MXPA05014095A - Nanoparticles of polyoxyethylenated derivatives - Google Patents

Abstract

The invention relates to nanoparticles of polyoxyethylenated derivatives, having a size of less than 1 micrometer, for the administration of pharmaceutically- or cosmetically-active ingredients. The inventive nanoparticles comprise a biodegradable polymer, a polyoxyethylene-derived block copolymer and at least one pharmaceutically- or cosmetically-active ingredient. The invention further relates to the method of obtaining the aforementioned nanoparticles and to compositions containing same.

Description

NANOPARTICLES OF POLIOXYMENTED DERIVATIVES FIELD OF THE INVENTION The present invention relates to nanoparticles (size less than 1 μm) with a new composition, which are suitable for the administration of active molecules. The new composition comprises two polymers: a biodegradable polymer and a block copolymer derived from polyoxyethylene. BACKGROUND OF THE INVENTION Polymeric nanoparticles are receiving special attention due to their interest in improving stability and promoting the transport and controlled release of drugs to certain regions of the body. The most used biodegradable polymers for their formation are derivatives of polylactic acid (PLA) and their copolymers with glycolic acid (PLGA) due to their biodegradability, biocompatibility and innocuity (Johansen et al., Eur. J. Pharm. Biopharm., 2000, 50; 129-146). Other biodegradable polymers that also offer a promising future in this line are polyesters such as poly (e-caprolactone) (Losa et al., Pharm. Res., 1993, 10, 1, 80-87) and polyanhydrides (Mathiowitz et al., Nature, 1997, 386, 410-414). The micro and nanoparticles of PLA and PLGA have been extensively studied for encapsulation and release REF. 169051 of a large number of therapeutic molecules (Quintanar-Guerrero et al., Drug Dev. Ind. Pharm., 1998, 24 (12), 1113-1128, Sánchez et al., Int. J. Pharm., 1993, 99, 263 -273, Sturesson et al, J. Control, Reí., 1999, 59, 377-389, Hsu et al, J. Drug Targ., 7 (4), 313-323). A remarkable feature of these particles lies in the fact that their ability to control the release of active molecules depends on their degradation profile. In this way, a control in the degradation rate of the polymer has a direct impact on the control of the release of the active molecule associated therewith. It is known that the degradation of the polyesters leads to the formation of acid oligomers that can accumulate inside the particles, thus causing an acidification of the polymeric framework and with it a significant reduction of the internal pH of the particles (Belbella et al., Int. J. Pharm., 1996, 129, 95-102). This acid microclimate caused by the accumulation of polymer degradation products within the particles has a very negative effect on the stability of the active molecule incorporated in them and represents a limitation in the use of these polymeric systems for controlled release of macromolecules such as proteins and DNA plasmids (Zhu et al., Nature Biotech., 18, 52-57).

Poloxamers are polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) triblock copolymers which, depending on their PEO: PPO ratio, vary in their characteristics of molecular weight, hydrophobicity, etc. Poloxamines are copolymers formed by 4 chains of PEO -PPO joined by an ethylene diamine bridge. Analogously to poloxamers, their characteristics may change when the PEO-PPO ratio changes. One of the applications that has recently been proposed for this family of copolymers derived from polyoxyethylene is to be promoters of drug transport through the blood-brain barrier (BBB) (Kabanov et al., Adv. Drug. Deliv. Rev., 2003, 55, 151-164). Likewise, recent studies have shown their interest in transferase studies of plasmids DNA (Lemieux et al., Gene Ther., 2000, 7, 986-991). On the other hand, PEO: PPO block copolymers have been widely studied as coating agents that allow modifying the biodistribution of nanoparticles used as drug transporters. Thus, numerous studies have shown that the coating of nanoparticles with poloxamers and poloxamines affects their biodistribution and, therefore, their ability to transport drugs to different regions of the organism (Moghimi et al., FEBS Letters, 1994, 344, 25-30, Ha ley et al., FEBS Letters, 1997, 400, 319-323). There are several documents in which the use of PEO-PPO derivatives as nanoparticle coating agents is claimed (WO96120698 and US4904479). The objective has been to prolong the circulation time of the same after their intravenous injection and modify their biodistribution profile. In the compositions the poloxamer / poloxamine is not part of the constitutive polymer matrix of the particles but is adsorbed at the surface level. Therefore, the amount of poloxamer / poloxamine adsorbed is limited and its presence has no implications in the encapsulation or controlled release of the active molecule encapsulated in the particles, but its role is limited to the modification of the biodistribution profile of the particles. On the other hand, in another document, US5578325, the idea of chemically coupling said copolymers to polyesters has been proposed, thus forming multi-block copolymers. In these cases the polyoxyethylenated derivative is covalently bound to the polyester, thus leading to the formation of a new copolymer. These copolymers also make it possible to obtain nanoparticles coated with PEO-PPO that offer a long stay in the bloodstream after their intravenous administration.

