Field of invention

The present invention relates to a class of cell penetrating enhancers having corona-like structures consisting of (guanidyl)-oligosaccharidic derivatives and use thereof as carriers for intracellular delivery of macromolecules and colloidal systems covalently or physically conjugated thereto.

Background of the invention

The impermeable nature of the biological barriers to large hydrophilic structures is the main reason of poor absorption and inefficient intracellular delivery of proteins, oligonucleotides and colloidal supramolecular systems. Such a poor transmembrane permeability strongly limits the development of convenient non parenteral formulations, transmucosal drug delivery as well as the intracellular delivery of bioactive macromolecules and colloidal drug therapeutic systems, namely polymer conjugates and nanoparticles.

Recently, small regions of proteins termed protein transduction domains (PTDs) or cell penetrating peptides (CPPs) have been demonstrated to possess cell penetrating properties that can be properly exploited to enhance the performance of colloidal therapeutics.

Since about 15 years ago, when penetratin has been recognised as the first cell penetrating enhancer peptide, a variety of CPPs have been developed. They include Tat derived peptides, signal sequence derived peptides and synthetic or chimeric peptides. Most of the recognized CPPs contain cationic, amphiphilic or hydrophobic functions, which promote the cell surface interaction and translocation.

The Tat derived peptides have been demonstrated to be very efficient penetrating agents. They have been designed according to the structure of the HIV-1 Tat, an 86 amino acid nuclear transcription-activating protein constituted by three functional domains. The region responsible for cell uptake and nuclear import contains 6 arginine and 2 lysine residues within a linear sequence of 9 amino acids in random conformation. The presence of short peptide sequences containing basic residues, such as arginines and lysines, is a structural requisite to enable cell-penetrating activity and nucleus translocation of these oligoaminoacids (Green M, Loewenstein PM, 1988; Kalderon D et al., 1984). Accordingly, many homopolymers of arginine have been synthesised and investigated. Studies demonstrated that their internalization efficiency depends on the length of the peptide backbone. Stretches of six (R6) to eight (R8) arginine residues showed the highest cytoplasmic internalization ability (Futaki S et al., 2001 ). However, the mechanism of the cell translocation is still controversial as either passive or active mechanisms, receptor or absorptive mediated endocytosis have been claimed. Also, the correlation between the structural features of these peptides and the cell uptake behaviour is still under active investigation.

Although many aspects of CPP activity have not been fully elucidated yet, and a number of contrasting experimental data have been generated, these molecules have a wide relevance in the pharmaceutical and medical fields as they open up interesting perspectives either for enhanced transmucosal absorption, mostly for nasal and intestinal delivery, or for intracellular drug delivery and targeting (Fonseca S et al., 2009). Covalent linkages or simple physical interactions of CPPs have been demonstrated to promote the cell entry of a large variety of hydrophilic compounds ranging from small molecules to plasmid DNA and oligonucleotides (Fisher AA et al., 2009; Veldhoen S et al., 2008), peptides (Bonny C et al., 2001 ), proteins (Peitz M et al., 2002; Saalik P et al., 2004), as well as nanocarriers such as liposomes, micelles (Torchilin VP, 2008), polymeric particles and quantum-dots (Lewin M et al., 2000; Medintz IL et al., 2008). Nevertheless, many studies highlighted some pitfalls in the use of CPPs in drug delivery such as poor cell uptake, unspecific cell delivery, inconvenient in vivo performance, low stability and intrinsic biological activity. Therefore, the combination of these molecules may result ineffective or entail the risk of toxicity (Trehin R et al. 2004). Summary of the invention

Since the structural features of CPPs have been shown paramount to the penetration properties, in order to overcome the drawbacks to the exploitation of CPPs for drug delivery previously mentioned, the first purpose of the present invention is to provide non-peptide molecules with cell penetrating activity. Another purpose is to design molecules displaying higher stability and lower toxicity as compared to CCPs and being devoid of any biological activity. A further purpose is to properly build up unconventional chemical structures suitable for chemical conjugation or physical assembling to macromolecular or supramolecular therapeutic systems for efficient and selective cell targeting.

According to these purposes, the inventors have conceived a novel class of cell penetrating enhancers with unusual corona-like structure suitable to be covalently or physically combined with macromolecular and colloidal systems to promote their cell uptake.

Therefore, in a first aspect an object of present invention are molecules consisting of (guanidyl)-oligosaccharidic derivatives represented by the general formula (I)

(I)

wherein:

R ¹ , R ² , R ³ , R ⁴ can be equal or different from each other and can be:

a hydrogen -H;

a linker of formula -CO-R'-X, where R ¹ is a C ₂ -C ₃ alkyl branched chain and where X is a halogen; or

a guanidine terminating group of formula -CO-R ^l -[CH ₂ C(CH ₃ )-CO-NH-R ¹¹ - NHC(NH ₂ )=NH]-X, where R ¹ and X have the same meanings previously mentioned for the linker, and R" is a linear or branched C ₄ -C ₈ alkyl chain optionally bearing a -OH or a -COOH group,

with the proviso that R ¹ , R ² , R ³ , R ⁴ represent independently one from the other at least one guanidine terminating group of formula -CO-R'-[CH ₂ C(CH ₃ )-CO-NH-R"-

NHC(NH ₂ )=NH]-X;

R ⁵ is: a spacer: -(CH ₃ CO)N-R" -Y, where R'" is a linear or branched C ₈ -C ₂ 2 alkyl chain or an oligo(ethylene glycol) chain derivative of formula -[CH ₂ CH ₂ O] _m - (CH ₂ )p having different molecular weight and where m is an integer selected in the range from 2 to 1 36 and p is an integer selected in the range from 2 to 6, and Y is a terminating group selected from the carboxyl-, amino-, hydroxyl- or thiol- groups;

n is an integer ranging from 2 to 8,

and salts thereof.

In another aspect a further object of the invention is the use of (guanidyl)- oligosaccharidic derivatives represented by the general formula (I) as carriers for intracellular delivery of macromolecules and colloidal systems physically or covalently conjugated thereto.

Yet, in a further aspect another object of the present invention is the method for preparing said (guanidyl)-oligosaccharidic derivatives represented by the general formula (I) comprising at least the steps of :

oligosaccharides derivatization on the anomeric carbon of the terminal glycosidic unit with a spacer of formula -HN-R'"-Y, where R ¹ " and Y have the meanings previously mentioned;

N-acetylation of the oligoglycosylamino-derivative obtained;

- esterification of oligosaccharide hydroxyl groups with a linker of formula -CO-R'-X, where R ¹ and X have the meanings previously mentioned;

conjugation of guanidyl residue of formula -[CH ₂ C(CH ₃ )-CO-NH-R"- NHC(NH ₂ )=NH]-X, where R" has the meanings previously mentioned, to the linkers previously introduced on the hydroxyl groups of the oligosaccharidic backbone via atom transfer radical polymerization (ATRP).

Other characteristics and advantages of the present invention will be described in the following detailed description of possible, but not exclusive, embodiments of corona-like (guanidyl)-oligosaccharidic derivatives of general formula (I). The examples of corona-like (guanidyl)-oligosaccharidic derivatives according to the present invention and the method of preparation thereof are herein provided for illustrative purpose and then non-limiting.

Brief description of the figures

Figure 1 shows the NMR analysis of the corona-like (arginyl) ₄ -maltosyl-N-acetyl- amino-dodecanoic acid of example 1 .

Figure 2 shows the FT-IR analysis of the corona-like (arginyl) -maltosyl-N-acetyl- amino-dodecanoic acid of example 1 .

Figure 3 shows the NMR analysis of the corona-like (arginyl)4-lactosyl-N-acetyl- amino-dodecanoic acid of example 2.

Figure 4 shows the FT-IR analysis of the corona-like (arginyl) ₄ -lactosyl-N-acetyl- amino-dodecanoic acid of example 2.

Figure 5 shows the NMR analysis of the corona-like (arginyl) ₇ -maltotriosyl-N- acetyl- amino-dodecanoic acid of example 3.

Figure 6 shows the FT-IR analysis of the corona-like (arginyl) ₇ -maltotriosyl-N- acetyl- amino-dodecanoic acid of example 3.

Figures 7A-B show the synthesis of a corona-like (arginyl)-maltotriosyl-N-acetyl- amino-dodecanoic acid of example 3, namely the (arginyl) ₇ -maltotriosyl-N-acetyl- amino-dodecanoic acid: A. 1 . maltotriose derivatization with 12-aminododecanoic acid; 2. esterification of the maltotriosyl hydroxyl groups with 2-bromoisobutyryl bromide; B. 3. arginine conjugation via atom transfer radical polymerization (ATRP).

