BRPI0705630A2 - Functional liposomal configuration and process of obtaining functional liposomal configuration - Google Patents

Abstract

Functional Liposome Configuration and Process for Obtaining Functional Liposome Configuration The invention provides a product for therapy and vaccination consisting of a functional liposomal configuration containing complex polynucleotides preferably on the outer surface of DRV-like liposomes, and their production process. This functional liposomal configuration containing polynucleotides has mucoadhesion properties that allow administration via noninvasive routes such as the nasal route, and is capable of efficiently transporting polynucleotides into cells, releasing them into the cytoplasm. These properties enhance greater efficiency of in vivo action of carried polynucleotides, reduction of concentration and frequency of doses, and, in the case of vaccination produces protection against specific disease.

Description

DESCRIPTION REPORT OF THE INVENTION PATTERN FOR FUNCTIONAL LIPOSOMAL CONFIGURATION AND PROCEDURE FOR OBTAINING FUNCTIONAL LIPOSOMAL CONFIGURATION "

The present invention relates to the functional product as well as the process of obtaining a polynucleotide carrier liposomal composition for evacuation therapy. This liposomal configuration incorporating polynucleotides is more effective as compared to free polynucleotides as carried on other liposomal configurations. It is also economically viable, clinical and veterinary for various routes of administration, stable when stored under refrigeration and its production allows scaling up for application in the industrial sector.

Description of the prior art

Phospholipid liposomes or vesicles are iamellar aggregates of lipid bilayers, alternated by one or more internal aqueous domains, forming approximately spherical particles with diameters of the order of nanometers to tens of microns. These aggregates are capable of incorporating or encapsulating in their matrix charged or neutral compounds of different natures such as hydrophilic, which are positioned in the internal aqueous domains, lipophilic in the bilayers and amphiphilic between both domains. Iamelas act as membranes that protect the carried compounds from the environment to which they are exposed and promote their sustained release.

Some actives when exposed to air oxygen, light, heat or biological action, are subject to oxidation and / or decomposition or denaturation, losing their activity. Incorporation and / or encapsulation of these actives in liposomes circumvents the limitations of physicochemical and biological stability.

The protection of the biological environment and the sustained release of liposome-loaded bioactives increase their efficiency of action and allow the dose and frequency of administration to be reduced. As a result, there is also a reduction in the bioactivity and side effects of bioactives compared to their free forms. In addition, protection is also necessary for the commercialization and safe use of these products.

Liposomes belong to the class of nano and microcapsules that have been successfully applied for the incorporation and / or encapsulation of active compounds of various nature, such as flavors, enzymes, cosmetics and drugs, among others. Liposomes are generally biodegradable, non-toxic and non-immunogenic. Incorporation refers to the dispersion of the active compound throughout the matrix of nano or microparticles (interior and surface) and encapsulation to its only inner confinement of the structure. Nano or microcapsules refer to particles with nanometer (109m) or micrometer (106m) diameter, respectively, and which have two distinct domains composed of at least one aqueous nucleus surrounded by a matrix of structural material such as lipids, polymers, etc. and their blends. Active compounds may be internalized (encapsulated in the aqueous nucleus (s) or incorporated into the structural matrix) or located on the surface and / or in the outermost regions of the nano or microcapsules. The liposomal configuration is defined by positioning the active compounds in their structural matrix. Polynucleotides are double stranded nucleotide strands that constitute the nucleic acids DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).

Compared to other sustained release systems such as polymeric gels and matrices, liposomes allow for better interaction with cells by mimicking structure and composition. Depending on the composition, liposomes may also function as functional carriers directing the compounds to specific target organs and / or intracellular spaces, performing co-adjuvant function. In the case of RNA and DNA nucleic acids the specific targets are cytoplasm and nucleus respectively.

Effective DNA transport into cells was only achieved with its association with cationic lipids in a mechanism called lipofection (FELGNER, PL; GADEK, TR; HOLM1 M .; ROMAN, R.; CHAM1 HW; WENZ, M .; NORTHROP , JP; RINGOLD, GM; DANIELSEN, M. Proceedings of the National Academy of Sciences of the United States of America, v. 84, pp. 7413-7417, 1987). In this context, cationic lipids were used to carry DNA through simple electrostatic complexation and to facilitate its transport into cells, because they have an oppositely charged load. This fact has initiated several research fronts, focusing on aspects such as the development of new cationic lipids and the development of gene vaccines. Among the documents that describe these developments can be cited: (LASIC, DD, Boca Raton ^ FloridaiCRC Press1 1997) . The first studies were done with cationic stearylamine olipide, but did not proceed due to its toxicity (FRANLEY, R; PAPAHADJOPOULOS, D., Current topics in microbiology andimmunology, v. 96, pp. 171-191, 1981). Currently several other cationic synthetic lipids of lower toxicity have been used, among which the best known as nucleic acid carriers are quaternary ammonium salts such as dimethyldioctadecyl ammonium bromide (DDAB), 1,2-dioleoyl! -3-trimethylammonium propane (DOTAP) and 1,2-diacyl-3-dimethylammonium propane (DOTAP) (LASIC, D.D., Boca Raton-Florida: CRC Press, 1997).

In the process of transport into cells, polynucleotides associated with cationic liposomes are initially internalized preferentially through the endocytosis mechanism. In a second step it is necessary that the polynucleotides be released from the endosomal membrane to the cytoplasm, possibly by rupturing the membrane that is destabilized due to interactions between the cationic lipids and their anionic molecules (WATTIAUX, R; JADOT, M.; WARNIER-PIRROTTE, WATTIAUX-DE CONINCK, S., FEBS Letters, v. 417, p.199-202, 1997; ZHOU, X., Huang, L., Biochimica et Biophysica Acta, v. 1189, pp. 195-203, 1994). The destabilization of the endosomal membrane results in a flip-flop of its anionic lipids, predominantly present in the monolayer that interfaces the cytoplasm. Anionic lipids diffuse into the lipidiocathionic / nucleotide complex and form with the cationic lipids ion pairs with carganeutra, displacing the nucleotides and releasing them into the cytoplasm (XU, Y .; SZOKA, FCJ, Biochemistry, v. 35, pp. 5616-5623, 1996). Within this mechanism, the presence of phosphatidolethanolamines (PEs), also known as "helpers", intensifies the release process because they have fusogenic characteristics that facilitate endosomal membrane stabilization (FELGNER, JH; KUMAR, R; SRIDHAR, CN; WHEELER, CJ TSAI, YJ; BORDER, R.; MARTIN, M.; FELGNER, PL, Journal of Biological Chemistry, v. 269, pp. 2550-2561, 1994; ZABNER, J.; FASBENDER, AJ; MONINGER, T.; POELLINGER; , KA; WELSHI M., Journal of Biological Chemistry, v.270, pp. 18997-19007, 1995; FARHOOD, H.; SERBINA, N.; HUANG, L., Biochimica et Biophysica Acta, v.1235, p.289 (1995).

However, the fusion of DNA-associated lipids with the PE-induced endosomal membrane does not occur directly with the cytoplasmic membrane, and the initial stage of endocytosis is necessary (WROBEL, I .; COLLINS, D., Biochimica et Biophysica Acta, v. 1235, p. 296-304, 1995) = For this reason PEs are also referred to in the literature as co-lipids in relation to cationic lipids.

Among the PEs, the fusogenic action of 1,2 dioleoyl-sn-glycero-3-phospho ethanolamine (DOPE) is better known. The importance of DOPE in the studies of potentiation of "in vitro" nucleotide release with cationic lipids, is due to its ability to pass to the hexagonal phase Hllj facilitating the destabilization of the endosomal membrane and promoting the release of polynucleotides in the cytoplasm through the similar lipid lipid exchange. endosome with the liposomal membrane as illustrated for the DNA in Figure 1. The released DNA must still be internalized in the nucleus to define gene expression of proteins. RNA performs its functions in the cellular cytoplasm itself.

Most works in the literature focus on the simple electrostatic complexation of DNA with binary cationic lipid systems consisting of cationic lipids and helper lipids as reported in US5552157, US7105574, W00024428, W0200045849-A2.

Several in vitro DNA transfection kits consisting of cationic lipids in binary systems with PEs are commercially available. Typical examples are: "Lipofectin" (DOTMA / DOPE), "Lipofectamine" (DOSPA / DOPE) and "Lipofectace" (DODAB / DOPE), where DOTMA is dioleoxy propyl trimethyl ammonium chloride and DOSPA is 2,3 dioleoyloxy lipid. -N - [(sperminecarboxamino) ethyl] -N, N-dimethyl-1-propanaminium and DODAB is dioctadecyl dimethylammonium bromide. These binary structures composed of cationic lipids and DOPE are called Iipoplexes (WASAN, EK; HARVIE P.; EDWARDS, K.; KARLSSON, G .; BALLY1 MB, Biochimicaet Biophysica Acta, v.1461, p. 27-46, 1999) . However, there are several works in the literature that call them liposomes simply because they have lipid bilayers.

A very important parameter to characterize the electrostatic complexation between polynucleotides and cationic lipids is the molar ratio between charges (R +/-) established by (RÀDLER, JO; KOLTOVER, I .; JAMIELSON, A.; SALDITT, T.; SAFINYA, CR , Langmuir, v. 14, pp. 4272-4283, 1998). This parameter relates the number of moles of positive charges from the positively charged polar heads of cationic lipids and the number of moles of negatively charged phosphate groups present in the polynucleotide molecule skeleton.

