ITRM20120480A1 - MULTICOMPONENT LIPID NANOPARTICLES AND PROCEDURES FOR THEIR PREPARATION. - Google Patents

Description

"MULTICOMPONENT LIPID NANOPARTICLES AND PROCEDURES FOR THE

THEIR PREPARATION "

DESCRIPTION

The present invention relates to the field of gene therapy, in particular it relates to new multicomponent lipid nano particles for the non-viral transport of nucleic acids and processes for their preparation and their use in gene therapy.

STATE OF THE PRIOR ART

Gene therapy consists in the transfer of genetic material inside a cell in order to temporarily or permanently alter its phenotype. It offers new treatment options for numerous diseases, both inherited and acquired, which conventional clinical procedures cannot effectively cure. It is therefore considered the therapy of the future and in recent years it has been the subject of intense study and development for its possible use on a large scale in the clinical setting (â € œClinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacyâ €. Chang, H .; Yeh, M. Int. J. Nanomed. 2012, 7, 49-60). The first step consists in identifying the single gene or the different genes responsible for the genetic disease. This is possible thanks to the great progress of molecular biology methods developed since the 1980s. These techniques allow the cloning and sequencing of various genes. This entailed the precise identification of many gene alterations in different pathologies and the ability, thanks to recombinant DNA techniques, to modify microorganisms (such as bacteria or fungi) in order to make them express molecules of interest. The next step is to evaluate the possibility of transfecting the somatic cells of an individual with a genetic disease with a segment of DNA containing the healthy allele. This approach was subsequently extended to diseases such as tumors, HIV infections and other pathologies in which a defective gene is not replaced but one is added that can set in motion a therapeutically useful phenomenon.

A decisive component of gene transport consists in the conveyance of the DNA inside the target cells by means of vectors that protect it from degradation and ensure its transcription. The vectors used are currently divided into viral and non-viral. Viruses have an excellent tendency to infect cells and insert their DNA into them both by integrating it and in the form of an episome. On the other hand, viral vectors have some drawbacks that limit their use such as the activation of the immune system, toxicity and the reduced size of the transportable genetic material (usually <40,000 base pairs). This last property is very limiting considering that many important human genes are longer than 40,000 base pairs if we also consider the regulatory sequences and non-coding introns. Among the non-viral vectors, cationic liposomes, vesicles with a double lipid layer made up of cationic lipids and neutral lipids, represent an attractive opportunity for molecular engineering since they are configured as real self-assembling biomaterials in micro / nanoscale (Elsabahy et al. â € œNon-Viral Nucleic Acid Delivery: Key Challenges and Future Directionsâ €. Current Drug Delivery, 8, 2011, 235-244). In particular, cationic liposome-DNA complexes (lipoplexes) represent the most common lipid-based materials. Lipoplexes have some considerable advantages over viral vectors such as, for example, ease of large-scale preparation, low toxicity, virtually no limit on the size of the transportable DNA. This last aspect is significant in the hypothesis of transferring entire chromosomes into the nucleus of the cell (the average size of human chromosomes is between 50 and 280 million base pairs). In spite of the above advantages, the main limitation of lipoplexes is the low transfer efficiency of the genetic material (transfection efficiency, TE). To date, the primary approach to increase the transfection efficiency of lipoplexes is the synthesis of new amphipathic molecules or the use of adjuvant non-cationic lipid species. Recently, lipid nanoparticles are emerging as a potential alternative to lipoplexes as they have chemical-physical properties (size, surface charge, ability to condense genetic material, etc.) that are easier to control. These particles consist of a nucleus of DNA (or short-interfering RNA, siRNA) condensed by divalent cations (Ca2 +, Mg2 +, Mn2 +; Fe2 +), poly-cations (spermine, spermidine), cationic amino acids (arginine, lysine, histidine) and cationic proteins (protamine, histones) coated by a lipid shell consisting of one to a few lipid double layers. The preparation method is consolidated and consists of two successive steps: (i) the formation of the DNA nucleus / condensing agent; (ii) the subsequent coating of the DNA core / condensing agent with a lipid shell. The condensation of DNA occurs as a result of the electrostatic interaction between the negative charges of the DNA and the positive charges of the cationic condenser. By varying the mixing ratio between the DNA and the condensing agent, it is possible to obtain DNA / condensing agent nuclei with different charges. This allows to cover the DNA / condensing agent nucleus with lipid shells which vary according to the charge of the aforementioned nucleus.

The net charge of the surface of the nanoparticles allows a non-covalent functionalization by electrostatic interaction with ligands of opposite charge. The activity of the lipid nanoparticles is strictly connected to the specific chemical-physical properties of the formulation adopted.

There are numerous publications in the field of lipid nanoparticles, such as the publications of the group of dr. L. Zhang of the University of California at San Diego (UCSD).

In the publication â € œProgrammed packaging of multicomponent envelope-type nanoparticle system for gene deliveryâ € Daniela Pozzi, Carlotta Marianecci, Maria Carafa, Cristina Marchini, Maura Montani, Augusto Amici and Giulio Caracciolo. Appl. Phys. Lett. 96, 183702 (2010); doi: 10.1063 / 1.3427354 it is shown that a â € œprogrammed packagingâ € procedure allows to obtain lipid nanoparticles of controlled size and charge. The lipids used are anionic lipids.

In the publication â € œFactors Determining the Superior Performance of Lipid / DNA / Protammine Nanoparticles over Lipoplexesâ € Giulio Caracciolo, Daniela Pozzi, Anna Laura Capriotti, Carlotta Marianecci, Maria Carafa, Cristina Marchini, Maura Montani, Augusto Amici, Heinz Amenitsch, Michelle A. Digman, Enrico Gratton, Susana S. Sanchez, and Aldo LaganÃ. J. Med. Chem. 54 (12), pp 4160â € “4171 (2011) nanoparticles are described consisting of a single cationic lipid species (DOTAP / protamine / DNA). In this same publication, the factors that determine the higher efficiency of DOTAP lipid nanoparticles compared to DOTAP / DNA lipoplexes are discussed.

