EP0938580A1 - Artificial polymeric membrane structure, method for preparing same, method for preparing this polymer, particle and film containing this structure - Google Patents

Artificial polymeric membrane structure, method for preparing same, method for preparing this polymer, particle and film containing this structure

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Publication number
EP0938580A1
EP0938580A1 EP97911315A EP97911315A EP0938580A1 EP 0938580 A1 EP0938580 A1 EP 0938580A1 EP 97911315 A EP97911315 A EP 97911315A EP 97911315 A EP97911315 A EP 97911315A EP 0938580 A1 EP0938580 A1 EP 0938580A1
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EP
European Patent Office
Prior art keywords
membrane
substrate
structure according
bifunctional
membrane structure
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97911315A
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German (de)
French (fr)
Inventor
Daniel Samain
Etienne Perochon
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Toulouse III Paul Sabatier
Original Assignee
Universite Toulouse III Paul Sabatier
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Publication of EP0938580A1 publication Critical patent/EP0938580A1/en
Withdrawn legal-status Critical Current

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    • 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—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/645—Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to an artificial membrane structure analogous to natural plasma membranes fixed on a cytoskeleton, as found in eukaryotic cells, in particular in erythrocytes and derivatives of eukaryotic cells, which are enveloped viruses.
  • Natural plasma membranes include a lipid bilayer (mainly phospholipid) forming a stable lamellar lyotropic phase incorporating amphiphilic molecules and proteins held together by stable non-covalent lyotropic interactions (mainly involving so-called "hydrophobic" bonds) Amphiphilic molecules are arranged in a continuous double layer having a thickness of the order of 4 to 5 n in the state of liquid crystal and fluid mosaic (cf.
  • the natural plasma membranes of eukaryotic cells are fixed on a cytoskeleton (network of filamentary proteins) which - stabilizes the plasma membrane from the inside. Binding involves a set of specific bonds between proteins anchored in the membrane and proteins of the cytoskeleton.
  • the envelopes of the enveloped viruses are formed from a plasma membrane fixed to the capsid of the virus by specific proteins of viral envelope capable of binding with the proteins of the capsid.
  • the erythrocyte membrane is fixed on a fibrous cytoskeleton, via a protein, band 3, capable of binding with the cytoskeletal nkyrin, which gives it a biconcave form (which turns into a spherocyte when the fixing is faulty).
  • the fixed plasma membranes not only possess the fundamental properties specific to lipid bilayers, but also they can have a complex morphology (in particular with a considerable specific surface, for example intestinal cells provided with microvilli, or the axons of neurons) and possess, due to the fixation of the bilayer on the cytoskeleton, remarkable mechanical properties, in particular of resistance and stability.
  • the fixing system preserves the lateral fluidity of the bilayer and the fixed membranes retain the property of being self-sealing as are the liposomes, that is to say are capable of closing spontaneously, in particular after having undergone a perforation. Given the importance of these properties, which are specific to fixed plasma membranes, it would be highly desirable to be able to obtain similar artificial structures.
  • the first method makes it possible to obtain only stabilized spherical polymerized liposomes, and does not authorize the production of a membrane structure of any shape and self-sealing.
  • the bilayer has poly erized portions in which the lateral fluidity is absent, and non-polymerized portions which remain fragile.
  • this first method provides a structure which cannot exhibit the various properties mentioned above of a fixed plasma membrane.
  • the second method also only makes it possible to obtain spherical liposomes with a solid core. surrounded by a lipid bilayer which is not linked to the solid core. This method therefore does not make it possible to obtain any structure of any shape and / or having the above-mentioned properties and the advantages of a fixed plasma membrane.
  • the third method requires the use of a bilayer of unusual ionic specific phospholipids which alter the fundamental properties (selective tightness and permeability and lateral fluidity) of the bilayer.
  • membrane linked by ionic and / or covalent and / or lyotropic bonds on a substrate membrane linked by ionic and / or covalent and / or lyotropic bonds on a substrate
  • - artificial structure analogous to a fixed natural plasma membrane structure obtained by synthesis presenting the fundamental functional properties of a natural membrane fixed on a cytoskeleton: selective sealing and permeability, structure of fluid mosaic (lateral fluidity), polymorphism, resistance, stability, autoscellable character.
  • the invention aims to propose an artificial membrane structure analogous to fixed natural plasma membranes having a stable functional membrane (that is to say having two-dimensional lateral fluidity properties, and selective sealing and permeability) fixed to a solid phase substrate, in particular a porous substrate formed from a network of polymers, the nature and forms of which can be diverse.
  • the invention also aims to propose a process for the preparation of such a simple membrane structure and compatible with industrial constraints of implementation; a particle or a supramolecular synthetic film comprising such a membrane structure and their applications; a polymer which can serve as a bifunctional fixing compound in such a membrane structure; a process for the preparation of this polymer; a drug ; and the therapeutic applications of this membrane structure, and of these particles.
  • the invention relates to an artificial membrane structure analogous to fixed natural plasma membranes, characterized in that it comprises: - a solid phase substrate having a surface with a surface density of electrical charges,
  • a stable functional membrane of amphiphilic compounds which has a free surface extending opposite the substrate, said surface being adapted to be able to be placed in contact with a medium, said external medium, with a shape such that it do not circumscribe this external environment, at least one bifunctional compound for fixing the functional membrane to the substrate, inserted between the functional membrane and the substrate, and the chemical structure of which comprises: at least one polyionic chain adapted to cooperate by polyelectrolytic complexation with the surface density of electrical charges of the substrate, at least one membrane ligand linked by covalent bond to such a polyionic chain, and adapted to form a stable lyotropic noncovalent bond with the amphiphilic compounds of the functional membrane, without significantly affecting the functional properties of the functional membrane.
  • Such an artificial membrane structure thus presents both the properties of the functional membrane (selective sealing and permeability and lateral fluidity) and the polymorphism and mechanical properties which depend on the nature of the substrate chosen.
  • the free surface of the functional membrane extending in contact with the external medium makes it possible to manage the exchanges with this external medium.
  • This free surface does not circumscribe the external medium, that is to say is not closed around this medium with which it is in contact, and this in contrast to the case of a cationic liposome surrounded by a poly eric structure .
  • the free surface of the membrane of a membrane structure according to the invention is distinct from a concave sphere, and is intended to come into contact with an external medium which is distinct from a closed internal spherical cavity.
  • the free surface of the membrane of the membrane structure according to the invention is such that it can have a theoretical envelope surface which extends entirely opposite the substrate and which is planar or convex with oriented convexity in the opposite direction to the substrate.
  • the free surface of the membrane will itself be flat or convex with convexity oriented opposite "to the substrate.
  • this free surface may have concave parts with concavity oriented towards the outside.
  • the functional membrane of the membrane structure according to the invention thus achieves a protective barrier with predetermined selective tightness and permeability which separates and protects the bifunctional fixing compounds and the substrate as well as their polyelectrolytic interactions, from the external medium.
  • the functional membrane and more generally the membrane structure, is perfectly stable and can be handled without special precautions, including when the substrate is porous.
  • the functional membrane of a membrane structure is artificial, that is to say it does not come from a living organism.
  • the amphiphilic compounds of the functional membrane can be formed of all amphiphilic compounds, preferably generally neutral (not carrying a net electrical charge), capable of forming a functional membrane, in particular a bilayer, having the state of fluid mosaic.
  • the membrane is mainly made up of amphiphilic compounds organized in a bilayer, but can nevertheless incorporate any other compound, in more or less significant proportions, having specific properties, and which is compatible with the bilayer, that is to say preserves them. functional properties and stability.
  • the amphiphilic compounds of the membrane are phospholipid compounds of natural or synthetic origin - in particular of the family of phosphatidylcholines - with fatty chains comprising between 12 and 22 saturated or unsaturated carbon atoms, in particular between 16 and 18 carbon atoms.
  • Amphiphilic membrane compounds may include, in addition to phospholipids, a greater or lesser proportion " of the compounds belonging to the following families, alone or as a mixture: sphingomyelin; gangliosides; glycolipids; ceramides; di O-alkyl; sterols (cholesterol, ergosterol ).
  • the membrane structure according to the invention may comprise a single bifunctional fixing compound, or as a variant, a mixture of several bifunctional fixing compounds of different natures.
  • the membrane structure comprises at least one bifunctional fixing compound, the chemical structure of which comprises at least a plurality of membrane ligands, in particular a plurality of similar or identical membrane ligands.
  • these membrane ligands are distributed over the molecule of the bifunctional fixing compound at a distance from each other which is greater than that separating the amphiphilic compounds which adjoin in a layer of the functional membrane, so that this membrane presents amphiphilic compounds which are not linked to a membrane ligand.
  • the bifunctional fixing compound has a number of unit ion charges of the same sign capable of cooperating by polyelectrolytic complexation with the surface density of electrical charges of the substrate, which is greater than the number of membrane ligands.
  • the ionic charges are distributed over the molecule of the bifunctional fixing compound at a distance from each other which is less than the smallest distance separating two membrane ligands.
  • the membrane ligands are chosen from phospholipids, fatty acids, isoprenoids, peptides, fatty amines, ethers, sterols, terpenes, glycolipids, shingolipids, gangliosides, ceramides .
  • the membrane ligands can be composed of a lipid branch - in particular phospholipid - similar to the amphiphilic compounds of the functional membrane, and of a connecting branch connecting by covalent bonds this lipid branch to the polyionic chain of the bifunctional fixing compound. . In this way, the lipid branch of the membrane ligand is inserted into the layer opposite the bilayer forming the functional membrane, and is therefore linked by lyotropic interactions within this functional membrane.
  • the bifunctional fixing compounds are formed from oligomers or from polyionic polymers.
  • bifunctional polycationic fixing compounds which can be used in the invention, mention may be made of: polycationic proteins and peptides; polycationic oligo and polysaccha ⁇ des; polyammes; polycationic synthetic polymers.
  • bifunctional polyanionic fixing compounds which can be used in the invention, mention may be made of: polyanionic proteins and peptides; polyanionic oligo and polysaccharides; polyacids; polyanionic synthetic polymers.
  • the electrical charges of the substrate are negative, and the bifunctional fixing compounds have a polycationic structure.
  • the reverse is possible.
  • the structure comprises at least one bifunctional fixing compound whose chemical structure comprises at least one group chosen from a peptide, a polypeptide, a protein or an oside.
  • all the bifunctional compounds for fixing the membrane structure according to the invention have this chemical structure, so that the membrane structure is biocompatible.
  • the structure membrane according to the invention is characterized in that it comprises at least one polyamine as a bifunctional fixing compound.
  • a polyamine is a relatively common compound and easy to handle, including on an industrial scale.
  • a polyamine is used, the chemical structure of which comprises at least one group chosen from a peptide, a polypeptide, a protein or an oside.
  • the bifunctional fixing compound has the advantage of good biocompatibility and is easy to handle on an industrial scale and of low cost.
  • a membrane structure according to the invention advantageously comprises a polylysine-succinophospholipid, that is to say a part of the amino groups of which carries a succinophospholipid ligand - in particular N-succinyl - phosphatidylethanolamine - as bifunctional fixing compound.
  • the polyamine - in particular the polylysine succinophospholipidic - exhibits a rate of grafting of the amino functions by the membrane ligands of between 1% and 20%.
  • the succinophospholipid polylysine has a starting polylysine molecular weight of between 10,000 and 50,000.
  • the solid substrate of a membrane structure according to the invention can be chosen from the following solids:
  • crosslinked polymers nucleic acids, DNA, RNA; polyanionic proteins: polyaspartate, polyglutamate, sial proteins; polyanionic polysaccharides: hyaluronic acid, alginic acid, xanthan, heparin, and acid derivatives (phosphate, sulfonate, carboxymethyl sulfate, succinate, etc.) of neutral polysaccharides such as cellulose, starch, dextran; synthetic polymers (nylon, silicone, etc.) derived by anionic functions. All these polymers can only be used in solid form and therefore crosslinked.
  • the crosslinking can be of covalent or ionic nature. This crosslinking must be carried out before the establishment of the functional membrane.
  • Crosslinking can in particular be caused by a polyelectrolytic complexation between the polyanionic polymer and a polycationic polymer, the latter possibly being a polycationic chain of the bifunctional compounds themselves.
  • polycationic substrates polycationic proteins: polylysine, poly-arginine, protamine, histone, polycationic polysaccharides: chitosan, DEAE dextran, synthetic polymers derived by basic functions (DEAE Nylon), alumina, cationic tectosilicates, particles and cationic anion exchange membranes.
  • the functional membrane of a membrane structure according to the invention can have at least one compound for interaction with said external medium.
  • This interaction compound is linked to the functional membrane by any suitable bond so as to extend within the external medium from the free surface of the functional membrane.
  • the membrane structure comprises an interaction compound chosen from a peptide, a protein, a carbohydrate, a glycoprotein.
  • These compounds of interaction with the external environment can be mono or polyclonal antibodies, recognition ligands (transferri ⁇ e, growth factor, hormone, sugar, immunological markers), receptors, transport proteins, enzymes or even fusion proteins.
  • An interaction compound can be linked to at least one membrane ligand of a bifunctional fixing compound by a covalent bond, or on the contrary can be linked by stable non-covalent lyotropic interactions with amphiphilic compounds within the functional membrane.
  • the functional membrane of a membrane structure according to the invention formed of a bilayer of amphiphilic compounds extends over a thickness of less than 5 nm, in particular of the order of 4 to 5 nm.
  • the substrate has pores of average dimensions greater than 5 nm and less than 0.5 ⁇ m. In this way, in particular, an invasion of the pores of the substrate is avoided by the bilayer of amphiphilic compounds.
  • Such a membrane structure according to the invention can be used for obtaining a medicament.
  • the membrane structure according to the invention being analogous to natural plasma membranes of the fixed type, has the properties thereof, and can therefore be used as an artificial plasma membrane in medicinal products or therapeutic compositions, in particular for gene therapy.
  • a membrane structure according to the invention can be used to prepare synthetic supramolecular particles.
  • the invention extends to a synthetic supramolecular particle, characterized in that it comprises a membrane structure according to the invention forming its external periphery and delimiting an internal volume, the functional membrane of the membrane structure having a free surface which extends outside the particle, and which is intended to be placed in contact with an external medium.
  • the substrate occupies at least substantially the entire internal volume of the particle, or alternatively, only part of the internal volume of the particle.
  • the substrate can advantageously be formed from a synthetic porous polymer matrix - in particular DNA or crosslinked RNA.
  • the particle according to the invention may contain a liquid composition, - in particular a therapeutic composition - in its internal volume.
  • the internal volume is entirely occupied by a substrate formed from a porous synthetic polymer matrix which incorporates within its pores a liquid composition.
  • the functional membrane in a particle according to the invention, can be adapted to present the kinetics of release of the liquid composition according to a predetermined profile. It suffices in fact to choose the constitution of the functional membrane to obtain the selective sealing and permeability sought with a view to obtaining this kinetics, and this in a manner known per se (for example in the case of liposomes).
  • a particle according to the invention can have an average dimension of between 10 nm and 5 mm.
  • a particle according to the invention can be the subject of various applications, in particular as a medicament.
  • the invention also extends to a medicament, characterized in that it comprises at least one particle according to the invention.
  • the invention also extends to a supramolecular synthetic film characterized in that it comprises a membrane structure according to the invention.
  • the film according to the invention can also incorporate an ionophore making it possible to selectively transport ions through the bilayer.
  • a film according to the invention can be formed of a portion of a membrane structure according to the invention which is not closed in on itself, that is to say in the general form of a sheet.
  • a film according to the invention is advantageously at least substantially planar, but can have a certain flexibility.
  • a film according to the invention can be the subject of various applications in particular as a separator or extractor of compounds. The invention therefore also extends to the application of a film according to the invention for extracting or separating salts and / or ions from a liquid solution by filtration.
  • the invention also extends to a polycationic polymer of purity greater than 95% formed of a polycationic polyamine endowed with a plurality of lipid ligands - in particular phospholipidic - grafted on part of the nitrogen atoms of the amino functions, and capable forming a stable, non-covalent lyotropic bond with a stable functional membrane of amphiphilic compounds, this polymer possibly acting as a bifunctional compound for fixing the functional membrane to the substrate of a membrane structure according to the invention.
  • this polyamine has a rate of grafting of the amino functions by the lipid membrane ligands which is between 1% and 20%.
  • the polymer is formed from polylysine, in particular a succinophospholipid L-polylysine such as the polylysine N-succinyl-phosphatidylethanolamine.
  • the succinophospholipid polylysine has a molecular weight of starting polylysine of between 10,000 and 5,000.
  • the invention also extends to the process for the preparation of a polycationic polymer - in particular a polymer according to the invention - provided with a plurality of lipid ligands capable of forming a stable non-covalent lyotropic bond with a stable functional membrane of amphiphilic compounds, so that this polymer can act as a bifunctional compound for fixing the functional membrane to the substrate of a membrane structure according to the invention, characterized in that after having carried out the chemical synthesis operations allowing the obtaining of the polymer molecule, it is brought into contact with a citrate in a polar solvent so as to obtain precipitation of the polymer.
  • bifunctional fixing compounds such as polylysines-NSPE
  • polar organic solvents such as DMSO.
  • DMSO polar organic solvents
  • precipitation with citrate has the advantage of being rapid and quantitative.
  • the precipitate obtained is stable and can be easily washed with several types of solvent to remove contaminants.
  • precipitation is selective for polycations and does not lead to ancillary products.
  • precipitation by citrate is easily reversible, quantitatively. Reversion takes place either on the pH or on the ionic strength and leads to a perfectly functional unaltered molecule. The released citrate is easily removed by dialysis and does not disturb the subsequent use of the compound.
  • citrate is a natural product, inexpensive, completely non-toxic and remarkably easy to use.
  • the citrate purification process therefore makes it possible to envisage carrying out without problem the extraction and purification of bifunctional fixing compounds such as polylysines-NSPE on a scale. industrial which was not possible with traditional processes.
  • the invention also extends to the process for preparing a membrane structure according to the invention, which is characterized in that an aqueous suspension of the bifunctional fixing compounds is first prepared in the following manner:
  • a solution of the bifunctional fixing compounds in DMSO is prepared, - an aqueous solution is prepared comprising at least one nonionic detergent, at a concentration higher than its critical micellar concentration,
  • the bifunctional fixing compounds such as the polylysines-NSPE are amphiphilic compounds insoluble in water and in non-polar organic solvents. When these compounds are obtained in the dry state after purification with citrate, it is practically impossible to dissolve them or even to suspend them directly in aqueous solutions even in the presence of a high concentration of nonionic detergent. In addition, the small amount of compound which seems to disperse does not appear to have the expected properties, that is to say the ability to form polyelectrolytic complexes.
  • the inventors have surprisingly found that it is possible to obtain a solubilization of the bifunctional fixing compounds in an aqueous medium by first dissolving it in DMSO and then by injecting this solution, with stirring, into an aqueous detergent solution.
  • the bifunctional fixing compounds such as polylysine-NSPE, thus solubilized in DMSO do indeed have the expected polyelectrolytic complexing properties.
  • the method is further characterized in that one adds then in said aqueous suspension a composition of polyionic polymers capable of forming a solid substrate by polyelectrolytic crosslinking with the polyionic chains of the bifunctional fixing compounds.
  • the method is further characterized in that:
  • amphiphilic compounds capable of forming a functional membrane either in the aqueous solution of the detergent, or in the aqueous suspension before or after addition of the composition of polyionic polymers, and so that the concentration of the detergent remains higher than its critical micellar concentration
  • the solid substrate is therefore formed of a polyionic polymer crosslinked during the preparation of the membrane structure, by the bifunctional fixing compounds themselves.
  • This first variant of the process for preparing the membrane structure according to the invention is more particularly applicable when the substrate is of relatively small dimension, that is to say of average overall dimension of less than 1 ⁇ m.
  • this first variant of the preparation process according to the invention makes it possible to produce particles of average size of the order of 50 to 200 nm incorporating a core of DNA as a substrate, on which a functional membrane is attached. These particles are therefore artificial viruses.
  • the invention also extends to another variant of the process for preparing the membrane structure according to the invention which is more particularly applicable in the case where the substrate is of larger dimension, that is to say of overall dimension average greater than 1 ⁇ m.
  • the preparation process is characterized in that:
  • Amphiphilic compounds capable of forming a functional membrane are introduced either into said aqueous solution of the detergent, or into said aqueous suspension, - then, the concentration of the detergent in said aqueous suspension is reduced to a concentration below its critical micellar concentration ,
  • this aqueous suspension is placed in contact with a solid phase substrate,
  • membranes called “supported membranes” which are formed of a lipid bilayer on a flat solid substrate such as quartz (cf. "Supported planar membrane in studies of cell-cell recognition in the immune System” HM Me Connell and al, Biochimica and Biophysica Acta 864 (1986) 95-106).
  • the bilayer is not not fixed on the substrate, so that the system has a very high brittleness, which considerably reduces its practical interest.
  • hydrophobic chains are grafted on the surface by covalent bonds on the solid support, on which a monolayer of phospholipids is deposited, then optionally a succession of bilayers.
  • a stable functional membrane is not formed, since the monolayer associated with the hydrophobic chains linked to the solid support cannot exhibit the fundamental properties of a bilayer.
  • the lipid bilayers possibly present above the monolayer are not fixed and are therefore, here again, very fragile and unstable.
  • the polymer has not been purified or characterized and cannot be obtained in practice.
  • Their authors mistakenly consider that the total consumption of the NHS-ester of NGPE demonstrates the obtaining of lipopolylysine, and do not envisage purification.
  • the compound obtained is capable of forming a clear solution in water in the absence of detergent, which is not the case, and cannot be the case, of the lipopolylysine that they describe.
  • the structure of the product obtained in this document does not correspond to the lipopolylysine that they claim to have obtained which, because it carries two lipid groups NGPE, would be insoluble in water, and could at best only disperse therein.
  • the DNA and cationic liposome complexes described by these authors in the second document comprise DNA adsorbed by polyelectrolytic bond to the outside of liposomal complexes (which are not in fact true liposomes) formed of lipopolylysine (LPLL) with non-phospholipid DPSG chains and of dioleoylphosphatidylethanolamine (DOPE).
  • LPLL lipopolylysine
  • DOPE dioleoylphosphatidylethanolamine
  • the LPLL is therefore not linked to a functional membrane formed by a stable bilayer of amphiphilic compounds since DOPE does not form such a functional bilayer, but a hexagonal structure.
  • the DPSG triglyceride chains (considered preferable to the NGPE chains which the authors have given up in this second document) cannot be inserted into a functional bilayer.
  • the invention in fact provides the only means of establishing and fixing a functional membrane continuously covering a porous substrate.
  • the inventors have in fact found that, in a membrane structure according to the invention, the bifunctional fixing compound restricts the relative mobility of the phospholipid bilayer with respect to the substrate by preventing its penetration inside the pores.
  • FIG. 1 is a reaction scheme for the preparation of a polycationic polymer according to the invention
  • FIG. 2 is a diagram illustrating the results of tests of Example 3 of inhibition of fluorescence by L-polylysine-NSPE in solution in DMSO and added to an aqueous solution of DNA, detergent and BET,
  • FIG. 3 is a diagram illustrating the general structure of an artificial viral particle according to the invention obtained in example 4,
  • FIG. 4 is a diagram illustrating the results of tests of example 5 of inhibition by the
  • FIG. 5 is a diagram illustrating the kinetics of hemoglobin release from the particles according to the invention in accordance with Example 7,
  • FIG. 6 is a partial sectional view of a membrane structure according to one embodiment of the invention.
  • EXAMPLE 1 Preparation of a polycationic polymer according to the invention: an L-polylysine N-succinylphosphatidylethanolamine.
  • EYPE egg yolk phosphatidylethanolamine compound (I) FIG. 1
  • 824.2 mg of EYPE egg yolk phosphatidylethanolamine compound (I) FIG. 1
  • 163 ⁇ l of TEA triethylamine are added with magnetic stirring.
  • 176.7 mg of succinic anhydride (II) is added and the reaction is allowed to continue for two hours.
  • the disappearance of free amines is monitored by chromatography on silica gel with a chloroform / methanol / water mixture (1/2 / 0.9; v / v / v) as eluent.
  • the product (III) is dissolved in 15 ml of chloroform and 560 mg of N-hydroxysuccinimide (compound (IV)) are added with magnetic stirring. Then weighed 1.457 g of dried N, N '-dicyclohexylcarbodiimide (DCCD) and solubilized in 6 ml of chloroform. To the solution of III + IV, 1 ml of DCCD solution is gradually added every 10 min with magnetic stirring at room temperature. After the last addition of DCCD, the mixture is allowed to incubate overnight at room temperature. The reaction medium is filtered on glass wool to remove the precipitate of dicyclohexylurea.
  • DCCD N-hydroxysuccinimide
  • the reaction scheme for these first three synthesis steps is illustrated in FIG. 1. It allows the synthesis of the product (VI) which is an L-polylysine N-succinyl-phosphatidylethanolamine, that is to say an L-polylysine of which certain amino groups carry the phospholipid ligands NSPE.
  • such a phospholipid polylysine is designated by polylysine-NSPE or, when it is desired to specify its molecular weight and its rate of grafting by phospholipid ligands, by the designation: L-polylysine (x) -NSPE-dsy where x is the molecular weight (in kilodaltons) of L-polylysine in the form of starting hydrobromide and y is the grafting rate expressed as a percentage of substituted amino functions.
  • the precipitate is taken up in 2 ml of DMSO, then 1 ml of H 0 and finally, 100 ⁇ l of 1N HCl allowing the pH to be lowered under the pK of citric acid. Homogenized with magnetic stirring. The solution becomes clear.
  • the lyophilized sample is taken up in 2 ml of DMSO in which it dissolves perfectly.
  • the sample is subjected to a traditional protein and phospholipid assay. It is noted that the sample obtained contains 7.55 mg of proteins and 180 ⁇ g of phosphorus which corresponds to a grafting rate of L-polylysine of 10% of the amino groups, that is to say to L- polylysine (19, 2) - NSPE-ds10.
  • EXAMPLE 2 variation of the grafting rate:
  • the number of amino groups carrying a phospholipid ligand on the total number of amino groups of the polymer (VI) constitutes its grafting rate.
  • the purification procedure is identical to that described in Example 1 steps 4) to 6) for L-polylysine (19, 2) -NSPE-ds10.
  • the products (VI) obtained by synthesis in the form of a powder are essentially insoluble in water unlike the starting L-polylysine (19,2), thus highlighting the chemical modification of L-polylysine. These products are also insoluble in a buffer containing a nonionic detergent such as HECAMEG at pH 7.
  • the capacity of the L-polylysines-NSPE synthesized as described previously in Example 1 to interact with DNA is more precisely studied by their property in displacing a fluorescent probe, ethidium bromide (BET), which is intercalated naturally between the bases of DNA.
  • BET ethidium bromide
  • the BET is displaced from the DNA / BET complex and loses its fluorescence. This loss of fluorescence is represented by the curves of FIG. 2 as a function of the amount of added L-polylysine expressed on the abscissa by the ratio (+/-) between the positive charges of L- polylysine and the negative charges of 1 'AD ' N.
  • Curve A represents the displacement of BET by L-polylysine (19, 2) not phospholidated
  • curve B represents the displacement of BET by L-polylysine (19, 2) -NSPE-ds1
  • curve C represents displacement of BET by L-polylysine (19, 2) - NSPE-ds1,7
  • curve D represents the displacement of BET by L-polylysine (19, 2) -NSPE-ds3
  • curve E represents the displacement of BET by L-polylysine (19, 2) -NSPE-ds10
  • curve F represents the displacement of BET by L- polylysine (19, 2) -NSPE-ds21
  • curve G represents displacement of BET by L- polylysine (19, 2) -NSPE-ds30.
  • EXAMPLE 4 Preparation of artificial viral particles according to the invention: In a 50 ml flask, a lipid solution is prepared in 1 ml of chloroform containing 250 ⁇ g of egg yolk lecithin (L- ⁇ -phosphatidylcholine from yolk). egg, EPC - Lipoid) and 25 ⁇ g of cholesterol. This solution is dried under nitrogen and then lyophilized for 12 h.
  • the DNA core 31 can be considered as an artificial nucleocapsid.
  • the artificial viral particles are stable for at least 15 days.