In general, the molecular geometry of cationic amphiphiles and DOPE alone does not favor aggregation in Iamelar bilayers under physiological conditions. The formed aggregates are unstable and co-exist between the Iamelar and hexagonal phases (RÀDLER, JO; KOLTOVER, I.; SALDITT, T.; SAFINYA, CR, Science, v.275, p. 810-814, 1997). DNA interspersed in the structures (Figure 2). The association of cationic lipids and DOPE with structural phospholipids such as phosphatidylcholines (PCs) is essential for the formation of liposomes, which are constituted by regular layered or lamellas that aggregate between aqueous domains and generate approximately spherical colloidal particles. Evaluations of various phospholipids as carriers of DNA in the "in vivo" immune response reported in the literature (TOMLINSON, E.; ROLLAND, AP, Journalof Controlled Release, v. 39, p. 357-372, 1996; FARHOOD, H.; SERBINA, N .; HUANG, L., Biochimica et Biophysia Aeta1 v.1235, p.289-295, 1995; FELGNER, JH; KUMAR, R.; SRIDHAR, CN; WHEELER, CJ; TSAI, YJ; BORDER, R .; MARTIN, M .; FELGNER, PL, Journal of Biological Chemistry, v. 269, pp. 2550-2561, 1994; WROBEL, I.; COLLINS, D., Bioehimiea et Biophysiea Acta, v. 1235, p. 296-304, 1995), disclosed. that the presence of DOPE or PC / PE as neutral phospholipids is important for enhancing the response unological. However, the association with high temperature phase-shift (Tc) PCs, such as distearoylphosphatidylcholine (DSPC), decreases transfection efficiency by making the bilayer more rigid (less fluid), reducing its alteration with the endosomal membrane. The existence of a direct correlation between bilayer fluidity and transfection activity has been studied by (DUZGUNES, N.; GOLDSTEIN, J.A.; FRIEND, D.S .; FELGNER, P.L. Biochemistry, v. 28, p. 9179-9184,1989). Therefore, PCs should have a low phase transition temperature, such as natural egg phosphatidylcholine (EPC), so that at body temperature liposomal lipids are in the crystalline liquid state, giving it the fluidity needed to facilitate DOPE and DOTAP in transporting polynucleotides into cells.

From the above it can be concluded that stable liposomal formulations capable of transporting polynucleotides into cells must form ternary systems containing functional lipid components:

(i) Structural lipids, which ensure the liposomal aggregation of the bilayers. A typical representative of this category with low Tc is naturally occurring egg La-Phosphatidylcholine (EPC), composed of a mixture of phospholipids with fatty acids, saturated in 16: 0 (34%) and 18: 0 (11) ratios. %) and unsaturated 16: 1 (2%), 18: 1 (32%), 18: 2 (18%), 20: 4 (3%), where the largest ratios are 16 carbon atoms ( 16: 0) and mono-unsaturated with 18 carbon atoms (18: 1).

(ii) Cationic lipids for complexation with nucleotides such as 1,2-dioleoyl-3-trimethylammonium propane (DOTAP).

(iii) Helper lipids or co-lipids such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). Ternary structure of polynucleotide-containing liposomal structure may be prepared by simple incubation for similar electrostatic complexation. However, the internalization of polynucleotides in the liposomal structure is hampered by their molecular size. Liposomes with high encapsulation efficiency of DNA1 between 88 and 97% were developed by (PERRIE, Y .; GREGORIADIS, G., Biochimica et Biophysica Acta, v. 1475, p. 125-132, 2000), by the dehydration method. rehydration (KIRBY, C.; GREGORIADIS, G., Biotechnology, v. 2, p. 979-984, 1984). In this method, previously prepared (hydrated) liposomes were dehydrated by lyophilization and then rehydrated. During rehydration the liposomes fuse to form new liposomal structures called dehydrated-rehydrated vesicles (DRVs). In this step, the DNA that has been lyophilized, interleaves into the aqueous domains between the DRV strands, forming a liposomal configuration with the encapsulated DNA, designated by the abbreviation DRV (DNA). The use of radiolabeled DNA demonstrated its localization in the interlamellar spaces of liposomes with a diameter of approximately 1 pm.

These studies also report that the levels of DNA encapsulation in DRV configuration (DNA) were not influenced by the amount of cationic and neutral lipids. However, the increased proportion of cationic lipid resulted in smaller diameter liposomes due to the mutual repulsion of the charged surfaces, disfavoring fusion during the dehydration-hydration process. The DRV (DNA) configuration, composed of EPC / DOPE / DOTAP lipids encapsulating the plasmid pRc / CMV HBS, which encodes hepatitis B antigen expression, showed better immunological response when compared to naked DNA (PERRIE, Y .; FREDERIK, G. GREGORIADIS, G. Li., Vaccine, v. 19, pp. 3301-3310, 2001). The optimum EPC / DOPE / DOTAP molar composition was 16: 8: 4 for 10 pg DNA in 2 ml liposomal formulation with molar ratio was (R +/- 20) generating a dosage of 5 pg depDNA per 100 µg. pL of liposomal solution. Results were obtained by immunization of Bab / c mice by intramuscular route between 2 and 4 applications. The efficiency of action of encapsulated DNA has been attributed to the protection exerted by lipids on encapsulated DNA, preserving it from degradation of nucleases and attack by anionic demolecules that could act competitively by displacing the DNA from the liposome or lipoplex surface (GREGORIADIS, G .; SAFFIE, R., HART, SL, Journal of Drug Targeting, v. 3, pp. 469-475, 1996; PERRIE, Y .; GREGORIADIS, G. Biochimica et Biophysica Acta, v. 1475, pp. 125-132, 2000) . These studies originated document W09810748 and subsequent developments involving the administration of oral cationic liposomes encapsulating DNA in the DRV (DNA) configuration (US7008791).

An alternative liposomal configuration, designated as DRV-DNA, was obtained through DNA complexation with empty DRV liposomes, producing particles with diameters ranging from 4-20 μηι (PERRIE, Y .; GREGORIADIS, G. Biochimicaet Biophysica Acta, v. 1475, pp 125-132, 2000). However, the large size, 4μιη or larger, and the heterogeneity of the liposomes in the DRV-DNA configuration led the authors to discard them from their research.

As seen, the work developed comprises the production of binary (Iopopexex) and ternary (Iiposome) lipid structures encapsulating DNA and RNA.

DRV-like liposomal structures encapsulated DNA with high efficiency producing DRV-like liposomal configuration (DNA) with a diameter of the order of Ιμηι, whose transfection was characterized "in vivo". The work described, however, suggests that the production and biological effects of the reduced-size (1-2 μιη) DRV-DNA liposomal configuration are not desired. In addition, DRVs containing DNA in both DRV (DNA) or DRV-DNA configurations were produced only with a cationic lipid to DNA (R +/-) charge ratio of 20 or greater. The prophylactic and / or external therapeutic effects of these configurations were not investigated with dose reduction in relation to naked DNA, nor as a function of the administration route. Cholinucleotide-based medicines have recognized benefits for human health and high added value. Therefore, increasing its efficiency of action becomes necessary.

The invention is directed to the presentation of a functional DRV-DNA liposomal configuration incorporating polynucleotides that provides greater efficacy of action than both free and loaded polynucleotides in other hitherto known liposomal configurations used in the administration of nucleic acids. The invention also provides a method for obtaining this liposomal configuration which, by employing specific parameters, enables the formation of a DRV-DNA liposomal configuration having reduced size and low solid dispersion, important factors when using these configurations in gene vaccines.

Brief Description of the Invention:

The present invention describes a functional liposomal configuration incorporating polynucleotide as well as a process for producing such a configuration.

The liposomal configuration referred to in this invention comprises: (a) a ternary lipid system comprising cationic lipid, structural lipid and adjuvant lipid and,

(b) a polynucleotide.

The invention further relates to a process for obtaining the functional liposomal configuration comprising the following steps:

a) Obtaining Iamellar liposomes from a mixture containing lipidiocathionic, structural lipid and adjuvant lipid.

b) freeze drying or drying of empty liposomes

c) obtaining dehydrated-rehydrated vesicles (DRVs)

d) complexing a polynucleotide with DRVs by mixing DRVs with polynucleotides at a ratio of 1.1 to 50 moles of cationic lipid positive charges to 1 mol of polynucleotide negative charge.

The present invention also relates to processes for obtaining functional liposome configuration that include the process described in this invention and pharmaceutical compositions containing liposomal configuration as described in the invention.

The invention further relates to a liposomal configuration defined in the invention obtained according to a process defined in the invention.

Brief Description of the Figures

Following, reference is made to the Figures accompanying this descriptive report, for a better understanding and illustration of it, where it can be seen:

Figure 1 illustrates the DNA release mechanism of the DNA / liposomatic complex DNA. STEP 1: Endocytosis of the cationic liposome / DNA complex. STEP 2: Flip-flop of anionic lipids in the endosomal membrane destabilize the endosome. STEP 3: Formation of neutral ionic pairs between anionic cationic lipids. STEP 4: DNA diffusion into the cytoplasm (Adapted from Xu & Szoka, 1996).

Figure 2 illustrates the equilibrium between the Iamelar Lai0 and inverse hexagonal HMC phases of Iipoplexes (Adapted from Radler, 1997).