Figure 8 shows the chemical formula of the corona-like (arginyl) ₇ -maltotriosyl-N- acetyl-amino-dodecanoic acid of example 3.

Figures 9A-B show the synthesis of fluorescent (arginyl) ₇ -maltotriosyl-N-acetyl- amino-dodecanoyl-BSA.

Figure 10 shows the relative fluorescence of untreated (black), FITC-BSA treated (white) and (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA treated (grey) lysated MCF-7 cells and MC3T3-E1 cells. Mean values and standard deviations were calculated on the basis of values obtained from five experiments. Figure 11 shows the side scatter (SSC-H) versus fluorescence (FL1-H) plots obtained by flow activated cytometry sorting analysis of MCF-7 (A) MC3T3-E1 (B) cells after incubation with plain buffer (A1 and B1 ), FITC-BSA (A2 and B2) and (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA (A3 and B3). Figure 12 shows the confocal microscope images obtained with MCF-7 (A) and MC3T3-E1 (B) incubated for 30 min with FITC-BSA (A1 and B1 ) and (arginyl) ₇ - maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA conjugate (A2 and B2) (back = background; dark grey = blue fluorescence; light grey = green fluorescence).

Figure 13 shows the cytotoxicity profiles obtained by MCF-7 (A) and MC3T3-E1 (B) incubation with increasing concentrations of 2 kDa polyethylenimine (o) and (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid (·). Mean values and standard deviations were calculated on the basis of values obtained from six experiments.

Detailed description of the invention

The corona-like cell penetration enhancers consist in non linear structures different from oligopeptides (i.e. cell penetrating peptides). Their structure is characterized by guanidine group grafting to oligosaccharide cores.

The cell-penetrating enhancers of the invention consisting of corona-like non-linear (guanidyl)-oligosaccharidi (I)

(I)

wherein:

R ¹ , R ² , R ³ , R ⁴ can be equal or different from each other and can be:

a hydrogen -H; or

a linker of formula -CO-R -X, where R is a C ₂ -C ₃ alkyl branched chain and where X is a halogen; or

a guanidine terminating group of formula -CO-R'-[CH ₂ C(CH ₃ )-CO-NH-R"- NHC(NH ₂ )=NH]-X, where R ¹ and X have the same meanings previously mentioned for the linker, and R" is a linear or branched C ₄ -C ₈ alkyl chain optionally bearing a -OH or a -COOH group, with the proviso that R ¹ , R ² , R ³ , R ⁴ represent independently one from the other at least one guanidine terminating group of formula -CO-R'-[CH ₂ C(CH ₃ )-CO-NH-R"- NHC(NH ₂ )=NH]-X;

R ⁵ is:

- a spacer: -(CH ₃ CO)N-R ^lll -Y, where R ^m is a linear or branched C ₈ -C ₂₂ alkyl chain or a linear oligo(ethylene glycol) chain derivative of formula - [CH ₂ CH ₂ 0] _m -(CH ₂ )p having different molecular weight and where m is an integer selected in the range from 2 to 1 36 and p is an integer selected in the range from 2 to 6, and Y is a terminating group selected from the carboxyl-, amino-, hydroxyl- or thiol- groups;

n is an integer selected in the range from 2 to 8,

have been designed keeping in mind the oligo-arginyl structure of the Tat protein (SwissProt P04608; GenBank K03455) fragment 49-57, which is involved in the trans-membrane translocation. Said corona-like non-linear (guanidyl)- oligosaccharidic derivatives of general formula (I) are specifically conceived for conjugation to large size systems, namely proteins, oligonucleotides or colloidal drug carriers in order to promote their cell entry. The synthesis of the new cell penetrating enhancer was set up in order to obtain a non-conventional architecture by introducing guanidyl moieties into a core anchoring structure. The oligosaccharides were selected as anchoring molecules, since their multifunctional structure can be properly derivatised through simple chemical protocols with guanidyl residues and a spacer arm to produce a non peptide "corona-like" construct for conjugation to macromolecules or supramolecular drug delivery systems.

In fact, the (guanidyl)-oligosaccharidic derivatives of formula (I) can easily conjugate bioactive macromolecules, both specific macromolecular or supramolecular colloidal structures, by means of a covalent binding through the spacer represented by R ⁵ bearing the latter a terminating functional group, whilst the cationic features of these (guanidyl)-oligosaccharidic derivatives can be exploited for a physical combination with said macromolecules or colloidal systems. Said macromolecules or supramolecular colloidal structures can be peptides, proteins, oligonucleotides, siRNA, genes, polymer therapeutics, namely dendrimers and other bioconjugates, or supramolecular cargoes, namely liposomes, micelles, nanoparticles.

For the purposes of the invention, the oligosaccharide core is at least formed by two and up to 8 (and preferably 2 or 3) glycosidic units and said oligosaccharides are preferably selected from the group consisting of lactose, mannose, cellobiose, maltose, isomaltose, maltotriose, maltotetraose, maltopentose, maltohexaose, maltohepatose, maltooctaose. The preferred oligosaccharides are lactose, maltose, maltotriose, maltoheptaose and the most preferred oligosaccharides are lactose, maltose and maltotriose.

Moreover, for the corona-like (guanidyl)-oligosaccharidic derivatives of formula (I) in R ¹ , R ² , R ³ , R ⁴ R ¹ is preferably an isopropyl group and X is preferably CI or Br, and when in R ¹ , R ² , R ³ , R ⁴ are guanidyl residue of formula -[CH ₂ C(CH ₃ )-CO-NH- R"-NHC(NH ₂ )=NH]-X R" is a C ₄ alkyl chain bearing a -COOH group. As for the preferred meanings of R ⁵ , R ^M is preferably a C-I _O -C-I _S alkyl chain and Y is a terminating functional group preferably selected from -COOH or -NH ₂ . When R ¹ " is a linear oligo(ethylene glycol) chain -[CH ₂ CH ₂ O] _m - its preferred molecular weight is from 200 to 3400 Da and m is selected from 5 to 77. The most preferred linker of formula - CO-R'-X is 2-bromoisobutyryl group and the most preferred spacer R ⁵ is N-acetyl-12-aminododecanoyl acid.

In preferred embodiments in the (guanidyl)-oligosaccharidic derivatives of general formula (I) R ¹ , R ² , R ³ , R ⁴ , independently one from the other, are at least 2 and preferably from 4, when the oligosaccharidic core is a disaccharide, to 20, when the oligosaccharidic core is a heptasaccharide, guanidine terminating group of formula -C0-R'-[CH ₂ C(CH ₃ )-C0-NH-R"-NHC(NH ₂ )=NH]-X. In most preferred embodiments R ¹ , R ² , R ³ , R ⁴ are all equal to guanidine terminating groups of formula -C0-R'-[CH ₂ C(CH ₃ )-C0-NH-R"-N HC(NH ₂ )=NH]-X. The preferred guanidine terminating groups of formula -CO-R ^l -[CH ₂ C(CH ₃ )-CO-NH-R"- NHC(NH ₂ )=NH]-X are selected from 4 to 7, when the oligosaccharidic core is a disaccharide, and from 4 to 20, when the oligosaccharidic core is a heptasaccharide, where R ¹ , X and R" have the preferred meanings above indicated. The corona-like (guanidyl)-oligosaccharidic derivative of the invention was synthesised according to the multi-step chemical protocol involving as herein previously indicated:

oligosaccharides derivatization on the anomeric carbon of the terminal glycosidic unit with a spacer of formula -NH-R ^m -Y selected from -NH-(linear or branched C ₈ -C ₂ 2 alkyl chain)-COOH, -NH-(linear or branched C ₈ -C ₂ 2 alkyl chain)-OH, -NH-(linear or branched C ₈ -C ₂ 2 alkyl chain)-SH, -NH-(linear or branched C ₈ -C ₂ 2 alkyl chain)-NH ₂ , - NH-(CH ₂ CH ₂ 0) _m (CH ₂ )p-COOH _! -NH-(CH ₂ CH ₂ 0) _m (CH ₂ ) _p -OH _! -NH- (CH ₂ CH ₂ 0)m(CH ₂ )p-SH, -NH-(CH ₂ CH ₂ 0) _m (CH ₂ ) _p -NH ₂ [m=2-136; p=2-

6],

N-acetylation of the oligoglycosylimino-group function obtained;

esterification of the remaining oligosaccharide hydroxyl groups with the linker of formula X-CO-R'-X, where R ¹ and X have the meanings previously mentioned, and preferably with 2-bromoisobutyryl bromide;

guanidyl conjugation to the arms introduced on the hydroxyl groups of the oligosaccharidic backbone with the linkers via atom transfer radical polymerization (ATRP).