In this continuously developing sector, but still subject to extensive improvements, the main problem encountered by the researchers consists in identifying a nanocarrier capable of efficiently overcoming the barriers of cell transfection. With a view to marketing and medical use that can be approved and standardized, a nano-carrier must have the following characteristics: (i) controlled size and charge; (ii) effective cell internalization; (iii) cytoplasmic release of the complete genetic load; (iv) efficient entry into the nucleus; (v) high transfection efficiency; (vi) high cell viability.

SUMMARY OF THE INVENTION

In order to solve the technical problem indicated above, the inventors have analyzed numerous formulations of nano particles and have identified a specific formulation of nanoparticles more efficient than the reagents commonly used for lipid-based transfection currently on the market. In fact, a multicomponent lipid formulation has been created that incorporates the advantages of each lipid species. The object of the present invention is in fact a multicomponent lipid nanoparticle consisting of a nucleus consisting of one or more nucleic acid molecules and one or more condensing agents of nucleic acids and an external coating consisting of cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and from the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC); a process for the preparation of multicomponent lipid nanoparticles comprising the following steps:

to. nucleic acid molecules and a set of one or more condensing agents the nucleic acids are dissolved separately at the same concentration in distilled H2O and are then mixed together at a ratio between 1: 1 and 1: 0.5 thus obtaining a complex negatively charged (For slight agitation);

b. a desired volume of the complex obtained in step a. It is left to equilibrate for a period between 30 and 180 minutes at room temperature;

c. multicomponent cationic liposomes are prepared by individually dissolving the cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) phospholipids in a suitable organic solvent and desired quantities of each dissolved lipid are mixed until a homogeneous mixture is obtained;

d. the organic solvent is completely removed from the mixture obtained at point c. thus obtaining a lipid film;

And. the lipid film obtained in point d. It is hydrated with a suitable buffer at cellular physiological pH until a desired final concentration is obtained;

f. the lipid solution obtained in point e. It is sonicated until a clear solution is obtained (which indicates the formation of uni-lamellar vesicles here also referred to as cationic liposomes);

g. the balanced complex obtained in point b. It is made to interact with an appropriate volume of the solution obtained at point f. at a charge ratio rho (Ï) mole of cationic lipid / nucleic acid base of between 2 and 3 extremes inclusive and is incubated at about 37 ° C until the formation of nano particles and called nanoparticles for use in gene therapy, or a gene therapy method comprising a step for administering said nanoparticles.

The inventors have shown (data reported in the experimental section) that, compared to the most common reagents for cell transfection existing on the market (Lipofectamine, Lipofectamine 2000, Lipofectamine Plus), the multicomponent lipid nanoparticles object of the present invention show numerous advantages relative to the problems indicated over. The main advantages are the following:

(i) The nanoparticles have controlled dimensions and extremely monodisperse;

(ii) The surface charge of the nanoparticles is positive and extremely reproducible;

(iii) Cell internalization is fast and extremely effective;

(iv) The cytoplasmic release of the genetic load is complete;

(v) The released genetic material effectively enters the nucleus;

(vi) The transfection efficiency of the nanoparticles is higher than that of the reagents commonly used in lipotransfection (such as Lipofectamine, Lipofectamine 2000, Lipofectamine Plus) (â ‰ ˆ 10 <10> RLU / mg protein) in different cell lines (NIH 3T3; HeLa; CHO; A17).

(vii) The nanoparticles are not toxic (cell viability> 70%).

GLOSSARY

By lipid nanoparticles for the purposes of the present invention we mean particles consisting of a nucleus of nucleic acids coated with one or more lipid layers (lipid shell) suitable for use in gene therapy.

With the term condensing agent nucleic acids are meant positively charged molecules commonly known for their ability to condense nucleic acids, endowed with positive charge, to this category belong different classes of substances known in literature as described in the state of technique.

By Lipids for the purposes of the present invention we mean both lipids as commonly understood in scientific dictionaries, and their derivatives such as cholesterol derivatives and phospholipids.

As nucleic acids we mean: dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof. These nucleic acids may be single and / or double-stranded, in the form of vectors that can be integrated into the genome, transposable, minichromosomes (that is, with centromere and telomeres or more generally, with elements that allow autonomous replication), provided with a ... ™ origin of replication etc.

By gene transport we mean those methodologies that allow to transfer an exogenous nucleic acid inside a cell in order to temporarily or permanently alter its phenotype such as transfection techniques that allow to express or silence specific genes in transformed cells .

The term gene silencing is used in the present description in accordance with the state of the art.

Gene therapy means the insertion of genetic material as defined above (nucleic acids) inside cells in order to be able to cure pathologies. This insertion procedure, known as transfection, allows the transfer of one or more healthy genes into a diseased cell, in order to treat a disease caused by the absence or defect of one or more (mutated) genes or of one or more nucleic acids capable of silencing genes that are over-expressed or expressed in the diseased cell.

The acronym DOTAP indicates the cationic lipid 1,2-dioleoyl-3-trimethylammoniumopropane. The acronym DC-Chol indicates the cationic lipid derived from cholesterol 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride.

The acronym DOPE indicates the phospholipid dioleoylphosphatidylethanolamine.

The acronym DOPC indicates the phospholipid dioleylphosphocoliin.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1 shows in 1 (A) the transfection efficiency in CHO ovarian cells of different nanoparticles according to the invention (the relative ratios between the cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP) are shown, 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) under each histogram most common reagents for cell transfection. The gray histograms refer to experiments carried out using 5 µg of sample (for sample we mean the solution of cationic liposomes described in point f. Of the above method) per well, those in black using 10 µg of sample per well. In 1 (B) the figure shows the transfection efficiency in NIH 3T3 fibroblasts of different nanoparticles according to the invention (the relative ratios between the cationic lipids 1,2-dioleoyl-3-trimethylammoniopropane (DOTAP), 3beta are shown - [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) under each histogram) and of Lipofectamine. The gray histograms refer to experiments carried out using 5 µg of sample per well, those in black using 10 µg of sample per well.