Another application of the PEO-PPO block copolymers has been the stabilization of proteins encapsulated in PLGA particles and the modification of their release from them. Thus, in previous studies carried out in our laboratory, we have been able to verify that the incorporation of block copolymers PEO-PPO, more specifically poloxamers, in micro and nanoparticles of polyacid lactic acid / glycolic acid (PLGA) allows to improve the stability of proteins nanoencapsulated in such particles. In this initial study, for the incorporation of poloxamer in the particles we opted for the double emulsion method (water / organic solvent / water), according to which, the hydrophilic poloxamer is dissolved in the aqueous internal phase of the emulsion (Blanco et al., Eur. J. Pharm. Biopharm., 1997, 43, 287-294, Blanco y collaborators Eur. J. Pharm. Biopharm., 1998, 45, 285-294). This method allows the incorporation of very small amounts of poloxamer in relation to the amounts of PLGA (normally the ratio is 10: 1 PLGA: poloxamer). This is due to two fundamental reasons: on the one hand, the volume of the aqueous internal phase in which the poloxamer is dissolved is much lower than the volume of organic solvent in which the hydrophobic polymer (PLGA) is dissolved; on the other hand, the poloxamer tends to diffuse, during the emulsification process, from the aqueous internal phase to the aqueous external phase, thus hindering the formation of the particles. This difficulty has been overcome by using an anhydrous microencapsulation method, consisting of the formation of an emulsion of an organic solvent (in which the PLGA and the poloxamer are to be dissolved) in an external oil phase in which an organic solvent is dissolved. surfactant agent. Thanks to this method, high amounts of poloxamers have been incorporated into microparticles of PLGA (up to 50%) forming mixed matrices PLGA: poloxamer. This mixed microparticular system formed by an intimate mixture of poloxamer and PLGA has allowed the controlled release of proteins (Tobío et al., Pharm. Res., 1999, 16, 5, 682-688). However, the most notable drawback of this method lies in the difficulty to obtain nanometric particles, the average size of these populations being greater than 1 miera (1000 nanometers). Furthermore, given the need to use oils as the external phase of the emulsion, the isolation of the microspheres becomes very laborious and the use of significant amounts of organic solvents is necessary to achieve the elimination of the oil. Therefore, until now, no process has been described that allows the incorporation of high amounts of poloxamer in mixed poloxamer nanoparticles: PLGA.

The first reference found regarding the use of poloxamers in the formation of mixed matrices with polyesters based on the physical union of both polymers is described in the publication US5330768. In the document, the use of the mixtures is proposed in order to achieve a modification in the release of the active molecule incorporated in these systems. It refers to the formation of films by co-dissolution of both polymers in a common organic solvent and subsequent evaporation of the solvent or by the joint fusion of both polymers and also to the formation of microparticles by the double emulsion method (water / solvent). organic / water); however, the formation of nanoparticles is not mentioned. It should be noted that the aforementioned process of formation of particles in aqueous external phase, allows only the incorporation of limited amounts of hydrophilic poloxamers due to its logical tendency to diffuse to the aqueous external phase; this fact has been confirmed in our previous studies (Blanco et al., Eur. J. Pharm. Biopharm., 1997, 43, 287-294, Blanco et al. Eur. J. Pharm. Biopharm., 1998, 45, 285- 294). Also, the document US5330768 does not mention the use of lipophilic poloxamers or poloxamines in the formation of said mixtures.