The first step of derivatization of a selected oligosaccharide is obtained by reaction of the amino group of the selected spacer R ⁵ , preferably 12-amino dodecanoic acid, with the anomeric carbon of selected oligosaccharide (preferably maltose or maltotriose). The reaction is carried out in methanol/acetic acid (95/5 by volume) at 50-65 °C. The resulting amino group is stabilised by amino acetylation using acetic anhydride to avoid the detachment of the amino-spacer residue. The acetylation is carried out for up to 96 hours at room temperature. The oligosaccharide derivative obtained is purified by precipitation in ethyl ether.

In the second step said N-acetylated glycosyl-amino-spacer derivative is esterificated at the saccharides OH functions with a selected linker (preferably 2- bromo-isobutyryl-bromide) in order to obtain a multi-armed oligosaccharide initiator for ATRP polymerization. The linker conjugation is carried out in chloroform at 0°C in the presence of triethylamine. The conjugation of guanidyl residues to the multi- armed oligosaccharide initiator is carried out in dimethylsulfoxide by ATRP polymerization using methacryloyl-guanidyl derivative (preferentially methacryloyl- arginine) as monomer and tris[(2-pyridyl)methyl]amine (TPMA) as ligand for Cu(l). Both methacryloyl-arginyl derivative and TPMA are synthesised according to modified protocols reported in the literature known to an artisan skilled in the art. The ATRP reaction is carried out under nitrogen atmosphere to avoid the catalyst inactivation by oxygen oxidation of Cu(l) to Cu(ll).

The evaluation assays for cell-penetrating activity and cytotoxicity were performed on the lead derivative (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid.

The cell penetrating properties of the new (arginyl) ₇ -maltotriosyl-N-acetyl-amino- dodecanoyl derivative were evaluated using BSA as cargo molecule because this hydrophilic macromolecule is not taken up by cells. The human MCF-7 breast adenocarcinoma and murine MC3T3-E1 embryonic fibroblast cell lines were chosen because they do not have phagocytic properties.

The cell culture analysis by fluorescence spectrometry, fluorescence activated cell sorting and confocal microscopy showed that the corona-like (arginyl) ₇ - maltotriosyl-N-acetyl-amino-dodecanoyl derivative possesses excellent cell penetration properties.

The quantitative fluorescence analysis showed that, although both cell lines undergo extensive (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA uptake, the protein internalization obtained with MC3T3-E1 was slightly higher than that obtained with MCF-7. On the other hand, the cytofluorimetric data showed that higher fraction of MC3T3-E1 cells were involved in the (arginyl) ₇ - maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA uptake with respect to the MCF-7 cells. These results seem to suggest that the novel cell penetration enhancer display cell dependent translocation properties possibly exploiting different penetration mechanisms.

The confocal microscopy confirmed the remarkable cell uptake of (arginyl) ₇ - maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA. Furthermore, the confocal images showed a punctuated fluorescence distribution, which corroborates the hypothesis that the macromolecular cargo is taken up by endocytosis pathway rather than by simple passive diffusion. Also, the lack of fluorescence in the nucleus seems to indicate that the transmembrane translocation is limited to the cytoplasm compartment. Finally, the novel non-linear (arginyl) ₇ -oligosaccharide derivative was found to possess negligible toxicity. The IC50 was in fact higher that 1 00 μΜ, which corresponds to a very high concentration.

This is a relevant issue in favour of these new cell-penetrating products, which undergoes intracellular localisation. Furthermore, low toxicity is required because these products can be used also in high doses to enhance the drug absorption of non-invasive formulations. In any case, absence or low toxicity is a requisite for its exploitation in drug delivery.

Therefore, the corona-like (guanidyl)-oligosaccharidic derivatives provided by the present invention fully fulfil the purposes of the present invention and can be usefully employed as carriers for intracellular delivery of macromolecules or colloidal nanoparticles through a covalent binding or a physical combination with the same.

The invention is further illustrated by the following examples.

EXAMPLES

Materials

Maltose monohydrate [4-O-cc-D-glucopyranosyl-D-glucose], Lactose monohydrate [4-0- -D-galactopyranosyl-cc, -D-glucose], Maltotriose [O-cc-D-glucopyranosyl- (1 ,4)-0-cc-D-glucopyranosyl-(1 ,4)-D-glucose], Maltoheptaose {O-cc-D- glucopyranosyl-[(1 -4)-0-cc-D-glucopyranosyl] ₅ (1 -4)-D-glucose}, 1 2- aminododecanoic acid, acetic anhydride, 2-bromo-isobutyryl bromide, triethylamine, ascorbic acid, 1 -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), polyethylenimine (PEI, 2 kDa), fetal bovine serum (FBS), RPMI-1 640 medium, Dulbecco's modified essential medium (D-MEM), penicillin (1 0000 UI/mL) -streptomycin (1 0 mg/mL) - amphotericin B (25 μg/mL) stabilised solution, L-glutamine (200 mM), Dulbecco's phosphate buffered saline (D-PBS), trypsin-EDTA solution (2.5 %w/v), 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Amino/carboxyl terminating heterobifunctional poly(eththlene glycol) (NH ₂ -PEG- COOH, MW 3.4 kDa) was obtained by Nektar (Huntsville, AL, USA). L-Arginine hydrochloride was furnished by SERVA Electrophoresis (Heidelberg, Germany).

Fluorescein isothiocyanate (FITC), 2-picolyl chloride hydrochloride, 2-picolylamine, copper(l) bromide (CuBr), bovine serum albumin - fraction V (BSA), Triton X-100, paraformaldehyde were purchased from Fluka (Buchs SG, Switzerland). Vectashield ^® Mounting Medium with DAPI was from Vector Laboratories (Peterborough, UK).

All other reagents were obtained from Fluka Analytical or Sigma-Aldrich.

The solvents of analytical or HPLC grade were furnished by Carlo Erba (Milan, Italy), VWR International (Lutterworth, UK) and Sigma-Aldrich (St. Louis, MO, USA).

Cell culture plates and flask, 96-well flat bottomed microassay plate and four-well chambered slides were purchased from BD Falcon (BD Bioscience, NJ, USA). As example of embodiments of the invention novel corona-like (guanidyl)- oligosaccharidic derivatives were synthesised according to the multi-step chemical protocol previously described in detail. The intermediate and final products were characterised by ¹ H-NMR, FT-IR, mass spectrometry, colorimetric assays and elemental analysis.

In the examples 1 and 4 are given in full details the preparations of the (arginyl) ₄ - maltosyl-N-acetyl-amino-dodecanoic acid, (arginyl) ₄ -lactosyl-N-acetyl-amino- dodecanoic acid, (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid and oligo(arginyl)-maltoheptaosyl-N-acetyl-amino-poly(ethylene glycol)-carboxyl acid. Example 1. Synthesis of the corona-like (arginylk-maltosyl-N-acetyl-amino- dodecanoic acid

Synthesis of maltosyl-N-acetyl-amino-dodecanoic acid

One gram of maltose monohydrate (2.775 mmoles) was dissolved in 20 mL of CH ₃ OH/1 .5% acetic acid containing 1 .195 g of 12-aminododecanoic acid (5.55 mmoles). The suspension was thermostated at 65 °C and magnetically stirred. After 24 hours, the reaction mixture was cooled to 0°C and added of 2.5 mL of acetic anhydride (26.47 mmoles). The solution was maintained under stirring for 24 hours at 40 °C and then cooled to room temperature and left under stirring. The precipitate was removed by centrifugation and the solution was reduced to 4 mL under vacuum and dropped into 200 mL of diethyl ether. The precipitate was collected by filtration and desiccated under reduced pressure. The product yield was 1 .6 g.

The purified material was analyzed by mass spectrometry, ¹ H-NMR, and RP- HPLC using a Luna C18 column (250x4.6mm, Phenomenex, Torrance, CA, USA) eluted with a gradient of H ₂ O/0.05% trifluoroacetic acid (A) and acetonitrile/0.05% trifluoroacetic acid (B) (0-3 min 15% B, 3-27 min from 15% to 90% B), the UV detector was set at 220 nm.

