Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7088—Compounds having three or more nucleosides or nucleotides
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/10—Dispersions; Emulsions
  • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant

Definitions

• the present invention relates to the functional product as well as to a method for obtaining a polynucleotide- carrier liposomal composition for therapy and vaccination.
• This liposomal configuration incorporating polynucleotides is more effective both regarding free polynucleotides and polynucleotides carried in other liposomal configurations. It is also economically feasible, capable of clinical and veterinary use by several administration routes, stable when stored under refrigeration, and its production allows increasing the production scale for application in the industrial field.
• Liposomes or phospholipid vesicles are lamellar aggregates of lipid bilayers, alternated by one or more internal aqueous domains, forming roughly spherical particles having diameters on the order of nanometers to tens of micra. These aggregates are able to incorporate or encapsulate into their matrix charged or neutral compounds of different natures, such as hydrophilic, which are located on the internal aqueous domains, lipophilic, on the bilayers, and amphiphilic, between both domains.
• the lamellae act as membranes, which protect the carried compounds from aggressions from the medium to which they are exposed and promote their sustained release.
• Some active compounds when exposed to the oxygen from air, light, heat, or the action of biological means, are subjected to oxidation and/or breakdown or denaturation, thereby losing their activity.
• the incorporation and/or encapsulation of these active compounds into liposomes bypasses the physicochemical and biological stability limitations.
• Liposomes belong to the class of nano- and microcapsules, which have been employed successfully in the incorporation and/or encapsulation of active compounds of various natures, such as scents, enzymes, cosmetic products, drugs, among others. In general, liposomes are biodegradable, atoxic and non-immunogenic.
• Nano- or microcapsules refer to particles having diameters in the range of nanometers (10 "9 m) or micrometers (10 "6 m), respectively, and which have two distinct domains composed of at least one aqueous core enveloped by a matrix of structural material, such as lipids, polymers, etc. and blends thereof.
• the active compounds may be internalized (encapsulated in the aqueous core(s) or incorporated into the structural matrix) or located on the surface and/or at the outermost areas of the nano- or microcapsules.
• the liposomal configuration is defined by the positioning of the active compounds in their structural matrix.
• Polynucleotides are double- stranded nucleotide chains, which constitute the nucleic acids DNA (deoxyribonucleic acid) and RNA (ribonucleic acid).
• liposomes allow for a better interaction with cells by mimicking the structure and composition thereof.
• liposomes may also act as functional carriers by directing the compounds to the target organs and/or specific intracellular spaces, thus performing a helper function.
• the specific targets are the cytoplasm and the nucleus, respectively.
• cationic lipids were used to carry the DNA by means of simple electrostatic complexation and to facilitate its transportation into the interior of the cells, since they have opposed charges.
• nucleic acid carriers are the quaternary ammonium salts, such as dimethyldioctadecyl- ammonium bromide (DDAB), l,2-dioleoyl-3- trimethylammonium-propane (DOTAP), and l,2-diacyl-3- trimethylammonium propane (DOTAP) (LASIC, D.D., Boca Raton-Florida:CRC Press, 1997).
• DDAB dimethyldioctadecyl- ammonium bromide
• DOTAP l,2-dioleoyl-3- trimethylammonium-propane
• DOTAP l,2-diacyl-3- trimethylammonium propane
• the polynucleotides associated with cationic liposomes are initially internalized preferably through the endocytosis mechanism.
• the destabilization of the endosomal membrane results in a flip-flop of its anionic lipids, predominantly present in the monolayer which interfaces the cytoplasm.
• the anionic lipids diffuse into the cationic lipid/nucleotide complex and form, with the cationic lipids, ion pairs with neutral charge, displacing the nucleotides and releasing them into the cytoplasm (XU, Y.; SZOKA, F.C.J., Biochemistry, v. 35, p. 5616-5623, 1996).
• PEs phosphatidylethanolamines
• helpers enhance the release process thanks to their fusogenic characteristics, which facilitate the destabilization of the endosomal membrane
• PEs are also referred to in the literature as colipids with respect to cationic lipids.
• DOPE 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine
• Typical examples include: Lipofectin (DOTMA/DOPE), Lipofectamine (DOSPA/DOPE), and Lipofectace (DODAB/DOPE), in which DOTMA is dioleoxy propyl trimethyl ammonium chloride and DOSPA is the lipid 2,3- Dioleyloxy-N-[(spermine-carboxamide)ethyl]-N,N-dimethyl- 1 - propanaminium and DOBAB is dioctadecyldimethylammonium bromide.
• DOSPA dioleoxy propyl trimethyl ammonium chloride
• DOSPA is the lipid 2,3- Dioleyloxy-N-[(spermine-carboxamide)ethyl]-N,N-dimethyl- 1 - propanaminium
• DOBAB dioctadecyldimethylammonium bromide.
• These binary structures composed of cationic lipids and DOPE are called lipoplexes (WASAN, E.
• a very important parameter for characterizing the electrostatic complexation between polynucleotides and cationic lipids is the molar ration between charges (R+/-) established by (RADLER, J.O.; KOLTOVER, L; JAMIELSON, A.; SALDITT, T.; SAFINYA, C.R., Langmuir, v.14, p. 4272- 4283, 1998).
• This parameter relates the number of moles of positive charges from the positively-charged polar heads of the cationic lipids to the number of moles of negative charges from phosphate moieties present in the skeleton of the molecule of the polynucleotides.
• the molecular geometry of cationic amphiphilics and DOPE by itself does not further the aggregation into lamellar bilayers at physiologic conditions.
• the formed aggregates are unstable, occurring co-existence between the lamellar and hexagonal phases (RADLER, J.O.; KOLTOVER, I.; SALDITT, T.; SAFINYA, C.R., Science, v. 275, pp. 810-814, 1997), with the DNA intercalated in the structures ( Figure 2).
• PCs must have a low phase transition temperature, such as the natural egg phosphatidylcholine (EPC), such that at body temperature the lipids of the liposomal membrane are in the crystalline liquid state, giving it the necessary fluidity to facilitate the action of DOPE and DOTAP in the transportation of polynucleotides into the interior of the cells.
