dendrimer : a highly branched polymer in which the repeating units are organised into generations from a single focal point . their nanometric size and multivalency mean that these macromolecules are ideal for use in receptor - ligand interaction processes . block copolymer : a copolymer is a polymer formed from two or more monomeric species . those copolymers formed from two blocks of different polymerised monomers are known as block copolymers . bioactive proteins : macromolecules formed from linear chains of amino acids that regulate or are involved in a biological process . bioactive proteins are characterised by their ability to organise themselves in four structural levels known as the primary , secondary , tertiary and quaternary structure . the primary structure is a description of the covalent bonds that join the various amino acids in a protein chain . the secondary structure refers to particularly stable arrangements of the amino acids that lead to repetitive structural patterns . the tertiary structure describes all aspects of the three - dimensional folding of the protein . finally , when a protein possesses two or more polypeptide sub - units , their spatial arrangement is known as the quaternary structure . the bioactivity of a protein depends on these four organisational levels . thus , if the three - dimensional structure of a protein is destroyed ( denaturation ), its function is also destroyed . bioactive proteins are also characterised by their low charge density as only five of the 20 proteinaceous amino acids contain ionisable groups . isoelectric point ( p1 ): the isoelectric point is the ph at which an amphoteric substance has zero nett charge . this concept is particularly important for bioactive proteins as they are practically insoluble at their isoelectric point . once the isoelectric point is known , the nett charge of a protein can be varied by varying the ph . thus , a protein with a high isoelectric point such as lysozyme ( p1 ˜ 11 ) will have a nett positive charge in aqueous solution at a ph below 11 and a nett zero charge at ph 11 . pic micelles : a type of polymeric micelle characterised in that their formation is mainly based in electrostatic interactions between oppositely charged polymers or macromolecules . they are unusual in that the ratio between their charges is stoichiometric , thus meaning they have a nett zero charge . they were first described by kataoka ( 1995 ), who named them “ polyion complex micelles ” ( pic micelles ). enhanced permeability and retention ( epr ) effect : an effect by which macromolecules ( for example polymeric micelles ) tend to passively accumulate in tumour tissues . this fact has been used in the context of anticancer therapy in order to selectively accumulate drug toxicity in cancerous tissues whilst minimising the effects in healthy tissues . it is known that this effect occurs as a result of two main factors , namely ( i ) the hyperpermeability of the tumour vasculature , which allows the transport of macromolecules to the tumour , and ( ii ) poor lymphatic drainage , which results in high retention of macromolecules in the tumour . in order to overcome the problems encountered in the prior state - of - the - art , the present invention provides the following improvements . the present invention provides a type of pic micelle formed by the electrostatic interaction between a charged dendritic block copolymer and a bioactive protein . a key advantage of the present invention is the proposed solution to the problem of the instability of pic micelles containing bioactive proteins under physiological saline conditions , which involves the incorporation of charged dendritic block copolymers . in this manner , the low stability with respect to the ionic force resulting from the low charge density of bioactive proteins is compensated by the greater stability provided by the dendritic block . the present invention is the first example of a dendritic pic micelle containing bioactive proteins that is stable under physiological conditions . this increased stability is reflected by the ability to lyophilise these micelles , thereby facilitating their conservation and storage . a further key advantage offered by the present invention is that , in contrast to other protein delivery systems , the bioactive protein itself is one of the constituents of the micelle . this means that a system in which the protein is subsequently encapsulated need not be prepared . another key advantage of the present invention is that the bioactive protein - containing pic micelles are formed in an aqueous medium , with no need for covalent bond formation , under very mild conditions , thus meaning