ITUB20154631A1 - Vesicular systems formed by asymmetric bilayers with double structure for the transmission of genetic material - Google Patents

Description

Description of the patent application for Industrial Invention entitled: "Vesicular systems formed by asymmetric bilayers with double structure for the conveyance of genetic material"

Technical field of the invention

The present invention relates to the production of vesicular structures or systems consisting of a double lipid layer capable of forming a double structure consisting of an internal bilayer and an external bilayer. The internal bilayer is made up of positively charged phospholipids capable of forming electrostatic complexes with the genetic material present inside. The external bilayer is neutral or negatively charged and is made up of phospholipids able to fuse with biological membranes, and / or hydrophilic and / or amphipathic polymers conjugated to phospholipids. Such vesicular systems are characterized by improved stability and biopharmaceutical properties with respect to the vesicular systems known up to now. The AVs of the invention can be used in the delivery of genetic material with pharmacological activity, such as genes, double-stranded DNA and RNA, oligonucleotides, miRNA, siRNA.

Aite note

The therapeutic activity of genetic material represents an important experimental approach in anti-cancer therapy and for the treatment of numerous other pathologies. The recent discovery of RNA synthesis and transcription mediators, in particular, micro RNA (miRNA) and small interference RNA (siRNA) has revolutionized therapeutic protocols in gene therapy. The mediators of RNA synthesis, transcription and translation can in fact be used to modulate the activity of genes and control the processes related to their activation. However, the use of RNAi in therapy, in particular, miRNA and siRNA, is severely limited by their loss of efficacy after systemic administration and by the poor efficacy of transcription vectors generally used for their delivery. In fact, several formulations have been developed for the treatment of genetic diseases in which the administration of genetic material devoid of vectors for its delivery or as single compounds did not produce any therapeutic effect (Tiramt et al., 2014; Ando et al., 2013; Molinaro et al., 2013; Zhang et al., 2013).

In the pharmaceutical field, the technical problems to be overcome are therefore linked to the in vivo administration of nucleotide material such as, but not limited to, siRNA and miRNA. Said technical problems, in particular, include their rapid degradation after systemic administration, the uptake by macrophages of the reticuloendothelial system and poor cellular internalization.

For this reason, several strategies have been developed to overcome these issues. Among the potential choices, non-viral vectors have been widely accepted and used by the scientific community as nanocarriers for gene therapy as they are considered non-immunogenic with respect to viral vectors. For this reason, the use of liposomes and vesicular systems in general, in particular nanosystems formed by lipid and / or polymeric biomaterials, for the systemic delivery of miRNA and siRNA has been extensively studied. The main disadvantage of miRNA and siRNA in clinical use is their poor transfection capacity in vivo.

Liposomes are drug delivery and directing systems consisting of an aqueous core and a hydrophobic phospholipid bilayer capable of encapsulating drugs with different chemical-physical properties. Hydrophilic and liposoluble compounds can be encapsulated in liposomes individually or in combination and their ine ap sul action allows to obtain different therapeutic advantages (Gentile et al., 2013; Cosco et al., 2012; Cosco et al., 2011; Torchilin, 2005). Indeed, liposomes improve the biopharmaceutical and pharmacokinetic properties of encapsulated compounds, their chemical-physical and biological stability both in in vitro and in vivo experimental models (Biswas et al., 2012; Ratei et al., 2011; Celia et al. , 2011; Paolino et al., 2010; Blume and Cevc, 1990).

In the last decade, new liposomal formulations have been proposed for the delivery of genetic material (Endo-Takahashi et al., 2014; Hatakeyama et al., 2014; Takahashi et al., 2013; Rai et al., 2011; Kibria et al. , 2011).

Recently, lipoplexes have been developed and suggested as potential non-viral vectors for the delivery of genetic material. Complexation of the genetic material with lipoplexes improves transfection both in vitro and in vivo. However, their use is linked to the onset of numerous side effects. In fact, the positively charged groups favor the non-specific interaction with the tissue surface near the administration site, limiting its systemic distribution and consequently, also the conveyance of the encapsulated genetic material (Dan and Danino, 2014; Li et al., 2014; Pozzi et al., 2014; Sheng et al., 2014). Furthermore, lipoplexes stimulate several ree ectory pathways, in particular Tolldike Receptors (TLRs) 4, generating an inflammatory response mediated by cytokines and interferon (Hashimoto et al., 2014; Schworer et al., 2014; Yang et al., 2014; al., 2014).

An ideal non-viral vector, in addition to favoring a high encapsulation / complexation efficiency of the genetic material, allows you to choose highly biocompatible biomaterials for their formulation.

Biomaterials such as phospholipids can be used to create supramolecular systems for the delivery of genetic material. The chemical-physical properties of phospholipids, in particular the possibility of having a neutral, positive or negative charge, can influence their interaction with the cell surface and their intracellular accumulation. US patent 5,252,26.3 describes vesicular systems adapted to modulate the distribution of positively charged lipids and protein molecules through a phospholipid bilayer. The patent describes the migration rate of positively charged proteins and lipids through the phospholipid bilayer of the vesicles as a function of the pH variation and the ionic gradient produced by an aqueous medium added to the lipid formulation. In this patent the asymmetric distribution of the phospholipid bilayer of the vesicles is represented by a single bilayer. The vesicles thus obtained do not guarantee to encapsulate gene material and to obtain, at the same time, the properties of long circulation.

US patent 7,005,140 describes the procedure for preparing lipid nanoparticles with asymmetric structure by removing, under specific reaction conditions, charged phospholipids from the external bilayer, thus obtaining asymmetric lipid particles, or liposomes, which have an internal structure formed by at least one positively charged lipid, capable of complexing the therapeutic agent, genetic material, proteins or peptides, and an external structure formed by neutral lipids or lipids modified with hydrophilic polymers, such as for example polyethylene glycol (PEG), with and without targeting agents. The process described for the formation of the asymmetric bilayer described in this patent has the disadvantage of removing part of the complexed therapeutic agent in the cationic liposomes during the incubation step with the neutral, negative or pegylated liposomes in the aqueous reaction medium, thus reducing the activity of the formulation. Consequently, the process does not allow to obtain an adequately high yield of double bilayers.

US patent application 2011/0117026 describes a lipid formulation formed by an internal structure (core) consisting of polycationic lipids complexed with DNA and an external structure (bilayer) formed by lipids / polycationic compounds / DNA (LPD-NPs) which covers the central core (core). The preparation method used to create the asymmetric vesicles involves the mixing of a lipid suspension A, formed by liposomes (DOTAP / Cholesterol = 1/1 in a molar fraction) at the final lipid concentration of 8.3 mM and 0.2 mg / ml of solubilized protamine in 150 µl of nuclease-free aqueous solution; and a solution B, formed by 0.16 mg / ml of siRNA and 0.16 mg / ml of co-solubilized cali thymus DNA 150 µl of aqueous solution without nuclease. The obtained mixture was subsequently kept under continuous stirring at room temperature for 10 minutes. LPDs coated with PEG chains were obtained by co-incubating the suspension of LPD (300 µl) with 37.8 µl of my eli and obtained using DSPE-PEG2000 or DSPE-PEG2000 conjugated with a compound capable of promoting active targeting (10 mg / mi). PEGylated systems must be used within 20 minutes of their preparation. The process described in US 2011/0117026 does not allow to obtain two asymmetric bilayers organized in such a way as to have an internal phospholipid double layer, positively charged and capable of complexing the genetic material, and an external phospholipid bilayer formed by phospholipids capable of fusing with cell membranes or covered with polyethylene glycol (PEG) and therefore capable of favoring the entry of AVs into cells and a long systemic circulation. These structural differences imply that the LPD-NPs have a structure similar to those of the lipopoliplexes mentioned above, rather than a structure formed by a phospholipid bilayer characteristic of liposomes. Furthermore, the coating of the outer surface with polyethylene glycol (PEG) units make them easy to remove after systemic administration.

In the patent application US20 13/149374 the authors describe asymmetric liposomes, which are not comparable to the asymmetric liposomes of the present invention since the preparation involves the mixing of preformed inverse micelles. The method described in US 2013/149374 provides for a phase inversion stage that prevents the direct addition of gene material. Consequently, the therapeutic agents must be added after the phase inversion, the evaporation of the organic solvent and the dilution of the fused inverse micelles with buffer. This implies that only a part of the gene material can be complexed inside the nano-cells, thus greatly reducing the complexable quantity and the overall efficiency of the systems.

The need was therefore felt to have available systems capable of complexing a large amount of genetic material within the internal bilayer, formed by positively charged lipids, able to remain in circulation for a long time, i.e. such as to allow carry out their pharmacological function. The need was also felt to obtain these results with an easy-to-implement production technology with high yields.

The inventors have now found a way to obtain double bilayer liposomes which provide a simple preparation method and give an efficient delivery such as to overcome the drawbacks of the known art.

Summary of the Invention

The object of the present invention is the realization of vesicular or liposomal systems, called AVs, having not only optimal characteristics of biocompatibility and biodegradability for the conveyance of genetic material, for example fragments of genes, siRNA, DNA and double-stranded RNA, oligonucleotides, miRNA , but, above all, having considerably improved characteristics with respect to the known art as regards the protection of DNA, RNA and oligonucleotides from enzymatic degradation after systemic administration and their cellular internalization.

The AVs of the invention have a structure consisting of two concentric asymmetrical bilayers, an internal bilayer and an external bilayer, each of said bilayers having in turn an internal layer and an external layer, the internal layer of the internal bilayer being provided with a charge. net positive; the outer layer of the outer bilayer being neutral or having a net negative charge (Figure 1). The internal bilayer (also called IL) is positively charged due to the presence of cationic phospholipids, such as DOTAP and DC -cholesterol, capable of forming electrostatic complexes with the genetic material and increasing its action. The external bilayer (also called EL) is negatively charged and formed by phospholipids capable of interacting with cell membranes and merging with them, for example DOPE, cholesterol, hydrophilic and / or amphipathic polymers conjugated to phospholipids, such as PEG, in able to improve the stability and biopharmaceutical properties of the supramolecular system. A schematization of the vesicular systems of the invention is illustrated in Fig. 1, where it can be seen that each vesicular system is formed by two double layers (or bilayers) concentric with each other, the innermost layer of the internal bilayer showing a substantially positive charge towards the of the vesicular system, the outermost layer of the external bilayer showing a substantially negative or neutral charge towards the outside of the vesicular system. The intermediate layers remain free to have an arrangement of charges such as to ensure the neutrality of the entire vesicular system.

Another object of the invention is the method for obtaining said vesicular systems. According to the method of the invention, the two asymmetrical bilayers are prepared separately using the method of evaporation on a thin layer (Thin Layer Evaporation, TEE) of the phospholipids in order to obtain a phospholipid film on the reactor walls. The two asymmetric bilayers thus obtained are then used for the preparation of the vesicular systems of the invention after hydration in aqueous buffer. In particular, the preparation method includes the stages of carrying out:

<•> an internal bilayer or IL formed by cationic liposomes which complex the genetic material;

<•> an external bilayer or EL negatively charged and formed by phospholipids able to fuse across biological membranes, for example DOPE, cholesterol, hydrophilic and / or unpleasant polymers conjugated to phospholipids, for example PEG, able to improve stability and the biopharmaceutical properties of the supramolecular system;

IL and EL are subsequently incubated with each other and extruded with an extruder through polycarbonate filters with cut-off (between 800 and 200 nm).

A further object are the pharmaceutical compositions comprising the AVs of the invention, together with pharmaceutically acceptable excipients. Such compositions can be formulated for administration by parenteral route, in particular by intradermal route, subcutaneous route, intramuscular route, intravenous route and sub-arachnoid route.

Still another object are the uses of AVs or pharmaceutical compositions containing them for the treatment of pathologies which benefit from the administration of gene material.

Further objects will become evident from the following detailed description of the invention.

The method of preparation, characterization, applications and other peculiar characteristics of the A \ <7> s will be described in detail in the detailed description of the invention below, where application examples and experimental data are also reported, summarized in the attached Figures.

Brief description of the Figures

Figure 1. Schematic description of vesicular systems formed by double-structured asymmetric bilayers (AVs). The structure of the AVs is formed by a positively charged internal bilayer, and a neutral and / or negatively charged external bilayer (in this non-limiting example) covered with unpleasant polymer chains (polyethylene glycol; PEG) able to improve the biopharmaceutical properties of the AVs.

Figure 2. In vitro cytotoxicity assays of AVs. Cytotoxicity experiments were performed using cervical cancer cells (HeLa cells) using the MTT (cell viability assay) method. As shown in Figures 2A, 2B and 2C, the cells were treated at different incubation times (respectively 24, 48 and 72 h), using scalar concentrations (30 - 120 pg / ml) of the different formyl lipid actions. AVs (·); cationic liposomes (o); FuGene® (À). The experimental data obtained are the mean of six different experiments ± the standard deviation. Figure 3. Interaction between AVs and HeLa cells. Images made using the confocal microscopy (CLSM) technique. The cells were treated at different incubation times (6 - 24 h) with AVs (panel between A - D) or cationic liposomes (panel between E - H) prepared using the fluorescent lipid DHPE-rhodamine. CLSM images were acquired at the excitation wavelength of rhodamine (panels A and E), green fluorescent protein (GFP) (panels B and F) and 4 ', 6-diamidine-2-phenylindole (DAPI ) (panels C and G). Images D and H were obtained by superimposing cell staining obtained using rhodamine and DAPI.

