the present invention relates to nanoparticles which comprise a biodegradable polymer , preferably a polyester and a polyoxyethylene - derived block copolymer , preferably poloxamer and poloxamine . the invention further relates to a preparation method which permits the incorporation of high percentages of poloxamers and poloxamines in nanoparticles , the biodegradable polymer : polyethylenated derivate ratio being between 1 : 0 . 1 and 1 : 3 . therefore , according to a first aspect , the invention relates to a method of preparing nanoparticles , having a size of less than 1 • m , for the administration of active ingredients , comprising the steps of : a ) dissolving a biodegradable polymer together with a polyoxyethylene - derived block copolymer in an organic solvent , the biodegradable polymer : block copolymer weight ratio being between 1 : 0 . 1 and 1 : 3 ; b ) adding , with stirring , the solution obtained to a polar phase , wherein the biodegradable polymer has low solubility , precipitating the polymers and forming the nanoparticles ; c ) eliminating the organic solvent ; d ) isolating the particles . the active ingredient can be directly dissolved in the non - polar organic solvent ( lipophilic molecules ) or it can be previously dissolved in a small volume of aqueous phase ( water - soluble molecules ) and then dispersed in the organic solvent , before or after stage a ). preferably , the organic solvent in a ) will be a non - polar solvent . according to a preferred embodiment , the preparation of nanoparticle formulations of intimate mixture may additionally include a lyophilisation stage . in lyophilised form , nanoparticles can be stored for long periods of time and can be easily regenerated by simply adding an optimum volume of water . the nanoparticle lyophilisation has been optimised by incorporating a cryoprotective excipient ( glucose or trehalose ) in the formulations &# 39 ; suspension medium . according to another form of preferred embodiment , in the previous method , the biodegradable polymer is a polyester , which is selected from the group of polyesters such as polylactic acid , polylactic co - glycolic acid and their copolymers , polycaprolactone or the group of polyanhydrides . the polylactic co - glycolic acid polymer 50 : 50 resomer ® rg 503 mw : 3500 ( boehringer ingelheim ) has been used to prepare the nanoparticles of intimate mixture . according to other forms of preferred embodiment , the block copolymer is selected from poloxamers and polyoxamines . poloxamers are polyoxyethylene - polyoxypropylene - polyoxyethylene - type triblock copolymers ( peo - ppo - peo ) which , depending on their peo : ppo ratio , vary in their characteristics of molecular weight , hydrophobicity , etc . preferably , the poloxamers used will have a molecular weight between 1 , 000 and 25 , 000 daltons . these polymers can be obtained from basf corporation with the trade name pluronic .™. for the preparation of nanoparticles of intimate mixture , we have used the following poloxamers : pluronic .™ f68 with molecular weight 8350 and hlb = 29 , pluronic .™ with molecular weight 4400 and hlb = 1 . poloxamines are copolymers formed from 4 chains of peo - ppo bound by an ethyl diamine bridge . as with the poloxamers , their characteristics may vary when the peo - ppo ratio is changed . preferably , the poloxamines used will have a molecular weight between 1 , 000 and 25 , 000 daltons . these polymers can be obtained from basf corporation with the trade name tetronic .™. for the preparation of nanoparticles of intimate mixture , we have used the following poloxamines : tetronic .™ 908 , with molecular weight 25000 and hlb = 30 . 5 , tetronic .™ 904 , with molecular weight 6700 and hlb = 14 . 5 and tetronic .™ 901 , with molecular weight 4700 and hlb = 2 . 5 according to another preferred embodiment , the proportion by weight of the biodegradable polymer is between 1 : 1 and 1 : 3 . according to a second aspect of the present invention , it relates to both lyophilised and non - lyophilised nanoparticles obtained according to the aforementioned method . these nanoparticles offer innovative , distinctive characteristics given their capacity for the encapsulation and controlled release of very delicate active molecules , such as proteins and dna plasmids . furthermore , due to the presence of important amounts of poloxamers and poloxamines in their composition , said nanoparticles can present a differentiated biodistribution profile , in comparison with the classic particles formed from polyesters . due to their nanoparticulate size , these new systems can be administered to the human organism by any administration route , including intravenous route , whilst microparticles cannot be administered by this route due to the obstruction they would cause in the blood capillaries . there is also a great quantity of documentation which shows that nanoparticles can overcome biological barriers ( mucous membranes , epithelials ) whilst microparticles cannot . the physicochemical properties of the formulations of different composition and different polymer ratio have been characterised using photon correlation spectroscopy ( pcs ) and laser doppler anemometry techniques . the