  • EXAMPLE 5 Demonstration of the Transfecting Properties of Artificial Viral Particles. Coupling of a cellular and intracellular targeting interaction compound, a defective adenovirus, to the outer surface of artificial viral particles. Viral particles are synthesized in the same manner as in Example 4 but by adding, to the phospholipid composition, 5 mol% of egg yolk phosphatidylethanolamine N - ⁇ - 4- (N- maleimidomethyl) cyclohexane-1 -carbonyl ⁇ (MCC-EYPE).
  • MCC-EYPE Neutravidin substituted with N-Succinimidyl-3- (2-pyridyldithio) propionate (SPDP) is grafted onto the MCC-EYPE residues present on the external surface of the particles.
  • MCC-EYPE was obtained from EYPE (egg yolk phosphatidylethanolamine) and SMCC (Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 - carboxylate) as follows.
  • EYPE 113.7 mg of EYPE are dissolved in 5 ml of anhydrous chloroform. 24 ⁇ l of triethyla ine (TEA) are added then 50 mg of SMCC in solution in 0.5 ml of dimethylsulfoxide (DMSO). The mixture is incubated for 2 hours at 40 ° C with shaking. The appearance of MCC-EYPE is followed by thin layer chromatography on silica gel. The product is extracted with a chloroform / methanol / water mixture. After centrifugation for 10 min at 4000 rpm, the aqueous phase is eliminated and the chloroform phase containing the MCC-EYPE is evaporated. The structure of the MCC-EYPE is characterized by nuclear magnetic resonance.
  • N-propionyl-thiol-neutravidin thiolated neutravidin: 10 mg of neutravidin are dissolved in 1 ml of 200 mM Hepes buffer, 300 mM NaCl, pH 7.9. The suspension is passed through a column of SEPHADEX® G25 filtration gel at the outlet from which 500 ⁇ l fractions are collected. 95% of the protein is recovered in fractions 7 to 10.
  • a solution of SPDP N-Succinimidyl-3- (2-pyridyldithio) propionate
  • SPDP N-Succinimidyl-3- (2-pyridyldithio) propionate
  • the mixture is incubated for one hour at room temperature and then washed on G25 with 0.1M phosphate buffer, pH 7.2.
  • the neutravidin-PDP is treated with 100 ⁇ l of a 0.1M solution of dithiothreitol for 10 min at room temperature.
  • the thiolated neutravidin is washed on a SEPHADEX® G25 column with 0.1M phosphate buffer, pH 7.2.
  • the grafting rate is 10 thiols per molecule of neutravidin.
  • Biotin fixation on defective adenovirus particles a 400 ⁇ M solution of biotin- NHS is prepared in a 5 mM Hepes buffer, 150 mM NaCl, glycerol 10%, pH 7, 9. To 1 ml of this solution, 2.5 ⁇ 10 9 adenoviral particles are added and the whole is left for 3 hours at room temperature with gentle stirring. The unreacted biotin is eliminated by three successive passages in ultrafiltration (10 min at 1500 g). The biotinyl adenoviruses are taken up in 1 ml of PBS buffer (Phosphate Buffer Saline, 10mM phosphate, 150mM NaCl, pH 7.4).
  • PBS buffer Phosphate Buffer Saline, 10mM phosphate, 150mM NaCl, pH 7.4
  • Coupling of biotinyl defective adenoviruses with neutravidinylated artificial viral particles an amount of artificial viral particles corresponding to 5 ⁇ g of DNA is incubated for one hour at room temperature with gentle agitation with 8.10 adenoviral particles. The suspension is adjusted to 500 ⁇ l with PBS.
  • Transfections are carried out in 35 mm diameter dishes or in multi-well plates where the wells are the same size. Cells are transfected at 80% confluence (approximately 8.10 cells per well). The 500 ⁇ l of the suspension obtained in the preceding step are deposited homogeneously on the cells. After 1 hour of incubation, the medium is replaced by 2 ml of culture medium supplemented with serum. The cells are incubated for 48 hours at 37 ° C. to observe a transient expression.
  • modified transferrin 1 ⁇ mol of modified transferrin is mixed dissolved in a 100 mM phosphate buffer, pH 7.8 with particles containing 0.1 ⁇ mol of 3-mercaptopropionate DPPE and dispersed in a 20 mM sodium acetate buffer. The preparation is stirred for 24 h at room temperature, then is ultrafiltered through a 100KD membrane to remove the excess transferrin.
  • the viral particles are obtained as represented in FIG. 3 provided with ligands 34 formed of transferrin.
  • L-polylysine of molecular weight 19200 is weighed, which is dissolved in 10 ml of DMSO with magnetic stirring. 40 ⁇ l of triethylamine are added and wait 10 min. 1.1 mg of fluorescein isothiocyanate (FITC) are then added in solution in 149 ⁇ l of dimethylformamide (DMF). The reaction continues at 30 ° C for 2 h. The product is analyzed by chromatography on silica gel which shows the disappearance of the free FITC and the appearance of a protein and fluorescent product on deposition. L-polylysine-fluorescein is purified as follows.
  • the DMSO of the reaction medium is dialyzed twice for 2 h against distilled water at pH 6.5.
  • the dialysis product is then incubated with 500 mg of SEPHADEX® C50 in 100 ml of distilled water, then deposited on a column.
  • the column is first washed with 100 ml of distilled water pH 7.
  • the L-polylysine-fluorescein is eluted from the column with 100 ml of a 2M NaCl solution, pH 9. This solution is dialyzed against water distilled.
  • the final solution contains 35 mg of protein.
  • the quantity of fluorescein is estimated by spectrometry at 496 nm with a molecular extinction coefficient of 90,000 m ⁇ 1 cm ⁇ 1 .
  • the grafting rate is 1/233 of the amino functions.
  • polylysines-NSPE synthesized as described above to allow the establishment of a membrane structure impermeable to cupric ions (Cu ++ ) surrounding the complexed DNA is studied by the method of inhibiting the fluorescence of a fluorescent probe linked to DNA complex.
  • This probe is L-polylysine (19, 2) -fluorescein-dsO, 4 obtained in 1).
  • the viral particles are prepared as described in Example 4 with the only difference that L-polylysine-NSPE-dsO, 1 is replaced by a mixture of 90% of L-polylysine (19, 2) -NSPE- ds (n ) and 10% L-polylysine (19, 2) -fluorescein-dsO, 4.
  • the mixture (phospholipids + cholesterol + detergent + L (polylysine-NSPE-ds (n) + L-polylysine (19, 2) - fluorescein- ds0,4 + DNA) is then dialyzed and the fluorescence of the particles is analyzed with a spectrofluorimeter.
  • the intensity of the fluorescence is analyzed during the progressive addition of Cu ++ .
  • the value represented on the ordinate in the figure 4 represents the fluorescence inhibition rate, expressed as a percentage and calculated as follows: (Ig-If) / I / ' or ⁇ 0 es ⁇ the fluorescence intensity in the absence of copper, and If is the intensity of the fluorescence in the presence of copper.
  • Curve A represents the rate of inhibition of the fluorescence of the particles obtained from L-polylysine-NSPE-ds10 but in the presence of detergent which prevents the establishment of a functional membrane around the particle. It is observed that the inhibition rate of the fluorescence of L-polylysine (19, 2) -fluorescein- ds0.4 reaches a value close to 100% for a Cu + + concentration of 50 ⁇ M.
  • Curve B represents the rate of inhibition of the fluorescence of the particles obtained from L-polylysine-NSPE-dsl after dialysis of the detergent.
  • Curve D represents the rate of inhibition of the fluorescence of the particles obtained from L-polylysine-NSPE-ds10 after dialysis of the detergent. It is observed that the rate of inhibition of the fluorescence of L- polylysine (19, 2) -fluorescein-dsO, 4 reaches a value close to 25% for a Cu + + concentration of 50 ⁇ M.
  • Curve E represents the rate of inhibition of the fluorescence of the particles obtained from L-polylysine-NSPE-ds21 after dialysis of the detergent. It is observed that the inhibition rate of the fluorescence of L-polylysine (19, 2) - fluorescein-dsO, 4 reaches a value close to 20% for a Cu ++ concentration of 50 ⁇ M.
  • the artificial viral particles can be used for therapeutic purposes to ensure the intracellular delivery in vivo of therapeutic genes, for example for the treatment of genetic diseases (cystic fibrosis, etc.), certain cancers or for the preparation of gene vaccines.
  • micellar preparation is sonicated for 1 min in an ultrasonic bath, then is dialyzed against distilled water to give a suspension of the polylysine-NSPE and of the bilayer-forming phospholipids. The presence of particles of average size between 300 and 600 nm is observed in the particle analyzer.
  • preparation of neutral control liposomes proceed as indicated in a), but without adding the polylysine-NSPE solution to the dispersion of the lipids in the detergent. The presence of liposomes of average size between 200 e: 400 nm is observed.
  • absorption of hemoglobin on the porous substrate 5 mg of porous particle (SEPHADEX®
  • SPC50 with a diameter of 150 ⁇ m derived by suifopropyl groups and the size of the pores of which is sufficient to allow molecules of a maximum molecular weight of 250,000 to penetrate are dispersed in 5 ml of 10 mM bistris buffer at pH 6.5 (where hemoglobin is cationic is drawn into the anionic sites of porous particles). 20 mg of hemoglobin extracted from human red blood cells by the lysis method in a hypotonic medium are added to this dispersion. The preparation is stirred for 24 h at 4 ° C on a sun shaker. The particles are then decanted and the amount present in the supernatant is determined by UV spectrometry at 410 nm.
  • the particles loaded with hemoglobin are incubated in 5 ml of 150 mM PBS buffer at pH 7.4 (where
  • FIG. 5 illustrates the curves of kinetics of hemoglobin release obtained over time with the ordinate the mass in mg of hemoglobin released and on the abscissa the time in hours.
  • Curve C51 corresponds to the result obtained with the particles simply charged with hemoglobin from step c) of Example 7.
  • Curve C53 corresponds to the results obtained with the particles obtained in Step e) of Example 8 (neutral liposomes obtained in b) brought into contact in the polylysine-NSPE / phospholipid suspension obtained in a)). Curve C52 corresponds to the results obtained with the particles according to the invention obtained in d) of Example 8.
  • the particles loaded with doxorubicin are dispersed in 4 ml of distilled water. Then 1 ml of the polylysine-NSPE suspension and of the bilayer phospholipids obtained in step a) of example 7 are added, and the mixture is gently stirred for 2 h at 4 ° C. Then 625 ⁇ l of solution of 40 mM non-ionic detergent HECAMEG, stirred for 5 min, and the particles are decanted and the pellet is washed with distilled water to remove the detergent and the excess polylysine-NSPE / phospholipid complexes.
  • Example 8 a control experiment is carried out using a suspension of neutral liposomes composed solely of phospholipids (EYPC) and cholesterol.
  • EYPC phospholipids
  • the particles are dispersed in 150 ml of 150 mM PPS buffer at pH 7.1 and gently stirred. Aliquots are taken at regular time intervals and the concentration of doxorubicin in the supernatant is measured by UV spectrophotometry at 480 nm.
  • the results obtained indicate that, after 1 hour, 45% by weight of the doxorubicin has been released from the particles of the control experiment and from the particles loaded with doxorubicin used before being brought into contact with the polylysine-NSPE / phospholipid suspension, that is to say particles free of a fixed functional membrane.
  • the particles according to the invention comprising a fixed functional membrane, only 5% of the doxorubicin was released.
  • the fixed functional membrane formed of phospholipids makes it possible to effectively retain doxorubicin inside the particles according to the invention.
  • porous matrix SEPHADEX® C25 is dispersed in 100 ml of distilled water and the preparation is ground for 15 min with a propeller mill. The preparation is then centrifuged at 3000 g for 10 min, the supernatant is recovered and then subjected to another centrifugation at
  • the size of the particles obtained, measured with the particle analyzer, is between 300 and 500 nm.
  • small size complexes are prepared. To do this, 9 mg of phospholipids EYPC and 1 mg of cholesterol are dissolved in 1 ml of chloroform which is evaporated under reduced pressure on a rotary evaporator. The lipids are then dispersed in 2.5 ml of an aqueous solution of nonionic detergent
  • micellar preparation is sonicated for 1 min in an ultrasonic bath , then is quickly diluted in 10 ml of distilled water and dialyzed against water distilled to give a preparation of small polylysine-NSPE / phospholipid complexes. The presence of particles with an average size of 50 nm is observed with the particle analyzer. The porous particles of small sizes according to the invention are then prepared.
  • 625 ⁇ l of 40 mM HECAMEG® nonionic detergent solution are added to 2.5 ml of the suspension of polylysine-NSPE / phospholipids prepared previously, so as to obtain a concentration of 10 mM in detergent.
  • the suspension is sonicated for 1 min in an ultrasonic bath and is added slowly to 2.5 ml of the suspension of small porous particles loaded with doxorubicin prepared above, then placed in a continuous dialysis cell, kept under stirring.
  • Example 12 The particles prepared in Example 12 are dispersed in 150 ml of PBS buffer at pH 7.4 150 mM and gently stirred. Aliquots are taken at regular time intervals, the concentration of doxorubicin released is measured by UV spectrophotometry at 480 nm after ultrafiltration on a membrane whose filtration threshold is at 50,000.
  • EYPC 9 mg of EYPC, 1 mg of cholesterol and 0.05 mg of dipalmitoyl phosphatidyl ethanolamine rhodamine (DPPERd) are dissolved in 1 ml of chloroform and then evaporated under reduced pressure. The residue is taken up in 10 ml of a 40 m HECAMEG® solution. When the solution has become completely clear, it is dialyzed against distilled water. The fluorescent liposomes obtained are very difficult to distinguish upon observation and appear, owing to their small size, in the form of a uniform background noise.
  • DPPERd dipalmitoyl phosphatidyl ethanolamine rhodamine
  • Preparation of polylysine-NSPE / fluorescent phospholipid complexes The procedure is as above by adding 100 ⁇ l of a 40 mM HECAMEG® solution containing 1 mg of L- polylysine (19, 2) -NSPE-d6 to the solution of lipids. detergent. The preparation is sonicated for 1 min in an ultrasonic bath and then dialyzed against distilled water. The polylysine-NSPE / fluorescent phospholipid complexes are larger than the control liposomes and are visible in the form of small fluorescent spots.
  • the particles of SEPHADEX ® are first visualized by phase contrast microscopy. They are in the form of regular spheres with a diameter between 100 and 150 ⁇ m. These particles (0.1 mg in 0.5 ml are then incubated with 20 ⁇ l of the preparation of the polylysine-NSPE / fluorescent phospholipid complexes.
  • the particles obtained are then incubated with 2 ml of 150 mM NaCl PBS buffer for 10 min. It is observed that the appearance of the particles remains unchanged.
  • the energy involved in the formation of polyelectrolytic complexation is, indeed, considerable and this complexation remains stable over a large range of pH and ionic strength.
  • the preparation is then decanted and incubated with 2 ml of a 5 mM HECAMEG® solution for 10 min. It can be seen that the appearance of the crown has been modified and is becoming completely regular. This result indicates that the entities which had hung individually on the surface of the particles have now merged with each other to form a continuous phospholipid bilayer fixed to the particulate substrate.
  • the preparation is then decanted again and incubated with 2 ml of a 40 mM HECAMEG® solution for 10 min.
  • a control is, moreover, produced with neutral fluorescent liposomes incubated with particles of SEPHADEX® SPC50. There is no interaction between the particles which appear in black and the liposomes which appear in a uniform fluorescent background. This result indicates that the adhesion properties of the polylysine-NSPE / phospholipid complexes are indeed due to the presence of the polylysine-NSPE / phospholipid.
  • the particles are incubated in the same way for 5 min with the fluorescent polylysine solution. They are then washed with distilled water, incubated with 2 ml of 150 mM PBS buffer NaCl for 10 min and finally washed with 2 times 2 ml of PBS buffer. The results of the observation indicate that the fluorescence has apparently been maintained quantitatively around the particle. Likewise, no modification is made by incubation with a 40 mM HECAMEG® solution.
  • silica particles pore size 60 Angstroms, particle size 40 ⁇ m
  • 0.1 mg of silica particles are incubated with 200 ⁇ l of the fluorescent polylysine solution for 5 min, then decanted and washed with 2 ml of distilled water.
  • the results are observed in phase contrast and fluorescence. They indicate that polylysine has a strong affinity for silica.
  • a incubation of these particles with 2 ml of PBS 150 mM NaCl buffer indicates that this affinity is not affected by this buffer, the stability of the polyelectrolytic interactions between polylysine and silica is therefore also very strong with a material such as silica.
  • any glass-based material can be covered with a functional membrane according to the invention and be used as a solid substrate.
  • a functional phospholipid membrane is fixed on a flat porous substrate formed by an ion exchange filter.
  • a polylysine-NSPE / phospholipid suspension is prepared as indicated in step a) of Example 6, but with 20% by weight of cholesterol relative to the phospholipids EYPC. 2 ml of this suspension are dissolved in 200 ml of distilled water, and the mixture is placed in an ultrafiltration cell 47 mm in diameter and equipped with a Gelman anionic filter (reference 60943) with a porosity of 0.445 ⁇ m and equal diameter at 47 mm. This anionic filter therefore forms the solid porous substrate of the film. The cell pressure is adjusted to obtain an initial flow rate of 2 ml / min.
  • a control experiment is carried out using neutral liposomes as in Example 8.
  • Another experiment is also prepared by fixing a functional membrane containing an ionophore (agent facilitating the diffusion of ions) on the filter.
  • a suspension of polylysine-NSPE / phospholipids is prepared as indicated previously in this example, but by adding 0.1 mg of monensin (ionophore) to the chloroform solution of phospholipids EYPC (1% by weight of the total). The suspension is then used to establish the membrane on the filter in the same manner as previously indicated.
  • EXAMPLE 16 Study of the impermeability to ions of films. 200 ml of a 25 g / l NaCl solution, the resistivity of which is 55 mS / cm, are introduced into the ultrafiltration cell.
  • the pressure is established to obtain a flow rate of 0.5 ml / min and fractions of 1 ml are collected on which the conductivity is measured.
  • the experiments are carried out under different conditions with: a filter alone, a filter resulting from the control experiment associated with neutral liposomes, a synthetic film according to the invention formed of the filter and the fixed functional membrane, and a film according to the invention formed of the filter provided with a fixed functional membrane containing an ionophore.
  • the results obtained are expressed in the following table: Conductivity
  • the functional membranes fixed according to the invention on planar supports retain the same selective permeability properties as the plasma membranes of eukaryotic cells, and can like them, extend over considerable surfaces while remaining functional.
  • a film according to the invention can be used to extract or separate salts and / or ions from a liquid solution by filtration.
  • FIG. 6 illustrates in detail but schematically the composition of a membrane structure according to the invention, and therefore a portion of film according to the invention.
  • This structure comprises a bilayer 63 forming a functional membrane, polycationic L-polylysine-NSPE 62, and the porous substrate 61 " formed of the filter.
  • the L-polylysine-NSPE form polycationic polymer chains 64 and carry membrane ligands 65, the phospholipid chains 66 are inserted by lyotropic interaction within the functional membrane 63.
  • the invention can be subject to numerous variants and applications.
  • polycationic or polyanionic polymers can be used as bifunctional compounds; other amphiphilic compounds can be used to form the membrane; other solid polyionic substrates can be used as soon as they have a surface density of positive and / or negative electrical charges.

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Abstract

The invention concerns an artificial membrane structure analogous to fixed plasmic membranes comprising a solid substrate (61); a functional membrane (63) which does not circumscribe the external medium; and at least one bifunctional fixing compound (62) inserted between the membrane (63) and the substrate (61), co-operating by polyelectrolytic complexing with the substrate (61) and by lyotropic bonds with the membrane (63). The invention also concerns the use of this structure for obtaining a medicine, a particle, a film, its method of preparation and a lipidic polycationic polymer, and its method of preparation, acting as bifunctional compound (62).

Description

STRUCTURE MEMBRANAIRE ARTIFICIELLE POLYMERIQUE, PROCEDE POUR SA PREPARATION PROCEDE DE PREPARATION DE CE POLYMERE, PARTICULE ET FILM COMPRENANT CETTE STRUCTUREARTIFICIAL POLYMERIC MEMBRANE STRUCTURE, PROCESS FOR ITS PREPARATION PROCESS FOR THE PREPARATION OF THIS POLYMER, PARTICLE AND FILM COMPRISING THE SAME
L'invention concerne une structure membranaire artificielle analogue aux membranes plasmiques naturelles fixées sur un cytosquelette, telles qu'on les retrouvent chez les cellules eucaryotes, notamment chez les erythrocytes et les dérivés des cellules eucaryotes que constituent les virus enveloppés.The invention relates to an artificial membrane structure analogous to natural plasma membranes fixed on a cytoskeleton, as found in eukaryotic cells, in particular in erythrocytes and derivatives of eukaryotic cells, which are enveloped viruses.
Les membranes plasmiques naturelles (cf. par exemple MOLECULAR BIOLOGY OF THE CELL, third Edition, 1994, Bruce Alberts et al , Garland Publishmg, New-york, USA ou THE MEMBRANES OF CELLS , Philip YEAGLE, 1987, Académie Press Inc San Diego, USA) comprennent une bicouche lipidique (principalement phospholipidique) formant une phase lyotrope lamellaire stable incorporant des molécules amphiphiles et des protéines maintenues ensemble par des interactions lyotropiques stables non covalentes (faisant intervenir principalement des liaisons dites "hydrophobes" ) Les molécules amphiphiles sont arrangées en une double couche continue ayant une épaisseur de l'ordre de 4 à 5 n à l'état de cristal liquide et de mosaïque fluide (cf. Singer et Nicholson, Science, Vol 175, 720 (1972)) qui sert de barrière imperméable à la plupart des grosses molécules polaires neutres dont les sucres, et aux ions minéraux, tout en étant perméable aux molécules hydrophobes (notamment l'oxygène), et aux petites molécules polaires neutres (notamment l'eau, l'urée, le glycérol). Les protéines transmembranaires des membranes naturelles permettent notamment de transporter de façon sélective à travers la bicouche les ions, les sucres... Les membranes plasmiques naturelles portent également des systèmes gérant les échanges avec l'extérieur (en général des protéines ou des sucres).Natural plasma membranes (cf. for example MOLECULAR BIOLOGY OF THE CELL, third Edition, 1994, Bruce Alberts et al, Garland Publishmg, New-york, USA or THE MEMBRANES OF CELLS, Philip YEAGLE, 1987, Académie Press Inc San Diego, USA) include a lipid bilayer (mainly phospholipid) forming a stable lamellar lyotropic phase incorporating amphiphilic molecules and proteins held together by stable non-covalent lyotropic interactions (mainly involving so-called "hydrophobic" bonds) Amphiphilic molecules are arranged in a continuous double layer having a thickness of the order of 4 to 5 n in the state of liquid crystal and fluid mosaic (cf. Singer and Nicholson, Science, Vol 175, 720 (1972)) which serves as an impermeable barrier to the most large neutral polar molecules including sugars, and mineral ions, while being permeable to hydrophobic molecules (including oxygen), and x small neutral polar molecules (especially water, urea, glycerol). The transmembrane proteins of natural membranes make it possible in particular to selectively transport ions, sugars, etc. through the bilayer. Natural plasma membranes also carry systems that manage exchanges with the outside world (generally proteins or sugars).
Les membranes plasmiques naturelles des cellules eucaryotes sont fixées sur un cytosqueletre (réseau de protéines filamentaires ) qui - stabilise la membrane plasmique de l'intérieur. La fixation fait intervenir un ensemble de liaisons spécifiques entre des protéines ancrées dans la membrane et des protéines du cytosquelette .The natural plasma membranes of eukaryotic cells are fixed on a cytoskeleton (network of filamentary proteins) which - stabilizes the plasma membrane from the inside. Binding involves a set of specific bonds between proteins anchored in the membrane and proteins of the cytoskeleton.
Ainsi, les enveloppes des virus enveloppés sont formées d'une membrane plasmique fixée sur la capside du virus par des protéines spécifiques d'enveloppe virale capables de se lier avec les protéines de la capside. Egalement, la membrane des erythrocytes est fixée sur un cytosquelette fibreux, par l'intermédiaire d'une protéine, la bande 3, capable de se lier avec 1 ' nkyrine du cytosquelette, ce qui lui confère une forme biconcave (qui se transforme en sphérocyte lorsque la fixation est défaillante ) .Thus, the envelopes of the enveloped viruses are formed from a plasma membrane fixed to the capsid of the virus by specific proteins of viral envelope capable of binding with the proteins of the capsid. Also, the erythrocyte membrane is fixed on a fibrous cytoskeleton, via a protein, band 3, capable of binding with the cytoskeletal nkyrin, which gives it a biconcave form (which turns into a spherocyte when the fixing is faulty).
Dès lors, les membranes plasmiques fixées possèdent non seulement les propriétés fondamentales propres aux bicouches lipidiques, mais également elles peuvent présenter une morphologie complexe (notamment avec une surface spécifique considérable, par exemple les cellules intestinales dotées de microvillosités , ou les axones des neurones) et possèdent, du fait de la fixation de la bicouche sur le cytosquelette, des propriétés mécaniques remarquables, notamment de résistance et de stabilité. En outre, le système de fixation préserve la fluidité latérale de la bicouche et les membranes fixées conservent la propriété d'être autoscellables comme le sont les liposomes, c'est-à-dire sont capables de se refermer spontanément, notamment après avoir subi une perforation. Compte-tenu de l'importance de ces propriétés, propres aux membranes plasmiques fixées, il serait hautement souhaitable de pouvoir obtenir des structures artificielles analogues.Consequently, the fixed plasma membranes not only possess the fundamental properties specific to lipid bilayers, but also they can have a complex morphology (in particular with a considerable specific surface, for example intestinal cells provided with microvilli, or the axons of neurons) and possess, due to the fixation of the bilayer on the cytoskeleton, remarkable mechanical properties, in particular of resistance and stability. In addition, the fixing system preserves the lateral fluidity of the bilayer and the fixed membranes retain the property of being self-sealing as are the liposomes, that is to say are capable of closing spontaneously, in particular after having undergone a perforation. Given the importance of these properties, which are specific to fixed plasma membranes, it would be highly desirable to be able to obtain similar artificial structures.
Or, si l'on sait depuis longtemps réaliser des structures artificielles formées d'une bicouche lipidique (par exemple les liposomes), on n'est pas parvenu jusqu'à maintenant, de façon simple et compatible avec des contraintes industrielles, à préparer des structures membranaires artificielles mimant les membranes plasmiques naturelles des cellules eucaryotes fixées. En effet, le cytosquelette, les protéines permettant de fixer la bicouche sur le cytosquelette, et les liaisons spécifiques de ces protéines avec le cytosquelette sont extrêmement complexes à manipuler et à mettre en oeuvre ar ificiellement .However, if it has been known for a long time to make artificial structures formed from a lipid bilayer (for example liposomes), until now, we have not succeeded, in a simple manner and compatible with industrial constraints, in preparing structures artificial membranes mimicking the natural plasma membranes of fixed eukaryotic cells. Indeed, the cytoskeleton, the proteins making it possible to fix the bilayer on the cytoskeleton, and the specific bonds of these proteins with the cytoskeleton are extremely complex to handle and to implement ar ificially.
Ainsi, la publication "Molecular Architecture and Function of Polymenc Oriented Systems : Models for the Study of Organization, Surface Récognition, and Dynamics of Biomembranes", Helmut Ringsdorf et al., Ange . Chem. Int. Ed. Engl . 27 (1988) 113-158 décrit les systèmes supramoléculaires artificiels connus et indique les procédés envisageables pour simuler une membrane fixée sur un cytosquelette (§ 4.8.215 p 136-138) :Thus, the publication "Molecular Architecture and Function of Polymenc Oriented Systems: Models for the Study of Organization, Surface Récognition, and Dynamics of Biomembranes", Helmut Ringsdorf et al., Ange. Chem. Int. Ed. Engl. 27 (1988) 113-158 describes the known artificial supramolecular systems and indicates the possible methods for simulating a membrane fixed on a cytoskeleton (§ 4.8.215 p 136-138):
1 - associer par liaisons covalentes directes des réseaux de polymères linéaires faisant office de squelette à l'une des couches de la bicouche lipidique,1 - associating, by direct covalent bonds, networks of linear polymers acting as skeleton with one of the layers of the lipid bilayer,
2 - remplir l'intérieur d'un liposome d'un gel polymère formant un réseau tridimensionnel qui n'est pas lié à la bicouche lipidique,2 - fill the inside of a liposome with a polymer gel forming a three-dimensional network which is not linked to the lipid bilayer,
3 - associer par interactions électrostatiques directes des réseaux de polymères linéaires poly- îoniques faisant office de squelette à l'une des couches de la bicouche lipidique dont les têtes polaires sont également ioniques.3 - associating, by direct electrostatic interactions, networks of polyionic linear polymers acting as skeleton with one of the layers of the lipid bilayer whose polar heads are also ionic.