Figure 3 comparatively illustrates the steps for preparing the DRV-DNA configuration compared to the DRV (DNA) configuration and the delipoplex binary configuration. Multilamellar liposomes were produced by hydration of a lipid dry film; size reduction and homogenization by extrusion; liposome drying by lyophilization. For the DRV (DNA) configuration, DNA was lyophilized along with empty DRVs, later rehydrated. For DRV-DNA configuration empty DRVs were first rehydrated and then complexed with DNA. For Iipoplexes complexation was performed after obtaining lipid aggregates (multilamellar liposomes).

Figure 4 shows the transmission electron microscopy of empty DRV-like liposomes (PC / DOPE / DOTAP 50: 25: 25 mol% in saline). The bars indicate

A) 1000 nm, B) 100nm.

Figure 5 shows the transmission electron microscopy of DRV-type liposomes complexed with DR1-DNA configuration, at ratios (R + /.) 10 (PC / DOPE / DOTAP 50: 25: 25 mol% in saline). Bars indicate A) 400 nm,

B) 200 nm, C) 200 nm, D) 40 nm.

Figure 6 shows the transmission electron microscopy of DRV-like liposome-encapsulating DNA, DRV configuration (DNA) ratio (R + /.) 10 (PC / DOPE / DOTAP 50: 25: 25 mol% in saline). Bars indicate A) 1000 nm, B) 200nm, C) 100 nm, D) 100 nm.

Figure 7 shows transmission electron microscopy of lipid aggregates, binary composition (DOPE / DOTAP 50: 50 mol% in saline). Bars indicate A) 400 nm, B) 100nm.

Figure 8 shows transmission electron microscopy of Iipoplexes (DNA complexed lipid aggregates at R + / 10 charge ratio) (DOPE / DOTAP50: 50 mol% in saline). Bars indicate A) 400 nm, B) 200nm, C) 200 nm, D) 40 nm.

Figure 9 shows the size distribution of cationic liposomes composed of PC / DOPE / DOTAP (50/25/25 mol%) in the various processing steps for formation of DRVs structures. A) After hydration of the dry film with standard water for injection. B) Liposomes extruded into pore-polycarbonate membranes with a nominal diameter of 100 nm. C) Empty liposomes produced by the DRV method in saline (0.9% NaCl). D) Liposomes produced by the DRV method encapsulating DNA, DRV configuration (DNA) at a load ratio10. in saline (0.9% NaCl). Each graph shows the size distribution of three independent samples. E) Empty liposomes produced by the DRV method and DNA-complexed, DRV-DNA configuration, at a load ratio of 10, in saline solution (0.9% NaCl).

Figure 10 shows agarose gel electrophoresis for evaluation of plasmid pVAX-hsp65 integrity in DRV-type 1 DRV-DNA and lipid-like lipid structures in saline (R + /.) 10 in saline (0.9% NaCl) ). The plasmid was separated from each lipid preparation by the addition of organic solvent (chloroform / methanol 9: 1 v / v), purified with ethanol and digested with restriction enzyme Bam H1. Standard and extracted plasmids were subjected to 0.8% deagarose gel electrophoresis. Each line represents: M) 1Kb marker; 1) DNA separated from delipoplexes; DNA separated from 2) DRV-DNA, 3) DRV (DNA) 4) Standard DNA.

Figure 11 shows agarose gel electrophoresis for various cationic lipid structures obtained in saline (0.9% NaCl) and complexed with DN 10 minutes at room temperature at various charge ratios (R +/-): A) Liposome Complexes DNA extruded (LEs-DNA) composed of PC / DOPE / DOTAP50 / 25/25 (molar). Each line represents: 1) DNA (pVAXhsp65); 2) R + /. 0.5; 3) R +/- 1.0; 4) R +/- 1.5; 5) R + /. 2.0; 6) R + /. 2.5; 7) R + /. 3.0; 8) R +/- 3.5; 9) R + /. 4.0; 10) R + /. 4,5. B) Lipoplexes (Lipid-DNA aggregates composed of DOPE / DOTAP 50/50% (molar). Each line represents: 1) DNA (pVAXhsp65); 2) R + /. 0.5; 3) R + /. 1.0; 4) R + /. 1.5; 5) R + /. 2.0; 6) R +/- 2.5; 7) R + /. 3.0; 8) R +/- 3.5; 9) R +/- 4.0; 10) R + /. 4,5. C) DRV-DNA (DRV-DNA Liposomes) composed of PC / DOPE / DOTAP 50/25/25 (molar). Each line represents: 1) DNA (pVAXhsp65); 2) R + /. 0.5; 3) R + /. 1.0; 4) R + /. 1.5; 5) R + /. 2.0; 6) R +/- 2.5; 7) R + /. 3.0; 8) R +/- 3.5; 9) R + /. 4.0; 10) R + /. 4,5. Comparatively, the electrophoresis with the minimum charge ratio (R +/-) for complexation of all DNA pVax hsp65 plasmid DNA into extruded liposomes (LEs) (A), lipoplexes (B) and DRVs (C) .

Figure 12 shows the comparative thermogram between the various lipid structures.

The lipid compositions are: (i) empty DRV, PC / DOPE / DOTAP 50/25/25 mol%. (ii) DRV (DNA) and DRV-DNA, PC / DOPE / DOTAP 50/25/25 mol%, R + /. 10 .. (iii) Aggregate lipid, DOPE / DOTAP 50/50 mol%, R + /. 10. (iv) Lipoplex, DOPE / DOTAP 50/50 mole%, R +/- 10. Heating rate of 10 ° C / min. Fluorescent probe access to pVax hsp65 plasmid DNA incorporated in the DRV-DNA configuration compared to lipoplexes and Naked DNA.

Figure 13 shows the fluorescence intensity decay profiles of the PicoGreen probe (% of initial - referring to free DNA) as a function of R + /. for the DRV-DNA (-b-XEPC / DOPE / DOTAP, 50:25:25 mole%) and Lipoplex (- · -) (DOPE / DOTAP, 50:50 mole%) structures for complexations performed at various loading ratios in ultra pure water (Milli-Q). The samples were excited at 480 nm wavelength. The exploratory scanning calorimetry spectra of the DRV-DNA configuration compared to the spectra of the same composition configurations containing the DRV encapsulated DNA (DNA) and Iipoplexes.

Figure 14 shows the variation profile of the size distribution and hydrodynamic diameter of the lipid structures during storage under refrigeration. A) DRV empty; B) DRV encapsulating DNA [DRV (DNA)]; C) DNA complexing DRV [DRV-DNAD) Lipid aggregates; E) Lipoplexes.

Figure 15 shows the in vitro cytotoxicity assay on J774 macrophages using a methodology based on reduction of thiazolyl- (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide - MTT tetrazolium salt for the different empty lipid constructs containing the plasmid pVAX-hsp 65 or containing only the commercial pVAX receptor comparatively the toxicity levels of the lipid formulations of the same composition incorporating the plasmid pVax hsp65 DNA in the different configurations.

Figure 16 shows message detection for hsp65 in vitro using RT-PCR. J774 macrophages were transfected with the different liposomal formulations for 72 hours and their total RNA extracted. The presence of messages from these cells was detected by RT-PCR using the hsp65-specific primers. The quality of cDNA used was checked by D-actin amplification. Amplification of DNase I treated samples is identified by DNAse (-), indicating whether or not there is still DNA in these treated samples. After the PCR1 reaction, the samples were submitted to agarose gel electrophoresis (1%) and the bands visualized by ethidium bromide staining. bp: base pairs

Figure 17 shows the comparison between recovery of viable lung bacilli from vaccinated animals with different doses, configurations and routes and challenged with M. tuberculosis (Mtb). BALB / c mice were immunized with either a 50 μg dose of Hsp65 DNA or 2 doses of 50 μg of Hsp65 DNA intramuscularly, or with a 25 μg dose of Hsp65 DNA by intranasal instillation. Control groups were administered with the vector served or not, or with the empty liposome. Thirty days after administration, the mice were challenged with 105 M. tuberculosis bacilli via the intratracheal route. Thirty days after the challenge, the animals were killed and their lungs extracted for the recovery of colony-forming units. The results are represented in Iog10 ± standard deviation of CFU / g of each group. Statistical difference was considered significant when * p <0.05 in relation to Mtb. Detailed Description of the Invention

The invention provides a product for therapy and vaccination consisting of a functional liposomal configuration containing polynucleotides preferentially complexed on the outer surface of DRV-like liposomes and their production process. This functional liposomal configuration containing polynucleotides has mucoadhesion properties that allow administration via noninvasive routes such as the nasal route, and is able to efficiently carry polynucleotides to the interbody cells, releasing them into the cytoplasm. These properties enhance greater in vivo action efficiency of the loaded polynucleotides, reduced concentration and frequency of doses, and, in the case of vaccination, produces protection against specific disease. The present invention relates to a liposomal carrier-like configuration of cholinolinotides as well as to the process of obtaining such a configuration. Polynucleotide liposomal carrier for therapy and vaccination. The present invention treats the prophylactic and / or therapeutic activity of cholinucleotides, incorporated in a liposomal carrier, forming a biologically more stable functional configuration with respect to free polynucleotides and with greater effectiveness of action in relation to both free and carreached polynucleotides. The liposomes present in the embodiment of the present invention contain structural lipid functionalities, DNA incorporation and electrostatic attraction to the cell surface and enhancement of DNA release in the cell cytoplasm.

