composition of the invention is a pharmaceutical composition. The concentration of the fluorescent marker of formula I in said composition can be adjusted depending on the type of fluorescent marker of the present invention. In one embodiment, the concentration of the fluorescent marker may be in the range from 0.5 micromolar to 5 millimolar. The excipient to be contained in said composition can be adjusted such that the desired dosage of the fluorescent marker is obtained. The composition of the invention is preferably in the form of a sprayable composition, an oftalmic composition, a composition for aerosol, for intranasal administration, eye drops, ophthalmic ointment and even dry powder.

[0128] The ophthalmic composition according to the invention can be in the form of eye drops, ophthalmic ointment, dry powder or ophthalmic lotion.

[0129] Any suitable carrier or excipient known in the art for the preparation of compositions suitable ophthalmic compositions can be used by the skilled person.

[0130] The composition in liquid form, NFM-HumAfFt-BT1 can be suitably dissolved in a liquid formulation comprising one or more additional excipient, immediately before use.

[0131] The composition can be provided already aliquoted in single dosages or in single dosage fractions, apt to release a pre-defined amount of the solution.

[0132] Among the vehicles and / or acceptable diluents and / or excipients, according to the administration route and the practice of the pharmaceutical / nutritional standards the following are suitable for carrying out the invention although the person skilled in medical composition preparations will know also other suitable vehicles and / or acceptable diluents and / or excipients:

[0133] - for the liquid formulations, preferably a vehicle comprising: glycerin and / or mannitol and / or sorbitol, polysorbate 20, PEG, propylene glycol; one or more humectant agents such as: lecithin, polysorbate, lauryl sulphate; known substances, inactive and not toxic from the pharmaceutical / nutraceutic point of view.

[0134] The carriers, diluents and excipients mentioned above are meant exclusively by way of example, however not constituting an exhaustive list.

[0135] COMPOSITION EXAMPLE #1

[0136] NFM-HumAfFt-BT1 1 mg, polysorbate 80, 0.5 g, propylene glycol, 0,1 g, purified water 0.84 g, Sodium phosphate bibasic, 1,5 mg, Zinc sulphate, 0,5 mg. Benzalkoniumchloride 0.1 mg. COMPOSITION EXAMPLE #2

[0137] NFM-HumAfFt-BT1 2 mg, polysorbate 80, 0.5 g, propylene glycol, 0.1 g, purified water 0.84 g, Sodium phosphate bibasic, 1.5 mg, Magnesium sulphate, 0,5 mg. Benzalkoniumchloride 10 mg.

[0138] COMPOSITION EXAMPLE #3

[0139] NFM-HumAfFt-BT1 4 mg, polysorbate 80, 0.5 g, propylene glycol, 0.1 g, purified water 0.84 g, Sodium phosphate bibasic, 1.5 mg, Calcium sulphate, 0,5 mg. Benzalkoniumchloride 10 mg. One aspect of the present invention is referred to the use of the delivery system of the invention, or of the pharmaceutical composition of the invention for the detection of neurofibrillary tangles of the Tau protein.

[0140] In other words, the invention is also referred to a delivery system as herein defined or to a composition comprising said delivery system, for use in the detection of neurofibrillary tangles of the Tau protein in a subject in vivo.

[0141] In accordance with an embodiment of the invention, any of the systems or compositions taught herein can be administered to a subject in need thereof for use in the detection of neurofibrillary tangles of the Tau protein. As stated above, said detection can be in a subject, and it can be carried out in vivo without invasive tools. In a further embodiment of the invention the detection can be carried out in vitro on suitable samples, including organoids.

[0142] The invention further provides a method for the detection of neurofibrillary tangles of the Tau protein comprising the steps of: contacting the delivery system according to the invention or the composition according to the invention with a biological sample under conditions wherein said fluorescent marker binds to the neurofibrillary tangles of the tau protein; detecting said fluorescent marker bound to the biological sample.

[0143] In other words, the method can also be defined as a method for the detection of neurofibrillary tangles of the Tau protein comprising the step of: detecting said fluorescent marker bound to a biological sample contacted with the delivery system or with the composition according to the description or the claims, under conditions wherein said fluorescent marker binds to the neurofibrillary tangles of the tau protein. Non-limiting examples of biological samples that can be analysed by using the above method include an ocular tissue, a brain tissue or olfactive epithelia. In one preferred embodiment, said biological sample is an ocular tissue, preferably a retina. In one embodiment, the contacting step of the above method can be carried out at a temperature of 37°C. Said contacting step has a duration comprised between 10 and 120 minutes so that the marker released by the delivery system selectively binds the neurofibrillary tangles of the tau proteins present in the biological sample to be analysed.

[0144] The period of incubating the biological sample in the presence of said delivery system depends on the amount of the delivery system applied but falls within the above-mentioned range. In one embodiment, the contacting time is 30 minutes.

[0145] Preferably, in said contacting step, the fluorescent marker of formula I is in a concentration ranging from 0.5 micromolar to 5 millimolar.

[0146] The above method may further comprise, after said contacting step, a washing step in which any excess of said fluorescence marker of formula I is removed from the biological sample.

[0147] In one embodiment, the presence and / or amount of any of the fluorescent markers of formula I bound to the neurofibrillary tangles of the Tau protein within a biological sample can be determined by fluorescence measurements, preferably by fluorescence imaging.

[0148] Fluorescence imaging can be performed according to any of the fluorescence imaging techniques known in the art. For example, a qualitative and / or quantitative evaluation of the binding of the fluorescent marker of the invention to the neurofibrillary tangles of the tau protein within said biological sample can be accomplished using microscopy techniques.

[0149] In some embodiments, the detection step of the method of the present invention as described above further comprises the following steps:

[0150] - illuminating said biological sample with a light source, the light source having a wavelength (A) appropriate to determine the emission of fluorescence from the bound fluorescent marker of formula I; and

[0151] - detecting and optionally quantifying the fluorescence emitted by said fluorescent marker of formula I, wherein said emitted fluorescence has a wavelength in the range of from about 450 and 800 nm.