Figure 4. Intracellular internalization studies of AVS performed using CLSM. MCF-7 human breast cancer cells were used as a cell model during the experiment. After 24 h of incubation, the cells were treated with Pre-miRNA-FAM / FuGene® encapsulated in AVs or with the simple complex formed by Pre-miRNA-F AM / F u G and n e ®. The cells were subsequently collected at different incubation times (0.5, 1, 3 and 6 h). The internalization in MCF-7 cells of Pre -miRNA- FAM / FuGene® encapsulated in AVs was evaluated by measuring the effects of the pre-encapsulation of miRNAs with FuGene®. The internalization studies of the AVs were performed by evaluating the distribution of the carrier systems within the early cell organelles, as reported in the protocol provided by the manufacturer of the reaction kit. Figure 5. \ <7> evaluation of the stability in serum of the Pre-miRNA-FAM / FuGene® complex, cationic liposomes and AVs, respectively. The studies were conducted by evaluating the dynamic light scattering (DLS) of the different formulations (panels i, ii and iii) incubated in the presence of a solution consisting of fetal bovine serum and phosphate buffer (FBS / PBS, 70:30 v / v , pH 7.4) at 37 ° C and for different incubation times (1, 6, 12, 24 and 48 h) (panels A, B and C). It was evaluating the variation of their mean size as a function of time (1, 6, 12, 24 and 48 h). The dimensions of the different formulations obtained at time zero (before incubation) were used as a control.

Figure 6. Enzymatic degradation assays in the presence of RNAseA. The ability of A \ <7> s to protect encapsulated miRNAs from enzymatic degradation was evaluated by electrophorethical analysis. The miRNAs encapsulated in AVs or the bare miRNAs (scramble) were incubated for 1 h at 37 ° C in the presence of RNAse A and subsequently treated for 10 minutes with a mixture of EDTA, sodium dodecyl sulfate and sodium hydroxide. The samples were then incubated for one hour and the electrophorethical analysis was performed on a 1% w / v agarose gel, in Trisborate-EDTA (TBE) buffer for 45 minutes at 100 Λ <7> . bare miRNA (scramble (serious); line 1) and the scrl / FuGene® complex were used as controls during the experiments.

Figure 7. Evaluation of the in vitro transfection efficiency of AVs. The experiments were performed in vitro using FAM-miRNA. The amount of F AM -miRNA transfected in He La cells, used as a cell model, was evaluated by measuring the fluorescence intensity, using a FACSCaliburTM flow cytometer (BD Biosci ences, San Hose, CA, LISA), as a function of the number of cells seeded during the experiment. The Fu Gene® / F AMmiRNA complex was used as a positive control during the experiments. The transfection efficiency of FAM-miRNA / FuGene® encapsulated in AVs (panel C) is compared to that of FuGene® / FAM-miRNA (panel B) and control (untreated cells) (panel A). The fluorescence shift measured by FACSCaliburTM flow cytometer was measured on HeLa cells after 24 h of incubation.

Figure 8. In vitro therapeutic efficacy of miRNAs encapsulated in AVs. The anti-miRNA-18a antibody (anti-18a) was encapsulated in AVs and the antitumor activity was evaluated in vitro using MCF-7 cells. The anti-18a / FuGene® complex was used as a control in cytotoxicity experiments. Cell toxicity was measured using the MTT method (cell viability assay) on treated cells at different incubation times (24, 48 and 72 h). The anti -miRNA-18a was used at a final concentration equal to 10 nM. The in vitro antitumor activity was determined as a reduction in the cell viability of MCF-7 cells treated with the different formulations at the indicated incubation times.

Figure 9. Western blotting analysis of the efficacy of miRNA complexed with AVs. The formulations used to perform the experiments illustrated in Figure 8 were used for the western blotting experiments. After treatment for 72 h with anti-18a / FuGene® complex and with anti-miRNA-18a encapsulated in AVs, MCF-7 cells were harvested from cell culture plates, centrifuged, lysed and the amount of protein present in each sample was measured. 50 µg of proteins were subsequently separated using the SDS-PAGE method. The analysis by western blotting was carried out using anti-Estrogen Receptor-α (anti-Era) and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH) antibodies during the experiments. The expression levels of ERa and GAPDH (control) in MCF-7 cells were assessed by comparing the expression levels of both antibodies, reported above, for untreated cells (control) (line 1); anti -miRNA- 18a / FuGene® (line 2); anti -miRNA- 18a / FuGene® encapsulated in AVs (line 3); anti -miRNA-18a / FuGene® scramble (line 4), anti-miRNA-18a / FuGene® scramble encapsulated in AVs (line 5) and empty AVs (line 6).

Figure 10. Evaluation of the in vitro immune response for AVs in specific cell models. The potential in vitro immune response of the AVs was evaluated using a macrophage cell line of murine origin (Raw 264.7 cells). Cells were treated for 24 h with lipopolysaccharide (LPS) (1 Lig / inl), empty AVs and anti-miRNA-18a (100 nM) encapsulated in AVs. The production of pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-Ιβ, was evaluated using an ELISA kit according to the manufacturer's instructions. All experiments were performed in triplicate.

Detailed description of the invention Within the scope of the present invention, the terms "structures", "lipid systems", "vesicular systems" and "liposomal systems" are to be considered synonyms, and are collected under the term AVs (Asymmetries Vesicles; asymmetrical vesicles) and refer to structures consisting of two double layers or bilayers. As schematized in Figure 1, in the AVs of the invention the innermost positively charged layer is able to quantitatively complex the negatively charged gene material inserted inside it; while the outermost layer, which overall carries a neutral or negative charge, is able to interact with the biological membranes, allowing to obtain a very effective conveyance of the gene material. with good safety and biocompatibility features ..

The AVs of the invention represent a promising approach for the systemic delivery of RNAi, thanks to their biocompatibility, transfection efficiency and the possibility of large-scale production. The asymmetrical structure of the phospholipid bilayer presents considerable versatility since it can be prepared using different biocompatible materials and can have different dimensions. The positively charged internal bilayer favors complexation with gene material such as miRNA and siRNA; while the external bilayer, thanks to its composition which confers a negative or neutral charge, favors a prolonged systemic circulation of the AVs of the invention, improving the stability in biological fluids and regulates the interaction of the AVs with the target cells and tissues, and therefore the their therapeutic efficacy in diseases that benefit from the administration of gene material. In fact, the double bilayer structure of the vesicular systems of the invention is such that, thanks to the lipids that constitute the external bilayer EL, highly compatible with the cell membranes, the internalization of the vesicles is favored and the entry of the encapsulated gene material is facilitated. inside the internal bilay er IL. The coating of the external surface of the AVs, for example with PEG chains, can be performed after pre-complexation of the genetic material to be conveyed with a commercial transfection agent, for example FuGene®. In fact, the pegylation of liposomal suspensions prevents the complexation of the genetic material within the colloidal system, for this reason the pegylation is carried out after obtaining the double bilayer. The process for preparing the AVs according to the invention includes the following stages:

(i) prepare a positively charged internal bilayer (IL). The preparation can be carried out by solubilizing one or more cationic phospholipids, for example selected from the group of list II below, alone or in association with other cationic and / or neutral phospholipids, for example selected from the group of list I, in solvents or mixtures of apolar, polar aprotic or polar p rotic solvents. The mixture is then dried by eliminating the solvent, for example using the TLE (Thin Layer Evaporation) method and subsequently it is hydrated with water or aqueous solutions. Step (i) can be carried out under mechanical stirring conditions, for example with a speed comprised between 10 and 1000 rpm, preferably between 10 and 700 rpm, more preferably between 10 and 300 rpm.

The preferred method for preparing the IL phase of the AVs is the following. Cationic lipids are used, listed in list II at different molar fractions (varying between 1: 9 and 9: 1). The mixture of phospholipids used is solubilized in a solvent or mixture of solvents as mentioned above. The solvent mixture is then removed, for example by means of a vacuum evaporator (for example pressure between 100 and 700 mBar) at a temperature between 30 and 80 ° C. The removal of the residual traces of the solvent mixture from the obtained lipid film can then be carried out using a dryer, programmed at a temperature range between 30 and 80 ° C and connected to a vacuum pump programmed at a pressure range between 100 and 700 mBar.

The lipid film thus obtained is subsequently hydrated with an aqueous solution and the lipid suspension thus obtained is subjected to a number of alternating cycles of continuous stirring, between 3 and 20, and heating in a thermostated water bath, at a temperature between between 30 and 80 ° C, for a number of cycles between 3 and 20.

The final lipid concentration obtained for the IL liposomes is between 1 and 13.8 mM. The IL liposomes are then diluted using the same aqueous solution used for their preparation.

By transition temperature, Tm, we mean a temperature, characteristic for each phospholipid, below which the resulting bilayer is in the gel phase (rigid structure) and above which the resulting bilayer is in the liquid-crystalline phase (more fluid structure). Tm is measured using the differential scanning calorimetry (DSC) method. The Tm of the AVs has a value included in an interval consisting of the Tm values of the phospholipids that make up IL and EL. The value of Tm for AVs is between - 10 and 65 ° C.

The miRNAs, pre-miRNAs and siRNAs are complexed with IL liposomes during the hydration phase of the lipid film. This procedure can be performed as described in the literature (Maccarrone et al., 1992; Annesini et al., 1994).

(ii) prepare a negatively charged external bilayer (EL). The preparation can be carried out by solubilizing one or more negatively charged phospholipids, for example selected from the group of list III below, and / or selected from the group of phospholipids conjugated with polyethylene glycol of different molecular weight, as reported in list IV and / or polyethylene glycol of different molecular weight, alone or in combination, in solvents or mixtures of apolar, polar aprotic or polar protic solvents, as listed below. The mixture is then dried by eliminating the organic solvent by means of the TLE (Thin Layer Evaporation) method. Then follows the subsequent hydration step with double distilled water or aqueous solutions. Step (ii) can be carried out under conditions of mechanical stirring.

The obtainable bilayer, or EL phase, thanks to the negative or neutral charge is able to interact with cell membranes, merging with them.

LIST I - Neutral phospholipids: cholesterol and cholesterol derivatives; L-a-phosphatidylcholine (Egg, Chicken); L-aphosphatidylcholine, hydrogenated (Egg, Chicken); L-aphosphatidylcholine (95%) (Egg, Chicken); L-a-Phosphatidylcholine (Egg, Chicken -60%) (Total Egg Phosphatide Extract); L-alysophosphatidylcholine (Egg, Chicken); L-a-phosphatidylcholine (Soy); L-a-phosphatidylcholine, hydrogenated (Soy); L-aphosphatidylcholine (95%) (Soy); L-a-Phosphatidylcholine (Soy-40%); L-a-Phosphatidylcholine, 20% (Soy); L-alysophosphatidylcholine (Soy); L-α -phosphatidylcholine (Heart, Bovine); L-a-phosphatidylcholine (Brain, Porcine); L-aphosphatidylcholine (Liver, Bovine); 1,2-dilauroyl-sn-glycero-3-phosphocholine; 1,2-ditridecanoyl-sn-glycero-3-phosphocholine; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; 1,2-dipentadecanoyl-snglycero-3-phosphocholine; 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; 1,2-diphytanoyl-sn-glycero-3-phosphocholine; 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine; 1,2-distearoyl-snglycero-3-phosphocholine; 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine; 1,2-diarachidoyl-sn-glycero-3-phosphocholine; 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine; 1,2-dibehenoyl-snglycero-3-phosphocholine; 1,2-ditricosanoyl-sn-glycero-3-phosphocholine; 1,2-dilignoceroyl-sn-glycero-3-phosphocholine; Cisl, 2-dinervonoyl-sn-glycero-3-phosphocholine; l-myristoyl-2-pahnitoyl-sn-glycero-3-phosphocholine; l-myristoyl-2-stearoyl-snglycero-3-phosphocholine; 1-palmitoyl-2-acetyl-sn-glycero-3-phosphocholine; l-palinitoyl-2-stearoyl-sn-glycero-3-phosphocholine; 1-palinitoyl-2-linoleoyl-sn-gl3'Cero-3-phosphocholine; l-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; l-stearoyl-2-palinitoyl-sn-glycero-3-phosphocholine; l-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine; l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 1-stearoyl-2-docosahexaenoyl-sn-gl3 <r> cero-3-phosphocholine; 1-oleoyl-2myristoyl-sn-glycero-3-phosphocholine; l, 2-bis (10, 12-tr ì c o s ad iy noy 1) - s n - g h'c ero - 3 - p ho sp h oc h ol ι n e; 1,2-bis (10, 12-tricosadiynoyl) -sn-glyceiO-3-phosphoethanolamine; l-palmitoyl-2- (10, 12 - tri co s a diy noy 1) - s n - g ly c e ro - 3 - p ho sp ho c hol ι n e; l-palmitoyl-2- (10, 12- tri c o s a diy noy 1) - s n - g ly c e ro - 3 - p ho sp ho eth ano 1 a mi n e.

