Functionalization of pegylated surface

Abstract

The invention relates to a compound of the formula (i); Py-X (i) wherein: Py is a polycyclic aromatic hydrocarbon moiety, X is a therapeutic moiety or a diagnostic moiety or a bioactive moiety, and wherein the therapeutic moiety or the diagnostic moiety or a bioactive moiety is not an osmium complex.

Description

[0001] FUNCTIONALIZATION OF PEGYLATED SURFACE

[0002] Field of the invention

[0003] The invention relates to compounds for the functionalization of a pegylated surface, to a method for the functionalization of a pegylated surface and to the use of a vesicle or an object comprising a pegylated surface functionalized by the method of the invention.

[0004] State of the art

[0005] A pegylated surface can be an important component used in different applications as in medical implantable devices (to reduce the risk of thrombosis and immune response), in drug delivery carriers, in biosensors (to minimize nonspecific binding of biomolecules, improve selectivity and sensitivity), in cell culture (to create non-adherent environment), in tissue engineering scaffolds (encapsule and control the release of cell or bioactive molecules), in diagnostic or imaging assays and in vaccine.

[0006] A known example in the medical field is the use of vesicles and nanovesicles which have a pegylated surface and are drug delivery carriers encapsulating and protecting sensitive molecules, such as drugs, enzymes, other proteins and peptides, and DNA and RNA fragments.

[0007] The functionalization of a pegylated surface is a key step for creating more versatile systems. Several technologies have been already developed for the functionalization of pegylated surfaces.

[0008] Regarding the technologies developed for the functionalization of pegylated surface of nanovesicles, a first example is the conjugation of functional ligands onto the surface of preformed nanovesicles. In this approach the nanovesicle is prepared comprising a reacting group which will be used, once the nanovesicle is formed, in a non-covalent interactions or covalent interaction with a functional molecule. The functional molecule may be a biomolecule (e.g. Ligand: streptavidin, Cell targeting by a generic receptor-targeted polymer nanocontainer platform, J. Controlled Release, 2005, 102, 475-488).

[0009] A second example is a self-assembly end-group functionalized block copolymers, where targeting ligands are grafted to the end group of polymers, followed by mixing with non-modified polymers in a certain ratio. Also in this approach the nanovesicle is pre-formed comprising the reacting group which will be used, once the nanovesicle is formed (Ligand: mannose, Carbohydrate-coated nanocapsules from amphiphilic rod-coil molecule: binding to bacterial type 1 pili, Chem Commun, 2005, 2035-2037).

[0010] A third example, in which the nanovesicle is pre-formed comprising the reacting group, is the incorporation of bio-functional blocks into polymers. Glycopolymers, carbohydrate chains, peptides, and oligonucleotides can be incorporated into the polymer backbone as side chain groups (Biomimetic Doxorubicin Loaded Polymersomes from Hyaluronan-block-Poly(y-benzyl glutamate) Copolymers, Biomacromolecules 2009, 10, 10, 2802-2808. Ligand: Hyaluronan).

[0011] However, these prior art methods suffer from low efficiency, resulting in incomplete functionalization or low density of functional groups on the nanovesicle surface. Covalent conjugation usually involves the use of reactive agents or organic solvents, potentially destabilizing vesicles, causing cargo leakage, or altering other properties of the nanovesicles. In addition, the attachment of the targeting ligand to the amphiphilic block copolymer can affect the balance of hydrophilia and hydrophobicity, which is critical for the self-assembly behavior of nanovesicles. Additionally, during the self-assembly of end-group functionalized polymers, part of the functional moieties will inevitably be oriented towards the interior of nanovesicles or buried within the folded structure. Such undesirable molecule distribution further limits the accessibility and efficiency of its interactions and biological activities.

[0012] Surface functionalization has also been explored by an adaptive insertion of a hydrophobic anchor conjugated via a poly(ethylene glycol) spacer to an osmium complex (Daniela A. Wilson et al., Nature Chemistry volume 15, pages 240-247 (2023)). However, this method does not allow for an efficient spatiotemporal selective surface functionalization showing not optimal exposure or presentation of the functional module on the surface.

[0013] LIM E.-K. et al. “Preparation of pyrenyl-based multifunctional nanocomposites for biomedical applications”, Nature Protocols, februari 2016, vol. 11 , nr. 2, pag. 236-251 , discloses in Figure 2 a pyrene conjugated to HA (polysaccharide). The molecular mass of the HA used in Figure 2 can be calculated to be around 1 ,000 kDa. This leads to a greater tendency for Py-HA to form intramolecular aggregates, which hinders the exposure of pyrene and its subsequent insertion into the PEG corona. As a result, both the insertion efficiency and the stability of the system will be undesirably low.

[0014] Hence, spatiotemporal selective surface functionalization is still a challenge. There is still a need for a suitable, highly efficient, economically viable and environmental friendly technology for the functionalization of pegylated surfaces which solves at least part of the problems of the prior art.

[0015] The aim of the invention is to develop a technology for the functionalization of pegylated surfaces which is highly efficient, economically viable and environmental friendly.

[0016] After extensive studies, the inventor(s) solved the above-mentioned problems and developed novel compounds for the functionalization of a pegylated surface and a method for the functionalization of a pegylated surface.

[0017] Advantageously the pegylated surface functionalized by the method of the invention may be used in the medical field or in diagnostic field for example for a receptor mediated endocytosis, mitochondrial targeting and promotion of cell migration based on the type of therapeutic or diagnostic moiety used for the functionalization of the pegylated surface.

[0018] Summary of the invention

[0019] In a first aspect, the present invention is directed to a compound of the formula (i);

[0020] Py-X (i) wherein:

[0021] Py is a polycyclic aromatic hydrocarbon moiety,

[0022] X is a therapeutic moiety or a diagnostic moiety or a bioactive moiety, and wherein the therapeutic moiety or the diagnostic moiety or a bioactive moiety is not an osmium complex.

[0023] In a second aspect, the present invention is directed to a method for the functionalization of a pegylated surface comprising the steps of: a. Providing a surface comprising a layer of poly(ethylene glycol), b. Adding the compound of the invention to the surface comprising a layer of poly(ethylene glycol), wherein the addition of step b) is performed in aqueous solution.

[0024] In a third aspect, the present invention is directed to an object comprising a pegylated surface and the compound of the invention bound to the surface, wherein the amount of the compound is between 0.001- 0.1 molecules / nm2.

[0025] In a fourth aspect, the present invention is directed to a container comprising the compounds of the invention.

[0026] In a fifth aspect, the present invention is directed to a kit of parts for the functionalization of a pegylated surface, the kit comprising at least the first container of the invention and a second container comprising an aqueous solution.

[0027] The inventors surprisingly found that the compound of the invention can be easily used for the functionalization of a pegylated surface and that the activity of the loaded compounds is optimal providing an optimal spatiotemporal selective surface functionalization. Moreover, using the method of the invention, the loading of the compound of the invention can be easily controlled and it can be performed in a short time. Further, the compound of the invention showed a good affinity with different types of pegylated surfaces and when used to functionalize vesicles showed to provide functionalize vesicles having a good stability in vivo. Moreover, the compound of the invention and the functionalization of a pegylated surface can be performed with methods that are economically efficiency and excluding a negative environmental impact. Finally the kit of the invention allows the functionalization of a pegylated surface just before its use solving the problems related to storage and stability of a functionalized layer of poly(ethylene glycol).

[0028] Description of the drawings

[0029] Figure 1 shows a figurative representation of a polymersome PEG corona functionalized with Py-X compound.

[0030] Figure 2 shows the distribution of fluorescence on the membrane.

[0031] Figure 3 shows the fluorescent emission spectrums of Py-X, FITC labeled polymersomes (FITC / Poly.), FITC labeled polymersomes loaded with Py-X (Py-X / FITC / Poly.- 1), and FITC labeled polymersomes loaded with Py-X after centrifuge (Py-X / FITC / Poly.-2).

[0032] Figure 4 shows the loading stability of each Py-X (Py-TPP, Py-FA, Py-HA, and Py- RGD).

[0033] Figure 5 shows the cell migration promoted by Py-HA

[0034] Figure 6 shows the mitochondria targeting facilitated by Py-TPP.

[0035] Figure 7 shows the endocytosis mediated by Py-FA

[0036] Detailed description of the invention

[0037] The present invention is directed to a compound of the formula (i);

[0038] Py-X (i) wherein:

[0039] Py is a polycyclic aromatic hydrocarbon moiety,

[0040] X is a therapeutic moiety or a diagnostic moiety or a bioactive moiety, and wherein the therapeutic moiety or the diagnostic moiety or a bioactive moiety is not an osmium complex.

[0041] The term “polycyclic aromatic hydrocarbon moiety” according to the present invention refers to the a class of organic compounds that is composed of multiple aromatic rings which may comprise one or more substituents able to chemical react with the X moiety allowing the formation of a direct chemical bond between Py and X providing Py-X.

[0042] The direct chemical bond may be an ester bond, an amido bond, an ether bond or a thiol-maleimide bond. The direct chemical bond is not a polyethylene glycol moiety. An ester bond is formed through an esterification reaction. In particular by a reaction between a -COOH functional group and an -OH functional group. The -COOH functional group or the -OH functional group can be present on Py or on X.

[0043] An amido bond, is formed through a reaction between a -COOH functional group and an amino functional group. The -COOH functional group and the amino functional group can be present on Py or on X.

[0044] An ether bond is formed through a Williamson ether synthesis. In particular by a reaction between a -Br or -Cl functional group and a -OH functional group. The -Br or -Cl functional group or the -OH functional group can be present on Py or on X.

[0045] A thiol-maleimide bond is formed through a reaction between a -SH functional group and a maleimide functional group. The -SH functional group and the maleimide functional group can be present on Py or on X.