[0152] It is preferable that the excitation light source has a narrow emission range so as to avoid the excitation of any other constituents of the biological sample to be analysed. In one embodiment, the light source has a wavelength in the range of from about 350 to 650 nm, preferably is equal to 559 nm.

[0153] Another embodiment of the present invention is directed to an imaging method comprising the steps of:

[0154] - administering the delivery system or the composition as defined in the present specification,

[0155] - carrying out a non-invasive fluorescence imaging of the retina of said subject, wherein the detection of fluorescence from said fluorescent marker of formula I indicates the binding of said marker to the retina.

[0156] In other words, the method can be also defined as an imaging method comprising the step of:

[0157] - carrying out a non-invasive fluorescence imaging of the retina of a subject to which the fluorescent marker or the composition according to the description or the claims has been administered, wherein the detection of fluorescence from said fluorescent marker indicates the binding of said marker to the retina.

[0158] In one embodiment, the delivery system comprising the fluorescent marker of formula I or the composition comprising said fluorescent marker of formula I, according to the present invention, can be administered to a subject by oral administration or by ophthalmic administration or by an intranasal administration.

[0159] In a preferred embodiment, any of the delivery system or compositions comprising said fluorescence markers taught herein is administered to said subject from at least 30 minutes to one day prior to the fluorescence measurement. By way of example, the administration is at least 1 hour, 2 hours, at least 4 hours, at least 8 hours, ably at least 12 hours, at least 16 hours prior to the fluorescence measurement. Thanks to its capability to efficiently penetrate the blood-brain barrier, the delivery system of the invention is capable of reaching the retina following administration and of releasing the marker that selectively binds the neurofibrillary tangles of the tau protein present therein.

[0160] The time needed for said binding varies depending on the method selected for the administration of the marker or of the composition. A topical administration, in the form of an ophthalmic administration will need a shorter time for the subsequent fluorescence detection stem compared to an oral administration. In one embodiment, the fluorescent marker of formula I is administered through the delivery system of the invention, in an amount comprised between 0.5 micromolar to 5 millimolar per unit dosage.

[0161] Fluorescence imaging of the retina of a subject according to the methods of the present invention can be performed by means of any fluorescence imaging technique known in the art, as long as it is non-invasive. Non-limiting examples of non-invasive fluorescence imaging techniques include scanning laser ophthalmoscopy (SLO), confocal scanning laser ophthalmoscopy (cSLO), or fluorescence lifetime imaging ophthalmoscopy (FLIO). The fluorescence imaging can be qualitative or quantitative, in other terms it can indicate merely the presence or absence of fluorescence or it can quantify the amount of fluorescence detected.

[0162] By way of example, non-invasive fluorescence imaging of the retina can be carried out by employing a light source to illuminate the retina of said subject, together with means for detecting and / or quantifying the fluorescence emitted by the fluorescence marker of the invention bound to the neurofibrillary tangles of the tau protein within the retina, wherein said emitted fluorescence has an emission wavelength in the range of from about 450 to 800 nm.

[0163] As used herein, a “light source” may be any light source that can be configured to illuminate the retina of a subject, having a wavelength appropriate to determine the emission of fluorescence from the bound fluorescent marker in the delivery system of the invention within the retina of said subject. In one preferred embodiment, said light source has a wavelength comprised between 350 to 650 nm, preferably equal to 559 nm.

[0164] Hence, in one embodiment of the invention, the above imaging method may comprise determining the peak intensity of fluorescence produced by the fluorescent marker of formula I bound to the NFTs of the tau protein within the retina. The amount of the fluorescent marker bound to the NFTs of the tau protein may be determined based on said peak intensity.

[0165] By way of example, the fluorescent markers of formula I in the delivery system of the present invention bound to the NFTs of the tau protein within the retina may be excited by picosecond laser pulses and the fluorescence emission can be detected using time correlated single photon counting (TCSPC) technology.

[0166] Advantageously, images obtained with the detection of fluorescence of the retina of a subject with the imaging method of the invention, can be used for the diagnosis of tauopathies. As neurofibrillary tau tangles are associated to tauopathies, the detection of neurofibrillary tangles in the retina can be used for the diagnosis or to assist in the diagnosis of tauopathies.

[0167] As used herein, the term “tauopathy” encompasses the class of neurodegenerative diseases involving the aggregation of tau proteins into neurofibrillary or gliofibrillary tangles (NFTs), such as Alzheimer’s disease, Down syndrome, amyotrophic lateral sclerosis, Pick’s disease, Parkinson’s disease, primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CDB), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), lytico-bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), as well as lead encephalopathy, tuberous sclerosis, Pantothenate kinase-associated neurodegeneration, and lipofuscinosis.

[0168] In one preferred embodiment, said tauopathy is selected from Alzheimer’s disease, Down syndrome, amyotrophic lateral sclerosis, Pick’s disease, or Parkinson’s disease, preferably is Alzheimer’s disease.

[0169] In accordance with an embodiment of the invention, the method for detecting neurofibrillary tangles of tau protein in a biological sample or any of the imaging methods as described in the present specification can be combined with results obtained by other imaging techniques, for example, a technique for detecting amyloid proteins, in order to aid in a diagnosis of a tauopathy.

[0170] Another object of the present invention is a method for determining a fluorescence graph comprising the steps of:

[0171] - administering to a subject the delivery system or the composition according to the invention and carrying out a non-invasive quantitative fluorescence imaging on the retina of said subject, at a plurality of successive time instants ti, where i is 0 to n, so obtaining corresponding fluorescence values;

[0172] - using the obtained fluorescence values for determining a graph of the fluorescence as a function of time.

[0173] In an embodiment, said successive time instants are separated by a time period of one or more weeks or one or more months between each time instant and the subsequent one. Furthermore, the non-invasive fluorescence imaging can be carried out after 30 minutes to one day from said administration. In an embodiment of the invention, said delivery system or said composition is administered orally or intranasally, or by aerosol, or in the form of an ophthalmic ointment, dry powder or eyedrops.

[0174] In implementing the method described above, the time instant tj with i=0 represents the time in which monitoring is initiated. The successive time instants tj in which i is an integer value increasing from 0+1 to n, are instants of time subsequent to each other, their succession in time being indicated by the increase of the i value.

