FIELD OF THE INVENTION

The invention relates to the use of nanoassembled complexes as carriers for intracellular delivery of molecules, equal or different from one another, selected from molecules for diagnostic use and bioactive molecules for therapeutic use, such as for example chromophores or fluorophores, radiotracers, drugs, antibodies, peptides, proteins, enzymes, single or double-chained oligonucleotides and their analogues (PNA, LNA).

PRIOR ART

The development of technologies based on nanoparticles of various types has been attracting important prospects for their application in both diagnostic and therapeutic field.

The objective pursued with the most recent research is to develop multi-functional transport nanosystems capable of transporting molecules having different functions (i.e. bioactive and/or tracer molecules) specifically to the desired anatomical site in a more efficient manner. The technical problem relates to the possibility of charging onto the nanoparticles the various compounds to be carried, and of controlling their individual charge in a reproducible and convenient manner as well.

Of particular interest in this context are nanocomplexes based on the high-affinity interaction between avidin and biotin.

Indeed, in practice, avidin's property of having an elevated and multiple affinity for biotin represents the basis for its use as a molecular instrument in a very large number of biotechnological applications. Owing to this property, avidin is in fact able to function as a molecular bridge for stably combining various biological and chemical units together with the proviso that they are covalently bound to a biotin molecule (Wilchek M and Bayer EA, 1988; Wilchek M and Bayer EA, 1990).

The most common applications of avidin-biotin technology fall within the scope of analysis, more precisely in detection and quantification systems usually based on the possibility of combining an antibody, or any other molecule having a high affinity to the analyte (ligand/antigen), with a signal system (a fluorophore, an enzyme capable of emitting light/colour, a radionuclide etc). Other applications include the functionalisation of surfaces with specific chemical/biochemical entities, a procedure that is frequently performed using the molecular bridge consisting of the avidin-biotin complex; another application consists in the directing of parenterally administered drugs or diagnostic elements to specific sites of the body (Goldenberg DM et al., 2006).

However, one of the main drawbacks of conventional avidin: biotin technology consists in the maximum number, which is equal to four, of biotins that may be combined with an individual molecule of avidin, which constitutes the central "nucleus" of the system, being avidin as known a tetrameric protein. The possibility of having a central nucleus capable of binding a greater number of biotin molecules to itself should theoretically enable the potentiality of the system to be increased.

This increased capacity can be achieved by joining together a plurality of avidin molecules into a single unit definable as a poly-avidin unit.

The substantial impulse towards the development of a poly-avidin-type technology derives from the discovery of the ability of avidin to also bind to nucleic acids with high-affinity interactions with the nucleobases thereof (Morpurgo M, et al. 2004). This has contributed to the development of nanocomplexes formed of nucleic acids/avidin/biotin self-assembling themselves around a central nucleus consisting of a nucleic acid and a plurality of avidin units in a stoichiometric ratio between avidin and the nucleic acid base pairs of 1 :18 ± 4. The avidin assembled on the nucleic acid can in turn bind biotin. In the nucleic acid : avidin interactions, in fact, are involved specific regions of the protein but not the biotin binding site, which therefore remains free to bind biotinylated compounds, that is, compounds derived from covalent conjugation with biotin according to the previously cited known avidin : biotin technology.

However, the basic problem with these nano-assembled complexes is their poor stability with formation of aggregates in aqueous saline environment, such as the physiological environment, since the nano-assembled complexes are obtained only on the basis of high-affinity interactions and not by means of stable bonds, for example, of the covalent type. In order to obviate this technical problem, which actually renders these nanoparticles unusable for applications within the therapeutic and/or diagnostics field, nanoparticles derived from the double interaction of nucleic acid: avidin and of avidin: biotin, wherein the biotin binds with a covalent bond a hydrophilic polymer capable of ensuring protection at the surface of these nanocomplexes, have been developed. These nanocomplexes are represented by the general formula NB _n Av _y (B-X _a -PAb)z (NB = nucleobase; Av = avidin; B = biotin; X = linker; PA = hydrophilic polymer) (Morpurgo M et al., 2009; Pignatto M et al., 2010).

This enabled to obtain nanoparticles consisting of these nanocomplexes, which are soluble in an aqueous environment and stable, and which have a highly defined composition, being obtainable by means of high-affinity interactions on the basis of stoichiometric ratios between nucleobase: avidin : biotin.