These liposomes are prepared by the method of dehydration and rehydration under specific conditions defined in the invention, in which the preformed liposomes are initially obtained, then dehydrated by lyophilization and then dehydrated under controlled conditions. Polynucleotides are associated with liposomes and are preferentially located on their surface by electrostatic complexing under tightly controlled agitation and temperature conditions to generate particle sizes of the order of nanometers to 1-2 micrometers and low polydispersity. In this formulation called DRV-DNA, all polynucleotides are complexed to the liposomal system at a specific molar charge ratio between nucleotide and cationic lipid. This feasible configuration for clinical and veterinary use by the various administration routes is stable when stored under refrigeration and its production allows scaling up for application in the industrial sector.

The liposomal configuration of the present invention ensures a greater efficiency of internalizing DNA or polynucleotides in cells, resulting in greater efficiency in the formulation of a much lower dose liposomal formulation, producing therapeutic prophylactic effects. In addition, this liposomal configuration is non-cytotoxic even at high concentrations, has high polynucleotide carrying capacity, and is composed of nanometer-sized particles up to 1-2 micrometers that combines polydispersity and mucoadhesive properties so that they can be administered non-invasively mucosally. like the nasal route. Moreover, the configuration of the present invention is reproducible and economically viable for industrial production.

A plasmid construct of proven prophylactic and therapeutic activity when bound to these particles is capable of producing prophylactic and therapeutic effect. Currently, delivery of polynucleotides within cells from a lipid structure is accomplished in many cases with the use of lipid aggregates, lipoplexes. , binary systems that have only two functional lipids in their structure (cationic lipid, which allows electrostatic complexation with the nucleotide to enter cells and co-lipid, facilitating the release of the nucleotide inside the cell). These structures, existing on the market, are often unstable, simultaneously forming two structures organized in the hexagonalinverse and Iamelar phases giving greater instability in the formulations, and depending on the concentration they are also cytotoxic, which limits the carryover of more DNA. They are generally used for "in vitro" assays. Therefore, no gene devacin formulations or gene therapies on the market that act effectively in vivo are stable and reproducible to allow safe commercialization.

Over the past 10 years, a structurally acting lipid has been added to the binary formulation, constituting a ternary system of liposomal nature. The production method provides uniform nucleotide distribution throughout the liposome structure, as polynucleotides are encapsulated in liposomes by the conventional method of dehydration and rehydration. However, such formulations are not yet marketed. Non-cytotoxic configurations such as the present invention, non-cytotoxic, with high carrying capacity and particle size ranging from nanometers to 2 micrometers are neither found in the literature nor in the market.

The liposome configuration of the present invention has a maximum diameter of 2 micrometers and comprises:

(a) a ternary lipid system composed of cationic lipid, structural lipid and supporting lipid and,

(b) a polynucleotide.

The liposomal ternary lipid system of the invention comprises the molar ratio of 20% to 30% cationic lipid, 40% to 60% structural lipid and 20% to 30% adjuvant lipid. Preferably, the liposomal ternary delipid system of the invention comprises the molar ratio of 25% cationic lipid, 50% structural lipid and 25% adjuvant lipid.

The cationic lipid present in the liposome configuration of the invention is selected from 1,2-Dimyristoyl-3-Trimethylammonium-Propane; 1,2-Dipalmitoyl-3-Trimethylammonium-Propane; 1,2-Distearoyl-3-Trimethylammonium-Propane; 1,2-Dioleoyl-3-Trimethylammonium-Propane; 1,2-Diacyl-3-Dimethylammonium-Propane; DC-CholesterolHCI; Dimethyldioctadecylammonium Bromide; 1,2-Dilauroyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dimyristoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dipalmitoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Distearoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dioleoyl-sn-Glycero-3-Ethylphosphocholine; 1 -Palmitoyl-2-Oleoyl-sn-Glycero-3-

Ethylphosphocholine; 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, Ndimethyl-1-propanaminium trifluoroacetate; N- [1- (2,3-dioleyloxy) propyl] -n, n, n-trimethylammoniumchloride and 1,2-Dioleoyl-sn-Glycero-3- [Phospho-L-Serine]. Preferably, the lipidiocathionic present in the liposomal configuration of the invention is 1,2-Dioleoyl-3-Trimethylammonium-Propane.

Structural lipid is selected from synthetic lipids with asymmetric fatty acid chains, synthetic lipids with symmetrical unsaturated fatty acid chains, synthetic lipids with unsaturated symmetrical fatty acid chains and natural ellipids. Synthetic lipid with asymmetric fatty acid chains is selected from 1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine; 1-Myristoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine; 1-Palmytyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-

Phosphocholine; 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Linoleoyl-sn-Glycero-3-

Phosphocholine; 1-Stearoyl-2-Arachidonyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphocholine; 1-Oleoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine; 1-Oleoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine; 1-Oleoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine. Synthetic lipid with symmetrical saturated fatty acid chains is selected from 1,2-Diacyl-sn-Glycero-3-Phosphocoline where the acyl groups have carbon numbers ranging from C3 to C24. Synthetic lipid with unsaturated symmetrical fatty acid chains is selected from 1,2-Diacyl-sn-Glycero-3-Phosphocoline where acyl groups have carbon numbers: unsaturation ranging from C14: 1 to C24: 1, C18: 2 to C18: 3, C20: 4 or C22: 6. Natural olipide is selected from soybean phosphatidylcholine or egg phosphatidylcholine.

Preferably, the natural lipid is egg phosphatidylcholine.

The liposomal adjuvant lipid of the invention is selected from cholesterol and L-alpha-Dioleoyl Phosphatidylethanolamine. Preferably, the adjuvant olipid of the liposomal configuration of the invention is L-alpha-DioleoylPhosphatidylethanolamine.

The polynucleotide present in the liposomal configuration of the present invention corresponds to a DNA and / or RNA structure.

The invention also relates to a process of obtaining functional liposomal configuration comprising the following steps and parameters:

a) Obtaining Iamellar liposomes from a mixture containing lipidiocathionic, structural lipid and adjuvant lipid.

b) freeze drying or drying of empty liposomes

c) obtaining dehydrated-rehydrated vesicles (DRVs)

d) complexing a polynucleotide with DRVs by admixing DRVs with polynucleotides in the ratio of 1.1 to 50 moles of cationic lipid positive charge to 1 mol of negatively charged dopolynucleotide.

The obtention of multilamellar liposomes occurs through hydration of a lipid mixture. Hydration of the lipid mixture may initially occur through lipid solubilization in an organic solvent, preferably chloroform being used as an organic solvent. Evaporation of the organic solvent is then promoted and then water is added under intense stirring, thereby favoring the formation of multilamellar hydrated liposomes. Alternatively, a lipid mixture may be hydrated by the direct addition of lipids (powder) into water under agitation under controlled temperature and agitation conditions using a high pressure homogenizer to disperse the lipids in aqueous medium. The delipid mixture which is hydrated according to the procedure described above comprises cationic lipid, structural lipid and adjuvant lipid.

The cationic lipid present in the hydrated lipid mixture is selected from 1,2-Dimyristoyl-3-Trimethylammonium-Propane; 1,2-Dipalmitoyl-3-Trimethylammonium-Propane; 1,2-Distearoyl-3-Trimethylammonium-Propane; 1,2-Dioleoyl-3-Trimethylammonium-Propane; 1,2-Diacyl-3-Dimethylammonium-Propane; DC-CholesterolHCI; Dimethyldioctadecylammonium Bromide; 1,2-Dilauroyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dimyristoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dipalmitoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Distearoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dioleoyl-sn-Glycero-3-Ethylphosphocholine; 1-Palmitoyl-2-Oleoyl-SA? -Glycero-3-Ethylphosphocholine; 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, Ndimethyl-1-propanaminium trifluoroacetate; N- [1- (2,3-dioleyloxy) propyl] -n, n, n-trimethylammoniumchloride and 1,2-Dioleoyl-sn-Glycero-3- [Phospho-L-Serine]. Preferably, the lipid di-cationic present in the liposomal configuration of the invention is 1,2-Dioleoyl-3-Trimethylammonium-Propane.

The structural lipid present in the hydrated lipid mixture is selected from synthetic lipids with asymmetric fatty acid chains, synthetic lipids with saturated symmetrical fatty acid chains, synthetic lipids with unsaturated symmetrical fatty acid chains and natural lipids. Synthetic lipid with asymmetric fatty acid chains is selected from 1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine; 1-Myristoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Stearoyl-SA? -Glycero-3-

Phosphocholine; 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Palmitoyl-s Glycero-3-Phosphocholine; 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Arachidonyl-s Glycero-3-

Phosphocholine; 1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphocholine; 1-OoyoyI-2-Myristoyl-sn-Glycero-3-Phosphocholine; 1-Oleoyl-2-Palmitoyl-sn-Glycero-3-

Phosphocholine; 1-Oleoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine. Synthetic lipid with saturated symmetrical fatty acid chains is selected from 1,2-Diacyl-sn-Glycero-3-Phosphocoline where acyl groups have carbon numbers ranging from C3 to C24- Synthetic lipid with unsaturated symmetrical fatty acid chains is selected from 1,2-Diacyl-sn-Glycero-3-Phosphocoline where acyl groups have carbon numbers: unsaturation ranging from C14: 1 to C24: 1, C18: 2 to C18: 3, C20: 4or C22: 6. Natural lipid is selected from soybean phosphatidylcholine or egg phosphatidylcholinade. Preferably, the natural lipid is egg phosphatidylcholine.