[0175] Hence, the instant of time tj with i=0 is the instant in which the monitoring is initiated, and the fluorescence value of the fluorescent marker of formula I in the retina at this tj can be considered as the starting fluorescence value from which the progression in time of said fluorescence within the retina can be assessed.

[0176] Non-invasive quantitative fluorescence imaging of the retina can be repeated in time instants subsequent to tj with i=0 and subsequent to each other in progression from i= 1 to n, so that the time instant ti precedes the time instant t2, which precedes the time instant t3and so on.

[0177] Comparison of the fluorescence values in the images obtained for each subsequent ti according to the above method can hence be elaborated in a graph of the fluorescence as a function of time.

[0178] A graph as the one provided by the method above, can be advantageously used to assess the effectiveness of a medical treatment against a tauopathy or to assess the progression in time of a tauopathy.

[0179] In this case, the time tj with i=0 corresponds to the time at which medical treatment outcome is monitored, this can advantageously be before the start of the treatment itself but can be introduced at any time during the treatment even after the treatment has already started.

[0180] A progressive decrease or a stability of the fluorescence in the graph provided by the method above, can be indicative of the effectiveness of the medical treatment in ameliorating the tauopathy. The meaning of the graph and the related extent of the effectiveness can be evaluated by the interpretation of the graph by the clinical expert.

[0181] A progressive increase of the fluorescence in the graph provided by the method above can be indicative of the ineffectiveness of the medical treatment. The meaning of the graph and the related extent of the ineffectiveness can be evaluated by the interpretation of the graph by the clinical expert. In some cases, changing dietary habits and lifestyle may be suggested to a subject before undergoing a medical treatment. In these cases, it may be useful to monitor the progression of the disease over time, to see, for example, if changes in dietary habits and lifestyle have a positive effect on the disease.

[0182] The graph provided by the method above can be used to monitor the effects of said changes on the disease.

[0183] Anyway, monitoring the progression of a tauopathy on a subject, regardless of whether or not a possible therapeutic efficacy is evaluated, may be of interest for a medical doctor.

[0184] What has been previously described regarding the administration modes and dosages of the delivery system or compositions comprising said fluorescent marker in the explanation of the general imaging method, also applies to any of the other methods described above.

[0185] Non-invasive quantitative fluorescence imaging of the retina of a subject to be analyzed may be performed according to any one of the non-invasive fluorescence imaging techniques known in the art, including those as previously described in the present specification. In particular, according to one preferred embodiment of the invention, in any of the above imaging methods, said fluorescence imaging is carried out by submitting the retina of said subject to irradiation with a light source having a wavelength (A) comprised between 350 and 650 nm, and detecting and / or quantifying the fluorescence emitted by said fluorescent marker.

[0186] Any of the above methods comprising the fluorescence imaging of the retina, may further comprise "normalizing" a determined amount or the fluorescence level of the fluorescence marker of the invention bound to the NFTs of the tau protein, based on the background autofluorescence emitted from the retina of said subject. As used herein, such "normalizing" can include subtracting the amount of background autofluorescence from the amount of fluorescence emitted from the fluorescent marker of the invention bound to the NFTs of the tau protein; it can also include determining a ratio of such quantities; and can include using such a normalized result as a normalized measure of the amount of the fluorescent marker of formula I of the present invention bound to the NFTs of the tau protein.

[0187] The detection of neurofibrillary tangles of tau protein in the retina allows an early and non-invasive diagnosis of a tauopathy, the high specificity of the fluorescent markers of the present invention renders the detection of the tangles more sensitive and accurate, thereby improving an early diagnosis of the disease.

[0188] The imaging method of the invention thereby can be used in methods of treatment of tauopathies in which the early diagnosis of the disease is followed by appropriate treatments of the same at stages in which normally no diagnosis is possible.

[0189] Therefore, the method for determining a fluorescence graph as described in the present specification can be advantageously used for monitoring the progression of a tauopathy on a subject. Hence, object of the present invention is the use of the delivery system or the composition according to the invention in the diagnosis of a tauopathy.

[0190] As used herein, the term “tauopathy” encompasses the class of neurodegenerative diseases involving the aggregation of tau proteins into neurofibrillary or gliofibrillary tangles (NFTs), such as Alzheimer’s disease, Down syndrome, amyotrophic lateral sclerosis, Pick’s disease, Parkinson’s disease, primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CDB), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), lytico-bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), as well as lead encephalopathy, tuberous sclerosis, Pantothenate kinase-associated neurodegeneration, and lipofuscinosis.

[0191] In one preferred embodiment, said tauopathy is selected from Alzheimer’s disease, Down syndrome, amyotrophic lateral sclerosis, Pick’s disease, or Parkinson’s disease, preferably is Alzheimer’s disease.

[0192] In accordance with an embodiment of the invention, the method for detecting neurofibrillary tangles of tau protein in a biological sample or any of the imaging methods as described in the present specification can be combined with results obtained by other imaging techniques, for example, a technique for detecting amyloid proteins, in order to aid in a diagnosis of a tauopathy.

[0193] Further object of the present invention is a method for the diagnosis of neurodegenerative diseases characterised by tau tangles comprising the steps of:

[0194] - administering to a subject the delivery system or the composition according to the invention ; carrying out a non-invasive quantitative fluorescence imaging on the retina of said subject, wherein the presence of tangles indicates the presence of a neurodegenerative disease. In other words, the method can be defined also as a method for the diagnosis of neurodegenerative diseases characterised by tau tangles comprising the steps of: -carrying out a non-invasive quantitative fluorescence imaging on the retina of said subject, wherein the presence of tangles indicates the presence of a neurodegenerative disease in a subject to whom the delivery system or the composition according to the invention has been previously administered.

[0195] The present invention also relates to a method for the preparation of a delivery system as defined in the description and in the claims, comprising the following steps: i) reacting a solution comprising a moiety comprising a thiol reacting group with cysteine 54 of the HumAfFt of SEQ ID NO 1 thereby obtaining a moiety comprising a thiol reacting group -HumAfFt with a hydrophobic surface within the protein nanocage; ii) adding a solution of a fluorescent marker of formula I to the solution obtained in step i) thereby obtain a moiety comprising a thiol-reactive group- HumAfFt conjugated with a fluorescent marker of formula I; iii) at the end of the reaction, as determined by MALDI mass spectrometry, adding a divalent cation salt in order to close the HumanAfFt as 24-mer, hence creating an internal cavity.