These nanocomplexes have been compared with analogue complexes based on the conventional avidin: biotin interaction in an in vitro analytical study based on immunoassay, and have been demonstrated to be much more efficient in detection of the analyte (Morpurgo M et al., 2012).

However, the properties of these nanoassembled complexes with reference of their internalisation within the cells is unknown.

On the other hand, the presence of hydrophilic polymers on the surface casts doubt on this possible capability, because it is known that the biological barriers, such as cell membranes, are thereby more impermeable or barely permeable to hydrophilic structures and that this is the main reason for their poor absorption and inefficient intracellular transport. The poor trans-membrane permeability due to elevated hydrophilicity, in fact, greatly limits the intracellular transport of macro- molecules used as carriers for bioactive molecules, such as polymeric conjugates and nanoparticles. In the case of nanoparticle systems, it is generally accepted that cellular internalisation can occur only following endocytic processes, which, however, need to be triggered by an initial reaction of the nanoparticle with specific surface receptors (receptor-induced internalisation). Moreover, it is generally accepted that in the absence of these initial interactions, cellular internalisation is highly inefficient. It is also known that covering the surface of the nanoparticles with hydrophilic polymers further diminishes their capacity to interact with the tissues and to be internalised into the cells (Gupta AK, Gupta M, 2005; Chuang KH et al., 20 0; Koren E et al., 2012).

This is, for example, the principle underlying drugs formulated in pegylated liposomes (for example the injectable drug doxorubicin HCI in Doxil® liposomes), in which the hydrophilic polymer PEG at the surface has the specific purpose of promoting free circulation of the nanosystem by reducing the capacity thereof to interact in a non-specific manner with the tissues (Torchilin VP, 2005).

SUMMARY

However, the inventors have now found that, notwithstanding the hydrophilic characteristic of the nanoassembled complexes previously mentioned due to the presence of hydrophilic polymers at the surface, they can be internalised in cells by a mechanism which is not yet known.

Therefore, in an aspect, it is an object of present invention nanoassembled complexes comprising a nucleus obtained by means of a high-affinity interaction between one or more units of avidin and one or more molecules of a nucleic acid and wherein said nucleus is stabilised by a biotinylated surface-protecting agent represented by the general formula (I)

ΝΒ _η Αν^Β-Χ ₃ -ΡΑ _ϋ ) _ζ

(I)

wherein:

NB are the single nucleobases of a single or double-stranded nucleic acid selected from among any sequence of a single or double-stranded deoxyribonucleic acid (DNA), any sequence of a ribonucleic acid (RNA) in form of a single strand or hybridised with an RNA or complementary DNA chain, or a sequence of the two in which part of or all the bases have been chemically modified;

Av is a tetrameric avidin unit;

B-Xg-PAfc is a biotinylated surface-protecting agent, wherein PA is a polymer unit having one or two functionalisable residues, one of which binds, by a covalent bond either directly or through an X linker, a biotin residue B by means of the carboxyl functional group thereof, selected from among polyethylene glycol (PEG), optionally substituted, and a polyethylene glycol and polyoxypropylene copolymer (PEG-PPO);

n is a number higher than 16 and up to 100,000;

y is an integer equal to or higher than 1 and being relative to n is comprised from (0.0001 )·η to (0.0357)·η with the proviso that, if (0.0001 -0.0357)·η is less than 1, y is equal to 1 ;

z is an integer equal to or higher than 1 and being related to y is comprised from (0.02)*y to (4) ^» y with the proviso that, if (0.02-4) ^» y is less than 1 , z is equal to 1 ;

- a is a number comprised from 0 to 5;

b is a number comprised from 1 to 9,

for use as carriers in the intracellular delivery of molecules, equal or different one from the other, selected from molecules for diagnostic use and bioactive molecules for therapeutic use.

The advantages obtainable with the present invention will become clearer to a person skilled in the art by means of the following detailed description of specific embodiments, which is given by way of non-limiting example and with reference to the following drawings.