The adjuvant lipid present in the hydrated lipid mixture is selected from cholesterol and L-alpha-Dioleoyl Phosphatidylethanolamine. Preferably, the adjuvant lipid present in the mixture is L-alpha-Dioleoyl Phosphatidylethanolamine.

The process of obtaining functional liposomal configuration of the present invention may further comprise a step of reducing and homogenizing liposomes after obtaining them in step a. The liposome size reduction occurs through the extrusion step, in which the liposomes must cross overlapping polycarbonate membranes, with nominal diameter of 100 nm, under nitrogen pressure in several passages. Under these conditions the size reduction of these particles occurs.

Size reduction is not necessary when lipids are hydrated by the direct addition of lipids (in powder form) in water under agitation, under controlled temperature and agitation conditions, or by the use of a high pressure homogenizer to disperse lipids in aqueous medium. .

Obtaining dehydrated-rehydrated vesicles (DRVs) in step c is by hydration of the empty liposomes obtained in step b under stirring at a temperature of 0 ° C to 12 ° C. Preferably, the hydration temperature of the empty liposomes is from 2 ° C to 5 ° C.

In step d of obtaining the functional liposomal configuration of the invention, the lipid concentration (DRVs) to be complexed with polynucleotide is 0.02M to 0.2M. Preferably, the concentration of lipids to be complexed with compolinucleotide is from 0.05M to 0.1 M. Still in step d, mixing of compolinucleotide DRVs occurs preferably at a ratio of 5 to 15 moles of cationic lipid loading charges to 1 mol. negative charge of the polynucleotide. Even more preferably, the mixing of DRVs with polynucleotides occurs at a ratio of 10 moles of cationic lipid positive charges to 1 mol of dopolynucleotide negative charge. Mixing DRVs with polynucleotides in step d occurs at 0 ° C at 12 ° C. Preferably the mixture of compolinucleotide DRVs occurs at a temperature of 2 ° C to 5 ° C.

The subject invention further relates to processes for obtaining functional liposomal configuration which include the process described in this invention and pharmaceutical compositions containing liposomal configuration as described in the invention.

The invention also relates to a liposomal configuration defined in the invention obtained according to a process defined in the invention.

As an example of the functionality of the present invention and for purposes of comparison with other liposomal configurations, a functional liposomal configuration containing the vaccine and therapeutic bioactive plasmid pVax hsp65 was created according to the purpose of the present invention and constituted as a non-toxic formulation. stable and reproducible that will be called DRV-DNA. This plasmid construction, carrying the M. leprae Hsp65 gene, has proven prophylactic and therapeutic action (Lowrie1D.B .; Tascon, RE; Colston, MJ; Silva, CL, Vaccine, v.12, p. 1537-1540, 1994; Tascon Colston, MJ Ragno, S. Stavropoulos, E. Lowrie, DB, Nat.Med., V.2, pp. 888-892, 1996; Lowrie, DB; Silva, CL; Tascon, RE. SpringerSeminars in Immunopathology, v. 19, pp. 161-173, 1997; Bonato, VLD; Lima, VMF; Tascon, RE; Lowrie, DB; Silva, CL, Infected. Immun., V.66, pp. 169-175 , 1998; Lowrie, DB; Tascon, RE; Bonato, VDL; Lima, VMF; Faccioli, LH; Stavropoulos, E.; Colston, MJ; Hewinson, RG; Moelling, K.; Silva, CL, Nature, v. 400 269-271, 1999) .The following are characterizations that identify physicochemical and biological properties of the DRV-DNA functional liposomal configuration containing the plasmid pVax hsp65 construct.

The schematic of Figure 3 comparatively illustrates the steps for preparing the DRV-DNA configuration compared to the DRV (DNA) configuration and the lipoplex binary configuration. The preparation method is simple, consisting of steps that can be scaled for commercial production. Tight control of operating conditions ensures reproducibility of the configuration produced.

In the DRV-DNA liposomal configuration, as well as in the other configurations used for comparison, DRV (DNA) and lipoplex, a loading ratio (R +/- 10) was used, required to achieve a DNA dosage lower than previously determined for the Naked DNA for therapy and vaccination (3 dosages of 100μg). Lipid formulations contained a total concentration of 256 mM lipids, which produced a total concentration of 50 μg of DNA in 100 μί of lipid preparation.

Figures 4 to 8 show the morphological differences between the various liposomal configurations incorporating plasmid DNA through transmission electron microscopy (METs) images. Thus, Figures 4A and B show the empty DRV structure of the functional DRV-DNA-like liposomal configuration with the DNA positioned preferentially on the surface (Figures 5A to D), compared to configurations containing the DRV encapsulated DNA (DNA) (Figures 6A to D), lipid aggregates (Figures 7A and B) and Iipoplexes (Figures 8A to D).

In Figures 4A and B showing the empty liposomal DRV-like structures, approximately spherical morphology can be observed, showing some degree of fusion and aggregation, identified by the presence of larger and polydisperse structures. The process of dehydration and rehydration (DRV) has, as its main limitation, the production of large diameter liposomes and heterogeneous population, also allowing the formation of multilamellar liposomes (KIRBY, C.; GREGORIADIS, G., Biotechnology, ν. 2, ρ. 979-984, 1984). High polydispersity can be seen from the size distribution obtained by quasi-elasticIight scattering (QLS) (Figure 9C), where two populations are usually found: one smaller than about 175 nm and one larger than about 932 nm. The smaller population may be related to non-aggregated or fused vesicles, while the second largest population may be related to larger colloidal structures from fusions or aggregations.

The DRV-DNA structures shown in Figures 5 (A to D) show changes in morphology in relation to empty DRV due to DNA encapsulation in the R +/- 10 load ratio. Tubular structures can be identified in regions close to the liposomal surface, suggesting that the DNA is located in the outermost regions of the liposomes (Figures 5B, C and D). The white images that appeared in these microscopies, generated by artifacts in the sample preparation, did not compromise the morphological evaluation of the structures. Higher load ratios (smaller amounts of DNA) are not sufficient to alter DRV morphology as reported by Perrie et al (2001) for R +/- 20 liposomes. The structural differences observed in Figures 5 can also be identified from the average hydrodynamic diameters and size distribution (Table 1).

TABLE 1

Lipid Structure

<table> table see original document page 21 </column> </row> <table> (i) SD: Standard deviation for number of samples ranging from 3 to 4.

The varying sizes of the structures are the result of a specific rearrangement that occurs after aggregation (contact) and subsequent mixing of lipids between vesicles (intermediate stage) of the liposome membranes, resulting in the formation of a new layer. These interactions facilitate cross-linking of vesicles via DNA1 bridges and may lead to liposomal membrane changes favoring fusions or other forms of bilayer destabilization, as reported by (WASAN, EK; HARVIE P.; EDWARDS, K .; KARLSSON, BALLY, MB, Biochimica et BiophysicaActa, v.1461, pp. 27-46, 1999).

The difference between the morphologies found for DRV (DNA) and DRV-DNA1 structures Figures 5 and 6, respectively, are significant, especially on the outer surface of the liposomes. DRV-like structures (DNA) have some morphological irregularities (Figure 6 C), but DRV-DNA structures have a high degree of irregularity (Figure 5 B, C and D), forming tortuous structures on their surface. Comparison between these two structures suggests that DRV (DNA) presents the most homogeneous DNA distribution along the lipid bilayers, promoted by the encapsulation process (dehydration and controlled rehydration), while DRV-DNA allows DNA to be located on the outermost surfaces. The more externalized DNA localization allows fusions and aggregations between vesicles to occur more easily, promoting bilayer ruptures, thus forming the tubular or tortuous structures shown in Figure 5D.

METs of the lipid aggregates are shown in Figure 7. The presence of spherical structures can be identified (Figure 7A), but there are also structures resulting from fusions and aggregations. Figure 7B reveals some structures with irregularities in their shape when compared to the empty DRV-like liposome morphology (Figure 4A and 4B).

Wasan, E. K .; Harvie P .; Edwards, K .; Karlsson, G .; Bally, Μ. B. Biochimica et Biophysica Acta, v.1461, p. 27-46, 1999, obtained cryo-fracture electron microscopy images on DODAC (dioleyldimethylammoniumchloride) / DOPE (50/50 mole%) lipid structures in 150 mM NaCI and found dabicamate instability and could even see which part of the structure it is organized in the hexagonal inverse phase (Figure 2). The identification of this instability may justify the polymorphism found in DOPE / DOTAP (50/50 mole%) structures prepared in 154 mM NaCl and visualized by microscopy (Figure 7A and 7B). The microscopes of empty DRVs and lipid aggregates (Figures 4 and 7 , respectively) show that both lipid systems are highly dispersed. On the other hand, the size distribution analysis obtained through dynamic light scattering (QLS) shows us that these structures have bimodal distribution and also very polydisperse. The second population identified in the size distribution (Figure 9) may probably be a consequence of some fusions that occur, as shown in Figures 4A and 4B for empty DRVs and 7A and 7B for lipid aggregates. These results show that lipid distribution and packaging in the liposomal structure depends on the preparation method.