• EPC natural egg phosphatidylcholine
• EPC egg L- ⁇ - Phosphatidylcholine
• saturated-chain fatty acids at ratios of 16:0 (34%) and 18:0 (11%) and unsaturated- chain fatty acids at ratios of 16:1 (2%), 18:1 (32%), 18:2 (18%), 20:4 (3%), where the highest ratios are those of saturated fatty acid with 16 carbon atoms (16:0) and monounsaturated fatty acid with 18 carbon atoms (18:1).
• Lipids with cationic characteristic for complexation with the nucleotides such as the monocationic 1 ,2- dioleoyl-3-trimethylammonium-propane (DOTAP).
• DOTAP monocationic 1 ,2- dioleoyl-3-trimethylammonium-propane
• Helper lipids or colipids such as 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).
• DOPE 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine
• Liposomal-structure ternary systems containing polynucleotides may be prepared by simple incubation for electrostatic complexation, similar to binary systems, but the internalization of the polynucleotides into the liposomal structure is hindered by their molecular size. Liposomes with high DNA encapsulation efficiency, between 88 and 97%, were developed by (PERRIE, Y.; GREGORIADIS, G., Biochimica et Biophysica Acta, v. 1475, pp.
• the DRV(DNA) configuration composed by the lipids EPC/DOPE/DOTAP encapsulating the plasmid pRc/CMV HBS, which encodes the antigen expression for hepatitis B, showed better immunological response, when compared to the naked DNA (PERRIE, Y.; FREDERIK, P.M.; GREGORIADIS, G.
• DRV-DNA An alternative liposomal configuration, referred to as DRV-DNA, was obtained by complexing the DNA with empty DRV liposomes, producing particles with diameters ranging from 4-20 ⁇ m (PERRIE, Y.; GREGORIADIS, G. Biochimica et Biophysica Acta, v. 1475, pp. 125-132, 2000).
• PERRIE Y.
• GREGORIADIS G. Biochimica et Biophysica Acta
• v. 1475 pp. 125-132
• the developed works comprise the production of binary (lipoplexes) and ternary (liposomes) lipid structures encapsulating DNA and RNA.
• the described works fall short of the expectations as to the production and the biological effects of the DRV-DNA liposomal configuration of reduced size (1-2 ⁇ m).
• DRVs containing DNA in both configurations DRV(DNA) or DRV-DNA were produced only with a charge ratio between cationic lipid and DNA, (R+/-), equal to 20 or higher.
• the prophylactic and/or therapeutic effects of these configurations were not investigated with reduction of the dose with respect to the naked DNA, nor as a function of the administration route.
• Polynucleotide-based drugs have recognized benefits to human health and are high value-added.
• the present invention aims to provide a functional liposomal configuration DRV-DNA incorporating polynucleotides which provides for higher action efficiency both regarding free polynucleotides and polynucleotides carried in other liposomal configurations known so far and used in the administration of nucleic acids.
• the invention also provides a method for obtaining said liposomal configuration which, by employing specific parameters, allows the formation of a liposomal configuration DRV-DNA showing reduced size, important factor when using these configurations in gene vaccines.
• the present invention describes a functional liposomal configuration incorporating a polynucleotide, as well as a method for producing this configuration.
• the liposomal configuration to which this invention relates comprises:
• the invention still relates to a method for obtaining said functional liposomal configuration comprising the following steps of: a) obtaining pre-formed liposomes from a mixture containing cationic lipid, structural lipid and helper lipid. b) freeze-drying or drying the empty liposomes c) obtaining dehydrated-rehydrated vesicles (DRVs) d) complexing a polynucleotide with the DRVs by mixing the DRVs with polynucleotides at a ratio of 1.1 to 50 moles of positive charges from the cationic lipids to 1 mol of negative charge from the polynucleotide.
• the subject invention also relates to methods for obtaining a functional liposomal configuration, which includes the method described in this invention and to pharmaceutical compositions containing a liposomal configuration such as that described in the invention.
• the invention still relates to a liposomal configuration defined in the invention obtained according to the method defined in the invention.
• the present invention still relates to a liposomal configuration for specific prevention of tuberculosis.
• This product consists of a gene vaccine, comprising a polynucleotide-carrier liposomal composition, which polynucleotides encode the heat shock protein HSP65.
• Figure 1 shows the DNA release mechanism of the DNA/cationic liposome complex
• STEP 1 Endocytosis of the cationic liposome/DNA complex.
• STEP 2 Flip-flop of the anionic lipids present in the endosomal membrane destabilizes the endosome.
• STEP 3 Formation of neutral ionic pairs between cationic and anionic lipids.
• STEP 4 Diffusion of the DNA into the cytoplasm (adapted from Xu & Szoka, 1996).
• Figure 2 shows the balance between the lamellar L and reverse hexagonal Hn phases of the lipoplexes (Adapted from Radler , 1997).
• Figure 3 comparatively shows the steps of preparing the DRV-DNA configuration in comparison with the DRV(DNA) configuration and the binary lipoplex configuration.
• the multilamellar liposomes were produced by hydrating a dry lipid film; size reduction and homogenization was carried out by extrusion; drying of the liposomes was carried out by freeze- drying.
• the DNA was freeze- dried jointly with the empty DRVs, subsequently rehydrated.
• empty DRVs were firstly rehydrated and then complexed with the DNA.
• the lipoplexes the complexation was performed after obtaining the lipid aggregates (multilamellar liposomes).
• Figure 4 shows the transmission electron microscopy of empty DRV liposomes (PC/DOPE/DOTAP 50:25:25 mol % in salt solution). Bars indicate A) 1000 nm, B) lOOnm.
• Figure 5 shows the transmission electron microscopy of empty DRV liposomes complexed with DNA, DRV-DNA configuration, at a charge ratio (R +/ .) of 10 (PC/DOPE/DOTAP 50:25:25 mol % in salt solution). Bars indicate A) 400 nm, B) 200nm, C) 200 nm, D) 40 nm.