Figure 2 shows the results of a cell viability assay conducted on CHO ovarian cells as described in the experimental section (the relative ratios between the cationic lipids 1,2-dioleoyl-3-trimethylammoniumpropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) under each histogram).

Figure 3 shows representative overlays of fluorescence images and Nomarski images in CHO cells. The white signal is due to the presence of the fluorescent DNA.

For each image a scan was performed along the z axis. As seen in the two representative images, the DNA is uniformly diffused in the cytoplasm and nucleus of the cell. Compared to what is observed with the most common lipid-based transfection reagents, no perinuclear aggregates with confined DNA are visible.

DETAILED DESCRIPTION OF THE INVENTION

The present invention therefore relates to multicomponent lipid nano particles consisting of a nucleus consisting of one or more nucleic acid molecules and one or more condensing agents of nucleic acids and an external coating consisting of cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane ( DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and by the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC).

According to the present invention, since the nanoparticle must be suitable for use in gene therapy, the nucleic acid molecules can be chosen from dsDNA (double strand DNA), ssDNA (single strand DNA) or double or single stranded DNA, RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof.

The nucleic acid molecules can be inserted into suitable vectors that allow their incorporation into the host genome (for example transposable vectors) or that allow their replication and expression or transcription in the host cell (eukaryotic cell), such as for example suitable cloning vectors, expression vectors, minichromosomes, etc. Nucleic acids (also defined here as `` genetic material '' in general) may be genes for gene replacement therapy (i.e. for gene therapy in which the disease is caused by a lack of expression of a certain gene or in which it is expressed a non-functional gene) or they may be sequences aimed at interfering with the expression of one or more genes expressed or over-expressed in diseased cells, such as in cancer cells, and not normally expressed in healthy cells. The inserted genetic material may also consist of tRNA where the dysfunction is related to the absence or malfunction of a particular tRNA.

These nucleic acids are also defined in the present description as â € œsequences of interestâ € since in the realization of the invention as taught here it is not relevant, for the purposes of obtaining a particle having the technical advantages listed above, such as sequences nucleic acids / nucleic acid condensing agent are inserted in the nucleus, any sequence can be inserted without changing the chemical-physical characteristics of the nano particles.

It is evident that in the preferred embodiment such sequences will have a recognized function in gene therapy.

The person skilled in the art will be able to â € œfillâ € the nanoparticles of the invention with the nucleic acids suitable for the medical objective to be pursued without the need for further teaching in the present description which provides effective â € œmeans of transportâ € for such nucleic acids and procedures. detailed to make them.

The ratio between the volume of said one or more nucleic acid molecules and of said nucleic acid condensing agents in the nanoparticles according to the invention may be comprised between about 1: 1 and about 1: 0.5, in a form of realization this ratio will be approximately 1: 0.75.

One or more nucleic acid condensers commonly known in the art can be used to carry out the present invention. For example, such agents can be selected from polyamines (for example spermine or spermidine), cationic peptides (for example?), Cationic lipids (for example?), Cationic surfactants (for example?), Positively charged amino acids (for example arginine and lysine) , positively charged proteins, multivalent metal cations (e.g. Ca <2+>, Mg <2+>, Mn <2+>; Fe <2 +>, Fe <3+>).

The examples indicated above are not limitative of the realization of the invention but have the purpose of providing the reader of the present invention with concrete examples of suitable molecules among those known to those skilled in the art.

In a particular embodiment, the nucleus of the nanoparticle will be made using protamine as the condenser.

In this embodiment the core will have a hydrodynamic diameter, D = 230 ± 18 nm; and a surface charge = - 19.5 ± 2.5 mV.

As far as the outer shell of the nanoparticle is concerned, the cationic lipids and the phospholipids may be in variable reciprocal ratios, according to some embodiments called cationic lipids 1,2-dioleoyl-3-trimethylammonopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and said phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) will be present in a reciprocal ratio chosen between 1.5: 0.5: 1.5: 0.5 or 1: 1: 1: 1 or 0.5: 1.5: 0.5: 1.5.

In a particular embodiment the above mentioned reciprocal ratio will be 0.5: 1.5: 0.5: 1.5.

In the realization of the present invention, given the choice of the coating lipids, the surface charge of the nanoparticle is positive and, in the embodiment in which the reciprocal ratio between said cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and said phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) Ã ̈ 0.5: 1.5: 0, 5: 1.5 the surface charge of the nanoparticle will have a z potential between about 40 and 45 mV, a non-limiting example is represented by a z potential of about 42mV.

The lipid nanoparticle made as described here will have a diameter between 200 and 250 nm and may have a polydispersity index <0.2.

In the embodiment in which the reciprocal ratio of said cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC- Chol) and said phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) is 0.5: 1.5: 0.5: 1.5 the diameter D of the particle will be about 210 ± 12 nm and the polydispersity index it will be <0.2.

The aforementioned nanoparticles exhibit the following characteristics

(i) The nanoparticles have controlled dimensions and extremely monodisperse (polydispersity index <0.2)

(ii) They have a positive and extremely reproducible surface charge;

(iii) Cell internalization is fast and extremely effective;

(iv) The cytoplasmic release of the genetic load is complete;

(v) The released genetic material effectively enters the nucleus;

(vi) The transfection efficiency of nanoparticles is higher than that of commonly used lipotransfection reagents, such as lipofectamines (â ‰ ˆ 10 <10> RLU / mg protein) in different cell lines (NIH 3T3; HeLa; CHO ; A17).

(vii) The nanoparticles are not toxic (cell viability> 70%).