The first document found that refers to the formation of a mixed microparticular system formed by an intimate mixture of poloxamer and PLGA aimed at improving the stability of icroencapsulated proteins, also allowing their controlled release, is the one published by Tobio and collaborators (Pharm. ., 1999, 16, 5, 682-688). More recently, US6465425 also describes the formation of biodegradable microparticles containing poloxamer for the same purpose. Likewise, in the composition, an excipient of the acid type and at least one polysaccharide are incorporated for the same purpose. According to the document, the amount of poloxamer that can be included in this composition can vary between 1-40% with respect to the total weight of the composition. The form of presentation of this composition is that of films, obtained by simple evaporation of the solvent, or of microparticles obtained by atomization. However, no reference is made to the formation of nanoparticles, which is understood if we take into account that the atomization technique does not allow obtaining particles as small as nanoparticles. In the same line, with the aim of improving the stability of proteins, the document presented by Schwendeman et al. (Document US2002 / 0009493), which describes the use of hydrophilic poloxamers of molecular weight between 500 and 30,000 Da, as agents. of pores in systems made from polyesters. The document claims the presentation of these compositions in the form of cylinders or microparticles, with a size comprised between 10-100 μm. These particles are obtained by the technique of double emulsion in aqueous external phase, which allows only the incorporation of small amounts of hydrophilic poloxamers, as noted in previous studies (Blanco et al, Eur. J. Pharm. Biopharm., 1997, 43, 287-294; Blanco et al. Eur. J. Pharm. Biopharm., 1998, 45, 285-294), or alternatively, by the emulsification technique of organic solvent / oil, which, as indicated above ( Tobío and colleagues Pharm. Res., 1999, 16, 5, 682-688), does not allow obtaining nanoparticles. In relation to the documents that explicitly refer to the formation of nanoparticles containing poloxamers, mention may be made of document US5962566. However, this document points out the incorporation of cholesterol as an essential ingredient for the formation of nanoparticles. The training method also indicates the need to melt the set of materials and their subsequent dispersion in an aqueous phase. A document describing the formation of nanoparticles incorporating poloxamers and poloxamines in addition to a stabilizing lipid agent (document US20030059465) can also be cited. These nanoparticles are directed to the release of the cytostatic agent camptothecin and are obtained by a process of hydration of previously freeze-dried lipids. Although the possible incorporation of polyesters such as PLGA is claimed in the document, the fact is that the technique described is not applicable to this type of polymers. In any case, the incorporation of lipids in the structure is shown as an essential element of the nanoparticular composition. As a consequence of the review of the previous documents, it is worth noting that despite the significant number of documents that refer to the formation of PLGA and poloxamer particles, the truth is that none of the cited documents describes the formation of mixed matrices containing high amounts of poloxamers and poloxamines, with different characteristics of hydrophilicity / lipofilia and that appear under the nanoparticular form. This last aspect is of critical importance since the microencapsulation techniques intended for the formation of microparticles generally differ from the nanotechnologies applied to the formation of nanoparticles. LikewiseIt should be noted that the published documents related to obtaining nanoparticles use only hydrophilic poloxamers incorporated in very low proportions in the nanoparticular system. BRIEF DESCRIPTION OF THE INVENTION The present invention relates to nanoparticles comprising a biodegradable polymer, preferably a polyester and a block copolymer derived from polyoxyethylene, preferably poloxamer and poloxamine. Also, the present invention relates to a method of preparation that allows the incorporation of high percentages of poloxamers and poloxamines in nanoparticles, the ratio being biodegradable polymer: polyoxyethylenated derivative between 1: 0.1 and 1: 3. Therefore, according to a first aspect, the invention relates to a process for the preparation of nanoparticles smaller than 1 μm, for the administration of active ingredients, comprising the steps of: a) dissolving a biodegradable polymer together with a block copolymer derived from polyoxyethylene in an organic solvent, the weight ratio being biodegradable polymer: block copolymer between 1: 0.1 and 1: 3; b) adding, under stirring, the solution obtained to a polar phase, in which the biodegradable polymer has a low solubility, precipitating the polymers and forming the nanoparticles; c) remove the organic solvent; d) isolate the particles. The active ingredient can be dissolved directly in the non-polar organic solvent (lipophilic molecules) or can be previously dissolved in a small volume of aqueous phase (water-soluble molecules) and then dispersed in the organic solvent, before or after the step, preferably the organic solvent in a) will be a non-polar solvent. According to a preferred embodiment, the preparation of the intimate mixture nanoparticle formulations can additionally include a lyophilization step. In lyophilized form, nanoparticles can be stored for long periods of time and easily regenerated, simply by adding an optimal volume of water. The lyophilization of the nanoparticles has been optimized with the incorporation of a cryoprotective excipient (glucose or trehalose) in the suspension medium of the formulations. According to another preferred embodiment, in the above process the biodegradable polymer is a polyester, which is selected from the group of polyesters such as polylactic acid, polylactic-co-glycolic acid and its copolymers, polycaprolactone or the group of polyanhydrides. For the preparation of intimate nanoparticles, the polyactic-co-glycolic acid polymer 50:50 Resomer RG 503 Mw: 35000 (Boehringer Ingelheim) was used. According to other preferred embodiments, the block copolymer is selected from poloxamers and poloxamines. Poloxamers are polyoxyethylene-polyoxypropylene-polyoxyethylene (PEO-PPO-PEO) triblock copolymers which, depending on their PEO: PPO ratio, vary in their molecular weight, hydrophobicity, etc. characteristics. Preferably, the poloxamers used will have a molecular weight between 1,000 and 25,000 Daltons. These polymers can be obtained from BASF Corporation under the trade name Pluronic.TM. For the preparation of intimate mixture nanoparticles we used the following poloxamers: Pluronic.TM F68 with molecular weight 8350 and HLB = 29, Pluronic.TM with molecular weight 4400 and HLB = 1. Poloxamines are copolymers formed by 4 chains of PEO-PPO joined by an ethylene diamine bridge. Analogously to poloxamers, their characteristics may change when the PEO-PPO ratio changes. Preferably, the poloxamines used will have a molecular weight between 1,000 and 25,000 Daltons. These polymers can be obtained from BASF Corporation under the trade name Tetronic.TM. For the preparation of intimate mixture nanoparticles we used the following poloxamines: Tetronic.TM 908 with molecular weight 25000 and HLB = 30.5, Tetronic.TM 904 with molecular weight 6700 and HLB14.5, Tetronic.TM 901 with molecular weight 4700 and HLB = 2.5. According to another preferred embodiment, the weight ratio of biodegradable polymer is between 1: 1 and 1: 3. According to a second aspect of the present invention, this refers to nanoparticles obtained according to the process described above, both lyophilized and non-lyophilized. These nanoparticles offer innovative and distinctive features given their capacity for the encapsulation and controlled release of very sensitive active molecules, such as proteins and DNA plasmids. In addition, due to the presence of important amounts of poloxamers and poloxamines in their composition, the nanoparticles can present a profile of differentiated biodistribution, in comparison to the classic particles constituted from polyesters. Due to their nanoparticular size these new systems will be able to be administered to the human organism by any route of administration, including the intravenous route, while the microparticles can not be administered by this route due to the obstruction that they would cause in the blood capillaries. Also, there is abundant documentation that shows that nanoparticles are able to overcome biological barriers (mucous membranes), epithelia) while the microparticles are not. The physico-chemical properties of the formulations of different composition and different polymer ratio have been characterized using the techniques of photon correlation spectroscopy (PCS) and laser-Doppler anemometry. The morphology of the nanoparticles was studied by transmission electron microscopy (TEM) and 1 H NMR. These studies confirmed the formation of the intimate mixing system described above. In order to verify the applicability of these intimate mixture nanoparticles for the release of delicate macromolecules, we have encapsulated the plasmid pEGFP-Cl (encoding a green fluorescent protein) in the different formulations. The results of these "in vitro" release studies have demonstrated the potential of the formulations as controlled release vehicles during extended times. The cytotoxicity of the nanoparticles of different compositions, at different concentrations, has been tested in cell cultures with the colorimetric test of MTS ((3- (4,5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 - (4-sulfophenyl) -2H-tetrazolium) in the MCF-7 cell line grown in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS); It can be concluded that none of the formulations produces toxic effects in the cells. According to a third aspect, the present invention relates to compositions, especially pharmaceutical and cosmetic, that incorporate the nanoparticles according to the present invention. In the following, the invention will be explained in more detail on the basis of a series of examples, without limiting the scope of the invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1: 1H NMR spectra of the PLG / Pluronic.TM F68 nanoparticle formulations with different polymer ratios Figure 2: TEM images of the nanoparticle formulation PLGA / Pluronic.TM F68 with polymer ratio 1: 1 Figure 3: 1H NMR spectra of the PLGP / Tetronic nanoparticle formulations. TM 908 with different polymer ratios Figure: TEM images of PLGA / Tetronic .TM 908 nanoparticle formulations with polymer ratio 1: 1 Figure 5: size of nanoparticles PLGA / poloxamer and PLGA / poloxamine as a function of the PLGA / polymer ratio and the poloxamer or poloxamine type Figure 6: surface charge of the PLGA / poloxamer nanoparticles and PLGA / poloxamine depending on the ratio PLGA / polymer and type of poloxamer or poloxamine Figure 7: effect of cryoprotective agents on the size of lyophilized PLGA / poloxamer nanoparticles Figure 8: Effect of cryoprotective agents on the size of lyophilized PLGA / poloxamer nanoparticles Figure 9: "in vitro" release profile of plasmid DNA encapsulated in the nanoparticles PLGA / F68, PLGA / LI21, PLGA / 908 and PLGA / T904 with polymer ratio 1: 1 Figure 10: results of the cytotoxicity assay of the nanoparticles PLGA / F68, PLGA / L121, PLGA / T908 and PLGA / T904 with polymer ratio 1: 1 in MCF-7 cell culture DETAILED DESCRIPTION OF THE INVENTION EXAMPLES EXAMPLE 1 Intimate mixture nanoparticles were prepared with the modified solvent diffusion technique described above. More specifically: 50 mg of the polylactic-co-glycolic acid and 25, 50 or 75 mg of the poloxamer Pluronic.TM F68 (HLB = 29) were dissolved in 2 ml of dichloromethane and this organic solution was mixed for 30 s by vortex (2400 mm "1, Heidolph) with a small volume of aqueous phase The emulsion thus obtained was added to 25 ml of ethanol under moderate magnetic stirring The formulation was diluted with 25 ml of water and stirring was maintained for a further 10 min. After evaporation of the solvent at 30 ° C and under vacuum (Rotavapor, Büchi R-114), the nanoparticles were collected and concentrated in aqueous medium, optionally, for subsequent analysis, the nanoparticles were centrifuged (1 h, 8000xg, 15 ° C). C, Avanti 30, Beckman) and lyophilized (48 hours at -34 ° C, Labconco Corp) The size and polydispersity of the nanoparticles were measured with photon correlation spectroscopy (PCS) and the surface charge was determined by laser-Doppler anemometry (Zetasizer 3000 HS, Malvem Instruments) (TABLE 1). The composition of the matrices was analyzed using 1 H NMR spectroscopy (Bruker AMX-300) from lyophilized samples and dissolved in deuterated chloroform, which confirmed the presence of poloxamer / poloxamine in the nanoparticle matrix. from the corresponding peaks it can also be concluded that the quantity of the polyoxyethylene-polyoxypropylene block copolymer can be changed by adjusting the parameters of the preparation (FIGURE 1.) The morphological analysis of the nanostructures was carried out by transmission electron microscopy (CM 12 Philips) using samples stained with a 2% phosphotungstic acid solution (Figure 2).