nanoparticle morphology was studied using transmission electronic microscopy ( tem ) and 1 h nmr . these studies confirmed the formation of the aforementioned intimate mixture system . in order to check the applicability of these nanoparticles of intimate mixture for the release of derivate macromolecules , we have encapsulated the pegfp - c1 plasmid ( coder for a green fluorescent protein ) in the different formulations . the results of these in vitro release studies have revealed the potential of the formulations as controlled release vehicles during extended periods of time . the cytotoxicity of nanoparticles of different compositions , at different concentrations , has been tested in cell cultures with the mts calorimetric test (( 3 -( 4 , 5 - dimethylthiazol - 2 - yl )- 5 -( 3 - carboxymethoxyphenyl )- 2 -( 4 - sulphophenyl )- 2h - tetrazolium ) in the mcf - 7 cell line grown in dulbecco &# 39 ; s modified eagle &# 39 ; s medium ( dmem ) supplemented with 10 % foetal bovine serum ( fbs ). it can be concluded that none of the formulations produce toxic effects in the cells . according to a third aspect , the present invention relates to compositions , especially pharmaceutical and cosmetic , which incorporate nanoparticles according to the present invention . below , the invention will be explained in greater detail , based on a series of examples , without limitative character for the scope of the invention . fig1 : 1h nrm spectra of the plga / pluronic .™ f68 nanoparticles formulations with different polymer ratios . fig2 : tem images of the plga / pluronic .™ f68 nanoparticle formulations with polymer 1 : 1 ratio . fig3 : 1h nrm spectra of the formulations of the plga / tetronic .™ 908 nanoparticles with different polymer ratios . fig4 : tem images of the plga / tetronic .™ 908 nanoparticle formulations with 1 : 1 polymer ratio . fig5 : plga / poloxamer and plga / poloxamine nanoparticle size in accordance with the plga / polymer ratio and the type of poloxamer or poloxamine . fig6 : surface charge of the plga / poloxamer and plga / poloxamine nanoparticles in accordance with the plga / polymer ratio and the type of poloxamer or poloxamine . fig7 : effect of the cryoprotective agents on lyophilised plga / poloxamer nanoparticle size . fig8 : effect of the cryoprotective agents on lyophilised plga / poloxamer nanoparticle size . fig9 : in vitro release profile of plasmidic dna encapsulated in the plga / f68 , plga / l121 , plga / t908 and plga / t904 nanoparticles with 1 : 1 polymer ratio . fig1 : results of the cytoxicity test of the plga / f68 , plga / l121 , plga / t908 and plga / t904 nanoparticles with 1 : 1 polymer ratio in the mcf - 7 cell culture . nanoparticles of intimate mixture were prepared with the aforementioned modified solvent diffusion technique . more specifically : 50 mg of polylactic co - glycolic acid and 25 , 50 or 75 mg of pluronic .™ f68 ( hlb = 29 ) poloxamer were dissolved in 2 ml of dichloromethane and this organic solution was mixed for 30 sec . by vortex ( 2400 min − 1 , heidolph ), with a small volume of aqueous phase . the emulsion thus obtained was added to 25 ml of ethanol with moderate magnetic stirring . the formulation was diluted with 25 ml of water and it was stirred for a further 10 minutes . after solvent evaporation at 30 ° c . and in a vacuum ( rotavapor , büchi r - 114 ), the nanoparticles were collected and were concentrated in aqueous medium . optionally , for their later analysis , the nanoparticles were centrifuged ( 1 h , 8000 × g , 15 ° c ., avanti 30 , beckman ) and they were lyophilised ( 48 hours at − 34 ° c ., labconco corp ). the size and polydispersion of the nanoparticles were measured using photon correlation spectroscopy ( pcs ) and the surface charge was determined by laser doppler anemometry ( zetasizer 3000 hs , malvern instruments ) ( table 1 ). the matrix composition was analysed using 1 h nmr spectroscopy ( bruker amx - 300 ) from lyophilised samples dissolved in deuterated chloroform . these studies confirmed the presence of the poloxamer / poloxamine in the nanoparticulate matrix . from the intensities of the corresponding peaks , we can also conclude that the quantity of polyoxyethylene - polyoxypropylene block copolymers can be changed by adjusting the preparation parameters . ( fig1 ). the morphological analysis of the nanostructures was performed using transmission electronic microscopy ( cm 12 philips ) using samples stained with a 2 % phosphotungstic acid solution . ( fig2 ). nanoparticles of intimate mixture were prepared with the aforementioned modified solvent diffusion technique , but changing the type of polyoxyethylene - polyoxypropylene copolymer . the plga and the different quantities of pluronic .™ l121 ( hlb = 1 ) poloxamer 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 to ethanol with stirring . the formulations were diluted with water and it was stirred for a further 10 minutes . after the solvent evaporation , the nanoparticles were concentrated in aqueous medium . optionally , for their later analysis , the nanoparticles were centrifuged and lyophilised . the size and polydispersion of the nanoparticles were measured using pcs and the surface charge was determined by laser doppler anemometry ( table 2 ). the morphology and composition of the matrices were studied using 1 h nmr spectroscopy and tem microscopy . nanoparticles of intimate mixture were prepared with the aforementioned modified solvent diffusion technique , but changing the type of polyoxyethylene - polyoxypropylene copolymer : the plga and the different quantities of tetronic .