Field of the invention

The present invention origins in the biomedical and pharmaceutical field.

The invention relates to star-like (guanidyl)x-oligosaccharidic compounds as delivery carriers of diagnostic and therapeutic molecules.

In particular the invention concerns with polycationic macromolecules having the capacity of carrying and delivering small-sized drugs or biological molecules such as oligonucleotides, polypetides and proteins across biological membranes.

Background of the Invention

In the last years, several efforts have been made to deliver oligo- and polynucleotides (ONs) into cells in order to modify the biological activity of cells by regulation of gene expression including the expression or silencing of protein material. Therapeutic ONs, such as asRNA (single stranded antisense RNA), mRNA (messanger RNA) [1 ], snRNA (small nuclear RNA) [2], siRNA (small interfering RNA) [3, 4], miRNA (micro RNA) [5], or pDNA (plasmidic DNA) are good candidates for treatment of a variety of common genetic diseases (i.e. cancer [6], cystic fibrosis [7], muscular dystrophy [8], vascular disease [9], neurogenerative disorder [10], etc.). Unfortunately, due to their anionic nature, high molecular weight and structural fragility, ONs administration suffers from poor bioavailability and biological efficiency. These macromolecules can in fact undergo fast degradation by serum nucleases, negligible transmembrane transport, off- target profile [1 1 ] and elicit immunogenic response [12].

So far, several strategies have been investigated to simultaneously protect oligo- and poly-nucleotides from nucleases, yield cell targeting and promote the specific cell targeting and enhance the cell uptake.

Encapsulation into viral scaffolds, non-covalent complexation and chemical conjugation with lipids, polymers and cell penetration enhancers, are few of the approaches developed to enhance the ON biopharmaceutical and pharmacokinetic profiles. Engineered viral vectors are also used to enhance the cell uptake of ONs. Viruses have evolved efficient mechanism to bypass the cell membrane to reach the cytoplasm and nucleus. Most used viral carrier for gene therapy are adenovirus, lentivirus, herpes simplex virus (HSV) or retrovirus. However, the cost- effectiveness and availability of these carriers and their high capacity to activate the immune system have limited their use. Generally, the use of viruses as drug carriers, with few exceptions, entails the risks reported above [13, 14].

Another strategy involves the use of cationic materials, including cationic polymers, lipids and cell penetration peptides, which have been used to complex the anionic ONs by coulombic interactions [15-18]. These nano-complexes are stable in the blood stream and represent a shield to degradation of ONs from nucleases. Compared to viral vectors, they have low antigenicity and risks of insertional oncogenesis.

A typical example of cationic material is polyethyleneimine (PEI), a linear or branched polymer containing primary, secondary and tertiary amines that has been successfully used to deliver various ONs with high efficiency. Nevertheless, due to its high toxicity, PEI cannot be used in vivo [19].

Chitosan is a cationic polysaccharide with high content of amino group able to condense ONs [20].

Cationic lipids, namely, 1 ,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 1 ,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dimethyldioctadecylammonium (DDAB) and their colloidal formulations have been also used to complex ONs [21 ]. Lipofectamine, a liposomal formulation containing cationic lipids is regarded as a 'gold standard' for in vitro cell transfection. However, even these systems are inadequate for therapeutic application because, in general, cationic/ONs complexes are unstable, immunogenic and difficult to be manufactured.

Cationic cyclodextrins are cyclo-oligosaccharidic vehicles grafted with poly-cationic polymers such as PEI or poly-lysines that have been successfully used to deliver ONs or plasmids [22, 23]. A class of cyclodextrin-based gene delivery systems targeted to tumours by grafting transferrin to a PEG chain conjugated with adamantane that forms host-guest complexes with cationic cyclodextrins have been developed [24].

Covalent bioconjugates have been obtained by ON derivatization with several molecules including lipids, polymers and cell penetration enhancers.

The conjugation of cholesterol, estrone, a-tocopherol (vitamin E) and other lipophilic molecules promotes the cell up-take of ONs via high- and low-density lipoprotein (HDL and LDL) binding and transportation, α-tocopherol (vitamin E, a natural and lipophilic molecule) bioconjugation has been found to promote the brain delivery of ONs [25].

Cell penetration enhancers have been conjugated to anionic ONs without significant effect on transfection, while the conjugation of cell penetration enhancers to neutral ON derivatives has been found to yield high cell internalization of ONs [26, 27].

Human serum albumin (HSA) was also used as a carrier of ONs. HSA is a high biocompatible macromolecule that can be derivatized with modifiers including stimuli sensitive and targeting moieties to yield supramolecular systems with peculiar biopharmaceutical behaviour [28].

The covalent attachment of polymers, namely poly(ethylene) glycol (PEG), has been found to prolong the circulation time, decrease the renal clearance and reduce the immunogenicity of ONs in mice [29] [30]. The non-cleavable attachment of polymers have been often found to inhibit the ON activity. Conversely, cleavable conjugates have been found to prevent the ON inactivation [31 ].

The conjugation of cationic polymers has been also developed, even though these materials present often intrinsic significant toxicity similarly to PEL Poly(butyl amino vinyl ether) (PBAVE), a polymer that disrupts endosomal membranes, modified with PEG and targeting agents has been successfully conjugated to ONs via cleavable bonds to yield selective cell targeting. The derivative containing N- acetylgalactosamine (GalNAc) has been found to efficiently deliver the conjugated ONs to hepatocytes [32].

Bioconjugates have been obtained by attaching targeting agents to the ONs in order to yield specific cell targeting and cell up-take. Among the targeting agents used for this purpose there are: asialoglycoproteins for hepatocyte delivery [33], small peptides such as RGD that targets α\/β3 integrin receptor [34], bombesin that targets the BB2 G-protein overexpressed in prostate cancer cells [35], insulin growth factor-1 (IGF-1 ) peptide mimetic that targets the IGF-1 receptor of breast cancer cells [36], antibodies and antibody fragments, such as herceptin antibody, anti-Lewis-Y monoclonal antibody, antibody against a-CD19, a biomarker against acute lymphoblastic leukaemia [33].

Despite many strategies have been investigated to enhance the biopharmaceutical properties of ONs and produce efficient systems for therapeutic and diagnostic use and for cell manipulation, the delivery of ONs into the intracellular site, combined with the poor biopharmaceutical, immunological and pharmacokinetic profiles and inadequate cell trafficking, remains a significant challenge.

In particular, most of developed technologies have shown a good efficiency in cell- culture models, while the translation to medicine remains an unmet need for this class of therapeutics.

Therefore at present there is the need for new molecules acting as intracellular delivery vectors of biological molecules or drugs.

Summary of the invention

One of the purposes of the present invention is to provide a class of molecules acting as intracellular vehicle of therapeutic or diagnostic molecules.

Another aim of the invention resides in the provisions of drug delivery systems for transporting therapeutic molecules in the intracellular target sites.

Another aim of the invention is the provision of a class of polycationic macromolecules (pOG-M-PEG), without interferring anionic residues, acting as cell permeation enhancers.

In accordance with a first aspect of the present invention, the inventors have found that the insertion of guanidine moieties on a saccharide core provides a class of polycationic molecules with delivery features making them useful as intracellular delivery vectors of biological molecules such as oligo- and polynucleotides, aptamers and peptides for therapeutic or diagnostic purposes.