A BRIEF DESCRIPTION OF THE FIGURES

Figure 1. The figure shows the overview of the biotinylated fluorescent compounds described in examples 1 and 2: (A) compound 2 of example 1

Biotinamido-hexylamido-L-lys-( PEG _5kda -fluorescein)2; (B) compound 3 of example

2 Biotinamido-hexylamido-Alexa ⁶³³ .

Figure 2. The figure shows the stability of the nanoassembly of example 3 plasmid DNA:Av:[B-C ₆ -Lys-(PEG-fluorescein) ₂ , B-C ₆ -Lys-Alexa ⁶³³ ] following incubation under different conditions simulating the physiological environment. (A) Size exclusion chromatograms (in FPLC, TM-6 superose column) recorded at 495 nm and 631 nm of the fresh-prepared assembly (1 ) and after 15 hours of incubation at 37°C in 10 mM phosphate buffer, NaCI 150 mM PBS) (2), spleen (3) and liver (4) extracts and plasma (5); (B) for each chromatogram, the area of the peak relating to the nanoassembly (7.6 mL) has been normalised respect to that calculated for the control nanoassembly incubated in PBS; (C) the areas of the peaks detected at 495 nm and 631 nm, which elute between 14 and 17 ml_, relative to degradation products of the nanoassembly, have been normalised with respect to that detected for the freshly prepared nanoassembly.

Figure 3. The figure shows the images by confocal microscopy that are representative of HeLa cells incubated with the nanocomplex of example 3 plasmid DNA :Av:[B-C ₆ -Lys-(PEG-fluorescein) ₂ , B-C ₆ -Lys-Alexa ⁶³³ ] (30 pg/ml in a medium containing 10% foetal calf serum). Similar results have been obtained at lower concentrations (6 pg/ml). The panels A - E show the superimposition of the images taken in the three channels (red for the Alexa ⁶³³ channel, green for the fluorescein signal, yellow for the two superimposed signals red and green, and blue for the nuclei coloured with the Hoechst 33258 chromophore). The panel A shows a representative image of the control cells (treated only with medium). The panels B, C and D show the cells after 2 hours (B), 6 hours (C) e 24 hours (D) of treatment with the nanocomplexes. The panel E shows a size-enlarged image (2D) of the sample treated for 24 hours, and demonstrates how the nanoparticles localise spot-wise within the cytoplasm, close to the nuclei, and also as macroaggregates partly associated to the cell membrane. In panel F, one can see a 3D reconstruction of an image obtained from the same sample at 24 h.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The terms "nanocomplexes", "nanoassemblies" and "nanoassembly complexes" are used to indicate the compounds obtained by double self-assembly between the nucleobases (NB) of an oligonucleotide sequence and the tetrameric protein avidin (Av), in what follows also referred to as an avidin unit, and between the avidin and biotin (B) of an agent covering the central nucleus obtained from the first NB:Av self-assembly. These compounds are in nanoparticulate form, for which reason they are also referred to herein as "nanoparticles".

The term avidin is intended to define both the native tetrameric protein originating from hens' eggs or from some other analogous source (birds' eggs in general), and the recombinant one, and in the glycosylated and the deglycosylated form. Also intended are other, chemically or genetically modified, forms of avidin, provided that they are capable of assembling on a single or double-stranded nucleic acid as previously defined.

The term "dendrimer" means a symmetrical macromolecular compound consisting of repeated ramifications around a central core consisting of a smaller molecule or a polymeric nucleus. The functional groups present on the outside of the dendrimer, the number of which is dependent upon the number of its ramifications, are in turn functionalisable with other molecules including, for example, the PA polymers.

The term "nucleic acid(s)" is used to mean single or double-chain nucleic acids of DNA or RNA as defined previously, and which can occur in a linear or circular form, in a relaxed, coiled or supercoiled state.

The terms "linker" and "spacer" use here are to be considered equivalent for the purposes of the present description of the invention.

Description

The nanoassembled complexes of general formula (I) NB _n AVy(B-X _a -PA,b) _z , where NB, Av, X. PA and n, y, z, a and b have the meanings mentioned previously, have a well-defined composition based on the stoichiometric ratios between NB:Av:B and present in the form of nanoparticles that are discrete in terms of their size, wherein the protection and stability of the central nucleus in aqueous saline solutions is guaranteed by the hydrophilic polymers present on the surface.