• Figure 6 shows the transmission electron microscopy of empty DRV liposomes encapsulating DNA, DRV- DNA configuration, at a charge ratio (R +/ .) of 10 (PC/DOPE/DOTAP 50:25:25 mol % in salt solution). Bars indicate A) 1000 run, B) 200nm, C) 100 nm, D) 100 nm.
• Figure 7 shows the transmission electron microscopy of lipid aggregates, binary composition (DOPE/DOTAP 50:50 mol % in salt solution). Bars indicate A) 400 nm, B) lOOnm.
• Figure 8 shows the transmission electron microscopy of lipoplexes (lipid aggregates complexed with DNA in charge ratio R +/ . 10) (DOPE/DOTAP 50:50 mol % in salt solution). Bars indicate A) 400 nm, B) 200nm, C) 200 nm, D) 40 nm.
• Figure 9 shows the size distribution of cationic liposomes composed by PC/DOPE/DOTAP (50/25/25 mol %) in the various processing steps for forming the DRV structures.
• Each graph represents the size distribution of three independent samples.
• FIG. 10 shows an agarose gel electrophoresis for evaluating the integrity of the plasmid pVAX-hsp65 in lipid structures of the type DRV(DNA), DRV-DNA and lipoplexes at a charge ratio (R +/ .) of 10 in salt solution (0.9% NaCl).
• the plasmid was separated from each lipid preparation by adding an organic solvent (chloroform/methanol 9:1 v/v), purified with ethanol and digested with the restriction enzyme Bam HI.
• Each line represents: M) Marker of 1Kb; 1) DNA separated from lipoplexes; 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 salt solution (0.9% NaCl) and complexed with DNA during 10 minutes at room temperature, at different charge ratios (R +/ .):
• 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 +7 . 3.5; 9) R +/ .
• A) Lipoplexes Lipid aggregates-DNA 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.
• Each line represents: 1) DNA (pVAXhsp65); 2) R +/ . 0.5; 3) R +/ . 1.0; 4) R +7 . 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 to electrophoreses with the minimum charge ratio (R+/-) for the complexation of the entire DNA, the plasmid DNA pVax hsp65 in extruded liposomes (LEs) (A) , lipoplexes (B) and DRVs (C).
• FIG 12 shows a comparative thermogram between the various lipid structures.
• Lipid compositions include: (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) Lipid aggregate, DOPE/DOTAP 50/50 mol %, R +1 . 10. (iv) Lipoplex, DOPE/DOTAP 50/50 mol %, R+/- 10. Heating rate of 10°C/minute.
• Figure 13 shows fluorescence intensity decay profiles of the PicoGreen probe (% of the initial value - relating to the free DNA) as a function of R +/ . for the structures DRV- DNA (-B-XEPC/DOPE/DOTAP, 50:25:25 mol %) and Lipoplexes (-•-)(DOPE/DOTAP, 50:50 mol %) for complexations carried out at different charge ratios, in ultrapure water (Milli-Q). The samples were excited at a wavelength of 480 nm.
• FIG. 14 shows the variation profile of the size distribution and hydrodynamic diameter of the lipid structures during storage under refrigeration.
• Figure 15 shows the in vitro citotoxicity assay in macrophages J774 by employing a methodology based on the reduction of thiazolyl-(3-[4,5-Dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide - Tetrazolium salt MTT for the different empty lipid constructs, containing the plasmid pVAX- hsp 65 or containing only the commercial vector pVAC comparatively to the toxicity levels of the lipid formulations having the same composition incorporating the plasmid DNA pVax hsp65 in the different configurations.
• Figure 16 shows the message detection for hsp65 in vitro using RT-PCR.
• Macrophages from the J774 strain were transfected with the different liposomal formulations during 72 hours and had their total RNA extracted. The presence of messages in these cells was detected by PT-PCR using hsp65- specific primers. The quality of the used cDNA was checked by amplification of ⁇ -actin. The amplification of the samples treated with DNase I is identified by DNAse (-), which indicates if there is still DNA in these treated samples or not.
• FIG. 17 shows a comparison between the recovery of viable bacilli from the lung of animals vaccinated with different doses, configurations and routes, and challenged with M. tuberculosis (Mtb).
• BALB/c mice were immunized with a dose of 50 ⁇ g of DNA Hsp65 or 2 doses of ⁇ g of DNA Hsp65 intramuscularly, or with a dose of 25 ⁇ g of DNA Hsp65 by intranasal instillation. Control groups were administered with the vehicled vector or not, or with the empty liposome.
• mice 30 days after administration, mice were challenged with 10 5 bacilli of M. tuberculosis intratracheally. Thirty days after the challenge, the animals were killed and their lungs extracted in order to recover the colony forming units. Results are represented in logio ⁇ standard deviation of CFU/g of each group. The statistical difference was considered significant when *p ⁇ 0.05 with respect to Mtb.
• Figure 18 A shows the production of antibodies in mice vaccinated with a triple dose of DNA pVAXl HSP65 intramuscularly.
• Figure 18B shows the production of antibodies in mice vaccinated with a single dose of DNA pVAXl HSP65 vehicled in liposome intramuscularly.
• Figure 19 shows the proliferation of whole cells of the spleen.
• Figure 20 shows the dosage of nitrite in a supernatant from the lung homogenizate of mice vaccinated with DNA pVAXl HSP65 or vehicled in liposomal configurations and challenged with M. tuberculosis.
• Figure 21 shows the recovery of viable bacilli from the lung of animals vaccinated with different constructs and challenged with M. tuberculosis.
• This invention provides a product for therapy and vaccination, constituted by a functional liposomal configuration containing complexed polynucleotides preferably on the outer surface of DRV liposomes, and the production method thereof
• This functional liposomal configuration containing polynucleotides has mucoadhesion properties which allow administration by non-invasive routes, such as the nasal route, and is able to efficiently carry the polynucleotides to the interior of cells, releasing them in the cytoplasm. These properties allows for higher in vivo action efficiency of the carried polynucleotides, reduced concentration and dose frequency, and, in the case of vaccination, give protection against the specific disease.
• the present invention relates to a polynucleotide-carrier liposomal configuration, as well as to a method for obtaining said polynucleotide-carrier liposomal configuration for therapy and vaccination.