[0046] Example of organic compounds composed of multiple aromatic rings are naphthalene, having two aromatic rings, anthracene and phenanthrene having three aromatic rings, pyrene, having four aromatic rings and coronene, having six aromatic rings.

[0047] Preferably, the polycyclic aromatic hydrocarbon moiety is selected from the group consisting of naphthalene, anthracene, phenanthrene, pyrene and coronene moiety, more preferably naphthalene, anthracene, phenanthrene and pyrene moiety. The most preferred polycyclic aromatic hydrocarbon moiety is pyrene moiety.

[0048] Preferably, the organic compound composed of multiple aromatic ring used in the current invention is a pyrene moiety in which the pyrenyl part comprises one or more substituents able to chemical react with the X moiety allowing the formation of a direct chemical bond providing Py-X. Surprisingly, although a pyrenyl is hydrophobic, Py-X can be advantageously dissolved easily in water, further a pyrenyl is uncharged, non-polar and planar and showed a remarkable affinity and loading ability with an adaptive insertion into a pegylated surface of vesicles, in particular of polymersome vesicles.

[0049] Examples of pyrene moieties are 1 -Pyrenecarboxylic acid, 1 -Pyreneacetic acid, 1- Pyrenepropanoic acid, 1 -Pyrenebutyric acid; 1 -Pyrenemethylamine, 2-(Pyren-1-yl)ethan-1- amine, 3-pyren-1-yl-propylamine; Pyrene-1 -thiol, Pyrene-2-thiol, 1-(1-pyrenyl)-1 -methanethiol, 1-(2-mercaptoethyl)pyrene; 1 -bromopyrene, 1 -bromomethylpyrene, 1-(2-bromoethyl)pyrene, 1-(3-bromopropyl)pyrene; N-(1-Pyrenyl)maleimide, N-(1-methylpyrene)maleimide, 1- hydroxypyrene, 1 -pyrenemethanol, 2-(pyren-1-yl)ethanol, 3-(1-pyrenyl)propanol and 1- (bromoacetyl)pyrene. The term “therapeutic moiety” according to the present invention refers to a class of organic compounds that can be used to treat or ameliorate a specific disease, symptom, state of health, or medical- or health-related condition in humans and / or animals which may comprise one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X.

[0050] Example of organic compounds that can be used to treat or ameliorate a specific disease, symptom, state of health, or medical- or health-related condition in humans and / or animals are enzyme or co-enzyme as coenzyme Q10 (Ubiquinone) and ubiquinol (reduced CoQ10), NAD+ (Nicotinamide Adenine Dinucleotide), proteases, kinases, polymerases; vitamins as vitamin A, vitamin B as vitamin B1 (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid ,FA), B12 (cobalamin), vitamin C, vitamin D, vitamin E, vitamin K; polysaccharide as hyaluronic acid (HA), heparin, chitosan, dextran, beta-glucans, glycosaminoglycans, extracellular matrix polysaccharides; peptides as arginyl- glycyl-aspartic acid (RGD), insulin, glucagon-like peptide-1 (GLP-1), cell-penetrating peptides, oxytocin, somatostatin analogs, natriuretic peptides, enkephalins, opioid peptides analogs, calcitonin; proteins as monoclonal antibodies, recombinant insulin, interferons, growth hormones, botulinum toxin, immunoglobulins and interleukins.

[0051] Preferably, the therapeutic moiety is selected from the group consisting of an enzyme or co-enzyme, a vitamin, a polysaccharide, a peptide and a protein.

[0052] Preferably, the therapeutic moiety as indicated above is a small molecule, for instance, having a molecular mass between 100 Da and 50,000 Da, more preferably between 200 Da and 40,000 Da, most preferably between 300 Da and 20,000 Da.

[0053] In a specific embodiment, the therapeutic moiety is a polysaccharide, preferably the polysaccharide has a molecular mass between 500 Da and 50,000 Da, more preferably between 1000 Da and 20,000 Da, most preferably between 1000 Da and 10,000 Da.

[0054] In another specific embodiment, the therapeutic moiety is selected from the group consisting of an enzyme or co-enzyme, a vitamin, a peptide and a protein.

[0055] Preferably, the enzyme or co-enzyme, the vitamin, the peptide and the protein, have a molecular mass between 100 Da and 50,000 Da, more preferably between 200 Da and 40,000 Da, most preferably between 300 Da and 20,000 Da.

[0056] Preferably the enzyme or co-enzyme is selected from the group consisting of coenzyme Q10 (Ubiquinone), ubiquinol (reduced CoQ10), NAD+ (Nicotinamide Adenine Dinucleotide), proteases, kinases and polymerases. More preferably the enzyme or co- enzyme is selected from the group consisting of Q10 (Ubiquinone), ubiquinol (reduced CoQ10) and proteases. The most preferred enzyme or co-enzyme is Q10 (Ubiquinone).

[0057] So, preferably, the enzyme or co-enzyme moiety used in the current invention is a ubiquinone moiety which is the ubiquinone compound comprising one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X. Ubiquinone is a parabenzoquinone with methoxy groups on carbons 2 and 3 and a methyl group on carbon 5, plus a polyisoprene chain the length of which varies among species. Hence, examples of ubiquinone moiety is any possible derivative of ubiquinone compound.

[0058] Preferably, the vitamin is selected from the group consisting of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin K. More preferably, the vitamin is selected from the group consisting of vitamin B1 (thiamine), B2 (riboflavin), B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid ,FA) and B12 (cobalamin). The most preferred vitamin is the vitamin B9 (folic acid ,FA).

[0059] Among many vitamins, the vitamin B9 (folic acid, FA) can be used as a targeting moiety to cells that overexpress folate receptor (FR), which is a high-affinity membrane folate- binding protein. The prevalence of FR overexpression in human tumors makes FR an ideal marker for targeting delivery and cancer imaging. Upon binding with its ligand, clathrin is recruited and assembled on the cell membrane, leading to the formation of clathrin-coated pits and endosomes for the following endocytosis.

[0060] The vitamin B9 (folic acid, FA) can be coupled to Py moiety via amide reaction.

[0061] So, preferably, the vitamin moiety used in the current invention is a folic acid moiety which is a folic acid compound comprising one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X. Examples are (2S)-2-[[4-[(2-amino-4-oxo-1 H-pteridin-6- yl)methylamino]benzoyl]amino]pentanedioic acid, (2S)-2-[[4-[(2-amino-5-formyl-4-oxo-3, 6,7,8- tetrahydropteridin-6-yl)methylamino]benzoyl]amino]pentanedioic acid.

[0062] Preferably, the polysaccharide is selected from the group consisting of hyaluronic acid (HA), heparin, chitosan, dextran, beta-glucans, glycosaminoglycans. More preferably, the polysaccharide is selected from the group consisting of hyaluronic acid (HA), heparin, chitosan and dextran. Even more preferably the polysaccharide is selected from the group consisting of hyaluronic acid (HA) and heparin. The most preferred polysaccharide is hyaluronic acid (HA). Hyaluronic acid is widely distributed in eye vitreous, connective tissues, joints and skins, etc. Hyaluronic acid can bind to the extracellular domain of CD44 promoting the interaction with multiple cytoskeletal proteins (e.g. ankyrin, RhoGTPases, etc.), subsequently regulating the cytoskeletal dynamics and cell motility. As a result, Hyaluronic acid is used in targeting CD44 and tumor migration.

[0063] Preferably, the hyaluronic acid (HA) has a molecular mass between 500 Da and 50,000 Da, more preferably between 1000 Da and 20,000 Da, most preferably between 1000 Da and 10,000 Da.

[0064] Hyaluronic acid can be coupled to Py moiety via a amide reaction.

[0065] So, preferably, the polysaccharide moiety used in the current invention is a hyaluronic acid (HA) moiety which comprises one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X. Examples are poly{[(2S,3R,4R,5S,6R)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxane-2,4- diyl]oxy[(2R,3R,4R,5S,6S)-6-carboxy-3,4-dihydroxyoxane-2,5-diyl]oxy}, including other possible derivatives.

[0066] Preferably, the peptide is selected from the group consisting of as arginyl-glycyl- aspartic acid (RGD), insulin, glucagon-like peptide-1 (GLP-1), oxytocin, somatostatin analogs, natriuretic peptides, enkephalins, opioid peptides analogs and calcitonin. More preferably the peptide is selected from the group consisting of arginyl-glycyl-aspartic acid (RGD), insulin and glucagon-like peptide-1 (GLP-1). Even more preferably the peptide is selected from the group consisting of arginyl-glycyl-aspartic acid (RGD) and insulin. The most preferred peptide is arginyl-glycyl-aspartic acid (RGD).

[0067] Arginyl-glycyl-aspartic acid (RGD) can recognize and bind to integrin receptor triggering a conformational change in the integrin receptor, resulting in endocytosis through multiple pathways such as clathrin-mediated endocytosis, caveolin-mediated endocytosis, or clathrin- and caveolin-independent pathways, etc.

[0068] Arginyl-glycyl-aspartic acid moiety can be coupled to Py moiety via a thiol-maleimide reaction.