In a first aspect the present invention provides for (guanidyl)x-oligosaccharidic compounds represented by the general formula (I):

wherein:

Ri , R2, R3, R4, are independently

H;

a group of formula -CO- R °-X, wherein R ° is a C2-C3 alkyl branched chain and X is an halogen; or

a guanidine terminating group of formula -C0- R ^o -[CH ₂ CX( R')-C0-N H- R"- NHC(N H2)=NH], where R ° and X are as defined above, R' is a linear or branched C-i -Cs alkyl group or H , R" is a linear or branched C ₄ -Cs alkyl group optionally bearing an hydroxyl or carboxylic group;

R5 is a linker or spacer having formula -(Y)N- R"'-C, wherein

R'" is

a linear or branched C2-C22 alkyl or C2-C22 alkenyl,

a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C where a is an integer selected from 2 to 454, b is an integer selected in the range from 2 to 6, or a linear or branched C2-C22 alkyl or C2-C22 alkenyl linked to a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C as defined above,

Y is an hydrogen or an acyl moiety having formula R*-CO-, wherein R* is a linear or branched C1 -C22 alkyl or C1 -C22 alkenyl chain or a steroidic scaffold,

C is a terminating group selected from C-i -Cs alkoxy-, halogen-, vinyl-, carboxyl-, amino-, aldehyde-, hydroxyl-, thiol-, maleimido-, biotin-, alkine- or azido- group or CO-W-R'", wherein W is N H , O, and R'" has the same meaning defined above; Re, if present, is a group selected from

-a linear or branched C2-C22 alkyl or C2-C22 alkenyl,

-a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C where a is an integer selected from 2 to 454, b is an integer selected in the range from 2 to 6, or -a linear or branched C2-C22 alkyl or C2-C22 alkenyl linked to a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C as defined above,

- a group CO-W-Rcp, wherein W is NH, O, and wherein

Rep is

-H

-a linear or branched C2-C22 alkyl or C2-C22 alkenyl,

-a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C where a is an integer selected from 2 to 454, b is an integer selected in the range from 2 to 6, or -a linear or branched C2-C22 alkyl or C2-C22 alkenyl linked to a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C as defined above;

n is an integer ranging from 1 -8 sugar units, and salts thereof.

In accordance with certain embodiments the substituent R5 or Re if present, is bound, typically by covalent bonds, to a functional moiety which is a compound having biological, physicochemical, biopharmaceutical and/or targeting activity or a fluorophore or a radionuclide or macrosurfaces. In accordance with this embodiment, the compounds of formula (I) of the invention forms a conjugate with a functional moiety which make them useful as targeting or labelling compounds. Thus, according to an aspect the invention provides a conjugate comprising a compound of formula (I) bound by either the R5 or Re substituent group to a functional moiety selected from i) macromolecules or ii) a physicochemical and biopharmaceutical modifier iii) a fluorophore or iv) a radionuclide or v) macrosurfaces or a vi) targeting agent. In accordance with this aspect, the compound of Formula (I) is bound, typically by a covalent bond, to the compound having biological activity or fluorophore or radionuclide or targeting agent via either R5 or Re substituent group.

Typically, the above conjugate is used to target said functional moiety in a in vivo or in vitro biological site where it exerts its action.

The inventors have also found that the cationic (guanidyl)x-oligosaccharidic compounds of formula (I) interact, especially by means of non-covalent bounds, with organic molecules bearing an anionic group (anionic macromolecules) and preferably having biological activity, In accordance with this aspect of the invention, the cationic (guanidyl)x- oligosaccharidic compounds of formula (I) may interact or bound anionic macromolecules such as ONs, polypeptides, proteins, anionic macromolecules having biological activity, especially biological drugs, or cell membranes, typically by interaction with one or more of the substituents groups Ri, R2, R3, R4.

According to these aspects the invention also provides a complex comprising a cationic (guanidyl)x-oligosaccharidic compound of formula (I) as defined above and biological anionic macromolecules, especially ONs, peptides, proteins, plasmids, poly-nucleic acids or drugs. Typically, said conjugate is bound with non- covalent bonds with the compound of formula (I) via the substituents groups Ri,

In certain embodiments the conjugate defined above is encapsulated or bond with a delivery system such as liposomes, micelles, polymeric or inorganic nanoparticles, micro- and nanocapsules or micro- or nanospheres or other matrices.

In accordance to a further aspect the invention provides the use of the cationic (guanidyl)x-oligosaccharidic compounds of Formula (I) as carriers for delivering molecules having biological activities such as macromolecules, colloidal systems, peptides, proteins, drugs into a target cell, tissue or organ of a mammal, especially a human body or in vitro transfect agent.

In accordance with an additional aspect a compound of Formula I is provided which is bound with non-covalent bonds by substituents group Ri, R2, R3, R4 to a molecule having biological activity especially ONs, peptides, proteins, plasmids or poly-nucleic acids or drugs, and typically with covalent bounds by either the substituent group R5 or R6 to a compound having biological activity for example a drug, an antibody, a peptide, protein or nucleic acids, a targeting agent, a fluorophore or a radionuclide.

Brief description of the figures

Figure 1 illustrates the synthesis of maltotriosyl-N-acetyl-amino-hexanoic acid of the example 1 .

Figure 2 shows the synthesis of the star-like (2-bromoisobutyryl)6-maltotryosyl-N- acetyl-amino-hexanoic acid of the example 1 . Figure 3 shows the ¹ H NMR analysis of star-like (agmatinyl)6-maltotriosyl-N-acetyl- amino-hexanoic acid of the example 1 .

Figure 4 illustrates the synthesis of star-like (agmatinyl)6-maltotriosyl-N-acetyl- amino-hexanoic acid of the example 1 .

Figure 5 shows the molecular structure of star-like (agmatinyl)6-maltotriosyl-N- acetyl-amino-hexanoic acid of the example 1 .

Figure 6 illustrates the synthesis of star-like (agmatinyl)6-maltotriosyl-N-acetyl- amino-hexanoate-PEGekDa-NH2 of the example 2.

Figure 7 illustrates the synthesis of star-like (agmatinyl)6-maltotriosyl-N-acetyl- amino-hexanoate-oleylamide of the example 3.

Figure 8 shows the gel electrophoretic profiles of dsDNA/pOG-M-PEG complexes with N/P ratio between 0.5-5 at pH 7.4. The samples were run in polyacrylamide gel using TBE as running buffer.

Figure 9 shows the size and zeta potential of dsDNA/pOG-M-PEG complexes. Data are reported as mean values ± SD, with n= 3 experiments.

Figure 10A shows the TEM images of dsDNA/pOG-M-PEG complexes with 3 N/P ratio.

Figure 10B shows the TEM images of dsDNA/pOG-M-PEG complexes with 5 N/P ratio.

Figure 1 1 A shows the stability of dsDNA/pOG-M-PEG complexes with 3 N/P ratio in PBS, pH 7.4. The data are reported as mean values ± SD, with n= 3 experiments.

Figure 1 1 B shows the size of dsDNA/pOG-M-PEG complexes with 5 N/P ratio in PBS, pH 7.4. The data are reported as mean values ± SD, with n= 3 experiments. Figure 12A shows the size of dsDNA/pOG-M-PEG complexes with 3 N/P ratio in DMEM medium supplemented with 15% FBS (Fetal Bovine Serum). Data are reported as mean values ± SD, with n= 3 experiments.

Figure 12B shows the size of dsDNA/pOG-M-PEG complexes with 5 N/P ratio in DMEM medium supplemented with 15% FBS (Fetal Bovine Serum). Data are reported as mean values ± SD, with n= 3 experiments.

Figure 13 shows the hemolytic profiles of dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio at pH 7.4. PBS pH 7.4 and ON solution in PBS, pH 7.4, were used as negative control whereas 1 % w/v of Triton X-100 was employed as a positive control. Data are reported as mean values ± SD, with n= 3 experiments.

Figure 14 shows the isothermal calorimetry profiles of pOG-M-PEG titrated with dsDNA in 10 mM TRIS, 150 mM NaCI, pH 7.4 and relative table.

Figure 15 A shows the viability of KB cells incubated with dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio. The cells were incubated with the complexes for 24 and 48 hours. Data are reported as mean values ± SD, with n= 6 experiments. Figure 15 B shows the viability of MCF-7 cells incubated with dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio. The cells were incubated with the complexes for 24 and 48 hours. Data are reported as mean values ± SD, with n= 6 experiments. Figure 15 C. Viability of MC3T3-E1 cells incubated with dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio. The cells were incubated with the complexes for 24 and 48 hours. Data are reported as mean values ± SD, with n= 6 experiments. Figure 16 A. Cell cytometric profiles of MCF-7 cells after 6 hour cell treatment with Cy3-dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio. Untreated cells are used as control.