The present invention also relates to a process for the preparation of multicomponent lipid nanoparticles comprising the following steps:

to. nucleic acid molecules and a set of one or more condensing agents the nucleic acids are dissolved separately at the same concentration in distilled H2O and are then mixed together at a ratio between 1: 1 and 1: 0.5 thus obtaining a complex negatively charged (For slight agitation); b. a desired volume of the complex obtained in step a. It is left to equilibrate for a period between 30 and 180 minutes at room temperature;

c. multicomponent cationic liposomes are prepared by individually dissolving the cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) phospholipids in a suitable organic solvent and desired quantities of each dissolved lipid are mixed until a homogeneous mixture is obtained;

d. the organic solvent is completely removed from the mixture obtained at point c. thus obtaining a lipid film;

And. the lipid film obtained in point d. It is hydrated with a suitable buffer at cellular physiological pH until a desired final concentration is obtained; f. the lipid solution obtained in point e. It is sonicated until a clear solution is obtained (which indicates the formation of uni lamellar vesicles); g. the balanced complex obtained in point b. It is made to interact with an appropriate volume of the solution obtained at point f. at a charge ratio rho mole of cationic lipid / base of nucleic acid between about 2 and 3, extremes included, and it is incubated at about 37 ° C until nanoparticles are formed.

In the procedure described here, the concentration of the solution of nucleic acids and of the solution containing the agent or agents condensing the nucleic acids may have a concentration chosen according to the needs of the operator, this concentration, which will be the same for the two different solutions, may be between 0.5 and 1.5 mg / ml for example, ie about 0.5; about 0.6; about 0.7; about 0.8; about 0.9; about 1; about 1.1; about 1.2; about 1.3; about 1.4; about 1.5 mg / ml by way of example, even if the present invention does not exclude that the skilled in the art can select different concentrations.

The volume ratio between the nucleic acid solution and the nucleic acid condensing agent solution in step a. it will be, as described, in a range between 1: 1 and 0.5: 1. By way of non-limiting example, this ratio may be, in the order indicated above (volume of the nucleic acid solution: volume of the solution of nucleic acid condensing agents) about 0.5: 1: 0.55: 1; 0.6: 1; 065: 1; 0.7: 1; 0.75: 1; 0.8: 1; 0.85: 1; 0.9: 1; 0.95: 1; 1: 1.

The nucleic acid molecules that can be used for the preparation of the nucleus of the nanoparticles of the invention are those indicated previously in the part relating to the description of the nanoparticles themselves and can therefore be chosen from DNA, RNA, siRNA, shRNA, mRNA; miRNA; dsRNA.

In the process described above, the condensing agents of nucleic acids are selected from polyamines, cationic peptides, cationic lipids, cationic surfactants, positively charged amino acids, positively charged proteins, multivalent metal cations. Non-limiting examples of suitable molecules are provided here in the part relating to the description of the nanoparticles themselves and may therefore be: for the polyamines selected from spermine and spermidine, for the positively charged amino acids selected from arginine and lysine, for the positively charged proteins selected between protamine and histones, for the multivalent metal cations selected from Ca <2+>, Mg <2+>, Mn <2+>; Fe <2+>; Fe <3+>.

In a particular embodiment, protamine is selected as the condensing agent of nucleic acids.

In the process described above, a desired volume of the complex obtained in a is allowed to equilibrate. (which will correspond to the â € œnucleusâ € of the nanoparticles), for a period between 30 and 180 minutes approximately, this period could be, for example, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120 , 130, 140, 150, 160, 170, 180 minutes approximately. In general, a period of about 30 to 60 minutes, including extremes, is also sufficient.

The nuclei thus obtained can be stored for a period from a few hours to about 24 hours at about 4 ° C.

Since the nucleus obtained in b. containing nucleic acids, it is more delicate than the unilamellar vesicles obtained in step f., which can be stored in the refrigerator (at about 4 ° C) for about a week or two, it is advisable to prepare the vesicles first or otherwise prepare the nucleus and the vesicles simultaneously in order to use the nucleus obtained in b. as soon as possible and avoid possible degradation of the same in order to obtain multicomponent nanoparticles with the greatest effectiveness.

In the described process, for the preparation of the vesicles to be used for the coating of the core, the above mentioned lipids (cationic and phospholipids) will be used, in the desired reciprocal ratios. It has been found that the various ratios tested are all effective in producing vesicles with good transfection properties and low toxicity. In particular, in point c. cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the phospholipids dioleoylphosphatidylethanol ) and dioleylphosphocoliin (DOPC) in a reciprocal ratio chosen between 1.5: 0.5: 1.5: 0.5 or 1: 1: 1: 1 or 0.5: 1.5: 0.5: 1, 5.

In a particularly interesting embodiment, the aforementioned lipids can be mixed, and therefore present in the coating of the nucleic acid / condensing core of the nanoparticles described here, in the reciprocal ratio (in which the order of the lipids is that indicated in the previous paragraph ) 0.5: 1.5: 0.5: 1.5.

Each lipid will be dissolved in a suitable organic solvent and then mixed in the desired ratios or indicated above.

Any suitable organic solvent can be used, for example, suitable organic solvents, commonly used by the skilled in the art, can be chloroform or ethanol.

The reagents will be mixed until a homogeneous solution is obtained, at room temperature for a period of between 1 and 5 minutes, such as approximately 1-2 minutes. In an embodiment of the invention, the multicomponent cationic liposomes consist of the four lipid species which are obtained at point f. they can be prepared with a molar fraction of neutral lipid in the bilayer Φ = (neutral lipid / total lipid) (mol / mol) = between 0.4 and 0.6, for example 0.5. The lipids dissolve in the appropriate solvent, for example chloroform, and mix until obtaining a homogeneous mixing of the four lipid species (in organic solvent the mixing of lipid molecules is a homogeneous mixing). The amount of solvent to be used depends on the solubility of the lipid species used, for example, the solutions can be prepared at approximately 10/20 mg lipid / ml chloroform.