[0196] In one embodiment of the present invention, the moiety comprising the thiol-reactive group to which humanised ferritin from Archaelogus fulgidus of SEQ ID NO 1 is covalently bound, is selected from N-substituted maleimide, N-substituted halobenzene sulphonamide, and haloacetoamide.

[0197] In preferred embodiments said halo group is selected from iodine, bromine and fluorine.

[0198] In further preferred embodiments said N-substituted maleimide hydrophobic moieies are selected from the group consisting of N-alkyl maleimide in which alkyl group may be composed of 1 to 12 carbon atoms, and of rings particularly aromatic rings such as N-benzyl maleimide; N-cycloalkyl maleimide, N-cyclohexyl maleimide; and said N-substituted halobenzene sulphonamide hydrophobic moieties are selected from halobenzene sulphonamide, where hydrogens atoms may be substituted with 1 to 5 halogens such as fluorine, chlorine, bromine and the N-substituted compound may be composed of alkyl groups from 1 to 12 carbon atoms, aliphatic rings such as N-cyclohexyl and of fused rings of various size including some that are not aromatic, such as benzyl, biphenyl, pyrenyl and perylenyl group.. In a particularly preferred embodiment said N-substituted maleimide is N-(1- Pyrenil)maleimide (NPM).

[0199] Suitable divalent cations are all ions with a double positive charge including the elements of the second group of the periodic table such as beryllium, calcium, Magnesium, barium, radio, strontium, cesium, or the transition metals including iron, cobalt, copper, zinc, manganese, nickel.

[0200] In a preferred embodiment, said divalent cation salt is MgCh, CaCh, ZnCh, FeCI2.

[0201] In a preferred embodiment, the method comprises, before step i), a step of reduction reaction of the HumanAfFt of SEQ ID NO.1 with tris(2- carboxyethyl)phopshine hydrochloride (TCEP). Preferably, said reaction is carried out by reacting 96 pM of HumAfFt monomers (4 pM in 24-mer, in 20 mM HEPES pH 7.4) with 10 equivalents of TCEP, for 30 minutes at 55°C under mild agitation.

[0202] In another embodiment, the step i) is carried out in 10% v / v Dimethylsulfoxide (DMSO) to favor NPM solubility.

[0203] In an embodiment, the step i) further comprises the removal of NPM excess by gel filtration chromatography (G25 Desalting, eluent 20 mM HEPES pH 7.4).

[0204] In a preferred embodiment, said step ii) is carried out by addition of 10 equivalents of the fluorescent marker in DMSO solution, while the NPM- HumAfFt is in a 20 mM Hepes buffer solution at pH 7.4. The sample was kept overnight at RT under mild agitation.

[0205] In an embodiment, in said step iii) a final concentration of 50 mM of MgCI2 was added to the sample. Said step iii) may further comprise a step of removal of the fluorescent marker through centrifugation at 10000 rpm for 30 minutes, and the sterilization with a 0.22 pm filter.

[0206] In any part of this description and the claims, the term comprising may be replaced by the term "consisting of".

[0207] Reported below are some examples which have the purpose of better illustrating the methodologies disclosed in this description, these examples are in no way to be considered a limitation of the previous description and the subsequent claims.

[0208] EXAMPLES

[0209] 1. HumAfFT Ferritin protein production The chimeric HumAfFt ferritin of SEQ ID NO 1 was designed and produced as previously reported in the material and methods disclosed in deTurris et al. 2017 and its assembly properties were verified by size exclusion chromatography (SEC). Briefly, the gene encoding for bacterial ferritin from HumAfFt was cloned into the expression vector pET22b (Novagen). The recombinant plasmids were transformed into E. coli TOP 10 cells and the resulting colonies were screened by DNA sequencing. Plasmids bearing the desired mutations were transformed into BL21(DE3) E. coli strain for protein expression. For each mutant, protein overexpression was obtained as follows: 1 L LB broth medium was inoculated with 2 ml overnight culture of a single colony and the gene expression was induced with 1 mM IPTG when the absorbance at 600 nm reached 0.6. Cells were harvested by centrifugation after overnight induction at 37 °C and the cell pellets were stored at - 20 °C.

[0210] 2. Protein purification

[0211] Harvested cells form 1 I colture over-expressing HumAfFt were resuspended in 20 ml buffer A (20 mM HEPES pH 7.5, 50 mM MgCI2) containing a complete™ Mini Protease Inhibitor Cocktail Tablet (Roche) and disrupted by sonication. The soluble fraction was thermally purified by heating at 80 °C for 10 min followed by removal of denatured proteins by centrifugation at 14000 rpm for 30 min at 4 °C. The supernatant was fractioned by ammonium sulfate precipitation. 70% ammonium sulfate pellet containing highly purified protein was resuspended in buffer A, dialysed versus the same buffer, sterile filtered and stored at 4 °C. The final yield of highly purified (> 98,7 %) HumAfFt was about 100 mg per liter of bacterial colture.

[0212] 3 Pyrene-labeled ferritin preparation

[0213] HumAfFt of SEQ ID NO 1 thus purified, was readily modified by direct reaction of N- (l-Pyrenyl)maleimide(NPM) with cysteine 54 of the protein in order to obtain a hydrophobic surface within the protein nanocage. Such modification has been accurately described in Benni et al., 2018 and is resumed below.

[0214] HumAfFt (96 pM of HumAfFt monomers (4 pM in 24-mer, in 20 mM HEPES pH 7.4) was reduced with tris(2-carboxyethyl)phosphine hydrochloride (TCEP)(10 eq.) for 30 minutes at 55°C under mild agitation. The conjugation reaction was carried out in 10% v / v Dimethylsulfoxide (DMSO) to favor NPM solubility. The linker (5 eq. with respect to monomer concentration) was slowly added to the protein solution and the reaction mixture was left under stirring for 2 hours at 30 °C. NPM excess was removed by gel filtration chromatography (G25 Desalting, eluent 20 mM HEPES pH 7.4). The conjugation of NPM to HumAfFt (NPM-HumAfFt) was analyzed by means of MALDI mass spectrometry as shown in Fig 2.