• the present invention relates to polynucleotides having prophylactic and/or therapeutic activity, incorporated into a carrier of liposomal nature forming a functional configuration, biologically more stable with respect to free polynucleotides and with increased action efficiency both regarding free polynucleotides and polynucleotides carried in other liposomal configurations.
• the liposomes present in the configuration of the present invention contain lipids with the following functionalities: structural, DNA incorporation and electrostatic attraction with the surfaces of cells and enhancement of the DNA release into the cell cytoplasm.
• These liposomes are prepared by the dehydration/rehydration method under specific conditions defined in the invention, in which pre-formed liposomes are firstly obtained, and then dehydrated by freeze- drying and subsequently rehydrated under controlled conditions.
• the polynucleotides are associated with the liposomes, locating preferably onto their surface, by means of an electrostatic complexation under strictly-controlled stirring and temperature conditions such as to generate particles with sizes on the order of nanometers up to 1-2.
• DRV- DNA all polynucleotides are complexed with the liposomal system at a specific molar charge ratio between the nucleotide and the cationic lipid.
• This configuration is capable of clinical and veterinary use by several administration routes, is stable when stored under refrigeration, and its production allows increasing the production scale for application in the industrial field.
• the liposomal configuration of the present invention ensures a higher internalization efficiency of the DNA or polynucleotides into the cells, resulting in higher action efficiency of liposomal formulation with a much lower dose, producing the prophylactic and therapeutic effects.
• this liposomal configuration is not cytotoxic even at high concentrations, has high polynucleotide-carrying capacity and is constituted by particles having sizes on the order of nanometers up to 1-2 micrometers with mucoadhesion properties, such that they can be administered by a non-invasive mucosal route, such as the nasal route.
• the configuration of the present invention is reproducible and economically feasible for industrial production.
• a plasmid construct with proved prophylactic and therapeutic activity, when carried in these particles, is able to produce prophylactic and therapeutic effect.
• lipid aggregates which allows electrostatic complexation with the nucleotide and entrance to the interior of the cells, and colipid, which aids in releasing the nucleotide into the cell.
• cationic lipid which allows electrostatic complexation with the nucleotide and entrance to the interior of the cells
• colipid which aids in releasing the nucleotide into the cell.
• These commercially- available structures are mostly unstable, and simultaneously form two structures organized in the lamellar and reverse hexagonal phases, giving higher instability in the formulations, and, depending on the concentration, are also cytotoxic, which limits the carrying of a higher amount of DNA. They are generally used for in vitro assays. Therefore there are no formulations of gene vaccines or gene therapies in the market which act efficiently in vivo and which are stable and reproducible such as to allow their safe marketing.
• the liposomal configuration of the present invention shows a maximum diameter of 2 micrometers and comprises:
• the ternary lipid system of the liposomal configuration of the invention comprises the molar ratio of 20% to 30% of cationic lipid, 40% to 60% of structural lipid and 20% to 30% of helper lipid.
• the ternary lipid system of the liposomal configuration of the invention comprises the molar ratio of 25% of cationic lipid, 50% of structural lipid, and 25% of helper lipid.
• the cationic lipid present in the liposomal configuration of the invention is selected from l,2-Dimyristoyl-3- Trimethylammonium-Propane; 1 ⁇ -DipalmitDyl-3-Trimethylammonium- Propane; l ⁇ -DisteaiOyl-3-Trimethylammonium-Propane; l,2-Dioleoyl-3- Trimethylammonium-Propane; l ⁇ -Diacyl-3-Dimethylammonium-Propane; DC-CholesterolHCl; Dimethyldioctadecylammonium Bromide; 1 ⁇ 2- Dilauroyl-5 «-Glycero-3-Ethylphosphocholine; 3-Ethylphosphocholine; 1 ⁇ -Dipalmitoyl-5 « ⁇ lycero-3-Ethylphosphocholine; 1 ⁇ -Distearoyl-5 ⁇ Glycero-3-Ethylphosphocholine; 1 -2-Di
• Ethylphosphocholine 23 ⁇ ioleyloxy-N-[2(speiminecarboxarnido)ethyl]- NJSfdimethyl-1-piOpanaminium trifluoroacetate; N-[l-(2,3- dioleyloxy)piOpyl]-nAn-trimethylammonium chloride e l ⁇ -Dioleoyl-sw- Glycero-3-[Phospho-L-Serine].
• the cationic lipid present in the liposomal configuration of the invention is l,2-Dioleoyl-3- Trimethylammonium-Propane.
• the structural lipid is selected from synthetic lipids with asymmetric fatty acid chains, synthetic lipids with saturated symmetric fatty acid chains, synthetic lipids with unsaturated symmetric fatty acid chains, and natural lipids.
• the synthetic lipid with asymmetric fatty acid chains is selected from 1 -Myristoyl-2-Palmitoyl-5' «-Glycero-3-Phosphocholine; 1 -
• Myristoyl-2-Stearoyl-5 «-Glycero-3-Phosphocholine; 1 -Palmitoyl- 2-Myristoyl-sr ⁇ -Glycero-3-Phosphocholine; 1 -Palmitoyl-2- Stearoyl-sw-Glycero-S-Phosphocholine; 1 -Palmitoyl-2-Oleoyl-.sH- Glycero-3-Phosphocholine; 1 -Palmitoyl-2-Linoleoyl-5 i «-Glycero- 3-Phosphocholine; 1 -Palmitoyl ⁇ -Arachidonoyl- ⁇ -Glycero-S-
• Phosphocholine l-Stearoyl ⁇ -Myristoyl- ⁇ -Glycero-S-Phosphocholine; 1- Glycero-3-Phosphocholine; l-Stearoyl ⁇ -Linoleoyl-sr ⁇ -Glycero-S-
• Phosphocholine l-Stearoyl ⁇ -Arachidonyl- ⁇ w-Glycero-S-Phosphocholine; 1- Stearoyl ⁇ -DcxxDsahexaenoyl- ⁇ w-Glycero-S-Phosphocholine; 1 -Oleoyl-2- Myristoyl-sr ⁇ -GlyceiO-3-Phosphocholine; 1 -Oleoyl-2-Palmitoyl-sr ⁇ -GlyceiO- 3-Phosphocholine; l-Oleoyl ⁇ -Stearoyl- ⁇ -GlyceiO-S-Phosphocholine.