The polymorphic characteristic of Iipoplexes can be easily seen in Figure 8. Random shapes can be identified (Figures 8A and 8B), as well as tubular structures similar to those of the DRV-DNA type structures (Figure 8C and 8D).

Significant morphological changes were also observed by (Wasan, EK; Harvie P.; Edwards, K.; Karlsson, G .; Bally, MB. Biochimica et Biophysica Acta, v.1461, p. 27-46, 1999), but identified that the surface was of the finger print type for lipid structures formed by DODAC / DOPE (50/50 mol%), but due to charges 1,6. Similar structures were also found by Gustafsson et al. (1995) and Smisterová et al. (2001) in systems containing DOPE / DOTAP and SAINT-2 (pyrimidine-derivatized cationic lipids / DOPE 1: 1), respectively. The presence of these "fingerprints" was found only in formulations containing DOPE.

Comparison of DRV-DNA and Iipoplex microscopies (Figures 5 D and 7C) allows identification of regions that have morphology similar to the "fingerprints" described. The estimation of the peridiocities found reveals values in the range of 2.5 to 5.5 nm, of the same order of magnitude as the previously mentioned study, indicating that the same type of packaging is occurring in the identified regions. However, global amorphology found in DRV-DNA and Iipoplexes is not the same as found by Kasan, E. K .; Harvie P .; Edwards, K .; Karlsson, G .; Bally, Μ. B. Biochimica et BiophysicaActa, v.1461, p. 27-46, 1999, suggesting that differences in process parameters during complexation may alter the morphology of the various types of complexes.

Figure 10 shows the DRV-DNA configuration electrophoresis compared to free DNA, showing maintenance of plasmid integrity after preparation.

The configuration produced, called DRV-DNA, also has flexibility in molar charge ratio (R +/-) as a function of the required DNA dose, respecting the experimentally determined minimum charge ratio for total DNA complexation. This property allows the liposomal configuration to be used for other plasmid DNAs.

The minimum charge ratio (R +/-) for total DNA complexation depends on cationic lipid exposure, and thus characterizes the aggregate configuration. The electrophoresis of Figure 11 comparatively show the minimal (R +/-) charge ratio for total DNA complexation in the DRV-DNA (A) configuration compared to the encapsulated DNA [DRV (DNA)] (B) and Iipoplex configuration. (C) The values obtained from (R +/-) are presented comparatively in Table 2 for the various DRV-associated DNA configurations, including the configuration resulting from the association of DNA with extruded liposomes (DNA-LE) (obtained by the Bangham method and extruded for size homogenization) and lipoplexes.

TABLE 2

<table> table see original document page 24 </column> </row> <table>

The load ratio 1.5 identified for the DNA-LE configuration was the same as for the lipoplex-forming lipid aggregates. The extruded liposomes, with an average diameter of 136 nm and unimodal distribution, due to their size allow the exposure of the largest number of positive charges, causing 1.5 loading beats already to promote total DNA incorporation. In the case of lipid aggregates, despite the larger size (396.5 and 1792.8 nm), it also allows that in this load (R +/- 1.5) all the DNA is already complexed. This behavior shows the instability of the lipid structure containing only DOPE / DOTAP to complex DNA.

When DRVs are used for DNA complexation, it is found that the molar ratio of charges (R +/-) required to promote complete incorporation of DNA into the DRV-DNA configuration is 4. Comparing this value with that obtained for DNA-LE, It differs only in hydrodynamic diameter and size distribution, but for structures that have the EPC structural lipid, in addition to size, lipid distribution in the liposomal structure are important factors in the ratio of DNA uptake to incorporation. Through electron microscopy and cryo-fracture, Perrie, Y .; Frederik, Ρ.Μ .; Gregoriadis1 G. Vaccine1 ν. 19, ρ. 3301-3310, 2001, had an indication that DRV structures are multilamellar. This information explains the higher load ratio of the DRV-DNA configuration compared to DRV-LE, due to the presence of cationic lipid in the innermost lamosomes of the liposomes, hindering the initial contact with DNA and its subsequent complexation. Because EPC acts in a structural way, stabilizing lipid aggregation in bilayers, it becomes more difficult for DNA to complex with cationic lipids present in the innermost layers.

DRV-like structures (DNA) using similar plasmid (pcDNA3-hsp65) in TRIS-Edta buffer demonstrated DNA uptake capacity at up to 3.5 loading ratios.

The large difference between the loading ratio used in the lipid preparations for the in vitro and in vivo testing of this example (R +/- 10) and the ratio in which full DNA incorporation is promoted (Table 2) identifies the great flexibility of the system for adjustments. DNA concentration to meet dose requirements as applicable.

The differences in DNA / lipid packaging in liposomal and lipoplex configurations, characterized by the ratio of charges and observed in microscopies, are reflected in the surface charge measured by the zeta potential technique. Depending on the degree of complexation, the resulting configuration has a different surface charge. Table 3 comparatively shows the surface charge of the lipid formulations of the same composition incorporating the pvax hsp65 plasmid DNA into the liposome (A and B) and lipoplex (C) configurations.

TABLE 3

<table> table see original document page 25 </column> </row> <table>

(i) SD: Standard deviation for 3 samples.

(ii) Measurements used 0.9% saline as diluent medium.

All lipid structures produced in 0.9% NaCI are of cationic character because they have positive zeta potential (ζ) values. The presence of EPC structural lipid in DRV liposomes promotes a significant decrease in the zeta potential value, probably due to the greater lipid internalization that occurs due to its aggregation with DOPE and DOTAP, as well as fusion after rehydration and DNA complexation.

In the case of lipid aggregates, cationic lipids become more exposed on the colloidal surface compared to DRVs.

The presence of DNA in the charge ratio (R +/-) 10 does not significantly influence the cationic characteristic of DRVs and lipoplexes. This fact indicates that the surface of these scoloids remains cationic, with charge density values in the same order of magnitude when DNA is simply complexed (DRV-DNA and lipoplexes) or else incorporated [DRV (DNA)], suggesting that, together with the electrophoretic evaluation, that the DNA present in these structures is totally associated.

The differential exploratory calorimetry technique was used to evaluate the influence of the lipid composition and the presence of DNA at the phase transition temperature of the different lipid structures. The sample preparation consisted of a previous lyophilization step and a desiccator conditioning step under vacuum and cooling. The thermograms for the empty lipid structures and the incorporated DNA (Figure 12) allow the identification of the main transition temperatures and their enthalpy values, as shown in Table 4.

TABLE 4

<table> table see original document page 26 </column> </row> <table>

(iii) Enthalpy variation

(iv) Variation of EntropyRVD-like structures (Figure 12) had the phase transition occurring at a larger temperature range, generating wider and asymmetric transition bands when compared to lipid and lipoplex aggregates, caused by the presence of ECPC1 which is of natural origin and which contains a mixture of phospholipids with various fatty acids.

The thermograms referring to lipid and lipoplex aggregates (Figure 12), which contain only DOTAP and DOPE as lipids in their composition, have a more definite peak, probably due to the higher purity, as they are lipid-synthetics.

The phase transition temperatures for empty DRV and lipid aggregates were 12.97 and 1.22 ° C, respectively. This phase transition temperature variation occurs due to the differences in lipid compositions found in each structure, consequently reflecting differences in the aggregation form of each structure.

In the case of lipid aggregates, the phase transition temperature is attributed only to DOPE and DOTAP lipids, both in 0.5 molar fraction. The same study by KOLTOVER, I .; SALDITT, T .; RADLER, J.O .; SAFINYA, C.R. Science, v. 281, p. 78-81, 1998, lipid formulations under these conditions in water tend to form two phases simultaneously, the inverse hexagonal (H0I1) and the Iamelar (Lca). Thus, the phase transition temperature may be related to the crystalline to inverse hexagonal liquid phase transition (Lca Hcn).

The presence of EPC in empty DRV structures allows for greater hydrophobic interaction, generating a lower level of enthalpy variation (ΔΗ = 2010 cal / mol), while the presence of only DOPE and DOTAP for lipid aggregates decreases these interactions, contributing to a higher level. enthalpy ((ΔΗ = 4485cal / mol)). Entropy ((AS)) variations for empty DRV and lipid aggregates were 7.03 and 16.36 cal / mol.K, respectively. Lipid aggregates may indicate further disorganization of this structure, possibly due to the presence of inverse hexagonal lipid organization.

For DNA incorporated into [DRV (DNA)] structures, the Tm value is 12.87 ° C, remaining very close to the value of the respective empty DRV structure (Tm = 12.97 ° C).

This suggests that DNA is not causing major disruption in the lipid dabicamal organization, indicating that the process of dehydration and rehydration, in which vesicle fusion occurs, allows for good accommodation of DNA possibly in the fasacosa between the lamellae, in a sandwich-like configuration. as proposed by RÀDLER, OJ; KOLTOVER, I .; SALDITT, T .; SAFINYA, C.R. Science, v. 275, p. 810-814.1997.

This assessment is also consistent with the enthalpy and entropy variation values, as they do not change significantly, indicating that the presence of DNA does not significantly alter hydrophobic interactions and the level of dabicamate organization.