ESI-MS [m/z]: 580.28 (M-H ⁺ ) ^{1 "} [calcd for C ₂₆ H ₄₇ N0 ₁₃ : 581 .30].

1 H-NMR (D ₂ 0): δ ppm 5.22 d [1 H, H anomeric], ppm 3.97-3.60 broad m [14H, H maltose], ppm 3.43 t [2H, J = 9.3 Hz, -NCH ₂ -], ppm 2.30 t [2H, J = 7.3 Hz, - CH ₂ COOH), ppm 2.16 s [3H, -NCOCH ₃ ], ppm 1 .58 broad s [4H, - NCH ₂ CH ₂ (CH ₂ ) ₇ CH ₂ CH ₂ COOH], ppm 1 .30 s [14H, -N(CH ₂ ) ₂ (CH ₂ )7(CH ₂ ) ₂ COOH]. RP-HPLC (C18): 14.6 min retention time.

Synthesis of (2-bromoisobutyryl) ₄ -maltosyl-N-acetyl-amino-dodecanoic acid

The synthesis of (2-bromoisobutyryl) ₄ -maltosyl-N-acetyl-amino-dodecanoic acid was carried out according to a modified procedure reported in the literature (Stenzel-Rosenbaum MH et al., 2001 ). Maltosyl-N-acetyl-amino-dodecanoic acid previously prepared (200 mg, 0.34 mmoles) was suspended in 5 mL of anhydrous dichloromethane containing 380 μΙ of triethylamine (2.75 mmoles). The solution was cooled in an ice bath and added of 408 μΙ of 2-bromo-isobutyryl-bromide (3.30 mmoles) in 15 min, and left under stirring at room temperature. After 60 hours the mixture was filtered and the organic solution treated three times with 15 mL iced water, three times with 10 mL of 0.1 N NaOH and finally three times with 15 mL of iced water. The organic phase was dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under vacuum. The resulting red-brown solid was dissolved in 2 mL acetone and added of 250 mL cold water under vigorous stirring. Acetone was removed under vacuum and the aqueous suspension was lyophilised. The yield of the product was 285 mg.

The purified material was analysed by elemental analysis and ¹ H-NMR. Elemental analysis: found C, 42.4%; H, 5.6%; N, 1 .2%; Br, 25.8% (O, 25.7%) [calcd for (2-bromoisobutyryl) ₄ -N-acetyl-maltosyl-amino-dodecanoic acid (C ₄₂ H ₆₇ Br ₄ N0 ₁₇ ): C, 42.8%; H, 5.7%; N, 1 .2%; Br, 27.1 % (O, 23.2%)].

1 H NMR (CDCIs): δ ppm 4.60-3.30 (bm, 12H of glycosyl moiety), 3.29 (bm, 2H, λ- CH ₂ -), 2.15 (m, 2H, a-CH ₂ -), 1 .96 (bs, 24H, CH ₃ ), 1 .94 (s, 3H, acetyl-CH ₃ ), 1 .63 (bm, 4H, 1-CH2- and β-ΟΗ ₂ -), 1 .33-1 .22 (bm, 14H, -CH ₂ - of alkyl chain).

Synthesis of methacryloyl-arginine

Methacryloyl-arginine was synthesised according to the procedure reported in literature [US 6,703,468]. Briefly, 2.1 g of arginine hydrochloride (10 mmoles) dissolved in 20 mL of saturated NaHC0 ₃ water solution was cooled to 0°C and added dropwise of 970 μΙ of methacryloyl chloride (10 mmoles) under vigorous stirring. After 1 hour the pH of mixture was adjusted to 1 with hydrochloric acid and the solution was saturated with sodium chloride, filtered, washed three times with 30 mL of ethyl acetate, and finally extracted three times with 20 mL of 1 :1 of ethyl acetate/isopropanol mixture. The ethyl acetate/isopropanol volumes were pooled and concentrated under reduced pressure. The resulting suspension was filtered to remove the precipitate and the solution was desiccated. The colourless syrup was dissolved in 50 mL water and freeze-dried. The final product yield was 2.4 g. The product was analysed by ¹ H NMR, FT-IR and ESI-MS.

1 H-NMR (D ₂ 0): δ ppm 5.69 (s, 1 H, H ₂ C=C), 5.47 (s, 1 H, H ₂ C=C), 4.41 (dd, J = 9.18, 5.09 Hz, 1 H, a-CH), 3.20 (t, J = 6.77, 2H, 5-CH ₂ ), 1 .91 (s, 3H, CH ₃ -), 1 .85- 1 .72 (m, 2H, γ-ΟΗ ₂ ), 1 .72-1 .58 (m, 2H, β-ΟΗ ₂ ).

FT-IR (KBr): v (cm ^"1 ) 3368 (OH), 3150 (NHC(NH ₂ ) ₂ ⁺ ), 1654 (C=C, C=O and C=N), 1528 (CNH ₂ or CNH ₃ ⁺ ).

ESI-MS [m/z]: 243.13 (M+H ⁺ ) ^{1 +} [calcd for Ci ₀ H ₁₈ N ₄ O ₃ : 242.14].

The purity of the compound was assessed by the integration of the ¹ H-NMR triplet at 3.20 ppm compared with methacryloyl singlets at 5.69 and 5.47 ppm (99 %). Synthesis of tris[(2-pyridyl)methyl]amine (TPMA)

Tris[(2-pyridyl)methyl]amine (TPMA) was prepared according to a modified protocol described by Tyeklar et al. (Tyeklar Z et al. 1993). Ten grams of 2-pycolil chloride (61 .0 mmoles) were dissolved in 25 mL of distilled water and the mixture was cooled to 0°C and added of 12 mL of 5 N NaOH. The solution was added of 3.2 ml_ of 2-(aminomethyl)pyridine (30.5 mmoles) and 50 ml_ of dichloromethane. The reaction mixture was allowed to reach room temperature under vigorous stirring and the pH 9.5 was maintained by 5.0 N NaOH addition. After 48 hours the organic phase was washed three times with 20 ml_ of 2.5 N NaOH and then dried with anhydrous Na ₂ SO ₄ and filtered. The organic solvent was eliminated under vacuum. The TPMA was recovered by treating the resulting waxy red-brown product three times with 25 ml_ of boiling petroleum ether. The organic phase was then desiccated under vacuum. The final product yield was 5.6 g.

The product was analysed by ¹ H-NMR and ESI-MS.

1 H-NMR (CDCIs): δ ppm 8.53 (d, J = 4.86, 3H, Py-H ₆ ), 7.65 (t, J = 7.71 , 3H, Py- H ₄ ), 7.58 (d, J = 7.71 , 3H, Py-H ₃ ), 7.14 (t, J = 6.06, 3H, Py-H ₅ ), 3.88 (s, 6H, -CH ₂ -). ESI-MS [m/z]: 291 .15 (M+H ⁺ ) ^{1 +} and 313.14 (M+Na ⁺ ) ^{1 +} [calcd for Ci ₈ H ₁₈ N ₄ : 290.15]. Synthesis of arginy -maltosyl-N-acetyl-amino-dodecanoic acid

TPMA (45.5 mg, 0.16 mmoles), CuBr (22.49 mg, 0.16 mmoles) and ascorbic acid (0.34 mg, 1 .96x10 ^"3 mmoles) were dissolved in 2.0 ml_ of nitrogen purged DMSO and added to 0.5 ml_ of 0.32 g/mL methacryloyl-arginine previously prepared (0.66 mmoles) in DMSO under stirring at 55 °C. The solution was added of 0.5 ml_ of 92.7 mg/mL of (2-bromoisobutyryl) ₄ -maltosyl-N-acetyl-amino-dodecanoic acid (0.04 mmoles) in DMSO. The reaction mixture was maintained under nitrogen atmosphere for 12 hours and then was exposed to the air. After 30 min the solution was added dropwise to 200 ml_ of cold 1 :1 acetone/diethyl ether mixture containing 1 % acetic acid. The precipitate was desiccated under vacuum, redissolved in 5 ml_ of warm methanol and re-precipitated by dropping into 200 ml_ of the acetone/diethyl ether/acetic acid mixture. The product yield was 47.8% corresponding to 40.2 mg.

The arginine content in the final product was evaluated by Sakaguchi assay (Sakaguchi S, 1925). The experimental data were referred to a calibration curve obtained with standard solutions of 0-0.6 μΜ guanidinium content (y = 7.3955x - 0.0386, R ² =0.99). The final product was also analysed by elemental analysis, ¹ H- NMR (FIG. 1 ).