Figure 16 B shows the Cell cytometric profiles of KB cells after 6 hour cell treatment with Cy3-dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio. Untreated cells are used as control.

Figure 16 C shows the cell cytometric profiles of MC3T3-E1 cells after 6 hour cell treatment with Cy3-dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio. Untreated cells are used as control.

Figure 17 A shows the confocal microscopy images of MCF-7 cells treated with Cy3-dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio. Untreated cells are used as control.

Figure 17 B shows the confocal microscopy images of KB cells treated with Cy3- dsDNA/pOG-M-PEG with 3 and 5 N/P ratio. Untreated cells are used as control. Figure 17 C shows the confocal microscopy images of MC3T3-E1 cells treated with Cy3-dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio. Untreated cells are used as control. Detailed description of the invention

In accordance with certain aspect of the invention, the inventors have found that cationic (guanidyl)x-oligosaccharidic compounds of Formula (I) are useful as carriers of therapeutic and diagnostic molecules, in particular oligonucleotides, such as asRNA (single stranded antisense RNA), mRNA (messanger RNA), snRNA (small nuclear RNA), siRNA (small interfering RNA), miRNA (micro RNA), pDNA (plasmidic DNA) or aptamers.

In accordance with these aspects, the compounds of Formula (I) are useful as agents to formulate and deliver the above mentioned biological molecules through physical or covalent conjugation and optional encapsulation into a delivery system such as liposomes, micelles, polymeric or inorganic nanoparticles, micro- and nanocapsules or micro- and nanospheres or other matrices.

In accordance with a first aspect, the invention concerns with compounds of Formula (I)

wherein :

Ri , R2, R3, R4, are independently:

H;

a linker of formula -CO-R°-X, wherein R° is a C2-C3 alkyl branched chain and X is an halogen ; or

a guanidine terminating group of formula -CO-R°-[CH ₂ CX(R')-CO-NH-R"- NHC(NH2)=NH], where R° and X are as defined above, R' is a linear or branched C-i-Cs alkyl group or H, R" is a linear or branched C ₄ -Cs alkyl group optionally bearing an hydroxyl or carboxylic group;

R5 is a linker or spacer having formula -(Y)N-R"'-C, wherein

R'" is a linear or branched C2-C22 alkyl or C2-C22 alkenyl,

a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C where a is an integer selected from 2 to 454, b is an integer selected in the range from 2 to 6, or a linear or branched C2-C22 alkyl or C2-C22 alkenyl linked to a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C as defined above,

Y is an hydrogen or an acyl moiety having formula FT-CO-, wherein R* is a linear or branched C1-C22 alkyl or C1-C22 alkenyl chain or a steroidic scaffold,

C is a terminating group selected from C-i-Cs alkoxy-, halogen-, vinyl-, carboxyl-, amino-, aldehyde-, hydroxyl-, thiol-, maleimido-, biotin-, alkine- or azido- group or CO-W-R'", wherein W is NH, O, and R'" has the same meaning defined above; R6, if present, is a linker selected from

-a linear or branched C2-C22 alkyl or C2-C22 alkenyl,

-a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C where a is an integer selected from 2 to 454, b is an integer selected in the range from 2 to 6, or -a linear or branched C2-C22 alkyl or C2-C22 alkenyl linked to a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C as defined above,

- a group CO-W-Rcp, wherein W is NH, O, and wherein

Rep is:

-H

-a linear or branched C2-C22 alkyl or C2-C22 alkenyl,

-a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C where a is an integer selected from 2 to 454, b is an integer selected in the range from 2 to 6, or -a linear or branched C2-C22 alkyl or C2-C22 alkenyl linked to a poly(ethylene glycol) group having formula -[CH2CH20] _a -(CH2)b-C as defined above.

n is an integer ranging from 1 -8 sugar units, and salts thereof.

In accordance with certain embodiments the substituents R5 or Re if present, is bond preferably by covalent bounds to a functional moiety such as a molecule having biological activity or a fluorophore or radionuclide. In accordance with these embodiments, the inventor have found that the (guanidyl)x-oligosaccharidic compounds of Formula (I) are useful to target molecules having biological activity and a fluorophore or a radionuclide or a targeting agent, optionally beared to the molecules, in the desired site. In accordance with these aspects, the invention provides the use of the compounds of Formula (I) bound to a functional moiety, such as a molecule having biological activity, to target the functional moiety at the desired site of action.

Typically, said functional moiety is covalently bound to the substituent Rs or Re, if present, of the compounds of Formula (I) of the invention.

In certain embodiments R6 is linked to Rs with a functional group selected from phenyl-, alkoxy-, halogen-, vinyl-, carboxyl-, amino-, amido-, aldehyde-, hydroxyl-, thiol-, maleimido-, biotin-, alkine- or azido-group.

Typically, the functional moieties include low and high molecular weight molecules having biological activity or physicochemical and biopharmaceutical modifiers such as phospholipids, natural or synthetic, linear or branched oligomers, polymers, copolymers or conjugates, a polyethylene glycol or a cyclic structure, a peptide, a protein, a drug, organic and/or inorganic surfaces.

In certain embodiments, the functional moiety is a polymer or a compound having biological activity.

Exemplary polymers or molecules/compounds having biological activity or acting as physicochemical and biopharmaceutical modifiers bound to either Rs or Re substituent of the compounds of formula (I) include:

- a phospholipid such as 1 ,2-distearoyl-sn-glycero-3- phosphoethanolamine;

- natural or synthetic, linear or branched oligomers, copolymers or polymers such as dextran, pullulan, chitosan, hyaluronic acid, sialic acid, poly-glutamic (PGA) acid, polyoxazolidine (POX), poly(ethylene glycol) (PEG) derivatives or a cyclic structure such as cyclodextrin or conjugates, PEG being preferred;

- peptides, polypeptides and/or proteins, for example transferrin, albumin, lysozyme, antibody or its fragments, or peptides;

- a drug or a targeting agent;

- organic and/or inorganic surfaces such as liposomes, polymeric particles, micelles, inorganic particles such as gold nanoparticles, silica nanoparticles, carbon nanotubes, macrosurfaces. In certain embodiments Ri, R2, R3 and R ₄ substituents are -CO-R°-X, wherein R° is a C2-C3 alkyl branched group and X is bromine or chlorine and preferably -CO- R°-X is a 2-bromoisobutyryl group.

In certain embodiments the substituents Ri, R2, R3 and R ₄ , is an isopropyl group and X is represented by an halogen preferably CI or Br.

In certain embodiments, Ri, R2, R3 and R ₄ substituents are a group of formula - CO-R°-[CH2CX(R')-CO-NH-R"-NHC(NH2)=NH] wherein the R° substituent represent a methyl group, R' represents bromine and R" represents a C ₄ alkyl chain.

In certain preferred embodiments Ri, R2, R3 and R ₄ are a group of formula -CO- R°-[CH ₂ CX(R ^, )-CO-NH-R"-NHC(NH2)=NH] _J wherein the substituent R" is a liner or branched C ₄ -Cs alkyl group which is not bearing or linked to a carboxylic group. In accordance with some embodiments R5 is bound to or Re is a polymer selected from dextran, pullulan, chitosan, PGA, POX and PEG. The latter being preferred. In certain embodiments the poly(ethylene glycol) derivative, linear or branched, of formula -[CH2CH2O] _a -(CH2)b-C has an average molecular weight (Mw) from 132 to 20000 Da, preferably in the range of 4000 to 8000 MW.

For example, a is 136 b2 and the molecular weight is 6000.

The (average) molecular weight of the above polymers may be determined with standard methods such as those disclosed in Ueno et al., 1988, Chem Pharm Bull. 36, 4971 -4975; Wyatt 1993, Anal Chim Acta 272: 1 -40; Watt Technologies 1999 "Light scattering University Dawn Course Manual and "Dawn Eos Manual" Wyatt Technology Corp. Santa Barbara CA (USA).

In certain embodiments, the alkyl chain is oleylamine.