[0069] So, preferably, the peptide moiety used in the current invention is an arginyl-glycyl- aspartic acid moiety which is an arginyl-glycyl-aspartic acid compound comprising one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X. Examples are Arginyl-Glycyl-Aspartic acid, cyclo(Arg-Gly-Asp-D-Phe-Lys), Arg-Gly-Asp-D-Phe-Lys, Gly-Arg-Gly-Asp-Ser-Pro, including other possible derivatives. Preferably the protein is selected from the group consisting of transmembrane proteins such as cadherins, Growth Factors, CXCR4 and other trans-membrane G coupled receptor binding agents, antibodies including monoclonal antibodies (e.g. pembrolizumab, trastuzumab, Rituximab), insulin, hormones (e.g. growth hormone, thyroid hormones), neurotransmitters (e.g. vasopressin, neurokinins). More preferably, the protein is selected from the group consisting of a transmembrane proteins, antibodies (e.g. a monoclonal antibody) and hormones. So, preferably, the protein moiety used in the current invention is a transmembrane proteins or a monoclonal antibody moiety or hormone moiety which is a transmembrane protein or an antibody compound or an hormone compound comprising one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X.

[0070] The term “diagnostic moiety” according to the present invention refers to a class of compounds that can be used in the field of identification, diagnosis or prognosis of any disease or medical condition in humans or other animals using a licensed product (s) or developed product (s) which may comprise one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X.

[0071] Example of compounds that can be used in the field of identification, diagnosis or prognosis of any disease or medical condition in humans or other animals are fluorescent proteins, fluorodeoxyglucose, technetium-99m, iodine-based and gadolinium-based agents and Barium based agents.

[0072] Preferably, the diagnostic moiety is selected from the group consisting of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), fluorodeoxyglucose, technetium-99m, iodine-based and gadolinium-based agents and barium based agents. More preferably the diagnostic moiety is selected from the group consisting of green fluorescent protein moiety (GFP), fluorodeoxyglucose and iodine-based agent. The most preferred diagnostic moiety is green fluorescent protein moiety (GFP).

[0073] Green fluorescent protein (GFP) can be coupled to a Py moiety through the specific binding of nitrilotriacetic acid-nickel complexation (NTA-Ni2+) and oligohistidine sequences of GFP. The fluorescence of GFP makes it possible to observe its attachment on the PEG surface.

[0074] So, preferably, the diagnostic moiety used in the current invention is a green fluorescent protein moiety which is a green fluorescent protein compound comprising one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X. Examples are derivatives of green fluorescent protein

[0075] The term “bioactive moiety” according to the present invention refers to a class of organic compounds that exhibit specific effects on biological systems, such as living organisms, cells, or biomolecules.

[0076] Example of compounds that can be used in the field of bioactive moiety are antibiotics (Penicillin, Erythromycin, Streptomycin, etc.), anticancer agents (Doxorubicin, Paclitaxel, Cisplatin, etc.), antioxidants (Resveratrol, Curcumin, etc.), natural products (Morphine, Quinine, Caffeine, etc.) and triphenylphosphine (TPP).

[0077] Preferably, the bioactive moiety is selected from the group consisting of antibiotics, anticancer agents, antioxidants, natural products and triphenylphosphine (TPP). More preferably the bioactive moiety is selected from the group consisting of triphenylphosphine (TPP), Penicillin, Erythromycin, Streptomycin, Doxorubicin, Paclitaxel and Cisplatin. The most preferred bioactive moiety is triphenylphosphine (TPP).

[0078] Triphenylphosphine (TPP) can be coupled to a Py moiety through an ester bond.

[0079] So, preferably, the bioactive moiety used in the current invention is a triphenylphosphine moiety which is a triphenylphosphine compound comprising one or more substituents able to chemical react with the Py moiety allowing the formation of a direct chemical bond between X and Py providing Py-X. Examples are triphenylphosphine, Triphenylphosphine oxide, Triphenylphosphine dibromide, (3- Bromopropyl)triphenylphosphonium bromide, (3-Bromopropyl)triphenylphosphonium bromide, (4-Carboxybutyl)triphenylphosphonium bromide, (3-Carboxypropyl)triphenylphosphonium bromide, including other possible derivatives.

[0080] The inventor of the current invention found that the compound Py-X as described above can be used for the functionalization of a pegylated surface. The compound Py-X is able to form a van der Waals interaction with the pegylated surface allowing for a non- covalent binding with the pegylated surface after the pegylated surface formation. The inventor believes that the insertion of Py-X in the pegylated surface (loading) requires the breakage of hydrogen bonds present between the polyethylene glycol molecules and the removal of water molecules bonded to the polyethylene glycol molecules from the insertion site. The dehydration of the pegylated surface is typically very difficult due to the energy loss in breaking the hydrogen bonds and the chain conformational entropy loss. Surprisingly, the compound Py-X of the current invention makes the dehydration of the pegylated surface very easy. A possible explanation may be that the binding energy of Py-X is sufficient in breaking the hydrogen bonds which compensate for the chain conformational entropy loss, turning the dehydration of the pegylated surface into an energy-driven process.

[0081] Therefore, the inventor believe that the insertion of the compound Py-X is thermodynamically favored and therefore it is easily performed. Hence, vesicles including nanovesicles can advantageously first be formed and after their formation they can be functionalized as desired.

[0082] Several factors should be considered when performing a functionalization of a pegylated surface. The activity of the functional molecules loaded on the surface which is related to the conformation of the loaded molecules, the quantity control of functional molecules loaded on the surface, the time needed to functionalize the surface, the affinity of the molecules with different types of pegylated surfaces, the economic efficiency of the functionalization method, the environmental impact of the functionalization method and a suitable kit for performing the functionalization of a pegylated surface.

[0083] The peculiarity of the compound Py-X of the invention is in the fact that it does not comprise any spacer linker between Py and X. The absence of a linker surprisingly provides for a more rigid structure which is believed to avoid the problem of folding of the X moiety on the pegylated surface maintaining the full exposure of the X moiety on the pegylated surface (Figure 1). The conformation of the loaded Py-X compounds of the invention surprisingly makes the surface accessibility of the X moiety easier than a loaded Py-linker-X molecules. The activity of the loaded Py-X compounds of the invention is therefore optimal providing fully active Py-X compounds and solving therefore the spatiotemporal selective surface functionalization problems. The activity of Py-X compounds is of extreme importance in biological applications. The interaction between the X moiety and a receptor requires the Py- X to fully expose the X moiety in a fixed conformation avoiding the freedom of spinning, rotating, moving, or flipping as observed for the X moiety of Py-linker-X compounds.

[0084] Moreover, the loading quantity of the compound Py-X of the invention can be easily controlled. Dependently from the type of X moiety a higher or lower loading may be desired. For example in case X is an anticancer moiety the desired quantity loaded must be a balance between the toxicity of the anticancer moiety, the price and the activity of the anticancer moiety. For a certain type of anticancer moiety (e.g. very expensive but with strong activity) a lower loading may be desired compared to a different anticancer moiety (e.g. not so expensive and having low activity). For example, for small organic molecules, polysaccharide, peptides and protein, it is desired a loading efficiency of around 40-80%. Hence, the control of loading quantity of the compound Py-X is essential in the functionalization of a pegylated surface. For an optimal insertion into the pegylated surface, small organic molecules are preferred.

[0085] As the molecular weight of the polysaccharide, for example of hyaluronic acid (HA) increases, the inventor observed that the hydrophilic portion of the molecule also expands. This leads to a greater tendency for Py-X to form intramolecular aggregates, which hinders the exposure of the Py and its subsequent insertion into the pegylated surface. As a result, both the insertion efficiency and the stability of the system is not optimal.

[0086] The time needed to functionalize a pegylated surface is an important factor when performing the method for the functionalization of a pegylated surface. Ideally the time is short, within few minutes (e.g. around 2 minutes) which advantageously allows the functionalization of the pegylated surface while avoiding interference or a reaction between the therapeutic moiety or the diagnostic moiety with other molecules which may be used during the functionalization method. Also, short inserting time reduces the preparation time of pegylated surface and simplifies the operation, thus decreasing the production cost. Last, the rapid surface functionalization makes on-the-spot mix-and-match in any application scenario possible.

[0087] Surprisingly the compound Py-X of the invention is loaded onto a pegylated surface within nanoseconds or few minutes. Preferably the loading method is allowed within 10 minutes, more preferably within 5 minutes, most preferably within 2 minutes ensuring that the system reached the equilibrium state. Surprisingly the loading does not need the assistance of any catalysts or binding groups.

[0088] Table 1 below reassumes the data in relation to the loading efficiency and loading time of the compounds Py-X of the invention on polymersome vesicles.

[0089] The water solution concentration of the compound Py-X is 20 pM

[0090] Regarding the affinity of the compound of the invention to different types of pegylated surfaces the inventor found that the functionalization with the compound of the invention is efficiently achieved on different types of pegylated surfaces as polymersome vesicles surface, microparticles made of polyethylene glycol and gold nanoparticles with polyethylene glycol surface.

[0091] Regarding the economic efficiency of the preparation method of the compound of the invention, the inventor found that the compound of the invention Py-X can be more easily prepared and purified compared to the compounds Py-linker-X. Moreover, the inventor surprisingly found that the compound of the invention Py-X can be used for the functionalization of a pegylated surface without the need of coupling agents, organic solvents, or other complex reacting devices. Further, the preparation method of the compound of the invention and the functionalization method can be performed in a very tiny scale minimizing unnecessary waste of compounds and chemicals.

[0092] Regarding the environmental impact of the functionalization method of the invention, the inventor found that the invented method avoids the use of toxic and harmful chemicals. In fact, the method of the invention does not require organic solvent and other coupling agents which are usually toxic and harmful and may be performed in aqueous environment such water, phosphate buffer solution (PBS) or cell culture medium.

[0093] Regarding a suitable kit for performing the functionalization of a pegylated surface, the inventor of the current invention found that a very simple kit could be used for the preparation of the compound Py-X of the invention.