Elemental analysis: found C, 44.9%; H, 6.9%; N, 10.3%; Br, 21 .3% (O, 16.6%); [calcd for (arginyl) ₄ -maltosyl-N-acetyl-amino-dodecanoic acid (C82H-i ₃ 9Br ₄ N-i ₇ O29): C, 45.9%; H, 6.5%; N, 1 1 .1 %; Br, 14.9% (O, 21 .6%) and calcd for (arginyl) ₄ - maltosyl-N-acetyl-amino-dodecanoic acid HBr salt (C8 ₂ H-i39Br ₄ N ₇ 0 ₂ 9-4HBr): C, 39.9%; H, 5.8%; N, 9.6%; Br, 25.9% (O, 18.8%)].

FT-IR: v (cm ^"1 ) 3353 and 3176 [NHC(NH ₂ ) ₂ ⁺ ], 2931 (CH), 1733 (C=0), 1664 (C=N). (FIG. 2)

Example 2. Synthesis of the corona-like (arainyl)4-lactosyl-N-acetyl-amino- dodecanoic acid

Synthesis of lactosyl-N-acetyl-amino- dodecanoic acid

Two hundred and fifty milligrams of lactose monohydrate (0.694 mmoles) were dissolved in 10 mL of CH ₃ OH/1 .5% acetic acid containing 299 mg of 12- aminododecanoic acid (1 .39 mmoles). The suspension was thermostated at 65 °C and magnetically stirred. After 24 hours, the reaction mixture was cooled to 0°C and added of 0.62 mL of acetic anhydride (6.59 mmoles). The solution was maintained under stirring for 24 hours at 40 °C and then cooled to room temperature and left under stirring. The precipitate was removed by centrifugation and the solution was reduced to 4 mL under vacuum and dropped into 200 mL of diethyl ether. The precipitate was collected by filtration and desiccated under reduced pressure. The product yield was 294 mg.

The purified material was analyzed by mass spectrometry, elemental analysis, ¹ H- NMR and RP-HPLC using a Luna C18 column (250x4.6mm, Phenomenex,

Torrance, CA, USA) eluted with a gradient of H ₂ O/0.05% trifluoroacetic acid (A) and acetonitrile/0.05% trifluoroacetic acid (B) (0-3 min 15% B, 3-27 min from 15% to 90% B), the UV detector was set at 220 nm.

ESI-MS [m/z]: 580.28 (M-FT) ^{1 "} [calcd for C ₂₆ H ₄₇ NOi ₃ : 581 .30].

Elemental analysis: found C, 53.6%; H, 8.3%; N, 2.7% (O, 35.4%); [calcd for lactosyl-N-acetyl-amino-dodecanoic acid (C ₂₆ H ₄₇ NOi ₃ ): C, 53.7%; H, 8.1 %; N,

2.4% (O, 35.8%)].

¹ H-NMR (D ₂ 0): δ ppm 4.55 d [1 H, H anomeric], ppm 3.97-3.60 broad m [14H, H lactose], ppm 3.50 t [2H, -NCH ₂ -], ppm 2.35 t [2H, -CH ₂ COOH), ppm 2.18 s [3H, - NCOCHg], ppm 1 .60 broad s [4H, -NCH ₂ CH ₂ (CH ₂ ) ₇ CH ₂ CH ₂ COOH], ppm 1 .30 bs [14H, -N(CH ₂ ) ₂ (CH ₂ ) ₇ (CH ₂ ) ₂ COOH].

RP-HPLC (C18): 16.8 min retention time. Synthesis of (2-bromoisobutyryl) ₄ -lactosyl-N-acetyl-amino-dodecanoic acid

The synthesis of (2-bromoisobutyryl) ₄ -lactosyl-N-acetyl-amino-dodecanoic acid was carried out according to a modified procedure reported in the literature [ref. cit.]. Lactosyl-N-acetyl-amino-dodecanoic acid previously prepared (200 mg, 0.34 mmoles) was suspended in 5 mL of anhydrous dichloromethane containing 380 μΙ of triethylamine (2.75 mmoles). The solution was cooled in an ice bath and added of 408 μΙ of 2-bromo-isobutyryl-bromide (3.30 mmoles) in 15 min, and left under stirring at room temperature. After 60 hours the mixture was filtered and the organic solution treated three times with 15 mL iced water, three times with 10 mL of 0.1 N NaOH and finally three times with 15 mL of iced water. The organic phase was dried with anhydrous Na ₂ S0 ₄ , filtered and concentrated under vacuum. The resulting red-brown solid was dissolved in 2 mL acetone and added of 250 mL cold water under vigorous stirring. Acetone was removed under vacuum and the aqueous suspension was lyophilised. The yield of the product was 359 mg.

The purified material was analysed by elemental analysis and ¹ H-NMR.

Elemental analysis: found C, 41 .3%; H, 5.9%; N,1 .4%; Br, 26.4% (O, 25.0%) [calcd for (2-bromoisobutyryl) ₄ -N-acetyl-maltosylamino-dodecanoic acid (C ₄₂ H ₆₇ Br ₄ N0 ₁₇ ): C, 42.8%; H, 5.7%; N, 1 .2%; Br, 27.1 % (O, 23.2%)].

1 H NMR (CDCIs): δ ppm 4.60-3.30 (bm, 12H of glycosyl moiety), 3.29 (bm, 2H, λ- CH ₂ -), 2.15 (m, 2H, a-CH ₂ -), 1 .96 (bs, 24H, CH ₃ ), 1 .94 (s, 3H, acetyl-CH ₃ ), 1 .63 (bm, 4H, i-CH ₂ - and β-ΟΗ ₂ -), 1 .33-1 .22 (bm, 14H, -CH ₂ - of alkyl chain.

Synthesis of methacryloyl-arginine

Methacryloyl-arginine was synthesised as described in example 1 .

Synthesis of tris[(2-pyridyl)methyl]amine (TPMA)

Tris[(2-pyridyl)methyl]amine (TPMA) was prepared as described in example 1 . Synthesis of (arginyl) ₄ -lactosyl-N-acetyl-amino-dodecanoic acid

TPMA (49.3 mg, 0.17 mmoles), CuBr (24.4 mg, 0.17 mmoles) and ascorbic acid (0.37 mg, 2.12x10 ^"3 mmoles) were dissolved in 2.0 mL of nitrogen purged DMSO and added to 0.5 mL of 0.34 g/mL methacryloyl-arginine previously prepared (0.7 mmoles) in DMSO under stirring at 55 °C. The solution was added of 0.5 mL of 98.6 mg/mL of (2-bromoisobutyryl) ₄ -lactosyl-N-acetyl-amino-dodecanoic acid (0.042 mmoles) in DMSO. The reaction mixture was maintained under nitrogen atmosphere for 12 hours and then was exposed to the air. After 30 min the solution was added dropwise to 200 mL of cold 1 :1 acetone/diethyl ether mixture containing 1 % acetic acid. The precipitate was desiccated under vacuum, redissolved in 5 mL of warm methanol and re-precipitated by dropping into 200 mL of the acetone/diethyl ether/acetic acid mixture. The product yield was 25.3 mg. The arginine content in the final product was evaluated by Sakaguchi assay (Sakaguchi S, 1925). The experimental data were referred to a calibration curve obtained with standard solutions of 0-0.6 μΜ guanidinium content (y = 7.3955x - 0.0386, R ² =0.99). The final product was also analysed by elemental analysis, ¹ H- NMR (FIG. 3).

Elemental analysis: found C, 45.3%; H, 6.3%; N, 10.9%; Br, 15.8% (O, 21 .7%); [calcd for (arginyl) ₄ -lactosyl-N-acetyl-amino-dodecanoic acid (C8 ₂ H-i ₃ 9Br ₄ N-i ₇ O ₂ 9) : C, 45.9%; H, 6.5%; N, 1 1 .1 %; Br, 14.9% (O, 21 .6%) and calcd for (arginyl) ₄ - lactosyl-N-acetyl-amino-dodecanoic acid HBr salt (C ₈₂ Hi ₃₉ Br ₄ N ₇ 0 ₂ 9-4HBr): C, 39.9%; H, 5.8%; N, 9.6%; Br, 25.9% (O, 18.8%)].