• the synthetic lipid with saturated symmetric fatty acid chains is selected from l ⁇ -Diacyl-sn-Glycero-S-Phosphocoline, where the acyl groups have a number of carbon ranging from C 3 to C 24 .
• the synthetic lipid with unsaturated symmetric fatty acid chains is selected from l,2-Diacyl-sn-Glycero-3-Phosphocoline, where the acyl groups have a number of carbons :unsaturations ranging from C14:l to C24:l, C18:2 to C18:3, C20:4 or C22:6.
• the natural lipid is selected from soy phosphatidylcholine or egg phosphatidylcholine.
• the natural lipid is egg phosphatidylcholine.
• the helper lipid of the liposomal configuration of the invention is selected from cholesterol and L-alpha-Dioleoyl Phosphatidylethanolamine.
• the helper lipid of the liposomal configuration of the invention is L-alpha-Dioleoyl Phosphatidylethanolamine.
• 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 method for obtaining a functional liposomal configuration comprising the following steps and parameters: a) obtaining pre-formed liposomes from a mixture containing cationic lipid, structural lipid and helper lipid. b) freeze-drying or drying the empty liposomes c) obtaining dehydrated-rehydrated vesicles (DRVs) d) complexing a polynucleotide with the
• DRVs by mixing the DRVs with polynucleotides at the ratio of 1.1 to 50 moles of positive charges from the cationic lipids to 1 mol of negative charge from the polynucleotide.
• the obtainment of multilamellar liposomes occurs by hydrating a mixture of lipids.
• the hydration of the mixture of lipids may occur by firstly solubilizing the lipids in an organic solvent, with chloroform being preferably used as an organic solvent. Next, the evaporation of the organic solvent is promoted such that water is then added under strong stirring, thus furthering the formation of multilamellar hydrated liposomes.
• a mixture of lipids may be hydrated by directly adding (powdered) lipids into water under stirring, under controlled stirring and temperature conditions with the use of a high-pressure homogenizer in order to disperse the lipids in the aqueous medium.
• the mixture of lipids, which is hydrated according to the procedure described above comprises a cationic lipid, a structural lipid and a helper lipid.
• the cationic lipid present in the mixture of lipids which is hydrated is selected from l ⁇ -Dimyristoyl-3-Trimethylammonium- Propane; l ⁇ -Dipalmitoyl-3-Trimethylammonium-Propane; l ⁇ -Distearoyl-3- Trimethylammonium-Propane; 1 ⁇ -Dioleoyl-3-Trimethylammonium-
• Ethylphosphocholine 23 ⁇ ioleyloxy-N-[2(speiminecarboxamido)ethyl]- trifluoroacetate; N-[l-(2,3- dioleyloxy)piOpyl]-n ⁇ i ⁇ -1rimethylammonium chloride e 1 ,2-Dioleoyl-sr ⁇ - Glycero-3-[Phospho-L-Serine].
• the cationic lipid present in the liposomal configuration of the invention is 1,2- Dioleoyl-3-Trimethylammonium-Propane.
• the structural lipid present in the mixture of lipids, which is hydrated is selected from synthetic lipids with asymmetric fatty acid chains, synthetic lipids with saturated symmetric fatty acid chains, synthetic lipids with unsaturated symmetric fatty acid chains, and natural lipids.
• the synthetic lipid with asymmetric fatty acid chains is selected from l-Myristoyl-2- Palmitoyl-sr ⁇ -Glycero-3-Phosphocholine; 1 -Myristoyl-2-Stearoyl- .s «-Glycero-3-Phosphocholine; 1 -Palmitoyl-2-Myristoyl-sr ⁇ - Glycero-3-Phosphocholine; 1 -Palmitoyl ⁇ -SteaiOyl-sr ⁇ -Glycero ⁇ -
• Phosphocholine l-Palmitoyl ⁇ -Oleoyl- ⁇ -Glycero-S-Phosphocholine; 1- Palmitoyl ⁇ -Linoleoyl- ⁇ w-Glycero-S-Phosphocholine; 1 -Palmitoyl-2-
• Arachidonoyl-5 «-Glyceix)-3-Phosphocholine
• 1 -Palmitoyl-2- Docosahexaenoyl- ⁇ -Glycer ⁇ -S-Phosphocholine
• Arachidonyl-OT-Glycero-S-Phosphocholine 1 -Stearoyl-2-Docosahexaenoyl- ⁇ -Glycero-3-Phosphocholine; l-01eoyl-2-Myristoyl-sr ⁇ jlycero-3-
• Phosphocholine l-01eoyl-2-Palmitoyl-5 «-Glycer ⁇ -3-Phosphocholine; 1- 01eoyl-2-Stearoyl-OT-GlyceiO-3-Phosphocholine.
• the synthetic lipid with saturated symmetric fatty acid chains is selected from 1,2- Diacyl-sn-Glycero-3-Phosphocoline, where the acyl groups have a number of carbon ranging from C 3 to C 24 .
• the synthetic lipid with unsaturated symmetric fatty acid chains is selected from 1,2- Diacyl-sn-Glycero-3-Phosphocoline, where the acyl groups have a number of carbons :unsaturations ranging from C 14: 1 to C24:l, C18:2 to C18:3, C20:4 or C22:6.
• the natural lipid is selected from soy phosphatidylcholine or egg phosphatidylcholine.
• the natural lipid is egg phosphatidylcholine.
• the helper lipid present in the mixture of lipids which is hydrated is selected from cholesterol and L-alpha- Dioleoyl Phosphatidylethanolamine.
• the helper lipid present in the mixture is L-alpha-Dioleoyl Phosphatidylethanolamine.
• the method for obtaining a functional liposomal configuration of the present invention may further comprises a step of reducing and homogenizing the liposomes after obtaining the same in step a.