Le premier procédé ne permet d'obtenir que des liposomes polyménsés sphériques stabilisés, et n'autorise pas la réalisation d'une structure membranaire de forme quelconque et autoscellable. En outre, la bicouche présente des portions poly érisées dans lesquelles la fluidité latérale est absente, et des portions non polymérisées qui restent fragiles. Ainsi, ce premier procédé fournit une structure qui ne peut pas présenter les diverses propriétés sus-mentionnées d'une membrane plasmique fixée.The first method makes it possible to obtain only stabilized spherical polymerized liposomes, and does not authorize the production of a membrane structure of any shape and self-sealing. In addition, the bilayer has poly erized portions in which the lateral fluidity is absent, and non-polymerized portions which remain fragile. Thus, this first method provides a structure which cannot exhibit the various properties mentioned above of a fixed plasma membrane.
Le deuxième procédé ne permet aussi d'obtenir que des liposomes sphériques à coeur solide entouré d'une bicouche lipidique qui n'est pas liée au coeur solide. Ce procédé ne permet donc pas d'obtenir une structure de forme quelconque et/ou possédant les propriétés sus-mentionnées et les avantages d'une membrane plasmique fixée.The second method also only makes it possible to obtain spherical liposomes with a solid core. surrounded by a lipid bilayer which is not linked to the solid core. This method therefore does not make it possible to obtain any structure of any shape and / or having the above-mentioned properties and the advantages of a fixed plasma membrane.
Le troisième procédé nécessite l'utilisation d'une bicouche de phospholipides ioniques spécifiques peu courants qui altèrent les propriétés fondamentales (étanchéité et perméabilité sélectives et fluidité latérale) de la bicouche.The third method requires the use of a bilayer of unusual ionic specific phospholipids which alter the fundamental properties (selective tightness and permeability and lateral fluidity) of the bilayer.
En outre, la combinaison du premier et du troisième procédé décrite à la figure 43 de cette publication, cumule les inconvénients de chacun d'eux. Dans tous les cas, ces procédés imposent l'utilisation de monomères et de polymères spécifiques et ne permettent donc pas l'obtention d'une structure embranaire artificielle comprenant un substrat solide de nature et de formes diverses, pouvant être adaptées selon les applications.In addition, the combination of the first and third methods described in Figure 43 of this publication, combines the disadvantages of each of them. In all cases, these methods require the use of specific monomers and polymers and therefore do not make it possible to obtain an artificial branch structure comprising a solid substrate of various kinds and shapes, which can be adapted according to the applications.
Dans tout le texte, on adopte la terminologie et les définitions suivantes : membrane fonctionnelle : toute phase lamellaire lyotrope, notamment une bicouche de composés amphiphiles ,Throughout the text, the following terminology and definitions are adopted: functional membrane: any lyotropic lamellar phase, in particular a bilayer of amphiphilic compounds,
- membrane fixée sur un substrat : membrane liée par des liaisons ioniques et/ou covalentes et/ou lyotropiques sur un substrat,- membrane fixed on a substrate: membrane linked by ionic and / or covalent and / or lyotropic bonds on a substrate,
- liaisons, complexations , interactions lyotropiques : toutes interactions de faible énergie entre composés amphiphiles, s 'organisant en une structure ordonnée en présence d'eau, notamment hydrophobes, ioniques, hydrogène, van der aals, ou combinant de telles interactions ,- bonds, complexations, lyotropic interactions: all low energy interactions between amphiphilic compounds, organizing themselves into an ordered structure in the presence of water, in particular hydrophobic, ionic, hydrogen, van der aals, or combining such interactions,
- liaison, complexation ou interaction polyélectrolytique : liaison mettant en eu en milieu polaire une pluralité de dipôles électrostatiques formés par des groupes ionisés,- polyelectrolytic bonding, complexing or interaction: bonding putting in a polar medium a plurality of electrostatic dipoles formed by ionized groups,
- structure artificielle analogue à une membrane plasmique naturelle fixée : structure obtenue par synthèse présentant les propriétés fonctionnelles fondamentales d'une membrane naturelle fixée sur un cytosquelette : étanchéité et perméabilité sélectives, structure de mosaïque fluide (fluidité latérale), polymorphisme, résistance, stabilité, caractère autoscellable .- artificial structure analogous to a fixed natural plasma membrane: structure obtained by synthesis presenting the fundamental functional properties of a natural membrane fixed on a cytoskeleton: selective sealing and permeability, structure of fluid mosaic (lateral fluidity), polymorphism, resistance, stability, autoscellable character.
Dans ce contexte, l'invention vise à proposer une structure membranaire artificielle analogue aux membranes plasmiques naturelles fixées présentant une membrane fonctionnelle stable (c'est-à-dire possédant des propriétés de fluidité latérale bidimensionnelle, et d'étanchéité et de perméabilité sélectives) fixée sur un substrat en phase solide, notamment un substrat poreux formé d'un réseau de polymères, dont la nature et les formes peuvent être diverses.In this context, the invention aims to propose an artificial membrane structure analogous to fixed natural plasma membranes having a stable functional membrane (that is to say having two-dimensional lateral fluidity properties, and selective sealing and permeability) fixed to a solid phase substrate, in particular a porous substrate formed from a network of polymers, the nature and forms of which can be diverse.
L'invention vise également à proposer un procédé de préparation d'une telle structure membranaire simple et compatible avec des contraintes industrielles de mise en oeuvre ; une particule ou un film synthétique supramoléculaire comprenant une telle structure membranaire et leurs applications ; un polymère pouvant servir de composé bifonctionnel de fixation dans une telle structure membranaire ; un procédé de préparation de ce polymère ; un médicament ; et les applications thérapeutiques de cette structure membranaire, et de ces particules.The invention also aims to propose a process for the preparation of such a simple membrane structure and compatible with industrial constraints of implementation; a particle or a supramolecular synthetic film comprising such a membrane structure and their applications; a polymer which can serve as a bifunctional fixing compound in such a membrane structure; a process for the preparation of this polymer; a drug ; and the therapeutic applications of this membrane structure, and of these particles.
Pour ce faire, l'invention concerne une structure membranaire artificielle analogue aux membranes plasmiques naturelles fixées, caractérisée en ce qu'elle comprend : - un substrat en phase solide présentant une surface dotée d'une densité superficielle de charges électriques ,To do this, the invention relates to an artificial membrane structure analogous to fixed natural plasma membranes, characterized in that it comprises: - a solid phase substrate having a surface with a surface density of electrical charges,
- une membrane fonctionnelle stable de composés amphiphiles et qui présente une surface libre s 'étendant à l'opposé du substrat, ladite surface étant adaptée pour pouvoir être placée au contact d'un milieu, dit milieu externe, avec une forme telle qu'elle ne circonscrit pas ce milieu externe, - au moins un composé bifonctionnel de fixation de la membrane fonctionnelle sur le substrat, inséré entre la membrane fonctionnelle et le substrat, et dont la structure chimique comprend : . au moins une chaîne polyionique adaptée pour coopérer par complexation polyélectrolytique avec la densité superficielle de charges électriques du substrat , au moins un ligand membranaire lié par liaison covalente à une telle chaîne polyionique, et adapté pour former une liaison lyotropique stable non covalente avec les composés amphiphiles de la membrane fonctionnelle, sans affecter significativement les propriétés fonctionnelles de la membrane fonctionnelle. Une telle structure membranaire artificielle présente ainsi tout à la fois les propriétés de la membrane fonctionnelle (étanchéité et perméabilité sélectives et fluidité latérale) et les propriétés de polymorphisme et les propriétés mécaniques qui dépendent de la nature du substrat choisi.- A stable functional membrane of amphiphilic compounds and which has a free surface extending opposite the substrate, said surface being adapted to be able to be placed in contact with a medium, said external medium, with a shape such that it do not circumscribe this external environment, at least one bifunctional compound for fixing the functional membrane to the substrate, inserted between the functional membrane and the substrate, and the chemical structure of which comprises: at least one polyionic chain adapted to cooperate by polyelectrolytic complexation with the surface density of electrical charges of the substrate, at least one membrane ligand linked by covalent bond to such a polyionic chain, and adapted to form a stable lyotropic noncovalent bond with the amphiphilic compounds of the functional membrane, without significantly affecting the functional properties of the functional membrane. Such an artificial membrane structure thus presents both the properties of the functional membrane (selective sealing and permeability and lateral fluidity) and the polymorphism and mechanical properties which depend on the nature of the substrate chosen.
Il est à noter que selon l'invention la surface libre de la membrane fonctionnelle s 'étendant au contact du milieu externe permet de gérer les échanges avec ce milieu externe. Cette surface libre ne circonscrit pas le milieu externe, c'est-à-dire n'est pas refermée autour de ce milieu avec lequel elle est en contact, et ce contrairement au cas d'un liposome cationique entouré d'une structure poly érique. En particulier, la surface libre de la membrane d'une structure membranaire selon l'invention est distincte d'une sphère concave, et est destinée à venir au contact d'un milieu externe qui est distinct d'une cavité sphérique interne close. Autrement dit, la surface libre de la membrane de la structure membranaire selon l'invention est telle qu'elle peut présenter une surface d'enveloppe théorique qui s'étend intégralement à l'opposé du substrat et qui est plane ou convexe à convexité orientée dans le direction opposée au substrat. En général, la surface libre de la membrane sera elle-même plane ou convexe à convexité orientée à l'opposé" du substrat. Néanmoins, dans certains cas particuliers, il est possible que cette surface libre puisse présenter des parties concaves à concavité orientée vers l'extérieur. La membrane fonctionnelle de la structure membranaire selon l'invention réalise ainsi une barrière de protection à étanchéité et à perméabilité sélectives prédéterminées qui sépare et protège les composés bifonctionnels de fixation et le substrat ainsi que leurs interactions polyélectrolytiques , du milieu externe. En outre, la membrane fonctionnelle, et plus généralement la structure membranaire, est parfaitement stable et peut être manipulée sans précaution particulière, y compris lorsque le substrat est poreux. Avantageusement et selon l'invention, la membrane fonctionnelle d'une structure membranaire est artificielle, c'est-à-dire n'est pas issue d'un organisme vivant. Les composés amphiphiles de la membrane fonctionnelle peuvent être formés de tous composés amphiphiles, de préférence globalement neutres (ne portant pas de charge électrique nette), aptes à former une membrane fonctionnelle, notamment une bicouche, présentant l'état de mosaïque fluide. La membrane est principalement constituée des composés amphiphiles organisés en bicouche, mais peut incorporer néanmoins tout autre composé, en proportions plus ou moins importantes, présentant des propriétés spécifiques, et qui est compatible avec la bicouche, c'est-à-dire en préserve les propriétés fonctionnelles et la stabilité. Avantageusement et selon l'invention, les composés amphiphiles de la membrane sont des composés phospholipidiques d'origine naturelle ou synthétique - notamment de la famille des phosphatidylcholines- à chaînes grasses comprenant entre 12 et 22 atomes de carbone saturées ou insaturées, notamment entre 16 et 18 atomes de carbone .It should be noted that according to the invention the free surface of the functional membrane extending in contact with the external medium makes it possible to manage the exchanges with this external medium. This free surface does not circumscribe the external medium, that is to say is not closed around this medium with which it is in contact, and this in contrast to the case of a cationic liposome surrounded by a poly eric structure . In particular, the free surface of the membrane of a membrane structure according to the invention is distinct from a concave sphere, and is intended to come into contact with an external medium which is distinct from a closed internal spherical cavity. In other words, the free surface of the membrane of the membrane structure according to the invention is such that it can have a theoretical envelope surface which extends entirely opposite the substrate and which is planar or convex with oriented convexity in the opposite direction to the substrate. In general, the free surface of the membrane will itself be flat or convex with convexity oriented opposite "to the substrate. However, in certain particular cases, it is possible that this free surface may have concave parts with concavity oriented towards the outside. The functional membrane of the membrane structure according to the invention thus achieves a protective barrier with predetermined selective tightness and permeability which separates and protects the bifunctional fixing compounds and the substrate as well as their polyelectrolytic interactions, from the external medium. In addition, the functional membrane, and more generally the membrane structure, is perfectly stable and can be handled without special precautions, including when the substrate is porous. Advantageously and according to the invention, the functional membrane of a membrane structure is artificial, that is to say it does not come from a living organism. The amphiphilic compounds of the functional membrane can be formed of all amphiphilic compounds, preferably generally neutral (not carrying a net electrical charge), capable of forming a functional membrane, in particular a bilayer, having the state of fluid mosaic. The membrane is mainly made up of amphiphilic compounds organized in a bilayer, but can nevertheless incorporate any other compound, in more or less significant proportions, having specific properties, and which is compatible with the bilayer, that is to say preserves them. functional properties and stability. Advantageously and according to the invention, the amphiphilic compounds of the membrane are phospholipid compounds of natural or synthetic origin - in particular of the family of phosphatidylcholines - with fatty chains comprising between 12 and 22 saturated or unsaturated carbon atoms, in particular between 16 and 18 carbon atoms.
Les composés amphiphiles de la membrane peuvent comprendre, outre des phospholipides , une proportion plus ou moins importante " des composés appartenant aux familles suivantes, seuls ou en mélange : la sphingomyéline ; les gangliosides ; les glycolipides ; les céramides ; le di O-alkyl ; les stérols (cholestérol, ergostérol ... ) .Amphiphilic membrane compounds may include, in addition to phospholipids, a greater or lesser proportion " of the compounds belonging to the following families, alone or as a mixture: sphingomyelin; gangliosides; glycolipids; ceramides; di O-alkyl; sterols (cholesterol, ergosterol ...).
La structure membranaire selon l'invention peut comprendre un seul composé bifonctionnel de fixation, ou en variante, un mélange de plusieurs composés bifonctionnels de fixation de natures distinctes. Avantageusement et selon l'invention, la structure membranaire comprend au moins un composé bifonctionnel de fixation dont la structure chimique comprend au moins une pluralité de ligands membranaires, notamment une pluralité de ligands membranaires semblables ou identiques. Selon l'invention, ces ligands membranaires sont répartis sur la molécule du composé bifonctionnel de fixation à une distance les uns des autres qui est supérieure à celle séparant les composés amphiphiles qui se jouxtent dans une couche de la membrane fonctionnelle, de sorte que cette membrane présente des composés amphiphiles qui ne sont pas liés à un ligand membranaire.The membrane structure according to the invention may comprise a single bifunctional fixing compound, or as a variant, a mixture of several bifunctional fixing compounds of different natures. Advantageously and according to the invention, the membrane structure comprises at least one bifunctional fixing compound, the chemical structure of which comprises at least a plurality of membrane ligands, in particular a plurality of similar or identical membrane ligands. According to the invention, these membrane ligands are distributed over the molecule of the bifunctional fixing compound at a distance from each other which is greater than that separating the amphiphilic compounds which adjoin in a layer of the functional membrane, so that this membrane presents amphiphilic compounds which are not linked to a membrane ligand.
En outre, avantageusement et selon l'invention, le composé bifonctionnel de fixation présente un nombre de charges ioniques unitaires de même signe aptes à coopérer par complexation polyélectrolytique avec la densité superficielle de charges électriques du substrat, qui est supérieur au nombre de ligands membranaires.In addition, advantageously and according to the invention, the bifunctional fixing compound has a number of unit ion charges of the same sign capable of cooperating by polyelectrolytic complexation with the surface density of electrical charges of the substrate, which is greater than the number of membrane ligands.
Avantageusement et selon l'invention, les charges ioniques sont réparties sur la molécule du composé bifonctionnel de fixation à une distance les unes des autres qui est inférieure à la plus petite distance séparant deux ligands membranaires.Advantageously and according to the invention, the ionic charges are distributed over the molecule of the bifunctional fixing compound at a distance from each other which is less than the smallest distance separating two membrane ligands.
Avantageusement et selon l'invention, les ligands membranaires sont choisis parmi les phospholipides, les acides gras, les isoprénoïdes , les peptides, les aminés grasses, les éthers, les stérols, les terpènes, les glycolipides, les shingolipides, les gangliosides, les céramides . En particulier, les ligands membranaires peuvent être composés d'une branche lipidique -notamment phospholipidique- similaire aux composés amphiphiles de la membrane fonctionnelle, et d'une branche de liaison reliant par liaisons covalentes cette branche lipidique à la chaîne polyionique du composé bifonctionnel de fixation. De la sorte, la branche lipidique du ligand membranaire s'insère dans la couche en regard de la bicouche formant la membrane fonctionnelle, et est donc liée par des interactions lyotropiques au sein de cette membrane fonctionnelle.Advantageously and according to the invention, the membrane ligands are chosen from phospholipids, fatty acids, isoprenoids, peptides, fatty amines, ethers, sterols, terpenes, glycolipids, shingolipids, gangliosides, ceramides . In particular, the membrane ligands can be composed of a lipid branch - in particular phospholipid - similar to the amphiphilic compounds of the functional membrane, and of a connecting branch connecting by covalent bonds this lipid branch to the polyionic chain of the bifunctional fixing compound. . In this way, the lipid branch of the membrane ligand is inserted into the layer opposite the bilayer forming the functional membrane, and is therefore linked by lyotropic interactions within this functional membrane.
Avantageusement et selon l'invention, les composés bifonctionnels de fixation sont formés d'oligomères ou de polymères polyioniques . A titre de composés bifonctionnels de fixation polycationiques utilisables dans l'invention, on peut citer : les protéines et les peptides polycationiques ; les oligo et polysacchaπdes polycationiques ; les polyammes ; les polymères synthétiques polycationiques. A titre de composés bifonctionnels de fixation polyanioniques utilisables dans l'invention, on peut citer : les protéines et peptides polyanioniques ; les oligo et polysaccharides polyanioniques ; les polyacides ; les polymères synthétiques polyanioniques.Advantageously and according to the invention, the bifunctional fixing compounds are formed from oligomers or from polyionic polymers. As bifunctional polycationic fixing compounds which can be used in the invention, mention may be made of: polycationic proteins and peptides; polycationic oligo and polysacchaπdes; polyammes; polycationic synthetic polymers. As bifunctional polyanionic fixing compounds which can be used in the invention, mention may be made of: polyanionic proteins and peptides; polyanionic oligo and polysaccharides; polyacids; polyanionic synthetic polymers.
En outre, avantageusement et selon l'invention, les charges électriques du substrat sont négatives, et les composés bifonctionnels de fixation présentent une structure polycationique. Néanmoins, l'inverse est possible.In addition, advantageously and according to the invention, the electrical charges of the substrate are negative, and the bifunctional fixing compounds have a polycationic structure. However, the reverse is possible.
Avantageusement et selon l'invention, la structure comprend au moins un composé bifonctionnel de fixation dont la structure chimique comprend au moins un groupe choisi parmi un peptide, un polypeptide, une protéine ou un oside. En particulier, avantageusement et selon l'invention, tous les composés bifonctionnels de fixation de la structure membranaire selon 1 ' invention présentent cette structure chimique, de sorte que la structure membranaire est biocompatible .Advantageously and according to the invention, the structure comprises at least one bifunctional fixing compound whose chemical structure comprises at least one group chosen from a peptide, a polypeptide, a protein or an oside. In particular, advantageously and according to the invention, all the bifunctional compounds for fixing the membrane structure according to the invention have this chemical structure, so that the membrane structure is biocompatible.
Par ailleurs, avantageusement la structure membranaire selon l'invention est caractérisée en ce qu'elle comprend au moins une polyamine à titre de composé bifonctionnel de fixation. En effet, une telle polyamine est un composé relativement courant et facile à manipuler, y compris à l'échelle industrielle.Furthermore, advantageously the structure membrane according to the invention is characterized in that it comprises at least one polyamine as a bifunctional fixing compound. Indeed, such a polyamine is a relatively common compound and easy to handle, including on an industrial scale.
Avantageusement et selon l'invention, on utilise une polyamine dont la structure chimique comprend au moins un groupe choisi parmi un peptide, un polypeptide, une protéine ou un oside. De la sorte, le composé bifonctionnel de fixation présente l'avantage d'une bonne biocompatibilité et est facilement manipulable à l'échelle industrielle et de faible coût. En particulier, une structure membranaire selon 1 ' invention comprend avantageusement une polylysine-succinophospholipidique, c'est-à-dire dont une partie des groupes aminés porte un ligand succinophospholipidique -notamment N-succinyl- phosphatidyléthanolamine- à titre de composé bifonctionnel de fixation.Advantageously and according to the invention, a polyamine is used, the chemical structure of which comprises at least one group chosen from a peptide, a polypeptide, a protein or an oside. In this way, the bifunctional fixing compound has the advantage of good biocompatibility and is easy to handle on an industrial scale and of low cost. In particular, a membrane structure according to the invention advantageously comprises a polylysine-succinophospholipid, that is to say a part of the amino groups of which carries a succinophospholipid ligand - in particular N-succinyl - phosphatidylethanolamine - as bifunctional fixing compound.
Avantageusement et selon l'invention, la polyamine -notamment la polylysine succinophospholipidique- présente un taux de greffage des fonctions aminés par les ligands membranaires compris entre 1 % et 20 %. Avantageusement et selon l'invention, la polylysine succinophospholipidique présente un poids moléculaire de polylysine de départ compris entre 10000 et 50000.Advantageously and according to the invention, the polyamine - in particular the polylysine succinophospholipidic - exhibits a rate of grafting of the amino functions by the membrane ligands of between 1% and 20%. Advantageously and according to the invention, the succinophospholipid polylysine has a starting polylysine molecular weight of between 10,000 and 50,000.
Le substrat solide d'une structure membranaire selon l'invention peut être choisi parmi les solides suivants :The solid substrate of a membrane structure according to the invention can be chosen from the following solids:
- des substrats polyanioniques : . les polymères réticulés : les acides nucléiques, ADN, ARN ; les protéines polyanioniques : polyaspartate, polyglutamate, protéines sialées ; les polysaccharides polyanioniques : acide hyaluronique , acide alginique, xanthane, héparine, et dérivés acides (phosphate, sulfonate, sulfate carboxyméthyl, succinate ... ) de polysaccharides neutres tels que la cellulose, l'amidon, le dextrane ; les polymères synthétiques (nylon, silicone...) dérivés par des fonctions anioniques . Tous ces polymères ne peuvent être utilisés que sous forme solide et donc réticulée. La reticulation peut être de nature covalente ou ionique. Cette reticulation doit être réalisée avant 1 ' établissement de la membrane fonctionnelle. La reticulation peut en particulier être provoquée par une complexation polyélectrolytique entre le polymère polyanionique et un polymère polycationique, ce dernier pouvant être éventuellement une chaîne polycationique des composés bifonctionnels eux- mêmes . portions de peau, cuir, muqueuses, phanères (poil...), membrane cellulaire, fibres naturelles (coton, laine, papier...),- polyanionic substrates:. crosslinked polymers: nucleic acids, DNA, RNA; polyanionic proteins: polyaspartate, polyglutamate, sial proteins; polyanionic polysaccharides: hyaluronic acid, alginic acid, xanthan, heparin, and acid derivatives (phosphate, sulfonate, carboxymethyl sulfate, succinate, etc.) of neutral polysaccharides such as cellulose, starch, dextran; synthetic polymers (nylon, silicone, etc.) derived by anionic functions. All these polymers can only be used in solid form and therefore crosslinked. The crosslinking can be of covalent or ionic nature. This crosslinking must be carried out before the establishment of the functional membrane. Crosslinking can in particular be caused by a polyelectrolytic complexation between the polyanionic polymer and a polycationic polymer, the latter possibly being a polycationic chain of the bifunctional compounds themselves. portions of skin, leather, mucous membranes, integuments (hair ...), cell membrane, natural fibers (cotton, wool, paper ...),
. le verre, la silice, . les tectosilicates anioniques, les particules et membranes anioniques échangeuses de cations .. glass, silica,. anionic tectosilicates, anionic cation exchange particles and membranes.
- des substrats polycationiques : les protéines polycationiques : polylysine, poly-arginine , protamine, histone, les polysaccharides polycationiques : chitosan, DEAE dextrane, polymères synthétiques dérivés par des fonctions basiques (DEAE Nylon), l'alumine, les tectosilicates cationiques, les particules et membranes cationiques échangeuses d'anions.- polycationic substrates: polycationic proteins: polylysine, poly-arginine, protamine, histone, polycationic polysaccharides: chitosan, DEAE dextran, synthetic polymers derived by basic functions (DEAE Nylon), alumina, cationic tectosilicates, particles and cationic anion exchange membranes.
En outre, la membrane fonctionnelle d'une structure membranaire selon l'invention peut présenter au moins un composé d'interaction avec ledit milieu externe. Ce composé d'interaction est lié à la membrane fonctionnelle par toute liaison appropriée de façon à s ' étendre au sein du milieu externe à partir de la surface libre de la membrane fonctionnelle. Avantageusement, la structure membranaire comporte un composé d'interaction choisi parmi un peptide, une protéine, un glucide, une glycoprotéine. Ces composés d'interation avec le milieu extérieur peuvent être des anticorps mono ou polyclonaux, des ligands de reconnaissance ( transferriήe, facteur de croissance, hormone, sucre, marqueurs immunologiques ) , des récepteurs, des protéines de transport, des enzymes ou encore des protéines de fusion. Un composé d'interaction peut être lié à au moins un ligand membranaire d'un composé bifonctionnel de fixation par une liaison covalente, ou au contraire peut être lié par interactions lyotropiques stables non covalentes avec des composés amphiphiles au sein de la membrane fonctionnelle. La membrane fonctionnelle d'une structure membranaire selon l'invention formée d'une bicouche de composés amphiphiles s'étend sur une épaisseur inférieure à 5 nm, notamment de l'ordre de 4 à 5 nm. En outre, avantageusement et selon l'invention, le substrat présente des pores de dimensions moyennes supérieures à 5 nm et inférieures à 0,5 μm . De la sorte, on évite notamment une invasion des pores du substrat par la bicouche de composés amphiphiles .In addition, the functional membrane of a membrane structure according to the invention can have at least one compound for interaction with said external medium. This interaction compound is linked to the functional membrane by any suitable bond so as to extend within the external medium from the free surface of the functional membrane. Advantageously, the membrane structure comprises an interaction compound chosen from a peptide, a protein, a carbohydrate, a glycoprotein. These compounds of interaction with the external environment can be mono or polyclonal antibodies, recognition ligands (transferriήe, growth factor, hormone, sugar, immunological markers), receptors, transport proteins, enzymes or even fusion proteins. An interaction compound can be linked to at least one membrane ligand of a bifunctional fixing compound by a covalent bond, or on the contrary can be linked by stable non-covalent lyotropic interactions with amphiphilic compounds within the functional membrane. The functional membrane of a membrane structure according to the invention formed of a bilayer of amphiphilic compounds extends over a thickness of less than 5 nm, in particular of the order of 4 to 5 nm. In addition, advantageously and according to the invention, the substrate has pores of average dimensions greater than 5 nm and less than 0.5 μm. In this way, in particular, an invasion of the pores of the substrate is avoided by the bilayer of amphiphilic compounds.
Une telle structure membranaire selon l'invention peut être utilisée pour l'obtention d'un médicament. En effet, la structure membranaire selon 1 ' invention étant analogue aux membranes plasmiques naturelles de type fixées, en présente les propriétés, et peut donc être utilisée comme membrane plasmique artificielle dans des médicaments ou des compositions thérapeutiques, en particulier pour la thérapie génique.Such a membrane structure according to the invention can be used for obtaining a medicament. Indeed, the membrane structure according to the invention being analogous to natural plasma membranes of the fixed type, has the properties thereof, and can therefore be used as an artificial plasma membrane in medicinal products or therapeutic compositions, in particular for gene therapy.