TABLE 1 Pluronic.TM F68 PLGArpoloxamer size (nm) P.I. pot ? (mV) 1: 0 191.5 ± 7.1 0.046 -60.1 ± 7.4 1: 0.5 162.8 ± 4.4 0.079 -50.2 ± 0.8 1: 1 163.2 ± 5.1 0.135 -43.1 ± 6.4 1: 1.5 159.8 + 6.5 0.163 -38.5 ± 0.6 EXAMPLE 2 Intimate mixture nanoparticles were prepared with the modified solvent diffusion technique described above, but changing the type of the polyoxyethylene-polyoxypropylene copolymer; The PLGA and the various amounts of the poloxamer Pluronic.TM L121 (HLB = 1) were dissolved in dichloromethane and this organic solution was vortexed with a small volume of aqueous phase. The emulsion thus obtained was added with stirring to ethanol. The formulations were diluted with water and the stirring was maintained for a further 10 minutes. After evaporation of the solvent the nanoparticles were concentrated in aqueous medium. Optionally, for its subsequent analysis, the nanoparticles were centrifuged and lyophilized. The size and polydispersity of the nanoparticles were measured by PCS and the surface charge was determined with laser-Doppler anemometry (TABLE 2). The morphology and composition of the matrices were studied using 1H NMR spectroscopy and TEM microscopy. TABLE 2 Plutonic.TML121 PLGA: poloxamer size (nm) P.I. pot ? (mV) 1: 0 191.5 ± 7.1 0.046 -60.1 ± 7.4 1: 0.5 164.5 ± 6.3 0.156 -27.3 ± 7.1 1: 1 185.5 ± 6.0 0.195 -30.0 ± 8.0 1: 1.5 257.3 ± 10.0 0.179 -24.5 ± 5.5 EXAMPLE 3 Intimate mixture nanoparticles were prepared with the modified solvent diffusion technique described above, but by changing the type of the polyoxyethylene-polyoxypropylene copolymer: the PLGA and the different amounts of the poloxamine Tetronic.TM 908 (HLB = 30.5) were dissolved in dichloromethane and this organic solution was mixed by vortex with a small volume of aqueous phase. The emulsion thus obtained was added with stirring to ethanol: The formulations were diluted with water and the stirring was maintained for a further 10 minutes. After evaporation of the solvent the nanoparticles were concentrated in aqueous medium.