[0094] In a specific embodiment of the invention the compound of the formula (i);

[0095] Py-X is selected from compounds having the formula:

[0096] In a further specific embodiment of the invention the compound of the formula (i);

[0097] Py-X (i) ted from compounds having the formula:

[0098] In a further aspect, the invention is directed to a method for the functionalization of a pegylated surface comprising the steps of: a. Providing a surface comprising a layer of poly(ethylene glycol) b. Adding the compound Py-X to the surface comprising a layer of poly(ethylene glycol), wherein the addition of step b) is performed in aqueous solution.

[0099] The addition may be performed in any aqueous solution / solvent / medium. Preferably the addition is performed in water, phosphate buffer solution (PBS) or in cell culture medium. More preferably the addition is performed in phosphate buffer solution (PBS) when polar and charged X moieties are used in the compound Py-X. More preferably the addition is performed in a cell culture medium when X is a protein moiety in the compound Py-X. The most preferred aqueous solution / solvent / medium is water.

[0100] Presence of small amount of organic solvent (e.g. DMSO) is allowed. Preferably the organic solvent is used in a percentage less than 10 wt%, more preferably less than 5 wt%, even more preferably less than 3 wt%.

[0101] The temperature at which the method of the invention is performed is preferably at 15- 35 °C, more preferably at 18-30 °C, mostly preferably at 20-25 °C.

[0102] Preferably the addition of Py-X of step b. is performed using Py-X in an amount which is in excess compared to the amount of the surface comprising a layer of poly(ethylene glycol).

[0103] Any layer of poly(ethylene glycol) may be used to be functionalized with the method of the current invention. Examples of a layer of poly(ethylene glycol) are poly(ethylene glycol) layer of a vesicle, polymeric nanoparticles with pegylated surface (e.g. liposomes, micelles and dendrimers), biomaterial implants (e.g. orthopedic implants, stents and dental materials), biosensors (e.g. electrodes, microarrays, and optical sensors), biomedical coatings (e.g. catheters, prosthetics and contact lenses), microfluidic devices and tissue engineering scaffolds.

[0104] Preferably the layer of poly(ethylene glycol) is selected from poly(ethylene glycol) layer of vesicle, polymeric nanoparticles with pegylated surface (e.g. liposomes, micelles and dendrimers), biomaterial implants (e.g. orthopedic implants, stents and dental materials), biosensors (e.g. electrodes, microarrays and optical sensors), biomedical coatings (catheters, prosthetics, and contact lenses).

[0105] The term “vesicle” according to the present invention refers to a natural or non-natural supramolecular assembly of lipid molecules wherein the outer membrane is a poly(ethylene glycol) surface which allows the formation of the self-contained structure as hollow spheres that may enclose for example a fluid, a gas, a drug. Examples of vesicle are giant polymeric vesicles and nano polymeric vesicles.

[0106] The term “giant polymeric vesicle” according to the present invention refers to polymeric vesicle having radii ranging from 10 micrometers to 40 micrometers or more. Examples of a giant polymeric vesicle are vesicles made of PEG-PS-PAA, PEG-PEO-PPO- PEO, PEG-PLA, PEG-POL, PEG-PB-PEO.

[0107] The term “nano polymeric vesicle” according to the present invention refers to polymeric vesicle having radii ranging from 10 nm to 1000 nm. Examples of a nano polymeric vesicle are polymeric nanoparticles, polymeric micelles, polymeric nanogel particles, polymeric lipid hybrid nanoparticles, polymeric nanoparticles, and other polymeric nanocarriers.

[0108] Preferably, the vesicle of the invention is a liposome vesicles or a polymersome vesicle. More preferably the vesicle of the invention is a polymersome vesicle.

[0109] Liposomes are vesicles formed from naturally occurring lipids. Based on vesicle structure, there are seven main categories for liposomes: multilamellar large (MLV), oligolamellar (OLV), small unilamellar (SUV), medium-sized unilamellar (MUV), large unilamellar (LUV), giant unilamellar (GUV) and multivesicular vesicles (MW) which contains one or more smaller vesicles. The major types of liposomes are the multilamellar vesicle (MLV, with several lamellar phase lipid bilayers), the small unilamellar liposome vesicle (SUV, with one lipid bilayer), the large unilamellar vesicle (LUV), and the cochleate vesicle.

[0110] Polymersomes are made using amphiphilic synthetic block copolymers to form the vesicle membrane, and have radii ranging from 50 nm to 5 pm or more. The membrane of polymersomes consists of a hydrophobic polystyrene core and two hydrophilic poly(ethylene glycol) coronas (PEG-coronas). Typically, polymersomes contain an aqueous solution in their core and are useful for encapsulating and protecting sensitive molecules, such as drugs, enzymes, other proteins and peptides, and DNA and RNA fragments. The polymersome membrane provides a physical barrier that isolates the encapsulated material from external materials, such as those found in biological systems.

[0111] Different types of polymersomes can be prepared. For example using polyethylene glycol (PEG), Poly(2-oxazoline)-b-Poly(£-caprolactone) (POx-b-PCL), Poly(N- vinylpyrrolidone)-b-Poly(E-caprolactone) (PNVP-b-PCL), Poly(sarcosine)-b-Poly(L-lactic acid) (PSar-b-PLLA), Poly(zwitterionic)-b-Poly(caprolactone) (e.g., PCB-b-PCL), Poly(2-methyl-2- oxazoline)-b-Poly(D,L-lactide) (PMOx-b-PLA), Poly(N-isopropylacrylamide)-b-Poly(lactide) (PNIPAM-b-PLA), Poly(2-methacryloyloxyethyl phosphorylcholine)-b-PLA (PMPC-b-PLA), Poly(hydroxypropyl methacrylamide)-b-PCL (PHPMA-b-PCL), and Poly(glycidol)-based amphiphiles. Preferably, the polymersomes of the current invention are prepared using polyethylene glycol (PEG) as hydrophilic block and therefore they have a pegylated surface.

[0112] Advantageously, liposomes and polymersomes are biocompatible, have low toxicity, are stable against chemical degradation and show site-specific targeting. These characteristics allow to use them as drug carriers, for the treatment and prevention of diseases or for vaccine delivery.

[0113] Polymersome are in general preferred to liposomes because beside having many of the properties of natural liposomes, polymersomes exhibit increased stability and reduced permeability. Furthermore, the use of synthetic polymers enables to manipulate the characteristics of the membrane and thus to control permeability, release rates, stability and other properties of the polymersome. The most preferred Polymersome are the PEG-PS polymersome. The PEG-PS polymersome advantageously have well-defined structure and high stability due to their glassy polystyrene core in aqueous condition.

[0114] The non-covalent binding method of the invention comprises adding the compound Py-X as described above to the surface comprising a layer of poly(ethylene glycol) as described above. In the addition the Py-X molecule is allowed to get in contact with the surface comprising a layer of poly(ethylene glycol). The addition may be performed by any method known to the skilled person in the art. For example the addition may be performed incubating the Py-X molecule with a pre-formed layer of poly(ethylene glycol) (e.g. a polymersome). The addition / incubation is performed by solubilizing the Py-X in a solvent (e.g. water) and mixing the solubilized Py-X and the polymersome for several minutes, preferably from 1 minute to 10 minutes. Preferably the addition / incubation is from 1 minute to 5 minutes, more preferably from 1 minute to 3 minutes. The mixing is continued during the indicated time. Surprisingly, the inventor found that a layer of poly(ethylene glycol) which is thought to be "inert" and "stealthy" can be functionalized by the Py-X compound which embed into the inert poly(ethylene glycol) layer so easily.

[0115] Surprisingly with the non-covalent binding method of the invention the addition / incubation is not only very fast but also provides an homogeneous distribution of the Py-X compound loaded without affecting the morphology of the surface comprising a layer of poly(ethylene glycol). For example, in case a vesicle is used, the addition / incubation is performed with a pre-formed vesicle and surprisingly the morphology of the vesicle remained unaffected. Meanwhile, the maximum amount of X moiety is exposed on the surface, allowing for the efficient and spatial-controlled surface functionalization of the vesicle.

[0116] The method of the invention as described above allows the insertion of the compound Py-X as described above in the poly(ethylene glycol) surface (loading). When X is an enzyme or coenzyme, the loading may be between 20%-50% as ratio of remained amount of Py-X compared to the initial amount of Py-X, when X is a vitamin the loading may be between 30%-70% as ratio of remained amount of Py-X compared to the initial amount of Py-X, when X is a polysaccharide the loading may be between 30%-70% as ratio of remained amount of Py-X compared to the initial amount of Py-X, when X is a peptide the loading may be 25-70% as ratio of remained amount of Py-X compared to the initial amount of Py-X, when X is a protein the loading may be between 20%-50% as ratio of remained amount of Py-X compared to the initial amount of Py-X when measured by a UV-vis spectrometer wherein the water solution concentration of the compound Py-X is 20 pM.

[0117] The method of the invention as described above allows the insertion in the poly(ethylene glycol) surface (loading) of at least 40% of the compound Py-X as described above when measured as ratio of remained amount of Py-X compared to the initial amount of Py-X, by a UV-vis spectrometer (JASCO V-630 UV-Vis spectrophotometer) at wavelength of Aex of 384 wherein the water solution concentration of the compound Py-X is 20 pM.

[0118] The loading behavior may be influenced at a concentration higher than the critical micelle concentration. When the concentration of the Py-X compounds of the invention in aqueous solution is higher than the critical micelle concentration, the inventor believes that the Py-X compounds of the invention will self-assemble to generate micelle, instead exist in single molecule form. Hence, the loading of the Py-X compounds of the invention in aqueous solution may be negatively affected.