En outre, une structure membranaire selon 1 ' invention peut servir pour préparer des particules synthétiques supramoléculaires . Ainsi, l'invention s'étend à une particule synthétique supramoléculaire , caractérisée en ce qu'elle comprend une structure membranaire selon l'invention formant sa périphérie externe et délimitant un volume interne, la membrane fonctionnelle de la structure membranaire présentant une surface libre qui s'étend à l'extérieur de la particule, et qui est destinée à être placée au contact d'un milieu externe. Dans une particule selon l'invention, le substrat occupe au moins substantiellement tout le volume interne de la particule, ou en variante, une partie seulement du volume interne de la particule. En outre, le substrat peut avantageusement être formé d'une matrice polymerique poreuse synthétique - notamment d'ADN ou d 'ARN réticulé-. La particule selon l'invention peut renfermer une composition liquide, - notamment une composition thérapeutique- dans son volume interne. Avantageusement et selon l'invention, le volume interne est entièrement occupé par un substrat formé d'une matrice polymerique poreuse synthétique qui incorpore au sein de ses pores une composition liquide.In addition, a membrane structure according to the invention can be used to prepare synthetic supramolecular particles. Thus, the invention extends to a synthetic supramolecular particle, characterized in that it comprises a membrane structure according to the invention forming its external periphery and delimiting an internal volume, the functional membrane of the membrane structure having a free surface which extends outside the particle, and which is intended to be placed in contact with an external medium. In a particle according to the invention, the substrate occupies at least substantially the entire internal volume of the particle, or alternatively, only part of the internal volume of the particle. In addition, the substrate can advantageously be formed from a synthetic porous polymer matrix - in particular DNA or crosslinked RNA. The particle according to the invention may contain a liquid composition, - in particular a therapeutic composition - in its internal volume. Advantageously and according to the invention, the internal volume is entirely occupied by a substrate formed from a porous synthetic polymer matrix which incorporates within its pores a liquid composition.
En outre, dans une particule selon l'invention, la membrane fonctionnelle peut être adaptée pour présenter une cinétique de libération de la composition liquide selon un profil prédéterminé. Il suffit en effet de choisir la constitution de la membrane fonctionnelle pour obtenir l' étanchéité et la perméabilité sélectives recherchées en vue de l'obtention de cette cinétique, et ce de façon connue en soi (par exemple dans le cas des liposomes). Une particule selon l'invention peut présenter une dimension moyenne comprise entre 10 nm et 5 mm.In addition, in a particle according to the invention, the functional membrane can be adapted to present the kinetics of release of the liquid composition according to a predetermined profile. It suffices in fact to choose the constitution of the functional membrane to obtain the selective sealing and permeability sought with a view to obtaining this kinetics, and this in a manner known per se (for example in the case of liposomes). A particle according to the invention can have an average dimension of between 10 nm and 5 mm.
Une particule selon l'invention peut faire l'objet de diverses applications, notamment à titre de médicament. Ainsi, l'invention s'étend également à un médicament caractérisé en ce qu'il comprend au moins une particule selon l'invention.A particle according to the invention can be the subject of various applications, in particular as a medicament. Thus, the invention also extends to a medicament, characterized in that it comprises at least one particle according to the invention.
L'invention s'étend également à un film synthétique supramoléculaire caractérisé en ce qu'il comprend une structure membranaire selon l'invention. Le film selon l'invention peut en outre incorporer un ionophore permettant de transporter sélectivement des ions à travers la bicouche. Ainsi, un film selon l'invention peut être formé d'une portion d'une structure membranaire selon l'invention non refermée sur elle-même, c'est-à-dire en forme générale de nappe. Un film selon l'invention est avantageusement au moins sensiblement plan, mais peut présenter une certaine flexibilité. Un film selon l'invention peut faire l'objet de diverses applications notamment à titre de séparateur ou d'extracteur de composés. L'invention s'étend donc aussi à l'application d'un film selon l'invention pour extraire ou séparer des sels et/ou des ions à partir d'une solution liquide par filtration.The invention also extends to a supramolecular synthetic film characterized in that it comprises a membrane structure according to the invention. The film according to the invention can also incorporate an ionophore making it possible to selectively transport ions through the bilayer. Thus, a film according to the invention can be formed of a portion of a membrane structure according to the invention which is not closed in on itself, that is to say in the general form of a sheet. A film according to the invention is advantageously at least substantially planar, but can have a certain flexibility. A film according to the invention can be the subject of various applications in particular as a separator or extractor of compounds. The invention therefore also extends to the application of a film according to the invention for extracting or separating salts and / or ions from a liquid solution by filtration.
L'invention s'étend également à un polymère polycationique de pureté supérieure à 95 % formé d'une polyamine polycationique dotée d'une pluralité de ligands lipidiques -notamment phospholipidiques- greffés sur une partie des atomes d'azote des fonctions aminés, et aptes à former une liaison lyotropique stable non covalente avec une membrane fonctionnelle stable de composés amphiphiles, ce polymère pouvant faire office de composé bifonctionnel de fixation de la membrane fonctionnelle sur le substrat d'une structure membranaire selon l'invention.The invention also extends to a polycationic polymer of purity greater than 95% formed of a polycationic polyamine endowed with a plurality of lipid ligands - in particular phospholipidic - grafted on part of the nitrogen atoms of the amino functions, and capable forming a stable, non-covalent lyotropic bond with a stable functional membrane of amphiphilic compounds, this polymer possibly acting as a bifunctional compound for fixing the functional membrane to the substrate of a membrane structure according to the invention.
Avantageusement et selon l'invention, cette polyamine présente un taux de greffage des fonctions aminés par les ligands lipidiques membranaires qui est compris entre 1 % et 20 %. En particulier et selon l'invention, le polymère est formé de polylysine, notamment une L-polylysine succinophospholipidique telle que la polylysine N-succinyl-phosphatidyléthanolamine . Avantageusement et selon l'invention, la polylysine succinophospholipidique présente un poids moléculaire de polylysine de départ compris entre 10 000 et 5 000.Advantageously and according to the invention, this polyamine has a rate of grafting of the amino functions by the lipid membrane ligands which is between 1% and 20%. In particular and according to the invention, the polymer is formed from polylysine, in particular a succinophospholipid L-polylysine such as the polylysine N-succinyl-phosphatidylethanolamine. Advantageously and according to the invention, the succinophospholipid polylysine has a molecular weight of starting polylysine of between 10,000 and 5,000.
L'invention s'étend également au procédé de préparation d'un polymère polycationique -notamment d'un polymère selon l'invention- doté d'une pluralité de ligands lipidiques aptes à former une liaison lyotropique stable non covalente avec une membrane fonctionnelle stable de composés amphiphiles, de sorte que ce polymère peut faire office de composé bifonctionnel de fixation de la membrane fonctionnelle sur le substrat d'une structure membranaire selon l'invention, caractérisé en ce qu'après avoir effectué les opérations de synthèse chimique permettant l'obtention de la molécule du polymère, on le met en contact d'un citrate dans un solvant polaire de façon à obtenir une précipitation du polymère.The invention also extends to the process for the preparation of a polycationic polymer - in particular a polymer according to the invention - provided with a plurality of lipid ligands capable of forming a stable non-covalent lyotropic bond with a stable functional membrane of amphiphilic compounds, so that this polymer can act as a bifunctional compound for fixing the functional membrane to the substrate of a membrane structure according to the invention, characterized in that after having carried out the chemical synthesis operations allowing the obtaining of the polymer molecule, it is brought into contact with a citrate in a polar solvent so as to obtain precipitation of the polymer.
On a en effet constaté avec surprise qu'un tel polymère peut être purifié de façon extrêmement simple par simple adjonction de citrate dans un solvant polaire en présence de ce polymère, ce qui entraîne sa précipitation.It has indeed been surprisingly found that such a polymer can be purified in an extremely simple manner by simple addition of citrate in a polar solvent in the presence of this polymer, which causes its precipitation.
La purification des composés bifonctionnels de fixation tels que les polylysines-NSPE est délicate car ces composés présentent des parties polaires ioniques et des parties hydrophobes. En conséquence, ils ne sont solubles ni dans 1 ' eau ni dans les solvants organiques apolaires. Ils sont en revanche solubles dans les solvants organiques polaires comme le DMSO. Ces molécules interagissent de plus très fortement avec tous les supports chromatographiques usuels et ce beaucoup plus fortement, par exemple, que les polylysines de départ. Les techniques d'extraction et de purification usuelles par extraction liquide ou par chro atographie sont donc pratiquement inutilisables .The purification of bifunctional fixing compounds such as polylysines-NSPE is delicate because these compounds have polar ionic parts and hydrophobic parts. Consequently, they are neither soluble in water nor in non-polar organic solvents. On the other hand, they are soluble in polar organic solvents such as DMSO. These molecules also interact very strongly with all the usual chromatographic supports and this much more strongly, for example, than the starting polylysines. The usual extraction and purification techniques by liquid extraction or by chromatography are therefore practically unusable.
Au contraire, la précipitation par le citrate présente l'avantage d'être rapide et quantitative. Le précipité obtenu est stable et peut être lavé facilement avec plusieurs types de solvant pour éliminer les contaminants. De plus, la précipitation est sélective des polycations et n'entraîne pas de produits annexes. II est à noter en outre que la précipitation par le citrate est facilement réversible et ce quantitativement. La réversion se fait en jouant soit sur le pH soit sur la force ionique et conduit à une molécule non altérée parfaitement fonctionnelle. Le citrate libéré s'élimine facilement par dialyse et ne vient pas perturber l'utilisation ultérieure du composé.On the contrary, precipitation with citrate has the advantage of being rapid and quantitative. The precipitate obtained is stable and can be easily washed with several types of solvent to remove contaminants. In addition, precipitation is selective for polycations and does not lead to ancillary products. It should also be noted that precipitation by citrate is easily reversible, quantitatively. Reversion takes place either on the pH or on the ionic strength and leads to a perfectly functional unaltered molecule. The released citrate is easily removed by dialysis and does not disturb the subsequent use of the compound.
Enfin, le citrate est un produit naturel, bon marché, complètement non toxique et d'une simplicité d'emploi remarquable. Le procédé de purification au citrate permet donc d'envisager de réaliser sans problème l'extraction et la purification des composés bifonctionnels de fixation tels que les polylysines-NSPE à une échelle industrielle ce qui n'était pas envisageable avec les procédés traditionnels.Finally, citrate is a natural product, inexpensive, completely non-toxic and remarkably easy to use. The citrate purification process therefore makes it possible to envisage carrying out without problem the extraction and purification of bifunctional fixing compounds such as polylysines-NSPE on a scale. industrial which was not possible with traditional processes.
L'invention s'étend aussi au procédé de préparation d'une structure membranaire selon l'invention, qui est caractérisé en ce qu'on prépare tout d'abord une suspension aqueuse des composés bifonctionnels de fixation de la façon suivante :The invention also extends to the process for preparing a membrane structure according to the invention, which is characterized in that an aqueous suspension of the bifunctional fixing compounds is first prepared in the following manner:
- on prépare une solution des composés bifonctionnels de fixation dans le DMSO, - on prépare une solution aqueuse comprenant du moins un détergent non ionique, à une concentration supérieure à sa concentration micellaire critique,- a solution of the bifunctional fixing compounds in DMSO is prepared, - an aqueous solution is prepared comprising at least one nonionic detergent, at a concentration higher than its critical micellar concentration,
- on ajoute la solution des composés bifonctionnels de fixation dans la solution aqueuse.- Adding the solution of the bifunctional fixing compounds in the aqueous solution.
Il est à noter que les composés bifonctionnels de fixation tels que les polylysines-NSPE sont des composés amphiphiles insolubles dans l'eau et dans les solvants organiques apolaires. Lorsque ces composés sont obtenus à l'état sec après purification au citrate, il est pratiquement impossible de les dissoudre ou même de les suspendre directement dans des solutions aqueuses même en présence d'une concentration élevée de détergent non ionique. De plus, le peu de composé qui semble se disperser ne paraît pas présenter les propriétés attendues c'est-à- dire la capacité à former des complexes polyélectrolytiques .It should be noted that the bifunctional fixing compounds such as the polylysines-NSPE are amphiphilic compounds insoluble in water and in non-polar organic solvents. When these compounds are obtained in the dry state after purification with citrate, it is practically impossible to dissolve them or even to suspend them directly in aqueous solutions even in the presence of a high concentration of nonionic detergent. In addition, the small amount of compound which seems to disperse does not appear to have the expected properties, that is to say the ability to form polyelectrolytic complexes.
Les inventeurs ont constaté avec surprise qu'il est possible d'obtenir une solubilisation des composés bifonctionnels de fixation en milieu aqueux en le dissolvant d'abord dans le DMSO puis en injectant sous agitation cette solution dans une solution aqueuse de détergent. En outre, les composés bifonctionnels de fixation, tels que la polylysine-NSPE, ainsi solubilisés dans le DMSO possèdent bien les propriétés de complexation polyélectrolytique attendues.The inventors have surprisingly found that it is possible to obtain a solubilization of the bifunctional fixing compounds in an aqueous medium by first dissolving it in DMSO and then by injecting this solution, with stirring, into an aqueous detergent solution. In addition, the bifunctional fixing compounds, such as polylysine-NSPE, thus solubilized in DMSO do indeed have the expected polyelectrolytic complexing properties.
Dans une première variante de l'invention, le procédé est en outre caractérisé en ce qu'on ajoute ensuite dans ladite suspension aqueuse une composition de polymères polyioniques aptes à former un substrat solide par reticulation polyélectrolytique avec les chaînes polyioniques des composés bifonctionnels de fixation. Avantageusement, dans cette première variante, le procédé est en outre caractérisé en ce que :In a first variant of the invention, the method is further characterized in that one adds then in said aqueous suspension a composition of polyionic polymers capable of forming a solid substrate by polyelectrolytic crosslinking with the polyionic chains of the bifunctional fixing compounds. Advantageously, in this first variant, the method is further characterized in that:
- on introduit des composés amphiphiles aptes à former une membrane fonctionnelle soit dans la solution aqueuse du détergent, soit dans la suspension aqueuse avant ou après adjonction de la composition de polymères polyioniques, et de façon à ce que la concentration du détergent reste supérieure à sa concentration micellaire critique,- Introducing amphiphilic compounds capable of forming a functional membrane either in the aqueous solution of the detergent, or in the aqueous suspension before or after addition of the composition of polyionic polymers, and so that the concentration of the detergent remains higher than its critical micellar concentration,
- on élimine ensuite le détergent de la suspension.- then detergent is removed from the suspension.
Dans cette première variante, le substrat solide est donc formé d'un polymère polyionique réticulé lors de la préparation de la structure membranaire, par les composés bifonctionnels de fixation eux-mêmes. Tel est le cas en particulier d'un substrat formé d'acide nucléique (ADN ou ARN) réticulé. Dans cette variante, il est nécessaire de prévoir initialement une concentration de détergent supérieure à la CMC pour assurer au préalable la reticulation du substrat par formation des complexations polyélectrolytiques avec les chaînes polyioniques des composés bifonctionnels, puis, dans un deuxième temps lors de l'élimination du détergent, la formation de la membrane fonctionnelle. Il est à noter que cela permet en particulier de former la membrane à l'extérieur du substrat solide réticulé et non l'inverse.In this first variant, the solid substrate is therefore formed of a polyionic polymer crosslinked during the preparation of the membrane structure, by the bifunctional fixing compounds themselves. This is the case in particular of a substrate formed of crosslinked nucleic acid (DNA or RNA). In this variant, it is necessary to initially provide a concentration of detergent greater than the CMC to ensure beforehand the crosslinking of the substrate by formation of polyelectrolytic complexes with the polyionic chains of the bifunctional compounds, then, in a second step during the elimination detergent, the formation of the functional membrane. It should be noted that this makes it possible in particular to form the membrane outside the crosslinked solid substrate and not the reverse.
Cette première variante du procédé de préparation de la structure membranaire selon l'invention est plus particulièrement applicable lorsque le substrat est de relativement faible dimension, c'est-à-dire de dimension globale moyenne inférieure à 1 μm. Par exemple, cette première variante du procédé de préparation selon l'invention permet de réaliser des particules de dimension moyenne de l'ordre de 50 à 200 nm incorporant un noyau d'ADN à titre de substrat, sur lequel une membrane fonctionnelle est fixée. Ces particules sont donc des virus artificiels .This first variant of the process for preparing the membrane structure according to the invention is more particularly applicable when the substrate is of relatively small dimension, that is to say of average overall dimension of less than 1 μm. For example, this first variant of the preparation process according to the invention makes it possible to produce particles of average size of the order of 50 to 200 nm incorporating a core of DNA as a substrate, on which a functional membrane is attached. These particles are therefore artificial viruses.
L'invention s'étend également à une autre variante du procédé de préparation de la structure membranaire selon l'invention qui est plus particulièrement applicable dans le cas où le substrat est de plus grande dimension, c'est-à-dire de dimension globale moyenne supérieure à 1 μm. Dans cette deuxième variante, le procédé de préparation est caractérisé en ce que :The invention also extends to another variant of the process for preparing the membrane structure according to the invention which is more particularly applicable in the case where the substrate is of larger dimension, that is to say of overall dimension average greater than 1 μm. In this second variant, the preparation process is characterized in that:
- on introduit des composés amphiphiles aptes à former une membrane fonctionnelle soit dans ladite solution aqueuse du détergent, soit dans ladite suspension aqueuse, - puis, on diminue la concentration du détergent dans ladite suspension aqueuse jusqu'à une concentration inférieure à sa concentration micellaire critique,- Amphiphilic compounds capable of forming a functional membrane are introduced either into said aqueous solution of the detergent, or into said aqueous suspension, - then, the concentration of the detergent in said aqueous suspension is reduced to a concentration below its critical micellar concentration ,
- puis on place cette suspension aqueuse au contact d'un substrat en phase solide,- then this aqueous suspension is placed in contact with a solid phase substrate,
- puis on élimine le détergent non ionique. Il est à noter que l'on connaît déjà des liposomes polycationiques utilisés pour complexer des molécules polyanioniques telles que 1 ' ADN . Mais les charges électrostatiques de ces liposomes étant situées à l'extérieur, la complexation électrolytique se propage dans le milieu, ce qui entraîne une agrégation progressive des liposomes et des molécules polyanioniques. Ce procédé produit donc des entités de structure évolutive et aléatoire présentant des propriétés finales non reproductibles et mal maîtrisées.- then the non-ionic detergent is removed. It should be noted that polycationic liposomes are already known which are used to complex polyanionic molecules such as DNA. But the electrostatic charges of these liposomes being located outside, the electrolytic complexation propagates in the medium, which involves a progressive aggregation of liposomes and polyanionic molecules. This process therefore produces entities of evolving and random structure having final properties that are not reproducible and poorly controlled.
On connaît aussi des membranes dites "membranes supportées" qui sont formées d'une bicouche lipidique sur un substrat solide plan tel que le quartz (cf. "Supported planar membrane in studies of cell-cell récognition in the immune System" H. M. Me Connell et al, Biochimica et Biophysica Acta 864 (1986) 95-106). Dans de telles membranes simplement supportées, la bicouche n'est pas fixée sur le substrat, de sorte que le système présente une très grande fragilité, ce qui en réduit considérablement l'intérêt pratique.Also known are membranes called "supported membranes" which are formed of a lipid bilayer on a flat solid substrate such as quartz (cf. "Supported planar membrane in studies of cell-cell recognition in the immune System" HM Me Connell and al, Biochimica and Biophysica Acta 864 (1986) 95-106). In such simply supported membranes, the bilayer is not not fixed on the substrate, so that the system has a very high brittleness, which considerably reduces its practical interest.
Dans une variante de membrane supportée décrite dans le document sus-cité (voir également WO 89/11271), des chaînes hydrophobes sont greffées en surface par des liaisons covalentes sur le support solide, sur lesquelles on dépose une monocouche de phospholipides, puis éventuellement une succession de bicouches. Avec ce système, on ne forme pas une membrane fonctionnelle stable, puisque la monocouche associée aux chaînes hydrophobes liées au support solide ne peut pas présenter les propriétés fondamentales d'une bicouche. En outre, les bicouches lipidiques éventuellement présentes au-dessus de la monocouche ne sont pas fixées et sont donc, là encore, très fragiles et instables.In a variant of the supported membrane described in the abovementioned document (see also WO 89/11271), hydrophobic chains are grafted on the surface by covalent bonds on the solid support, on which a monolayer of phospholipids is deposited, then optionally a succession of bilayers. With this system, a stable functional membrane is not formed, since the monolayer associated with the hydrophobic chains linked to the solid support cannot exhibit the fundamental properties of a bilayer. In addition, the lipid bilayers possibly present above the monolayer are not fixed and are therefore, here again, very fragile and unstable.
Par ailleurs, il est aussi à noter que les documents "Lipophilic polylysine médiate efficient DNA transfection in mammalian Cells", Xiaohuai Zhou et al., Biochimica et Biophysica Acta 1065 (1991) 8-14 et "DNA Transfection ediated by cationic liposomes containing lipopolylysine : characterisation and mechanism of action" Xiaohuai Zhou, Leaf Huang, Biochimica et Biophysica Acta 1189 (1994) 195-203, décrivent une lipopolylysine qui est un polymère polycationique doté de chaînes lipidiques (NGPE ou DPSG), qui serait obtenue à partir d'une polylysine de poids moléculaire de 3300.Furthermore, it should also be noted that the documents "Lipophilic polylysine mediate efficient DNA transfection in mammalian Cells", Xiaohuai Zhou et al., Biochimica and Biophysica Acta 1065 (1991) 8-14 and "DNA Transfection ediated by cationic liposomes containing lipopolylysine : characterization and mechanism of action "Xiaohuai Zhou, Leaf Huang, Biochimica and Biophysica Acta 1189 (1994) 195-203, describe a lipopolylysine which is a polycationic polymer endowed with lipid chains (NGPE or DPSG), which would be obtained from a polylysine with a molecular weight of 3300.
Néanmoins, dans le premier de ces documents, le polymère n'a pas été purifié ni caractérisé et ne peut pas être obtenu en pratique. Leurs auteurs considèrent en effet à tort que la consommation totale du NHS-ester de NGPE démontre l'obtention de la lipopolylysine, et n'envisagent pas de purification. Au demeurant, selon ces auteurs, le composé obtenu est susceptible de former une solution claire dans l'eau en l'absence de détergent, ce qui n'est pas le cas, et ne peut pas être le cas, de la lipopolylysine qu'ils décrivent. En pratique, la structure du produit obtenu dans ce document ne correspond pas à la lipopolylysine qu'ils prétendent avoir obtenu qui, du fait qu'elle porte deux groupements lipidiques NGPE, serait insoluble dans l'eau, et ne pourrait au mieux que s'y disperser. En outre, il est à noter que les complexes d'ADN et de liposomes cationiques décrits par ces auteurs dans le deuxième document comprennent de 1 'ADN adsorbé par liaison polyélectrolytique à l'extérieur de complexes liposomiques (qui ne sont pas en fait de véritables liposomes) formés de lipopolylysine (LPLL) à chaînes DPSG non phospholipidiques et de dioléoylphosphatidyléthanolamine (DOPE). Dans cette structure, la LPLL n'est donc pas liée à une membrane fonctionnelle formée d'une bicouche stable de composés amphiphiles puisque la DOPE ne forme pas une telle bicouche fonctionnelle, mais une structure hexagonale. En outre, les chaînes DPSG triglycérides (considérées comme préférables aux chaînes NGPE auxquelles les auteurs ont renoncé dans ce deuxième document) ne peuvent pas s'insérer dans une bicouche fonctionnelle.However, in the first of these documents, the polymer has not been purified or characterized and cannot be obtained in practice. Their authors mistakenly consider that the total consumption of the NHS-ester of NGPE demonstrates the obtaining of lipopolylysine, and do not envisage purification. Moreover, according to these authors, the compound obtained is capable of forming a clear solution in water in the absence of detergent, which is not the case, and cannot be the case, of the lipopolylysine that they describe. In practice, the structure of the product obtained in this document does not correspond to the lipopolylysine that they claim to have obtained which, because it carries two lipid groups NGPE, would be insoluble in water, and could at best only disperse therein. In addition, it should be noted that the DNA and cationic liposome complexes described by these authors in the second document comprise DNA adsorbed by polyelectrolytic bond to the outside of liposomal complexes (which are not in fact true liposomes) formed of lipopolylysine (LPLL) with non-phospholipid DPSG chains and of dioleoylphosphatidylethanolamine (DOPE). In this structure, the LPLL is therefore not linked to a functional membrane formed by a stable bilayer of amphiphilic compounds since DOPE does not form such a functional bilayer, but a hexagonal structure. In addition, the DPSG triglyceride chains (considered preferable to the NGPE chains which the authors have given up in this second document) cannot be inserted into a functional bilayer.
Le document "Drug delivery : Piercing vesicles by their adsorption onto a porous médium" Marie- Alice GUEDEAU-BOUDEVILLE et al., Proc . Natl . Sci . USA Vol.92, pp 9590-9592 (1995), démontre par ailleurs qu'il n'est pas possible de former une membrane continue étanche avec une bicouche phospholipidique sur un substrat poreux par complexation polyélectrolytique directe entre des phospholipides anioniques et un support cationique puisque, dans ce cas, les bicouches phospholipidiques pénètrent dans les pores et en tapissent les parois.The document "Drug delivery: Piercing vesicles by their adsorption onto a porous medium" Marie-Alice GUEDEAU-BOUDEVILLE et al., Proc. Natl. Sci. USA Vol. 92, pp 9590-9592 (1995), also demonstrates that it is not possible to form a continuous tight membrane with a phospholipid bilayer on a porous substrate by direct polyelectrolytic complexation between anionic phospholipids and a cationic support. since, in this case, the phospholipid bilayers penetrate into the pores and line the walls.
Au contraire, l'invention procure en fait le seul moyen d'établir et de fixer une membrane fonctionnelle recouvrant continûment un substrat poreux. Les inventeurs ont en effet constaté que, dans une structure membranaire selon l'invention, le composé bifonctionnel de fixation restreint la mobilité relative de la bicouche phospholipidique par rapport au substrat en empêchant sa pénétration à l'intérieur des pores. D'autres caractéristiques et avantages de 1 ' invention apparaîtront à la lecture de la description des exemples suivants et des figures annexées dans lesquelles :On the contrary, the invention in fact provides the only means of establishing and fixing a functional membrane continuously covering a porous substrate. The inventors have in fact found that, in a membrane structure according to the invention, the bifunctional fixing compound restricts the relative mobility of the phospholipid bilayer with respect to the substrate by preventing its penetration inside the pores. Other characteristics and advantages of the invention will appear on reading the description of the following examples and the appended figures in which:
- la figure 1 est un schéma réactionnel de préparation d'un polymère polycationique selon l'invention,FIG. 1 is a reaction scheme for the preparation of a polycationic polymer according to the invention,
- la figure 2 est un diagramme illustrant les résultats d'essais de l'exemple 3 d'inhibition de la fluorescence par la L-polylysine-NSPE en solution dans le DMSO et ajoutée à une solution aqueuse d'ADN, de détergent et de BET,FIG. 2 is a diagram illustrating the results of tests of Example 3 of inhibition of fluorescence by L-polylysine-NSPE in solution in DMSO and added to an aqueous solution of DNA, detergent and BET,
- la figure 3 est un schéma illustrant la structure générale d'une particule virale artificielle selon l'invention obtenue à l'exemple 4,FIG. 3 is a diagram illustrating the general structure of an artificial viral particle according to the invention obtained in example 4,
- la figure 4 est un diagramme illustrant les résultats d'essais de l'exemple 5 d'inhibition par leFIG. 4 is a diagram illustrating the results of tests of example 5 of inhibition by the
Cu++ de la fluorescence de particules virales artificielles selon l'invention,Cu ++ of the fluorescence of artificial viral particles according to the invention,
- la figure 5 est un diagramme illustrant la cinétique de libération d'hémoglobine des particules selon l'invention conformément à l'exemple 7,FIG. 5 is a diagram illustrating the kinetics of hemoglobin release from the particles according to the invention in accordance with Example 7,
- la figure 6 est une vue partielle en coupe d'une structure membranaire selon un mode de réalisation de l'invention.- Figure 6 is a partial sectional view of a membrane structure according to one embodiment of the invention.