Optionally, for its subsequent analysis, the nanoparticles were centrifuged and lyophilized. The size and polydispersity of nanoparticles they were measured by PCS and the surface charge was determined with laser-Doppler anemometry (TABLE 3). The morphology and composition of the matrices were studied using 1H NMR spectroscopy and TEM microscopy (FIGURE 3 and 4). TABLE 3 Pluronic.TM 908 PLGArpoloxamer size (nm) P.I. pot ? (mV) 1: 0 191.5 ± 7.1 0.046 -60.1 ± 7.4 1: 0.5 189.2 + 4.6 0.202 -30.9 ± 3.9 1: 1 174.0 ± 5.4 0.271 -26.9 ± 1.2 1: 1.5 171.213.2 0.235 -24.1 ± 1.0 EXAMPLE 4 Intimate mixture nanoparticles were prepared with the modified solvent diffusion technique described above, but changing the type of the polyoxyethylene-polyoxypropylene copolymer: the PLGA and the different amounts of the poloxamine Tetronic.TM 904 (HLB = 14.5) were dissolved in dichloromethane and this organic solution was mixed by vortex with a small volume of aqueous phase. The emulsion thus obtained was added with stirring to ethanol. The formulations were diluted with water and the stirring was maintained for a further 10 minutes. After evaporation of the solvent the nanoparticles were concentrated in aqueous medium. Optionally, for its subsequent analysis, the nanoparticles were centrifuged and lyophilized. The size and polydispersity of the nanoparticles were measured by PCS and the surface charge was determined with laser-Doppler anemometry (TABLE 4). The morphology and composition of the matrices were studied using 1H NMR spectroscopy and TEM microscopy (FIGURE 3 and 4). TABLE 4 Pluronic.TM 904 PLGA: poloxamer size (nm) P.I. pot ? (mV) 1: 0 191.5 ± 7.1 0.046 -60.117.4 1: 0.5 160.2 + 5.6 0.188 -40.0 + 4.6 1: 1 168.7 ± 9.4 0.179 -38.4 ± 3.3 1: 1.5 168.812.5 0.160 -39.6 ± 2.0 EXAMPLES Intimate mixture nanoparticles were prepared with the modified solvent diffusion technique described above, but changing the type of the polyoxyethylene-polyoxypropylene copolymer: the PLGA and the different amounts of the poloxamine Tetronic.TM 904 (HLB = 14.5) were dissolved in dichloromethane and this organic solution was mixed by vortex with a small volume of aqueous phase. The emulsion thus obtained was added with stirring to ethanol. The formulations were diluted with water and the stirring was maintained for a further 10 minutes. After evaporation of the solvent the nanoparticles were concentrated in aqueous medium. Optionally, for its subsequent analysis, the nanoparticles were centrifuged and lyophilized. The size and polydispersity of the nanoparticles were measured by PCS and the surface charge was determined with laser-Doppler anemometry (TABLE 5). The morphology and composition of the matrices were studied using 1H NMR spectroscopy and TEM microscopy. BOARDS .