FT-IR: v (cm ^"1 ) 3392 and 3185 [NHC(NH ₂ ) ₂ ⁺ ], 2935 (CH), 1734 (C=0), 1659 (C=N). (FIG. 4)

Example 3. Synthesis of the corona-like (arginvD -maltotriosyl-N-acetyl- amino- dodecanoic acid

Synthesis of maltotriosyl-N-acetyl-amino-dodecanoic acid

One gram of 12-aminododecanoic acid (4.6 mmoles) was dissolved in 15 mL of CH ₃ OH/5% acetic acid. The solution was thermostated at 60 °C and added of 1 .2 g maltotriose (2.4 mmoles). After 24 hours, 7.5 mL of acetic anhydride (79.4 mmoles) were added to the mixture and the solution was maintained under stirring for 96 hours at room temperature. The volume was reduced to 4 mL under vacuum and added of 3 mL of 30% ammonia solution. After 12 hours, the solution was desiccated under vacuum, and the dried product was redissolved in 3 mL of methanol and added dropwise to 200 mL of diethyl ether. The precipitate was collected by filtration and desiccated under vacuum. The reaction yield was 1 .2 g. The final product was analyzed by RP-HPLC using a Luna C18 column (250x4.6mm, Phenomenex, Torrance, CA, USA) eluted with a gradient of H ₂ O/0.05% trifluoroacetic acid (A) and acetonitrile/0.05% trifluoroacetic acid (B) (0- 3 min 10% B, 3-27 min from 10% to 80% B). The UV detector was set at 220 nm. The product was also characterized by elemental analysis, mass spectrometry (ESI-MS; ESI-TOF Applied Biosystems Mariner Foster City, CA, USA), ¹ H-NMR (AMX 300 MHz, Bruker Spectrospin, Fallanden, Switzerland) and FT-IR (FT-IR 1600, Perkin-Elmer, Norworlk, CT, USA).

RP-HPLC (C18): 16.3 min retention time.

Elemental analysis: C, 51 .2%, H, 7.9%, N, 1 .7% (O, 39.2%) [calcd for C ₃₂ H ₅₇ NO ₁₈ :

C, 51 .7%, H, 7.7%, N, 1 .8% (O, 39.8%)].

ESI-MS [m/z]: 742.34 (Μ-ΗΓ) ^{1 "} [calcd for C ₃₂ H ₅₇ NO ₁₈ : 743.36].

1 H-NMR (D ₂ 0): δ ppm 5.40 (d, J = 3.26, 2H, anomeric), 5.23 (d, J = 3.79, 1 H, anomeric), 4.20-3.60 (bm, 18H of glycosyl moiety), 3.55 (t, J = 9.3, 2H, λ-ΟΗ ₂ -) _> 2.33 (t, J = 7.2, 2H, cc-CH ₂ -), 2.18 (bs, 3H, acetyl-CH ₃ ), 1 .59 (bm, 4H, i-CH ₂ - and p-CH ₂ -), 1 .29 (bs, 14H, -CH ₂ - of alkyl chain). NMR signals were indicated as singlet (s), broad singlet (bs), doublet (d), triplet (t), multiplet (m) and broad multiplet (bm); coupling constants of peak multiplicities (J) have been expressed in Hz.

FT-IR (KBr): v (cm ^-1 ) 3396 (OH), 2929 (CH), 1717 (CO-OH), 1612 (acetyl CO-N). Synthesis of (2-bromoisobutyryl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid (2-bromoisobutyryl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid was synthesised according to a modified procedure of Stenzel-Rosenbaum et al. [ref. cit.]. Briefly, 1 g of maltotriosyl-N-acetyl-amino-dodecanoic acid (1 .35 mmoles) were suspended in 20 mL of anhydrous chloroform containing 3.7 mL of triethylamine (26.7 mmoles). The suspension was chilled to 0°C, added of 3.3 mL of 2-bromo- isobutyryl-bromide (26.7 mmoles) in 30 min and left under stirring for 72 hours. The mixture was filtered and the organic solution treated three times with 30 mL cold water, three times with 20 mL of 0.1 N NaOH and three times with 30 mL of cold water. The organic phase was finally dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting red-brown solid was dissolved in 5 mL acetone and added of 100 mL cold water under vigorous stirring. Acetone was removed under reduced pressure and the aqueous suspension was lyophilised. The product yield was 1 .47 g. The product was analysed by elemental analysis, ¹ H-NMR and FT-IR. Elemental analysis: C, 40.8%; H, 5.1 %; N, 1 .0%; Br, 30.4% (O, 22.7%) [calcd for (2-bromoisobutyryl)^maltotriosyl-N-acetyl-amino-dodecanoic acid (C ₆ oH ₉₂ Br ₇ N0 ₂ 5) : C, 40.3%; H, 5.2%; N, 0.8%; Br, 31 .3% (O, 22.4%)].

1 H NMR (CDCIs): δ ppm 4.60-3.30 (bm, 18H of glycosyl moiety), 3.29 (m, 2H, λ- CH ₂ -), 2.15 (m, 2H, a-CH ₂ -), 1 .96 (bs, 42H, CH ₃ ), 1 .94 (s, 3H, acetyl-CH ₃ ), 1 .59 (bm, 4H, 1-CH ₂ - and β-ΟΗ ₂ -) _> 1 .33-1 .22 (bm, 14H, -CH ₂ - of alkyl chain).

FT-IR: v (cm ^"1 ) 3437 (OH), 2932 (CH), 1743 (isobutyryl CO-O), 1619 (acetyl CO- N).

Synthesis of methacryloyl-arginine

Methacryloyl-arginine was synthesised as described in example 1 .

Synthesis of tris[(2-pyridyl)methyl]amine (TPMA)

Tris[(2-pyridyl)methyl]amine (TPMA) was prepared as described in example 1 .

Synthesis of (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid

TPMA (1 .31 g, 4.5 mmoles), CuBr (0.65 g, 4.5 mmoles) and ascorbic acid (7.93 mg, 0.045 mmoles) were dissolved in 5 mL of nitrogen purged DMSO and added to 15 mL of 0.1 g/mL methacryloyl-arginine solution (6.2 mmoles) in DMSO under stirring at 55 °C. The solution was added of 3 mL of 0.27 g/mL of (2- bromoisobutyryl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid (0.45 mmoles) in nitrogen purged DMSO. The reaction mixture was maintained under nitrogen atmosphere for 12 hours and then was exposed to the air. After 30 min the solution was added dropwise to 200 mL of cold 1 :1 acetone/diethyl ether mixture containing 1 % acetic acid. The precipitate was desiccated under reduced pressure, redissolved in 5 mL of warm methanol and re-precipitated by dropping into 200 mL of the acetone/diethyl ether/acetic acid mixture. The product yield was 90% corresponding to 1 .54 g.

The arginine content in the final product was evaluated by Sakaguchi assay [ref. cit.]. The experimental data were referred to a calibration curve obtained with standard solutions of 0-0.6 μΜ guanidinium content (y = 7.3955x - 0.0386, R ² =0.99). The final product was also analysed by elemental analysis, ¹ H-NMR (FIG. 5), FT-IR. Elemental analysis: found C, 39.3%; H, 5.9%; N, 10.0%; Br, 28.1 % (O, 16.7%); [calcd for (arginyl)T-N-acetyl-maltotriosylamino-dodecanoic acid HBr salt (C ₁₃ oH2i8Br ₇ N2 ₉ 0 ₄ 6-7HBr): C, 38.6%; H, 5.6%; N, 10.0%; Br, 27.6% (O, 18.2%)]. FT-IR: v (cm ^"1 ) 3373 and 3182 [NHC(NH ₂ ) ₂ ⁺ ], 2938 (CH), 1735 (C=0), 1657 (C=N) (FIG 6).

The synthetic scheme adopted the synthesis of (arginyl)T-maltotriosyl-N-acetyl- amino-dodecanoic acid of is summarized in figures 7A-B and the chemical formula of the product are reported in figure 8.

The epta-guanidyl derivative prepared as described in example 3 was conjugated to fluorescein labelled bovine serum albumin (FITC-BSA) used as cargo molecule model for testing the cell penetrating properties in cell cultures. The preparation of the (arginyl)T-maltotriosyl-N-acetyl-amino-dodecanoyl-(FITC-BSA) is described in full detail in the following example 5. The synthetic scheme is summarized in figures 9A-B.