EXEMPLE 1 : Préparation d'un polymère polycationique selon l'invention : une L-polylysine N-succinyl- phosphatidyléthanolamine .EXAMPLE 1 Preparation of a polycationic polymer according to the invention: an L-polylysine N-succinylphosphatidylethanolamine.
1 ) Synthèse de la N-succinyl-phosphatidyléthanolamine (NSPE) :1) Synthesis of N-succinyl-phosphatidylethanolamine (NSPE):
Dans un ballon de 25 ml, on pèse 824,2 mg de phosphatidylethanolamine de jaune d'oeuf EYPE, (composé (I) figure 1) que l'on solubilise dans 10 ml de chloroforme. On ajoute 163 μl de triéthylamine TEA sous agitation magnétique. On ajoute ensuite 176,7 mg d'anhydride succinique (II) et on laisse la réaction se poursuivre pendant deux heures. On suit la disparition d'aminés libres par chromatographie sur gel de silice avec du mélange chloroforme / méthanol / eau (1/2/0.9; v/v/v) comme éluant . Au milieu réactionnel, on ajoute 20 ml de méthanol et 9 ml d'eau sous agitation magnétique et enfin 60 μl de HC1 5N. On vérifie que le pH a une valeur de 3 à 4. On laisse sous agitation pendant 10 min à température ambiante puis on ajoute 10 ml de chloroforme et 10 ml d'H2θ et on agite. On centrifuge le mélange pendant 10 min à 4000 tours par minute (418,9 rad/s) et on aspire la phase supérieure. On prélève la phase organique et on lave une fois la phase aqueuse avec 10 ml de chloroforme. On centrifuge à nouveau 10 min, on élimine la phase aqueuse et on récupère la phase organique. On mélange les deux phases chloroformiques d'extraction et de lavage. On évapore le solvant à 1 ' évaporateur rotatif et on obtient 1 g de N-succinyl-phosphatidyléthanolamine d'aspect cireux (produit (III) sur la figure 1). 2) Synthèse de l'ester activé N-succinyl- phosphatidyléthanolamine-N-succinimide (NSPE-NS) :In a 25 ml flask, 824.2 mg of EYPE egg yolk phosphatidylethanolamine (compound (I) FIG. 1) are weighed, which is dissolved in 10 ml of chloroform. 163 μl of TEA triethylamine are added with magnetic stirring. Then 176.7 mg of succinic anhydride (II) is added and the reaction is allowed to continue for two hours. The disappearance of free amines is monitored by chromatography on silica gel with a chloroform / methanol / water mixture (1/2 / 0.9; v / v / v) as eluent. To the reaction medium, 20 ml of methanol and 9 ml of water with magnetic stirring and finally 60 μl of 5N HC1. It is checked that the pH has a value of 3 to 4. It is left under stirring for 10 min at room temperature then 10 ml of chloroform and 10 ml of H 2 O are added and the mixture is stirred. The mixture is centrifuged for 10 min at 4000 revolutions per minute (418.9 rad / s) and the upper phase is aspirated. The organic phase is taken and the aqueous phase is washed once with 10 ml of chloroform. Centrifuged again for 10 min, the aqueous phase is eliminated and the organic phase is recovered. The two chloroform extraction and washing phases are mixed. The solvent is evaporated on a rotary evaporator and 1 g of N-succinyl-phosphatidylethanolamine with a waxy appearance is obtained (product (III) in FIG. 1). 2) Synthesis of the activated ester N-succinylphosphatidylethanolamine-N-succinimide (NSPE-NS):
On solubilise le produit (III) dans 15 ml de chloroforme et on ajoute 560 mg de N-hydroxysuccinimide (composé (IV)) sous agitation magnétique. On pèse ensuite 1,457 g de N, N' -dicyclohexylcarbodiimide (DCCD) séchée et on la solubilise dans 6 ml de chloroforme. A la solution de III + IV, on rajoute progressivement 1 ml de solution de DCCD toutes les 10 min sous agitation magnétique à température ambiante. Après la dernière addition de DCCD, on laisse incuber le mélange pendant la nuit à température ambiante. On filtre le milieu réactionnel sur laine de verre pour éliminer le précipité de dicyclohexylurée . On réduit le volume de chloroforme à 3 ml par évaporation sous pression réduite et on filtre à nouveau le milieu réactionnel. On ajoute 20 ml d'acétone, on agite et on stocke le mélange pendant 12 h à -20°C. On centrifuge ensuite le précipité et on récupère le surnageant que l'on évapore. Le mélange contient 800 mg de NSPE-NS (V_) que l'on reprend dans 10 ml de chloroforme. 3) Synthèse de la L-polylysine N-succinyl- phosphatidyléthanolamine (VI) :The product (III) is dissolved in 15 ml of chloroform and 560 mg of N-hydroxysuccinimide (compound (IV)) are added with magnetic stirring. Then weighed 1.457 g of dried N, N '-dicyclohexylcarbodiimide (DCCD) and solubilized in 6 ml of chloroform. To the solution of III + IV, 1 ml of DCCD solution is gradually added every 10 min with magnetic stirring at room temperature. After the last addition of DCCD, the mixture is allowed to incubate overnight at room temperature. The reaction medium is filtered on glass wool to remove the precipitate of dicyclohexylurea. The volume of chloroform is reduced to 3 ml by evaporation under reduced pressure and the reaction medium is again filtered. 20 ml of acetone are added, the mixture is stirred and the mixture is stored for 12 h at -20 ° C. The precipitate is then centrifuged and the supernatant is recovered which is evaporated. The mixture contains 800 mg of NSPE-NS (V_) which is taken up in 10 ml of chloroform. 3) Synthesis of L-polylysine N-succinylphosphatidylethanolamine (VI):
On pèse 20,4 mg de L-polylysine de poids moléculaire 19200 et on la dissout dans 3 ml de DMSO sous agitation magnétique. On ajoute 13,8 μl de triéthylamine TEA et 12 mg de N,N-diméthyl aminopyridine (DMAP). Au mélange, on ajoute 870 μl de chloroforme puis 130 μl de la solution de (V) . On incube sous agitation magnétique pendant 30 min à température ambiante puis pendant 10 min à 50° C.20.4 mg of L-polylysine of molecular weight 19200 are weighed and dissolved in 3 ml of DMSO under magnetic agitation. 13.8 μl of TEA triethylamine and 12 mg of N, N-dimethyl aminopyridine (DMAP) are added. To the mixture, 870 μl of chloroform is added, then 130 μl of the solution of (V). The incubation is carried out with magnetic stirring for 30 min at room temperature and then for 10 min at 50 ° C.
Le schéma réactionnel de ces trois premières étapes de synthèse est illustré sur la figure 1. Il permet la synthèse du produit (VI) qui est une L- polylysine N-succinyl-phosphatidyléthanolamine, c'est-à- dire une L-polylysine dont certains groupes aminés portent les ligands phospholipidiques NSPE.The reaction scheme for these first three synthesis steps is illustrated in FIG. 1. It allows the synthesis of the product (VI) which is an L-polylysine N-succinyl-phosphatidylethanolamine, that is to say an L-polylysine of which certain amino groups carry the phospholipid ligands NSPE.
Dans tout le texte, une telle polylysine phospholipidique est désignée par polylysine-NSPE ou, lorsque l'on veut préciser son poids moléculaire et son taux de greffage par les ligands phospholipidiques, par la désignation : L-polylysine (x)-NSPE-dsy où x est le poids moléculaire (en kilodaltons) de la L-polylysine sous forme de bromhydrate de départ et y est le taux de greffage exprimé en pourcentage de fonctions aminés substituées.Throughout the text, such a phospholipid polylysine is designated by polylysine-NSPE or, when it is desired to specify its molecular weight and its rate of grafting by phospholipid ligands, by the designation: L-polylysine (x) -NSPE-dsy where x is the molecular weight (in kilodaltons) of L-polylysine in the form of starting hydrobromide and y is the grafting rate expressed as a percentage of substituted amino functions.
4) Précipitation par le citrate de sodium :4) Precipitation by sodium citrate:
Au milieu réactionnel obtenu en 3) contenant 3 ml de DMSO et 1 ml de chloroforme, on ajoute 0,1 ml d'une solution de citrate trisodique 0,33 M, pH 7. Le précipité est placé pendant 12 h à + 4° C. On centrifuge ensuite le mélange pendant 10 min à 3000 tours par minute (314,16 rad/s). On élimine le surnageant et on lave le précipité avec 6 ml de DMSO contenant du citrate de Na (100 μl d'une solution 0,33 M, pH7 ) . On laisse 2 heures à - 20° C, puis on centrifuge à 3500 tours par minute (366,52 rad/s) pendant 10 min.To the reaction medium obtained in 3) containing 3 ml of DMSO and 1 ml of chloroform, 0.1 ml of a 0.33 M trisodium citrate solution, pH 7 is added. The precipitate is placed for 12 h at + 4 ° C. The mixture is then centrifuged for 10 min at 3000 revolutions per minute (314.16 rad / s). The supernatant is eliminated and the precipitate is washed with 6 ml of DMSO containing Na citrate (100 μl of a 0.33 M solution, pH7). It is left for 2 hours at -20 ° C., then it is centrifuged at 3500 revolutions per minute (366.52 rad / s) for 10 min.
5) reprise dans le DMSO :5) included in the DMSO:
On reprend le précipité dans 2 ml de DMSO, puis 1 ml d'H 0 et enfin, 100 μl de HC1 1N permettant d'abaisser le pH sous le pK de l'acide citrique. On homogénéise sous agitation magnétique. La solution devient limpide .The precipitate is taken up in 2 ml of DMSO, then 1 ml of H 0 and finally, 100 μl of 1N HCl allowing the pH to be lowered under the pK of citric acid. Homogenized with magnetic stirring. The solution becomes clear.
6) Dialyse du DMSO et du citrate et lyophilisation : On dialyse les échantillons après avoir ajouté 1 ml d'une solution 64 mM de détergent non ionique HECAMEG () à une concentration finale de 20 mM pendant la nuit à -4° C contre de l'eau pH 7, puis 3 h contre de l'eau acide (pH 3). On lyophilise l'échantillon (18,5 mg ) . On obtient ainsi le produit (VI) à l'état lyophilisé sec avec une grande pureté, supérieure à 95%.6) Dialysis of DMSO and citrate and lyophilization: The samples are dialyzed after adding 1 ml of a 64 mM solution of non-ionic detergent HECAMEG () at a final concentration of 20 mM overnight at -4 ° C. against water pH 7, then 3 h against acidic water (pH 3). The sample is lyophilized (18.5 mg). The product (VI) is thus obtained in the dry lyophilized state with high purity, greater than 95%.
7) Dosage des protéines et des phospholipides :7) Determination of proteins and phospholipids:
On reprend l'échantillon lyophilisé dans 2 ml de DMSO dans lequel il se dissout parfaitement. On soumet l'échantillon à un dosage traditionnel des protéines et des phospholipides. On constate que l'échantillon obtenu contient 7,55 mg de protéines et 180 μg de phosphore ce qui correspond à un taux de greffage de la L-polylysine de 10% des groupes aminés, c'est-à-dire à la L-polylysine ( 19 , 2 )- NSPE-ds10. EXEMPLE 2 : variation du taux de greffage :The lyophilized sample is taken up in 2 ml of DMSO in which it dissolves perfectly. The sample is subjected to a traditional protein and phospholipid assay. It is noted that the sample obtained contains 7.55 mg of proteins and 180 μg of phosphorus which corresponds to a grafting rate of L-polylysine of 10% of the amino groups, that is to say to L- polylysine (19, 2) - NSPE-ds10. EXAMPLE 2: variation of the grafting rate:
Le nombre de groupes aminés portant un ligand phospholipidique sur le nombre total de groupes aminés du polymère (VI) constitue son taux de greffage.The number of amino groups carrying a phospholipid ligand on the total number of amino groups of the polymer (VI) constitutes its grafting rate.
Des produits similaires ont été obtenus dont le taux de greffage des fonctions aminées varie entre 30% (L-polylysine(19,2)-NSPE-ds30) et 1% (L-polylysine- ( 19 , 2 )-NSPE-ds1 ) . Le procédé de synthèse est le même que celui décrit pour L-polylysine ( 19 , 2 )-NSPE-ds10 à la seule différence que le volume de la solution de (V) ajouté à la L-polylysine (19,2) varie avec le taux de greffage désiré.Similar products have been obtained, the grafting rate of the amino functions varying between 30% (L-polylysine (19.2) -NSPE-ds30) and 1% (L-polylysine- (19, 2) -NSPE-ds1) . The synthesis process is the same as that described for L-polylysine (19, 2) -NSPE-ds10 with the only difference that the volume of the solution of (V) added to L-polylysine (19.2) varies with the desired grafting rate.
Taux de greffage Vol. de Vol. de Taux de greffage désiré (V) Chloroforme mesuréGrafting rate Vol. flight. Desired grafting rate (V) Chloroform measured
L-polylysine-NSPE-dsl 13 987 L-polylysine-NSPE-ds 1L-polylysine-NSPE-dsl 13 987 L-polylysine-NSPE-ds 1
L-polylysine-NSPE-ds2 26 974 L-polylysine-NSPE-dsl ,7L-polylysine-NSPE-ds2 26 974 L-polylysine-NSPE-dsl, 7
L-polylysine-NSPE-ds4 52 948 L-polylysine-NSPE-ds3L-polylysine-NSPE-ds4 52 948 L-polylysine-NSPE-ds3
L-polylysine-NSPE-ds20 260 740 L-polylysine-NSPE-ds21L-polylysine-NSPE-ds20 260 740 L-polylysine-NSPE-ds21
L-polylysine-NSPE-ds50 651 349 L-polylysine-NSPE-ds30 La procédure de purification est identique à celle décrite à l'exemple 1 étapes 4) à 6) pour L- polylysine ( 19 , 2 )-NSPE-ds10.L-polylysine-NSPE-ds50 651 349 L-polylysine-NSPE-ds30 The purification procedure is identical to that described in Example 1 steps 4) to 6) for L-polylysine (19, 2) -NSPE-ds10.
EXEMPLE 3 : Caractérisation des propriétés des polylysine- NSPE.EXAMPLE 3 Characterization of the properties of polylysine-NSPE.
1 ) Solubilité :1) Solubility:
Les produits (VI) obtenus par synthèse sous forme d'une poudre sont essentiellement insolubles dans l'eau contrairement à la L-polylysine (19,2) de départ, mettant ainsi en évidence la modification chimique de la L- polylysine. Ces produits sont aussi insolubles dans un tampon contenant un détergent non ionique tel que 1 ' HECAMEG a pH 7.The products (VI) obtained by synthesis in the form of a powder are essentially insoluble in water unlike the starting L-polylysine (19,2), thus highlighting the chemical modification of L-polylysine. These products are also insoluble in a buffer containing a nonionic detergent such as HECAMEG at pH 7.
De plus, un produit obtenu lors d'une synthèse réalisée dans les mêmes conditions que celles décrites précédemment avec une L-polylysine de faible poids moléculaire (3900) et présentant un degré de substitution de 6 % (L-polylysine ( 3 , 9 )-NSPE-ds6 ) n'est que très partiellement soluble dans l'eau mais aussi dans une solution de détergent non ionique, 1 HECAMEG a la concentration 20 mM. Il a été aussi constaté que ce produit n'interaqit pas avec des substrats anioniques tels que le (R) (R) (R)In addition, a product obtained during a synthesis carried out under the same conditions as those described above with a low molecular weight L-polylysine (3900) and having a degree of substitution of 6% (L-polylysine (3, 9) -NSPE-ds6) is only very partially soluble in water but also in a solution of non-ionic detergent, 1 HECAMEG has a concentration of 20 mM. It has also been found that this product does not interact with anionic substrates such as (R) (R) (R)
SEPHADEX C50, SEPHADEX C25, SEPHADEX SPC25, l'ADN, ni avec le citrate de sodium. En revanche, lorsque le produit L~ polylysine ( 19 , 2 )-NSPE-ds10 , après avoir été lyophilisé, est dissout dans du DMSO à une concentration proche de 1 mg/ml, il retrouve sa propriété d'être précipité par le citrate de sodium. A savoir, lorsque 0,5 mg du produit L-polylysine (19, 2)-NSPE-ds10 est mis en solution dans 1 ml de DMSO, l'addition de 200 μl d'une solution 0,19 M de citrate de sodium entraîne la précipitation d'un complexe. Ce complexe est centrifugé et le contenu protéique du surnageant est dosé. Seul 1% de la L-polylysine ( 19 , 2 )-NSPE-ds10 est retrouvé dans le surnageant.SEPHADEX C50, SEPHADEX C25, SEPHADEX SPC25, DNA, nor with sodium citrate. On the other hand, when the product L ~ polylysine (19, 2) -NSPE-ds10, after having been lyophilized, is dissolved in DMSO at a concentration close to 1 mg / ml, it regains its property of being precipitated by citrate sodium. Namely, when 0.5 mg of the product L-polylysine (19, 2) -NSPE-ds10 is dissolved in 1 ml of DMSO, the addition of 200 μl of a 0.19 M solution of sodium citrate causes precipitation of a complex. This complex is centrifuged and the protein content of the supernatant is assayed. Only 1% of L-polylysine (19, 2) -NSPE-ds10 is found in the supernatant.
De plus, lorsque le produit L-polylysine (19, 2)-NSPE-ds10, après avoir été lyophilisé, est dissout dans du DMSO à une concentration proche de 1 mg/ml puis dialyse contre de l'eau pour éliminer lé DMSO et le remplacer par de l'eau, on obtient une solution limpide et homogène contenant le produit L-polylysine ( 19 , 2 )-NSPE-ds10 à une concentration correspondant à une concentration de protéines de 290 μg/ml. Si on ajoute 1 ml de cette solution a 5 mg de SEPHADEX C25, on ne retrouve, après homogénéisation et décantation du SEPHADEX C25, que 77% de la L-polylysine( 19 , 2 )-NSPE-ds10 dans le surnageant. Si on ajoute 1 ml de cette solution a 5 mg de SEPHADEX ® SPC25, on ne retrouve, après homogénéisation et décantation du SEPHADEX ® C25, que 33% de la L-polylysine ( 19 , 2 )-NSPE- ds10 dans le surnageant. Si on ajoute 1 ml de cette solution à 200 μl d'une solution de citrate de sodiumIn addition, when the product L-polylysine (19, 2) -NSPE-ds10, after having been lyophilized, is dissolved in DMSO at a concentration close to 1 mg / ml then dialysis against water to remove the DMSO and replace it with water, a clear and homogeneous solution is obtained containing the product L-polylysine (19, 2) -NSPE-ds10 at a concentration corresponding to a protein concentration of 290 μg / ml. If 1 ml of this solution is added to 5 mg of SEPHADEX C25, after homogenization and decantation of SEPHADEX C25, only 77% of the L-polylysine (19, 2) -NSPE-ds10 is found in the supernatant. If 1 ml of this solution is added to 5 mg of SEPHADEX® SPC25, after homogenization and decantation of SEPHADEX® C25, only 33% of the L-polylysine (19, 2) -NSPE- ds10 is found in the supernatant. If 1 ml of this solution is added to 200 μl of a sodium citrate solution
0,18 M, on ne retrouve, après homogénéisation et centrifugation du complexe, que 38% de la L- polylysine ( 19 , 2 )-NSPE-ds10 dans le surnageant. Si on ajoute 1 ml de cette solution à 200 μl d'une solution d'ADN à 1 mg/ml, on ne retrouve, après homogénéisation et centrifugation du complexe, que 31% de la L- polylysine( 19 , 2 )-NSPE-ds10 dans le surnageant. Si on ajoute0.18 M, there is found, after homogenization and centrifugation of the complex, only 38% of L-polylysine (19, 2) -NSPE-ds10 in the supernatant. If 1 ml of this solution is added to 200 μl of a 1 mg / ml DNA solution, after homogenization and centrifugation of the complex, only 31% of the L-polylysine (19, 2) -NSPE is found. -ds10 in the supernatant. If we add
1 ml de cette solution, complémentée avec un détergeant non ionique (HECAMEG ®) a. une concentration de 20 mM, a 200 μl d'une solution d'ADN à 1 mg/ml, on ne retrouve, après homogénéisation et centrifugation du complexe, que 30% de la L-polylysine( 19 , 2 )-NSPE-ds10 dans le surnageant.1 ml of this solution, supplemented with a non-ionic detergent (HECAMEG ®) a. a concentration of 20 mM, at 200 μl of a DNA solution at 1 mg / ml, there is found, after homogenization and centrifugation of the complex, only 30% of L-polylysine (19, 2) -NSPE-ds10 in the supernatant.
2) Capacité des polylysines-NSPE à former un complexe polyélectrolytique avec 1 'ADN :2) Ability of polylysines-NSPE to form a polyelectrolytic complex with DNA:
La capacité des L-polylysines-NSPE synthétisées comme décrit précédemment à l'exemple 1 à interagir avec de 1 ' ADN est plus précisément étudiée par leur propriété à déplacer une sonde fluorescente, le bromure d'éthidium (BET), qui s'intercale naturellement entre les bases de l'ADN. En présence de L-polylysine, le BET est déplacé du complexe ADN/BET et perd sa fluorescence. Cette perte de la fluorescence est représentée par les courbes de la figure 2 en fonction de la quantité de L-polylysine ajoutée exprimée en abscisses par le rapport (+/-) entre les charges positives de la L- polylysine et les charges négatives de 1 ' AD'N . La courbe A représente le déplacement du BET par la L-polylysine ( 19 , 2 ) non phospholidée, la courbe B représente le déplacement du BET par la L-polylysine ( 19 , 2 )-NSPE-ds1 , la courbe C représente le déplacement du BET par la L-polylysine ( 19 , 2 )- NSPE-ds1,7, la courbe D représente le déplacement du BET par la L-polylysine ( 19 , 2 )-NSPE-ds3 , la courbe E représente le déplacement du BET par la L-polylysine ( 19 , 2 )-NSPE-ds10 , la courbe F représente le déplacement du BET par la L- polylysine ( 19 , 2)-NSPE-ds21 , la courbe G représente le déplacement du BET par la L-polylysine ( 19 , 2 )-NSPE-ds30. On observe donc que l'efficacité de déplacement du BET est maximale pour la L-polylysine ( 19 , 2 )-NSPE-ds1 , 7 (courbe C) et pour la L-polylysine ( 19 , 2 )-NSPE-ds3 (courbe D). Par contre, si le taux de greffage est trop important (supérieur à 20%) la L-polylysine-NSPE ne s'associe pas à 1 ' ADN .The capacity of the L-polylysines-NSPE synthesized as described previously in Example 1 to interact with DNA is more precisely studied by their property in displacing a fluorescent probe, ethidium bromide (BET), which is intercalated naturally between the bases of DNA. In the presence of L-polylysine, the BET is displaced from the DNA / BET complex and loses its fluorescence. This loss of fluorescence is represented by the curves of FIG. 2 as a function of the amount of added L-polylysine expressed on the abscissa by the ratio (+/-) between the positive charges of L- polylysine and the negative charges of 1 'AD ' N. Curve A represents the displacement of BET by L-polylysine (19, 2) not phospholidated, curve B represents the displacement of BET by L-polylysine (19, 2) -NSPE-ds1, curve C represents displacement of BET by L-polylysine (19, 2) - NSPE-ds1,7, curve D represents the displacement of BET by L-polylysine (19, 2) -NSPE-ds3, curve E represents the displacement of BET by L-polylysine (19, 2) -NSPE-ds10, curve F represents the displacement of BET by L- polylysine (19, 2) -NSPE-ds21, curve G represents displacement of BET by L- polylysine (19, 2) -NSPE-ds30. We therefore observe that the displacement efficiency of BET is maximum for L-polylysine (19, 2) -NSPE-ds1, 7 (curve C) and for L-polylysine (19, 2) -NSPE-ds3 (curve D). On the other hand, if the grafting rate is too high (greater than 20%) L-polylysine-NSPE does not associate with DNA.
EXEMPLE 4 : Préparation de particules virales artificielles selon 1 ' invention : Dans un ballon de 50 ml, on prépare une solution lipidique dans 1 ml de chloroforme contenant 250 μg de lécithine de jaune d ' oeuf (L-α-phosphatidylcholine de jaune d'oeuf, EPC - Lipoïd) et 25 μg de cholestérol. On sèche cette solution sous azote puis on la lyophilise pendant 12 h .EXAMPLE 4 Preparation of artificial viral particles according to the invention: In a 50 ml flask, a lipid solution is prepared in 1 ml of chloroform containing 250 μg of egg yolk lecithin (L-α-phosphatidylcholine from yolk). egg, EPC - Lipoid) and 25 μg of cholesterol. This solution is dried under nitrogen and then lyophilized for 12 h.
A ce produit séché, on ajoute 4 ml de tampon contenant : Hepes (acide N-(2- hydroxyethyl )piperazine-N ' -( 2-éthanesulfonique ) 10 mM, détergent non ionique HECAMEG ® ( 6-0- (N-heptylcarbamoyl )- methyl-α-D-glucopyranoside) 20 mM, pH 7 et on soumet ce mélange aux ultrasons pendant 10 min. On obtient une solution homogène et limpide.To this dried product, 4 ml of buffer containing: Hepes (N- (2-hydroxyethyl) acid piperazine-N '- (2-ethanesulfonic) 10 mM, non-ionic detergent HECAMEG® (6-0- (N-heptylcarbamoyl) are added ) - methyl-α-D-glucopyranoside) 20 mM, pH 7 and this mixture is subjected to ultrasound for 10 min. A homogeneous and clear solution is obtained.
Tout en maintenant les ultrasons, on ajoute à l'aide d'une seringue Hamilton, 13,8 μg de L- polylysine (19, 2 )-NSPE-ds10 en solution dans 3 μl de DMSOWhile maintaining the ultrasound, 13.8 μg of L-polylysine (19, 2) -NSPE-ds10 dissolved in 3 μl of DMSO are added using a Hamilton syringe
(solution dans le DMSO à 4,6 mg/ml). La solution reste homogène et limpide.(solution in DMSO at 4.6 mg / ml). The solution remains homogeneous and clear.
On ajoute ensuite, à l'aide d'une seringue Hamilton, 38 μg d'ADN en solution dans 27 μl d'eau et laisse le mélange sous agitation pendant 30 min. Il se produit alors une reticulation de 1 'ADN avec la polylysine- NSPE. On dialyse ensuite le détergent contre 5 1 d'eau distillée pH 7 pendant 4 h et on renouvelle le bain de dialyse trois fois, ce qui entraîne la formation de la membrane fonctionnelle autour de 1 ' ADN et de la polylysine- NSPE. On obtient alors les particules virales artificielles telles que schématisé figure 3 comprenant un noyau 31 d'ADN faisant office de substrat solide polyanionique, un composé bifonctionnel de fixation 32 formé de L-polylysine-NSPE polycationique, et une membrane fonctionnelle 33 externe périphérique formée d'une bicouche de phospholipides. Le noyau 31 d'ADN peut être considéré comme une nucléocapside artificielle. Les particules virales artificielles sont stables pendant au moins 15 jours . EXEMPLE 5 : Mise en évidence des propriétés de transfection des Particules Virales artificielles. Couplage d' un composé d'interaction de ciblage cellulaire et intracellulaire, un adénovirus défectif, à la surface externe des particules virales artificielles. On synthétise des particules virales de la même manière que dans l'exemple 4 mais en ajoutant, à la composition de phospholipides, 5 % en moles de phosphatidylethanolamine de jaune d'oeuf N-{-4-(N- maleimidométhyl ) cyclohexane-1 -carbonyl } (MCC-EYPE). La neutravidine substituée avec du N-Succinimidyl-3- ( 2- pyridyldithio )propionate (SPDP) est greffée sur les résidus MCC-EYPE présents à la surface externe des particules . La MCC-EYPE a été obtenue à partir de EYPE (phosphatidylethanolamine de jaune d'oeuf) et de SMCC (Succinimidyl 4-(N-maleimidométhyl ) cyclohexane-1 - carboxylate) comme suit.Then add, using a syringe Hamilton, 38 μg of DNA in solution in 27 μl of water and leaves the mixture with stirring for 30 min. A DNA cross-linking then occurs with the polylysine-NSPE. The detergent is then dialyzed against 5 l of distilled water pH 7 for 4 h and the dialysis bath is renewed three times, which results in the formation of the functional membrane around the DNA and the polylysine-NSPE. We then obtain the artificial viral particles as shown diagrammatically in FIG. 3 comprising a DNA core 31 acting as a polyanionic solid substrate, a bifunctional fixing compound 32 formed from polycationic L-polylysine-NSPE, and a peripheral external functional membrane 33 formed from 'a bilayer of phospholipids. The DNA core 31 can be considered as an artificial nucleocapsid. The artificial viral particles are stable for at least 15 days. EXAMPLE 5 Demonstration of the Transfecting Properties of Artificial Viral Particles. Coupling of a cellular and intracellular targeting interaction compound, a defective adenovirus, to the outer surface of artificial viral particles. Viral particles are synthesized in the same manner as in Example 4 but by adding, to the phospholipid composition, 5 mol% of egg yolk phosphatidylethanolamine N - {- 4- (N- maleimidomethyl) cyclohexane-1 -carbonyl} (MCC-EYPE). Neutravidin substituted with N-Succinimidyl-3- (2-pyridyldithio) propionate (SPDP) is grafted onto the MCC-EYPE residues present on the external surface of the particles. MCC-EYPE was obtained from EYPE (egg yolk phosphatidylethanolamine) and SMCC (Succinimidyl 4- (N-maleimidomethyl) cyclohexane-1 - carboxylate) as follows.