LIST II - Cationic phospholipids: 1,2-dioleoyl-3-trimethylammonium-propane (chloride salt); 3β- [Ν- (Ν ', Ν’-dimethylaminoethane) -carbamoyl] cholesteiOl hydrochloride (DC-Cholesterol-HCl); 1,2- diteti 'a dee anoyl - 3 -trimethy 1 ammonmmpropane (chloride salt) (DMTAP); 1,2-dioleoyl-3-trimethylammonium-piOpane (methyl sulfate salt); 1,2-dihexadecanoyl-3-trimethylaminonium-propane (chloride salt); 1,2-dioctadecanoyl-3-trimethylamnionium-propane (chloride salt); 1-oleoyl-2- [6 - [(7-nitro-2- 1J3-benzoxadiazol-4-yl) amino] hexanoyl] -3-trimethylammonium propane (chloride salt); Fluorescent 1 -oleoyl-2- [6- [(7-nitiO-2-l; 3-benzoxadiazol-4-yl) amino] hexanoyl] -3-trimethylammonium propane (chloride salt); N- (4-carboxybenzyl) -N, N - di methyl - 2, 3 - bi s (ol eoyloxy) prop an - 1 - ammmm (DOBAQ); 1,2-dioleoyl-3-dimethylainmonium-propane (DODAP); 1,2-dimyristoyl-3-dimethylaminonium -propane; 1,2- di p to 1 to \ d - 3-dimethyl ammonium uni-prop ane; 1,2- di stearoyl - 3-dimethyl ammonium um-propane, dimethyl dioctadecyl animo ni uni (bromide salt) (DDA); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (chloride salt); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt); 1, 2 - di my r i sto 1 e oy 1 - s n - g ly c e r o 3-ethylphosphocholine (Tf salt); 1, 2 - d 1 p al mi toy 1 - sn - glycero - 3 -ethylphosphocholine (chloride salt); 1,2- di ste aroy 1 - sn - glycero - 3 -ethylphosphocholine (chloride salt); 1,2- diol eoy 1 - sn - glycero - 3 -ethylphosphocholine (chloride salt); l -palmitoyl-2-oleoyl-snglycero-3-ethylphosphocholine (chloride salt); 1,2- di - O -octadec enyl -3-trimethylaminonium propane (chloride salt) (DOT MA), NI - [2 - ((1 S) - 1 - [(3 - aminopropyl) amino] -4- [di (3 - aminop r o py 1) animo] b uty 1 c ar b ox ami do) e thy 1] - 3, 4 - di [o 1 y 1 oxy] - b enz ami d e.

List III. Anionic phospholipids: L-a-phosphatidic acid (Egg, Chicken); L-a-phosphatidic acid (Soy) (sodium salt); 1,2-dihexanoyl-sn-glycero-3-phosphate (sodium salt); 1,2-dioctanoyl-snglycero-3-phosphate (sodium salt); 1,2-didecanoyl-sn-glycero-3-phosphate (sodium salt); 1,2-dilauroyl-sn-glycero-3-phosphate (sodium salt); 1,2- dimyri stoyl - sn - glycero - 3 -p hosphate (sodium salt); 1.2-dimyristoyl-sn-glycero-3-phosphate (sodium salt); 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt); 1,2-diphytanoylsn - glycero -3 -p hosphate (sodium salt); 1,2-diheptadecanoyl-snglycero-3-phosphate (sodium salt); l, 2-distearoyl-sn-glycero-3-phosphate (sodium salt); 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt); 1, 2 - of 1 m ol e o \ d - sn - gly c er o - 3 - p ho s p h at e (sodium salt); 1.2-dilinoleoyl-sn-glycero-3-phosphate (sodium salt); 1,2-diarachidonoyl-sn-glycero-3-phosphate (sodium salt); 1,2-didocosahexaenoyl-sn-gl3 <r> cero-3-phosphate (sodium salt); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (sodium salt); 1-p al mi toy 1 - 2 - 1 mo 1 eo \ d - s n - g ly c e r o - 3 - p ho sp h at e (sodium salt); 1palmitoyl-2-arachidonoyl-sn-glycero-3-phosphate (sodium salt); 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphate (sodium salt); l-stearoyl-2-oleoyl-sn-glycero-3-phosphate (sodium salt); 1-stearoyl-2dinoleoyl-sn-glycero-3-phosphate (sodium salt); 1-steai'oyl-2-arachidonoyl-sn-glycero-3-phosphate (sodium salt); 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphate (sodium salt); 1-hexanoyl-2-hydroxy-sn-glycero-3-phosphate (ammonium salt); 1-nw ri s toy 1 - 2 - hy dro xy - s n - g ly c e ro - 3 - p ho sp h at e (sodium salt); 1-palmitoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt); 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt); 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt); 1-oleoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt); 1-linoleoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt); l -arachidonoyl-2-hy d roxy - sn - gly c e r o - 3 - p ho s p h at e (ammonium salt); 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphate (ammonium salt); 1-O-octadecyl-2-hydroxy-sn-glyceiO-3-phosphate (ammonium salt); 1- (9Z-octadecenyl) -2-hydroxy-sn-glycero-3-phosphate (ammonium salt); 1 - p al mi toy 1 - s n - g ly c e ro - 2, 3 - cy c 11 c - p ho sp h ate (ammonium salt); 1-h e p t a d ec an oy 1 - gh 'c ero - 2, 3 - cy c 11 c - p h o s p h at e (ammonium salt); 1-oleoyl-sn-glycero-2,3-cyclic-phosphate (ammonium salt); 1-0-h e x a d e cy 1 - sn - gly c e ro - 2, 3 - c \ <7> c 11 c - p ho sp h at e (ammonium salt); 1-0- (9 Z - oc t a cl e c eny 1) - s n - gly c ero - 2, 3 - c \ <7> c 11 c - p ho s p h at e (ammonium salt); l-oleoyl-2-methyl-sn-glycero-3-phosphothionate (ammonium salt); 1 - o 1 eoy 1 - 2 - methy 1 - sn - gly c er o - 3 - p ho s p ho thi o n at e (ammonium salt); (S) -phosphoric acid mono- {2-octadec-9-enoylamino-3- [4- (pyridin-2ylmethoxy) -phenyl] -propyl} ester (ammonium salt); (S) -phosphoric acid mono - {2 -octad ec - 9 - enoyl amino - 3 - [4- (pyn dm - 2 -y lmethoxy) -phenyl] -propyl} ester (ammonium salt); (R) -phosphoric acid mono- {2-octadec-9-enoylamino-3- [4- (pyridin-2-ylmethoxy) -phenyl] -propyl} ester (ammonium salt); N-palmitoyl-serine phosphoric acid (ammonium salt); N -p almi toyl-ty rosine phosphoric acid (ammonium salt); L-a-phosphatidylglycerol (Egg, Chicken) (sodium salt); L-a-phosphatidylglycerol (Soy) (sodium salt); L-aphosphatidylglycerol (E. coli) (sodium salt); 1,2- dihexanoyl - sn -glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2-dioctanoyl-snglyceiO-3-phospho- (l'-rac-glycerol) (sodium salt); 1, 2 - of d and c anoy 1 - s n -glyceiO-3-phospho- (r-rac-glycerol) (sodium salt); 1,2-dilauroyl-snglyceiO-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2-dimyristoylsn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2-dipentadecanoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); L2-dipalmitoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); L2-diphytanoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2-diheptadecanoyl-sn-glycero-3-phospho- (l'-racglycerol) (sodium salt); 1,2-distearoyl-sn-glycero-3-phospho- (r-racglycerol) (sodium salt); c i s - 1, 2 - di o 1 eoy 1 - s n - g ly c e ro - 3 - p ho sp ho - (1 '-rac-glycerol) (sodium salt); trans- 1,2-dielaidoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2-dihnoleoyl-sn-glycero-3-phospho- (r-rac-glycerol) (sodium salt); 1,2-dilinolenoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2- of arac hi donoyl - sn -glycero-3- [phospho-rac- (l-glycerol)] (sodium salt); 1,2didocosahexaenoyl-sn-glycero-3- [phospho-rac- (l-glyceiOl)] (sodium salt); l-palmitoyl-2-oleoyl-sn-glyceiO-3-phospho- (l'-rac-glycerol) (sodium salt); l-palmitoyl-2dinoleoyl-sn-glycero-3-phospho- (l'-racglycerol) (sodium salt); l-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); l-palmitoyl-2-docosahexaenoyl-sn-glyceiO-3-phospho- (l'-rac-glycerol) (sodium salt); l-steaiOyl-2-oleoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); l-stearoyl-2-linoleoyl-sn-glycero-3-phospho- (r-racglycerol) (sodium salt); l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); l-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho- (r-rac-glycerol) (sodium salt); 1 - my ri s toy 1 - 2 - hy d roxy - s n - g ly c e ro - 3 - p h o s p ho - (Γ - r ac - g ly c e rol) (sodium salt); l-palmitoyl-2-hydroxy-sn-glycero-3-phospho- (l'-racglycerol) (sodium salt); l-stearoyl-2-hydroxy-sn-glycero-3-phospho- (Γ-rac-glycerol) (sodium salt); l-oleoyl-2-hydroxy-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); L-a-phosphatidylserine (Brain, Porcine) (sodium salt); L-adysophosphatidylserine (Brain, Porcine) (sodium salt); L-a-phosphatidylserine (Soy, 99%) (sodium salt); 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-dioctanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-didecanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-di 1 aur oy 1 - sn - gh <7> c e r o - 3 - p ho s p ho - L - s e r m e (sodium salt); 1,2-dimyristoyl-sn-glyceiO-3-phospho-L-serine (sodium salt); 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2diheptadecanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-dilinoleoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-diarachidonoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-didocosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1-palmitoyl-2-oleoyl-sn-glyceiO-3-phospho-L-serine (sodium salt); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium salt); L-a-lysophosphatidylserine (Brain, Porcine) (sodium salt); l-palmitoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt); 1- (10Z-heptadecenoyl) -2-hydroxysn-glycero-3- [phospho-L-serine] (sodium salt); l-stearoyl-2-hydiOX3'-sn-glycero-3-phospho-L-serine (sodium salt); l-oleoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt); L-aphosphatidylinositol (Liver, Bovine) (sodium salt); L-alysophosphatidylinositol (Liver, Bovine) (sodium salt); L-aphosphatidylinositol (Soy) (sodium salt); L-alysophosphatidylinositol (Soy) (sodium salt); L-aphosphatidylinositol-4-phosphate (Brain, Porcine) (ammonium Salt); L-a-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt); 1,2-dioctanoyl-sn-glycero-3-phospho- (l'-myoinositol) (ammonium salt); 1,2-dipalmitoyl-sn-glycero-3-phospho- (Γ-myo-inositol) (ammonium salt); 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt); 1,2-dioleoyl-sn-glycero-3-phospho- (l'-myo-inositol) (ammonium salt); l-palmitoyl-2-oleoylsn-glycero-3-phosphoinositol (ammonium salt); l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol (ammonium salt); 1,2-dioctanoyl-sn-glycero-3- (phosphoinositol-3-phosphate) (ammonium salt); 1,2-dioctanoyl-sn-glyceiO-3-phospho- (l'-myo-inositol-4'-phosphate) (ammonium salt); 1, 2 - di o 1 eoy 1 - s n - g ly c e r o - 3 - p ho sp ho - (1 '-myo-inositol-3'-phosphate) (ammonium salt); 1,2-dioleoyl-snglyceiO-3-phospho- (l'-myo-inositol-4'-phosphate) (ammonium salt); 1,2-dioleoyl-sn-glyceiO-3-phospho- (l'-myo-inositol-5'-phosphate) (ammonium salt); 1,2-dihexanoyl-sn-glycero-3-phospho- (l'-myoinositol-3 ', 5'-bisphosphate) (ammonium salt); 1,2-dioctanoyl-snglyceiO-3-phospho- (l'-myo-inositol-3 ', 4'-bisphosphate) (ammonium salt); 1,2-dioctanoyl-sn-glyceiO-3-phospho- (l'-myo-inositol-3 ', 5'-bisphosphate) (ammonium salt); 1,2-dioleoyl-sn-glycero-3-phospho- (l'-myo-inositol-4 ', 5'-bisphosphate) (ammonium salt); 1,2-dioleo \ dsn-glycero-3-phospho- (l'-myo-inositol-3 ', 4'-bisphosphate) (ammonium salt); 1,2-dioleoyl-sn-glycero-3-phospho- (l'-myoinositol-3 ', 5'-bisphosphate) (ammonium salt); 1,2-dioleoyl-snglycero-3-phospho- (l'-myo-inositol-4 ', 5'-bisphosphate) (ammonium salt); l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho- (r-myo-

1 no yes tol - 3 ', 5' - b 1 sp h o s p h at e) (ammonium salt); l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho- (r-myo-inositol-4 ', 5'-bisphosphate) (ammonium salt); 1, 2 - di h ex anoy l-sn-glycero-3-phospho- (r-myo-inositol-3 ') 4') 5'-trisphosphate) (ammonium salt); l ^ -dioctanoyl-sn-glycero-S-phospho ^ r-myo-inositol-S ', ^^' - trisphosphate) (ammonium salt); 1, 2 - d ι ol e oy 1 - sn - gly c ero - 3 - p h o s p ho - (1 '- my o - 1 no si tol - 3 4', 5 '- tri s p ho s p h at e) (ammonium salt); 1-stearoyl-2-arachidonoyl-sn-glycero-3-phospho- (r-myo-inositol-3 ') 4') 5'-trisphosphate) (ammonium salt); L-adysophosphatidylinositol (Liver, Bovine) (sodium salt); L-adysophosphatidylinositol (Soy) (sodium salt); 1 - tri d ec an oy 1 - 2 - hy dro xy - sn - gly c er o - 3 - p ho s p h o - (Γ -myo-inositol) (ammonium salt); 1 -palmitoyl-2-hydroxy-sn-glycero-3-phosphoinositol (ammonium salt); l-stearoyl-2-hydroxy-snglycero-3-phosphoinositol (ammonium salt); l- (lt) Z-heptadecenoyl) -2-hydiOxy-sn-glyceiO-3-phospho- (l'-myo-inositol) (ammonium salt); l-oleoyl-2-hydroxy-sn-glyceiO-3-phospho- (l'-myo-inositol) (ammonium salt); l-arachidonoyl-2-hydroxy-sn-glycero-3-phosphoinositol (ammonium salt).