In certain embodiments of the invention the substituents Ri, R2, R3, R4 are independently from 2 to 20 guanidine terminating group having formula -CO-R°- [CH2CX(R')-CO-NH-R"-NHC(NH2)=NH] if the oligoglycosidic scaffold is maltotriose.

According to certain embodiments of the compounds of Formula (I) the substituents Ri, R2, R3, R4 are guanidine derivatives of formula -CO-R°-[CH2CX (R')-CO-NH-R"-NHC(NH ₂ )=NH]. Preferred guanidine terminating moieties are selected from 2 to 10 in a maltotriose backbone and has the formula -CO-R°- [CH2CX(R')-CO-NH-R"-NHC(N H2)=NH], where R°, R\ R" and X have the same meanings described in any of the above referred embodiments.

In certain embodiments R" is a C ₄ alkyl chain bearing a -COOH group.

In certain embodiments Rs is N-acetyl-12-aminododecanoyl acid.

Typically, the compounds of Formula (I) according to the invention have star-like (guanidyl)x-oligosaccharidic moieties and may physically form, typically by non- covalent bonds, complex with compounds having a negative charge, especially anionic compounds having a biological activity. Thus, the compounds of Formula (I) may form complexes with anionic macromolecules especially those having a biological activity and protect them by enzymatic degradation and improve the delivery and cell uptake. For example, the (guanidyl)x-oligosaccharidic molecules of the invention may form complexes with anionic therapeutic macromolecules such as ONs, especially asRNA (single stranded antisense RNA), mRNA (messenger RNA), snRNA (small nuclear RNA), siRNA (small interfering RNA), miRNA (micro RNA), pDNA (plasmidic DNA) or aptamers or with peptides, proteins, drugs, polymer therapeutics such as linear or branched bioconjugates, liposomes, micelles or nanoparticles.

Typically, the complexation may be exploited by anchoring the (guanidyl)x- oligosaccharidic compound of Formula (I) to hydrophilic polymers, lipids, proteins, surfaces to obtain colloidal systems suitable to promote the cell entry of therapeutic macromolecules or their use in in vitro tools.

Typically, the scaffold of the compounds of Formula (I) contains oligosaccharides moieties, especially from 1 to 8 saccharidic units, that provide the star-like shape, and guanidinium groups to provide positive charges.

In accordance with some embodiments, the saccharidic units are maltotriose, glucose, lactose, trealose, gentiobiose, mannose, cellobiose, maltose, isomaltose, maltotetraose, maltopentaose, maltohexaose, maltoheptaose, maltooctaose and preferably are lactose, maltose or maltotriose.

Typically, the star-like (guanidyl)x-oligosaccharidic compounds of Formula (I) have a pKa in the range of from 12 to 13 which is fully protonated in physiological environment. This feature enables the compounds of Formula (I) to complex negative macromolecules such as ONs. In accordance with certain aspects the invention concerns compounds of Formula (I) for use as carriers for intracellular delivery of macromolecules or supramolecular colloidal structures physically or covalently conjugated thereto. In certain embodiments, the compounds of Formula (I) are conjugated with hydrophilic polymer, such as a poly(ethylene glycol) to give stealth properties at the system, improving pharmacokinetic parameters such as half-life and degradation.

The star-like (guanidyl)x-oligosaccharidic compounds of Formula (I) may be complexed with ONs taking advantage of the natural attraction between opposite charges or may covalently bind functional moieties such as molecules or colloidal systems or anchoring to surfaces of molecules or products having biological activity.

Preparation of compounds of the invention

In accordance to another aspect, the present invention provides a method for the production of the (guanidyl)x-oligosaccharidic derivatives of formula (I) as defined above, said method comprising the steps of:

Oligoglycosidic functionalization of the anomeric carbon of the terminal saccharidic ring through derivatization with a spacer having formula -(Y)N-R"'-C, where R'" is a linear saturated or unsaturated or branched C ₄ -C22 alkyl chain or an poly(ethylene glycol) derivative, linear or branched, having formula

(CH2)b-C where a is an integer selected in the range from 2 to 454, b is an integer selected in the range from 2 to 6; Y is an hydrogen or an acyl moiety having general formula R*-CO-, where R* is a linear or branched with or without multiple bond when is possible C1 -C22 alkyl chain or a molecule derivative with a steroidic scaffold; C have the same meanings for both and is an terminating group which alkoxy-, halogen-, vinyl-, carboxyl-, amino-, aldehyde-, hydroxyl-, thiol-, maleimido- , biotin-, alkine- or azido- group;

Stabilization of the amino-oligosaccharide obtained derivative;

Functionalization of oligosaccharide hydroxyl groups with an agent having formula -CO-R°-X, where R° have the meanings of C2-C3 alkyl branched chain and X can be an halogen ; Conjugation of cationic guanidyl structure with formula -CO-R°-[CH2CX(R')- CO-NH-R"-NHC(NH ₂ )=NH], where R° and X have the same role that was described previously, R' can be an optionally alkylic group, R" is a linear or branched C ₄ -Cs alkyl chain that can bear an hydroxyl or carboxylic group or their derivatives; cationic structure was linked with the hydroxyl groups of the oligoglycosil scaffold by the use of copper salts, tris[(2-pyridyl)methyl]amine (TPMA) and ascorbic acid in appropriate amounts.

Conjugation of the cationic (guanidyl)x-oligosaccharidic derivatives to a molecule, macromolecule or surface;

- Application of the (guanidyl)x-oligosaccharidic derivative to condense and deliver drugs and diagnostics, namely small molecules, macromolecules such as peptides, proteins, ONs etc.

In accordance with certain embodiments the method for the production of compounds of formula (I) comprising the following steps:

1 . Oligoglycosidic functionalization on the anomeric carbon of the terminal saccharidic ring through derivatization with a spacer having formula -(Y)N-R"'-C. Suitable spacers are -NH-(linear or branched C ₄ -C22 alkyl chain)-OCH3; -NH- (linear or branched C ₄ -C22 alkyl chain)-X, where X is an halogen; -NH-(linear or branched C ₄ -C22 alkyl chain)-CH=CH2; -NH-(linear or branched C ₄ -C22 alkyl chain)-COOH; -NH-(linear or branched C ₄ -C22 alkyl chain)-NH ₂ ; -NH-(linear or branched C ₄ -C22 alkyl chain)-CHO; -NH-(linear or branched C ₄ -C22 alkyl chain)- OH; -NH-(linear or branched C ₄ -C22 alkyl chain)-SH; -NH-(linear or branched C ₄ - C22 alkyl chain)-Mal; -NH-(linear or branched C ₄ -C22 alkyl chain)-Biotin; -NH- (linear or branched C ₄ -C22 alkyl chain)-C≡CH; -NH-(linear or branched C ₄ -C22 alkyl chain)-N ₃ ; -NH-[CH2CH20] _a -(CH2)b-OCH ₃ ; -NH-[CH ₂ CH20] _a -(CH2)b-X, where X is an halogen; -NH-[CH2CH ₂ 0] _a -(CH2)b-C=CH2; -NH-[CH2CH ₂ 0] _a -(CH2)b- COOH; -NH-[CH2CH20] _a -(CH2)b-NH ₂ ; -NH-[CH2CH ₂ 0] _a -(CH2)b-CHO; -NH- [CH2CH ₂ 0] _a -(CH2)b-OH; -NH-[CH2CH ₂ 0] _a -(CH2)b-SI± -NH-[CH2CH ₂ 0] _a -(CH2)b- Mal; -NH-[CH2CH ₂ 0] _a -(CH2)b-biotin; -NH-[CH2CH ₂ 0] _a -(CH2)b-C≡CH; -NH- [CH2CH ₂ 0] _a -(CH2)b-N3 [a=3-454; p:2-6];

According to some embodiments the stabilization of the amino-oligosaccharide derivative obtained is carried out trough N-acetylation; 2. Functionalization of oligosaccharide hydroxyl groups with an agent having formula -CO-R°-X, where R° have the meanings of C2-C3 alkyl branched chain and X may be an halogen and preferably is 2-bromoisobutyryl bromide;

3. Conjugation of cationic guanidyl structure with formula -CO-R°-[CH2CX(R')-CO- NH-R"-NHC(NH ₂ )=NH], where R°, R\ R" and X have the same meanings as above described; cationic structure was linked with the hydroxyl groups of the oligoglycosil scaffold by the use of copper salts, tris[(2-pyridyl)methyl]amine (TPMA) and ascorbic acid;

4. Conjugation of the cationic (guanidyl)x-oligosaccharidic derivatives to a hydrophilic polymer. In certain embodiments a poly(ethylene glycol) chain is used having formula -NH-[CH2CH20] _a -(CH2)b-C where a, b and C have the same meaning described above. A suitable gycol is poly(ethylene glycol) with molecular weight of 6000 wherein a=136, b=2, C=-NH2.