Pluronic.TM 901 PLGA: poloxamer size (nm) P.I. pot ? (mV) 1: 0 191.5 ± 7.1 0.046 -60.117.4 1: 0.5 205.3154.5 0.162 -25.415.0 1: 1 277.4 ± 102.9 0.308 -28.9 ± 5.4 1: 1.5 333.7 ± 82.1 0.275 -38.2 ± 8.3 EXAMPLE 6 The Intimate Mixture Nanoparticles of PLGA / Pluronic.TM F68, PLGA / Pluronic.TM L121, PLGA / Tetronic. TM 908 and PLGA / Tetroñic 904 with polymer ratio 1: 1 were prepared as described in Examples 1, 2, 3 and 4. Two cryoprotective agents (glucose and trehalose) were incorporated into the nanoparticle suspension medium. The formulations, at different concentrations (1, 2.5, 5 mg / ml), were lyophilized in the presence of 5% or 10% of the cryoprotectant. The size and polydispersity of the nanoparticles were measured after the lyophilization-resuspension process and compared with the initial values. The effects of the concentration of the nanoparticles, type and concentration of the cryoprotectant have been evaluated. It can be concluded that in the presence of 5% cryoprotectant, all formulations can be lyophilized at relatively high concentrations (2.5 mg / ml) without significant aggregation (FIGURE 7 and 8). TABLE 6 cryoprotectant size ratio dilution resuspension / original NPs mg / ml F68 L121 T908 T904 % glucose 1 1. .2110.04 1.2510.21 1.1710.04 1.1810.01 2.5 1..15 + 0.06 1.65 + 0.29 1.12 + 0.09 1.5910.35 TABLE 6 (Continued) cryoprotectant size ratio dilution resuspension original NPs mg / ml F68 L121 T908 T904 5 1.11 + 0.09 2.6210.73 1.0310.05 3. .77 + 0.39 % glucose 1 1.28 ± 0.04 1.32 + 0.39 2.60 1.04 2.5 1.39 ± 0.15 1.6910.67 1.59 1.18 5 1.3010.10 2.2510.15 1.20 1.33 % trehalose 1 1.2210.17 3.58 + 2.47 1.1310.04 1, .2910.17 2.5 1.93 + 0.55 4.66 ± 2.9 1.21 ± 0.03 2, .27 + 0.90 5 1.5710.27 5.23 + 0.21 1.2310.04 5, .6614.48 % trehalose 1 1.21 + 0.12 2.3211.09 1.25 + 0.10 1.64 2.5 1.3010.07 5.46 + 0.99 1.4610.06 2.33 5 1.82 + 0.51 5.3510.52 1.4610.20 2.74 EXAMPLE 7: The Intimate Mixture Nanoparticles of PLGA / Pluronic.TM P68, PLGA / Pluronic.TM L121, PLGA / Tetronic. TM 908 and PLGA / Tetronic 904 with polymer ratio 1: 1 were prepared as described in Examples 1, 2, 3 and 4. The plasmid model pEGFP-Cl (coding for a green fluorescent protein) was incorporated in the aqueous internal formulations with a theoretical loading of 0.4%. The size, polydispersity and surface charge of the formulations loaded with DNA were measured using photon correlation spectroscopy and laser-Doppler anemometry "(TABLE 7) .The efficacy of encapsulation and" in vitro "release profiles were determined from the samples of supernatants from different times with fluorimetric assays using PicoGreen Quantitation Kit (Molecular Probes) in TE buffer. at pH = 7.5 (FIGURE 9).