LIST IV - Phospholipids conjugated to polyethylene glycol chains: L2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [me thoxy (p oly ethy lene glycol) -750] (ammonium salt); 1,2-dipalmitoyl-sn-glyceiO-3-phosphoethanolamine-N- [methoxy (poly ethy lene glycol) -750] (ammonium salt); 1,2-distearoyl-sn-glyceiO-3-phosphoethanolamine-N [methoxy (polyethylene glycol) -750] (ammonium salt); 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -750] (ammonium salt); 1,2- dimy ri stoy 1 - sn - glycero - 3 -phosphoethanolamine-N- [methoxy (polyethylene glycol) - 1000] (ammonium salt); 1,2- clip almitoy 1 - sn - glycero - 3 -phosphoethanolamine-N- [me th oxy (p oly ethy 1 e n e glycol) - 1000] (ammonium salt); 1,2- di ste aroyl - sn - glycero - 3 -phosphoethanolamine-N- [methoxy (polyethylene glycol) - 1000] (ammonium salt); 1, 2 - di ol e oy 1 - s n - g ly c ero - 3 - p ho sp h o e th anol ami n e -N- [methoxy (polyethylene glycol) - 1000] (ammonium salt); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (poly ethy lene glycol) -2000] (ammonium salt); 1,2-dip almitoyl - sn - glyc ero - 3 -pho sp ho ethanol amine -N - [methoxy (poly ethy lene glycol) -2000] (ammonium salt); 1,2-disteaiOyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (poly ethy lene glycol) -2000] (ammonium salt); 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt); 1, 2 -di my ri s toy 1 - sn - glycero - 3 -phosphoethanolamine-N- [methoxy (polyethylene glycol) -3000] (ammonium salt); 1,2- dip almitoyl - sn - glycero - 3 -phosphoethanolamine-N- [me th oxy (p oly ethy 1 e n e glycol) -3000] (ammonium salt); 1,2- di ste ai'o \ d - sn - glycero - 3 -phosphoethanolamine-N- [me th oxy (p oly ethy 1 e n e glycol) -3000] (ammonium salt); 1, 2 - di ol e o \ d - s n - g ly c er o - 3 - p ho sp h o e th anol ami n e -N - [me th oxy (p oly e th \ d e n e glycol) -3 000] (ammonium salt); 1,2dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -5000] (ammonium salt); 1,2-dip alirdtoyl - sn - glyc ero - 3 -pho sp ho ethanol ainine -N - [methoxy (polyethylene glycol) -5000] (ammonium salt); 1,2-distearoyl-sn-glycero-3-phosphoethanolainine-N- [methoxy (polyethylene glycol) -5000] (ammonium salt); 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -5000] (ammonium salt); N-palmitoyl-sphingosine-1- {succinyl [methoxy (polyethylene glycol) 750]}; N-palmitoylsphingosine- 1- {succinyl [methoxy (polyethylene glycol) 2000]}; N-palmitoyl-sphingosine- l- {succinyl [methoxy (polyethylene

gly col) 5000]}; 1, 2 - di s te aroy 1 - s n - g ly c e ro - 3 - p ho sp h o e th anolamme-N- [dibenzocyclooctyl (polyethylene glycol) -2000] (ammonium salt); 1,2-disteaiOyl-sn-glycero-3-phosphoethanolamine-N- [azido (polyethylene glycol) -2000] (ammonium salt); 1,2-distearoylsn - glycero - 3 - p ho s p h o e th anol ami n e - N - [sue c ι ny 1 (p oly ethy 1 e n e glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [carboxy (poly ethy lene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -p ho sp hoethanolamine-N- [maleimi de (poly ethy lene glycol) -2000] (ammonium s alt); 1,2- di ste aroyl - sn - glycero - 3 -pho sphoethanolamine-N- [PDP (polyethylene glycol) -2000] (ammonium s alt); 1,2- di ste aroyl - sn - glycero - 3 -pho sphoethanolamine-N- [amino (polyethylene glycol) -2000] (ammonium salt); 1,2- di st and ai'oy 1 - sn - glycero - 3 phosphoethanolamine-N- [biotinyl (polyethylene glycol) -2000] (ammonium salt); 1,2- di st and aroyl - sn - glycero - 3 -phosphoethanolamine-N- [cyanur (polyethylene glycol) -2000] (ammonium salt); 1,2- di st and aroyl - sn - glycero - 3 -phosphoethanolamine-N- [folate (polyethylene glycol) -2000] (ammonium salt); 1,2- di ste ai'oyl - sn - glycero - 3 -phosphoethanolamine-N- [folate (polyethylene glycol) -5000] (ammonium salt); 1,2- di st and aroyl - sn - glycero - 3 -phosphoethanolamine-N- [poly (ethylene glycol) 2000-N'-carboxyfluorescein] (ammonium salt).

Surfactants (surfactants) and co-polymers can be used in place of and / or in association with the phospholipids conjugated to polyethylene glycol chains, with different molecular weight, reported above, to prepare the external bilayer or EL described in point (ii): GM1 Ganglioside (Brain, Ovine-Sodium Salt); GM3 Ganglioside (Milk, Bovine -Ammonium Salt); GD3 Ganglioside (Milk, Bovine -Ammonium Salt); Total Ganglioside Extract (Brain, Porcine -Ammonium Salt); D-lactosyl-β-Ι, Γ N -1 auroy 1 - 1) - ery thro -sphingosine; D-lactosyl-β- 1, Γ N-palmitoyl-D -ery thro- sphingosine; D-Lactosyl-h-1, l'-N-Heptadecanoyl-D- ery thro-Sphingosine; D-lactosyl-β-Ι, Γ N-stearoyl-D-erythro -sphingosine; D-lactosyl-β-Ι, Γ N-lignoceroyl-D-erythro -sphingosine; I) -lactosyl -β 1 - Γ -N -neivonoyl -D-erythro-sphingosine; polyethylene glycol with different molecular weight, polypropylene glycol with different molecular weight and branching in the side chain, polysorbates with different molecular weight, co-polymers of polyethylene glycol and polypropylene glycol with different molecular weight, branching in the side chain and number of monomer units.

The solvents used to solubilize the phospholipids and prepare the internal bilayer (IL) described in point (i) and the external bilayer (EL) described in point (ii) can be of the apolar, aprotic polar and practical polar type.

In particular, the following types of solvents or mixtures of the same solvents at different percentages (1 to 99% v / v and from 99% to 1% v / v) are used to prepare the internal IL and external EL bilayer reported in points (i ) and (ii):

<•> non-polar solvents, for example selected from: diethyl ether, chloroform, ethyl acetate, dichloro methane;

Polar aprotic solvents, for example selected from: acetone, acetonitrile, dimethylformamide;

<•> practical polar solvents, for example selected from: butanol, 2-propanol, 2-propanol, ethanol, methanol, water and buffered and non-buffered aqueous solutions.

The preferred way to prepare the bilayer which constitutes the external phase (EL) is to solubilize the lipids in a mixture of solvents, as reported above. The solvent mixture can be removed by heating with a thermostated device programmed in the temperature range between 30 and 80 ° C, connected to a vacuum pump programmed at a pressure between 100 and 700 mBar. The removal of residual traces of the solvent mixture from the obtained lipid film can then be carried out overnight using a dryer, programmed at a temperature range between 30 and 80 ° C and connected to a membrane vacuum pump programmed to a pressure range between 100 and 700 mBar.

The lipid film obtained in step (ii) is then hydrated with an aqueous solution, and the lipid suspension thus obtained is subjected to a number of alternating cycles of continuous stirring, between 3 and 20, and heating in a water bath. thermostated, at a temperature between 30 and 80 ° C, for a number of cycles between 3 and 20. The final lipid concentration obtained for the EL liposomes is between 1 and 13.8 mM.

The EL liposomes are then diluted to a concentration of 0.1 mM using the same aqueous solution used for their preparation. The following technological procedure can be performed as reported in the literature (Pasut et al., 2015; Paolino et al., 2014; Paolino et al., 2010; Cosco et al., 2009).

(iii) mixing the EL suspension with the IL suspension, adding 1 ml of IL to 1 ml of EL previously prepared. The mixing of the two liposomal formulations can be advantageously carried out under mechanical stirring using a rotation speed in the range from 10 to 300 rpm, for a time in the range from 1 to 72 h, at a temperature in the range from 30 and 80 ° C, to obtain vesicular structures consisting of a double lipid layer which thus form a double vesicular structure for the conveyance of gene material such as plasmid DNA (pDNA) and dsRNA (miRNA and siRNA) and which can subsequently be extruded. In particular, the EL liposomes, obtained using the preparation method described in point (ii), are hydrated with the IL liposomes, obtained using the preparation method described in point (i).

The concentration of both IL liposomes and EL liposomes is in the range from 1 to 13.8 mM.

According to a preferred embodiment, the lipid suspension obtained from the combination of the liposomes IL and EL is subjected to a number of 3 alternating cycles of mechanical stirring under continuous flow, using a rotation speed in the range between 10 and 300 rpm, for a time between 1 and 72 h, at a temperature between 30 and 80 ° C. The lipid suspension of AVs thus obtained is then subjected to further alternating cycles of continuous agitation, between 3 and 20, and heating in a thermostated water bath, at a temperature between 30 and 80 ° C, for a number of cycles between 3 and 20. The combination of the liposomes IL and EL allows to form AVs which have a total lipid concentration in the range between 2 and 26.5 mM.

The obtained AVs are further incubated at a temperature between 30 and 80 ° C for a further time interval between 1 and 24 hours to favor the formation and stabilization of the supramolecular structure. (iv) extrusion of the mixture of IL and EL. The AVs are preferably extruded using a stainless steel extruder under nitrogen pressure, between 100 and 1500 kPa, with an extrusion chamber temperature between 30 and 80 ° C, and for an interval of time between between 10 minutes and 2 hours. It is also possible to proceed with extrusion through filters of synthetic material, polycarbonate, and / or polypropylene, and / or polystyrene. The size of the cut-off is between 30 and 1000 mn. The preferred cutoff is between 800 and 200 nm The number of passes for each filter is between a minimum of 1 and a maximum of 20. From 1 to 2 synthetic material filters are used for the extrusion process of the AVs. The final yield of the AVs after the extrusion process is between 30 and 100%. After the extrusion procedure, the yield of the AVs is greater than 94%.

(v) optional washing and final purification with water and / or buffers of the AVs.

The process of the invention allows to obtain asymmetrical vesicles formed by bilayers with a double structure, identified with the following acronym (AVs), for the conveyance of genetic material such as for example DNA pi asmic (pDNA) and dsRNA (miRNA, siRNA) . The AVs thus obtained can find applications in the pharmaceutical and diagnostic fields for the treatment and prevention of neoplastic, neuro-degenerative, metabolic, viral, fibro-proliferative, cardiovascular and psychosomatic pathologies in both animals and humans.

In particular, the AVs of the invention allow to improve in vivo the therapeutic action of gene material such as miRNA and siRNA after systemic administration. The pathologies that benefit from their administration are, in particular: carcinoma with and without metastases of: breast, lungs, kidneys, ovary, prostate, pancreas, liver, cervix, bone marrow, hematopoietic cells (leukemia, lymphoma and myeloma), pheochromocytoma, glioblastoma, neuroblastoma. Still in the context of the treatment of malignant tumors, the AVs of the invention also find application in the treatment of stromal tumor cells. Other diseases treatable with the AVs of the invention are: Huntington's chorea, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, viral infections, fibro-proliferative disorders of the liver, kidneys, heart and lungs, forms of induced diabetes. from other pathologies, cardiovascular pathologies and metabolic diseases.

The A \ <7> s of the invention allow to mitigate the side effects of the therapies used in the treatment of the aforementioned pathologies and which manifest themselves with toxicity affecting the kidneys, heart, liver, spleen, lungs, pancreas, nervous system central and peripheral.

The AVs of the invention can be formulated in pharmaceutical compositions comprising a pharmaceutically acceptable vehicle and improve the therapeutic delivery of gene material such as dsRNAs (miRNA and siRNA) which, unencapsulated, rapidly lose efficacy after systemic administration and overcome the problems of poor efficacy of viral transcription vectors currently used for the systemic administration of dsRNA (miRNA and siRNA). AYs can be administered using both topical routes (such as percutaneous, transmucosal and ophthalmic routes) and systemic routes such as, for example, intramuscular, intravenous, intradermal, intraperitoneal, intracardi ac a, oral, etc. They can also be formulated in the form of a suspension to be prepared at the time of use.

The innovation of the preparation method proposed here consists in not providing for the use of organic solvents and in also making it possible to obtain an aqueous trans-membrane gradient, with an acid internal pH, with respect to the neutral or basic external pH of the aqueous medium. in which the liposomes are dispersed. Furthermore, the AVs can be used months after their preparation due to their high stability. The stability of the suspension thus obtained is ensured for at least six months at 4 ° C (data not shown).