[0215] NPM-HumAfFt was then loaded with BT1 by addition of 10 equivalents in DMSO solution to the protein in 20 mM Hepes buffer solution at pH 7.4. The sample was kept overnight at RT under mild agitation. At the end of the reaction, a final concentration of 50 mM of MgCI2 was added to the sample, to close the HumAfFt as 24-mer, hence creating the internal cavity. The unbound BT 1 was removed through centrifugation at 10000 rpm for 30 minutes. The sample was sterilized with a 0.22 pm filter. In Fig.4, the emission spectra displayed the fluorescence intensity of BT1 in polar solvent as DMSO compared to the intensity of BT 1 in aqueous solution (20 mM Hepes, pH = 7.4) highlighted the dramatic decrease of intensity in aqueous solution, due to aggregation induced quenching. BT1 embedded in the hydrophobic environment of the NPM-HumAfFt showed a strong increase of fluorescence intensity compared to BT1 embedded in the unmodified HumAfFt.

[0216] The ability of BT1-loaded in NPM-HumAfFt to bind to Tau fibrils was evaluated in time-course experiments by fluorescence emission spectroscopy. Unfibrillated K18 domain (residues from 244 to 376) of human Tau protein containing the 4 microtubule-binding repeats and fibrillated K18 upon the addition of the aggregation promoter heparin (ratio 1:1) were analyzed after the addition of BT1 -loaded NPM- HumAfFT (5 pM in protein concetration). The fluorescence of the BT1-embedded in NPM-HumAfFt increases with the formation of K18 fibrils as shown in Fig. 5 where the maximum fluorescence (at Aem = 565 nm) is plotted as a function of time of fibrillogenesis.

[0217] 4. Retinal culture differentiation from human iPSC

[0218] Retinal cultures were differentiated from healthy human induced-pluripotent stem cell lines (iPSC) according to a previously published protocol (Sluch et al, 2015- 2017) with minor modifications. The multi-stage differentiation approach lasts about 30 days, and by using small molecules leads to a homogeneous and well-distributed 2D network of retinal neurons. Human iPSCs (iPSC0028) were dissociated into single cells with IxAccutase and plated on growth factor-reduced Matrigel coated plates (dilution 1:100) at a density of 1000 cells / mm2 in mTeSR Plus with 10 pM Rock Inhibitor. Afterwards, medium was completely replaced with neurogenic basal medium (N2B27w / oA) which consists of 50% DMEM / F12 [1:1], 50% Neurobasal with 1% GlutaMAX Supplement, 0.1% Pen-Strep, 1% NEEA, 1% N2 Supplement, and 2% B27 Supplement w / oA. The formation of a uniform confluent neuroepithelium-like sheet is visible at day 6, the clustering of potentially retinal progenitor macro-islands around day 20 and lastly the generation of ramified retinal neurons at day 30. Different mix of small molecules was added at specific time points: 1 pM Dorsomorphin and 2.5 pM IDE2 freshly added every day from day 0 to day 6 and 10 mM Nicotinamide (all from Sigma-Aldrich) until day 10 to improve retinal cells differentiation; 25 pM Forskolin (Sigma-Aldrich) was supplemented from day 0 to day 30 to promote the retinal progenitor cell proliferation and expansion; 10 pM DAPT (Prepotech) was supplemented from day 20 to day 30 to enhance the genesis of retinal neurons. For RNA extraction, retinal progenitors were dissociated with 1x Accutase and plated onto PLO / Laminin coated petri dish at the density of 1000 / mm2. Instead, for immunostaining, live / dead and internalization analysis they are plated onto a PLO / Laminin coated cover glasses circle (0.12mm, ThorLab) at a density of 100.000 cells per glass. The medium was replaced with N2B27w / o A medium supplemented with 10 pM Rl (only for the seeding), 10 pM DAPT (Prepotech), 25 pM Forskolin. The medium was renewed every 3 days until day SO- 35 when retinal neurons appeared in culture. For future analysis, 3 differentiations (3 biological replicates or batches) were carried out.

[0219] 5. RNA extraction and RT-qPCR

[0220] In order to confirm retinal differentiation, 3 batches of iPSC-derived neurons were analyzed by RT-qPCR. RNA samples were collected at day 0, 6, 20, 30 during differentiation. Total RNA was extracted with EZNA Total RNA Kit I (Omega Bio- Tek). RNA concentration and purity were assessed spectrophotometrically by the ratio A260 / 280 values obtained by Nanodrop (Thermo Fisher). RNA samples were retro-transcribed using the iScript Reverse Transcription Supermix (Bio-Rad). RT- qPCR was performed with iTaq Universal SYBR Green Supermix (Bio-Rad) on a ViiA 7 Real-Time PCR System (Applied Biosystems) and gene expression data was reported as relative expression (2-AACT), normalized to two internal control (GAPDH) and compared to control sample (day 0, undifferentiated iPSCs). Temporal gene expression toward retinal differentiation was carried out using the primers: GAPDH (FW: GGC CAT CCA CAG TCT TCT G, RV: TCA TCA GCA ATG CCT CCT G), BRN3a (FW:ACCACCATTATTACCACCTCCC, RV: CTCGCTCGTTTGGTTTTCGTT), RECOVERIN (FW:

[0221] TTCAAGGAGTACGTCATCGCC, RV: GATGGTCCCGTTACCGTCC), RLBP1 (FW: GGCAGGGAACAACCAAGACT, RV: AGTCAGGGCCAAGTTGTGAC), CD71 (FW: CGTGAGGCTGGATCTCAAA, RV: CCAGGATTCTCCACCAGGTA), NANOG (FW: GAAATACCTCAGCCTCCAGC, RV: GCGTCACACCATTGCTATTC)