™ 908 ( hlb = 30 . 5 ) poloxamine 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 to ethanol with stirring . the formulations were diluted with water and it was stirred for a further 10 minutes . after the solvent evaporation , the nanoparticles were concentrated in aqueous medium . optionally , for their later analysis , the nanoparticles were centrifuged and lyophilised . the size and polydispersion of the nanoparticles were measured using pcs and the surface charge was determined by laser doppler anemometry ( table 3 ). the morphology and composition of the matrices were studied using 1 h nmr spectroscopy and tem microscopy ( fig3 and 4 ) nanoparticles of intimate mixture were prepared with the aforementioned modified solvent diffusion technique , but changing the type of polyoxyethylene - polyoxypropylene copolymer : the plga and the different quantities of tetronic .™ 904 ( hlb = 14 . 5 ) poloxamine 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 to ethanol with stirring . the formulations were diluted with water and it was stirred for a further 10 minutes . after the solvent evaporation , the nanoparticles were concentrated in aqueous medium . optionally , for their later analysis , the nanoparticles were centrifuged and lyophilised . the size and polydispersion of the nanoparticles were measured using pcs and the surface charge was determined by laser doppler anemometry ( table 4 ). the morphology and composition of the matrices were studied using 1 h nmr spectroscopy and tem microscopy ( fig3 and 4 ) nanoparticles of intimate mixture were prepared with the aforementioned modified solvent diffusion technique , but changing the type of polyoxyethylene - polyoxypropylene copolymer : the plga and the different quantities of tetronic .™ 904 ( hlb = 14 . 5 ) poloxamine 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 to ethanol with stirring . the formulations were diluted with water and it was stirred for a further 10 minutes . after the solvent evaporation , the nanoparticles were concentrated in aqueous medium . optionally , for their later analysis , the nanoparticles were centrifuged and lyophilised . the size and polydispersion of the nanoparticles were measured using pcs and the surface charge was determined by laser doppler anemometry ( table 5 ). the morphology and composition of the matrices were studied using 1 h nmr spectroscopy and tem microscopy . nanoparticles of intimate mixture of plga / pluronic .™ f68 , plga / pluronic .™ l121 , plga / tetronic .™ 908 and plga / tetronic .™ 904 with 1 : 1 polymer ratio were prepared as has been described in examples 1 , 2 , 3 and 4 . two cryoprotective agents ( glucose and trehalose ) were incorporated in the nanoparticulate suspension medium . the formulations , at different concentrations ( 1 , 2 . 5 , 5 mg / ml ), were lyophilised in the presence of 5 % or 10 % of the cryoprotective agent . the nanoparticle size and polydispersion were measured after the lyophilisation / resuspension process and they were compared with the initial values . the effects of the nanoparticle concentration , the type and the concentration of the cryoprotective agent have been evaluated . it can be concluded that in the presence of 5 % cryoprotective agent , all the formulations can be lyophilised at relatively high concentrations ( 2 . 5 mg / ml ) without significant aggregation ( fig7 and 8 ). nanoparticles of intimate mixture of plga / pluronic .™ f68 , plga / pluronic .™ l121 , plga / tetronic .™ 908 and plga / tetronic .™ 904 with 1 : 1 polymer ratio were prepared as has been described in examples 1 , 2 , 3 and 4 . the pegfp - c1 plasmid model ( coder of a green fluorescent protein ) was incorporated in the internal aqueous phase of the formulations with a theoretical charge of 0 . 4 %. the size , polydispersion and surface charge of the formulations loaded with dna were measured using photon correlation spectroscopy and laser doppler anemometry ( table 7 ). the encapsulation efficiency and the in vitro release profiles were determined from the samples of supernatants of different times with fluorometric assays using the picogreen quantification kit ( molecular probes ) in te buffer at ph = 7 . 5 ( fig9 ). nanoparticles of intimate mixture of plga / pluronic .™ f68 , plga / pluronic .™ l121 , plga / tetronic .™ 908 and plga / tetronic .™ 904 with 1 : 1 polymer ratio were prepared as has been 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 % fbs . the cells were incubated with different nanoparticle concentrations ( from 1 to 5 mg / ml ) for 24 hours . the cell viability was measured with mts reagent after a 24 - hour recovery period . the results show that , despite the high concentrations and extended incubation times , none of the formulations produce toxic effects in the cells .
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 .