The following examples are to be considered illustrative and not limitative of the scope of the invention.

EXAMPLES

Example 1: Materials

The AVs were prepared using the following compounds purchased from Sigma-Aldrich Chemical. Co., St. Louis, Mo.

U.S.A .: 1,2-dipalmitoyl-sn-glyceiO-3-phosphatidylcholine (DPPC), 1,2-dioleoyl-sn-glyceiO-3-phosphotidylethanolamine (DOPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) , cholesterol (Chol) and 3β- [Ν- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-chol), 1,2-distearoyl-sn-glycerol-3-phosphatidylethanolamine-N- [methoxy ( polyethylene glycol) -2000] (DSPE-PEG 2000). The confocal microscopy experiments were performed using a lipid formulation containing the phospholipid (1,2-hexadecanoilsn - glycine - 3 -phosphates of ethanol amine -N-lissamin a ro damma B sulfonyl, ammonium salt (DHPE-rhodamine) labeled with the rhodamine fluorescent probe. The lipids containing the fluorescence marker were added to the liposomes during the preparation steps of the lipid film and were purchased from Molecular Probes®, Celi Labeling and Detection (Life Technologies ™, Grand Island, NY, USA). Cervical cancer cells (HeLa), human breast cancer (MCF-7) and Raw 264.7 mouse macrophage cells were purchased from the American Type Cells Collection (ATCC). All lines Cells listed above were maintained in culture using Dulbecco's Modified Eagle Medium (Gibco, Invitrogen Corporation, U.K.) supplemented with the addition of 10% v / v fetal bovine serum (FBS) and 1% v / v d i a penicillin / streptomycin solution (Invitrogen, Life Technologies ™, Grand Island, NY, LISA). Cells were incubated at 37 ° C with 5% CO <2>. The plasmid containing DNA encoding proteins that fluorescence at wavelengths of green light (pCMV-GFP) was purchased from Plasmid Factory (GmbH & Co. KG, Germany). FuGene <®> HD transfection agent was purchased by Promega corporation (Madison, USA). The FAM ™ dyedabeled Pre-miR Negative Control miRNA dye, used to identify the amount of miRNA and siRNA during the different phases of the trial, was purchased from Life TechnologiesTM (Grand Island, NY, USA). Rabbit polyclonal IgG antibodies used to identify ERa expression was purchased from Santa Cruz Biotechnology, Ine. (Santa Cruz, CA, USA); while the rat mAB, to identify GAPDH, was purchased by Celi Signaling (Merck Millipore S.p.a., Vimodrone (MI), Italy). Murine TNF-α, IL-6 and IL-Ιβ ELISA kits were purchased from eBioscience (eBioscience, Ine. San Diego, CA, LISA).

Example 2: Preparation of the AVs.

The AVs were prepared using the Thin Layer Evaporation (TLE) method. The detailed steps of the method are shown below.

The IL formulation was obtained using DOTAP / Cholesterol. In particular, 40 mg of a lipid formulation consisting of DOTAP / Cholesterol in a 7/3 mole fraction, or of DPPC / DC-Chol, in a 7/3 mole fraction, were solubilized in a glass flask with an emery neck using 2 ml of chloroform. The resulting lipid mixture was evaporated under pressure and the organic solvent was removed using a rotary evaporator (Buchi 461, Buchi Italy, Assago (MI), Italy) heated using a thermostated bath at a temperature of 65 ° C and connected to a diaphragm vacuum pump (Buchi Italy, Assago (MI), Italy) at a pressure of 200 mBar. To remove the excess organic solvent, the obtained lipid film was dried overnight using a thermostat-controlled dryer, programmed at a temperature of 45 ° C (Buchi mod. TO-51, Buchi Italy, Assago (MI), Italy), connected to a diaphragm vacuum pump (Buchi Italy, Assago (MI), Italy) at a pressure of 200 mBar. The lipid film IL was then hydrated with 500 µl of double distilled water and the resulting lipid suspension was subjected to 3 alternating cycles of continuous stirring and heating of 3 minutes each. In particular, the lipid suspension IL was first stirred continuously at 700 rpm using a mechanical bench shaker (Vortex, Heidolph Reax 2000) and then thermostated at 65 ° C in a thermostated bath, for 3 times (alternating cycles for each procedure) as previously reported. Another 500 µl of double distilled water was added to the IL formulation to obtain a liposomal formulation with a final volume of 1 ml, which was then used to hydrate the EL formulation. The final lipid concentration measured in the IL liposomes was 13., 8 mM. The miRNAs, pre-miRNAs and siRNAs were complexed with IL liposomes during the hydration phase of the lipid film. In particular, miRNA, pre-miRNA and siRNA were added to double distilled water devoid of the nucleic acid degradation enzyme (RNAase) and used to hydrate the lipid film of the ILs as previously reported.

The EL formulation was prepared using the following lipids: DOPE / Chol / DSPE-PEG (2000). In particular, 40 mg of a lipid formulation consisting of DOPE / Chol / DSPE-PEG (2000) in 5/3/2 molar fraction were solubilized in a glass ground flask using 2 ml of chloroform. The resulting lipid mixture was evaporated under pressure and the organic solvent was removed using a rotary evaporator (Buchi 461, Buchi Italy, Assago (MI), Italy) heated using a thermostated bath at a temperature of 65 ° C and connected to a diaphragm vacuum pump (Buchi Italy, Assago (MI), Italy) at a pressure of 200 mBar. To remove the excess organic solvent, the obtained lipid film was dried overnight using a thermostatted dryer programmed at a temperature of 45 ° C (Buchi mod. TO-51, Buchi Italy, Assago (MI), Italy), connected to a diaphragm vacuum pump (Buchi Italy, Assago (MI), Italy) at a pressure of 200 mBar. The lipid film EL was then hydrated with 1 ml of the liposomal formulation IL obtained as described above. The lipid suspension obtained from the combination of IL and EL liposomes was subjected to 3 alternating cycles of continuous stirring and heating of 3 minutes each. In particular, the lipid suspension obtained from the combination of IL and EL liposomes was first stirred continuously at 700 rpm using a bench mechanical stirrer (Vortex, Heidolph Reax 2000) and then thermostated at 65 ° C in a thermostated bath, to 3 times (alternating cycles for each procedure) as previously reported. The combination of IL and EL liposomes thus led to the formation of A \ <7> s which have a total lipid concentration equal to 26.5 mM. The obtained AVs were incubated at 60 ° C for another 2 hours to favor the formation and stabilization of the supramolecular structure. The A \ <7> s obtained were then extruded using a stainless steel extruder (Lipex Biomembranes, Northern Lipids Ine., Vancouver, BC, Canada) under nitrogen pressure. The extruder is equipped with a thermoregulation system connected to a thermostat (Grant Instruments Ltd, Cambridge, LIK). The extrusion temperature was programmed at 65 ° C. Before the extrusion process, the AVs were brought to the same extrusion temperature (65 ° C) for at least 45 minutes. The extrusion was carried out at a pressure of about 850 kPa through two superimposed polycarbonate filters (diameter of 25 mm; Nucleopore Corp., Pleasanton, CA, USA) with pores of 200 nm size. The AVs were extruded at 65 ° C at a pressure of 850 kPa ten times through the polycarbonate filters with pores of 200 nm diameter. After the extrusion procedure, the yield of the AVs was greater than 94%.

Example 3: Physico-chemical characterization of AYs

Table 1 reports the main chemical-physical parameters, such as average dimensions, dimensional homogeneity (PDI) and surface charge (zeta potential or ZP) of the internal bilayer IL.

Table 1. Physico-chemical characterization of IL. The values obtained represent the mean of 6 different analyzes ± the standard deviation (SD).

Average size PDI ± SD ZP (V) ± SD (nin) ± SD

DOTAP / Cholesterol <1> 65 ± 1.23 0.37 ± 0.01 47.5 ± 0.31 <1> Each analysis represents the mean of 6 different analyzes ± the standard deviation.

Table 2 reports the main chemical-physical parameters, such as average size, dimensional homogeneity (PDI) and surface charge (zeta potential; ZP) of the vesicular structure that forms the external bilayer (EL) of the AVs.

Table 2. Physico-chemical characterization of EL. The values obtained represent the mean of 6 different analyzes ± the standard deviation (SD).

Average size PDI ± SD ZP (mV) ± SD (nm) ± SD

D OPE / C olesterol / DSPE- 116 ± 1.05 0.24 ± 0.05 -46.9 ± 0.42 PEG 2000 <1>

<1> Each run represents the mean of 6 different runs ± the standard deviation.

The chemical-physical parameters obtained provide numerous data relating to the self-assembly of IL and EL within the AVs. Both the average size and the PDI of the IL and EL phospholipid bilayers show differences in their initial values and both the chemical-physical parameters depend on the lipid composition and the molar fractions of the lipids used to prepare the two colloidal formulations (Table 1 and 2) . The surface charge of opposite sign that distinguishes IL and EL favors the electrostatic interaction between the individual liposomal formulations, their self-assembly and their stabilization probably due to the strong electrostatic interaction that occurs between IL and EL. This attractive effect between the two bilayers with opposite surface charge, which favors the formation and stabilization of AVs, is confirmed by the reduction of the average size, of the PDI and of the ZP value in the A \ <7> s (Table 3). The AVs, in fact, have an average size of 146.0 nm, a PDI of 0.22 and a ZP value of -28.5 mV. The presence of a negative surface charge in the AVs shows that the external bilayer, and therefore the surface of the supramolecular system formed, is made up of EL that surrounds the inner layer (core) formed by IL (positive charge).

The measurements of dynamic light scattering (DLS) also show that 99% of the particles have the chemical-physical parameters described above and reported in Table 3.

The extrusion process led to a further reduction in both the average size (131 nm) and the PDI (0.12) of the AVs while ZP undergoes only a small reduction (-26.9 mV) of its values (Table 3).

Table 3. Physico-chemical characterization of AVs. The values obtained represent the mean of 6 different analyzes ± the standard deviation (SD).

F ormulations Dimensions SD PDI ± SD ZP (mV) ± SD Average (mn) ±

SD AVs <1> 146 ± 1.5 0.22 ± 0.01.-28.5 ± 1.1 AVs <1> 200 imi 131 ± 0.8 0.12 ± 0.01 -26.9 ± 0.5

<1> Each run represents the mean of 6 different runs ± the standard deviation.

The structure of the AVs, highlighted by analyzing the chemical-physical parameters, obtained from the analysis of dynamic light scattering (DLS), was confirmed by electron microscopy studies, which highlighted the presence of an internal bilayer, formed by IL liposomes, and an external bilayer formed by the EL liposomes. The obtained AVs showed the best chemical-physical properties and the best technological formulation parameters for the conveyance of the genetic material (average size -140 nm, good dimensional homogeneity and surface charge (zeta potential) equal to ~ -21 mV). AVs are capable of complexing large amounts of both pi asthmatic DNA and dsRNA (miRNA and siRNA); they are stable in serum for over 72 hours of incubation and protect dsRNAs (miRNA and siRNA) from enzymatic degradation mediated by RNase A.

The biocompatibility of AVs and their ability not to activate the immune system and not to provoke an immune response was evaluated in vitro using HeLa and Raw 264.7 cells. The experimental data obtained have shown that the AVs are biocompatible and do not stimulate the activation of the immune system in in vitro experimental models. The AVs are also internalized by the cells and favor the incorporation of the material both for the delivery of the plasmid DNA and of the dsRNA (miRNA and siRNA), thus favoring the intact release of the encapsulated genetic material inside the cells as demonstrated by the transfection experiments obtained using HeLa cells. The transfection efficiency and antitumor activity of AVs containing dsRNA (miRNA and siRNA) were evaluated using MCF-7 cells. The experimental data obtained have shown that AVs increase the transfection efficiency of miRNA and siRNA in MCF-7 cells compared to both the control (untreated cells) and the complex formed by dsRNA (miRNA and siRNA) and the agent of commercial transfection Fu Gene®. Furthermore, AVs containing dsRNA (miRNA and siRNA) decrease cell viability of MCF-7 cells compared to untreated (control) cells and dsRNA (miRNA and siRNA) / FuGene® complex. The reduction in cell viability on MCF-7 cells is a consequence of the inhibition of ERa gene expression promoted by AVs that contain dsRNA (miRNA and siRNA). The experimental results obtained show that AVs can represent innovative non-viral nanovectors for the delivery of plasmid DNA and dsRNA (miRNA and siRNA).

Example 4: AV cytotoxicity assays.

The cytotoxicity of AVs not complexed with siRNA and miRNA was evaluated using the MTT method (cell viability assay). The cells were seeded into 96-well cell culture plates at a concentration of 5 x 10 <3> cells / 0.2 ml and incubated for 24 hours at 37 ° C to promote their adhesion to the bottom of the cell culture plates. Once a confluence of 70% was obtained, the cell culture medium was removed and replaced with new medium (control) or the different formulations to be tested, such as cationic liposomes, FuGene® and AYs). The treated cells were incubated for 24, 48 and 72 hours. Cells treated with cell culture medium alone and cells not treated with any formulation were used as controls.