On solubilise 113,7 mg de EYPE dans 5 ml de chloroforme anhydre. On ajoute 24 μl de triéthyla ine (TEA) puis 50 mg de SMCC en solution dans 0,5 ml de dimethylsulfoxyde (DMSO). Le mélange est incubé 2 heures à 40°C sous agitation. L'apparition de MCC-EYPE est suivie par chromatographie sur couche mince sur gel de silice. Le produit est extrait par un mélange chloroforme/méthanol/eau . Après centrifugation 10 min à 4000 trs/min, on élimine la phase aqueuse et on évapore la phase chloroformique contenant la MCC-EYPE. La structure de la MCC-EYPE est caractérisée par résonnance magnétique nucléaire.113.7 mg of EYPE are dissolved in 5 ml of anhydrous chloroform. 24 μl of triethyla ine (TEA) are added then 50 mg of SMCC in solution in 0.5 ml of dimethylsulfoxide (DMSO). The mixture is incubated for 2 hours at 40 ° C with shaking. The appearance of MCC-EYPE is followed by thin layer chromatography on silica gel. The product is extracted with a chloroform / methanol / water mixture. After centrifugation for 10 min at 4000 rpm, the aqueous phase is eliminated and the chloroform phase containing the MCC-EYPE is evaporated. The structure of the MCC-EYPE is characterized by nuclear magnetic resonance.
Préparation de la N-propionyl-thiol- neutravidine (neutravidine thiolée) : on solubilise 10 mg de neutravidine dans 1 ml de tampon Hepes 200mM, NaCl 300mM, pH 7,9. La suspension est passée sur colonne de gel filtration SEPHADEX® G25 à la sortie de laquelle des fractions de 500 μl sont collectées. On récupère 95 % de la protéine dans les fractions 7 à 10. On prépare une solution de SPDP (N-Succinimidyl-3-( 2-pyridyldithio)propionate ) à 29 mM dans de 1 ' éthanol et on en ajoute 57 μl à la neutravidine. Le mélange est incubé une heure à température ambiante puis lave sur G25 avec un tampon phosphate 0,1M, pH 7,2. La neutravidine- PDP est traitée avec 100 μl d'une solution 0,1M de dithiothréitol pendant 10 min à température ambiante. La neutravidine thiolée est lavée sur colonne SEPHADEX® G25 avec du tampon phosphate 0,1M, pH 7,2. Le taux de greffage est de 10 thiols par molécule de neutravidine.Preparation of N-propionyl-thiol-neutravidin (thiolated neutravidin): 10 mg of neutravidin are dissolved in 1 ml of 200 mM Hepes buffer, 300 mM NaCl, pH 7.9. The suspension is passed through a column of SEPHADEX® G25 filtration gel at the outlet from which 500 μl fractions are collected. 95% of the protein is recovered in fractions 7 to 10. A solution of SPDP (N-Succinimidyl-3- (2-pyridyldithio) propionate) at 29 mM is prepared in ethanol and 57 μl are added to the neutravidin. The mixture is incubated for one hour at room temperature and then washed on G25 with 0.1M phosphate buffer, pH 7.2. The neutravidin-PDP is treated with 100 μl of a 0.1M solution of dithiothreitol for 10 min at room temperature. The thiolated neutravidin is washed on a SEPHADEX® G25 column with 0.1M phosphate buffer, pH 7.2. The grafting rate is 10 thiols per molecule of neutravidin.
Fixation de la neutravidine sur les particules virales artificielles : les 3 ml de neutravidine thiolée sont immédiatement incubés une nuit sous agitation douce à température ambiante avec 1 ml de suspension de particules virales artificielles préparées avec de la MCC- EYPE, comme décrit ci-dessus. La neutravidine n'ayant pas réagi est éliminée par gel filtration sur colonne de sépharose.Fixation of neutravidine on artificial viral particles: the 3 ml of thiolated neutravidine are immediately incubated overnight with gentle shaking at room temperature with 1 ml of suspension of artificial viral particles prepared with MCC-EYPE, as described above. The unreacted neutravidin is removed by gel filtration on a sepharose column.
Fixation de biotine sur les particules d'adénovirus défectifs : une solution à 400 μM de biotine- NHS est préparée dans un tampon Hepes 5mM, NaCl 150mM, glycérol 10 % , pH 7 , 9. A 1 ml de cette solution, 2,5.109 particules adénovirales sont ajoutées et l'ensemble est laissé 3 heures à température ambiante sous agitation douce. La biotine n'ayant pas réagi est éliminée par trois passages successifs en ultrafiltration (10 min à 1500 g). Les adenovirus biotinyles sont repris dans 1 ml de tampon PBS (Phosphate Buffer Saline, phosphate 10mM, NaCl 150mM, pH 7,4).Biotin fixation on defective adenovirus particles: a 400 μM solution of biotin- NHS is prepared in a 5 mM Hepes buffer, 150 mM NaCl, glycerol 10%, pH 7, 9. To 1 ml of this solution, 2.5 × 10 9 adenoviral particles are added and the whole is left for 3 hours at room temperature with gentle stirring. The unreacted biotin is eliminated by three successive passages in ultrafiltration (10 min at 1500 g). The biotinyl adenoviruses are taken up in 1 ml of PBS buffer (Phosphate Buffer Saline, 10mM phosphate, 150mM NaCl, pH 7.4).
Couplage des adenovirus défectifs biotinyles avec les particules virales artificielles neutravidinylées : une quantité de particules virales artificielles correspondant à 5 μg d'ADN est incubée une heure à température ambiante sous agitation douce avec 8.10 particules adénovirales. La suspension est ajustée a 500 μl avec du PBS.Coupling of biotinyl defective adenoviruses with neutravidinylated artificial viral particles: an amount of artificial viral particles corresponding to 5 μg of DNA is incubated for one hour at room temperature with gentle agitation with 8.10 adenoviral particles. The suspension is adjusted to 500 μl with PBS.
Transfection avec les complexes particules virales artificielles - adenovirus : les transfections sont réalisées dans des boîtes de 35 mm de diamètre ou dans de plaques multi-puits où les puits ont la même dimension. Des cellules sont transfectées à 80 % de confluence (environ 8.10 cellules par puits). Les 500 μl de la suspension obtenue à 1 ' étape précédente sont déposés de façon homogène sur les cellules. Après 1 heure d'incubation, le milieu est remplacé par 2 ml de milieu de culture complémenté en sérum. Les cellules sont incubées 48 heures à 37°C pour observer une expression transitoire.Transfection with artificial viral particle - adenovirus complexes: transfections are carried out in 35 mm diameter dishes or in multi-well plates where the wells are the same size. Cells are transfected at 80% confluence (approximately 8.10 cells per well). The 500 μl of the suspension obtained in the preceding step are deposited homogeneously on the cells. After 1 hour of incubation, the medium is replaced by 2 ml of culture medium supplemented with serum. The cells are incubated for 48 hours at 37 ° C. to observe a transient expression.
Résultats : aucune transfection n'a été observée avec les particules non reliées à 1 ' adenovirus . Un niveau de transfection significatif a, par contre, été observé (supérieur à 2 %) avec les complexes formés des particules virales artificielles et d ' adenovirus .Results: no transfection was observed with the particles not linked to the adenovirus. On the other hand, a significant level of transfection was observed (greater than 2%) with the complexes formed of artificial viral particles and of adenovirus.
Cette expérience montre que les particules virales artificielles sont bien capables de délivrer 1 ' ADN au niveau intracellulaire et de permettre son expression. Elle indique, de plus, que contrairement aux vecteurs synthétiques cationiques, la présence de ligands spécifiques (reconnaissance et fusion) à la surface des particules est indispensable pour la délivrance intracellulaire et l'expression du transgène. EXEMPLE 6 : Couplage d'un composé d'interaction de ciblage, la transferrine, à la surface externe des particules virales artificielles. On synthétise d'abord des particules virales de la même manière que dans l'exemple 4 mais en ajoutant 2 % de dipalmitoyl phosphatidylethanolamine (DPPE) à la composition phospholipidique.This experiment shows that the artificial viral particles are well capable of delivering DNA at the intracellular level and of allowing its expression. It also indicates that, unlike synthetic cationic vectors, the presence of specific ligands (recognition and fusion) on the surface of the particles is essential for delivery. intracellular and expression of the transgene. EXAMPLE 6 Coupling of a targeting interaction compound, transferrin, to the external surface of the artificial viral particles. First, viral particles are synthesized in the same manner as in Example 4 but by adding 2% of dipalmitoyl phosphatidylethanolamine (DPPE) to the phospholipid composition.
. Préparation des particules virales avec une membrane contenant du 3-mercaptopropionate DPPE. 12 mg de particules contenant 10 mg de phospholipides et 200 μg de DPPE (0,26 μmol ) sont dispersés dans 30 ml d'un tampon acétate de sodium 75 mM amené à pH 8,5 par addition d'un tampon bicarbonate. On ajoute 100 μl d'une solution 15 mM de succinnimidyl 3-(-2-pyridyldithio) propionate (SPDP) et on agite vigoureusement pendant 1 h . On ajoute alors 6 ml d'une solution d'acétate de sodium 1M. La suspension est ensuite dialysée contre un tampon 20 mM acétate de sodium. On ajoute 2,3 mg (15 μmol) de dithiothréitol dans un tampon bicarbonate de sodium et la suspension est maintenue, sous argon, à pH 7,5 pendant 1 h. Le pH est ensuite ajusté à 5,2 en ajoutant un tampon acétate de sodium et la suspension est ensuite dialysée contre un tampon acétate de sodium 20 mM . On obtient une préparation contenant 0,1 μmol de DPPE modifié par du mercapto propionate.. Preparation of the viral particles with a membrane containing 3-mercaptopropionate DPPE. 12 mg of particles containing 10 mg of phospholipids and 200 μg of DPPE (0.26 μmol) are dispersed in 30 ml of a 75 mM sodium acetate buffer brought to pH 8.5 by addition of a bicarbonate buffer. 100 μl of a 15 mM solution of succinnimidyl 3 - (- 2-pyridyldithio) propionate (SPDP) are added and the mixture is stirred vigorously for 1 h. 6 ml of a 1M sodium acetate solution are then added. The suspension is then dialyzed against a 20 mM sodium acetate buffer. 2.3 mg (15 μmol) of dithiothreitol are added in a sodium bicarbonate buffer and the suspension is maintained, under argon, at pH 7.5 for 1 h. The pH is then adjusted to 5.2 by adding a sodium acetate buffer and the suspension is then dialyzed against a 20 mM sodium acetate buffer. A preparation is obtained containing 0.1 μmol of DPPE modified with mercapto propionate.
Préparation de 3- ( 2-pyridyldithio) proprionate transferrine .Preparation of 3- (2-pyridyldithio) proprionate transferrin.
On ajoute 200 μl d'une solution éthanolique de SPDP (3,0 μmol) à une solution de 120 mg (1,5 μmol) de transferrine de poulet dans 3 ml d'un tampon phosphate de sodium 100 mM, pH 7,8. La solution est agitée vigoureusement pendant 1 h à température ambiante puis est soumise à une filtration sur gel sur SEPHADEX G25 pour donner 6 ml d'une solution de 1,4 μmol de transferrine modifiée par 2,8 μmol de dithiopyridine .200 μl of an ethanolic solution of SPDP (3.0 μmol) are added to a solution of 120 mg (1.5 μmol) of chicken transferrin in 3 ml of 100 mM sodium phosphate buffer, pH 7.8 . The solution is stirred vigorously for 1 h at room temperature and then is subjected to gel filtration on SEPHADEX G25 to give 6 ml of a solution of 1.4 μmol of transferrin modified with 2.8 μmol of dithiopyridine.
. Conjugaison de la transferrine avec les particules .. Conjugation of transferrin with particles.
On mélange 1 μmol de transferrine modifiée dissoute dans un tampon phosphate 100 mM, pH 7,8 avec des particules contenant 0,1 μmol de 3-mercaptopropionate DPPE et dispersées dans un tampon acétate de sodium 20 mM. La préparation est agitée pendant 24 h à température ambiante, puis est ultrafiltrée sur une membrane de 100KD pour éliminer la transferrine en excès.1 μmol of modified transferrin is mixed dissolved in a 100 mM phosphate buffer, pH 7.8 with particles containing 0.1 μmol of 3-mercaptopropionate DPPE and dispersed in a 20 mM sodium acetate buffer. The preparation is stirred for 24 h at room temperature, then is ultrafiltered through a 100KD membrane to remove the excess transferrin.
. On obtient les particules virales telles que représentées figure 3 dotées de ligands 34 formés de transferrine . EXEMPLE 7 : Caractérisation des particules virales artificielles :. The viral particles are obtained as represented in FIG. 3 provided with ligands 34 formed of transferrin. EXAMPLE 7 Characterization of the artificial viral particles:
1 ) Synthèse de L-polylysine fluorescente : L- polylysine( 19,2 )-fluorescéine-dsO , 4.1) Synthesis of fluorescent L-polylysine: L- polylysine (19,2) -fluorescein-dsO, 4.
Dans un ballon de 25 ml, on pèse 36,3 mg de L-polylysine de poids moléculaire 19200 que l'on dissout dans 10 ml de DMSO sous agitation magnétique. On ajoute 40 μl de triéthylamine et on attend 10 min. On ajoute ensuite 1,1 mg d ' isothiocyanate de fluorescéine (FITC) en solution dans 149 μl de diméthylformamide (DMF). La réaction se poursuit à 30°C pendant 2 h. Le produit est analysé par chromatographie sur gel de silice qui montre la disparition du FITC libre et l'apparition d'un produit protéique et fluorescent au dépôt. La L-polylysine-fluorescéine est purifiée comme suit. Le DMSO du milieu réactionnel est dialyse deux fois pendant 2 h contre de l'eau distillée à pH 6,5. Le produit dialyse est ensuite incubé avec 500 mg de SEPHADEX ® C50 dans 100 ml d , eau distillé,e puis dépose sur une colonne. La colonne est d'abord lavée avec 100 ml d'eau distillée pH 7. La L-polylysine-fluorescéine est éluée de la colonne avec 100 ml d'une solution NaCl 2M, pH 9. Cette solution est dialysée contre de l'eau distillée. La solution finale contient 35 mg de protéine. La quantité de fluorescéine est estimée par spectrométrie à 496 nm avec un coefficient d'extinction moléculaire de 90000 m~1cm~1. Le taux de greffage est de 1/233 des fonctions aminés.In a 25 ml flask, 36.3 mg of L-polylysine of molecular weight 19200 is weighed, which is dissolved in 10 ml of DMSO with magnetic stirring. 40 μl of triethylamine are added and wait 10 min. 1.1 mg of fluorescein isothiocyanate (FITC) are then added in solution in 149 μl of dimethylformamide (DMF). The reaction continues at 30 ° C for 2 h. The product is analyzed by chromatography on silica gel which shows the disappearance of the free FITC and the appearance of a protein and fluorescent product on deposition. L-polylysine-fluorescein is purified as follows. The DMSO of the reaction medium is dialyzed twice for 2 h against distilled water at pH 6.5. The dialysis product is then incubated with 500 mg of SEPHADEX® C50 in 100 ml of distilled water, then deposited on a column. The column is first washed with 100 ml of distilled water pH 7. The L-polylysine-fluorescein is eluted from the column with 100 ml of a 2M NaCl solution, pH 9. This solution is dialyzed against water distilled. The final solution contains 35 mg of protein. The quantity of fluorescein is estimated by spectrometry at 496 nm with a molecular extinction coefficient of 90,000 m ~ 1 cm ~ 1 . The grafting rate is 1/233 of the amino functions.
2) Caractérisation des particules virales par inhibition de la fluorescence :2) Characterization of the viral particles by inhibition of fluorescence:
La capacité des polylysines-NSPE synthétisées comme décrit précédemment à permettre la mise en place d'une structure membranaire imperméable aux ions cuivriques (Cu++) entourant 1 ' ADN complexé est étudiée par la méthode d'inhibition de la fluorescence d'une sonde fluorescente liée à 1 ' ADN complexé. Cette sonde est la L- polylysine( 19, 2 )-fluorescéine-dsO , 4 obtenue en 1). Les particules virales sont préparées comme décrit à l'exemple 4 à la seule différence que la L-polylysine-NSPE-dsO , 1 est remplacée par un mélange de 90% de L-polylysine ( 19 , 2 )-NSPE- ds(n) et de 10% de L-polylysine ( 19 , 2 )-fluorescéine-dsO, 4. Le mélange (phospholipides + cholestérol + détergent + L(polylysine-NSPE-ds (n) + L-polylysine ( 19 , 2 )-fluorescéine- ds0,4 + ADN) est ensuite dialyse et la fluorescence des particules est analysée avec un spectrofluorimètre . L'intensité de la fluorescence est analysée au cours de l'addition progressive de Cu++. La valeur représentée en ordonnée sur la figure 4 représente le taux d'inhibition de la fluorescence, exprimé en pourcentage et calculé comme suit : (Ig-If) / Iθ' ou ^0 es^ l'intensité de fluorescence en absence de cuivre, et If est l'intensité de la fluorescence en présence de cuivre.The capacity of polylysines-NSPE synthesized as described above to allow the establishment of a membrane structure impermeable to cupric ions (Cu ++ ) surrounding the complexed DNA is studied by the method of inhibiting the fluorescence of a fluorescent probe linked to DNA complex. This probe is L-polylysine (19, 2) -fluorescein-dsO, 4 obtained in 1). The viral particles are prepared as described in Example 4 with the only difference that L-polylysine-NSPE-dsO, 1 is replaced by a mixture of 90% of L-polylysine (19, 2) -NSPE- ds (n ) and 10% L-polylysine (19, 2) -fluorescein-dsO, 4. The mixture (phospholipids + cholesterol + detergent + L (polylysine-NSPE-ds (n) + L-polylysine (19, 2) - fluorescein- ds0,4 + DNA) is then dialyzed and the fluorescence of the particles is analyzed with a spectrofluorimeter.The intensity of the fluorescence is analyzed during the progressive addition of Cu ++ . The value represented on the ordinate in the figure 4 represents the fluorescence inhibition rate, expressed as a percentage and calculated as follows: (Ig-If) / I / ' or ^ 0 es ^ the fluorescence intensity in the absence of copper, and If is the intensity of the fluorescence in the presence of copper.
La courbe A représente le taux d'inhibition de la fluorescence des particules obtenues à partir de L- polylysine-NSPE-ds10 mais en présence de détergent qui empêche la mise en place d'une membrane fonctionnelle autour de la particule. On observe que le taux d'inhibition de la fluorescence de la L-polylysine ( 19 , 2 )-fluorescéine- ds0,4 atteint une valeur proche de 100% pour une concentration en Cu+ + de 50 μM. La courbe B représente le taux d'inhibition de la fluorescence des particules obtenues à partir de L-polylysine-NSPE-dsl après dialyse du détergent. On observe que le taux d'inhibition de la fluorescence de la L-polylysine ( 19 , 2 )-fluorescéine-dsO, 4 atteint une valeur proche de 30% pour une concentration en Cu++ de 50 μM. La courbe D représente le taux d'inhibition de la fluorescence des particules obtenues à partir de L- polylysine-NSPE-ds10 après dialyse du détergent. On observe que le taux d'inhibition de la fluorescence de la L- polylysine( 19 , 2 )-fluorescéine-dsO , 4 atteint une valeur proche de 25% pour une concentration en Cu+ + de 50 μM. La courbe E représente le taux d'inhibition de la fluorescence des particules obtenues à partir de L-polylysine-NSPE-ds21 après dialyse du détergent. On observe que le taux d'inhibition de la fluorescence de la L-polylysine ( 19 , 2 )- fluorescéine-dsO , 4 atteint une valeur proche de 20 % pour une concentration en Cu++ de 50 μM.Curve A represents the rate of inhibition of the fluorescence of the particles obtained from L-polylysine-NSPE-ds10 but in the presence of detergent which prevents the establishment of a functional membrane around the particle. It is observed that the inhibition rate of the fluorescence of L-polylysine (19, 2) -fluorescein- ds0.4 reaches a value close to 100% for a Cu + + concentration of 50 μM. Curve B represents the rate of inhibition of the fluorescence of the particles obtained from L-polylysine-NSPE-dsl after dialysis of the detergent. It is observed that the inhibition rate of the fluorescence of L-polylysine (19, 2) -fluorescein-dsO, 4 reaches a value close to 30% for a Cu ++ concentration of 50 μM. Curve D represents the rate of inhibition of the fluorescence of the particles obtained from L-polylysine-NSPE-ds10 after dialysis of the detergent. It is observed that the rate of inhibition of the fluorescence of L- polylysine (19, 2) -fluorescein-dsO, 4 reaches a value close to 25% for a Cu + + concentration of 50 μM. Curve E represents the rate of inhibition of the fluorescence of the particles obtained from L-polylysine-NSPE-ds21 after dialysis of the detergent. It is observed that the inhibition rate of the fluorescence of L-polylysine (19, 2) - fluorescein-dsO, 4 reaches a value close to 20% for a Cu ++ concentration of 50 μM.
Ces résultats montrent que lorsque le taux de substitution des L-polylysine-NSPE-ds (n ) augmente la protection du complexe ADN / L-polylysine-NSPE-ds (n ) par les phospholipides libres augmente ce qui démontre la mise en place d'une membrane fonctionnelle fixée autour de la particule virale. 3) Taille des particules virales artificielles :These results show that when the substitution rate of L-polylysine-NSPE-ds (n) increases the protection of the DNA / L-polylysine-NSPE-ds (n) complex by free phospholipids increases, which demonstrates the establishment of d 'a functional membrane fixed around the viral particle. 3) Size of artificial viral particles:
L'analyse de la taille des particules virales artificielles effectuée avec un microanalyseur de particules, démontre la présence de particules dont la dimension moyenne est de 100 nm. 4) Application des particules virales artificielles.The analysis of the size of the artificial viral particles carried out with a microanalyzer of particles, demonstrates the presence of particles whose average size is 100 nm. 4) Application of artificial viral particles.
Les particules virales artificielles peuvent servir à titre thérapeutique pour assurer la délivrance intracellulaire in vivo de gênes thérapeutiques, par exemple pour le traitement de maladies génétiques (mucoviscidose ... ) , de certains cancers ou pour la préparation de vaccins géniques .The artificial viral particles can be used for therapeutic purposes to ensure the intracellular delivery in vivo of therapeutic genes, for example for the treatment of genetic diseases (cystic fibrosis, etc.), certain cancers or for the preparation of gene vaccines.
Les résultats obtenus indiquent clairement qu'une membrane étanche aux ions a été établie autour des complexes ADN/polylysine . Ils démontrent donc la possibilité d'établir une membrane fonctionnelle selon l'invention. Ils indiquent également que les complexes polyélectrolytiques sont bien isolés du milieu extérieur et n'ont plus la possibilité de se propager dans le milieu extérieur . EXEMPLE 8 : Préparation de particules synthétiques supramoléculaires renfermant de l'hémoglobine absorbée dans un substrat poreux (erythrocytes artificiels).The results obtained clearly indicate that an ion-tight membrane has been established around the DNA / polylysine complexes. They therefore demonstrate the possibility of establishing a functional membrane according to the invention. They also indicate that the polyelectrolytic complexes are well isolated from the external environment and no longer have the possibility of propagating in the external environment. EXAMPLE 8 Preparation of synthetic supramolecular particles containing hemoglobin absorbed in a porous substrate (artificial erythrocytes).
Dans cet exemple, on prépare des particules synthétiques supramoléculaires à partir d'un polymère polycationique polylysine-NSPE . a) Association de la L-polylysine-NSPE et des phospholipides formant bicouche : On dissout 9 mg de phospholipides EYPCIn this example, we prepare particles supramolecular synthetics from a polycationic polylysine-NSPE polymer. a) Association of L-polylysine-NSPE and of the bilayer-forming phospholipids: 9 mg of EYPC phospholipids are dissolved
(phosphatidylécholine de jaune d'oeuf) et un 1 mg de cholestérol dans 1 ml de chloroforme que l'on évapore sous pression réduite à 1 ' évaporateur rotatif. Les lipides sont ensuite dispersés dans 10 ml d'une solution aqueuse de détergent non ionique HECAMEG ® a. 40 mM . Lorsque la dispersion est complète, on rajoute 100 μl d'une solution du détergent non ionique 40 mM contenant 1 mg de L- polylysine ( 19 , 2 )-NSPE-ds6 , 25 obtenue comme indiqué aux exemples 1 et 2. La préparation micellaire est soniquée 1 min au bain à ultrasons, puis est ensuite mise à dialyser contre de l'eau distillée pour donner une suspension de la polylysine-NSPE et des phospholipides formant bicouche. On observe à l'analyseur de particules la présence de particules de taille moyenne comprise entre 300 et 600 nm . b) préparation de liposomes témoins neutres : on procède comme indiqué en a), mais sans ajouter la solution de polylysine-NSPE à la dispersion des lipides dans le détergent. On observe la présence de liposomes de taille moyenne comprise entre 200 e: 400 nm . c) absorption de l'hémoglobine ur le substrat poreux : 5 mg de particule poreuse (SEPHADEX ®(egg yolk phosphatidylecholine) and a 1 mg of cholesterol in 1 ml of chloroform which is evaporated under reduced pressure on a rotary evaporator. The lipids are then dispersed in 10 ml of an aqueous solution of non-ionic detergent HECAMEG® a. 40 mM. When the dispersion is complete, 100 μl of a 40 mM solution of nonionic detergent containing 1 mg of L-polylysine (19, 2) -NSPE-ds6, 25 obtained as indicated in Examples 1 and 2 are added. The micellar preparation is sonicated for 1 min in an ultrasonic bath, then is dialyzed against distilled water to give a suspension of the polylysine-NSPE and of the bilayer-forming phospholipids. The presence of particles of average size between 300 and 600 nm is observed in the particle analyzer. b) preparation of neutral control liposomes: proceed as indicated in a), but without adding the polylysine-NSPE solution to the dispersion of the lipids in the detergent. The presence of liposomes of average size between 200 e: 400 nm is observed. c) absorption of hemoglobin on the porous substrate: 5 mg of porous particle (SEPHADEX®
SPC50) d'un diamètre de 150 μm dérivées par des groupements suifopropyles et dont la taille des pores est suffisante pour laisser pénétrer des molécules d'un poids moléculaire maximum de 250000 sont dispersés dans 5 ml de tampon bistris 10 mM à pH 6,5 (où l'hémoglobine est cationique est attirée à l'intérieur des sites anioniques des particules poreuses). On ajoute à cette dispersion 20 mg d'hémoglobine extraite d'hématies humaines par la méthode de lyse en milieu hypotonique. La préparation est agitée pendant 24 h à 4° C sur un agitateur soleil. Les particules sont ensuite décantées et la quantité présente dans le surnageant est dosée par spectrométrie UV à 410 nm. Les résultats obtenus indiquent que plus de 98 % de l'hémoglobine est présente à l'intérieur des particules. L'hémoglobine non absorbée est éliminée par décantation des particules et lavage par le tampon bistris 10 mM . d) fixation de la membrane fonctionnelle : Les particules chargées en hémoglobineSPC50) with a diameter of 150 μm derived by suifopropyl groups and the size of the pores of which is sufficient to allow molecules of a maximum molecular weight of 250,000 to penetrate are dispersed in 5 ml of 10 mM bistris buffer at pH 6.5 (where hemoglobin is cationic is drawn into the anionic sites of porous particles). 20 mg of hemoglobin extracted from human red blood cells by the lysis method in a hypotonic medium are added to this dispersion. The preparation is stirred for 24 h at 4 ° C on a sun shaker. The particles are then decanted and the amount present in the supernatant is determined by UV spectrometry at 410 nm. The results obtained indicate that more than 98% of the hemoglobin is present inside the particles. Unabsorbed hemoglobin is removed by settling of the particles and washing with the 10 mM bistris buffer. d) fixation of the functional membrane: Particles loaded with hemoglobin
(24 mg ) obtenues en c) sont dispersées dans 4 ml de tampon bistris 10 mM à pH 6,5. On ajoute 1 ml de la suspension de polylysine-NSPE/phospholipides obtenue en a), et on agite pendant 2 h sur l'agitateur soleil à 4° C. On ajoute ensuite 625 μl d'une solution de détergent non ionique HECAMEG ® 40 mM pour atteindre une concentration finale de 5 mM . La suspension est encore agitée 5 min, puis on décante les particules et on lave le culot à 1 ' eau distillée pour éliminer le détergent et les complexes polylysine-NSPE/phospholipides en excès. e) Une expérience témoin est réalisée comme indiqué en d) mais en utilisant les liposomes témoins neutres obtenus en b) à la place de la suspension obtenue en a) .(24 mg) obtained in c) are dispersed in 4 ml of 10 mM bistris buffer at pH 6.5. 1 ml of the polylysine-NSPE / phospholipid suspension obtained in a) is added, and the mixture is stirred for 2 h on the sun shaker at 4 ° C. Then 625 μl of a solution of non-ionic detergent HECAMEG® 40 is added mM to reach a final concentration of 5 mM. The suspension is further stirred for 5 min, then the particles are decanted and the pellet is washed with distilled water to remove the detergent and the excess polylysine-NSPE / phospholipid complexes. e) A control experiment is carried out as indicated in d) but using the neutral control liposomes obtained in b) in place of the suspension obtained in a).