The organic solvent is then removed at point d., This can be achieved with any suitable means known to the person skilled in the art, such as using a conventional rotary evaporator. This procedure leads to the formation of a thin lipid film on the bottom of the container used. The removal of the organic solvent must be complete, to ensure the complete removal of the organic solvent, the lipid film can for example be kept for 24 hours under vacuum.

Subsequently, the lipid film in point d. It is hydrated with a suitable buffer at cellular physiological pH until a desired final concentration is obtained.

Any buffer commonly suitable for use with viable cells, which stabilizes the pH at physiological levels, such as the physiological pH Tris-HCl buffer can be used.

For example, Tris-HCl (10 mM, pH 7.4) can be used, necessary to obtain a final concentration of 1 mg / ml.

At this point, the lipid solution obtained in step e. it will be sonicated until a clear solution is obtained, the clarification of the solution indicates the formation of lamellar vesicles consisting of the aforementioned lipids in the ratios selected by the operator, which will be used to coat the nucleus formed in point b.

Any suitable sonicator conventionally used by the person skilled in the art, such as a titanium tipped sonicator, can be used for a period of approximately 10 minutes. The technician in the sector will be able to choose the most effective on-off cycle sequence without the need for further training. By way of non-limiting example, a sequence of 8 s on - 6 s off cycles for t = 10 min can be used. The vesicles thus prepared can be stored at about 4 ° C for a period of up to 1 or 2 weeks, and can then be directly used for step f. of the process for preparing the nanoparticles of the invention.

In this step, a suitable volume of said vesicles is mixed with a suitable volume of the balanced complex obtained at point b. (in an exemplary form, obviously not limiting the invention, about 5 microliters of vesicles can be mixed with 2 microliters of condensant / nucleic acid) at a charge ratio (moles of cationic lipid / bases of nucleic acid Ï between about 2 and 3, extremes included.This ratio, in a non-limiting exemplary form, could be for example about 2.5.

The calculation of the charge ratio Ï can be done as follows (the example refers to an embodiment in which the nucleic acid is DNA):

the number of moles of cationic lipid is given by the calculation:

cationic lipid mass / molecular weight of cationic lipid;

the number of nucleotides is given by the calculation:

mass of DNA / 324.5 (molecular weight of a nucleotide that contains a negative charge).

Hence, rho = Number of moles of cationic lipid / Number of nucleotides (or bases).

The incubation can be carried out for a period between about 30 minutes and 3 hours, for example a period of 30, 40, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180 minutes approximately.

In a very effective embodiment, multicomponent nanoparticles are prepared in which the nucleus is formed by nucleic acid and protamine in a nucleic acid / protamine volume ratio equal to about 1: 0.5 starting from initial solutions having a concentration c = 1 mg / ml. The core has dimensions with diameter D = 230 ± 18 nm and surface charge = -19.5 ± 2.5 mV.

The solutions of DOTAP, DC-Chol, DOPE and DOPC are prepared in chloroform to a mole fraction of neutral lipid in the bilayer Ф = (neutral lipid / total lipid) (mol / mol) equal to 0.5 and the reciprocal ratio in which they are mixed in step c. (in the order indicated above) is chosen from

â € ¢ DOTAP-DC-Chol-DOPC-DOPE (1.5: 0.5: 1.5: 0.5)

â € ¢ DOTAP-DC-Chol-DOPC-DOPE (1: 1: 1: 1)

â € ¢ DOTAP-DC-Chol-DOPC-DOPE (0.5: 1.5: 0.5: 1.5).

The solution containing the nucleus and that containing the multicomponent vesicles obtained as described above are mixed in such a way that the charge ratio (mole of cationic lipid / nucleic acid base is equal to about 2.5.

The obtained nanoparticles have the lipid composition in the coating with reciprocal ratios equal to those used in the mixing step c.

The nanoparticles defined as â € œnanoparticles obtainable by means of the process of the invention as defined in the description and in the relative claims of said processâ € are obviously also object of the invention.

As already indicated, the object of the present invention is the nanoparticles as described herein for use in gene therapy.

In other words, the object of the invention is a method of gene therapy which includes the step of administering the nanoparticles of the invention to a patient who needs it.

It is evident that the nanoparticles will be made with the appropriate nucleic acids selected in relation to the desired gene therapy.

Without binding to specific theories, the inventors of the present invention have formulated the following hypotheses.

The electrostatic interactions are not sufficient to describe the formation and thermodynamic stability of the DNA / condensing agent nucleus, but it is necessary to refer to the mechanism of the release of the counter-ions. The Poisson-Boltzman condensation predicts that charged macromolecules are surrounded in solution by a diffuse layer of counter-ions. Under the formation of the complex, the macromolecules release their counter-ions in solution with a considerable entropic gain of the system (about 1 KT for each counter-ion released). The same mechanism of action is able to justify the formation of the nanoparticle. The most significant aspect is the extraordinary ability exhibited by the multicomponent lipid nanoparticles to release DNA into the cytoplasm and nucleus of the cell (see Figure 3). This property makes multicomponent lipid nanoparticles unique and is the most likely explanation for their high efficiency (Figure 1). This intrinsic ability to exit the endosomes and release DNA is probably due, among other factors, to the number of lipid species involved which are able to maximize the entropy of lipid mixing, making it energetically favored mixing with lipid species of endosomal membranes (Caracciolo et al. The Journal of Physical Chemistry B 110, 20829-20835 (2006)). This mixing seems to be necessary for the formation of the structural intermediates that lead to the fusion of the lipid bilayer of the nanoparticles and that of the membranes of the endosomes. In addition, the reduced number of layers of the lipid shell compared to the lipoplexes of the same composition is compatible with a simpler disassembly of the nanoparticles compared to the multilamellar lipoplexes.

The following examples have the purpose of illustrating, without however limiting it to their content, the invention in order to provide the skilled person with concrete examples of realization of the invention.