10 LI 1 of tetrazolium salts, solubilized in PBS (5 mg / ml) were added to each well and the cells were incubated for a further 3 hours. The cell culture medium containing the tetrazolium salts was subsequently removed and the formazan crystals, formed by the precipitation of these salts at the bottom of each well after mitochondria oxidative reaction to e, were solubilized using 200 µl of a solution consisting of DMSG. / ethanol (1: 1, v / v). The solution obtained was stirred for 20 minutes at 230 rpm using an orbiting shaker (IRA® KS 130 Control, IRA® WERRE GMBH & Co., Staufen, Germany). The formazan crystals obtained were then quantified using a bench spectrophotometer with reading plate (Multiscan MS 6.0, Labsystem) programmed at a wavelength of 540 nm, with a reference wavelength set at 690 nm. The viability percentage was calculated using the following equation:

Cell viability (%) = Abst / Absc x 100

In which, Abst represents the absorbance value of the cells treated with the different formulations and Absc represents the absorbance value of the untreated cells (control). The concentration of formazan crystals obtained is directly proportional to cell viability. The values obtained are the mean of 6 different experiments ± the standard deviation.

Figure 2 shows that the AVs do not produce toxic effects in the treated cells, at the same concentrations and at the same incubation times, compared to both FuGene® and cationic liposomes. Example 5: Interaction between AVs and cellular models used during the experimentation.

The interaction between the cell lines reported above and the AVs labeled with the rhodamine fluorescent probe was studied by confocal microscopy (CLSM). The DHPE-rhodamine phospholipid was solubilized in chloroform with the other phospholipids used to prepare the EL formulation during the preparation of the lipid film. CLSM measurements were performed by seeding cells at a density of 4 x 10 <5> cells / mL in 6-well cell culture plates. The cells were placed on a sterile slide inserted into each well and covered with DMEM culture medium. The cells were incubated for 24 hours and subsequently treated at different incubation times (6 and 24 hours) with AVs containing DHPE-rhodamine. At different incubation times, the different wells were washed with 2 ml of PBS (3 times) to remove excess AVs containing the fluorescent marker rhodamine and the cells were subsequently fixed on the slide using 1 mg of ethanol ( 70% v / v) in solution. Subsequently the slides were washed again with 2 ml of PBS (3 times) and finally the PBS (2 ml) was replaced with a fresh solution of the same buffer. The DAPI dye was used to label the cell nuclei. The 6-well cell culture plates containing the fixed cells were stored at 4 ° C until CLSM analysis. The slides with the cells adhered to their surface and fixed in advance were covered with other slides. A glycerol solution (70% v / v) was used to fix the cells and remove the air trapped between the two slides. Finally, the glass plates containing the samples were fixed with transparent enamel to avoid the inclusion of air in the sample. The measurements were performed using a Leika TCS SP2 MP CLSM microscope programmed at the excitation wavelength Xexc = 557 nm and the rhodamine emission Xeni = 571 nm. The microscope parameters are shown below: resolution between 4096 x 4096 pixels, Ar / Kr laser beam with a nominal power of 75 mW, correlated with a filter for fluorescence analysis. The samples were processed using the Micro Developer software package which allows a multimedia series acquisition and direct access to the digital control panel. A 100x immersion microscopy lens was used during the experiments.

Figure 3 shows that AVs interact better with HeLa cells (cell model) after 6 and 24 hours of incubation than cationic liposomes.

Example 6: Evaluation of the intracellular internalization mechanisms of AVs.

The internalization of AVs in the cell lines reported above was evaluated using CLSM (Confocal Laser Scanning Microscopy) analysis. Briefly, MCF-7 cells were seeded in 4 sections of a BD FalconTM chamber (Bedford, USA) at a concentration of 1 x 10 <5> cells / 500 id of DMED cell culture medium and incubated at 37 ° C, 5 % CO2. After 24 hours of incubation, the cells were treated with PremiRNA-FAM / FuGene® complexes encapsulated in AVs and with complexes formed by PL'e-miRNA-FAM / FuGene®. The cells were subsequently collected at different incubation times (0.5; 1, 3 and 6 hours). The cellular internalization of Pre-miRNA-FAM / FuGene® encapsulated in AVs was evaluated by measuring the effects of press ap on the action of miRNAs with Fu Gene® alone. For this reason, the internalization of AVs in lysosomes, late and early endosomes was monitored and evaluated, as reported in the protocol provided by the manufacturer of the reaction kit.

Figure 4 shows that AVs containing Pre-miRNA-FAM / FuGene® and labeled with the rhodamine fluorescent probe are better internalized within MCF-7 cell nuclei and increase miRNA nuclear transfection when compared to cationic liposomes.

Example 7: Evaluation of the serum stability of AVs.

The serum stability of the AVs was measured by Dynamic Light Scattering (DLS).

200 µl of A \ <7> s were added to 1 ml of a solution consisting of FBS / PBS (70:30 v / v, pii 7.4) and the suspension obtained was incubated at 37 ° C under continuous stirring (Li et al. 2012). The Pre-miRNA-FAM / FuGene® complex and the cationic liposomes were used as controls during the experiment. The analysis of the different formulations by DLS was performed at time zero (control) (Figures 5 - i, ii and iii) and at different incubation times (1, 6, 12, 24 and 48 hours) (Figures 5 - A , B and C). The average sizes of the different formulations were measured as a function of the intensity parameter at the different incubation times.

Figure 5 shows that AVs are the only stable formulation in serum for more than 24 hours of incubation (Figure 5 - D). The Pre-miRNA-FAM / FuGene® complex is degraded after only one hour of incubation due to the interaction of the genetic material with the whey proteins and is followed by the formation of aggregates of different sizes (Figures 5 - B). Cationic liposomes are stable for less than 24 hours (Figure 5 - C).

Example 8: Enzymatic degradation assays in the presence of RNase A,

The ability of AVs to protect encapsulated miRNAs from enzymatic degradation was evaluated by electrophoretic analysis (Yuan et al. 2010). Briefly, 0.2857 id of RNase A (2 units) was added to 4 iliof miRNAs encapsulated in AVs or 0.1 Lig of bare miRNA (scramble). The samples obtained were then incubated at 37 ° C under continuous stirring for 1 hour. To inactivate RNase A, the different samples were treated with 4 ul of EDTA (0.25 M) for 10 minutes and mixed with a 1.0% (v / v) sodium dodecyl sulfate (SDS) solution previously solubilized in a 1 M NaOH solution (pH 7.2 - 7.5). The obtained samples were brought to a final volume of 18 µl and, finally, they were incubated for 1 hour. Electrophorethical analysis was performed on a 1% w / v agarose gel, with an electrophoretic run performed in TBE buffer for 45 minutes at 100 V. bare miRNA (scramble (serious); Figure 6 - line 1) and the scrl / FuGene® complex were used as control samples during the experiments.

AVs protect (serious) miRNAs from degradation produced by the enzyme RNase A. Non-degraded (serious) miRNAs (Figure 6, line 1), are visible only when encapsulated in AVs (Figure 6, line 4), but not when are present in the miRNA (seri) / FuGen and <®> complex (Figure 6, line 6).

Example 9: Evaluation of the in vitro transfection efficiency of AVs.

The in vitro transfection efficiency of AVs was measured using FAM-miRNA. The transfection efficiency was assessed from a quantitative point of view using a FACSCaliburTM flow cytome ter (BD Bio Sciences, San Ho se, CA, LISA).

HeLa cells were seeded into 6-well cell culture plates at a concentration of 5 x 10 <5> cells / well and incubated overnight at 37 ° C in 2 ml of DMEM cell culture medium complete with penicillin / FBS. streptomycin. At 70-80% confluence, the cell culture medium was removed and the cells were washed 2 times with PBS, incubated with serum-free cell culture medium and treated for 6 hours with encapsulated FAM-miRNA / FuGene® in AVs (Figure 7 - C). The serum-free cell culture medium was then replaced with complete medium and the cells were incubated for an additional 24 hours. The FuGene <®> / FAM-miRNA complex was used as a positive control during the experiments (Figures 7 - B). The in vitro transfection efficiency of the different formulations was determined by measuring the shift in fluorescence intensity of the different samples with respect to the control (Figure 7 -A).

AVs have a transfection efficiency of 68.57%; while the Fu Gene® / FAM-miRNA complex has a transfection efficiency of approximately 52%.

Example 10: In vitro therapeutic efficacy of miRNAs encapsulated in AVs.

To evaluate the in vitro therapeutic efficacy of AVs, anti-miRNA-18a (anti-18a) was encapsulated in the AVs and its antitumor activity was evaluated using MCF-7 cells as a model. The cytotoxic activity of anti-18a encapsulated in AVs was evaluated using the MTT method (cell viability assay). The anti-18a / FuGene® complex was used as a control in in vitro cytotoxicity experiments on MCF-7 cells. Cells were seeded in 96-well cell culture plates (5 x 10 <3> cells / 0.2 ml) and incubated for 24 hours at 37 ° C. When a confluence of 70% was reached, the culture medium was removed, the cells were washed twice with PBS, incubated with serum-free culture medium and then treated using both the anti-18a / Fugene complex < ®> that the anti-18 encapsulated in AVs. After 6 hours of incubation, the serum-free culture medium was replaced with complete medium and the cells were incubated for another 24, 48 and 72 hours. Anti-18a was used at the final concentration of 10 nM. Untreated cells were used as controls during the experiments.

At different incubation times, 10 µl of tetrazolium salts solubilized in PBS (5 mg / ml) were added to each well and the cell culture plates were kept in incubation for another 3 h. The culture medium was then removed and the formazan crystals obtained were solubilized using 200 Lil of a mixture formed by DMSO / ethanol (1: 1, v / v). The suspension obtained was kept under constant stirring on an orbiting plate for 20 minutes at 230 rpm (IKA® KS 130 Control, IKA® WERKE GMBH & Co., Staufen, Germany). Cell viability was assessed using a spectrophotometer with cell culture plate housing (Multi se an MS 6.0, Labsystem) programmed at a wavelength of 540 nm, with a reference wavelength of 690 nm. The cell viability percentage was calculated using the following formula: Cellular htality (%) = Abst / Absc x 100

in which, Abst represents the absorbance of the cells treated with the different formulations and Absc represents the absorbance of the cells used as control (untreated cells). The concentration of formazan crystals is directly proportional to cell viability. The results obtained are the mean of 6 different experiments ± the standard deviation.

Anti-18 encapsulated in AVs are more effective in reducing cell viability of MCF-7 cells than anti-18 / FuGene <®> complex. The result obtained depends on the better transfection efficacy obtained using the AVs compared to the complex formed by anti-18 / FuGene <®>. Figure 8 demonstrates that anti-18 encapsulated in AVs reduce cell viability by up to 46%, compared to 60% using the anti18 / FuGene <®> complex.

Example 11: Western blotting analysis of the re-efficacy of miRNAs complexed in AVs.

To validate the results obtained in Example 8, the samples were analyzed by western blotting. Cells (5 x 10 <5> cells / 2 mL) were detached from cell culture plates using a trypsin solution, collected after 72 h incubation, centrifuged 1,200 x g for 5 min to remove excess medium, and finally washed twice with PBS. The obtained pellets were solubilized in lysis buffer (35 µl) and frozen for 15 minutes. The samples were then centrifuged at 4 ° C for 10 minutes at 14,800 rpm, the supernatant was collected and the total protein concentration contained in each sample was evaluated. The proteins were then separated using SDS-PAGE by loading 50 µg of proteins into each well. Anti-ERa and anti-GAPDH antibodies were used throughout the experiments. The anti-18a encapsulated in AVs reduce the expression levels of ERa (Figure 9 - line 3), demonstrating a greater efficacy of FuGene <®> (Figure 9 - line 2). This effect depends on the greater in vitro cytotoxic activity of anti-18a carried by AVs in MCF7 cells. No significant reduction of ERa expression levels were obtained using the complex formed by seri / FuGene <®> (Figure 9 - line 4), inserted / FuGene® encapsulated in AVs (Figure 9 - line 5) and empty AVs ( Figure 9 - line 6).

Example 12: Evaluation of the in vitro immune response for AVs in specific cell models.

Raw 264.7 cells (1.5 x 10 <4> cells / 100 µl) were seininated in 96-well cell culture plates, incubated for 24 hours and finally treated with lipopolysaccharide (EPS) (1 Lig / mL), Empty AVs and miRNAs -encapsulated in AVs (100 nM) at 37 ° C for 24 hours. The production of the following pro-inflammatory cytokines (TNF-α, IL-6 and IL-Ιβ) was evaluated using an ELISA kit (Figure 8). All experiments were performed in triplicate. Figure 10 shows that both empty AVs and those containing anti-18 miRNA do not produce any significant immune response after in vitro treatment of Raw 264.7 cells. This effect was evaluated quantitatively by measuring the expression of TNF-α, IL-6 and IL-Ιβ on Raw 264.7 cells after treatment with the different formulations. The results obtained are in agreement with those already reported in Example 2. The lack of immune response after treatment of Raw 264.7 cells with AVs justifies their use in vivo and supports their clinical potential, for example in the treatment of tumor pathologies.