TABLE 7 Type of Size (nm) P.I. Potential-Z Efficiency of poloxamer / poloxamine (mV) encapsulation Pluronic.TM F68 182.6 ± 6. 0 0.114 -50.813. 6 35.2 ± 8. 9 Pluronic.TM L121 216. 8 ± 5. 3 0.154 -23.511. 4 31,313. 8 Tetronic.TM T908 268.7111. 6 0.437 -35.010. 9 32,013. 7 Tetronic.TM T904 161.517. 6 0.154 -54.112. 0 44,114. 3 EXAMPLE 8 The Intimate Mixture Nanoparticles of PLGA / Pluronic.TM F68, PLGA / Pluronic.TM L121, PLGA / Tetronic. TM 908 and PLGA / Tetronic 904 with polymer ratio 1: 1 were prepared as described in Examples 1, 2, 3 and 4. The cytotoxicity of the formulations was studied in the MCF-7 cell culture in DMEM supplemented with 10 mg / ml. % FBS. The cells were incubated with different concentrations of nanoparticles (from 1 to 5 mg / ml) for 24 hours. Cell viability was measured with the MTS reagent after a recovery period of 24 hours. The results show that, in spite of the high concentrations and the extended incubation time, none of the formulations produces toxic effects in the. cells It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (15)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. Process for the preparation of nanoparticles smaller than 1 μm, for the administration of active ingredients, characterized in that it comprises the steps of: a) dissolving a biodegradable polymer together with a block copolymer derived from polyoxyethylene in an organic solvent, the ratio being by weight of both polymers between 1: 0.1 and 1: 3. b) adding, under stirring, the solution obtained to a polar phase, in which the biodegradable polymer has a low solubility, precipitating the polymer and forming the nanoparticles. c) remove the organic solvent; d) isolating the particles, wherein the active ingredient is dissolved in the organic solvent employed in a), before or after step a), or is dissolved in a small volume of aqueous phase, which is subsequently dispersed in the organic solvent employed in a), before or after step a).