In a first embodiment, the nanoassembly complexes of the general formula (I) NBnAvyiB-Xg-PAbJz can be used as such and can transport molecules, equal or different from one another, selected from molecules for diagnostic use and bioactive molecules for therapeutic use, bound by means of a covalent bond or equivalent bonds of hydrazonic type or a shift base to the polymer PA.

However, a significant feature of these nanoparticles is linked to the fact that the binding sites of biotin present on the avidin units of the central nucleus NB.Av cannot be completely saturated by the biotinylated surface Β-Χ ₉ -ΡΑ _ϋ protecting agent for reasons deriving both from the choice of preparation and of the physical type. In the first case, the incomplete saturation of the binding sites of biotin is a direct consequence of the stoichiometric ratios between the avidins and the biotins of protecting-agent used in the preparation of the nanoassemblies.

Instead, in the second case the presence of free binding sites of biotin can be a consequence of the steric hindrance of the polymers present on the surface, which could interfere with the access thereto to other B-X _a -PA _b covering agents. In each case, this leaves free biotin binding sites on avidin for further high-affinity interactions between avidins and other biotinyiated compounds capable of penetrating the nanoparticles, notwithstanding the presence of polymers at the surface.

This enables to deliver molecules, equal or different from one another, selected from molecules for diagnostic use and bioactive molecules for therapeutic use, both by binding them by means of a covalent bonds or the equivalent bonds previously cited to the polymer PA of the B-Xa-PA^ covering agent, and by binding them simply to another biotin different from that of the covering agent and capable of binding to the free binding sites of avidins.

This feature is very important for diagnostic and therapeutic applications, in that it enables nanoparticles to be designed and prepared according to the target diagnostic and/or therapeutic requirements.

In a first embodiment can be prepared ΝΒ:Αν:Β-Χ ₃ -ΡΑ _ϋ nanoparticles, where the polymer PA is not functionalised with molecules for diagnostic and/or therapeutic use, and the binding sites of biotin present on the avidin units are not saturated by the B-Xg-PAi, covering agent and, then, the free biotin binding sites bind biotinyiated compounds of molecules equal or different from one another for diagnostic and/or therapeutic use.

In a second possible embodiment of these nanoassemblies according to these methods of preparation, for example, nanoparticles having a double label can be prepared by binding a tracer with a bond to the polymer of the protecting agent B- X _a -PAi, and a second tracer to a further biotin different from that of the protecting agent. This second biotinyiated tracer may be taken up again by processes of absorption favoured by the high affinity between avidin and biotin in the nanoassembly formed by ΝΒ:Αν:Β-Χ ₉ -ΡΑί,. The high affinity between the free avidin of the NB:Av central nucleus and the biotin of the biotinylated tracer enables its self-assembly thereon.

In another realisation of this second embodiment, the nanoparticles can, instead, transport bioactive molecules, equal or different from one another, bound to the PA polymer of the B-X _a -PA _b protecting agent and directly to a further biotin. In this case, the nanoparticles can be used as multiple and/or differentiated-release vectors of bioactive molecules bound respectively to the polymer of the protecting agent or directly to a biotin different from that of the cover agent B-X _a -PA _b . Indeed, the different localisation of the bioactive molecules bound to the polymer of the protecting agent B-X _a -PA _b or to a biotin different from that of B-X _a -PAi, respect to the nanoparticle surface can produce a different susceptibility to degradation-type process with a differentiated release thereof.

Another different further embodiment is also possible, i.e. nanoparticles which are able to act as carriers for bioactive molecules for therapeutic use that are bound to the polymer PA of the B-X _a -PAt, protecting agent and molecules for diagnostic use, such as for example a tracer, bound to a biotin different from that of the B-X _a - PA covering agent.

It is also important to note that, for therapeutic or diagnostic purposes, nanoparticles can be variously functionalised in order to direct them in a targeted manner towards specific organs and/or cells by functionalising either the polymer PA of the B-X _a -PA _b protecting agent or another biotin different from that of the B- Xa-PA _b , protecting agent or both.

From these examples of possible embodiments of these nanoparticles, the extreme versatility and simplicity of preparation can be deduced.

For the purposes of the present invention, nanoparticles having predefined compositions are to be preferred, wherein the components NB,,, X _a and PA _b and y and z have the meanings given below.