[0119] The insertion in the poly(ethylene glycol) surface (loading) is evaluated as follows: an aqueous solutions of Py-X is mixed with 1 mL of PEG-PS polymersomes suspension. The final concentration of Py-X is 20 pM. The mixture is subjected to centrifugation (14000 rpm, 10 min) and the concentration of Py-X in supernatant is detected by UV-vis spectrometer (JASCO V-630 UV-Vis spectrophotometer, thus to obtain the remining concentration of Py-X loaded. Afterwards, repeated centrifugations are performed and the concentration of Py-Xs in the supernatant after each centrifugation is detected by a UV-vis spectrometer (JASCO V-630 UV-Vis spectrophotometer).

[0120] Preferably the method of the invention as described above allows the insertion in the poly(ethylene glycol) surface (loading) of at least 40% of Py-X added in step b, specifically of each of the Py-X compounds Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP. More preferably of at least 50% of Py-X compounds as Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP, most preferably of at least 60%, wherein the amount of inserted compound above the poly(ethylene glycol) surface is measured in the aqueous solution of step b. compared to the initial amount of the added compound, wherein the measure is performed by a UV-vis spectrometer ((JASCO V-630 UV-Vis spectrophotometer) at wavelength of Aex of 384 nm and wherein the aqueous solution concentration of the Py-X is 20 pM.

[0121] In a further aspect, the invention is directed to an object comprising a pegylated surface and the compound of the invention bound to the surface wherein the amount of the compound of the invention is between 0.001- 0.1 molecules / nm2. Preferably the amount of the compound of the invention is between 0.002- 0.8 molecules / nm2, more preferably is between 0.002- 0.5 molecules / nm2. Such a quantity allows to have a sufficiently functionalized surface of the object.

[0122] The term “object” according to the present invention refers to any object comprising a surface comprising a layer of poly(ethylene glycol). Examples of objects according to the current invention are vesicles, giant polymeric vesicle and nano polymeric vesicles as defined above; particles as metal particles (e.g. gold, titanium), lipid based particles, nanospheres, mesoporous nanoparticles, micelles, quantum dot nanocrystals; assay surface, biomaterial implants (orthopedic implants, stents and dental materials), biosensors (electrodes, microarrays, and optical sensors), biomedical coatings (catheters, prosthetics, and contact lenses), microfluidic devices and tissue engineering scaffolds.

[0123] Preferably the object of the invention is selected from the group consisting of a vesicle, a giant polymeric vesicle, a nano polymeric vesicle, a particle, a nanoparticle, a nanosphere, a micelle, a quantum dot nanocrystal, an assay surface, a biomaterial implant, a biosensor, a biomedical coatings, a microfluidic device and tissue engineering scaffold. More preferably the object of the invention is selected from the group consisting of a vesicle, a giant polymeric vesicle, a nano polymeric vesicle, a particle and an assay surface. Even more preferably the object of the invention is selected from the group consisting of a vesicle, a giant polymeric vesicle and a nano polymeric vesicle, more preferably the object is a nano polymeric vesicle. The most preferred object of the invention is a polymersome vesicle.

[0124] The inventor found that when a giant polymeric vesicles having a particle size around 15-20 urn or a nanovesicles having a particle size around 450 nm were functionalized with the method of the invention, a specific amount molecules / nm2of the compound of the invention bound to the surface.

[0125] Table 2 below reassumes the data in relation to the binding molecules / nm2for each of the Py- X compounds of the invention (Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP).

[0126] Table 2

[0127] The binding in the poly(ethylene glycol) surface (loading) is evaluated as follows: an aqueous solutions of Py-X is mixed with 1 mL of PEG-PS polymersomes suspension. The final concentration of Py-X is 20 pM. The mixture is subjected to centrifugation (14000 rpm, 10 min) and the concentration of Py-X in supernatant is detected by UV-vis spectrometer (JASCO V-630 UV-Vis spectrophotometer), thus to obtain the remining concentration of Py-X loaded. Afterwards, repeated centrifugations are performed and the concentration of Py-Xs in the supernatant after each centrifugation is detected by a UV-vis spectrometer (JASCO V-630 UV-Vis spectrophotometer).

[0128] The Area that one molecule takes (nm2) has been calculated by the following formular: wherein

[0129] D is the average diameter of polymersomes;

[0130] Cpis the concentration of polymersomes in the PEG-PS polymersomes suspension, as well as resuspended suspensions. This concentration has been measured by Nanosight LM10

[0131] Cm is the concentration of molecules in supernatants obtained above. NA = 6.02 xio23and k is the dilution ratio of polymersomes suspension during measurement.

[0132] The numbers of Py-X per one nm2can be calculated by the following formular:

[0133] 1 Number of Py — X per nm = - - - - - - - - —

[0134] Area that one molecule takes

[0135] Hence, the numbers of Py-X per one nm2has been measured according to the test as described above, as ratio of remained amount of Py-X compared to the initial amount of Py-X, wherein the measure is performed by a UV-vis spectrometer and wherein the aqueous solution concentration of the compound Py-X is 20 pM.

[0136] The object of the invention maybe an assay.

[0137] The surface of an assay is often taken for granted; however, its function is as crucial as the other components of the assay. The surface of an assay is in fact an integral component of the assay because may affect the diagnostic moiety as they attach or do not attach to the surface.

[0138] Surprisingly, the method of the invention allows the functionalization of a surface of an assay in a controlled manner without influencing the structure or the physical properties of the diagnostic moiety. So, with the method of the invention the surface of the assay will have diagnostic moieties homogeneously distributed within the surface and with a favorable efficient spatiotemporal exposure of the diagnostic moieties. Moreover, the method of the invention allows the functionalization of a layer of poly(ethylene glycol) (e.g. the surface of an assay) just before its use solving the problems related to storage and stability of a functionalized layer of poly(ethylene glycol) (e.g. functionalized surface of an assay).

[0139] The object of the invention maybe a particle.

[0140] The use of particles and nanoparticles in biosensing and in medicine and more specifically in drug delivery is set to spread rapidly. Currently many particles and nanoparticles are under investigation for example for drug delivery and more specifically for cancer therapy.

[0141] Surprisingly, the method of the invention allows the functionalization of a surface of a particle or of a nanoparticle in a controlled manner without influencing the structure or the physical properties of the particle or of the nanoparticle. In a further aspect, the invention is directed to an object as described above for use in the therapeutic field or diagnostic field.

[0142] For example, vesicles and particles may be used in the therapeutic field as drug delivery carriers encapsulating and protecting sensitive molecules, such as drugs, enzymes, other proteins and peptides, and DNA and RNA fragments while an assay may instead be used in diagnostic field.

[0143] Surprisingly, the inventor found that when a vesicle is functionalized with the method of the invention, the vesicle is characterized by a good stability in vivo.

[0144] As known to the person skilled in the art, the environment in the human body differs substantially from that of a matrix (formulation) of a drug product and may impact the stability of the therapeutic compound. In vivo the degradation of the therapeutic compound may alter efficacy and / or safety characteristics such as immunogenicity.

[0145] Therefore, a vesicle with improved in vivo stability faces less risks related to the efficacy and / or safety of the vesicle when used as therapeutic compound.

[0146] The practical applicability and effectiveness of the non-covalent binding method of the invention has been confirmed by performing a cell migration study, a mitochondria targeting study and an endocytosis study.

[0147] Cell migration

[0148] CD44 is a cell surface glycoprotein that is highly expressed in many cancer cells and plays important roles in various cellular processes, including cell adhesion, migration, proliferation, and signaling.

[0149] Hyaluronic acid (HA) is the natural ligand for CD44 receptor. CD44-HA interactions promote cell adhesion to the extracellular matrix and facilitate cell migration. Therefore, we performed cell scratch assay to investigate the migration capacity of SKOV-3 cells incubated with Py-HA functionalized polymersomes.

[0150] Surprisingly, the successfully engineered polymersome-Py-HA was able to promote cell migration as shown in Figure 5. The 12-hour-migration rate of polymersome-Py-HA was significantly higher than that of the negative control group (PBS) and cell incubated with only polymersomes. The migration rate of polymersome-Py-HA is similar to cell incubated with free HA.

[0151] Hence, the compound Py-HA of the invention can be used to functionalize any vesicle comprising a broad range of drugs, enzymes, proteins, peptides, DNA, RNA, pharmacophores, and imaging agent to target to CD44 receptor.

[0152] Mitochondria targeting

[0153] Mitochondria is one of the most crucial organelles in most eukaryotic cells and play essential roles in cellular energy metabolism and regulation of programmed cell death. Dysfunctions or malfunctions of mitochondria have been associated with various diseases, including neurological and cardiovascular diseases and cancer, characterized by enhanced anabolism, unlimited proliferation potential, and impaired autophagy. Hence, mitochondria is used as target for the treatment of many different types of disease.

[0154] Because of the negative membrane potential of the mitochondrial inner membrane, positively charged compounds can accumulate in the mitochondrial matrix. Various positively charged compounds as lipophilic cations, including triphenylphosphonium-compounds cations, rhodamine, cyanine cations, and cationic peptides, have been attached to the bioactive compound of interest and have been used to improve its mitochondrial uptake.

[0155] Hence, the compound Py-TPP of the invention has been inserted onto the surface of a polymersome and incubated with Hela cells to verify whether the invented insertion method allows the polymersome-Py-TPP to target mitochondria.

[0156] Surprisingly, the successfully engineered polymersome-Py-TPP was able to target mitochondria as shown in Figure 6.

[0157] Hence, the method of the invention can be used to functionalize with the compound Py-TPP any vesicle comprising a broad range of drugs, enzymes, proteins, peptides, DNA, RNA, pharmacophores, and imaging agent to target mitochondria.

[0158] Endocytosis is a cellular process in which substances are brought into the cell.