Les résultats obtenus démontrent que la structure membranaire des particules selon l'invention, comme dans le cas des erythrocytes naturels, est la seule barrière empêchant l'hémoglobine de sortir de la particule.The results obtained demonstrate that the membrane structure of the particles according to the invention, as in the case of natural erythrocytes, is the only barrier preventing hemoglobin from leaving the particle.
EXEMPLE 9 : Etude de la libération de l'hémoglobine :EXAMPLE 9 Study of the release of hemoglobin:
Dans cet exemple, on étudie de façon comparée la libération de l'hémoglobine absorbée à l'intérieur des particules poreuses obtenues à l'exemple 8.In this example, the release of the hemoglobin absorbed inside the porous particles obtained in example 8 is studied in a comparative manner.
Les particules chargées en hémoglobine sont incubées dans 5 ml de tampon PBS 150 mM à pH 7,4 (oùThe particles loaded with hemoglobin are incubated in 5 ml of 150 mM PBS buffer at pH 7.4 (where
1 ' hémoglobine est au-dessus de son point isoélectrique et a donc tendance à être exclue du substrat poreux). On agite à 4° C sur l'agitateur soleil. Des aliquots sont prélevés à intervalles de temps réguliers et la concentration d'hémoglobine présente dans le surnageant est dosée par spectrophotométrie . La figure 5 illustre les courbes de cinétique de libération de l'hémoglobine obtenue au cours du temps avec en ordonnée la masse en mg d'hémoglobine libérée et en abscisse le temps en heure. La courbe C51 correspond au résultat obtenu avec les particules simplement chargées d'hémoglobine issue de l'étape c) de l'exemple 7. La courbe C53 correspond aux résultats obtenus avec les particules obtenues à l'étape e) de l'exemple 8 (liposomes neutres obtenus en b) mis en contact dans la suspension polylysine-NSPE/phospholipides obtenue en a)). La courbe C52 correspond aux résultats obtenus avec les particules selon l'invention obtenues en d) de l'exemple 8.1 hemoglobin is above its isoelectric point and therefore tends to be excluded from the porous substrate). The mixture is stirred at 4 ° C. on the sun shaker. Aliquots are taken at regular time intervals and the concentration of hemoglobin present in the supernatant is determined by spectrophotometry. FIG. 5 illustrates the curves of kinetics of hemoglobin release obtained over time with the ordinate the mass in mg of hemoglobin released and on the abscissa the time in hours. Curve C51 corresponds to the result obtained with the particles simply charged with hemoglobin from step c) of Example 7. Curve C53 corresponds to the results obtained with the particles obtained in Step e) of Example 8 (neutral liposomes obtained in b) brought into contact in the polylysine-NSPE / phospholipid suspension obtained in a)). Curve C52 corresponds to the results obtained with the particles according to the invention obtained in d) of Example 8.
Comme on le voit, en l'absence de membranes fonctionnelles fixées (courbes C51 et C53) l'hémoglobine est quantitativement libérée des particules dès le début de l'incubation. Au contraire, avec les particules selon l'invention (courbe C52) dotées d'une membrane fonctionnelle fixée, l'hémoglobine n'est pas libérée et reste associée à l'intérieur des particules poreuses. Ces résultats démontrent donc que la membrane fonctionnelle fixée permet de retenir efficacement l'hémoglobine. EXEMPLE 10 : préparation de particules synthétiques supramoléculaires renfermant un anticancéreux :As can be seen, in the absence of fixed functional membranes (curves C51 and C53), the hemoglobin is quantitatively released from the particles at the start of the incubation. On the contrary, with the particles according to the invention (curve C52) endowed with a fixed functional membrane, the hemoglobin is not released and remains associated inside the porous particles. These results therefore demonstrate that the fixed functional membrane effectively retains hemoglobin. EXAMPLE 10 Preparation of synthetic supramolecular particles containing an anticancer agent:
On procède globalement comme dans 1 ' exemple 7, mais en incorporant de la doxorubicine (C27H29N011 ,PM543, 53) à la place de l'hémoglobine, 5 mg de chlorhydrate de doxorubicine sont dissous dans 1 ml d'eau distillée et incubés avec 5 mg de particules poreusesThe procedure is generally as in Example 7, but by incorporating doxorubicin (C27H29N011, PM543, 53) in place of hemoglobin, 5 mg of doxorubicin hydrochloride are dissolved in 1 ml of distilled water and incubated with 5 mg of porous particles
(SEPHADEX ® C25) dé ,rivées par des groupements carboxymethyles. Les pores des particules ont une taille permettant la pénétration des molécules de poids moléculaire maximum de 25000. La suspension est agitée 2 h à 4° C sur un agitateur soleil. Elle est ensuite décantée et la concentration de doxorubicine libre est mesurée par spectrophotométrie UV à 480 nm. Les résultats obtenus indiquent que plus de 97% de la doxorubicine est associé aux particules. La fraction de doxorubicine non absorbée est éliminée par décantation et lavage du culot à l'eau distillée.(SEPHADEX ® C25) dice, riveted by carboxymethyl groups. The pores of the particles have a size allowing the penetration of molecules with a maximum molecular weight of 25,000. The suspension is stirred for 2 h at 4 ° C. on a sun shaker. It is then decanted and the concentration of free doxorubicin is measured by UV spectrophotometry at 480 nm. The results obtained indicate that more than 97% of doxorubicin is associated with the particles. The fraction of doxorubicin which is not absorbed is eliminated by decantation and washing of the pellet with water. distilled.
Les particules chargées en doxorubicine sont dispersées dans 4 ml d'eau distillée. On ajoute ensuite 1 ml de la suspension de polylysine-NSPE et des phospholipides formant bicouche obtenue à l'étape a) de l'exemple 7, et on agite doucement pendant 2 h à 4° C. On ajoute alors 625 μl de solution de détergent non ionique HECAMEG 40 mM, on agite 5 min, et on décante les particules et on lave le culot à l'eau distillée pour éliminer le détergent et les complexes polylysine- NSPE/phospholipides en excès.The particles loaded with doxorubicin are dispersed in 4 ml of distilled water. Then 1 ml of the polylysine-NSPE suspension and of the bilayer phospholipids obtained in step a) of example 7 are added, and the mixture is gently stirred for 2 h at 4 ° C. Then 625 μl of solution of 40 mM non-ionic detergent HECAMEG, stirred for 5 min, and the particles are decanted and the pellet is washed with distilled water to remove the detergent and the excess polylysine-NSPE / phospholipid complexes.
Comme dans l'exemple 8, une expérience témoin est réalisée en utilisant une suspension de liposomes neutres composés uniquement de phospholipides (EYPC) et de cholestérol.As in Example 8, a control experiment is carried out using a suspension of neutral liposomes composed solely of phospholipids (EYPC) and cholesterol.
EXEMPLE 11 : Etude de la libération de la doxorubicine :EXAMPLE 11 Study of the release of doxorubicin:
On étudie la cinétique de libération de la doxorubicine par les particules obtenues à l'exemple 10.The kinetics of release of doxorubicin by the particles obtained in example 10 are studied.
Les particules sont dispersées dans 150 ml de tampon PPS 150 mM à pH 7,1 et agitées doucement. Des aliquots sont prélevés à intervalles de temps réguliers et la concentration de doxorubicine dans le surnageant est mesurée par spectrophotométrie UV à 480 nm.The particles are dispersed in 150 ml of 150 mM PPS buffer at pH 7.1 and gently stirred. Aliquots are taken at regular time intervals and the concentration of doxorubicin in the supernatant is measured by UV spectrophotometry at 480 nm.
Les résultats obtenus indiquent que, au bout de 1 h, 45 % en poids de la doxorubicine a été libéré des particules de l'expérience témoin et des particules chargées en doxorubicine utilisées avant la mise en contact avec la suspension polylysine-NSPE/phospholipides, c'est-à- dire des particules exemptes de membrane fonctionnelle fixée. Au contraire, avec les particules selon l'invention comprenant une membrane fonctionnelle fixée, seulement 5 % de la doxorubicine a été libéré. Ainsi, la membrane fonctionnelle fixée formée des phospholipides permet de retenir efficacement la doxorubicine à l'intérieur des particules selon l'invention.The results obtained indicate that, after 1 hour, 45% by weight of the doxorubicin has been released from the particles of the control experiment and from the particles loaded with doxorubicin used before being brought into contact with the polylysine-NSPE / phospholipid suspension, that is to say particles free of a fixed functional membrane. On the contrary, with the particles according to the invention comprising a fixed functional membrane, only 5% of the doxorubicin was released. Thus, the fixed functional membrane formed of phospholipids makes it possible to effectively retain doxorubicin inside the particles according to the invention.
EXEMPLE 12 : préparation de particules synthétiques supramoléculaires de petites tailles :EXAMPLE 12 Preparation of small supramolecular synthetic particles:
On procède comme dans l'exemple 10, mais on cherche à préparer des particules poreuses de plus petite taille. 1 g de matrice poreuse SEPHADEX ® C25 est disperse dans 100 ml d'eau distillée et la préparation est broyée pendant 15 min avec un broyeur à hélice. La préparation est ensuite centrifugée à 3000 g pendant 10 min, le surnageant est récupéré puis soumis à une autre centrifugation àWe proceed as in Example 10, but we seeks to prepare smaller porous particles. 1 g of porous matrix SEPHADEX® C25 is dispersed in 100 ml of distilled water and the preparation is ground for 15 min with a propeller mill. The preparation is then centrifuged at 3000 g for 10 min, the supernatant is recovered and then subjected to another centrifugation at
25000 g pendant 45 min. Le surnageant est éliminé, le culot est remis en suspension et on obtient 50 mg de produit sec après lyophilisation. La taille des particules obtenues, mesurée à l'analyseur de particules, est comprise entre 300 et 500 nm .25,000 g for 45 min. The supernatant is removed, the residue is resuspended and 50 mg of dry product are obtained after lyophilization. The size of the particles obtained, measured with the particle analyzer, is between 300 and 500 nm.
5 mg de ces particules poreuses de petite taille sont dispersés dans 5 ml d'eau distillée. On ajoute 5 mg de doxorubicine et on agite pendant 2 h. La concentration de doxorubicine non associée aux particules est déterminée par ultrafiltration d'un aliquot de la suspension sur une membrane d ' ultrafiltration dont le seuil de séparation est à 50000, et on mesure la concentration de la doxorubicine libre par spectrométrie UV à 480 nm . Les résultats obtenus indiquent que plus de 98 % de la doxorubicine est associé aux particules. Celles-ci sont ensuite utilisées telles quelles, sans purification supplémentaire .5 mg of these small porous particles are dispersed in 5 ml of distilled water. 5 mg of doxorubicin are added and the mixture is stirred for 2 h. The concentration of doxorubicin not associated with the particles is determined by ultrafiltration of an aliquot of the suspension on an ultrafiltration membrane whose separation threshold is at 50,000, and the concentration of free doxorubicin is measured by UV spectrometry at 480 nm. The results obtained indicate that more than 98% of doxorubicin is associated with the particles. These are then used as is, without further purification.
Pour associer ces particules poreuses de petites tailles à des complexes polylysine- NSPE/phopholipides aptes à former une membrane fonctionnelle fixée sur ces particules, on prépare des complexes de petites tailles. Pour ce faire, on dissout 9 mg de phospholipides EYPC et 1 mg de cholestérol dans un 1 ml de chloroforme que l'on évapore sous pression réduite à 1 ' évaporateur rotatif. Les lipides sont ensuite dispersés dans 2,5 ml d'une solution aqueuse de détergent non ioniqueIn order to associate these small porous particles with polylysine-NSPE / phopholipid complexes capable of forming a functional membrane fixed on these particles, small size complexes are prepared. To do this, 9 mg of phospholipids EYPC and 1 mg of cholesterol are dissolved in 1 ml of chloroform which is evaporated under reduced pressure on a rotary evaporator. The lipids are then dispersed in 2.5 ml of an aqueous solution of nonionic detergent
®®
HECAMEG a 40 mM . Lorsque la dispersion est complète, on ajoute 100 μl d'une solution de L-polylysine ( 19 , 2 )-NSPE- ds6,25 obtenue comme indiqué aux exemples 1 et 2. La préparation micellaire est soniquée pendant 1 min au bain à ultrasons, puis est ensuite diluée rapidement dans 10 ml d'eau distillée et mise à dialyser contre de l'eau distillée pour donner une préparation de complexes polylysine-NSPE/phospholipides de petite taille. On observe à 1 ' analyseur de particules la présence de particules dont la taille moyenne est de 50 nm. On prépare ensuite les particules poreuses de petites tailles selon l'invention. On ajoute 625 μl de solution de détergent non ionique HECAMEG ® 40 mM a 2,5 ml de la suspension de polylysine-NSPE/phospholipides préparée précédemment, de façon à obtenir une concentration de 10 mM en détergent. La suspension est soniquee 1 min au bain à ultrasons et est ajoutée lentement à 2,5 ml de la suspension de particules poreuses de petite taille chargées en doxorubicine préparée précédemment, puis placée dans une cellule de dialyse continue, maintenue sous agitation.HECAMEG at 40 mM. When the dispersion is complete, 100 μl of a solution of L-polylysine (19, 2) -NSPE- ds6.25 obtained as indicated in Examples 1 and 2 is added. The micellar preparation is sonicated for 1 min in an ultrasonic bath , then is quickly diluted in 10 ml of distilled water and dialyzed against water distilled to give a preparation of small polylysine-NSPE / phospholipid complexes. The presence of particles with an average size of 50 nm is observed with the particle analyzer. The porous particles of small sizes according to the invention are then prepared. 625 μl of 40 mM HECAMEG® nonionic detergent solution are added to 2.5 ml of the suspension of polylysine-NSPE / phospholipids prepared previously, so as to obtain a concentration of 10 mM in detergent. The suspension is sonicated for 1 min in an ultrasonic bath and is added slowly to 2.5 ml of the suspension of small porous particles loaded with doxorubicin prepared above, then placed in a continuous dialysis cell, kept under stirring.
EXEMPLE 13 : Etude de la libération de la doxorubicine :EXAMPLE 13 Study of the release of doxorubicin:
Les particules préparées à l'exemple 12 sont dispersées dans 150 ml de tampon PBS à pH 7,4 150 mM et agitées doucement. Des aliquots sont prélevés à intervalles de temps réguliers, la concentration de doxorubicine libérée est mesurée par spectrophotométrie UV à 480 nm après ultrafiltration sur une membrane dont le seuil de filtration est à 50000.The particles prepared in Example 12 are dispersed in 150 ml of PBS buffer at pH 7.4 150 mM and gently stirred. Aliquots are taken at regular time intervals, the concentration of doxorubicin released is measured by UV spectrophotometry at 480 nm after ultrafiltration on a membrane whose filtration threshold is at 50,000.
Les résultats obtenus démontrent que au bout de 4 h, 65 % de la doxorubicine a été libéré des particules sans membrane fixée (avant mise en contact avec la suspension de polylysine-NSPE/phospholipides) alors que seulement 10 % de la doxorubicine a été libéré des particules dotées d'une membrane fonctionnelle fixée selon 1 ' invention.The results obtained demonstrate that after 4 h, 65% of the doxorubicin has been released from the particles without a fixed membrane (before contacting with the suspension of polylysine-NSPE / phospholipids) while only 10% of the doxorubicin has been released. particles with a functional membrane attached according to the invention.
EXEMPLE 14 : Etude de la complexation polyélectrolytique de la polylysine-NSPE sur les particules poreuses de 1 ' exemple 8. . Méthodologie : On utilise un marqueur fluorescentEXAMPLE 14 Study of the polyelectrolytic complexation of polylysine-NSPE on the porous particles of Example 8. Methodology: We use a fluorescent marker
(commercialisé par la Société Molécular Probes) constitué d'un phospholipide, la dipalmitoylphosphatidyléthanolamine, dérivé par un groupement fluorescent, la rhodamine . Ce composé de nature phospholipidique permet de visualiser les phospholipides, soit sous forme de motifs lorsqu'ils sont associés entre eux, soit sous forme de fluorescence diffuse lorsqu'ils sont sous forme soluble ou très dispersés. L'observation se fait à la fois en contraste de phase et en microscopie à fluorescence (A ex 510-560 nm, λ em 590 nm) . . Préparation des liposomes fluorescents témoins :(marketed by the Molecular Probes Company) consisting of a phospholipid, dipalmitoylphosphatidylethanolamine, derived from a fluorescent group, rhodamine. This compound of phospholipid nature makes it possible to visualize phospholipids, either in the form of patterns when they are associated with each other, or in the form of diffuse fluorescence when they are in soluble or very dispersed form. The observation is made both in phase contrast and in fluorescence microscopy (A ex 510-560 nm, λ em 590 nm). . Preparation of control fluorescent liposomes:
9 mg d'EYPC, 1 mg de cholestérol et 0,05 mg de dipalmitoyl phosphatidyl ethanolamine rhodamine (DPPERd) sont dissouts dans 1 ml de chloroforme puis évaporé sous pression réduite. Le résidu est repris par 10 ml d'une solution d HECAMEG ® 40 m . Lorsque la solution est devenue complètement limpide, elle est mise à dialyser contre de l'eau distillée. Les liposomes fluorescents obtenus se distinguent très mal à l'observation et apparaissent, par suite de leur petite taille, sous forme d'un bruit de fond uniforme .9 mg of EYPC, 1 mg of cholesterol and 0.05 mg of dipalmitoyl phosphatidyl ethanolamine rhodamine (DPPERd) are dissolved in 1 ml of chloroform and then evaporated under reduced pressure. The residue is taken up in 10 ml of a 40 m HECAMEG® solution. When the solution has become completely clear, it is dialyzed against distilled water. The fluorescent liposomes obtained are very difficult to distinguish upon observation and appear, owing to their small size, in the form of a uniform background noise.
Préparation de complexes polylysine-NSPE/phospholipides fluorescents : On opère comme précédemment en ajoutant 100 μl d'une solution d ' HECAMEG ® 40 mM contenant 1 mg de L- polylysine ( 19 , 2 )-NSPE-d6 à la solution des lipides en détergent. La préparation est soniquee 1 min au bain à ultrasons puis mise à dialyser contre de l'eau distillée. Les complexes polylysine-NSPE/phospholipides fluorescents ont une taille supérieure à celle des liposomes témoins et sont visibles sous forme de petites taches fluorescentes.Preparation of polylysine-NSPE / fluorescent phospholipid complexes: The procedure is as above by adding 100 μl of a 40 mM HECAMEG® solution containing 1 mg of L- polylysine (19, 2) -NSPE-d6 to the solution of lipids. detergent. The preparation is sonicated for 1 min in an ultrasonic bath and then dialyzed against distilled water. The polylysine-NSPE / fluorescent phospholipid complexes are larger than the control liposomes and are visible in the form of small fluorescent spots.
Interaction entre les complexes polylysine- NSPE/phospholipides et les particules poreuses de SEPHADEX ® SPC50. Les particules de SEPHADEX ® sont d abord visualisées en microscopie à contraste de phase. Elles se présentent sous forme de sphères régulières d'un diamètre compris entre 100 et 150 μm. Ces particules (0,1 mg dans 0,5 ml sont ensuite mises à incuber avec 20 μl de la préparation des complexes polylysine-NSPE/phospholipides fluorescents .Interaction between the polylysine- NSPE / phospholipid complexes and the porous particles of SEPHADEX ® SPC50. The particles of SEPHADEX ® are first visualized by phase contrast microscopy. They are in the form of regular spheres with a diameter between 100 and 150 μm. These particles (0.1 mg in 0.5 ml are then incubated with 20 μl of the preparation of the polylysine-NSPE / fluorescent phospholipid complexes.
A l'observation, on perçoit nettement l'adhésion des premières entités fluorescentes à la surface de la particule. A un stade d'association plus avancé, on distingue une couronne fluorescente dense mais discontinue avec une juxtaposition de petits points fluorescents. Ces résultats indiquent que, grâce à leur charge positive, les complexes polylysine-NSPE/phospholipides sont attirées par la surface des particules qui est chargée négativement. Le fait d'avoir une couronne avec une structure discontinue indique, par ailleurs, que les entités ne fusionnent pas entre elles .Upon observation, we clearly perceive the adhesion of the first fluorescent entities to the surface of the particle. At a more advanced stage of association, there is a dense but discontinuous fluorescent ring with a juxtaposition of small fluorescent dots. These results indicate that, thanks to their positive charge, the polylysine-NSPE / phospholipid complexes are attracted to the surface of the particles which is negatively charged. The fact of having a crown with a discontinuous structure indicates, moreover, that the entities do not merge together.
. Les particules obtenues sont ensuite incubées avec 2 ml d'un tampon PBS 150 mM NaCl pendant 10 min. On constate à l'observation que l'aspect des particules reste inchangé. L'énergie impliquée dans la formation de la complexation polyélectrolytique est, en effet, considérable et cette complexation reste stable dans un grand intervalle de pH et de force ionique.. The particles obtained are then incubated with 2 ml of 150 mM NaCl PBS buffer for 10 min. It is observed that the appearance of the particles remains unchanged. The energy involved in the formation of polyelectrolytic complexation is, indeed, considerable and this complexation remains stable over a large range of pH and ionic strength.
. La préparation est alors décantée et incubée avec 2 ml d une solution d HECAMEG ® 5 mM pendant 10 min. On constate que l'aspect de la couronne a été modifié et devient complètement régulier. Ce résultat indique que les entités qui s ' étaient accrochées individuellement à la surface des particules ont maintenant fusionnées entre elles pour former une bicouche continue phospholipidique fixée au substrat particulaire .. The preparation is then decanted and incubated with 2 ml of a 5 mM HECAMEG® solution for 10 min. It can be seen that the appearance of the crown has been modified and is becoming completely regular. This result indicates that the entities which had hung individually on the surface of the particles have now merged with each other to form a continuous phospholipid bilayer fixed to the particulate substrate.
. La préparation est alors décantée à nouveau et incubée avec 2 ml d'une solution d ' HECAMEG ® 40 mM pendant 10 min.. The preparation is then decanted again and incubated with 2 ml of a 40 mM HECAMEG® solution for 10 min.
On constate que l'aspect des particules a, à nouveau, complètement changé. Il n'est plus possible de visualiser les particules grâce à la fluorescence. Au contraire, les particules semblent plutôt apparaître en noir sur un fond de fluorescence diffuse provenant de la solubilisation des phospholipides par la solution d ' HECAMEG ® 40 mM. Cette expérience indique que les phospholipides étaient bien attachés aux particules par des interactions hydrophobes.It can be seen that the appearance of the particles has again completely changed. It is no longer possible to visualize particles thanks to fluorescence. On the contrary, the particles seem rather to appear in black on a background of diffuse fluorescence originating from the solubilization of the phospholipids by the solution of HECAMEG® 40 mM. This experiment indicates that the phospholipids were well attached to the particles by hydrophobic interactions.
Un témoin est, de plus, réalisé avec de liposomes fluorescents neutres incubés avec des particules de SEPHADEX ® SPC50. On ne constate aucune interaction entre les particules qui apparaissent en noir et les liposomes qui apparaissent en fond uniforme fluorescent. Ce résultat indique que les propriétés d'adhésion des complexes polylysine-NSPE/phospholipides sont bien dues à la présence de la polylysine-NSPE/phospholipidée .A control is, moreover, produced with neutral fluorescent liposomes incubated with particles of SEPHADEX® SPC50. There is no interaction between the particles which appear in black and the liposomes which appear in a uniform fluorescent background. This result indicates that the adhesion properties of the polylysine-NSPE / phospholipid complexes are indeed due to the presence of the polylysine-NSPE / phospholipid.
. Efficacité de la complexation polyélectrolytique :. Efficiency of polyelectrolytic complexation:
Cette efficacité est illustrée par les deux expériences suivantes :This efficiency is illustrated by the following two experiences:
1) Réalisation d'un complexe polyélectrolytique entre des particules de SEPHADEX ® SPC25 (de taille environ 80 μm) dont la surface est couverte de charges négatives sont incubées avec 200 μl d'une solution de L-polylysine ( 19 , 2 )- fluorescine-dsO , 4 telle que préparée à l'exemple 5. Ces particules sont d'abord observées en contraste de phase. L'observation en fluorescence (A ex 458-1) Realization of a polyelectrolytic complex between particles of SEPHADEX ® SPC25 (of size approximately 80 μm) whose surface is covered with negative charges are incubated with 200 μl of a solution of L-polylysine (19, 2) - fluorescine -dsO, 4 as prepared in Example 5. These particles are first observed in phase contrast. Fluorescence observation (A ex 458-
490, A em 515-565 nm) après une incubation d'une durée de 10 s révèle déjà la présence de fluorescence autour de la particule .490, A em 515-565 nm) after an incubation for a period of 10 s already reveals the presence of fluorescence around the particle.
Dans une autre préparation, les particules sont incubées de la même façon pendant 5 min avec la solution de polylysine fluorescente. Elles sont ensuite lavées à l'eau distillée, incubées avec 2 ml de tampon PBS 150 mM NaCl pendant 10 min et enfin lavées avec 2 fois 2 ml de tampon PBS. Les résultats de l'observation indiquent que la fluorescence s'est apparemment maintenue quantitativement autour de la particule. De même, aucune modification n'est apportée par une incubation avec une solution d 'HECAMEG ® 40 mM .In another preparation, the particles are incubated in the same way for 5 min with the fluorescent polylysine solution. They are then washed with distilled water, incubated with 2 ml of 150 mM PBS buffer NaCl for 10 min and finally washed with 2 times 2 ml of PBS buffer. The results of the observation indicate that the fluorescence has apparently been maintained quantitatively around the particle. Likewise, no modification is made by incubation with a 40 mM HECAMEG® solution.