EXAMPLES and EXPERIMENTAL PART

The transfection experiments reported below were performed on commercial cell lines and

NIH 3T3 ATCC number CRL-1658

HeLa ATCC CCL-2 number

CHO (ATCC CCL-61 number)

On mouse cells here named A17 isolated by the inventors.

1. PROCEDURE OF PREPARATION OF LIPID NANOPARTICLES

The nanoparticles consist of a DNA / protamine nucleus obtained by mixing two solutions of DNA and protamine in a DNA / protamine volume ratio 1: 0.75 (the starting solutions of DNA and portamine have the same concentration, c = 1 mg / ml) . The DNA / protamine shell is allowed to equilibrate for one hour at room temperature. At the end of this procedure, the DNA / protamine shell is negatively charged (Zeta potential = -19.5 ± 2.5 mV) and has a characteristic size of approximately 230 nm (hydrodynamic diameter, D = 230 ± 18 nm). A volume DNA / protamine complex thus prepared is made to interact for 20 minutes with a solution of cationic liposomes consisting of a mixture of the commercial cationic lipid DOTAP, the cationic derivative of cholesterol DC-Chol and two neutral phospholipids such as DOPE and DOPC. The four molecular species are mixed in the ratio 0.5: 1.5: 0.5: 1.5. After 2 hours of incubation, the formation of the nanoparticles is complete. The surface charge is positive with the DNA / protamine core covered by a lipid shell consisting of one to a few lipid bilayers. The lipid formulation used in the present invention has never previously been used for the production of nanoparticles with a DNA / protamine core.

2. DETAILED PROCEDURE FOR PREPARING THE NANOPARTICLES USED IN THE EXPERIMENTS SHOWN IN THE FIGURES

The cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP) and 3ß- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and the two neutral phospholipids dioleoylphosphatidylethanolamine and dioleylphosphocoliin (DOPC) were purchased from Avanti Polar Lipids (Alabaster, AL) and used without further purification. For fluorescence microscopy measurements, DOPC-NBD and DOPE-NBD fluorescent neutral lipids purchased from Avanti Polar Lipids were used. The multicomponent cationic liposomes consisting of the four lipid species were prepared with a mole fraction of neutral lipid in the bilayer Φ = (neutral lipid / total lipid) (mol / mol) = 0.5. The lipids were dissolved in chloroform and mixed in order to obtain a homogeneous mixing of the four lipid species. The quantity of chloroform to be used depends on the solubility of the lipid species used, generally the solutions have been prepared at about 10/20 mg lipid / ml chloroform. The organic solvent was removed using a rotary evaporator which led to the formation of a thin lipid film on the bottom of an ampoule. To ensure complete removal of the organic solvent, the lipid film was kept under vacuum for 24 hours. Subsequently the lipid film was hydrated by adding the volume of Tris-HCl buffer (10 mM, pH 7.4) necessary to obtain a final concentration of 1 mg / ml. To obtain unilamellar vesicles, the lipid solutions were sonicated using a titanium-tipped sonicator with a sequence of cycles (8 s on - 6 s off) for t = 10 min. The lipid solutions used were prepared by varying the ratio between the cationic lipid species:

â € ¢ DOTAP-DC-Chol-DOPC-DOPE (1.5: 0.5: 1.5: 0.5)

â € ¢ DOTAP-DC-Chol-DOPC-DOPE (1: 1: 1: 1)

â € ¢ DOTAP-DC-Chol-DOPC-DOPE (0.5: 1.5: 0.5: 1.5)

Double-stranded DNA from calf thyme was purchased from Sigma (St. Louis, MO) and used without further purification. It was dissolved in distilled water at a concentration of 1 mg / ml and sonicated with a sonicator tip (t = 5 min., 8 s on - 6 s off) to obtain a homogeneous solution in length (length of the DNA from the 500 to 1000 base pairs). Linear DNA was used for all the chemical-physical characterization experiments of the nanoparticles.

For the cell transfection experiments, plasmid DNA (pGL3 encoding for luciferase) purchased from Promega (Madison, WI) and dissolved in double distilled water (Carlo Erba Reagenti, Milan, Italy) at a concentration of 1 mg / ml. Cy3-labeled 2.7-kbp plasmid DNA (Mirus Bio Corporation, Madison, WI) was used for the fluorescence microscopy experiments.

Salmon protamine sulphate (MW = 5.1 kDa), purchased from Sigma, was dissolved in distilled water at a concentration of 1 mg / ml. The nucleus of the nanoparticles was obtained by mixing the DNA (linear, plasmid and fluorescent) and the protamine solution at a volume ratio 1: 0.75 in order to obtain a negatively charged complex. A desired volume of DNA / protamine is left to equilibrate for one hour at room temperature and subsequently it has been made to interact with an appropriate volume of the cationic liposome solution at a charge ratio Ï (mole of cationic lipid / base of DNA ) = 2.5. After 2 hours of incubation, the formation of the nanoparticles is complete.

3. Protocol for a transfection experiment (conditions referred to one well of a 24-well plate) *; 1. 0.5 microliters of DNA (concentration 1 mg / ml) are mixed with 0.35 microliters of protamine (concentration 1 mg / ml) and allowed to incubate for 10 minutes. ;2. 49.1 microliters of Opti-MEM® (Life Technologies) are added to the formed DNA / protamine complex. ; 3. 5 microliters of the cationic liposome solution are diluted in 45 microliters of Opti-MEM®. ; 4. 50 μl of the DNA / protamine solution is mixed with 50 μl of the cationic liposome solution. The nanoparticles thus formed are left to incubate for 2 hours at room temperature. ; 5. 100 microliters of the nanoparticle solution are diluted in 400 microliters of Opti-MEM® (volume of 500 x well) and given to one well of a 24-well plate (cells at 70% confluence). ; 6. After 4 hours, the 500 microliters are removed from the well and 1 ml of culture medium (DMEM) complemented with serum is added. ;; * In this case the charge ratio of the nanoparticles is ̈ Ï = 2.5. To double the charge ratio it is necessary to use 10 microliters of the cationic liposome solution (step 3).