[0222] 6. Immunostaining, image acquisition and analysis

[0223] Differentiated day 30 neurons were then fixed with 4% paraformaldehyde (PFA, Sigma Aldrich) for 15 minutes at room temperature, permeabilized with PBS containing 0.2% Triton X-100 (Sigma Aldrich) for 10 minutes and then blocked for 45 minutes at room temperature with a PBS solution containing 0.1% Tween-20 (Sigma Aldrich) and 5% goat serum (Merck). Cells were incubated overnight at 4°C with primary antibodies. The day after, the primary antibody solution was washed out and the cells were incubated with secondary antibody Goat anti-rabbit, anti-chicken and anti-mouse Alexa Fluor (1:750, ThermoFisher Scientific) for 1 hour at room temperature. Washes after primary and secondary antibody staining were performed with PBS solution containing 0.1% Tween-20, adding Hoechst (1 :300, 33258, Merck) in the last wash to stain nuclei. In order to prepare the cells for analysis, round cover glasses with attached and stained cells were sealed with rectangular ones using the Prolong glass antifade mountant (P36984, Invitrogen). The specificity of the staining was tested by performing control experiments in the absence of primary antibody incubation. For fluorescent image analysis, three independent batches of cultures for each condition were carried out.

[0224] Primary antibodies used: TLIJ1 (1:1000 rabbit, T2200, Sigma), MAP2 (1:2000 chicken, ab5392, Abeam), BRN3a (1:50 mouse, sc-8429, Santa-CRuz); CONNEXIN-43 (1:500 mouse, SAB4200730, Sigma), CD71 (1:50 rabbit, ab214039, Abeam).

[0225] Confocal images of TLIJ1 , MAP2 and BRN3a staining were acquired with water objective 60X / NA 2.0 on Fluoview FV10i (Olympus) confocal laser scanning microscope.

[0226] Confocal images of CD71 , CONNEXIN-43 and MAP2 were acquired with oil objective 60X / NA 1.42 (Olympus) on an Olympus iX73 microscope equipped with an X-Light V3 spinning disc head (CrestOptics), an LDI laser illuminator (89 North), a PRIME CMOS camera and a MetaMorph software (Molecular Devices).

[0227] 7. Image analysis.

[0228] Fluorescence images of the probe BT 1 co-stained with the antibodies AT8, T22, and HT7 were analyzed through a custom code developed in MATLAB environment. Maximum Z-projections of image stacks were first subjected to a pre-processing step consisting of the following operations: Gaussian filtering (sigma = 3 pixels), background removal, contrast enhancement, and H-minima transformation. More precisely, the Gaussian filter was used to smooth the images and reduce the noise, the background level was identified with a histogram shape-based method beyond the peak of the intensity distribution, the top 0.01 % of the signal was saturated to enhance images contrast, and H-minima transform was additionally applied for further denoise.

[0229] Pre-processed images were normalized and binarized to select meaningful pixels with a global thresholding method. To take into account the high variability of the signal distribution among images, we developed a Matlab Guided User Interface to adjust the signal selection and eliminate unspecific signals accurately.

[0230] Finally, to quantify the colocalization of the probe BT 1 with the anti-bodies AT8, T22, and HT7, we evaluated both Pearson’s correlation coefficient (PC) and Manders’ overlap coefficients (M1 and M2). While PC measures pixel intensity spatial correlations among pre-processed images of BT1 and antibodies, M1 (resp. M2) quantifies the co-occurrence of BT 1 and antibodies as the fraction of the antibody (resp. probe) signal coinciding with the probe (resp. antibody) signal in the binarized images.

[0231] 8 Analysis of NPM-HumAfFt-BT1 complex internalization into hiPSCs-derived retinal neurons

[0232] To assess the internalization, differentiated day 30 retinal neurons were incubated with 150 nM of HumAfFt-rhodamine for different hours (6-8-16-24-28) at 37°C in 5% CO2. Subsequently, the cell culture medium was removed and neurons were washed one time before the staining solution was added (8pg / ml of FDA and Hoechst (1 :300). Cells were incubated at room temperature for 5 minutes in the dark. Then, neurons were washed once and round cover glasses were transferred into an I bidi glass bottom dish in HEPES-buffered external solution (NES) containing 140 mM NaCI, 2.8 mM KCI, 2 mM CaCI2, 2 mM MgCI2, 10 mM HEPES, 10 mM D- glucose (pH 7.3 with NaOH; 290 mOsm). Live imaging was performed with an air objective 20X / NA 0.45 (Olympus) on an Olympus iX73 microscope equipped with an X-Light V3 spinning disc head (CrestOptics), an LDI laser illuminator (89 North), a PRIME CMOS camera and a MetaMorph software (Molecular Devices). Stack images (z-step of 0.2 pm) were analyzed with the Imaged software (Schneider et al, 2012), flattened in a maximum intensity Z-projection of 13 slices. Stacked images were adjusted in the grayscale using an automatic thresholding method on Imaged (IsoData algorithm-Triangle) to subtract the background noise, and measured the integrated density. Data was plotted in GraphPad Prism version 8.0 for Mac (GraphPad Software, San Diego, California USA, www.graphpad.com). The analysis was performed on 15 FOV (field of view), 5 FOV per batch, and statistical significance was evaluated using one-way ANOVA.

[0233] 9. Cytotoxicity test of NPM-HumAfFt-BT1 complex

[0234] Toxicity of BT1 was evaluated using Fluorescence-based live-dead assays with fluorescein diacetate (FDA) and propidium iodide (PI), which stain viable cells and dead cells, respectively. The staining solution was freshly prepared with 8pg / ml of FDA, 20 pg / ml of PI, and Hoechst (1 :300) to stain nuclei. FDA is taken up by cells which convert the non-fluorescent FDA into the green fluorescent metabolite fluorescein. In contrast, PI dye passes through disordered areas of dead cell membranes, and intercalates with the DNA double helix of cells. To evaluate the cytotoxicity of BT1 , the cells were incubated with different concentrations of NPM- HumAfFt-BT1 (100nM, 150nM, 300nM, 600 nM) for 24 hours. Then, neurons were incubated with a staining solution at room temperature for 5 minutes in the dark. Round cover glasses with neurons were transferred into an I bidi glass bottom dish in HEPES-buffered external solution (NES) containing 140 mM NaCI, 2.8 mM KCI, 2 mM CaCI2, 2 mM MgCI2, 10 mM HEPES, 10 mM D-glucose (pH 7.3 with NaOH; 290 mOsm). Live imaging was performed with an air objective 20X / NA 0.45 (Olympus) on an Olympus iX73 microscope equipped with an X-Light V3 spinning disc head (CrestOptics), an LDI laser illuminator (89 North), a PRIME CMOS camera and a MetaMorph software (Molecular Devices). The analysis was performed counting the total number of cells, the live cells in the green channel and the dead cells in the red channel, using the Imagej Software. The percentage of live cells (Live Cells / Total Cells Number)* 100 and the percentage of dead cells (Dead Cells / Total Cells Number)* 100 was plotted in GraphPad Prism version 8.0. The analysis was performed on 9 / 3 / 3 (FOV / batches / cover slips) per conditions.