[0159] Folate receptor (FR) is a membrane receptor overexpressed in numerous kinds of tumor cells. Targeting of FR has demonstrated effectiveness in facilitating the uptake of drugs and nanovesicles. FR have strong affinity for folate so, folic acid interacts with FR receptors binding the FR and being first surrounded by an area of cell membrane, which then buds off inside the cell to form a vesicle containing the ingested compound internalizing it.

[0160] Hence, the compound Py-FA of the invention has been inserted onto the surface of a polymersome and the polymersome-Py-FA was incubated with normal Hela cells (Hela-) in an FA-deficient medium to induce upregulation of FR expression, resulting in Hela cells with upregulated (HelallFR). The successfully engineered polymersome-Py-FA showed to facilitate the endocytosis pathway as a molecule that undergoes internalization via the membrane, thereby guiding the polymersome into the cellular milieu as shown in Figure 7.

[0161] In a further aspect, the invention is directed to a container comprising the compounds of the invention as described above.

[0162] The term "container" as used herein refers to an object for holding or transporting a substance. Any type of containers suitable for holding or transporting substances may be used. Preferably, the container is suitable for holding or transporting a medical substance. The compound of the invention Py-X as described above may be transported in the container in his solid form or in aqueous solution. Preferably, the compound of the invention as described above may be transported in the container in his solid form.

[0163] The aqueous solution may be water, phosphate buffer solution (PBS) or cell culture medium. Preferably the aqueous solution is a phosphate buffer solution (PBS) when polar and charged X moieties are used in the compound Py-X. More preferably the aqueous solution is a cell culture medium when X is a protein moiety in the compound Py-X. The most preferred aqueous solution / solvent / medium is water.

[0164] In a final aspect, the invention is directed to a kit of parts for the functionalization of a pegylated surface, the kit comprising at least a first container as described above and a second container comprising an aqueous solution. The aqueous solution may be water, phosphate buffer solution (PBS) or cell culture medium.

[0165] Preferably, the content of the first container of the kit of parts of the invention is a solid.

[0166] When the content of the first container of the kit of parts of the invention is a solid, it may be advantageously dissolved in the aqueous solution of the second container to obtain a desired concentration.

[0167] Advantageously, the kit of the current invention allows the functionalization of a layer of poly(ethylene glycol) just before its use solving the problems related to storage and stability of a functionalized layer of poly(ethylene glycol).

[0168] Examples

[0169] Example 1 : Synthesis of Py-TPP

[0170] 1 -Pyrenecarboxylic acid (74 mg, 0.3 mmol) and K2CO3 (55 mg, 0.4 mmol) were dissolved in 3 mL of THF and stirred at room temperature for 30 minutes under N2. 3- Bromopropyl triphenylphosphonium bromide (97 mg, 0.2 mmol) was added to the reaction mixture and stirred overnight under N2 at room temperature. The crude product was purified by column chromatography on silica gel (heptane: ethyl acetate: ethanol = 20:56:24) providing the purified Py-TPP compound. The soild Py-TPP was obtained by vacuum drying. The TPP used in Py-TPP has molecular mass of 466.19 Da.

[0171] Example 2: Synthesis of Py-FA

[0172] Folic acid (88 mg, 0.2 mmol) was dissolved in 2 mL of anhydrous dimethylformamide (DMF), followed by the addition of N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC HCI, 37.2 mg, 0.24 mmol) and N-Hydroxysuccinimide (NHS, 46 mg, 0.4 mmol). The reaction mixture was stirred for 30 minutes at 40°C under N2. 1 -Pyrenemethylamine (46 mg, 0.2 mmol, 1 eq) was added, and the mixture was heated at 50°C overnight. The reaction mixture was precipitated with 30 mL of acetonitrile and the resulting yellow precipitate was washed three times with diethyl ether and Milli-Q water respectively providing the compound Py-FA as yellow solid. The soild Py-FA was obtained by vacuum drying plus. The FA used in Py-FA has molecular mass of 441 Da.

[0173] Example 3: Synthesis of Py-HA

[0174] Hyaluronic acid (HA, MW=5000, 150 mg, 0.03 mmol of HA), 1 -Pyrenemethylamine (313 mg, 1.17 mmol), EDC HCI (225 mg, 1.17 mmol), and NHS (254 mg, 1.17 mmol) was dissolved in a 20 mL mixture of DMSO and water (1:1, v / v). The pH of the reaction was adjusted to 7 by the addition of 0.1 M NaOH and the reaction proceeded overnight at 30°C. The crude product was purified by washing with methanol and water three times respectively providing the compound Py-HA. The soild Py-HA was obtained by lyophilization. The HA used in Py-HA has molecular mass of around 5000 Da.

[0175] Example 4: Synthesis of Py-RGD

[0176] CRGD (45 mg, 0.1 mmol) and N-(1-Pyrenyl)maleimide (14.85 mg, 0.05 mmol) were dissolved in 2 mL degassed milli-Q water and 2 mL degassed anhydrous DMSO, respectively. The CRGD solution was added dropwise to the N-(1-Pyrenyl)maleimide solution using a syringe pump (4 mL / h) under vigorous stirring at room temperature. The resulting solution was stirred for an additional 2 hours before purification using preparative HPLC providing the compound Py-RGD. The soild Py-RGD was obtained by vacuum drying and lyophilization. The RGD used in Py-RGD has molecular mass of 449.49 Da.

[0177] Example 5: Synthesis of Py-GFP

[0178] 1 -Pyrenecarboxylic acid (457 mg, 1.85 mmol) was dissolved in 27 mL anhydrous dimethylformamide (DMF). EDC (354 mg, 1.85 mmol) and NHS (426 mg, 3.7 mmol) were added and the mixture was stirred for 1 h at 40°C using a magnetic stirrer. Triethylamine (TEA, 317 pL, 2.3 mmol) was added to adjust the pH to 8 and ensure a basic environment. Nalpha,Nalpha-Bis(carboxymethyl)-L-lysine hydrate (NTA, 520 mg, 1.85 mmol) was added to the reaction mixture, followed by stirring for 72 h at 60°C. For purification, the reaction mixture was dissolved in 50 mL water and titrated with hydrochloric acid (HCI, 1 mol / L). Finally, 2,2'- ((1-carboxy-5-(pyrene-1-carboxamido)pentyl)azanediyl)diacetic acid (Py-NTA) was obtained after centrifugation and lyophilization.

[0179] Py-NTA (9.8 mg, 0.02 mmol) and NiCh (2.6 mg, 0.02 mmol) were dissolved in 5 mL water, respectively. A syringe pump was used to add NiCh solution to Py-NTA solution (10 mL / h), followed by stirring for 2 h at room temperature. Py-NTA-Ni2+was obtained by repeated washing by centrifuge (14000 rpm, 10 min). The supernatants were destructed by 1% nitric acid overnight and the concentration of Ni2+was detected by ICP-MS to allow the quantification of Py-NTA chelated with nickel ions.

[0180] 5 pL GFP solution (1500 pg / mL in PBS) was added to 1 mL of Py-NTA-Ni2+solution (2 mM in Milli-Q water) under vigorously stirring at room temperature and the mixture was stirred overnight providing Py-GFP in solution. The soild Py-GFP was obtained by lyophilization. The GFP used in Py-GFP has molecular mass of 27 kDa (27000 Dalton).

[0181] Example 6: Insertion of a Py-X onto PEG corona and confirmation of a Py-X loading onto PEG corona by confocal microscope

[0182] Preparation of micrometer-sized polymersomes and insertion of a Py-X

[0183] To visualize the insertion of a Py-X (Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP) onto PEG corona, the co-block polymer poly(ethylene oxide)-b-poly(1,2-butadiene) (PEG-b-PBD) has been adopted. This co-block polymer can self-assemble to form micrometer-sized polymersomes with PEG corona.

[0184] Specifically, 1.25 mg of PEG-b-PBD was dissolved in 1 mL of chloroform, and the organic solvent was removed by a direct stream of nitrogen. After desiccation in a vacuum for 4 h, the giant polymeric vesicle was formed upon the addition of 3 mL water and incubation at 60°C for 48 h. 1 mL of Micrometer-sized polymersomes suspensions was placed in a glass vial and certain amount of Py-X (Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP) aqueous solutions was added to the glass vial and the final concentration of Py-X is 20 uM. The mixture was vortexed preferably at 100-1200 rpm for 2 min. Afterwards the mixture is allowed to rest for 10 min providing micrometer-sized polymersomes with inserted Py-X. The process is performed at a temperature between 15-35 degrees

[0185] Confirmation of a Py-X loading onto the PEG corona of micrometer-sized polymersomes by confocal microscope

[0186] The giant vesicles themselves have no fluorescence while after the insertion, fluorescence of pyrene can be observed.

[0187] Images of micro-sized polymersomes were captured by confocal microscope. A wavelengths of Aex = 405 nm, Aem = 465 nm have been used for Py-TPP, Py-FA, Py-HA and Py-RGD loaded polymersomes, while for Py-GFP loaded polymersomes, excitation of different wavelengths has been used, in particular the DAPI channel (Aex = 405 nm, Aem = 465 nm) and GFP channel (Aex = 488 nm, Aem = 510 nm) have been used to obtain the fluorescence signal of pyrene and GFP, respectively.

[0188] When a Py-X (Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP) is inserted onto the PEG corona, the Py-X excited by the laser will emit fluorescent, making the micrometer-sized polymersomes visible under confocal microscopy. All micrometer-sized polymersomes loaded with Py-TPP, Py-FA, Py-HA, and Py-RGD showed clear fluorescence. Fluorescent intensity along the marked area suggested that the fluorescence was predominantly distributed on the membrane (Figure 2).

[0189] As for Py-GFP, the plot of fluorescent intensity revealed a notable coincidence between the fluorescence signals from the two channels, suggesting both pyrene and GFP were colocalized on the polymeric membrane.