Simply complexing DNA with empty DRVs generates DRV-DNA-like structures. In this case, an increase of Tm to 14,410C is observed, being greater than the respective empty structure (Tm = 12,97 ° C) and the DRV (DNA) (Tm = 12,87 ° C). This same behavior of Tm was also identified by SUBRAMANIAN, M .; Holopainen, J. M; PAUKKU, T .; ERIKSSON, O .; HUHTANIEM, I .; KINNUNEN, P. K.J., Biochimica et Biophysica Acta, v. 1466, p. 289-305, 2000, in characterization studies of DMPC / BPDAB (95/5 mole%) compound liposomes, where BPDAB refers to bis [2- (11-phenoxyundecanoate) ethyl] dimethylammonium bromide. The authors identified that even a small increase in DNA concentration elevates the phase transition temperature, also suggesting that DNA condenses cationic lipid, forming orderly lipid domains that alter Tm.

Lipid aggregates have phase transition temperature of 1.22 ° C (Table 4). When these structures are complexed with DNA, the phase transition temperature drops to -2,410 ° C. This indicates that the organization in lipid packaging has been disturbed, diminishing van der Waals' attracting interactions.

Fluorescent probe accessibility to DNA characterizes its placement in the structural lipid configuration. PicoGreen dormant fluorescence emission spectra after double stranded DNA (dsDNA), single stranded DNA (ssDNA) and RNA binding are illustrated by Figure 13, indicating the specificity of this double stranded DNA probe. This probe is specific for double-stranded DNA (dsDNA) quantitation, but the binding mechanism of this reagent is not yet fully clear, possibly occurring its DNA intercalation (SINGER, VL; JONES, LJ; YUE, ST; HAUGLAND, RP, Analitycal Biochemistry., V. 249, pp. 228-238, 1997).

When this probe is used in cationic lipid / DNA complexes, fluorescence intensity decay occurs, which is dependent on the charge ratio. This decay is caused by the barrier created by the cationic lipid and the lipid structure, thereby decreasing DNA probe accessibility (TSAI, JT; FURSTOSS, KJ; MICHNIK, T.; SLOANE, DL Biotechnology and Applied Biochemistry1v. 36, p 13-20, 2002.). Fluorescence intensity curves as a function of the charge ratio can be constructed, allowing the characterization of DNA probe accessibility in different lipid structures. In order to minimize the influence of media composition, ultrapure water (MiIIi-Q) was used as the reaction medium of decomplexation.

The fluorescence decay profiles emitted by the PicoGreen probe in the DRV and lipid aggregate structures as a function of the fluorescence charge ratio (percentage of initial) are shown in Figures 13. Initially the fluorescence intensity drops and reaches a constant value as it rises. arazão of cargoes. When higher levels of cationic lipid are employed, the amount of free DNA in the solution tends to disappear, and consequently decreases the amount of DNA accessible to the probe. R +/- values greater than 4 for DRV-DNA and 1.75 for Iipoplex already maintain the practically constant absolute fluorescence intensity, indicating that higher load ratios do not promote more influence, possibly because there is no longer accessible DNA in the colloidal suspension. This "residual" fluorescence value is then characteristic of each type of lipid structure, and at this point can identify the fluorescence intensity (percentage of initial) as the probe's accessibility to DNA. The accessibility values were 37 ± 8.7% and 12% for RVV-DNA and lipoplex, respectively. This indicates that DRV-DNA structures allow PicoGreen greater access to DNA. On the other hand, since DNA complexation with oPicoGreen strongly depends on the presence of the double stranded configuration of DNA, in these assays fluorescence intensity is a function not only of the amount of DNA but also of its conformation (double strand or single strand). .

Although lipoplex and DRV-DNA fluorescence measurements were not performed at R +/- 10, it is known that the value of fluorescence intensity is practically the same as found for R +/- 1.75 and 4, respectively, as observed in the behavior of Figure 13 (landing region). Thus, the accessibility and behavior of these buildings in R +/- 10 is the same as previously identified.

The fluorescence intensity for the DRV (DNA) construct in R +/- 10 (formulation used for "in vivo" test) was measured, enabling the identification of accessibility, generating a value of 27.6%. This value is lower than that found for RVDR-DNA (37%), possibly due to the more even distribution in RVDR (DNA) S caused by the dehydration and rehydration process. These differences suggest that only diffusive effects are influencing the intercalation of the DNA1 probe indicating that DRV (DNA) has the most internalized DNA in its structure, while DRV-DNA has DNA present in the outermost region, and that double-stranded configuration is present. maintained. The probe's accessibility to the DNA contained in each structure type can then be related by arranging them in descending order: DRV-DNA> DRV (DNA)> Lipoplex. This behavior allows distinguishing differences between the types of DRVs structures and lipid aggregates. DRVs allow greater probe access to DNA, possibly due to its higher bilayer organization, while lipid aggregates allow for tighter molecular interaction due to lower lipid structure (simultaneous presence of inverse Iamelar and hexagonal forms), hindering diffusion and intercalation of the probe to DNA.

The physical stability of the DNA-containing lipid structures and their "empty" structures was evaluated by monitoring the hydrodynamic diameter of the different populations over 70 days of storage, as shown in Figure 14.

The empty DRV-like structure remained virtually constant over 60 days of storage (Figure 14A) maintaining both populations (200 nm and 900 nm).

When DNA is encapsulated [DRV (DNA)], the two main populations are retained, although the larger hydrodynamic diameter population has a slight tendency to increase in size (Figure 14B). It is evident the presence of two populations with hydrodynamic diameters of the same order of magnitude as the respective populations found for the empty structure, but with larger size oscillation for population 2. This oscillation can be justified by the morphology change (eg sphericity change). found in the transmission electron microscopy (Figure 6) giving a certain reading variation of the diameters in the dynamic light scattering equipment.

The DRV-DNA structure (Figure 14C), in turn, already presents a higher aggregation tendency of the largest population. The smaller population with an average diameter close to 400 nm remained relatively stable. The larger population also remained stable for 30 days with a certain tendency of segregation after 60 days of storage.

Although some variation in hydrodynamic diameter occurs for DRV-type structures, the variability was considered acceptable as the maximum hydrodynamic diameter established by the Tuberculosis Research Center is 6 μπι for application in vaccines. Storage of lipid aggregates under refrigeration and consequent monitoring The size distribution and hydrodynamic diameter can be seen in Figure 14D. In this case, the lower stability of these structures can be verified through the initial presence of three populations, including aggregates of the 12000 nm order. Significant population changes occur during storage, as from the 15th day of storage the smallest population with an initial diameter of 100 nm disappears and the third population begins to decay in size, while the second population remains the most stable. The system is highly polydisperse throughout the period evaluated.

The hydrodynamic diameter behavior of the Iipoplex populations is shown in Figure 14E. There is a tendency to vary the hydrodynamic diameter of the largest population, indicating that around 15 days of storage this population assumes a minimum size around 2000 nm and then increases again. In addition, after 35 days of storage aggregates appeared that did not disperse with manual agitation, indicating that an irreversible state of aggregation was beginning.

Interestingly, all cationic lipid structures produced a population with a diameter in the range 200 to 500 nm which remains relatively stable over the evaluated storage period, regardless of composition (presence or absence of structural lipid) and method of DNA incorporation ( encapsulation or electrostatic complexation).

The different lipid constructs were evaluated for in vitro cytotoxicity in J774 macrophage cells using a methodology based on the reduction of thiazolyl - (3- [4,5-Dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide - MTT tetrazolium salt, performed only by living cells, from method developed by DENIZOT, F.; LANG, R. Journal of Immunological Methods, v. 89, p.271-277, 1986.

Figure 15 shows control-related viable cell curves for various lipid structures [empty DRV, DRV-DNA1 DRV (DNA), elipoplex lipid aggregate] in the presence of plasmid pVAX-hsp 65 or only the commercial vector pVAX at various concentrations. The abscissa of this graph has 2 directly related scales: the amount of DNA used for "in vitro" assays, proportional to the total volume of lipid solution employed. These dual scales are necessary as the evaluation was broad in scope, including naked DNA (in proportion to the amount used in lipid formulations) and lipid formulations containing or not DNA. For DRV-like structures or only naked DNA at dosages of up to 200 μς DNA or 400 μL lipid dissolution, 70% of cells still remain viable, while lipid aggregates and Iipoplexes present less than 50% of viable cells for dosage of 8.37 μg. of DNA or 17 μΙ_ of lipid solution. This behavior indicates high cytotoxicity of these lipid structures that do not contain natural egg phosphatidylcholine in its formulation. It is noteworthy that many in vitro transfection assays are performed with these structures, but the lipid concentrations are much lower than proposed in this work.

Through in vitro transfection in J774 macrophages, it was possible to identify whether the plasmid could reach the cell nucleus and performed the transcriptional step so that the synthesis of messenger RNA (mRNA) containing the hsp 65 protein coding could occur. The method employed consisted of incubating the lipid structures with macrophage cells and then extracting total RNA. Since it is difficult to identify hsp 65-specific mRNA, a reverse transcriptase-like enzyme is used to obtain total cDNA (complementary DNA). The PCR technique amplified the specific cDNA for hsp 65 or β-actin (control) for later identification in agarose gel electrophoresis. As β-actin is a constituent element of cellarase cells, it was used as a process control.