Example 4. Synthesis of the corona-like (arqinvDpn- altoheptaosyl-N-acetyl- poly '(ethylene plycoQ-carboxyl acid

Synthesis of maltoheptaosyl-N-acetyl-poly(ethylene glycol)-carboxyl acid

NH ₂ PEG-COOH (147 mg, 0.043 mmoles) was dissolved in 4 mL of CH ₃ OH/0.5% acetic acid. The solution was thermostated at 60°C and added of 50 mg maltoheptaose (0.043 mmoles). After 24 hours, 41 μΙ_ of acetic anhydride (0.43 mmoles) were added to the mixture and the solution was maintained under stirring for 96 hours at room temperature. The volume was reduced to 0.5 mL under vacuum and the solution was added dropwise to 40 mL of diethyl ether. The precipitate was collected by filtration and desiccated under vacuum. The reaction yield was 187 mg. The product was also characterized by elemental analysis and ¹ H-NMR (AMX 300 MHz, Bruker Spectrospin, Fallanden, Switzerland).

Elemental analysis: found C, 49.9%, H, 8.5%, N, 0.4% (O, 41 .2%) [calcd for C195H377NO113: C, 51 .5%, H, 8.4%, N, 0.3% (O, 39.8%)].

¹ H-NMR (D ₂ 0): δ ppm 5.08 (bd, 6H anomeric H), 4.20-3.2 0 (bm, H of glycosyl moiety and λ-ΟΗ ₂ -), 3.38 (bs, 300H OCH ₂ CH ₂ ), 2.40 (bs, 3H, acetyl-CH ₃ ), 2.33 (t, 2H, (X-CH2-). NMR signals were indicated as singlet (s), broad singlet (bs), doublet (d), triplet (t), multiplet (m) and broad multiplet (bm). Synthesis of (2-bromoisobutyryl) ₂ o-maltoheptaosyl-N-acetyl-poly(ethylene glycol)- carboxyl acid

(2-bromoisobutyryl) ₂ o-maltoheptaosyl-N-acetyl-poly(ethylene glycol)-carboxyl acid was synthesised according to a modified procedure of Stenzel-Rosenbaum et al. [ref. cit.]. Briefly, 150 mg of maltoheptaosyl-N-acetyl-poly(ethylene glycol)-carboxyl acid (0.03 mmoles) were suspended in 8 mL of anhydrous chloroform containing 104 μΙ_ of triethylamine (0.75 mmoles). The suspension was chilled to 0°C, added of 1 1 1 μΙ_ of 2-bromo-isobutyryl-bromide (0.9 mmoles) in 30 min and left under stirring for 96 hours. The mixture was filtered and the organic solution treated three times with 15 mL cold water, three times with 10 mL of 0.1 N NaOH and three times with 15 mL of cold water. The organic phase was finally dried with anhydrous Na ₂ SO ₄ , filtered and concentrated under reduced pressure. The resulting red-brown solid was dissolved in 3 mL acetone and added of 100 mL cold water under vigorous stirring. Acetone was removed under reduced pressure and the aqueous suspension was lyophilised. The product yield was 142 mg. The product was analysed by elemental analysis, ¹ H-NMR and FT-IR.

Elemental analysis: C, 42.1 %; H, 7.7%; N, 0.6%; Br,22.1 % (0,27.5%) [calcd for (2- bromoisobutyryl) ₂ o-maltoheptaosyl-N-acetyl-poly(ethylene glycol)-carboxyl acid (C ₂ 75H ₄ 77Br ₂ oNOi33): C,43.9%; H, 6.4%; N, 0.2%; Br, 21 .2% (O, 28.3%)].

Synthesis of methacryloyl-arginine

Methacryloyl-arginine was synthesised as described in example 1 .

Synthesis of tris[(2-pyridyl)methyl]amine (TPMA)

Tris[(2-pyridyl)methyl]amine (TPMA) was prepared as described in example 1 . Synthesis of (arg\ny\) ₂ o-maltoheptaosyl-N-acetyl-poly(ethylene glycol)-carboxyl acid

TPMA (76.6 mg, 0.264 mmoles), CuBr (37.9 mg, 0.264 mmoles) and ascorbic acid (0.18 mg, 0.67 10 ^"3 mmoles) were dissolved in 3 mL of nitrogen purged DMSO and added to 26 mL of 10 mg/mL methacryloyl-arginine solution (1 .06 mmoles) in DMSO under stirring at 55 °C. The solution was added of 3 mL of 33 mg/mL of (2- bromoisobutyryl) ₂₀ -maltoheptaosyl-N-acetyl-poly(ethylene glycol)-carboxyl acid (0.013 mmoles) in nitrogen purged DMSO. The reaction mixture was maintained under nitrogen atmosphere for 12 hours and then was exposed to the air. After 30 min the solution was added dropwise to 50 mL of cold 1 :1 acetone/diethyl ether mixture containing 1 % acetic acid. The precipitate was desiccated under reduced pressure, redissolved in 5 mL of warm methanol and re-precipitated by dropping into 50 mL of the acetone/diethyl ether/acetic acid mixture. The product yield was 82% corresponding to 134 mg.

The arginine content in the final product was evaluated by Sakaguchi assay [ref. cit.]. The experimental data were referred to a calibration curve obtained with standard solutions of 0-0.6 μΜ guanidinium content (y = 7.3955x - 0.0386, R ² =0.99).

Example 5. Bovine serum albumin labelling with fluorescein and conjugation of (argin vD -maltotrios yl-N-acetyl-amino-dodecanoic acid

Bovine serum albumin (BSA, 10 mg, 0.15 μΐτιοΐβε) was dissolved in 250 μΙ of 0.1 M borate buffer, pH 8.3, and added of 58 μΙ of 10 mg/mL of FITC (1 .5 μΐτιοΐβε) in DMSO. The protein solution was left at room temperature under mild stirring for 1 hour in the dark and then was added of 15 μΙ of ethanolamine (249 μΐτιοΐβε). The FITC labelled protein was purified by fast protein liquid chromatography (FPLC) using a Superose 12 gel filtration column (Amersham-Pharmacia Biotech, Uppsala, Sweden) isocratically eluted with 0.5 mL/min of 100 mM phosphate buffer, pH 7.0. The fractions containing the fluorescent protein were pooled, lyophilized and analysed by RP-HPLC using a Jupiter C4 column (250x4.6mm, Phenomenex, Torrance, CA, USA) eluted with a gradient of H ₂ O/0.05% trifluoroacetic acid (A) and acetonitrile/0.05% trifluoroacetic acid (B) (0-3 min 5% B, 3-23 min from 5% to 55% B). The UV detector was set at 280 nm.

A DMSO solution (0.15 mL) containing 9.5 mg of (arginyl) ₇ -maltotriosyl-N-acetyl- amino-dodecanoic acid (2.72 μΐτιοΐβε) of example 2 was added to 1 mL of water containing 10 mg of EDC (52.16 μΐτιοΐβε). After 30 min the solution was slowly added to 3 mL of in 100 mM morpholino-ethan-sulfonic acid buffer (MES), pH 4.6. containing 19.8 mg of FITC-BSA (0.3 μΐτιοΐβε). The reaction mixture was maintained overnight under stirring in the dark and then ultrafiltered using Amicon System equipped with a 10 kDa cut-off membrane (Millipore Amicon, Bedford, MA, USA). The product was finally purified by ion exchange chromatography using a TSK-Gel Chelate-5PW column (Tosho Bioscience LLC, PA, USA) eluted with a gradient of 20 mM phosphate buffer, 0.1 M NaCI, pH 6.5 (Eluent A) and 20 mM phosphate buffer, 0.7 M NaCI, pH 6.5 (Eluent B): 0-3 min Eluent A 1 00%: 9 min Eluent A 0%; 1 2 min Eluent A 0%. The flow rate was 1 mUmin. The UV detector was set at 280 nm and the Fluorescence detector was set at 494 nm and 520 nm Xem- The elution volume corresponding to the bioconjugate was collected, ultrafiltered with a 1 0 kDa cut-off membrane to eliminate the salts and finally lyophilized. The final product was characterized by RP-HPLC as reported above. The number of (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoyl moieties per FITC-BSA molecule was determined by Sakaguchi assay [ref. cit] using FITC- BSA as reference.

The cell penetrating properties of the novel epta-arginyl derivative were investigated in vitro by cell incubation with (arginyl) ₇ -maltotriosyl-N-acetyl-amino- dodecanoyl-FITC-BSA and FITC-BSA as control.