that the activity of the protein should be unaffected . finally , it should be noted that the overall preparation process for the bioactive protein - containing pic micelles described in the present invention is extremely simple due to the facile synthesis of the dendritic block copolymers , thus meaning that this is a cheap , and therefore ideal , process from an industrial viewpoint . the present invention refers to a polymeric micelle formed by the electrostatic interaction between : a ) a dendritic block copolymer represented by the general formula [ 1 ]; where p is a polymer , d is a dendritic structure , q represents the number of charged atoms at the periphery of the dendritic structure , which varies between 1 and 5000 , c represents a positive or negative charge , and b ) a bioactive protein a with a nett charge c ′ opposite to c . the present invention also relates to a polymeric micelle characterized in that the polymer p defined in claim 1 is preferably selected from linear polymers , block copolymers , copolymers , terpolymers , graft copolymers , graft terpolymers and amphiphilic copolymers . the present invention also relates to a polymeric micelle characterized in that the polymer p is preferably selected from natural polymers or polymers produced by chemical synthesis or biotechnological processes . in a particular embodiment , the polymer p is preferably selected from n -( 2 - hydroxypropyl ) methacrylamide ( hpma ), poly ( styrene - co - maleic acid / anhydride ) ( sma ), poly ( divinyl ether maleic anhydride ) ( divema ), poly ( n - vinyl pyrrolidone ) ( pvp ), poly ( n - acryloyl ) morpholine ( pacm ), poly ( ethylene glycol ) ( peg ), poly ( propylene oxide ) ( pop ) and their derivatives . in another particular embodiment , the polymer p is poly ( ethylene glycol ) ( peg ). the present invention also relates to a polymeric micelle characterized in that the bioactive protein a is preferably selected from antibodies , antibody fragments , heteroproteins , enzymes and hormones . in a particular embodiment , the bioactive protein a is preferably selected from myoglobin , lysozyme and insulin . in a preferred embodiment , dendritic structure d is a dendrimer , dendron or dendritic polymer constructed from one or more repeating units . the present invention also relates to a polymeric micelle characterized in that the dendritic structure d is preferably a dendron constructed from a repeating unit represented by general formula [ 2 ] wherein z represents a covalent bond between the repeating unit and the polymer p or the repeating unit and the previous generation , r 1 and r 2 are linear or branched chains which can be identical or different and which are characterized by containing alkyl , alcohol , thiol , azide , nitrile , amine , imide , imine , cyanate , isocyanate , isothiocyanate , ether , thioether , ketone , aldehyde , ester , carboxylic acid or aromatic groups ; x represents the repeating unit of the following generation or alternatively an anionic or cationic group . in another particular embodiment , the dendritic structure d is preferably a dendron constructed from a repeating unit represented by general formula [ 2 ], wherein z is preferably an amide bond ; r 1 and r 2 are identical and selected from poly ( ethylene glycol ) or oligo ( ethylene glycol ) chains , they are preferably triethylene glycol ; when x is an anionic group it is preferably selected from carboxylic acids and their derivatives , sulfates and their derivatives , sulfonates and their derivatives , phosphates and their derivatives , phosphonates and their derivatives , arylphosphonic acids and their derivatives , phenols and their derivatives ; when x is a cationic group it is preferably selected from amines , polyamines , oligoamines , preferably spermidines , spermines , anilines , benzylamines , imidazoles , morpholines , ammonium salts , primary , secondary or tertiary amines or guanidinium groups . a specific aspect of the invention relates to a polymeric micelle as defined above formed by the electrostatic interaction between a peg - dendritic block copolymer with benzoate groups at its periphery and the bioactive protein lysozyme , as shown schematically in fig1 . another specific aspect , the invention relates to a polymeric micelle as defined above formed by the electrostatic interaction between a peg - dendritic block copolymer with benzoate groups at its periphery and the bioactive protein myoglobin . another specific aspect , the invention relates to a polymeric micelle as defined above formed by the electrostatic interaction between a peg - dendritic block copolymer with positively charged aniline groups at its periphery and the