Lipid structures containing the plasmid pVAXhsp 65 or only the pVAX vector, in addition to the plasmid and naked vector, and except for lipid aggregates and Iipoplexes due to their cytotoxicity, were evaluated for the message for hsp 65. Through Figure 16, the control for the β-actin was positive for all constructs, indicating that the process of obtaining mRNA is possible. The presence of hsp65 mRNA was identified only in cells that were transfected with the DRV-DNAhsp65 structure, suggesting that this construct has a higher efficiency of invitro transfection when compared to the other formulations used. The lack of message detection for hsp65 with the other formulations may be due to the low transfection efficiency, which may be compensated by a longer period of in vitro culture for hsp65 message detection. Through the characterization of the DRV (DNA) S it can be observed that these structures are more compact, presenting smaller average population diameter. This feature may have been influenced during the transfection process, possibly requiring greater incubation time with macrophages to allow this structure to allow DNA incorporated into the innermost regions to be released. Message detection for hsp65 was also not observed for the Iipofectin complex and DNA under the conditions recommended by the manufacturer. One of the reasons attributed to this event was the presence of a considerable amount of cellulard lysis during transfection with the formulation of Iipofectin and DNA, which may have contributed to the degradation of messenger RNAs. Although it is possible to obtain a message for β-actin from these samples, indicating that at least part of the mRNA was in good quality, less frequent messages such as hsp65 may have their detection impaired in this situation.

Interestingly, like lipofectin, there was also a large presence of cell lysis during transfection into the lipid-coagulated transfected macrophages.

The prophylactic effect of the DRV-DNA configuration containing the plasmidealpVAX-Hsp65 construct was evaluated in comparison to the DRV (DNA) configuration and the nDNA by the intramuscular and nasal routes. In the analysis of the results, protection was defined as a reduction equal to or greater than 0.5 Iog in the number of decolonial forming units (CFU) in the lungs of animals challenged with M tuberculosis, maintaining the integrity of the pulmonary parenchyma. The experimental protocol used for rotaintramuscular treatment included the total administration of 300 μg of DNA in 3 doses of 100μg, and 50 μg of DNA in single dose or 100μg of DNA in 2 50 μg applications.

Nasal administration of the liposomal configurations clearly differentiates the structures and demonstrates the superiority of the DRV-DNA configuration in protecting against TB.

Figure 17 shows the result in terms of counting the number of colony forming units (CFU) in the lungs of the M.tuberculosis challenged animals when only 1 dose of 25 μg DNA was applied.

Claims (29)

1. Functional liposomal configuration characterized by the fact that it has a maximum diameter of 2 micrometers and comprises: a) a ternary lipid system composed of cationic lipid. Structural lipid and supporting lipid and, b) a polynucleotide Functional liposomal configuration according to claim 1, characterized in that the ternary lipid system comprises the molar proportion of 20% to 30% cationic lipid, 40% to 60% structural lipid and 20% to 30% adjuvant delipid. . Functional liposomal configuration according to any one of claims 1 and 2, characterized in that the ternary lipid system comprises the molar ratio of 25% cationic lipid, 50% structural lipid and 25% supporting lipid. Functional liposomal configuration according to any one of claims 1 to 3, characterized in that the cationic lipid is selected from 1,2-Dimyristoyl-3-Trimethylammonium-Propane; 1,2-Dipalmitoyl-3-Trimethylammonium-Propane; 1,2-Distearoyl-3-Trimethylammonium-Propane; 1,2-Dioleoyl-3-Trimethylammonium-Propane; 1,2-Diacyl-3-Dimethylammonium-Propane; DC-CholesterolHCI; Dimethyldioctadecylammonium Bromide; 1,2-Dilauroyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dimyristoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dipalmitoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Distearoyl-sn-Glycero-3-Ethylphosphocholine; 1,2-Dioleoyl-sn-Glycero-3-Ethylphosphocholine; 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Ethylphosphocholine; 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, Ndimethyl-1-propanaminium trifluoroacetate; N- [1- (2,3-dioleyloxy) propyl] -n, n, n-trimethylammoniumchloride and 1,2-Dioleoyl-sn-Glycero-3- [Phospho-L-Serine] Functional liposomal configuration according to claim 4, characterized in that the cationic lipid is 1,2-Dioleoyl-3-Trimethylammonium-Propane. Functional liposomal configuration according to any one of claims 1 to 5, characterized in that the structural lipid is selected from synthetic lipids with asymmetric fatty acid chains, synthetic lipids with saturated symmetrical fatty acid chains, synthetic lipids with unsaturated symmetrical fatty acid chains. and natural lipids. Functional liposomal configuration according to claim 6, characterized in that the synthetic lipid with asymmetric fatty acid chains is selected from 1-Myristoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine; -1-Myristoyl-2-Stearoyl- sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Arachidonoyl-sn-Glycero-3-Phosphocholine; 1-Palmitoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Myristoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Palmitoyl-s Glycero-3-Phosphocholine; 1-Stearoyl-2-Oleoyl-s / Glycero-3-Phosphocholine; 1-Stearoyl-2-Linoleoyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Arachidonyl-sn-Glycero-3-Phosphocholine; 1-Stearoyl-2-Docosahexaenoyl-sn-Glycero-3-Phosphocholine; 1-Oleoyl-2-Myristoyl-sn-Glycero-3-Phosphochoine; 1-Oleoyl-2-Palmitoyl-sn-Glycero-3-Phosphocholine; 1-Oleoyl-2-Stearoyl-sn-Glycero-3-Phosphocholine. Functional liposomal configuration according to claim 6 characterized in that the synthetic lipid with symmetrical saturated fatty acid chains is selected from 1,2.-Diacyl-sn-Glycero-3-Phosphocoline where the acyl groups have a number of carbons ranging from C3 to C24. Functional liposomal configuration according to claim 6 characterized in that the synthetic lipid with unsaturated symmetrical fatty acid chains is selected from 1,2-Diacyl-sn-Glycero-3-Phosphocoline where the acyl groups have number of carbons: unsaturation ranging from C14: 1 to C24: 1, C18: 2 to C18: 3, C20: 4 or C22: 6. Functional liposomal configuration according to claim 6, characterized in that the natural lipid is selected from phosphatidylcholine desoja or egg phosphatidylcholine. Functional liposomal configuration according to claim 10, characterized in that the natural lipid is egg phosphatidylcholine. Functional liposomal configuration according to any one of claims 1 to 11, characterized in that the adjuvant lipids are selected from cholesterol and L-alpha-Dioleoyl Phosphatidylethanolamine. Functional liposomal configuration according to claim 12, characterized in that the adjuvant lipid is L-alpha-DioleoylPhosphatidylethanolamine. Functional liposomal configuration according to any one of claims 1 to 13, characterized in that the polynucleotide corresponds to a DNA and / or RNA structure. 15. Process for obtaining functional liposomal configuration characterized by the fact that it comprises the following steps: e) obtaining Iamellar liposomes from a mixture containing lipidiocathionic, structural lipid and adjuvant lipid.f) lyophilization or drying of empty liposomesg) obtaining vesicles dehydrated-rehydrated (DRVs) h) complexing a polynucleotide with DRVs by mixing DRVs with polynucleotides in a ratio of 1.1 to 50 moles of cationic lipid-positive charge to 1 mol of dopolinucleotide negative charge. Process for obtaining a functional liposomal configuration according to claim 15, characterized in that the obtaining of multilamellar liposomes in step a occurs from the hydration of a lipid mixture. Process for obtaining the functional liposomal configuration according to claim 16, characterized in that the lipid mixture comprises cationic lipid, structural lipid and adjuvant lipid. Process for obtaining a functional liposomal configuration according to any one of claims 15 to 17, characterized in that it further comprises a step of reducing and homogenizing the liposomes after obtaining them in step a. Process for obtaining a functional liposomal configuration according to any one of claims 15 to 18 characterized in that the obtaining of dehydrated-rehydrated vesicles (DRVs) in step c occurs by hydration of the empty liposomes obtained in step b under agitation and temperature. 0 ° C to 12 ° C. Process for obtaining functional liposomal configuration according to claim 19, characterized in that the hydration temperature of the empty liposomes is from 2 ° C to 5 ° C. Process for obtaining a functional liposomal configuration according to any one of claims 15 to 20, characterized in that the concentration of lipids to be complexed with polynucleotide is from 0.02M to 0.2M. Process for obtaining functional liposomal configuration according to claim 21, characterized in that the concentration of lipids to be complexed with polynucleotide is from 0.05M to 0.1 M. Process for obtaining a functional liposomal configuration according to any one of claims 15 to 22, characterized in that in step d, the mixing of DRVs with polynucleotides occurs at a ratio of 5 to 15 moles of cationic lipid depositive charges per 1 molar charge. polynucleotide negative. Process for obtaining a functional liposomal configuration according to claim 23, characterized in that the mixture of compolinucleotide DRVs occurs at a ratio of 10 moles of positive lipid-cationic charges to 1 mol of negatively charged polynucleotide. Process for obtaining a functional liposomal configuration according to any one of claims 15 to 24, characterized in that in step d, the mixing of DRVs with polynucleotides occurs at a temperature of 0 ° C to 12 ° C. Process for obtaining a functional liposomal configuration according to claim 25, characterized in that the mixture of compolinucleotide DRVs occurs at a temperature of 2 ° C to 5 ° C. A process for obtaining a liposomal configuration comprising a pellet comprising a process as defined in any one of claims 15 to 26. Functional liposomal configuration as defined in any one of claims 1 to 15, characterized in that it is obtained according to the compromise defined in any one of claims 16 to 27. Pharmaceutical composition characterized in that it comprises functional liposomal configuration as defined in any one of claims 1 to 14,