• the size reduction of the liposomes occurs through the extrusion step, in which the liposomes must pass through superposed polycarbonated membranes, with a nominal diameter of 100 nm, under nitrogen pressure through several passages.
• step c the obtainment of dehydrated-rehydrated vesicles (DRVs) in step c occurs by hydrating the empty liposomes obtained in step b under stirring at a temperature of O 0 C a 12 0 C.
• the hydration temperature of the empty liposomes is from 2 0 C to 5 0 C.
• the concentration of lipids (DRVs) to be complexed with polynucleotides is from 0.02M to 0.2M.
• the concentration of lipids to be complexed with polynucleotides is from 0.05M a 0.1 M
• the mixture of DRVs with polynucleotides occurs preferably at the ratio of 5 to 15 moles of positive charges from the cationic lipids to 1 mol of negative charge from the polynucleotide.
• the mixture of DRVs with polynucleotides occurs preferably at the ratio of 10 moles of positive charges from the cationic lipids to 1 mol of negative charge from the polynucleotide.
• the mixture of DRVs with polynucleotides in step d occurs at a temperature of O 0 C a 12 0 C.
• the mixture of DRVs with polynucleotides occurs at a temperature of 2 0 C a 5 0 C.
• the subject invention further relates to methods for obtaining a functional liposomal configuration, which includes the method described in this invention and to pharmaceutical compositions containing a liposomal configuration such as that described in the invention.
• the invention further relates to a liposomal configuration defined in the invention obtained according to the method defined in the invention.
• a functional liposomal configuration was created and tested containing the plasmid bioactive compound pVax hsp65, with vaccinal and therapeutic action, constituting a non-toxic, stable and reproducible formulation that will be designated as DRV-DNA.
• This plasmid construct, carrier of the Hsp65 gene from M. leprae has proved prophylactic and therapeutic action (Lowrie,D.B.; Tascon, R.E.; Colston, MJ.; Silva, C.L., Vaccine, v. 12, pp.
• the scheme of Figure 3 comparatively shows the steps of preparing the DRV-DNA configuration in comparison with the DRV(DNA) configuration and the binary lipoplex configuration.
• the preparation method is simple and comprised of steps capable of being scaled for commercial production. The rigorous control of the operational conditions ensures the reproducibility of the produced configuration.
• a charge ratio (R+/- 10) required to achieve a DNA dosage lower than that previously determined for the naked DNA for therapy and vaccination (3 dosages of ) was used (lOO ⁇ g).
• the lipid formulations had a total lipid concentration of 64 mM, which produced a total concentration of approximately 50 ⁇ g of DNA in 100 ⁇ L of lipid preparation.
• Figures 4 and 8 show the morphological differences between the different liposomal configurations incorporating plasmid DNA, through images obtained by transmission electron microscopy (TEMs).
• Figures 4A and B show the structure of the empty DRV, the functional liposomal configuration of the DRV-DNA type with the DNA located preferably on the surface ( Figures 5A and D), as compared to configurations containing encapsulated DNA DRV(DNA) ( Figures 6A to D), lipid aggregates ( Figures 7A and B) and lipoplexes ( Figures 8A to D).
• FIGs 4A and B which show the empty liposomal structures of the DRV type, it can be observed a roughly spherical morphology, showing a certain degree of fusion and aggregation, identified by the presence of bigger and polydispersed structures.
• the dehydration-rehydration process has as its major limitation the production of liposomes with large diameter and heterogeneous population, also providing for the formation of multilamellar liposomes (KIRBY, C; GREGORIADIS, G., Biotechnology, v. 2, pp. 979-984, 1984).
• the high polydispersity may be evidenced from the size distribution obtained by means of quasi-elastic light scattering (QLS) ( Figure 9C), where two populations are usually found: a lower one having about 175 nm and a higher one having about 932 nm.
• QLS quasi-elastic light scattering
• Figure 9C quasi-elastic light scattering
• the DRV-DNA structures shown in Figures 5 exhibit alterations in their morphology in relation to empty DRVs as a result of the DNA encapsulation at a charge ratio R+/- of 10.
• Tubular structures can be identified in areas near the liposomal surface, suggesting that the DNA is located on the outermost areas of the liposomes ( Figures 5B, C and D).
• Higher charge ratios lower DNA amounts
• the structural differences observed in Figure 5 may also be identified from the mean hydrodynamic diameters and size distribution (Table 1).
• the different sizes of the structures result from a specific rearrangement that occurs after the aggregation (contact) and subsequent mixture of lipids between vesicles (intermediary step) of the liposomal membranes, resulting in the formation of a new structures.
• These interactions facilitate the cross-linking of the vesicles through DNA linkers, possibly leading to changes in the liposomal membranes which promote fusions or other forms of destabilization of the bilayers, as reported by (WASAN, E. K.; HARVIE P.; EDWARDS, K.; KARLSSON, G.; BALLY, M. B., Biochimica et Biophysica Acta, v.1461, p. 27-46, 1999).
• Figure 7 TEMs of the lipid aggregates are shown in Figure 7. It is also possible to identify the presence of spherical structures (Figure 7A), but there are also structures resulting from fusions and aggregations. Figure 7B shows some structures with irregularities in their shape, when compared to the morphology of empty DRV liposomes ( Figure 4A and 4B).
• the identification of this instability may account for the polymorphism found in the DOPE/DOTAP structures (50/50 mol %) prepared in 154 mM of NaCl and viewed through the microscopies ( Figure 7A and 7B).
• the microscopies of empty DRVs and lipid aggregates show that both lipid systems are highly polydispersed.
• the size distribution analysis obtained through quasi-elastic light scattering (QLS) shows us that these structures exhibit a bimodal and also very polydispersed distribution.
• the second population identified in the size distribution ( Figure 9) may be probably a consequence of some fusions that occur, as shown in Figures 4A and 4B for empty DRVs and 7 A and 7B for lipid aggregates.
• DRV-DNA and lipoplexes allows the identification of areas having a similar morphology to the described finger prints.
• the estimation of found periodicities reveals values in the range of 2.5 to 5.5 nm, on the same order of magnitude that the previously mentioned study, indicating that the same type of packing is occurring in the identified areas.