In the first step of synthesis, the oligosaccharide was functionalized with a spacer through the reaction of the amino group of the selected spacer R5, preferably ε- aminohexanoic acid with the anomeric carbon of the selected oligosaccharides, preferably maltotriose. The reaction was performed in methanol supplemented with of acetic acid (1 % v/v) at 50 °C. The bond between the selected sugar and the spacer R5 was stabilized preferably through the addition of acetic anhydride in order to prevent the separation of the spacer from the oligosaccharide bulk. The reaction of acetylation was stopped after 24 hours. The product of conjugation was purified by precipitation in a cold ethyl ether.

The hydroxyl groups of the N-acetylamino spacer oligosaccharide derivative were then functionalized by the use of an appropriate linker, preferably 2- bromoisobutyryl bromide. This functionalization was carried out in order to obtain a star shape oligosaccharide derivative. The N-acetylamino spacer oligosaccharide was dispersed in cold chloroform with trietylamine and the suspension was added of 2-bromoisobutyryl bromide. The reaction was stirred for 72 hours and the product was isolated by sequential extractions.

The (2-bromoisobutyryl) star-like oligosaccharide was then modified with the guanidine moiety. The conjugation was performed through the use of copper salts (preferably CuBr), TPMA (tris[(2-pyridyl)methyl amine], a ligand of Cu (I)) and ascorbic acid as reducing agent. The monomer employed in this synthesis was an acryloyl guanidyl derivative (preferably acryloyl agmatine, synthesized according with the literature). The reaction was carried out under nitrogen conditions for 3 days at 65 °C and the final product was precipitated in a solution of ethyl ethenacetone (1 :1 ) with the addition of 1 % v/v of acetic acid.

The last step of synthesis concerns the PEGylation of the star-like (guanidyl)x- oligosaccharidic derivative. The attachment of the polymeric side chain was performed on the lead derivative star-like (agmatinyl)6-maltotriosyl-N-acetyl-amino- hexanoic acid. The reaction was performed by dissolving the star-like (guanidyl)x- oligosaccharidic derivative and the poly(ethylene glycol) derivative (preferably the (NH2)2-PEG, 6 kDa) in a mixture 1 :2 DMSO and 100 mM morpholino-ethan- sulphonic acid buffer (MES), pH=4.7 by adding NHS and EDC as a coupling agent. The final product was purified by dialysis and recovered by lyophilization. The star-like (guanidyl)x-oligosaccharidic compound of formula (I), may be covalently or non-covalently linked to anionic molecules, preferably ONs.

An assay for assessing the capability of the compounds of formula (I) to link target anionic molecules was performed using a lead derivative the (agmatinyl)e- maltotriosyl-N-acetyl-amino-hexanoate-P EG6kDa-NH2.

As anionic molecule model was used a dsDNA with 1 1 kDa as model ON.

The assembly proprieties of the PEGylated star-like (guanidyl)x-oligosaccharidic derivative with an ON was explored by gel shift assay gel retardation and Isothermal Titration Calorimetry (ITC) analysis. The gel electrophoresis results showed a total complexation at 3N/P ratio. ITC analysis showed a spontaneous interaction between the negative charges of ONs and the positive charges of the PEGylated star-like (guanidyl)x-oligosaccharidic derivative that are the object of the present invention.

Dynamic light scattering was used to determine the hydrodynamic radii of complexes at various N/P ratio. Complexes were approximately of 70-100 nm in diameter. The zeta potential were also evaluated and the results showed that the charge is approximately neutral (± 4-8 mV). TEM images showed a rod like shape of the complexes having size in agreement with the DLS analysis. The formulation were stable in PBS, pH 7.4, and in cell culture medium in 12 hours. These features are very important for a gene delivery systems.

The hemolytic effect of the complexes were evaluated and the analysis showed a complete compatibility with the biomembranes of the red blood cells under physiological conditions.

Biological studies to evaluate the biological effect of complexes on cellular metabolism were performed on MCF-7, KB and MC3T3-E1 cells. In agreement with the hemolysis test, the MTT study showed a higher biocompatibility of the complexes in 24 h of incubation. A reduction of cell viability was shown for extended incubation (48 hours).

The capacity of this nanosystem to cell internalize ONs was investigated though cell uptake tests using labeled dsDNA. pOG-M-PEG complexes showed a very high uptake in 6 hour of incubation with MCF-7, KB and MC3T3-E1 cells.

The uptake studies were confirmed by confocal microscopy that showed a great uptake after 6 hours of incubation.

The assay confirms that the star-like (guanydil)x-oligosacharidic compounds of the invention are suitable for the intracellular delivery of macromolecules as described above.

Definitions

All technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which the claimed subject matter belongs, unless otherwise defined.

The following terms, used in the specification and claims of this application, have the meaning specified hereunder, unless otherwise defined.

The term "halogen", as used herein, indicates fluorine (F), chlorine (CI), bromine (Br) or iodine (I).

The term "alkyl", as used herein, indicates a saturated aliphatic hydrocarbon radical, including straight chain and branched chain radicals of 1 to 6 carbon atoms referred to as C-i -e alkyl. Non-limiting examples of alkyl are methyl, ethyl, propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-amyl, iso-amyl, n-hexyl, and the like. The alkyl group of the compounds described herein may be designated as "C1-C22, C-i-Cs or C1-C4 alkyl" or similar designations. By way of example only, "Ci-C ₄ alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from among methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl.

By way of an example, saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec- butyl, cyclohexyl, cyclohexylmethyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like.

An unsaturated alkyl group is one having one or more double bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2- isopentenyl, 2-butadienyl, 2,4-pentadienyl, 3-(1 ,4-pentadienyl), ethynyl, 1 - and 3- propynyl, 3-butynyl, and the higher homologs and isomers. Alkyl groups which are limited to hydrocarbon groups are termed "homoalkyl".

"Peptides" means products derived from condensation of two or more amino carboxylic acid molecules (the same or different) by formation of a covalent bond from the a-carbonyl carbon of one to the a-nitrogen atom of another with formal loss of water. The term is usually applied to structures formed from a-amino acids, but it includes those derived from any amino carboxylic acid.

"Proteins" means naturally occurring and synthetic polypeptides having molecular weights greater than about 5000 daltons.

The term "alkoxy", as used herein, means an unsubstituted or substituted alkyl chain linked to the remainder of the molecule through an oxygen atom. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propyloxy, isopropyloxy, benzyloxy and the like.

In general, the term "optionally substituted" or "substituted" means that the referenced group may be substituted with one or more additional group(s) individually and independently selected for example from C1-22 alkyl, cycloalkyl, halo, carbonyl, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. The term steroidic scaffold means naturally occurring compounds and synthetic analogues, based on the cyclopenta[a]phenanthrene carbon skeleton, partially or completely hydrogenated; there are usually methyl groups at C-10 and C-13, and often an alkyl group at C-17. By extension, one or more bond scissions, ring expansions and/or ring contractions of the skeleton may have occurred.

Typical compounds having steroidic scaffold comprise cholestanes such as cholesterol and cholanes such as cholic acid.

The term phospholipid means lipids containing phosphoric acid as mono- or di- esters, including phosphatidic acids and phosphoglycerides. Useful phospholipid include phosphatidylcholine, phosphatic acid, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, phosphatidylserine, lysophosphatidylcholine.