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Claims (10)

CLAIMS 1. Phospholipid-based vesicular system characterized by a structure consisting of two concentric bilayers, an internal bilayer and an external bilayer, each of said bilayers having an internal layer and an external layer, the internal layer of the internal bilayer being provided with a net positive charge; the outer layer of the outer bilayer being neutral or having a net negative charge. 2. Vesicular system according to claim 1, wherein the internal bilayer is formed by positively charged phospholipids selected from DOTAP and DC-cholesterol and the external bilayer is formed by lipids selected from DOPE, DSPE-mPEGs, or phospholipids conjugated to hydrophilic polymers and / or amphipathic. 3. Vesicular system according to any one of claims 1-2, which comprises within the internal bilayer genetic material with pharmacological activity of synthetic or natural origin selected from: oligonucleotides, single and double-stranded DNA and RNA, plasmid DNA, dsRNA, miRNA, siRNA. 4. Vesicular system according to any one of claims 1-3, to be used in the pharmaceutical field. 5. Pharmaceutical compositions comprising the vesicular systems according to any one of claims 1-4, together with pharmaceutically acceptable excipients. 6 Compositions according to claim 5, formulated for administration by parenteral route, intradermal route, subcutaneous route, intramuscular route, intravenous route and subarac us de a 7. Vesicular systems according to any one of claims 1-4 and compositions according to any one of claims 5-6, to be used in the treatment of pathologies of animals and humans which benefit from the administration of genetic material, in particular neoplastic, neurodegenerative pathologies , metabolic, fibroro-proliferative, cardiovascular and psychosomatic. Method for obtaining the vesicular system according to any one of claims 1-3, comprising the following steps: (i) preparing an internal bilayer by solubilizing an aliquot of one or more cationic fospolipids optionally in combination with an aliquot of one or more neutral phospholipids in the presence of a solvent; then dry by eliminating the solvent and rehydrate with water or an aqueous solution by adding an aliquot of the genetic material to be complexed in order to obtain liposomes containing the genetic material; (ii) prepare an external bilayer by solubilizing an aliquot of one or more anionic fospolipids possibly in combination with an aliquot of polyethylene glycol or one or more phospholipids conjugated with polyethylene glycol in the presence of a solvent; then dry by eliminating the solvent and rehydrate the vesicles thus obtained with water or an aqueous solution; (iii) mixing the vesicles obtained in step (ii) with the liposomes obtained in step (i); (iv) incubating at a temperature between 30 ° and 80 ° C for a time between 1 and 24 hours and then subjecting the mixture to extrusion with a cut-off between 800 and 200 nrn. 9. Method according to claim 8, wherein the neutral phospholipids are selected from: cholesterol and cholesterol derivatives; L-a-phosphatidylcholine (Egg, Chicken); L-aphosphatidylcholine, hydrogenated (Egg, Chicken); L-aphosphatidylcholine (95%) (Egg, Chicken); L-a-Phosphatidylcholine (Egg, Chicken-60%) (Total Egg Phosphatide Extract); L-alysophosphatidylcholine (Egg, Chicken); L-a-phosphatidylcholine (Soy); L-a-phosphatidylcholine, hydrogenated (Soy); L-aphosphatidylcholine (95%) (Soy); L-a-Phosphatidylcholine (Soy-40%); L-a-Phosphatidylcholine, 20% (Soy); L-alysophosphatidylcholine (Soy); L-a-phosphatidylcholine (Heart, Bovine); L-a-phosphatidylcholine (Brain, Porcine); L-aphosphatidylcholine (Liver, Bovine); 1, 2 - of 1 aur oy 1 - s n - g ly c er o - 3 -phosphocholine; 1,2-ditridecanoyl-sn-glycero-3-phosphocholine; 1,2-dimyristoyl-sn-glycero-3-phosphocholine; 1, 2 - d ι p en ta d ec anoy 1 - s n -glycero -3 -phosphocholine; 1, 2 -dipalmi toyl-sn-glycero-3-phosphocholine; 1,2-diphytanoyl-sn-glycero-3-phosphocholine; 1,2-diheptadecanoyl-sn-glycero-3-phosphocholine; 1,2-distearoyl-snglycero-3-phosphocholine; 1,2-dinonadecanoyl-sn-glycero-3-phosphocholine; 1,2-diarachidoyl-sn-glycero-3-phosphocholine; 1,2 dihenarachidoyl-sn-glycero-3-phosphocholine; 1,2- dib ehenoyl - sn -glycero-3-phosphocholine; 1,2- ditnco sanoyl - sn - glycero - 3 -phosphocholine; 1,2-dilignoceroyl-sn-glyceiO-3-phosphocholine; Cis-1,2- di n ervo noy 1 - sn - gly c e ro - 3 - p ho s p ho c ho 1 m e; l-myristoyl-2-palirdtoyl-sn-glycero-3-phosphocholine; l-myristoyl-2-stearoyl-snglycero -3 -phosphocholine; 1 - p almitoyl - 2 - ac ety 1 - sn - glycero - 3 -phosphocholine; 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine; l-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine; 1-stearoyl-2-palinitoyl-sn-glyceiO-3-phosphocholine; l-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine; 1 - s te aroy 1 - 2 - ar ac h ì d onoy 1 - sn - gly c ero - 3 - p ho sp h oc ho 11 n e; 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine; l-oleoyl-2-myristoyl-sn-glycero-3-phosphocholine; 1,2-bis (10, 12-tricosadiynoyl) -sn-glycero-3-phosphocholine; 1,2-bis (10, 12-tricosadiynoyl) -sn-glycero-3-phosphoethanolamine; l-palmitoyl-2- (10, 12-tricosadiynoyl) -sn-glycero-3-phosphocholine; l-palinitoyl-2- (10, 12 - tri c o s a diy noy 1) - s n - g ly c e ro - 3 - p ho sp ho eth ano 1 ami n e; the cationic phospholipids are selected from: 1,2-dioleoyl-3-trimethylainmonium-propane (chloride salt); 3β- [Ν-ί <'> Ν', Ν'-dimethylaininoethane) -carbamoyl] cholesterol hydrochloride (DC-Cholesterol-HCl); 1,2- diteti 'a dee anoyl - 3 -trimethyl ammonirmi -propane (chloride salt) (DMTAP); 1,2-dioleoyl-3-trimethylainmonimn-propane (methyl sulfate salt); 1,2-dihexadecanoyl-3-trimethylaininonimn-propane (chloride salt); 1,2dioctadecanoyl-3-trimethylammonium-piOpane (chloride salt); 1-oleoyl-2- [6 - [(7-nitro-2-l, 3-benzoxadiazol-4-yl) amino] hexanoyl] -3-trimethylainmonium propane (chloride salt); Fluorescent 1-oleoyl-2- [6 - [(7-nitro-2-l, 3-benzoxadiazol-4-yl) amino] hexanoyl] -3-trimethyl ammonium propane (chloride salt); N- (4-carboxybenzyl) -N, N - dimethyl -2, 3 -bis (ol eoyloxy) prop an - 1 - amini um (D OB AQ); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-dimyristoyl-3-dimethylammonium-propane; 1, 2 - di p al mi toy 1 - 3-dimethyl ammonium um-propane; 1,2-distearoyl-3-dimethyl ammonium um-propane, dimethyl dioctadecyl annuo ni um (bromide salt) (DDA); 1,2-dilauroyl-sn-glycero-3-ethylphosphocholine (chloride salt); 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (chloride salt); 1,2-dimyristoleoyl-sn-glycero-3-ethylphosphocholine (Tf salt); 1,2-dipalmitoyl-sn-glycero-3-ethylphosphocholine (chloride salt); 1,2-distearoyl-sn-glycero-3-ethylphosphocholine (chloride salt); 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (chloride salt); l-palmitoyl-2-oleoyl-snglycero-3-ethylphosphocholine (chloride salt); 1,2-di-G-octadecenyl-3-trimethylammonium propane (chloride salt) (DOTMA), Nl- [2 - ((1 S) - 1 - [(3 - aminopropyl) amino] -4- [di (3 - aminopropyl) amino] butylcarboxamido) ethyl] -3,4-di [oleyloxy] -benzamide; the anionic phospholipids are chosen from: L-a-phosphatidic acid (Egg, Chicken); L-a-phosphatidic acid (Soy) (sodium salt); 1,2-dihexanoyl-sn-glycero-3-phosphate (sodium salt); l, 2-dioctano3d-snglycero-3-phosphate (sodium salt); 1,2-didecanoyl-sn-glycero-3phosphate (sodium salt); 1, 2 - of 1 auroy 1 - s n - g ly c e ro - 3 - p ho s p h at e (sodium salt); 1,2- dimyri stoyl - sn - glycero - 3 -p hosphate (sodium salt); 1,2- di my ri s toy 1 - s n - g ly c e ro - 3 - p ho sp h at e (sodium salt); 1,2-dipalmitoyl-sn-glycero-3-phosphate (sodium salt); 1,2-diphytanoylsn-glycero-3-phosphate (sodium salt); 1,2-diheptadecanoyl-snglycero -3 -phosphate (sodium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphate (sodium salt); 1,2-dioleoyl-sn-glycero-3-phosphate (sodium salt); 1, 2 - of 1 m ol e oy 1 - sn - gly c ero - 3 - p ho s p h at e (sodium salt); 1, 2 - of 11 no 1 eoy 1 - s n - g ly c er o - 3 - p ho sp h at e (sodium salt); 1,2-di ar ac hi do noyl-sn-glyc ero -3 -phosphate (sodium salt); 1,2-didocosahexaenoyl-sn-glycero-3-phosphate (sodium salt); 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (sodium salt); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphate (sodium salt); 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphate (sodium salt); 1-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phosphate (sodium salt); l-stearoyl-2-oleoyl-sn-glycero-3-phosphate (sodium salt); 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphate (sodium salt); 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphate (sodium salt); 1-stearoyl-2-docosahexaenoyl-sn-glycero-3-phosphate (sodium salt); 1 - h e x an oy 1 - 2 - hy dr o xy - sn - gly c ero - 3 - p h o s p h a te (ammonium salt); 1-myristoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt); 1-p al mi toy 1 - 2 - hy dr o xy - s n - g ly c e r o - 3 - p i sp h at e (sodium salt); 1-heptadecanoyl-2-hydroxy -sn-glycero-3 -phosphate (sodium salt); 1-heptadecanoyl-2-hydroxy-sn-glycero-3-phosphate (sodium salt); 1-ol e o \ d - 2 - hy dr o xy <r> - s n - g ly c ero - 3 - p ho sp h at e (sodium salt); 1-linoleoyl2-hydroxy-sn-glycero-3-phosphate (sodium salt); l -arachidonoyl-2-hydroxy-sn-glycero-3-phosphate (ammonium salt); 1-O-hexadecyl-2-hydroxy-sn-glycero-3-phosphate (aimnonium salt); 1-O-octadecyl-2-hydiOxy-sn-glycero-3-phosphate (ammonium salt); 1- (9Z-octadecenyl) -2-hydroxy-sn-glycero-3-phosphate (ammonium salt); 1 - ρ al mi toy 1 - s n - g ly c and ro - 2, 3 - cy c 11 c - p ho sp h ate (ammonium salt); 1-h e p t a d ec an oy 1 - gly c ero - 2, 3 - cy c 11 c - p h o s p h at e (ammonium salt); 1-oleoyl-sn-glycero-2,3-cyclic-phosphate (ammonium salt); 1-0-h e x a d e cy 1 - sn - gly c e r o - 2, 3 - cy c 11 c - p ho sp h at e (ammonium salt); 1-0- (9 Z - oc t a cl e c eny 1) - s n - gly c ero - 2, 3 - cy c 11 c - p ho s p h at e (ammonium salt); l-oleoyl-2-methyl-sn-glycero-3-phosphothionate (ammonium salt); 1 - o 1 eoy 1 - 2 - methy 1 - sn - gly c ero - 3 - p ho s p ho thi o n at e (ammonium salt); (S) -phosphoric acid mono- {2-octadec-9-enoylainino-3- [4- (pyridin-2-y Ime thoxy) - p h eny 1] - p r opy 1} ester (ammonium salt); (S) -phosphoric acid mono- {2-octadec-9-enoylamino-3- [4- (pyridin-2-ylmethoxy) -phenyl] -propyl} ester (ammonium salt); (R) -phosphoric acid mono- {2-octadec-9-enoylainino-3- [4- (pyridin-2-ylmethoxy) -phenyl] -propyl} ester (ammonium salt); N-palmitoyl-serine phosphoric acid (ammonium salt); N-p almi toyl-ty rosine phosphoric acid (ammonium salt); L-a-phosphatidylglycerol (Egg, Chicken) (sodium salt); L-a-phosphatidylglycerol (Soy) (sodium salt); L-aphosphatidylglycerol (E. coli) (sodium salt); 1,2- clihexanoyl - sn -glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1, 2 - d ι oc t anoy 1 - s n -glyceiO-3-phospho- (r-rac-glycerol) (sodium salt); 1, 2 - eli d e c anoy 1 - s n -glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2-dilauroyl-snglyc ero- 3-phospho- (l'-rac-glyc heroi) (sodium salt); 1,2-dimyristoylsn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2-dipentadecanoyl-sn-glyceiO-3-phospho- (r-rac-glycerol) (sodium salt); 1, 2 -di p almi toy 1 - s n - g ly c e r o - 3 - p ho s p ho - (T - r ac - gly c eroi) (sodium salt); 1,2-diphytanoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1,2-diheptadecanoyl-sn-glyceiO-3-phospho- (r-racglycerol) (sodium salt); 1,2- di ste aroyl - sn - glyc ero - 3 -phospho - (1 '-r ac -glyc eroi) (sodium salt); c ι s - 1, 2 - di o 1 eoy 1 - s n - g ly c e r o - 3 - p ho sp ho - (1 '-rac-glycerol) (sodium salt); trans- 1,2-dielaidoyl-sn-glycero-3-phospho- (r-rac-glycerol) (sodium salt); 1,2-dilmoleoyl-sn-glycero-3-phospho- (l '-rac-glycerol) (sodium salt); 1, 2 - d 111 us and noy 1 - s n - g ly c ero -3-phospho- (r-rac-glycerol) (sodium salt); 1,2- of arac hi donoyl - sn -glycero-3- [phospho-rac- (l-glycerol)] (sodium salt); 1,2-didocosahexaenoyl-sn-glycero-3- [phospho-rac- (l-glycerol)] (sodium salt); l-palmitoyl-2-oleoyl-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); 1 -palmi toyl-2-linoleoy 1-sn-gly cero-3-phospho- (l'-racglycerol) (sodium salt); l -palmitoyl-2-arachidonoyl-sn-glycero-3-phospho- (l '-rac-glycerol) (sodium salt); l-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospho- (r-rac-glycerol) (sodium salt); 1 - s t e ar oy 1 - 2 - o 1 eoy 1 - sn - gly c er o - 3 - p h o s p ho - (Γ - r ac - g h <7> c e r ol) (sodium salt); 1 -ste aroyl -2 -linoleoyl-sn- glyc ero- 3 -phospho - (Γ -racglycerol) (sodium salt); l-stearoyl-2-arachidonoyl-sn-glycero-3-phospho- (r-rac-glycerol) (sodium salt); l-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho- (r-rac-glycerol) (sodium salt); l-myristoyl-2-hydroxy-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); l-palmitoyl-2-hydroxy-sn-glyceiO-3-phospho- (l'-racglycerol) (sodium salt); l-stearoyl-2-hydiOxy-sn-glycero-3-phospho- (Γ-rac-glycerol) (sodium salt); l-oleoyl-2-hydroxy-sn-glycero-3-phospho- (l'-rac-glycerol) (sodium salt); L-a-phosphatidylserine (Brain, Porcine) (sodium salt); L-adysophosphatidylserine (Brain, Porcine) (sodium salt); L-a-phosphatidylserine (Soy, 99%) (sodium salt); 1,2-dihexanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-dioctanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-didecanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-dilauroyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-dimyristoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-dipalmitoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-diphytanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-diheptadecanoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-distearoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-di 11 no 1 eoy 1 - s n - g ly c e r o - 3 - p ho sp ho - L - s e r ì n e (sodium salt); 1,2-diarachidonoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1,2-didocosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt); 1-palmitoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-palmitoyl-2-arachidonoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-palmitoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-stearoyl-2-linoleoyl-sn-glycero-3-phospho-L-serine (sodium salt); l-stearoyl-2-arachidonoyl-sn-glyceiO-3-phospho-L-serine (sodium salt); l-stearoyl-2-docosahexaenoyl-sn-glycero-3-phospho-L-serine (sodium salt); L-adysophosphatidylserine (Brain, Porcine) (sodium salt); l-palmitoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt); 1- (10Z-heptadecenoyl) -2-hydroxysn-glycero-3- [phospho-L-serine] (sodium salt); l-stearoyl-2-hydroxy-sn-glycero-3-phospho-L-serine (sodium salt); l-oleoyl-2-hydroxy-sn-glyceiO-3-phospho-L-serine (sodium salt); L-aphosphatidylinositol (Liver, Bovine) (sodium salt); L-alysophosphatidylinositol (Liver, Bovine) (sodium salt); L-aphosphatidylinositol (Soy) (sodium salt); L-alysophosphatidylinositol (Boy) (sodium salt); L-aphosphatidylinositol-4-phosphate (Brain, Porcine) (ammonium Salt); L-a-phosphatidylinositol-4,5-bisphosphate (Brain, Porcine) (ammonium salt); 1,2-dioctanoyl-sn-glyceiO-3-phospho- (l'-myoinositol) (ammonium salt); 1,2-dipalmitoyl-sn-glycero-3-phospho- (l'-myo-inositol) (ammonium salt); 1,2-distearoyl-sn-glycero-3-phosphoinositol (ammonium salt); 1,2-dioleoyl-sn-glycero-3-phospho- (l'-myo-inositol) (ammonium salt); 1 - p al mi toy 1 - 2 - ol and oy 1 -sn-glycero-3-phosphoinositol (ammonium salt); l-stearoyl-2-arachidonoyl-sn-glycero-3-phosphoinositol (ammonium salt); 1,2-dioctanoyl-sn-glycero-3- (phosphoinositol-3-phosphate) (ammonium salt); 1,2-dioctanoyl-sn-glycero-3-phospho- (l'-myo-inositol-4'-phosphate) (ammonium salt); 1,2-dioleoyl-sn-glycero-3-phospho- (myo-inositol-3'-phosphate) (aminonium salt); 1,2-dioleoyl-snglycero-3-phospho- (r-myo-inositol-4'-phosphate) (ammonium salt); 1,2-dioleoyl-sn-glycero-3-phospho- (r-myo-inositol-5'-phosphate) (ammonium salt); 1,2- di h e x an oy 1 - sn - glycero - 3 - p h o s p ho - (1 '- my o -ì no si tol - 3 5' - b ì sp h o s p h at e) (ammonium salt); 1,2-dioctanoyl-snglycero-3-phospho- (r-myo-inositol-3 ', 4'-bisphosphate) (ammonium salt); L2-dioctanoyl-sn-glyceiO-3-phospho- (r-myo-inositol-3 ', 5'-bisphosphate) (ammonium salt); 1, 2 - di ol e oy 1 - sn - gly c ero - 3 - p ho s p h o - (1 '- my o - 1 no si tol - 4', 5 '- b ì sp h o s p h at e) (ammonium salt ); 1,2-dioleoylsn-glycero-3-phospho- (r-myo-inositol-3 ', 4'-bisphosphate) (ammonium salt); 1, 2 - d i ol e oy 1 - sn - gly c e r o - 3 - p ho s p ho - (1 '- my o -ì no si tol - 3 5' - b ì sp h o s p h at e) (ammonium salt); 1,2-dioleoyl-sngly c er o - 3 - p ho s p ho - (1 '- my o - 1 no s ι tol - 4', 5 '- b ι s p ho sp h at e) (ammonium salt) ; l-stearoyl-2-arachidonoyl-sn-glyceiO-3-phospho- (l'-myoì no si tol - 3 5 '- b ì sp h o s p h at e) (ammonium salt); l-stearoyl-2-arachidonoyl-sn-glyceiO-3-phospho- (r-myo-inositol-4 ', 5'-bisphosphate) (ammonium salt); 1, 2 - di h ex anoy 1 - s n - g ly c e ro - 3 -phospho- (r-myo-inositol-3 ') 4') 5'-trisphosphate) (ammonium salt); l ^ -dioctanoyl-sn-glyceiO-S-phospho-il'-myo-inositol-S'jd'jB'-trisphosphate) (ammonium salt); 1, 2 -dioleo \ d - sn- glycero -3 -phospho - (1 '- myo - 1 no si tol - 3 4', 5 '- tr ì s p ho s p h at e) (ammonium salt); l-stearo \ d-2-arachidonoyl-sn-glyceiO-3-phospho- (r-myo-inositol-3 ') 4', 5'-trisphosphate) (ammonium salt); L-a-lysophosphatidylinositol (Li ver, Bovine) (sodium salt); L-a-lysophosphatidylinositol (Soy) (sodium salt); l-tridecanoyl-2-hydroxy-sn-glyceiO-3-phospho- (myyo-inositol) (ammonium salt); 1 -pahnitoyl-2-hydiOxy-sn-glycero-3-phosphoinositol (ammonium salt); l-stearoyl-2-hydroxy-snglycero-3-phosphoinositol (ammonium salt); l- (lOZ-heptadecenoyl) -2-hydiOxy-sn-glycero-3-phospho- (r-myo-inositol) (ammonium salt); l-oleoyl-2-hydroxy-sn-glycero-3-phospho- (l'-myo-inositol) (ammonium salt); l-arachidonoyl-2-hydroxy-sn-glyceiO-3-phosphoinositol (ammonium salt); the phospholipids conjugated to polyethylene glycol chains are chosen from: 1, 2 - di my ri s toy 1 - s n - g ly c e ro - 3 - p ho sp h o e th anol ami n e - N - [methoxy (polyethylene glycol) - 750] (ammonium salt); 1,2-dip almi toy 1 - sn - glyc ero - 3 -pho sp ho ethanol amine -N - [me thoxy (p oly ethy lene glycol) -750] (ammonium salt); 1,2-disteaiOyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (poly ethy lene glycol) -750] (ammonium salt); 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -750] (ammonium salt); 1,2- dimy ri stoy 1 - sn - glycero - 3 -phosphoethanolamine-N- [methoxy (polyethylene glycol) - 1000] (ammonium salt); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) - 1000] (ammonium salt); 1,2- di ste aroyl - sn - glycero - 3 -phosphoethanolamine-N- [methoxy (polyethylene glycol) - 1000] (ammonium salt); 1, 2 - di ol e oy 1 - s n - g ly c er o - 3 - p ho sp h o e th anol ami n e -N - [me th oxy (p oly e th \ d e n e glycol) - 1000] (ammonium salt ); 1,2-dimyristoyl-sn-glyceiO-3-phosphoethanolamine-N- [me thoxy (poty ethy lene glycol) -2000] (ammonium salt); 1,2dip almitoyl - sn - glyc ero - 3 -pho sp ho ethanol amine -N - [methoxy (polyethylene glycol) -2000] (ammonium salt); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt); 1,2-dioleoylsn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (ammonium salt); 1,2- dimyri stoy 1 - sn - glycero - 3 -phosphoethanolamine-N- [methoxy (polyethylene glycol) -3000] (ammonium salt); 1,2- dip almitoy 1 - sn - glycero - 3 -phosphoethanolamine-N- [me thoxy (polyethylene glycol) -3000] (ammonium salt); 1,2- di ste ai'oyl - sn - glycero - 3 -phosphoethanolamine-N- [me thoxy (polyethylene glycol) -3000] (ammonium salt); 1, 2 - di ol e oy 1 - s n - g ly c er o - 3 - p ho sp h o e th anol ami n e -N- [methoxy (polyethylene glycol) -3000] (ammonium salt); 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -5000] (ammonium salt); 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -5000] (ammonium salt); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -5000] (ammonium salt); 1,2-dioleoylsn - glycero - 3 -p hosphoeth anol amin e -N- [methoxy (polyethylene glycol) -5000] (ammonium salt); N-palmitoyl-sphingosine-1- {succinyl [methoxj · (polyethylene glycol) 750]}; N-palmitoylsphingosine- 1- {succinyl [methoxy (polyethylene glycol) 2000]}; N-palmitoyl-sphingosine- 1- {succinyl [methoxy (polyethylene gly col) 5000]}; 1, 2 - di s te ar oy 1 - s n - g ly c e r o - 3 - p ho sp h o e th anol ami n e - N [dibenzocyclooctyl (polyethylene glycol) -2000] (ammonium salt); 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [azido (polyethylene glycol) -2000] (ammonium salt); 1, 2 - di ste aroy 1-sn - glycero - 3 -p hosphoeth anol amin e - N- [succ inyl (poly ethyl ene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [carboxy (poly ethyl ene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [maleimi de (poly ethyl ene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [PDF (poly ethyl ene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [amino (poly ethyl ene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [b ioti ny 1 (p o ly ethy lene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [cyanur (poly ethyl ene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [folate (poly ethyl ene glycol) -2000] (ammonium salt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [folate (poly ethyl ene glycol) -5000] (ammonium s alt); 1,2- di ste aroy 1 - sn - glycero - 3 -phosphoethanolamine-N- [poly (ethyl ene glycol) 2000-N'-carboxyfluorescein] (ammonium salt); the solvents used to solubilize the phospholipids and prepare the internal bilayer (IL) described in point (i) and the external bilayer (EL) described in point (ii) are selected from: < > non-polar solvents, for example selected from: diethyl ether, chloroform, ethyl acetate, dichloro methane; polar aprotic solvents, for example selected from: acetone, acetonitrile, dimethylformamide; <> polar protic solvents, for example selected from: butanol, 2-propanol, 2-propanol, ethanol, methanol, water and buffered and non-buffered aqueous solutions. Method according to any one of claims 8-9, wherein surfactants and co-polymers are used in substitution and / or in association with phospholipids conjugated to polyethylene glycol chains, and are selected from: GM1 Ganglioside (Brain, (Ovine- So diurn Salt); GM3 Ganglioside (Milk, Bovine -Ammonium Salt); GD3 Ganglioside (Milk, Bovine -Ammonium Salt); Total Ganglioside Extract (Brain, Porcine -Ammonium Salt); D-lactosyl-β-Ι, Γ N - 1 auroy 1 - D - ery thro -sphingosine; D-lactosyl-β-Ι, Γ N-pahnitoyl-D-erythro -sphingosine; D-Lactosyl ^ -l, l'-N-Heptadecanoyl-D-erythro- Sphingosine; D-lactosyl-β-Ι, Γ N-stearoyl-D-erythiO-sphingosine; D-lactosyl-β-Ι, Γ N-lignoceroyl-D-erythiO -sphingosine; D-lactosyl ^ l-l'-N-nervonoyl -D - ery th r o - s p h m go sm e; polyethylene glycol with different molecular weight, polypropylene glycol with different molecular weight and with different branching in the side chain, polysorbates with different molecular weight, polyethylene glycol and polypropylene glycol co-polymers and with different molecular weight, side chain branches and number of monomer units.