[0235] 10. Induction of a tau hyperphosphorylated state and incubation of NPM-HumAfFt- BT1 complex Differentiated day 30 cells were treated with 100 pM of Okadaic acid (Merck Life Science) for 4 hours at 37°C in 5% CO2. Afterward, untreated and treated neurons were incubated with 150 nM of NPM-HumAfFt-BT1 complex for 24 hours at 37°C in 5% CO2 and thus fixed with 4% paraformaldehyde (PFA, Sigma Aldrich) for 15 minutes at room temperature. For immunostaining, fixed retinal neurons were incubated overnight at 4°C with primary antibodies HT7 (1:1000 mouse, MN1000, Invitrogen), AT8 (1:200 mouse, MN1020, Invitrogen), T22 (1 :200 rabbit, ABN454, Merck). The day after, the cells were incubated with secondary antibody Goat antirabbit or anti-mouse Alexa Fluor™ plus 647 (1:750, ThermoFisher Scientific) for 1 hour at room temperature. Hoechst was used to stain nuclei. Fluorescence images of 2048 x 2048 pixels (6,5 pM / pixel) were acquired with oil objective 60X / NA 1.42 (Olympus) in stack with z-step of 0.2 pm, on an Olympus iX73 microscope equipped with an X-Light V3 spinning disc head (CrestOptics), an LDI laser illuminator (89 North), a PRIME CMOS camera and a MetaMorph software (Molecular Devices).

Claims

CLAIMS1. A delivery system consisting of a nanocage of humanised ferritin from Archaelogus fulgidus (HumAfFt) of SEQ ID NO 1 , and the fluorescent marker of formula I:wherein X and Y are carbon atoms linked either by a double bond with E or Z configuration or by an aromatic or heteroaromatic para-substituted ring, or by an aromatic or heteroaromatic 1,4 disubstituted ring;R is hydrogen, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)s, or C5-7 aromatic or aliphatic heterocycle; and in each substituent, RA is: hydrogen; halogen, hydroxyl; CF3, a C1-7 saturated or unsaturated chain, linear or branched containing up to three independent heteroatoms selected among nitrogen oxygen and sulphur; C5-7 cycloalkyl; phenyl; C5-7 heterocycle or n-etilenglycol, and wherein said HumAfFt is covalently bound to moiety comprising a thiol-reactive group.

2. The delivery system of claim 1 whereinX and Y are carbon atoms linked by an aromatic para-substituted ring or by an aromatic 1,4 disubstituted ring;R is hydrogen, halogen, NH(RA), N(RA)2, NHC(=O)RA, ORA, OC(=O)RA, SRA, SO2RA, SO3RA, OSO2RA, OSO3RA, C(RA)3, or C5-7 aromatic or aliphatic heterocycle; and in each substituent, RA is: hydrogen, halogen hydroxyl CF3; a C1-7 saturated or unsaturated chain, linear or branched containing up to three independentheteroatoms selected among nitrogen oxygen and sulphur; C5-7 cycloalkyl; phenyl; C5-7 heterocycle or n-etilenglycol.

3. The delivery system according to claim 2, wherein R is NH2, NH(CH3), N(CH3)2, N(Ph)2, imidazole, morpholine or piperazine.

4. The delivery system according to claim 4 wherein said fluorescent marker is selected from3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1 ,2- c:2', 1 -f] [ 1 ,3,2]diazaborinin-4-ium-5-uide;3-((E)-4-((E)-4-(diphenylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1 ,2- c:2', 1 -f] [ 1 ,3,2]diazaborinin-4-ium-5-uide;3-((E)-4-((E)-4-aminostyryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2-c:2',1'- f][1,3,2]diazaborinin-4-ium-5-uide;5.5-difluoro-1-methyl-3-((E)-4-((E)-4-(methylamino)styryl)styryl)-5H-dipyrrolo[1 ,2- c:2', 1 -f] [ 1 ,3,2]diazaborinin-4-ium-5-uide;5.5-difluoro-1-methyl-3-((1E,3E,5E)-6-(pyrrolidin-1-yl)hexa-1,3,5-trien-1-yl)-5H- dipyrrolo[1 ,2-c:2', 1'-f][1 ,3,2]diazaborinin-4-ium-5-uide;5.5-difluoro-1-methyl-3-((E)-4-((E)-4-morpholinostyryl)styryl)-5H-dipyrrolo[1 ,2-c:2',T- f][1,3,2]diazaborinin-4-ium-5-uide;5.5-difluoro-1-methyl-3-((E)-4-((E)-4-(piperazin-1-yl)styryl)styryl)-5H-dipyrrolo[1 ,2- c:2', 1 -f] [ 1 ,3,2]diazaborinin-4-ium-5-uide;3-((E)-4-((E)-4-(1H-imidazol-1-yl)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1,2- c:2', 1 -f] [ 1 ,3,2]diazaborinin-4-ium-5-uide.

5. The delivery system according to claim 4, wherein said fluorescent marker is 3-((E)-4-((E)-4-(dimethylamino)styryl)styryl)-5,5-difluoro-1-methyl-5H-dipyrrolo[1 ,2- c:2', 1 -f] [ 1 ,3,2]diazaborinin-4-ium-5-uide.

6. The delivery system according to anyone of claims 1 to 5, wherein said thiolreactive group is selected from N-substituted maleimide, N-substituted halobenzene sulphonamide, and haloacetoamide.