2. Process according to claim 1, characterized in that it comprises an additional step after e) of lyophilizing the obtained nanoparticles.

3. Process according to any of claims 1 and 2, characterized in that the biodegradable polymer is a polyester.

Process according to any of claims 1 and 2, characterized in that the biodegradable polymer is a polyanhydride.

Process according to claim 3, characterized in that the polyester is selected from polycaprolactone, polylactic acid, polylactic-co-glycolic acid and mixtures thereof.

6. Process according to any of claims 1 to 5, characterized in that the block copolymer is poloxamer.

Process according to claim 6, characterized in that the poloxamer has a molecular weight between 1,000-25,000 Daltons.

8. Process according to any of claims 1 to 5, characterized in that the block copolymer is poloxamine.

9. Process according to claim 8, characterized in that the poloxamine has a molecular weight between "1, 000-25, 000 Daltons.

10. Process according to any of claims 1 to 9, characterized in that the active ingredient is selected from molecules with therapeutic properties, vaccines and cosmetic ingredients.

11. Process according to any of claims 1 to 10, characterized in that the weight ratio of both polymers is between 1: 1 and 1: 3.

12. Nanoparticles for the administration of pharmaceutically or cosmetically active ingredients, of size less than 1 μm, characterized in that they are obtainable by the process according to any of claims 1 and 3 to 10.

13. Lyophilized nanoparticles for the administration of ingredients pharmaceutically or cosmetically active, of size less than 1 μm, characterized in that they are obtainable by the process according to claim 2.

14. Compositions characterized in that they comprise nanoparticles according to any of claims 12 and 13.

15. Pharmaceutical or cosmetic compositions characterized because they comprise nanoparticles according to any of claims 12 and 13.