In a preferred embodiment, NB _n is a nucleic acid wherein n is the number of the base and is comprised between 3,000 and 50,000 and consequently y, which identifies the number of the avidin units Av, is comprised between 30 and 1785 and z, which identifies the number of the B-X _a -PA ₆ protecting agents, is comprised between 43 and 7140. When the linker X, which binds the biotin to the hydrophilic polymer PA, is bifunctional it is a spacer of the general formula (II) Y-R-Y', wherein:

Y, , equal or different from one another, are -COO-, -NH-, -0-; -S02-, -S-

, -SO-, -CO-, -COS-; -NH-CO-, -NH-COO-, -HN-SO-NH-, -HN-N=CH- ; - R can be an alkyl, an alkenyl, an alkynyl, a cycloalkyl and an aryl having a number of carbon atoms between 1 and 20, and preferably between 5 and

10, optionally substituted.

Therefore, the bond between the linker X and the biotin B, and that between the linker X and the hydrophilic polymer PA can be, without distinction, an amide bond, an amine bond, a carbamide bond, an ester bond, a ketone bond, an ether bond, a thioesteric bond, a thioether bond, an urea bond, a thiourea, sulphonic, sulphoxide and hydrazonic bond.

More preferably, the linker X is a spacer group wherein Y and Y' are -NH-CO- and -NH-COO- and R is a linear alkyl having 6 carbon atoms.

In the case wherein b is greater than 1 , and therefore PA represents a hydrophilic polymer consisting of at least 2 or more polymeric units, the latter are combined with one another by a different linker X having a number of derivatisable functionalities equal to or higher than 3 (> 3), one of which binds a first spacer X or directly the biotin B by means of its own carboxyl group, when a is equal to 0, and the others bind the polymeric units PA. This diverse linker X can be selected from lysine, glutamic acid, aspartic acid, cysteine and a dendrimer. For the purposes of the present invention, this further polyfunctional linker is preferably lysine.

With reference to the polymer PA, polymers in which the polymeric unit has a molecular weight between 400 and 20,000 and more preferably between 1 ,000 e 5,000 are to be preferred. The most preferable polymer unit is polyethylene glycol (PEG), optionally substituted, or a copolymer thereof having a molecular weight between 2,000 and 5,000 and with b comprised from 2 to 5.

When the polymer PEG is substituted it is characterised by the following general formula (III)

-(CR ¹ R ² CR ³ R ⁴ O) _m - (lll)

where: R ¹ , R ² , R ³ and R ⁴ can be, independently of one another, residues equal to hydrogen, alkyl, cycloalkyl, aryl, alkenyl, alkynyl, alkoxy, thio alkoxy, aryloxy, and thio aryloxy,

m is a number comprised from 2 to 500.

With the nanoassemblies derivable from the various combinations mentioned above, various compounds can be carried within the cells both by derivatisation of the polymer PA of the biotinylated surface protecting agent B-X _a -PA _b and by derivatisation of a further biotin different from that of the protecting agent B-Xa-PA*, of the nanoassemblies.

These biotinylated compounds, different from the B-Xa-PAb protecting agent of the nanocomplexes, represented by the general formula of (I) NBnAvy(B-Xa-

PAb)z, can be comprised therein in consequence of the high-affinity interactions between the biotin and avidin of the central nucleus NB:Av.

These biotinylated compounds represented by the general formula (IV)

B-X _a -A _f c (IV)

wherein:

- B is biotin;

- X is a bifunctional or polyfunctional linker as previously defined;

- A is a compound selected from molecules for diagnostic use and bioactive molecules for therapeutic use, and

where a is an integer comprised from 0 to 5 and b is an integer comprised from 1 to 9.

By way of example, according to both the first embodiment and the second embodiment, compounds for diagnostic use can be carried, such as chromophores or fluorophores, radiotracers and chelating agents therefor, and bioactive molecules such as drugs, antibodies, peptides, proteins, enzymes, sugars, single or double-chained oligonucleotides and their analogues (PNA, LNA).