7. The delivery system according to claim 6 wherein said halo is selected from iodine, bromine and fluorine or said N-substituted maleimide hydrophobic moietiesare selected from the group consisting of N-alkyl maleimide wherein alkyl group comprises 1 to 12 carbon atoms, and / or rings, preferably aromatic rings such as N- benzyl maleimide; N-cycloalkyl maleimide, N-cyclohexyl maleimide; or said N- substituted halobenzene sulphonamide hydrophobic moiety is halobenzene sulphonamide, wherein hydrogens atoms is substituted with 1 to 5 halogens and comprises alkyl groups from 1 to 12 carbon atoms, aliphatic rings and / or fused rings, preferably benzyl, biphenyl, pyrenyl and perylenyl group.

8. The delivery system according to claim 8 wherein said N-substituted maleimide is N-(1-Pyrenil)maleimide.

9. A composition comprising the delivery system according to anyone of claims 1 to 8 and one or more additional excipients and / or carriers.

10. The composition of claim 9 wherein said composition is a pharmaceutical composition.

11. The composition of claim 9 or 10 in the form of a sprayable composition, an ophthalmic composition, a composition for aerosol, for intranasal administration, eye drops, ophthalmic ointment or dry powder.

12. Use of the delivery system of anyone of claims 1 to 7 or of the pharmaceutical composition of anyone of claim 8 to 10 for the detection of neurofibrillary tangles of the Tau protein.

13. A method for the detection of neurofibrillary tangles of the Tau protein comprising the steps of: contacting the delivery system according to any one of claims 1 to 8 or the composition according to anyone of claims 9 to 11 with a biological sample under conditions wherein said fluorescent marker binds to the neurofibrillary tangles of the tau protein; detecting said fluorescent marker bound to the biological sample.

14. An imaging method comprising the steps of:- administering the delivery system according to any one of claims 1 to 8 or the composition according to anyone of claims 9 to 11 ,- carrying out a non-invasive fluorescence imaging of the retina of said subject, wherein the detection of fluorescence from said fluorescent marker indicates the binding of said marker to the retina.

15. A method for determining a fluorescence graph comprising the steps of:- administering to a subject the delivery system according to any one of claims 1 to 8 or the composition according to anyone of claims 9 to 11 and carrying out a non- invasive quantitative fluorescence imaging on the retina of said subject, at a plurality of successive time instants ti, where i is 0 to n, so obtaining corresponding fluorescence values;- using the obtained fluorescence values for determining a graph of the fluorescence as a function of time.

16. The method of claim 15 wherein said successive time instants are separated by a time period of one or more weeks or one or more months between each time instant and the subsequent one.

17. The method of anyone of claims 14 to 16 wherein the non-invasive fluorescence imaging is carried out after 30 minutes to one day from said administration.

18. The method of anyone of claims 14 to 17, wherein said fluorescence imaging is carried out by submitting the retina of said subject to irradiation with a light source having a wavelength (A) comprised between 350 and 650 nm, and detecting and / or quantifying the fluorescence emitted by said delivery system.

19. A method according to any one of claims 14 to 18, wherein said delivery system or said composition is administered orally or intranasally, or by aerosol, or in the form of an ophthalmic ointment, dry powder or eyedrops.

20. Use of the delivery system as defined in anyone of claims 1 to 8 or of the composition as defined in anyone of claims 9 to 11 in the diagnosis of a thauthopathy.

21. A method for the diagnosis of neurodegenerative diseases characterised by tau tangles comprising the steps of:- administering to a subject the delivery system or the composition according to the invention;-carrying out a non-invasive quantitative fluorescence imaging on the retina of said subject, wherein the presence of tangles indicates the presence of a neurodegenerative disease.

22. The use of claim 20 or the method of claim 21, wherein said tauopathy is a neurodegenerative disease involving the aggregation of tau proteins into neurofibrillary or gliofibrillary tangles (NFTs), selected from Alzheimer’s disease, Down syndrome, amyotrophic lateral sclerosis, Pick’s disease, Parkinson’s disease, primary age-related tauopathy (PART), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), corticobasal degeneration (CDB), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), lytico-bodig disease (Parkinson-dementia complex of Guam), ganglioglioma and gangliocytoma, meningiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), as well as lead encephalopathy, tuberous sclerosis, Pantothenate kinase-associated neurodegeneration, and lipofuscinosis.

23. The use or the method according to claim 22 wherein said tauopathy is selected from Alzheimer’s disease, Down syndrome, amyotrophic lateral sclerosis, Pick’s disease, or Parkinson’s disease, preferably is Alzheimer’s disease.

24. A method for the preparation of a delivery system according to anyone of claims 1 to 8, comprising the following steps: i) reacting a solution comprising a moiety comprising a thiol reacting group with cysteine 54 of the HumAfFt of SEQ ID NO 1 thereby obtaining a moiety comprising a thiol reacting group -HumAfFt with a hydrophobic surface within the protein nanocage; ii) adding a solution of a fluorescent marker of formula I to the solution obtained in step i) thereby obtain a moiety comprising a thiol reacting group- HumAfFt conjugated with a fluorescent marker of formula I;iii) at the end of the reaction as determined by MALDI mass spectroscopy, adding a divalent cation salt in order to close the HumanAfFt as 24-mer, thereby creating an internal cavity.

25. The method according to claim 24, wherein said thiol-reactive group is selected from N-substituted maleimide, N-substituted halobenzene sulphonamide, and haloacetoamide.

26. The method according to claim 25 wherein said halo is selected from iodine, bromine and fluorine or said N-substituted maleimide hydrophobic moieties are selected from the group consisting of N-alkyl maleimide wherein alkyl group comprises 1 to 12 carbon atoms, and / or rings, preferably aromatic rings such as N- benzyl maleimide; N-cycloalkyl maleimide, N-cyclohexyl maleimide; or said N- substituted halobenzene sulphonamide hydrophobic moiety is halobenzene sulphonamide, wherein hydrogens atoms is substituted with 1 to 5 halogens and comprises alkyl groups from 1 to 12 carbon atoms, aliphatic rings and / or fused rings, preferably benzyl, biphenyl, pyrenyl and perylenyl group.

Images