bioactive protein insulin . the present invention also refers to the encapsulation of a drug , diagnostic agent , molecule or macromolecule in the interior of a polymeric micelle as defined above . a further aspect of the present invention refers to the use of a micelle as defined above as a carrier for the therapeutic administration of a bioactive protein . in a further aspect , the present invention refers to the use of a micelle as defined above as a carrier for administering of a drug or diagnostic agent , molecule or macromolecule . another aspect of the present invention refers to a pharmaceutical composition comprising a polymeric micelle as defined above and an active ingredient . as described in detail , the present invention provides a type of polymeric micelle formed from bioactive proteins which can be stable under physiological conditions and is prepared following a fast , simple and cheap method . the type of micelle described can be used in nanomedicine , medical and biological engineering , protein and drug delivery processes , biotechnology , therapy and diagnostics . fig2 . formation of a lysozyme - containing pic micelle and the dls histogram . fig3 . afm image of a lysozyme - containing pic micelle formed from dendritic block copolymers with benzoate groups . fig4 . formation of a myoglobin - containing pic micelle and the dls histogram . fig5 . formation of an insulin - containing pic micelle and the dls histogram . fig6 . afm image of an insulin - containing pic micelle formed from dendritic block copolymers with positively charged aniline groups . preparation of the bioactive protein - containing micelles of this invention is illustrated by way of the following examples , which should nevertheless not be considered to limit the scope of said invention : the dendritic unit 2 was prepared from methyl gallate 1 as shown in scheme 1 . the first - generation block copolymer peg -[ g1 ]- n 3 was obtained by coupling dendritic unit 2 to peg - nh 2 ( 3 ). higher generation block copolymers were obtained by reduction of the terminal azide groups of the previous generation by catalytic hydrogenation and coupling of the resulting amines to dendritic unit 2 as shown in scheme 2 . general procedure for the anionic functionalisation of the peg - dendritic block copolymers ( peg -[ gn ]- n 3 ). functionalisation of the block copolymers with anionic groups was performed by a cu ( i )- catalysed azide - alkyne [ 3 + 2 ] cycloaddition reaction . this means that the anionic ligands to be introduced must be functionalised with terminal alkyne groups . the peg - dendritic block copolymer peg -[ gn ]- n 3 and the anionic ligand were dissolved in a mixture of t - buoh and h 2 o ( 1 : 1 ). catalytic amounts of cuso 4 and sodium ascorbate were then added ( scheme 3 ). general procedure for the cationic functionalisation of the peg - dendritic block copolymers ( peg -[ gn ]- n 3 ). two strategies can be used to introduce cationic groups at the periphery of the block copolymer : 1 . reduction of the terminal azides , for example by catalytic hydrogenation in an acidic medium , leads to ammonium salts of the corresponding primary amines at the periphery of the dendrimer . the formation of peg -[ g3 ]- nh 3 + is shown in scheme 4 . 2 . a cu ( i )- catalysed azide - alkyne [ 3 + 2 ] cycloaddition reaction , as described in scheme 3 , but using cationic ligands functionalised with terminal alkynes . preparation of pic micelles from a block copolymer functionalised with terminal benzoate groups and the bioactive protein lysozyme ( p1 & gt ; 7 ). the block copolymer peg -[ g3 ]- benzoate , obtained from peg - nh 2 ( m n 5219 ) following the procedure described in examples 1 and 2 , was dissolved in phosphate buffer ( pbs ) at ph 7 . 4 . the protein lysozyme ( commercial product ) was dissolved in the same ph 7 . 4 pbs buffer in a separate recipient . the two solutions were then filtered and mixed , in a stoichiometric charge ratio , considering only the positive charges on the protein at the working ph for this purpose , to produce pic micelles in a final solution with ph 7 . 4 . after stirring magnetically for 24 h , the resulting solution was filtered and analysed by dynamic light scattering ( dls ). the dls measurements confirmed the presence of polymeric micelles with a very low polydispersity index ( 0 . 1 ) and a size of around 120 nm . ( fig2 ). this size was confirmed by atomic force microscopy ( afm ). to determine the stability of the lysozyme - containing pic micelles under physiological conditions , nacl was added to the original micelle solution to a concentration of 150 mm , the mixture heated to 37 ° c . and the resulting micelle solution subsequently analysed by dls . no change in size or any other signs of destabilisation were observed . characterisation . in order to visualise the dendritic lysozyme - containing pic micelles by afm , an aliquot of a micelle solution with a concentration of 1 . 