Example 6. Assay of the cell penetrating properties of the corona-like (arginylb- maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA

Cell lines and culture

The human MCF-7 breast adenocarcinoma and murine MC3T3-E1 embryonic fibroblast cell lines were growth in RPMI 1 640 and DMEM medium, respectively, supplemented with 1 0% heated inactivated foetal bovine serum (FBS), 1 % L- glutamine, 1 % penicillin-streptomycin-amphotericin B mixture. All cells were maintained at 37 °C in humidified 5% C0 ₂ atmosphere and splitted every 2-3 day. Fluorescence analysis

MCF-7 and MC3T3-E1 cells were seeded in six-well plates (2.0x 1 0 ⁶ cells/well). After 48 hours (90% confluence) the medium was removed the cells were incubated at 37°C with FITC-BSA or (arginyl) ₇ -maltotriosyl-N-acetyl-amino- dodecanoyl-FITC-BSA (0.2 μΜ in culture medium supplied with 1 0% FBS). After 30 min the medium was removed and the cells were washed three times with D- PBS and lysed in 1 ml_ with Triton X-1 00 (0.1 %). The plates were gently shaken for 2 hours and the lysates were centrifuged at 12000 rpm (1 0625 g) for 5 min. The fluorescence intensity was measured at 520 nm by a FP-6500 Jasco Fluorescence Spectrophotometer (Kyoto, Japan) using Triton X-1 00 (0.1 %) as blank. A similar procedure was carried out with unseeded plates in order to quantify the fluorescence intensity due to unspecific interaction of the fluorescein labelled BSA products to the tissue culture plastic material. The relative fluorescence/cell was determined on the basis of the BCA protein assay (Pierce/Celbio, Pero, Italy).

Fluorescence-activated cell sorter (FACS) analysis

MCF-7 and MC3T3-E1 cells were seeded in six-well plates (1 ^χ 1 0 ⁶ cells/well, 1 .8 ml_ culture medium/well). After 48 hours, the medium was removed and the cells were rinsed with 2.0 ml_ of D-PBS and incubated with FITC-BSA or (arginyl) ₇ - maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA (0.2 μΜ in culture medium supplied with 10% FBS) as described above. After 30 min incubation, the cells were washed three times with D-PBS and treated with trypsin-EDTA. Trypsinized cells were recovered by centrifugation at 1500 rpm (166 g) for 2 min and washed twice with D-PBS. The cells were fixed with 1 % (w/v) paraformaldehyde in phosphate buffer and stored at 4°C protected from light overnight. Afterwards, the cell samples (100,000 cells in average count) were analysed by a FACSCalibur flow cytometer using CellQuest software package (BD Bioscience, Heidelberg, Germany) and gated using forward versus side scatter to exclude debris and dead cells.

Confocal microscopic observation

MCF-7 and MC3T3-E1 cells were plated on four-well chamber slides at a density of 1 x 1 0 ⁵ cells for chamber and incubated at 37 °C in humidified 5% C0 ₂ atmosphere. After 24 hours adhesion, the cells were incubated with FITC-BSA or (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA (0.2 μΜ in culture medium supplemented with 1 0% FBS) as described above. After 30 min incubation at 37 °C, the cells were extensively washed with D-PBS and fixed with 1 % (w/v) paraformaldehyde in phosphate buffer for 1 hour at 4°C. Afterwards, fixed cells were rinsed twice with D-PBS and the samples were mounted with Vectashield ^® containing 1 .5 μg/mL DAPI and kept at 4 °C and protected from light until microscopic examination. Confocal imaging was performed using a Leica TCS SP5 confocal laser-scanning microscope (Leica Microsystems, Mannheim, Germany) equipped with a Leica HCX PL APO 63x/1 .40 oil immersion objective. The Blue argon laser was set at 351 nm (blue) and 488 nm (green) to produce the excitation wavelength for DAPI and fluorescein, respectively.

Spectrofluorimetric analysis

The cell associated relative fluorescence intensities were examined after MCF-7 and MC3T3-E1 FITC-BSA incubation with (arginyl) ₇ -maltotriosyl-N-acetyl-amino- dodecanoyl-FITC-BSA and cell lysis with Triton X-100. Parallel studies were carried out using untreated cells to assess the basal cell fluorescence (negative control). The experimental values reported in figure 10 show that a similar behaviour is obtained with MCF-7 and MC3T3-E1 cells. The relative fluorescence obtained after MCF-7 incubation with FITC-BSA was similar to the basal florescence obtained with untreated cells (2.19±1 .52 and 1 .85±1 .06, respectively), while a negligible increase of cell fluorescence was observed with MC3T3-E1 incubated with FITC-BSA as compared to the untreated cells (9.62±0.64 and 5.17±0.51 , respectively). On the contrary, the fluorescence intensity values of MCF-7 and MC3T3-E1 cells incubated with (arginyl) ₇ -maltotriosyl-N-acetyl-amino- dodecanoyl-FITC-BSA were remarkably higher as compared to that obtained with FITC-BSA treated cells. The MCF-7 and MC3T3-E1 incubation with (arginyl) ₇ - maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA resulted in about 34 and 9 fold fluorescence increase, respectively, as compared to the fluorescence obtained after cell incubation with FITC-BSA.

Flow cytometry analysis

The dot plots of side scatter (SSC-H) versus fluorescence intensity (FL1 -H) obtained by fluorescence activated cell sorting are reported in figure 1 1 . The plots obtained from MCF-7 and MC3T3-E1 incubated with FITC-BSA (panels A2 and B2, respectively) show that the amount of cells with of higher fluorescence than the basal value is similar to that displayed by the untreated cells (panels A1 and B1 ). The percentage of FITC-BSA treated MCF-7 and MC3T3-E1 cells with higher fluorescence as compared to the basal fluorescence value were 0.2% and 10%, respectively. The dot plots reported in panels A3 and B3 show that incubation of (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA increases significantly the cell associated fluorescence. About 25% of the (arginyl) ₇ - maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA treated MCF-7 cells and 95% of epta-arginyl-FITC-BSA treated MC3T3-E1 cells display higher fluorescence as compared to the basal values.

Confocal microscopy

The confocal images obtained with MCF-7 and MC3T3-E1 cells incubated with FITC-BSA and (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoyl-FITC-BSA are reported in figure 12. The images obtained with FITC-BSA treated MCF-7 and MC3T3-E1 cells (A1 and B1 , respectively) show the intense blue fluorescence (dark grey on a black background in the figure) corresponding to the DAPI stained nuclei. No green fluorescence associated to FITC could be observed in these samples indicating that FITC-BSA was neither cell surface adsorbed nor cell internalized. On the contrary, the images obtained with (arginyl) ₇ -maltotriosyl-N- acetyl-amino-dodecanoyl-FITC-BSA treated MCF7 and MC3T3-E1 cells (A2 and B2, respectively) show intense green punctuated fluorescence in the cytosol (light grey surrounding the dark grey in the figure). The image elaboration did not revealed detectable green fluorescence in the nuclei.

Example 7. Citoxicitv test of (arginylb-maltotriosyl-N-acetyl-amino-dodecanoic acid MCF-7 and MC3T3-E1 cells were plated in 96-well flat bottomed microassay plates (1 x 1 0 ⁴ cells/well) and grown for 24 hours. After complete adhesion, (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid or 2 kDa polyethylenimine (PEI) as control were added to the culture media at increasing concentrations: from 0 to 100 μΜ. The cells were incubated for 24 hours at 37°C and then the medium was discarded by aspiration and replaced with 200 μΙ of fresh medium. The cells were grown for further 48 hours. Twenty microliters of 5 mg/mL of 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) in D-PBS were added to each well. After 3 hours the MTT containing medium was removed and 200 μΙ of DMSO were added to each well in order to dissolve the formazan crystal formed by alive cells (Mosmann T 1983). The plates were gently shaken for 30 min and absorbance was measured at 570 nm with a reference wavelength of 630 nm. The cell viability was expressed as a percent viability of treated cells against untreated cells used as control.

The cytotoxicity profiles reported in figure 13 show that the (arginyl) ₇ -maltotriosyl- N-acetyl-amino-dodecanoic acid has a very low toxicity towards both the examined cell lines as compared to PEL The IC50 calculated for PEI was 40 and 30 μΜ in the case of MCF-7 and MC3T3-E1 , respectively, while the (arginyl) ₇ -maltotriosyl- N-acetyl-amino-dodecanoic acid IC50 obtained with both cell lines was higher than 100 μΜ. Higher (arginyl) ₇ -maltotriosyl-N-acetyl-amino-dodecanoic acid concentrations could not be examined because of the solubility limit of this product in the medium used for the cell viability assay.

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