4. ASSESSMENT OF TRANSFECTION EFFECTIVENESS (FIGURE 3) QUANTIFICATION OF TRANSFECTION

The data relating to the transfection efficiency are reported in figure 1 as a histogram and in figure 3 as fluorescence images.

to. Collection of cell lysates

48 hours after transfection, cell lysates were collected to be used for luminometer and spectrophotometer analysis. In each well of the culture plate:

- The medium was aspirated and washed with PBS at room temperature (1 ml / well);

- 200 microliters of lysis buffer â € ̃Lysis Bufferâ € ™ (Promega) were added and the cell lysate was recovered and transferred to an appropriately marked eppendorf and kept on ice.

At this point each tube was mixed with vortex (about 10 seconds) and centrifuged (2 minutes, 12000 g, 4 ° C); the supernatant was then aliquoted into two new tubes and stored at â € “80 ° C.

b. Luminometer readings

For the quantitative analysis of luciferase activity, as an index of the transfection efficiency, the cell lysates were tested on the luminometer (Berthold AutoLumat luminometer LB-953). For this purpose, 100 microliters of substrate (luciferin) were added to 20 microliters of each sample and immediately read for 10 seconds (2 sec delay), following the protocol attached to the Promega's Luciferase Assay System kit. The reaction that takes place involves the conversion into light of the chemical energy released by the oxidation of luciferin into oxyluciferin, catalyzed by luciferase. The light intensity, remaining constant for at least one minute, is acquired as a numerical value which corresponds to the area under the emission curve in the set time interval. Each sample was measured in duplicate. To normalize the values obtained with respect to the total proteins present in the lysates, the same samples were then analyzed on the spectrophotometer.

Bioluminescent reaction catalyzed by Firefly Luciferase.

Spectrophotometer readings

Luminometer readings were normalized for milligrams of total cellular protein present in the lysates, using the Bio-Rad Protein Assay Dye Reagent (Bio-Rad), according to the Bradford method.

In order to obtain reference values with which to compare the experimental data, a calibration curve was constructed, starting from a stock solution of BSA (Bovine Serum Albumin) at a known concentration (2 mg / ml) and diluting 100 microliters of this. € ™ last in 2 final ml of H2O. Two series of 6 cuvettes containing scalar quantities of BSA, H2O and Bio-Rad were then tested:

- 800 microliters H2O 200 microliters Bio-Rad

- 20 microliters BSA 780 microliters H2O 200 microliters Bio-Rad

- 50 microliters BSA 750 microliters H2O 200 microliters Bio-Rad

- 100 microliters BSA 700 microliters H2O 200 microliters Bio-Rad

- 150 microliters BSA 650 microliters H2O 200 microliters Bio-Rad

- 200 microliters BSA 600 microliters H2O 200 microliters Bio-Rad

Plotted the curve, therefore, two cuvettes were prepared for reading for each lysate of transfected cells, containing:

- 5 microliters of sample

- 795 microliters of H2O

- 200 microliters of Bio-Rad

Immediately before reading at 595 nm, the contents of each cuvette should be vortexed and left in the dark for between 5 and 60 minutes.

Data analysis

The statistical analysis of the data obtained from the measurements was carried out using the Microsoft Excel program. TE (Relative Light Unit / mg protein) Transfection Efficiency values were calculated as the average of at least four experimental observations. The standard deviation and the S.E.M. were also determined on the same samples. (Standard Error of the Mean).

5. ASSESSMENT OF CYTO-TOXICITY

A histogram of the results obtained from the assay reported below is presented in Figure 2. Live cells are able to chemically reduce the compound MTT (a water-soluble tetrazolium salt) to a water-insoluble, violet-colored formazan salt (Mosmann , T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods, 1983. 65 (1-2): p. 55-63). The toxicity test of multicomponent lipid nanoparticles was performed on CHO and 3T3 ovarian cells seeded on a 96-well plate (8000 cells per well), in such a way as to have 16 wells per experimental condition. The treatments were carried out in 100 microliters of final Optimem per well 96: 80 microliters of Optimem 20 microliters multicomponent lipid nanoparticles (generated using 0.1 micrograms of DNA per well and maintaining the same ratios between DNA and protamine and between DNA / protamine and cationic liposomes described in paragraph 3: transfection protocol).

After incubation with the nanoparticles (19 hours), MTT (final concentration of 0.5 mg / ml) is added to the culture medium and the plate is placed in the dark at 37 ° C for 4 hours. Subsequently, the soil is removed from the wells, gently sucking it up. After making sure that there are no traces of solution left in the wells, 100 microliters of DMSO are added to each well and the plate is shaken for 15 minutes at room temperature in the dark to dissolve the formazan salt crystals. If the cells are alive and metabolically active, they carry out the reduction of MTT and when DMSO is added, a violet color is observed. Viability is quantified by reading absorbance with an ELISA plate reader, at a wavelength of 540 nm. The data are expressed as a percentage with respect to the control represented by cells not treated with the lipid mixtures.

BIBLIOGRAPHY

- Akita H., et al., Multi-layered nanoparticles for penetrating, Biomaterials 30 (2009).

2940â € “2949.