The term molecule having biological activity means a compound exerting a biological activity, such as a drug, a protein, a polypeptide, or a oligonucleotide. The term biological site includes biological matter such as cells, or tissues or organs of the human body.

The terms physicochemical and biopharmaceutical modifiers mean agents improving the pharmacokinetic features or the biopharmaceutical features such as solubility, carrier of an active ingredient in the target site.

EXAMPLES

The following Examples provide illustrative embodiments. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter.

Materials

Maltotriose, 6-aminocaproic acid, acetic anhydride, 2-bromoisobutyryl bromide, triethylamine, ascorbic acid, Agmatine sulfate, 1 -ethyl -3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), Ν,Ν'-dicyclohexylcarbodiimide (DCC), N- hydroxysuccinimide (NHS), fetal bovine serum (FBS), folic acid free Dulbecco's modified essential medium (DMEM) supplemented with 10% FBS, 2 mM L- glutamine, 100 lU/mL penicillin, 100 g/mL streptomycin and 0.25 g/mL of amphotericin B, tripsin-PBS solution (1 % w/v), 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT), xylen cyanol, oleylamine, acryloyl chloride, copper bromide, tris(2-pyridylmethyl)amine (TPMA), deuterated solvents (D2O, DMSO-d6, CDC ), glycerol, tris(hydroxymethyl)aminomethane chloride (Tris), boric acid, ethylenediaminetetraacetic acid were purchased from Sigma Aldrich (St. Louis, MO, USA).

Bis amino terminating Polyethylen glicole was purchased from Iris Biotech GmbH (Marktredwiz, Germany).

The double strand DNA (dsDNA, 19 nucleotides per strand) and cyanin-3 labeled dsDNA (C3-dsDNA, 19 nucleotides per strand) were purchased from Biomers.net GmbH (Ulm, Germany).

30% Acrylamide/Bis acrylamide 29:1 solution was purchased from Bio-Rad (Milan, Italy).

The dsDNA intercalating agent GelRed was purchased from SICHIM ( Rome, Italy).

Cell lines from human breast carcinoma (MCF-7), from human cervical carcinoma (KB) and from murine embryonic fibroblast (MC3T3-E1 ) were provided by the cell bank ATCC (Manassas, VA, USA).

The solvents were furnished by Carlo Erba (Milan, Italy), VWR International (Lutherworth, UK), Sigma Aldrich (St. Louis, MO, USA).

Cell cultures plates and flasks, 96-well plates, 6-well plates were purchased from BD Falcon (D Biosciences, NJ, USA).

All the other reagents or salts were obtained from Fluka Analitical or Sigma- Aldrich.

Example 1

Synthesis of the (agmatinyl)6-maltotriosyl-N-acetyl-amino-hexanoic acid

1 ) Synthesis of maltotriosyl-N-acetyl-amino-hexanoic acid

One gram of maltotriose (1 .98 mmol) was dissolved in 30 mL of methanol acidified with 1 % v/v of acetic acid. The solution was heated to 60 °C until complete dissolution of maltoriose. Then, the solution was added of 1040.1 mg of 6- aminohexanoic acid (7.93 mmol) and maintained under stirring for 14 h at 40 °C. The volume was reduced to half volume under vacuum and the solution was added of 22.6 mL of acetic anhydride (237.6 mmol) over 60 min. The solution was stirred at room temperature for 24 h and then the volume was reduced to ^« 5 mL. The reaction solution was dropped into cold diethyl ether and the precipitate was recovered by centrifugation at 4000 rpm for 5 min and then desiccated under reduced pressure. The product yield was 1 .1 grams. The final product was analyzed by ¹ H-NMR, ESI-TOF mass spectrometry, FT-IR and elemental analysis. Figure 1 shows the scheme of synthesis of maltotriosyl-N-acetyl-amino-hexanoic acid. ¹ H NMR (300 MHz, D ₂ 0) δ 5.39 (s, 1 H, anomeric proton), 5.20 (s, 1 H, anomeric proton), 4.12 - 3.33 (m, 18H, sugar region), 2.45 - 2.32 (m, 2H, ε-ΟΗ ₂ ), 2.06 (s, 3H, CH3-CO-), 1 .75 - 1 .44 (m, 4H δ- CH ₂ and β-ΟΗ ₂ ), 1 .41 - 1 .20 (m, 2H, a-CH ₂ ).

ESI-MS [m/z]: 658.23 (M-H ⁺ ) ^{1 "} (calcd for 659.26)

FT-IR (KBr): v (cm ^"1 ) 3340 (-OH), 2931 (-CH), 1716 (-COOH), 1601 (acetyl CO- N);

Elemental analysis: C, 44.92%; H, 6.92%; N, 2.78%; (O, 45.38%) [calcd for maltotriosyl-N-acetyl-amino-hexanoic acid C, 47.34%; H, 6.88%; N, 2.12 %; O, 43.66%.].

2) Synthesis of star-like (2-bromoisobutyryl)6-maltotriosyl-N-acetyl-amino- hexanoic acid

The synthesis of (2-bromoisobutyryl)-malytotryosyl-N-acetyl-amino-hexanoic acid was performed following according to the modified protocol described by Stenzel- Rosembaum et al. [37].

A suspension of 1 .0 g of maltotriosyl-N-acetyl-amino-hexanoic acid (1 .52 mmols) in 40 mL of anhydrous chloroform was added of trimethylamine (4.21 ml, 30.2 mmol) and maintained at 0°C under stirring for 15 min. The suspension was added of 3.48 mL of 2-bromo-isobutyryl-bromide (30.2 mmol) over 30 min. The reaction was stirred for 3 hours at 0°C and then for 72 hours at room temperature. The mixture was poured in a separating funnel containing 100 mL water and washed three times with cold water (100 mL x 3), three times with 0.1 N NaOH (100 mL x 3) and finally three times with cold water (100 mL x 3). The organic layer was recovered and dried over Na ₂ SO ₄ . The suspension was filtered and the solvent was removed under reduced pressure. The final product was a red-brown oil and the yield was 3.0 grams. The product was analyzed by ¹ H-NMR, FT-I R and elemental analysis Figure 2 shows the synthesis of (2-bromoisobutyryl)e- maltotryosyl-N-acetyl-amino-hexanoic acid. Analysis:

¹ H NMR (400 MHz, CDCIs) δ 5.58 (s, 1 H, anomeric proton), 5.25 (s, 1 H, anomeric proton), 4.63 - 2.47 (m, 20H, glycosyl scaffold and £-CH ), 2.1 5 - 1 .74 (m, 36H, CO-(CH ₃ ) ₂ ), 1 .60 (s, 3H,-CO-CH ₃ acetyl moiety), 1 .45 - 1 .06 (m, 4H, δ- CH ₂ and β-ΟΗ ₂ ), 1 .01 - 0.67 (m, 2H, 2H, a-CH ₂ ).

FT-I R: 3472 (-OH), 2978 (-CH), 1 744 (isobutyryl -CO-O), 1 650 (acetyl -CO-N). Elemental analysis: found C, 40.02%; H, 4.89%; Br, 31 .1 2%; N, 0.84%; (O, 23, 1 3%). [calcd for (2-bromoisobutyryl)6-maltotryosyl-N-acetyl-amino-hexanoic acid (CsoHysBreNO; ), C, 38.66%; H, 4.87%; Br, 30.86%; N, 0.90%; O, 24.72%.].

3) Synthesis of acryloyl-agmatine

Arcyloyl-agmatine was synthesized according to the protocol reported in the literature [patent US 6.703.468][38J. Briefly, 2.0 grams of agmatine sulfate (8.76 mmol) was dissolved in NaHC03 saturated water solution at 0 °C. Acryloyl chloride (749.18 μΙ_, 9.64 mmol) was added over 30 min under vigorous stirring. After 1 h, the pH of the solution was adjusted to 1 .0 using 1 .0 N HCI and the mixture was saturated using sodium chloride. After filtration, the solution was washed in a separated funnel using ethyl acetate (50 mL x 3) and finally extracted three times with 50 mL of a solution 1 :1 of isopropanol : ethyl acetate. The organic layer were collected and concentrated under reduced pressure. A pale yellow oil was recovered, dissolved in water and freeze-dried. The final product yield was 1 .0 g. The product was analyzed by ¹ H NMR and ESI-TOF mass spectrometry.