[0190] Example 7. Insertion of a Py-X onto PEG corona and confirmation of a Py-X loading onto PEG corona by fluorescence resonance energy transfer (FRET)

[0191] In this example the fluorophore FITC (Fluorescein isothiocyanate) is used in the vesicle membrane. When insertion of Py-X is successful, the energy will transfer from pyrene to FITC and it is possible to see the fluorescence of FITC without exciting it.

[0192] Preparation of nanometre-sized polymersomes

[0193] Fluorescein isothiocyanate (FITC) labeled PEG-PS polymersomes (FITC / Poly.) were prepared. PEG44-b-PS167 (10 mg) was dissolved in a 1 mL mixture of distilled THF and dioxane (4:1 , v / v) in a 15 mL vial with a magnetic stirring bar. 40 pL FITC solution (dissolved in THF, 10 mg / mL) was then added to the mixture. After 30 min stirring, 0.5 mL Milli-Q water was added via a syringe pump at a rate of 1 mL / h while stirring vigorously. Approximately 10 mL of Milli-Q was added to quench the polymersomes. Repeated centrifugation (14000 rpm, 10 min) was used to remove the organic solvent. Free FITC was removed by repeated centrifugation (14000 rpm, 10 min).

[0194] Confirmation of a Py-X loading onto the PEG corona of nanometer-sized polymersomes by fluorescence resonance energy transfer (FRET)

[0195] To validate the insertion behavior of Py-X (Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP) in the PEG corona a fluorescein isothiocyanate (FITC) was chosen as the acceptor due to its lipophilic nature that allows for labeling the membrane, as well as its applicable spectrum overlap with pyrene allowing the energy transfer. 1 mL of nanometer-sized polymersomes (1 mg / mL) was placed in a 1 mL eppendorf tube. Certain amount of Py-X aqueous solution was added to the eppendorf tube and the final concentration of Py-X was 20 uM. The eppendorf was placed at room temperature for 2 min before measured by a fluorospectro photometer. As shown in Figure 3, upon excitation at 340 nm - 350 nm (which excites pyrene but not the FITC), all Py-X of the invention (Py-TPP: Aex = 488 nm, Py-FA: Aex = 375.5 nm, Py-HA: Aex = 367.5 nm, Py-RGD: Aex = 394 nm and Py-GFP: Aex = 390 nm) exhibited strong emission spectrums while FITC labeled polymersomes (FITC / Poly.) showed minimal fluorescence. However, when each of the Py-X was mixed with the polymersomes suspension, the fluorescence of FITC was largely intensified, indicating successful energy transfer from pyrene to FITC. To exclude the interaction between Py-X dispersed in the solvent and FITC- labeled polymersomes, the system was centrifuged and resuspended in water before another measurement. As depicted by the light dot line with , the fluorescence intensities of Py-X after centrifugation (Py-X / FITC / Poly.-2) remained consistent with those before centrifugation (Py- X / FITC / Poly.-1 , dark dot line), thereby confirming the insertion of each Py-X onto the polymersomes.

[0196] Confirmation of a Py-X loading onto the PEG corona of micrometer-sized polymersomes by fluorescence resonance energy transfer (FRET)

[0197] The emission spectra of Py-X (Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP) solutions, FITC / Poly. suspension, and the mixture of Py-X (Py-TPP, Py-FA, Py-HA, Py-RGD and Py- GFP) and FITC / Poly. were determined by a fluorescence spectrometer at the optimal excitation wavelength of each molecule (Py-TPP: Aex = 488 nm, Py-FA: Aex = 375.5 nm, Py- HA: Aex = 367.5 nm, Py-RGD: Aex = 394 nm and Py-GFP: Aex = 390 nm). FITC-labeled giant polymeric vehicles were prepared by adding 5 pL of FITC solution (10 mg / mL in THF) to the PEG-b-PBD solution before desiccation. Free FITC was removed by dialysis (MWCO = 12000-14000 Da) against Milli-Q water for 48 h. After incubating with each Py-X (Py-TPP, Py-FA, Py-HA, Py-RGD and Py-GFP) (final concentrations: 20 pM), fluorescent images were obtained using the DAPI channel (Aex = 405 nm, Aem = 465 nm) and GFP channel (Aex = 488 nm, Aem = 510 nm) separately. The FITC incorporated in the bilayers of micrometersized polymersomes showed to be not fully excited under laser at 405 nm in the absence of insertion of each Py-X. By contrast, the fluorescence intensities of FITC-labeled polymeric membranes were significantly increased due to the energy transfer from pyrene to FITC with the presence of insertion of each Py-X.

[0198] Example 8: Loading stability

[0199] The quantitative methodology of each Py-Xs was established by plotting the linear curve of Py-Xs and corresponding UV absorption values (see Supplementary Information). PEG-PS polymersomes were resuspended in Milli-Q water, PBS buffer, and cell culture medium (phenol red-free DMEM supplemented with 10% of FBS, 100 U / rnL penicillin, and 100 pg / mL streptomycin), followed by mixing with of Py-Xs (final concentration: 20 pM). During repeated centrifugation and resuspending, the concentrations of Py-Xs in the supernatants after each centrifugation were detected by a UV-vis spectrometer. The concentration of polymersomes in PEG-PS polymersomes suspension, as well as resuspended suspensions, was quantified by Nanosight LM10.

[0200] The loading stability of each Py-X (Py-TPP, Py-FA, Py-HA, and Py-RGD) has been investigated by centrifugation (14000 rpm, 10 minutes) and resuspension in phosphate buffer (PBS, pH 7.2), milli-Q water (H2O) and complete medium for cell culture (CM) (Figure 4). During centrifugation, varying degrees of detachment of Py-X (Py-TPP, Py-FA, Py-HA, and Py-RGD) could be observed. Interestingly, after five cycles of centrifuging and resuspending, Py-TPP showed better stability in PBS compared to milli-Q water, with around 63% of Py-Xs remaining inserted (45.03 ± 3.59% vs 63.01 ± 5.93%, milli-Q vs PBS, p = 0.01). Overall, after subjecting the polymersomes to five cycles of centrifugation and resuspending, over 40% of Py-X (Py-TPP, Py-FA, Py-HA, and Py-RGD) remained inserted. Furthermore, to simulate the real environment where polymersomes interact with cells, a complete medium for cell culture (CM, phenol red-free DMEM supplemented with 10% of FBS, 100 U / rnL penicillin, and 100 pg / mL streptomycin) was used. Overall, no significant difference was found in the remaining amount of each molecule between milli-Q water and the CM.

[0201] Zeta

[0202] Zeta potential is the surface charge of nanoparticles. The zeta potential of naked polymersomes is around -30 mV. After surface functionalization by a Py-X the Zeta potential of the nanovesicle changes correspondingly to the nature of each X. After centrifugation some loose of X can happen so the zeta potential should gradually recover towards the original state (-30 mv). The zeta potential is therefore a paramether that can be used to evaluate loading stability.

[0203] 1 mL of polymersomes suspension containing 1 mg of polymer was mixed with each Py-X and incubated for 10 min. The final concentrations of each Py-X were 20 pM. Afterward, the sediment obtained by centrifugation (14000 rpm, 10 min) was repeatedly resuspended with milli-Q water, and the Zeta-potential of each suspension was detected by DLS.

[0204] The insertion of Py-TPP, Py-FA, and Py-RGD generated notable changes in the zeta potential of polymersomes.

[0205] The zeta potential of polymersomes after Py-TPP loading increased from -33.33 ± 0.70 to 26.03 ± 1.12 and remained stable after multiple centrifugation cycles showing that Py- TPP is loaded with a good stability.

[0206] Example 9: Quantification of Py-X loaded onto PEG corona

[0207] The amount and density of each Py-X (Py-TPP, Py-FA, Py-HA, and Py-RGD) on the surface of nanovesicles has been quantified considering the number of each Py-X that remained inserted on one polymersome after each round of centrifugation and resuspension. The aqueous solutions of each Py-X has been mixed with 1 mL of PEG-PS polymersome suspension. The final concentration of each Py-X was 20 pM. During repeated centrifugation and resuspending, the concentration of each Py-X in the supernatant after each centrifugation was detected by a UV-vis spectrometer.

[0208] The number of molecules (n) inserted on each polymersome has been calculated by the following formula: wherein

[0209] Cpis the concentration of polymersomes in the PEG-PS polymersomes suspension, as well as resuspended suspensions. This concentration has been measured by Nanosight LM10

[0210] Cm is the concentration of molecules in supernatants obtained above.

[0211] NA = 6.02 xio23and k is the dilution ratio of polymersomes suspension during measurement.

[0212] After three rounds of centrifugation, approximately 1.77x104, 1.11 xio4, and 1.16X 104Py-TPP, Py-FA, and Py-RGD remain inserted on each polymersome.

[0213] Due to the relatively large space occupied by the HA molecule, the number of Py-HA was found to be 1.26X103. In the case of GFP, a protein consisting of 238 amino acids with a molecular mass of 27 kDa, a smaller number of Py-GFP inserted onto PEG corona was expected. As a result, one polymersome was functionalized by around 7.94x103GFP.

[0214] Example 10: General method for cell culture experiments

[0215] All cells were purchased from the American Type Culture Collection (ATCC). Hela Cells and NIH / 3T3 cells were cultured in DM EM. SKOV-3 cells were cultured in RPMI 1640 medium. All mediums contain 10% fetal bovine serum (FBS) and antibiotics (penicillin 100 U / rnL and streptomycin 100 pg / mL) at 37°C with 5% CO2. Trypsin / EDTA was used to digest cells.