18 mg / ml in 10 mm pbs , ph 7 . 4 , was removed and diluted with milli - q water to a concentration of 0 . 05 mg / ml . a 10 - μl portion of this solution was deposited on a silicon surface and allowed to dry at room temperature . spherical particles with a diameter of approximately 140 nm , in good agreement with the dls measurements , were observed ( fig3 ). micelle stability . the dendritic lysozyme - containing pic micelles are stable for more than a month at 4 ° c . in 10 mm pbs , ph 7 . 4 , and for several weeks at this temperature in the presence of 150 mm nacl . furthermore , these micelles are stable for several weeks at room temperature in pbs 10 mm , ph 7 . 4 , although storage at this temperature is not recommended due to their proteinaceous nature . further proof of the stability of these micelle arises form the fact that they can be lyophilised and subsequently re - suspended in the same volume , with only a small decrease in their size ( by dls ). preparation of pic micelles from a block copolymer functionalised with terminal benzoate groups and the bioactive protein myoglobin ( p1 ˜ 7 ). the block copolymer peg -[ g3 ]- benzoate , obtained from peg - nh 2 ( m n 5219 ) following the procedure described in examples 1 and 2 , was dissolved in phosphate buffer ( pbs ) at ph 9 . the myoglobin ( commercial product ) was dissolved in pbs ph 3 in a separate recipient . the two solutions were then filtered and mixed , in a stoichiometric charge ratio , considering only the positive charges on the protein at the working ph for this purpose , to produce pic micelles at a final ph of 7 . 4 . after stirring magnetically for 24 h , the resulting solution was filtered and analysed by dls ( fig4 ). the dls measurements confirmed the presence of polymeric micelles with a low polydispersity index ( 0 . 15 ) and a size of around 50 nm . these values were confirmed by afm . preparation of pic micelles from a block copolymer functionalised with positively charged aniline groups and the bioactive protein insulin ( p1 & lt ; 7 ). the block copolymer peg -[ g3 ]- aniline hcl , obtained from peg - nh 2 ( m n 5219 ) following the procedure described in examples 1 and 3 , was dissolved in phosphate buffer ( pbs ) at ph 3 . the insulin ( commercial product ) was dissolved in pbs ph 12 in a separate recipient . the two solutions were then filtered and mixed , in a stoichiometric charge ratio , considering only the positive charges on the protein at the working ph for this purpose , to produce pic micelles at a final ph of 7 . 4 . after stirring magnetically for 24 h , the resulting solution was filtered and analysed by dls ( fig5 ). the dls measurements confirmed the presence of polymeric micelles with a low polydispersity index ( 0 . 15 ) and a size of around 45 nm . these values were confirmed by afm . in order to visualise the dendritic insulin - containing pic micelles by afm , an aliquot of a micelle solution with a concentration of 0 . 96 mg / ml in 10 mm pbs , ph 7 . 4 , was removed and diluted with milli - q water to a concentration of 0 . 05 mg / ml . a 10 - μl portion of this solution was deposited on a silicon surface and allowed to dry at room temperature . spherical particles with a diameter of approximately 45 nm , in good agreement with the dls measurements , were observed ( fig6 ). to determine the stability of the insulin - containing pic micelles under physiological conditions , nacl was added to the original micelle solution to a concentration of 150 mm , the mixture heated to 37 ° c . and the resulting micelle solution subsequently analysed by dls . no change in size or any other signs of destabilisation were observed . the dendritic insulin - containing pic micelles are stable for several weeks at room temperature , although storage at this temperature is not recommended due to their proteinaceous nature . furthermore , the micelles can be stored for more than a month at 4 ° c . in either 10 mm pbs , ph 7 . 4 , or 10 mm pbs , ph 7 . 4 , plus 150 mm nacl .
a polyion complex polymeric micelle is formed by interaction between a bioactive protein and a dendritic block copolymer of opposite charge . the conventional low stability of pic micelles with bioactive proteins vis - à - vis ionic strength is offset by the stability provided by the dendritic block . the overall process for preparing the micelles is facilitated by the simplicity of the drendritic block copolymer synthesis .