1 H NMR (300 MHz, D ₂ 0) : 5 6.27 (dd, J = 17.1 , 9.7 Hz, 1 H,CH ₂ =C-), 6.21 - 6.12 (m, 1 H, C=CH-), 5.76 (dd, J = 9.7, 1 .9 Hz, 1 H, CH ₂ =C-), 3.29 (dd, J= 1 0.6, 4.2 Hz, 2H, a-CH ₂ ), 3.03 (t, J = 6.9 Hz, 2H, 5-CH ₂ ), 1 .66 - 1 .54 (m, 4H, β,γ-ΟΗ ₂ ).

ESI-TOF [m/z] : 1 85.14 (M+H ⁺ ) ^{1 +} [calcd for 1 84.1 3].

4) Synthesis of star-like (agmatinyl)6-maltotriosyl-N-acetyl-amino-hexanoic acid

CuBr (460.47 mg, 3.21 mmol), of tris[(2-pyridyl)methyl]amine (TPMA, 932.05 mg, 3.21 mmol) and ascorbic acid (1 1 .3 mg, 0.0642 mmol) were dissolved in anhydrous DMSO under nitrogen flow over 30 min and added to a degassed DMSO solution of acryloyl-agmatine (1 181 .28 mg, 6.42 mmol) and heated to 65 °C. The solution was added of 500 mg of (2-bromoisobutyryl)6-malytotryosyl-N-acetyl- amino-hexanoic acid (0.321 mmol). All solutions were degassed. The mixture was stirred in these conditions for 72 hours and then was exposed to the air. After 30 min the solution was extensively poured in 1 :1 v/v diethyl ether/acetone mixture added of 1 % v/v of acetic acid. The precipitate was collected and desiccated under vacuum. The product yield was 542 mg, corresponding to 65%.

The guanidinium groups content in the product was measured by Sakaguchi essay

[39]■ The experimental data were referred to a calibration curve obtained with a standard solutions of 0-100 μΜ guanidinium content (y= 9.1724x-0.0075, R ² =0.9965).

The final product was analyzed by ¹ H-NMR (Figure 3), FT-IR and elemental analysis.

FT-IR : v (cm ^"1 ) 3373 and 3182,[NHC(NH ₂ )2 ⁺ ] ; 2930 (CH ₂ ) ; 1750 (C=0); 1300 (C- H bending).

Elemental Analysis: found, C 37.58 %, H, 5.51 %, N, 10.98 %; Br, 31 .90 % (O, 14.03%). [Calcd for (agmatinyl)6-maltotriosyl-N-acetyl-aminododecanoic acid HBr salt (C9 ₃ Hi6i Br ₆ N2503o-6HBr): C, 36.33%, H, 5.48%, N, 1 1 .39%, Br, 31 .19%, O, 15.61 %].

Figure 4 shows the synthesis of (agmatinyl)6-maltotriosyl-N-acetyl-amino- hexanoic acid. Figure 5 shows the general structure of the oligoguanidine derivative.

The (guanidyl)x-oligosaccharidic derivatives (pOG-M) synthesized according to the procedure described above, was conjugated to poly (ethylene glycol) and oleylamine.

Example 2

Synthesis of star-like (OligoGuanidyl)6-maltotriosyl-N-acetyl-amino-hexanoate- poly(ethylene glycol)-NH ₂ (pOG-M-PEG).

(Oligoguanidyl)6-maltotriosyl-N-acetyl-amino-hexanoic acid {pOG-M, 500 mg, 0.193 mmol) was dissolved in 10 mL of a mixture of 1 :2 DMSO and 100 mM morpholino-ethan-sulphonic acid buffer (MES), pH=4.7. The solution was added of 1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC , 300 mg, 1 .93 mmol) and N-hydroxysuccinimide (NHS, 222.1 mg, 1 .93 mmol). The solution was stirred over 30 min and then added of 6 kDa (NH ₂ )2-PEG (965 mg, 0.161 mmol) previously dissolved in 1 :2 DMSO: 100 mM MES buffer, pH=4.7 . The solution was stirred for 72 hours and then dialyzed against water for 48 hours using a 3.5-5.0 kDa MWCO dialysis membrane . The product was lyophilized and characterized by UV-Vis. The yield of final product was 600 mg. The yield of conjugation determined by Sakaguchi assay for the guanidine content and the iodine test for the PEG detection [40] was 96%. Figure 6 reports the conjugation of (agmatinyl)e- maltotriosyl-N-acetyl-amino-hexanoic acid with 6 kDa (NH2)2-PEG.

Example 3

(Oligoguanidyl)6-maltotriosyl-N-acetyl-amino-hexanoate-oleyl amide

conjugation(pOG-M-OI).

500 mg of pOG-M (500 mg, 0.193 mmol) were solubilized in 5 ml_ of DMSO anhydrous. At this solution was added a 2.5 ml_ solution of Ν,Ν'- Dicyclohexylcarbodiimide (DCC, 398.2 mg, 1 .93 mmol). The solution was stirred over 15 min. The mixture was added to N-hydroxysuccinimide (NHS, 222.1 mg, 1 .93 mmol) and the solution was heated to 40°C and stirred overnight. The activated pOG-M was separated to the byproducts by filtration. Then at the solution were added 635.0 μΙ_ of oleylamine (1 .93 mmol) and 269.2 μΙ_ of triethylamine (1 .93 mmol). The solution was stirred over 24 hours and then was precipitated in a mixture of diethyl ether. The product was lyophilized and characterized by ¹ H-NMR and by Sakaguchi assay. The guanidinium group content was measured as reported above (see Example 1 ). Figure 7 reports the synthesis of agmatinyl)6-maltotriosyl-N-acetyl-amino-hexanoate-oleylamide .

Example 4

Oligonucleotide (ON) condensation with (Oligo-guanidyl)6-maltotriosyl-N-acetyl- amino-hexanoate-Poly(ethylenglycol)-NH2 (pOG-M-PEG) and complex formation. 4.1 Preparation of ON/pOG-M-PEG) complexes

ON/pOG-M-PEG complexes were prepared by using a scrambled 1 1 kDa dsDNA (19 pb) as ON model. dsDNA/polymer mixtures [0.5, 1 .0, 2.0, 3.0, 4.0, 5.0, 7.0 and 10.0 nitrogen/phosphorous (N/P) molar ratio] were prepared by mixing dsDNA solutions in 10 mM phosphate buffer, 0.15 M NaCI (PBS), pH=7.4 with pOG-M-PEG solutions in PBS, pH=7.4. The dsDNA and pOG-M-PEG concentrations were selected in order to yield fixed N/P ratios required for the specific analysis. The dsDNA/pOG-M-PEG mixtures were maintained at room temperature for 30 min under gentle agitation to equilibrate before analysis.

The same protocol was used to prepare Cy3-dsDNA/pOG-M-PEG complexes. 4.2 Electrophoresis analyses

Electrophoretic analyses were performed to evaluate the complexation capacity of (Oligoguanidyl)6-maltotriosyl-N-acetyl-amino-hexanoate-poly( ethylenglycol)-NH2 with oligonucleotides.