[0216] Example 11 : Cell migration promoted by Py-HA

[0217] The migration capacity of Sloan-Kettering Ovarian Cancer cell line 3 (SKOV-3) cells incubated with Py-HA functionalized polymersomes (Py-HA / Poly.) has been investigated following this procedure: SKOV-3 cells were seed to culture-inserts (Culture-Insert 2 Well in p-Dishm, ibidi GmbH) at a density of 3x104 cells / mL. After 24 hours, the inserts were removed and cells were left in the treatment media (PBS, Poly. Py-Fa / Poly., and Free HA) for another 12 hours of incubation. Cell migration was evaluated by microscopy and Imaged was used to analyze the scratched areas. 10 pg / mL of anti-CD44 was used as a control.

[0218] The cells incubated with Py-HA functionalized polymersomes showed a significantly higher migration rate than cells treated with phosphate buffer (PBS, pH 7.2) within 12 hours (49.77 ± 2.32% vs 86.32 ± 1.13%, PBS vs Py-HA / Poly., p < 0.0001). In addition, no significant difference was found between the migration ratios of Py-HA / Poly. and free hyaluronic acid (Free HA, 50 pg / mL) (86.32 ± 1.13% vs 78.14 ± 4.48%, PBS vs Free HA, p = 0.1159) as described in Figure 5 (Semiquantitative analysis of migration rate of SKOV-3 cells by Imaged).

[0219] Example 12: Mitochondria targeting facilitated by Py-TPP

[0220] To verify whether the insertion of Py-TPP onto the surface of polymersomes still endows the vesicles with mitochondria targeting capacity, Py-TPP functionalized polymersomes was incubated with Hela cells.

[0221] Specifically, Hela cells were seeded in an 8-well plate (ibidi GmbH) with a density of 5x 104per well and incubated overnight for adherence. Py-TPP-modified polymersomes labeled with Nile red (Py-TPP / NR / Poly., 150 pg / mL) were incubated with Hela cells at 37°C for 6 h. After PBS rinsing (three times), green mitochondria dye staining (100 nM, 20 min), formaldehyde fixation (4% paraformaldehyde, 15 min), fluorescent images were obtained by confocal fluorescence microscopy. The colocalization of mitochondria and polymersomes was analyzed with Imaged software (NIH). Polymersomes labeled with Nile red (NR / Poly.) were used as control.

[0222] (Mitochondria: hex = 490 nm, Aem = 523 nm; Nile red-labeled polymersomes: Aex = 560 nm, Aem = 635 nm. Scale bars = 20 pm).

[0223] Upon 6 hours of incubation, the red fluorescence of Nile red-labeled polymersomes and the green fluorescence generated by mitochondrial fluorescent dye were distributed dispersedly. However, after Hela cells were incubated with Py-TPP functionalized polymersomes, the overlap of the two fluorescence signals could be observed, indicating the successful targeting of polymersomes to mitochondria. This phenomenon was further confirmed by intensity profiles of the two fluorescence signals in the marked area (Figure 5).

[0224] Example 13: Endocytosis mediated by Py-FA

[0225] The endocytosis mediated by Py-FA has been evaluated by normal Hela cells (Hela-) cultured in an FA-deficient medium to induce upregulation of Folate receptor (FR) expression, resulting in Hela cells with upregulated (HelaUFR).

[0226] The following procedure has been applied: Hela cells were cultured in folate aciddeficient medium (RPMI 1640 Medium, no folic acid, Thermo Fisher Scientific) for 10 passages to obtain HeLa cells with upregulated FR expression (HelaUFR). HelaUFR, normal Hela cells (Hela-), and NIH / 3T3 cells were seeded in 12-well plates with a density of 5x104 cells / mL. After incubation overnight, Py-FA-modified polymersomes labeled with Nile red (Py- Fa / NR / Poly.) and polymersomes labeled with Nile red (NR / Poly.) were added to each well with a final concentration of 300 mg / mL, followed by incubation at 37°C for 6 h, After PBS rinsing, and 4',6-diamidino-2-phenylindole (DAPI) staining (1 pg / mL) staining, fluorescent images were captured by confocal fluorescence microscopy. Imaged was used to count the number of polymersomes uptake by cells.

[0227] Hela cells were seeded in 8-well plates with a density of 5X 104cells per well. After overnight incubation, Py-RGD-modified polymersomes labeled with Nile red (Py- RGD / NR / Poly.) and polymersomes labeled with Nile red (NR / Poly.) were added to each well with a final concentration of 300 mg / mL, followed by incubation at 37°C for 6 h, After PBS rinsing and DAPI staining, fluorescent images were captured by confocal fluorescence microscopy. Imaged was used to count the number of polymersomes uptake by cells. Cells treated with 10 pg / mL of anti-integrin avp3 were used as a control.

[0228] Mouse embryonic fibroblasts NIH / 3T3 cells were employed as a negative control lacking FR expression. After incubation with Py-FA functionalized polymersomes (Py- FA / Poly.) for 6 hours, HelaUFRcells exhibited a higher uptake of polymersomes compared to normal Hela cells (Hela-). Conversely, NIH / 3T3 cells, which lack FR expression, showed minimal uptake of the polymersomes. For comparison, polymersomes without Py-FA functionalization were also incubated with the three types of cells. As a result, HelaUFR- and Hela- internalized similar amounts of polymersomes, indicating that the FA on the surface of polymersomes could mediate specific uptake by FR (see data in Figure 7). Flow cytometry results showed that cellls had a higher level of uptake of polymersomes functionalized by Py-RGD compared to polymersomes, while after blocking the integrin recptor by its antibody, the cellular uptake remained the same level as the unfunctionlized polymersomes. The results indicate that functionalization by Py-RGD can enhance the cellular take mediated by integrin receptor.

Claims

CLAIMS1. A compound of the formula (i);Py-X (i) wherein:Py is a polycyclic aromatic hydrocarbon moiety,X is a therapeutic moiety or a diagnostic moiety or a bioactive moiety, and wherein the therapeutic moiety or the diagnostic moiety or a bioactive moiety is not an osmium complex and wherein the therapeutic moiety is selected from the group consisting of an enzyme or co-enzyme, a vitamin, a peptide and a protein.

2. The compound according to claim 1, wherein the polycyclic aromatic hydrocarbon moiety is selected from the group consisting of naphthalene, anthracene, phenanthrene, pyrene and coronene moiety, preferably is naphthalene, anthracene, phenanthrene and pyrene moiety, more preferably is a pyrene moiety.

3. The compound according to claim 1 or 2, wherein the diagnostic moiety is selected from the group consisting of green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), fluorodeoxyglucose, technetium-99m, iodine-based and gadolinium-based agents and barium based agents, preferably is a green fluorescent protein moiety.

4. The compound according to claim 1 or 2, wherein the bioactive moiety is selected from the group consisting of antibiotics, anticancer agents, antioxidants, natural products and triphenylphosphine (TPP), preferably is a triphenylphosphine moiety.

5. The compound according to claim 1or 2, wherein the enzyme or co-enzyme is selected from the group consisting of coenzyme ubiquinone, ubiquinol, nicotinamide adenine dinucleotide, proteases, kinases and polymerases, preferably the enzyme or co-enzyme is ubiquinone.

6. The compound according to claim 1 or 2, wherein the vitamin is selected from the group consisting of vitamin A, vitamin B, vitamin C, vitamin D, vitamin E and vitamin K, preferably the vitamin is vitamin B9 (folic acid, FA).

7. The compound according to claim 1 or 2, wherein the peptide is selected from the group consisting of arginyl-glycyl-aspartic acid (RGD), insulin, glucagon-like peptide-1 (GLP- 1), oxytocin, somatostatin analogs, natriuretic peptides, enkephalins, opioid peptides analogs and calcitonin, preferably the peptide is arginyl-glycyl-aspartic acid (RGD).

8. The compound according to claim 1 or 2, wherein the protein is selected from the group consisting of transmembrane proteins, antibodies, insulin, hormones and neurotransmitters.

9. The compound according to claim 1 selected from compounds having the formula:

10. A method for the functionalization of a pegylated surface comprising the steps of: a. Providing a surface comprising a layer of poly(ethylene glycol), b. Adding the compound according to claims 1 to 9 to the surface comprising a layer of poly(ethylene glycol), wherein the addition of step b) is performed in aqueous solution.

11. The method according to claim 10, wherein at least 40% of the compound added in step b is inserted in the poly(ethylene glycol) surface, wherein the amount of inserted compound is measured in the aqueous solution above the poly(ethylene glycol) surface and compared to the initial amount of the added compound, wherein the measure is performed by a UV-vis spectrometer and wherein the aqueous solution concentration of the compound according to claims 1 to 11 is 20 pM.

12. An object comprising a pegylated surface and a compound according to claims 1-9 bound to the surface, wherein the amount of the compound is between 0.001- 0.1 molecules / nm2.

13. The object according to claim 12, wherein the object is selected from the group consisting of a vesicle, a giant polymeric vesicle, a nano polymeric vesicle, a particle, a nanoparticle, a nanosphere, a micelle, a quantum dot nanocrystal, an assay surface, a biomaterial implant, a biosensor, a biomedical coatings, a microfluidic device and tissue engineering scaffold.

14. The object according to claims 12-13, wherein the object is selected from the group consisting of a vesicle, a giant polymeric vesicle and a nano polymeric vesicle, more preferably the object is a nano polymeric vesicle.

15. The object according to claims 12-14, wherein the object is a polymersome vesicle.

16. The object according to claims 12-15 for use in the therapeutic field or diagnostic field.

17. A container comprising the compounds of any one of claims 1-9.

18. A kit of parts for the functionalization of a pegylated surface, the kit comprising at least a first container according to claim 17 and a second container comprising an aqueous solution.

19. The kit of parts according to claim 18, wherein the content of the first container is a solid.

Images