• the overall morphology found in DRV-DNA and in the lipoplexes is not the same found by Wasan, E. K.; Harvie P.; Edwards, K.; Karlsson, G.; Bally, M. B. Biochimica et Biophysica Acta, v.1461, pp. 27-46, 1999, suggesting that differences in process parameters during complexation may change the morphology of the various types of complexes.
• Figure 10 shows the electrophoresis of the DRV-DNA configuration in comparison with the free DNA, showing the maintenance of the integrity of the plasmid after preparation.
• the produced configuration designated by DRV- DNA, also has flexibility in the molar charge ratio (R+/-) as a function of the required DNA dose, respecting the minimum charge ratio, determined experimentally, for the full DNA complexation. This property allows the liposomal configuration to be used for other plasmid DNAs.
• the minimum charge ratio (R+/-) for the full DNA complexation depends on the exposure of the cationic lipid and, therefore, characterizes the aggregate configuration.
• the electrophoreses of Figure 11 comparatively shows the minimum charge ratio, (R+/-) for the full DNA complexation in the DRV- DNA configuration (A), in comparison with the configuration containing the encapsulated DNA [DRV(DNA)] (B) and lipoplexes (C).
• This information accounts for the increased charge ratio of the DRV-DNA configuration as compared to DRV-LE, due to the presence of a cationic lipid in the innermost lamellae of the liposomes, making the initial contact with the DNA and its subsequent complexation difficult.
• EPC acts in a structural way, stabilizing the lipid aggregation into bilayers, it becomes more difficult for the DNA to complex with the cationic lipids present in the innermost layers.
• DRV(DNA) structures using a similar plasmid (pcDNA3-hsp65) in a TRIS-Edta buffer showed the ability of incorporating DNA at charge ratios as high as 3.5.
• the major difference between the charge ratio used in the lipid preparations for the in vitro and in vivo tests of this example (R+/- 10) and the ratio at which the full DNA incorporation is promoted (Table 2) identifies the high flexibility of the system in adjusting the DNA concentration to meet the dose requirements intended for the application.
• Lipid aggregate +48.3 ⁇ 3.2
• lipid structures produced in 0.9% NaCl are of cationic characteristic, as they show positive zeta potential values.
• the presence of the structural lipid EPC in the DRV liposomes promotes a significant reduction of the zeta potential value probably due to the higher internalization of the lipids that occurs both by their aggregation with DOPE and DOTAP and by their fusion after rehydration and complexation with the DNA.
• the cationic lipids become more exposed on the colloidal surface, as compared with DRVs.
• the differential scanning calorimetry technique was used to evaluate the influence of the lipid composition and the presence of DNA on the phase transition temperature of the different lipid structures.
• the sample preparation consisted of a previous step of freeze-drying and another step of packing into a desiccator under vacuum and refrigeration.
• the thermograms for the DNA-incorporated and empty lipid structures (Figure 12) allow to identify the main transition temperatures and the respective enthalpy values, as shown in Table 4.
• thermograms related with the lipid aggregates and lipoplexes which contain only DOTAP and DOPE as lipids in their composition, show a more accurate peak probably due to the higher purity, since these are synthetic lipids.
• phase transition temperatures for empty DRV and lipid aggregates were 12.97 e 1.22 0 C, respectively. This phase transition temperature variation occurs due to lipid composition differences found in each structure, thereby resulting in differences in the aggregation form of each structure.
• phase transition temperature is contributed only by the lipids DOPE and DOTAP, both at a molar fraction of 0.5.
• lipid formulations under these conditions in water, tend to simultaneously form two phases, reverse hexagonal (H C DD ) and lamellar (L C D ).
• the phase transition temperature may be related with the phase transition from crystalline liquid into reverse hexagonal (L C D — > H DD)-
• the entropy variations ( ⁇ S) for empty DRV and lipid aggregates were 7.03 and 16.36 cal/mol.K, respectively.
• the higher value of ( ⁇ S) for the lipid aggregates may indicate higher disorganization of this structure, possibly due to the presence of the lipid organization in the reverse hexagonal form.
• the lipid aggregates exhibit phase transition temperatures of 1.22 0 C (Table 4). When these structures are complexed with the DNA, the phase transition temperature falls to -2.41 0 C. This indicates that the organization in the lipid packing underwent perturbations, decreasing van der Walls interactions.
• the fluorescent probe accessibility to the DNA characterizes its positioning in the structural lipid configuration.
• the fluorescence emission spectra of the PicoGreen reagent after binding with double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) and RNA are shown in Figure 13, indicating the specificity of this probe to the double-stranded DNA.
• This probe is specific for quantification of double-stranded DNA (dsDNA), however, the binding mechanism of this reagent is not completely clear, possibly occurring its intercalation with the DNA (SINGER, V.L.; JONES, LJ.; YUE, S.T.; HAUGLAND, R.P., Analitycal Biochemistry., v. 249, pp. 228-238, 1997).
• this "residual" fluorescence value is characteristic of each type of lipid structure, being able at this point to identify the fluorescence intensity (percentage of the initial value) as the accessibility of the probe to the DNA.
• the accessibility values were 37 ⁇ 8.7% e 12%, for DRV-DNA and lipoplex, respectively. This indicates that DRV-DNA structures allow higher access of PicoGreen to the DNA.
• the fluorescence intensity is a function not only of the DNA amount, but also of its conformation (double-stranded or single-stranded).
• DRVs allows higher access of the probe to the DNA possibly due to its larger bilayer organization, while the lipid aggregates allow a more intense molecular interaction due to the lower lipid structuring (simultaneous presence of lamellar and reverse hexagonal forms), making the diffusion and intercalation of the probe into the DNA harder.
• the different lipid constructs were evaluated as to their in vitro citotoxicity in macrophage cells of the J774 strain by employing a methodology based on the reduction of thiazolyl- (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide - Tetrazolium salt MTT, carried out only by live cells, from the method developed by DENIZOT, F.; LANG, R. Journal of Immunological Methods, v. 89, pp. 271-277, 1986.