The dsDNA/polymer mixtures were prepared by simple mixing 2.58 L of 10 μΜ dsDNA solution (300 ng, 2.58-10 ^"11 nmol) in PBS, pH=7.4, with 10 μΙ_ of 0.705- 7.05 pg of pOG-M-PEG solutions in PBS, pH=7.4 inorder to yield 0.5, 1 .0, 2.0, 3.0, 4.0, 5.0 nitrogen/phosphorous (N/P) ratio. At the complex were added 3 μΙ_ of loading buffer containing 30% glycerol and 0.25% xylene cyanol in water. The dsDNA/pOG-M-PEG samples were analyzed by gel-electrophoresis at 100 V for 1 hours on a 12% polyacrylamide gel in Tris-Borate-EDTA (TBE) 1 X buffer. The migrated dsDNA was visualized by immersion of the gel in a Gel Red solution previously prepared by dilution of 15 L of staining marker in 30 ml_ of milliQ water for 30 min. Figure 8 shows the gel image obtained using a UV-Transilluminator. 4.3 Dynamic Light Scattering (DLS) and Zeta potential analysis

Dynamic Light Scattering and Zeta Potential analysis were performed at 25 °C using a Malvern Zetasizer NanoZS (Malvern Instruments Ltd., U.K.) supported by Zetasizer Software (version 6.12). The 1 mL dsDNA/pOG-M-PEG samples in PBS, pH 7.4, contained 500 μg/mL of pOG-M-PEG and 9.12-0.912 μΜ dsDNA to dsDNA/pOG-M-PEG corresponding to 1 .0, 2.0, 3.0, 5.0, 7.0, 10.0 N/P molar ratio. Figure 9 shows the size and zeta potential of dsDNA/pOG-M-PEG complexes with N/P ratio in the range of 1 -10.

4.4 Transmission Electron Microscopy

dsDNA/pOG-M-PEG samples containing at 100 pg/mL of pOG-M-PEG and 3 and 5 N/P ratio were analyzed by Transmission Electron Microscopy (TEM). The samples were deposited on a small copper grid (400 mesh), covered by "holey film" carbon layer and analyzed in negative staining mode. The contrast agent used was uranyl acetate 1 % w/v. Figure 10A and Figure 10B show the TEM images of complexes prepared with 3 and 5 N/P ratio, respectively.

4.5 Stability test

dsDNA/pOG-M-PEG samples containing 500 pg/mL of pOG-M-PEG and 3 and 5 N/P ratio in PBS, pH 7.4 or in DMEM medium supplemented with 15% FBS (Fetal Bovine Serum), 2 mM L-glutamine, 100 lU/mL penicillin, 100 pg/mL streptomycin and 0.25 g/mL of amphotericin B 4 were analyzed by Dynamic Light Scattering for 12 hours at 37°C. Figure 1 1 A-B and Figure 12A-B show the size of the samples at the two NP ratios and in buffer or DMEM medium, respectively.

Example 5 Hemolytic activity of dsDNA/pOG-M-PEG.

5 ml_ mouse of blood were centrifuged at 500 g for 5 min. The plasma was removed and the red blood cells (RBC) were diluted with 4.5 ml_ of 150 mM of NaCI solution. The tube was gently shaked and centrifuged at 500 g for 5 min. The RBC were washed 3 times and then the supernatant was removed and the cells were diluted with PBS, pH 7.4, and the diluted blood was splitted into three volumes. After centrifugation, the pellets were suspended in PBS, pH= 7.4. In 96- well plates, at 190 μΙ_ of diluited RBC 10 μΙ_ complex of dsDNA/pOG-M-PEG at N/P ratio of 3 and 5 were added at the final concentration of 0.1 , 0.5, 1 .0, 1 .5 mg/mL of polymer. The plates were incubated at 37°C for 1 hour under gentle agitation. The samples were then centrifuged at 500 g for 5 min and the supernatants (150 μΙ_) were transferred into 96-well plates and the hemoglobin released was measured with a microplate reader at 570 nm. A Triton X-100 solution in PBS pH 7.4 (1 .0% w/v) was used as positive control (total release of hemoglobin, 100% hemolysis). PBS and PBS solution of ONs at the same concentration of the dsDNA/pOG-M-PEG samples used above were used as a positive control. All the assays were repeated 5 times. Figure 13 shows the hemolytic profiles of dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio at pH 7.4.

Example 6 Isothermal Titration Calorimetry

dsDNA (100 μΜ) and pOG-M-PEG (20 μΜ) solutions were prepared by dissolving dsDNA and pOG-M-PEG in 10 mM TRIS, 150 mM NaCI, pH 7.4. Both the solutions were degassed and thermostated at 25 °C before the analysis. At 5 minutes intervals, 10 μΙ_ of dsDNA solution was injected in 10 sec into the calorimeter cell containing 1 .5 ml_ pOG-M-PEG. Figure 14 shows the isothermal calorimetry profiles of pOG-M-PEG titrated with dsDNA in 10 mM TRIS, 150 mM NaCI, pH 7.4 and relative table.

Example 7 Cell culture studies

7. 1 Cell lines and cultures

The human MCF-7 breast adenocarcinoma, human KB cervical carcinoma and murine MC3T3-E1 embryonic fibroblast cell lines were grown at 37°C, in 5% CO2 atmosphere, using DMEM medium supplemented with 15% FBS (Fetal Bovine Serum), 2 mM L-glutamine, 100 lU/mL penicillin, 100 pg/mL streptomycin and 0.25 g/mL of amphotericin B.

7.2 Cytotoxicity of pOG-M-PEG and dsDNA/pOG-M-PEG complexes

MCF-7, KB, MC3T3-E1 cells were seeded in 96 well plate (5 x 10 ³ cell/well). After 24 hours the medium was removed and the cells were incubated at 37°C with increasing concentrations of pOG-M-PEG and increasing dsDNA concentrations of dsDNA/pOG-M-PEG samples at 3 and 5 N/P ratio. Figure 15 shows the viability of MCF-7 (Figure 15A), KB (Figure 15B) and MC3T3-E1 (Figure 15C) cells incubated with dsDNA/pOG-M-PEG complexes with 3 and 5 N/P ratio for 24 and 48 hours.

7.3 Flow cytometry analysis

Cell uptake study was performed using MCF-7, KB and MC3T3-E1 cells using flow cytometry analysis. In order to evaluate dsDNA uptake into the cells, the complexes were prepared with labeled Cyanine 3-dsDNA (Cy3-dsDNA). Cells were seeded in 6 well plate at a density of 1 .5 x 1 0 ⁶ cells/well. The DMEM medium was removed and the cells were washed twice with PBS, pH 7.4, and then treated with 500 μΙ_ of Cy3-dsDNA/pOG-M-PEG complex solutions at fixed concentrations of 125 nM labelled ONs. After 1 hours of incubation time at 37°C, the wells were washed three times with PBS, pH 7.4, and transferred into cytometer tubes. After centrifugation at 1500 rpm for 5 min, the supernatants were discharged and the pellets were re-dispersed in 300 μΙ_ of PBS, pH 7.4, and analyzed by using the mean fluorescence intensity. Data collection was carried out with 10000 counts per sample. Figure 16 shows the cell cytometric profiles after 6 hour cell treatment with complexes with 3 and 5 N/P ratio.

7.4 Confocal microscopy observation

MCF-7, KB and MC3T3-E1 cells were seeded at density of 5 x 10 ⁴ cells/cm ² and grown for 24 h at 37°C and 5% C02.

The medium was removed and the cells were washed with PBS, pH 7.4. 500 uL of Cy3-dsDNA/pOG-M-PEG complexes in cell culture medium at concentration of 125 nM of Cy3-dsDNA were prepared by simple mixing of Cy3-dsDNA with pOG- M-PEG as described in Exemple 4 section 1 and added in each wells. Cells were treated at 37°C in the dark for 6 hours and then gently washed three times with 500 μΙ_ PBS, pH 7.4. Cells were fixed with 500 μΙ_ of 1 % w/v paraformaldehyde solution in PBS, pH 7.4 for 15 min at room temperature. The wells were washed three times and incubated with 500 μΙ_ of 5.0 pg/mL of DAPI solution for nuclei staining for 15 min at room temperature. In order to stain the membranes, the fixed cells were treated with 4.5 ug/mL solution of wheat germ agglutinin 488 AlexaFluor. The wells were washed three times with PBS solution and the three times with milliQ water.

The samples images were acquired using a Zeiss LSM 800 confocal microscopy equipped of an immersion lens with 63 X magnification. The lasers were fixed at 405 nm, 488 nm and 561 nm to detect DAPI, 488 AlexaFluor and Cy3-dsDNA.

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