For purposes of non-limiting illustration, there follow a number of examples of preparation of the nanoassembled compounds according to the invention and their characterisation in respect of stability under simulated physiological conditions and the property of intracellular molecular delivery. EXAMPLES

Example 1. Biotinmidoexylamido-L-Lys (PEG _5KDa -fluorescein) ₂ (B-C ₆ -Lys-(PEG- fluorescein) ₂ , compound 2)

The derivative biotinamidohexylamido-L-Lys (HCI) ₂ was synthesised on the basis of the description in the literature (Pignatto M et al. 2010, ref. cit.). The compound was then dissolved in anhydrous dimethylformamide (DMF) and 2 equivalents of a-N-hydroxisuccinimidyl carboxy.co-Fmoc-amino-PEGsKDa (RAPP Polymere, Germany), followed by 4 equivalents of triethylamine (TEA), were added. After 1 hour, the quantitative modification of the primary amines was confirmed by ninhydrin assay and the product was isolated by precipitating with anhydrous diethyl ether. The protective group Fmoc was removed by treating with 20% piperidine, and the end product (compound 1 ) was again isolated by precipitating with diethyl ether.

Compound 1 was then dissolved in 0.1 M borate buffer pH 8.5 and 3 equivalents of fluorescein isothiocyanate (FITC) were added. After incubating in the dark overnight, the product (compound 2) was purified of the excess of fluorophore by gel filtration on a Sephadex G25 (GE Healthcare) column and brought to dryness by lyophilisation. The amount of fluoroscein bound to biotinylated PEG in the end product was determined by UV-Vis spectrophotometric analysis. The biotin and PEG content in the end product were determined by testing with HABA (Green NM, 1965) and iodine (Morpurgo M et al., 2004), respectively.

Example 2. Biotinamido hexylamido-alexa ⁶³³ (B-C6-Alexa ⁶³³ , compound 3)

The succinimidyl ester of Alexa Fluor ^® 633 (Alexa ⁶³³ -/V/-/S) was obtained by Molecular probes ^® (Invitrogen). The derivative biotinamidohexylamine was synthesised as described in the literature (Pignatto M et al., 2010). The conjugation of Alexa ⁶³³ - V/-/S and biotinamidohexilamine was conducted in anhydrous DMSO, by mixing in two reagents in equimolar ratios, in the presence of 2 equivalents of triethylamine (TEA). The reaction was left under stirring at room temperature for 1 hour and then stored at -20°C, without further purification. The quantitative yield of the reaction was confirmed by chromatographic analysis.

Example 3. Preparation of the nanocomplexes plasmid DNA: avidin:[B-C6-Lys- (PEG-fluorescein) ₂ , B-C ₆ -Alexa ⁶³³ ] Doubly fluorescent nanoparticles (by means of fluorescein and Alexa 3) were prepared in accordance with a protocol analogous to the one reported in the literature (Pignatto M et al. 2010; Morpurgo M et al., 2012). In particular, a "basic" composition was prepared containing only the fluorophore fluorescein (Avidin:B- C6-Lys-(PEG-fluorescein)2) by mixing avidin (Belovo Chemical), compound 2 of example 1 and plasmid DNA (pEGFP-C1 , 4,7kb, Clonetech) in the ratios NB:Av:B- C6-Lys-(PEG-fluorescein) ₂ corresponding to 9.400:1.000:460. After stabilisation, the nanoassemblies were purified of the excess of avidin by ultrafiltration or size exclusion chromatography. Then, the "basic" composition was added with biotinamido-hexylamido-Alexa ⁶³³ (B-C6-Alexa ⁶³³ added, compound 3, example 2) and was purified again as described above. The number of fluorophores stably attached to the nanoparticles before and after addition of the compound 3 Β-Ce- Alexa ⁶³³ was determined by spectrophotometry on the basis of their absorption at 280nm (contribution of avidin, DNA and fluorescein), 495nm and 631 nm (contribution of fluorescein and Alexa ⁶³³ , respectively). On the basis of the general formula NB _n Avy(B-Xa-PAb)z, the "basic" nanoassembiy, prior to the addition of B- Ce-Alexa ⁶³³ is characterised by: n =9400; y = 336; z =350, with a = 2 and b = 2. The number of B-C ₆ -Alexa ⁶³³ stably bound in the final composition was equal to 281 . The average size of the nanoparticles measured by means of dynamic light scattering was 1 10nm.