• Figure 15 shows viable cell curves relative to the control for the different lipid structures [empty DRV, DRV- DNA, DRV(DNA), lipid aggregated and lipoplex] in the presence of the pVAX-hsp 65 plasmid or the commercial vector pVAX only at different concentrations.
• the abscissa from 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 double scales are necessary, since the evaluation had a general scope, including the naked DNA (in a amount proportional to that used in lipid formulations) and lipid formulations containing DNA or not.
• mRNA messenger RNA
• the employed method consisted in realizing the incubation of the lipid structures with macrophage cells, thereafter realizing the total RNA extraction.
• a reverse transcriptase enzyme is used to obtain the total cDNA (complementary DNA).
• ⁇ -actin is a constitutive element of eukaryotic cells, it was used as a control of the process.
• the control for ⁇ -actin was positive for all constructs, indicating that the method for obtaining mRNA is possible.
• the presence of mRNA for hsp65 was identified only in the cells which were transfected with the DRV- DNA structure hsp65, suggesting that this construction exhibits a higher in vitro transfection when compared with the other formulations used.
• the absence of message detection for hsp65 with the other formulations may result from the low transfection efficiency, which can be compensated by a longer in vitro culture time for detecting the message for hsp65.
• DRV(DNA)S By characterizing the DRV(DNA)S, it can be observed that these structures are more compact, having a lower population mean diameter. This characteristic may have influenced during the transfection process, possibly requiring a longer incubation time with macrophages in order that this structure allows the DNA incorporated in the innermost areas to be released.
• the prophylactic effect of the DRV-DNA configuration containing the plasmid construct pVAX-Hsp65 was evaluated in comparison with the DRV (DNA) configuration and with the naked DNA intramuscularly and nasally.
• the protection was defined as the lowering equal to or higher than 0.5 log of the number of colony forming units (CFU) in the lung of animals challenged with M. tuberculosis, maintaining the integrity of the lung parenchyma.
• the experimental protocol used for the intramuscular route contemplated the total administration of 300 ⁇ g of DNA in 3 doses of 100 ⁇ g, and of 50 ⁇ g of DNA in a single dose or 100 ⁇ g of DNA in 2 applications of 50 ⁇ g.
• FIG. 17 shows the result in terms of counting of the number of colony forming units (CFU) in the lung of animals challenged with M. tuberculosis when only 1 dose of 25 ⁇ g of DNA was applied.
• CFU colony forming units
• Example of said composition applied to a gene vaccine for the treatment and prevention of Tuberculosis For the present embodiment, the liposomal carrying of DNA-HSP65 was used.
• the DNA-HSP65 in this liposomal configuration has the advantage of combining its potential against tuberculosis in a liposomal configuration having high transfection efficiency, without the need of adding immunostimulators, such as trehalose dimycolate.
• the plasmid vaccine construct, pVAXl, containing DNA-Hsp65 carried in functional liposomes, that is, composed of the functional lipids EPC, DOPE and DOTAP, is prepared by the dehydration-rehydration technique, and contains the DNA preferably located on its surface (Herein named LIPO-DNA-HSP65). Said vaccine obtained herein was evaluated as to its immunization intramuscularly (IM) and intranasally (IN).
• IM intramuscularly
• IN intranasally
• the liposomal gene vaccine, LIPO-DNA- HSP65 is effective in reducing the CFU of M. tuberculosis in a similar way to the currently-used commercial vaccine, BCG, administered in single dose, using a single antigen (instead of the attenuated microorganism - BCG), by a non-invasive route, without including other immunoadjuvants in the formulation (such as trehalose dimycolate - TDM).
• the plasmids pVAXl, HSP65 and pVAXl were previously prepared by means of usual techniques in the literature and subsequently evaluated as to their integrity, also by usual methods in the literature. Sequentially, plasmids were selected to be used in the liposomal preparations for use in the obtainment of the liposomal gene vaccine herein object of the present embodiment.
• the liposomal gene vaccine disclosed herein was produced by complexing functional liposomes with the plasmid DNA-Hsp65.
• DOTAP Dioleoyl-3-Trimethylammonium-Propane
• DOPE Dioleoyl-sn-Glycero-3-Phosphoethanolamine
• EPC Phosphatidylcholine
• the liposomal composition comprising the l o lipids DOTAP, EPC and DOPE did not show citotoxicity .
• a previously determined amount of the lipid solutions that is, of the stock solution from each lipid, was added into a suitable container, by which it was subjected to homogenization in a suitable apparatus during a time period
• said solutions were added into a round-bottom flask and homogenized in a rotary evaporator. After the homogenization period, the evaporation of chloroform was promoted, herein used as a solvent in the selected lipid solutions. Said evaporation 0 occurred under relative vacuum ranging between 250-760mmHg and at a temperature value superior to the higher phase transition temperature of the components until a dry film was formed. Once all the solvent of the mixture was evaporated, the dry film obtained is hydrated with an enough amount of deionized water,
• liposomal structures such as the standard for injection to obtain the liposomal structures.
• a step of size reduction which consists in a extrusion procedure, which comprises the sequential injection of a sample volume into the stainless steel extrusion equipment, with a thermal jacket for water circulation under high pressure value using a polycarbonate membrane having a mean pore diameter of about lOOnm.
• the liposomal structures obtained herein after extrusion are subjected to fast freezing and low temperature values and then subjected to freeze-drying for about 24 hours for subsequent use.
• the liposomal structures were frozen with liquid nitrogen, during a time period preferably of 3 to 25 minutes.
• the functional liposomes were rehydrated by means of usual techniques in the literature.
• the liposomes were rehydrated in a salt solution, such as a NaCl solution (1.44%) at a temperature higher than that of phase transition of the lipids under stirring in a suitable apparatus.
• the liposomal gene vaccine was obtained by associating/complexing the DNA (in water), under the form of plasmid, into the functional liposomes, under constant stirring and under temperature control for a varying time period of about 60 seconds.
• the mean diameter and distribution per number of colloidal particles for the gene vaccine obtained herein by the method disclosed by the present embodiment has a size on the order of nanometers up to 1-2 micrometers.

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