-Caracciolo G. et al â € œFactors Determining the Superior Performance of Lipid / DNA / Protammine Nanoparticles over Lipoplexesâ € J. Med. Chem. 54 (12), pp 4160â € “4171 (2011)

-Chang, H .; Yeh, M. â € œClinical development of liposome-based drugs: formulation, characterization, and therapeutic efficacyâ €. Int. J. Nanomed. 2012, 7, 49-60â €

- Elsabahy et al. â € œNon-Viral Nucleic Acid Delivery: Key Challenges and Future Directionsâ €. Current Drug Delivery, 8, 2011, 235-244

-Pozzi D. et al â € œProgrammed packaging of multicomponent envelope-type nanoparticle system for gene deliveryâ €. Appl. Phys. Lett. 96, 183702 (2010); doi: 10.1063 / 1.3427354

Claims (23)

CLAIMS 1. Multicomponent lipid nanoparticle consisting of a core consisting of one or more nucleic acid molecules and one or more nucleic acid condensing agents and an outer coating consisting of cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and by the phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC). 2. Lipid nanoparticle according to claim 1 wherein said nucleic acid molecules are selected from dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof. 3. Lipid nanoparticle according to claim 2 wherein said nucleic acid molecules are minicromosomes comprising sequences of interest, expression vectors comprising sequences of interest, transposable elements comprising sequences of interest. 4. A lipid nanoparticle according to any one of claims 1 to 3 wherein the ratio of the volume of said one or more nucleic acid molecules to said nucleic acid condensing agents is about 1: 0.75. 5. Lipid nanoparticle according to any one of claims 1 to 4 wherein said one or more nucleic acid condensing agents are selected from polyamines, cationic peptides, cationic lipids, cationic surfactants, positively charged amino acids, positively charged proteins, metal cations multivalent. 6. Lipid nanoparticle according to claim 5 wherein said polyamines are selected between spermine and spermidine, said positively charged amino acids are selected between arginine and lysine, said positively charged proteins are selected between protamine and histones, said multivalent metal cations are selected among Ca <2+>, Mg <2+>, Mn <2+>; Fe <2 +>; Fe <3+>. 7. Lipid nanoparticle according to any one of claims 1 to 6 wherein said nucleus consists of nucleic acid and protamine, having a hydrodynamic diameter, D = 230 ± 18 nm; and a surface charge = - 19.5 ± 2.5 mV. 8. A lipid nanoparticle according to claim 7 wherein said nucleic acid condensing agents are protamine. 9. Lipid nanoparticle according to any one of claims 1 to 8 wherein said cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and said phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) are present in a reciprocal ratio chosen between 1.5: 0.5: 1.5: 0.5 or 1: 1: 1: 1 or 0.5: 1.5: 0.5: 1.5. 10. Lipid nanoparticle according to any one of claims 1 to 9 wherein the surface charge of said nanoparticle is positive and has a z potential of about 42mV. 11. Lipid nanoparticle according to any one of claims 1 to 10 having a diameter between 200 and 250 nm and a polydispersity index <0.2. 12. Process for the preparation of multicomponent lipid nanoparticles comprising the following steps: to. nucleic acid molecules and a set of one or more condensing agents the nucleic acids are dissolved separately at the same concentration in distilled H2O and are then mixed together at a ratio between 1: 1 and 1: 0.5 thus obtaining a complex negatively charged (For slight agitation); b. a desired volume of the complex obtained in step a. It is left to equilibrate for a period between 30 and 180 minutes at room temperature; c. multicomponent cationic liposomes are prepared by individually dissolving the cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) phospholipids in a suitable organic solvent and desired quantities of each dissolved lipid are mixed until a homogeneous mixture is obtained; d. the organic solvent is completely removed from the mixture obtained at point c. thus obtaining a lipid film; And. the lipid film obtained in point d. It is hydrated with a suitable buffer at cellular physiological pH until a desired final concentration is obtained; f. the lipid solution obtained in point e. It is sonicated until a clear solution is obtained; g. the balanced complex obtained in point b. It is made to interact with an appropriate volume of the solution obtained at point f. at a charge ratio rho mole of cationic lipid / base of nucleic acid between about 2 and 3, extremes included, and is incubated at about 37 ° C until nanoparticles are formed. 13. Process according to claim 12, wherein said nucleic acid solutions and said set of one or more nucleic acid condensing agents have a concentration of about 1mg / ml. 14. Process according to any one of claims 12 or 13, wherein said ratio in point a. It is approximately 1: 0.75. 15. Process according to any one of claims 12 to 14 wherein said nucleic acid molecules are selected from dsDNA (double strand DNA), ssDNA (single strand DNA), RNA, siRNA (small interfering RNA), shRNA (short hairpin RNA) ), mRNA, dsRNA (double strand RNA), tRNA, miRNA (microRNA) or a mixture thereof. 16. Process according to any one of claims 12 to 15 wherein said one or more condensing agents of nucleic acids are selected from polyamines, cationic peptides, cationic lipids, cationic surfactants, positively charged amino acids, positively charged proteins, multivalent metal cations . 17. Process according to claim 16 in which said polyamines are selected between spermine and spermidine, said positively charged amino acids are selected between arginine and lysine, said positively charged proteins are selected between protamine and histones, said multivalent metal cations are selected from Ca <2+>, Mg <2+>, Mn <2+>; Fe <2+>; Fe <3+>. 18. The process of claim 17 wherein said nucleic acid condensing agents are protamine. 19. Process according to any one of claims 12 to 18 wherein said desired volume of the complex obtained in step a. It is left to equilibrate for a period of about 60 minutes at room temperature 20. Process according to any one of claims 12 to 19 wherein said cationic lipids 1,2-dioleoyl-3-trimethylammoniumopropane (DOTAP), 3beta- [N- (Nâ € ™, Nâ € ™ -dimethylaminoethane) -carbamoyl] cholesterol hydrochloride (DC-Chol) and said phospholipids dioleoylphosphatidylethanolamine (DOPE) and dioleylphosphocoliin (DOPC) are mixed in point c. in a reciprocal ratio chosen between 1.5: 0.5: 1.5: 0.5 or 1: 1: 1: 1 or 0.5: 1.5: 0.5: 1.5. 21. Process according to any one of claims 12 to 20 wherein said organic solvent at point c. It is chloroform or ethanol. 22. Process according to any one of claims 12 to 21 wherein said incubation at point g. It is carried out for a period between about 30 minutes and 3 hours. 23. Nano particles according to any one of claims 1 to 11 for use in gene therapy.