Example 4. Stability of the nanoassembiy plasmid DNAiavidin [B-Ce-Lys-(PEG- fluorescein) ₂ , Β-Ce-Alexa ⁶³³ ] in plasma, and liver and spleen homogenates

The nanoassembiy of example 3 (120 pg/rnl in PBS) was incubated (15 h, 37°C) with 0.1 volumes of plasma or liver or spleen homogenates (Lazzari SJ et al., 2012), and PBS buffer as the control. The samples were then centrifuged (16600 g) for 2 min (4°C) and the supernatants were analysed by size exclusion chromatography using an FPLC Akta ^® purifier (GE) integrated with a SuperoseTM-6 column, by monitoring the eluate at 280 nm, 495 nm and 631 nm. The chromatograms of the mixtures were compared with that of the nanoassembiy prior to treatment.

The results obtained and presented in Figure 2 show that, after 15h of incubation at 37°C in environments similar to the body fluids, the peak relating to the nanoassembly (7.6 mL) is still clearly visible at both the wavelengths measured for fluorescein (495 nm) and Alexa ⁶³³ (631 nm), demonstrating the fact that the nanosystem persists in the assembled form even after treatment. A certain degree of degradation is observed from the appearance of peaks at times of greater retention (approx 14.9 mL avidin; 16.4 mL PEG derivatives), the percentage area of which relative to the total signal (in each channel) prior to the treatment is presented in panel C. The data in panel B shows that the intensity of the peak relative to the nanoassembly after treatment in the liver and spleen homogenates is similar to that recorded following treatment in phosphate buffer (PBS), indicating a good stability in these environments. The peak of the nanoassembly treated in plasma is lower than that in PBS. Nevertheless, comparing the chromatographic data with the UV absorption of the solution prior to charging, it emerges that the recorded chromatographic drop is due to the treatment of the assembly on the side of the column prefilter, a phenomenon which indicates the formation of aggregates of larger size than the original nanoassembly, probably secondary to interaction with the plasma proteins. The data are therefore in accordance in saying that the nanoassembly slowly disintegrates in the media studied. In every case, the component which seems to separate off the most rapidly from the nanoparticles following the treatment is the one bound to PEG fluorescein (panel C).

Example 5. In vitro cellular internalisation assay of the nanoassembly plasmid DNA:avidin:[B-C ₆ -Lys-(PEG-Fluorescein) ₂ , B-C ₆ -Alexa ⁶³³ ]

HeLa cells (in DMEM with addition of 10% foetal bovine serum, 2 mM l-glutamine, 100 U penicillin/0.1 mg/ml streptomycin) were seeded onto glass cover plates positioned inside of a 24-well plate (10,000 cells/well). After 24 hours of incubation at 37°C, 5% CO2, the culture medium was removed and replaced with a solution containing the nanoassembly doubly labelled with fluorescein and Alexa ⁶³³ of example 3 (30 pg/rnl in the same culture medium). At predetermined times (2, 6 and 24 h), the cells were fixed with 4% paraformaldehyde and the nuclei were stained with the fluorescing blue chromophore Hoechst 33258. The cover plates were analysed using an Olympus Fluoview BX61 microscope integrated with an FV500 confocal system, and fitted with a laser at wavelength of Aexc = 405 nm, Aexc = 488 nm and Aexc = 635 nm, to visualise the signals relating to the chromophore Hoechst 33258, the fluorescein and the Alexa ⁶³³ , respectively. Two- and three-dimensional images were obtained (Figure 3). The superimposition of the images taken in the three channels mentioned above was performed automatically by the software of the Olympus fluoview. The 3-D reconstruction was performed using the Imaris 5.0 (Bitplane) software.

As shown in Figure 3, the internalisation within the cells is shown to be efficient and with a time-dependent kinetics. Indeed, in panel A one can see control cells (treated with medium only) in which the nucleus appears coloured blue, while panels B-D shows cells following treatment with the nanocomplex at 2, 6 and 24 h: in panel B, no colours other than the blue of the nuclei were ascertained; in panel C, a slight yellow and red colouration is already appearing alongside the blue colour; and in panel D there is a very intense yellow colouration around the blue nucleus. The panel E (2D) shows an enlarged image of the sample treated for 24 hours and demonstrates the localisation of the nanoparticles at spot within the cytoplasm in proximity to the nuclei (blue colouration of the nuclei with an intense yellow colouration around it), but also macroaggregates in regions associated with the cell membrane. In panel F a 3D reconstruction can be seen of an image obtained from the same sample at 24 h with red and green macroaggregates. REFERENCES

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