2) Réalisation d'un complexe polyélectrolytique entre des particules de silice poreuse et la L-polylysine (19,2) fluorescente .2) Realization of a polyelectrolytic complex between particles of porous silica and fluorescent L-polylysine (19,2).
0,1 mg de particules de silice (taille des pores 60 Angstrôm, taille des particules 40 μm) sont incubés avec 200 μl de la solution de polylysine fluorescente pendant 5 min puis décantées et lavées avec 2 ml d'eau distillée. Les résultats sont observés en contraste de phase et en fluorescence. Ils indiquent que la polylysine présente une forte affinité pour la silice. Une incubation de ces particules avec 2 ml de tampon PBS 150 mM NaCl indique que cette affinité n'est pas affectée par ce tampon, la stabilité des interactions polyélectrolytiques entre la polylysine et la silice est donc également très forte avec un matériau comme la silice.0.1 mg of silica particles (pore size 60 Angstroms, particle size 40 μm) are incubated with 200 μl of the fluorescent polylysine solution for 5 min, then decanted and washed with 2 ml of distilled water. The results are observed in phase contrast and fluorescence. They indicate that polylysine has a strong affinity for silica. A incubation of these particles with 2 ml of PBS 150 mM NaCl buffer indicates that this affinity is not affected by this buffer, the stability of the polyelectrolytic interactions between polylysine and silica is therefore also very strong with a material such as silica.
Ces résultats indiquent donc que les matériaux à base de silice sont capables d' interagir fortement avec des polycations comme la polylysine. Ainsi, en particulier, tout matériau à base de verre peut être recouvert d'une membrane fonctionnelle selon l'invention et être utilisé à titre de substrat solide.These results therefore indicate that materials based on silica are capable of interacting strongly with polycations such as polylysine. Thus, in particular, any glass-based material can be covered with a functional membrane according to the invention and be used as a solid substrate.
EXEMPLE 15 : Préparation d'un film synthétique supramoléculaire selon l'invention.EXAMPLE 15 Preparation of a supramolecular synthetic film according to the invention.
Dans cet exemple, on fixe une membrane fonctionnelle phospholipidique sur un substrat poreux plan formé d'un filtre d'échange d'ions.In this example, a functional phospholipid membrane is fixed on a flat porous substrate formed by an ion exchange filter.
On prépare une suspension polylysine- NSPE/phospholipides comme indiqué à l'étape a) de l'exemple 6, mais avec 20 % en poids de cholestérol par rapport au phospholipides EYPC. On dissout 2 ml de cette suspension dans 200 ml d'eau distillée, et on place le mélange dans une cellule d ' ultrafiltration de 47 mm de diamètre et équipée d un filtre anionique Gelman (référence 60943) de porosité de 0,445 μm et diamètre égal à 47 mm. Ce filtre anionique forme donc le substrat poreux solide du film. La pression de la cellule est ajustée pour obtenir un débit initial de 2 ml/min. Lorsque le débit devient inférieur à 0,3 ml/min, indiquant que les complexes polylysine- NSPE/phospholipides ont recouvert la surface du filtre et ont obturé les pores, on arrête 1 ' ultrafiltration . On élimine la plus grande partie du surnageant et on ajoute 100 ml d'une solution 5 mM de détergent non ionique. On réajuste la pression pour obtenir un débit de 1 ml/min jusqu'à ce que les deux tiers de la solution aient été filtrés. L'adjonction de la solution de détergent non ionique 5 mM a pour effet de former la bicouche de phospholipides sur le filtre anionique. En effet, ce détergent non ionique introduit en dessous de sa concentration micellaire critique permet de faire fusionner les complexes polylysine-NSPE/phospholipides à la surface du filtre. On élimine alors la plus grande partie du surnageant et on lave avec précaution le filtre plusieurs fois avec de l'eau distillée en remettant la pression pour faire passer l'eau distillée au travers du filtre.A polylysine-NSPE / phospholipid suspension is prepared as indicated in step a) of Example 6, but with 20% by weight of cholesterol relative to the phospholipids EYPC. 2 ml of this suspension are dissolved in 200 ml of distilled water, and the mixture is placed in an ultrafiltration cell 47 mm in diameter and equipped with a Gelman anionic filter (reference 60943) with a porosity of 0.445 μm and equal diameter at 47 mm. This anionic filter therefore forms the solid porous substrate of the film. The cell pressure is adjusted to obtain an initial flow rate of 2 ml / min. When the flow rate falls below 0.3 ml / min, indicating that the polylysine-NSPE / phospholipid complexes have covered the surface of the filter and have closed the pores, the ultrafiltration is stopped. Most of the supernatant is removed and 100 ml of a 5 mM solution of nonionic detergent is added. The pressure is readjusted to obtain a flow rate of 1 ml / min until two-thirds of the solution has been filtered. The addition of the 5 mM non-ionic detergent solution has the effect of forming the phospholipid bilayer on the anionic filter. Indeed, this non-ionic detergent introduced below its critical micellar concentration allows the polylysine-NSPE / phospholipid complexes to fuse on the surface of the filter. Most of the supernatant is then removed and the filter is carefully washed several times with distilled water, putting the pressure back to pass the distilled water through the filter.
Une expérience témoin est réalisée en utilisant des liposomes neutres comme dans l'exemple 8.A control experiment is carried out using neutral liposomes as in Example 8.
On prépare, en outre, une autre expérience en fixant sur le filtre une membrane fonctionnelle contenant un ionophore (agent facilitant la diffusion des ions). Pour ce faire, on prépare une suspension de polylysine-NSPE/phospholipides comme indiqué précédemment dans cet exemple, mais en ajoutant 0,1 mg de monensin (ionophore) à la solution chloroformique de phospholipides EYPC (1% en poids du total). La suspension est ensuite utilisée pour établir la membrane sur le filtre de la même manière que précédemment indiqué. EXEMPLE 16 : Etude de l'imperméabilité aux ions des films. On introduit dans la cellule d ' ultrafiltration 200 ml d'une solution de NaCl à 25 g/1 dont la résistivité est de 55 mS/cm. On établit la pression pour obtenir un débit de 0,5 ml/min et on collecte des fractions de 1 ml sur lesquelles on mesure la conductivité . Les expériences sont réalisées dans différentes conditions avec : un filtre seul, un filtre issu de l'expérience témoin associé à des liposomes neutres, un film synthétique selon l'invention formé du filtre et de la membrane fonctionnelle fixée, et un film selon l'invention formé du filtre doté d'une membrane fonctionnelle fixée contenant un ionophore. Les résultats obtenus sont exprimés dans le tableau suivant : ConductivitéAnother experiment is also prepared by fixing a functional membrane containing an ionophore (agent facilitating the diffusion of ions) on the filter. To do this, a suspension of polylysine-NSPE / phospholipids is prepared as indicated previously in this example, but by adding 0.1 mg of monensin (ionophore) to the chloroform solution of phospholipids EYPC (1% by weight of the total). The suspension is then used to establish the membrane on the filter in the same manner as previously indicated. EXAMPLE 16 Study of the impermeability to ions of films. 200 ml of a 25 g / l NaCl solution, the resistivity of which is 55 mS / cm, are introduced into the ultrafiltration cell. The pressure is established to obtain a flow rate of 0.5 ml / min and fractions of 1 ml are collected on which the conductivity is measured. The experiments are carried out under different conditions with: a filter alone, a filter resulting from the control experiment associated with neutral liposomes, a synthetic film according to the invention formed of the filter and the fixed functional membrane, and a film according to the invention formed of the filter provided with a fixed functional membrane containing an ionophore. The results obtained are expressed in the following table: Conductivity
(en % de la conductivité de la solution de NaC initiale)(in% of the conductivity of the initial NaC solution)
Fractions 1 2 3 4 5 6 7 8Fractions 1 2 3 4 5 6 7 8
Nature du filmNature of the film
Filtre seul 50 100 100 100 100 100 100 100Filter only 50 100 100 100 100 100 100 100
Filtre + liposomes neutres 55 100 100 100 100 100 100 100Filter + neutral liposomes 55 100 100 100 100 100 100 100
Filtre + membrane fixée 1 1 1 2 2 2 2 2Filter + membrane attached 1 1 1 2 2 2 2 2
Filtre + membrane fixéeFilter + membrane attached
+ ionophore 4 6 6 7 7 7 7 7+ ionophore 4 6 6 7 7 7 7 7
Ces résultats indiquent que la membrane fixée permet bien de retenir les ions alors que le filtre seul et le filtre associé aux liposomes neutres sont complètement inefficaces. Les résultats montrent aussi que cet effet est partiellement inversé par la présence au sein de la membrane d'un composé ionophore dont la fonction est de transporter les ions à travers les bicouches.These results indicate that the fixed membrane allows to retain ions while the filter alone and the filter associated with neutral liposomes are completely ineffective. The results also show that this effect is partially reversed by the presence within the membrane of an ionophore compound whose function is to transport the ions through the bilayers.
Ainsi, les membranes fonctionnelles fixées selon 1 ' invention sur des supports plans conservent les mêmes propriétés de perméabilité sélective que les membranes plasmiques des cellules eucaryotes, et peuvent comme elles, s ' étendrent sur des surfaces considérables tout en restant fonctionnelles. Un film selon l'invention peut être utilisé pour extraire ou séparer des sels et/ou des ions à partir d'une solution liquide par filtration.Thus, the functional membranes fixed according to the invention on planar supports retain the same selective permeability properties as the plasma membranes of eukaryotic cells, and can like them, extend over considerable surfaces while remaining functional. A film according to the invention can be used to extract or separate salts and / or ions from a liquid solution by filtration.
La figure 6 illustre en détail mais de façon schématique la composition d'une structure membranaire selon l'invention, et donc une portion de film selon l'invention. Cette structure comprend une bicouche 63 formant une membrane fonctionnelle, des L-polylysine-NSPE polycationiques 62, et le substrat poreux 61" formé du filtre. Les L-polylysines-NSPE forment des chaînes polymériques polycationiques 64 et portent des ligands membranaires 65 dont les chaînes phospholipidiques 66 s'insèrent par interaction lyotropiques au sein de la membrane fonctionnelle 63. L'invention peut faire l'objet de nombreuses variantes et applications. En particulier d'autres polymères polycationiques ou polyanioniques peuvent être utilisés à titre de composés bifonctionnels ; d'autres composés amphiphiles peuvent être utilisés pour former la membrane ; d'autres substrats solides polyioniques peuvent être utilisés dès lors qu'ils présentent une densité superficielle de charges électriques positives et/ou négatives. FIG. 6 illustrates in detail but schematically the composition of a membrane structure according to the invention, and therefore a portion of film according to the invention. This structure comprises a bilayer 63 forming a functional membrane, polycationic L-polylysine-NSPE 62, and the porous substrate 61 " formed of the filter. The L-polylysine-NSPE form polycationic polymer chains 64 and carry membrane ligands 65, the phospholipid chains 66 are inserted by lyotropic interaction within the functional membrane 63. The invention can be subject to numerous variants and applications. In particular, other polycationic or polyanionic polymers can be used as bifunctional compounds; other amphiphilic compounds can be used to form the membrane; other solid polyionic substrates can be used as soon as they have a surface density of positive and / or negative electrical charges.

Claims

REVENDICATIONS 1/ - Structure membranaire artificielle analogue aux membranes plasmiques naturelles fixées, caractérisée en ce qu'elle comprend : - un substrat (31, 61) en phase solide présentant une surface dotée d'une densité superficielle de charges électriques,CLAIMS 1 / - Artificial membrane structure analogous to fixed natural plasma membranes, characterized in that it comprises: - a substrate (31, 61) in solid phase having a surface with a surface density of electrical charges,
- une membrane fonctionnelle (33, 63) stable de composés amphiphiles et qui présente une surface libre s ' étendant à l'opposé du substrat, ladite surface libre étant adaptée pour pouvoir être placée au contact d'un milieu, dit milieu externe, avec une forme telle qu'elle ne circonscrit pas ce milieu externe,- A functional membrane (33, 63) stable of amphiphilic compounds and which has a free surface extending opposite the substrate, said free surface being adapted to be able to be placed in contact with a medium, said external medium, with a form such that it does not circumscribe this external medium,
- au moins un composé (32, 62) bifonctionnel de fixation de la membrane fonctionnelle (33,- at least one bifunctional compound (32, 62) for fixing the functional membrane (33,
63) sur le substrat (31, 61), inséré entre la membrane et le substrat, et dont la structure chimique comprend :63) on the substrate (31, 61), inserted between the membrane and the substrate, and the chemical structure of which comprises:
. au moins une chaîne (64) polyionique adaptée pour coopérer par complexation polyélectrolytique avec la densité superficielle de charges électriques du substrat ( 31 , 61 ) , au moins un ligand membranaire (65, 66) lié par liaison covalente à une telle chaîne polyionique, et adapté pour former une liaison lyotropique stable non covalente avec les composés amphiphiles de la membrane fonctionnelle (33, 63), sans affecter significative ent les propriétés fonctionnelles de la membrane fonctionnelle (33, 63).. at least one polyionic chain (64) adapted to cooperate by polyelectrolytic complexation with the surface density of electrical charges of the substrate (31, 61), at least one membrane ligand (65, 66) linked by covalent bond to such a polyionic chain, and suitable for forming a stable, non-covalent lyotropic bond with the amphiphilic compounds of the functional membrane (33, 63), without significantly affecting the functional properties of the functional membrane (33, 63).
2/ - Structure membranaire selon la revendication 1 , caractérisée en ce que les composés amphiphiles de la membrane fonctionnelle (33, 63) sont des composés phospholipidiques.2 / - Membrane structure according to claim 1, characterized in that the amphiphilic compounds of the functional membrane (33, 63) are phospholipid compounds.
3/ - Structure membranaire selon l'une des revendications 1 et 2, caractérisée en ce qu'elle comprend au moins un composé (32, 62) bifonctionnel de fixation dont la structure chimique comprend au moins une pluralité de ligands membranaires (65, 66).3 / - Membrane structure according to one of claims 1 and 2, characterized in that it comprises at least one bifunctional fixing compound (32, 62) whose chemical structure comprises at least a plurality of membrane ligands (65, 66 ).
4/ - Structure membranaire selon la revendication 3, caractérisée en ce que les ligands membranaires (65, 66) sont répartis sur la molécule du composé (32, 62) bifonctionnel de fixation à une distance les uns des autres qui est supérieure à celle séparant les composés amphiphiles qui se jouxtent dans une couche de la membrane fonctionnelle (33, 63), de sorte que la membrane fonctionnelle (33, 63) présente des composés amphiphiles qui ne sont pas liés à un ligand membranaire (65, 66).4 / - Membrane structure according to the claim 3, characterized in that the membrane ligands (65, 66) are distributed over the molecule of the bifunctional fixing compound (32, 62) at a distance from each other which is greater than that separating the amphiphilic compounds which are contiguous in a layer of the functional membrane (33, 63), so that the functional membrane (33, 63) has amphiphilic compounds which are not bound to a membrane ligand (65, 66).
5/ - Structure membranaire selon l'une des revendications 3 ou 4 , caractérisée en ce que le composé (32, 62) bifonctionnel de fixation présente un nombre de charges ioniques unitaires de même signe aptes à coopérer par complexation polyélectrolytique avec la densité superficielle de charges électriques du substrat, supérieur au nombre de ligands membranaires (65, 66).5 / - Membrane structure according to one of claims 3 or 4, characterized in that the bifunctional fixing compound (32, 62) has a number of unit ion charges of the same sign capable of cooperating by polyelectrolytic complexation with the surface density of electrical charges of the substrate, greater than the number of membrane ligands (65, 66).
6/ - Structure membranaire selon la revendication 5, caractérisée en ce que les charges ioniques sont réparties sur la molécule du composé (32, 62) bifonctionnel de fixation à une distance les unes des autres qui est inférieure à la plus petite distance séparant deux ligands membranaires (65, 66).6 / - Membrane structure according to claim 5, characterized in that the ionic charges are distributed over the molecule of the bifunctional fixing compound (32, 62) at a distance from each other which is less than the smallest distance separating two ligands membranes (65, 66).
7/ - Structure membranaire selon l'une des revendications 1 à 6, caractérisée en ce que les ligands membranaires (65, 66) sont choisis parmi les phospholipides, les acides gras, les isoprénoïdes , les peptides .7 / - Membrane structure according to one of claims 1 to 6, characterized in that the membrane ligands (65, 66) are chosen from phospholipids, fatty acids, isoprenoids, peptides.
8/ - Structure membranaire selon l'une des revendications 1 à 7, caractérisée en ce que les composés (32, 62) bifonctionnels de fixation sont formés d' oligomères ou de polymères.8 / - Membrane structure according to one of claims 1 to 7, characterized in that the bifunctional fixing compounds (32, 62) are formed of oligomers or polymers.
9/ - Structure membranaire selon l'une des revendications 1 à 8, caractérisée en ce que les charges électriques du substrat (31, 61) sont négatives, et en ce que les composés (32, 62) bifonctionnels de fixation présentent une structure polycationique.9 / - Membrane structure according to one of claims 1 to 8, characterized in that the electrical charges of the substrate (31, 61) are negative, and in that the bifunctional fixing compounds (32, 62) have a polycationic structure .
10/ - Structure membranaire selon l'une des revendications 1 à 9, caractérisée en ce qu'elle comprend au moins un composé (32, 62) bifonctionnel de fixation dont la structure chimique comprend au moins un groupe choisi parmi un peptide, un polypeptide, une protéine ou un oside.10 / - Membrane structure according to one of claims 1 to 9, characterized in that it comprises at least one bifunctional fixing compound (32, 62) of which the chemical structure comprises at least one group chosen from a peptide, a polypeptide, a protein or an oside.
11/ - Structure membranaire selon l'une des revendications 1 à 10, caractérisée en ce qu'elle comprend au moins une polyamine à titre de composé bifonctionnel de fixation .11 / - Membrane structure according to one of claims 1 to 10, characterized in that it comprises at least one polyamine as bifunctional fixing compound.
12/ - Structure membranaire selon la revendication 11 , caractérisée en ce que la polyamine est une polylysine succinophospholipidique. 13/ - Structure membranaire selon l'une des revendications 1 à 12, caractérisée en ce que la membrane fonctionnelle (33, 63) présente au moins un composé (34) d'interaction avec le milieu externe.12 / - Membrane structure according to claim 11, characterized in that the polyamine is a succinophospholipid polylysine. 13 / - Membrane structure according to one of claims 1 to 12, characterized in that the functional membrane (33, 63) has at least one compound (34) for interaction with the external medium.
14/ - Structure membranaire selon la revendication 13, caractérisée en ce qu'elle comporte un composé (34) d'interaction choisi parmi un peptide, une protéine, un glucide, une glycoprotéine .14 / - Membrane structure according to claim 13, characterized in that it comprises an interaction compound (34) chosen from a peptide, a protein, a carbohydrate, a glycoprotein.
15/ - Structure membranaire selon l'une des revendications 13 et 14, caractérisée en ce qu'un composé d'interaction est lié à au moins un ligand membranaire (65,15 / - Membrane structure according to one of claims 13 and 14, characterized in that an interaction compound is linked to at least one membrane ligand (65,
66) d'un composé bifonctionnel (32, 62) de fixation par une liaison covalente.66) of a bifunctional compound (32, 62) for fixing by a covalent bond.
16/ - Structure membranaire selon l'une des revendications 13 à 15, caractérisée en ce qu'un composé (34) d'interaction est lié par une liaison lyotropique stable non covalente avec des composés amphiphiles de la membrane fonctionnelle (33, 63).16 / - Membrane structure according to one of claims 13 to 15, characterized in that an interaction compound (34) is linked by a stable non-covalent lyotropic bond with amphiphilic compounds of the functional membrane (33, 63) .
17/ - Structure membranaire selon l'une des revendications 1 à 16, caractérisée en ce que la membrane fonctionnelle (33, 63) s'étend sur une épaisseur inférieure à 5 nm .17 / - Membrane structure according to one of claims 1 to 16, characterized in that the functional membrane (33, 63) extends over a thickness of less than 5 nm.
18/ - Structure membranaire selon l'une des revendications 1 à 17, caractérisée en ce que le substrat (31, 61) présente des pores de dimension moyenne supérieure à 5 nm et inférieure à 0,5 μm .18 / - Membrane structure according to one of claims 1 to 17, characterized in that the substrate (31, 61) has pores of average size greater than 5 nm and less than 0.5 μm.
19/ - Utilisation d'une structure membranaire selon l'une des revendications 1 à 18 pour l'obtention d'un médicament. 20/ - Particule synthétique supramoléculaire caractérisée en ce qu'elle comprend une structure membranaire selon l'une des revendications 1 à 18 formant sa périphérie externe et délimitant un volume interne, la membrane fonctionnelle (33) de la structure membranaire présentant une surface libre qui s'étend à l'extérieur de la particule et qui est destinée à être placée au contact d'un milieu externe.19 / - Use of a membrane structure according to one of claims 1 to 18 for obtaining a medicament. 20 / - Synthetic supramolecular particle characterized in that it comprises a membrane structure according to one of claims 1 to 18 forming its external periphery and delimiting an internal volume, the functional membrane (33) of the membrane structure having a free surface which extends outside the particle and which is intended to be placed in contact with an external medium.
21/ - Particule selon la revendication 20, caractérisée en ce que le substrat (31) occupe au moins substantiellement tout le volume interne de la particule.21 / - Particle according to claim 20, characterized in that the substrate (31) occupies at least substantially the entire internal volume of the particle.
22/ - Particule selon la revendication 20, caractérisée en ce que le substrat (31) occupe une partie seulement du volume interne de la particule. 23/ - Particule selon l'une des revendications 20 à 22, caractérisée en ce que le substrat (31) est formé d'ADN ou d ' ARN .22 / - Particle according to claim 20, characterized in that the substrate (31) occupies only part of the internal volume of the particle. 23 / - Particle according to one of claims 20 to 22, characterized in that the substrate (31) is formed of DNA or RNA.
24/ - Particule selon l'une des revendications 20 à 23, caractérisée en ce que le substrat (31) est formé d'une matrice polymerique poreuse synthétique .24 / - Particle according to one of claims 20 to 23, characterized in that the substrate (31) is formed of a synthetic porous polymeric matrix.
25/ - Particule selon l'une des revendications 20 à 24, caractérisée en ce qu'elle contient une composition liquide dans son volume interne. 26/ - Particule selon la revendication 25, caractérisée en ce que la membrane fonctionnelle (33) est adaptée pour présenter une cinétique de libération de la composition liquide selon un profil prédéterminé.25 / - Particle according to one of claims 20 to 24, characterized in that it contains a liquid composition in its internal volume. 26 / - Particle according to claim 25, characterized in that the functional membrane (33) is adapted to present kinetics of release of the liquid composition according to a predetermined profile.
27/ - Particule selon l'une des revendications 20 à 26, caractérisée en ce qu'elle présente une dimension moyenne comprise entre 5 nm et 5 mm .27 / - Particle according to one of claims 20 to 26, characterized in that it has an average dimension between 5 nm and 5 mm.
28/ - Médicament caractérisé en ce qu'il comprend au moins une particule selon l'une des revendications 20 à 27. 29/ - Film synthétique supramoléculaire, caractérisé en ce qu'il comprend une structure membranaire selon l'une des revendications 1 à 18.28 / - Drug characterized in that it comprises at least one particle according to one of claims 20 to 27. 29 / - Synthetic supramolecular film, characterized in that it comprises a membrane structure according to one of claims 1 to 18.
30/ - Application d'un film selon la revendication 29 pour extraire ou séparer des sels et/ou des ions à partir d'une solution liquide par filtration.30 / - Application of a film according to the claim 29 for extracting or separating salts and / or ions from a liquid solution by filtration.
31 / - Procédé de préparation d'un polymère polycationique doté d'une pluralité de ligands (65, 66) lipidiques aptes à former une liaison lyotropique stable non covalente avec une membrane fonctionnelle (33, 63) stable de composés amphiphiles, de sorte que ce polymère peut faire office de composé (32, 62) bifonctionnel de fixation de la membrane fonctionnelle (33, 63) sur le substrat (31, 61) d'une structure membranaire selon l'une des revendications 1 à 18, caractérisé en ce qu'après avoir effectué les opérations de synthèse chimique permettant l'obtention de la molécule du polymère, on le met en contact d'un citrate dans un solvant polaire de façon à obtenir une précipitation du polymère.31 / - Process for the preparation of a polycationic polymer provided with a plurality of lipid ligands (65, 66) capable of forming a stable non-covalent lyotropic bond with a functional membrane (33, 63) stable of amphiphilic compounds, so that this polymer can act as a bifunctional compound (32, 62) for fixing the functional membrane (33, 63) to the substrate (31, 61) of a membrane structure according to one of claims 1 to 18, characterized in that that after performing the chemical synthesis operations allowing the polymer molecule to be obtained, it is brought into contact with a citrate in a polar solvent so as to obtain precipitation of the polymer.
32/ - Polymère polycationique de pureté supérieure à 95 % formé d'une polyamine polycationique dotée d'une pluralité de ligands (65, 66) lipidiques greffés sur une partie des atomes d'azote des fonctions aminés, et aptes à former une liaison lyotropique stable non covalente avec une membrane fonctionnelle (33, 63) stable de composés amphiphiles, ce polymère pouvant faire office de composé (32, 62) bifonctionnel de fixation de la membrane fonctionnelle (33, 63) sur le substrat (31, 61) d'une structure membranaire selon l'une des revendications 1 à 18.32 / - Polycationic polymer of purity greater than 95% formed of a polycationic polyamine endowed with a plurality of lipid ligands (65, 66) grafted on part of the nitrogen atoms of the amino functions, and capable of forming a lyotropic bond stable non-covalent with a functional membrane (33, 63) stable of amphiphilic compounds, this polymer being able to act as a bifunctional compound (32, 62) for fixing the functional membrane (33, 63) on the substrate (31, 61) d 'a membrane structure according to one of claims 1 to 18.
33/ - Polymère selon la revendication 32, caractérisé en ce qu'il présente un taux de greffage des fonctions aminés par les ligands lipidiques compris entre 1 % et 20 %.33 / - Polymer according to claim 32, characterized in that it has a rate of grafting of the amino functions with the lipid ligands of between 1% and 20%.
34/ - Polymère selon l'une des revendications 32 et 33, caractérisé en ce qu'il est formé d'une L-polylysine succinophospholipidique.34 / - Polymer according to one of claims 32 and 33, characterized in that it is formed of a succinophospholipid L-polylysine.
35/ - Procédé de préparation d'une structure membranaire selon l'une des revendications 1 à 18, caractérisé en ce qu'on prépare tout d'abord une suspension aqueuse des composés bifonctionnels de fixation de la façon suivante : - on prépare une solution des composés (32, 62) bifonctionnels de fixation dans le DMSO,35 / - Process for the preparation of a membrane structure according to one of claims 1 to 18, characterized in that an aqueous suspension of the bifunctional fixing compounds is first prepared in the following manner: a solution of the bifunctional fixing compounds (32, 62) in the DMSO is prepared,
- on prépare une solution aqueuse comprenant au moins un détergent non ionique à une concentration supérieure à sa concentration micellaire critique,an aqueous solution is prepared comprising at least one nonionic detergent at a concentration greater than its critical micellar concentration,
- on ajoute la solution des composés (32, 62) bifonctionnels de fixation dans la solution aqueuse.- adding the solution of the bifunctional fixing compounds (32, 62) in the aqueous solution.
36/ - Procédé selon la revendication 35, caractérisé en ce qu'on ajoute ensuite dans ladite suspension aqueuse une composition de polymères polyioniques aptes à former un substrat (33) solide par reticulation polyélectrolytique avec les chaînes polyioniques des composés bifonctionnels de fixation. 37/ - Procédé selon la revendication 36, caractérisé en ce que :36 / - Process according to claim 35, characterized in that a composition of polyionic polymers capable of forming a solid substrate (33) by polyelectrolytic crosslinking with the polyionic chains of the bifunctional fixing compounds is then added to the said aqueous suspension. 37 / - Method according to claim 36, characterized in that:
- on introduit des composés amphiphiles aptes à former une membrane fonctionnelle (33) soit dans la solution aqueuse du détergent, soit dans la suspension aqueuse avant ou après adjonction de la composition de polymères polyioniques, et de façon à ce que la concentration du détergent reste supérieure à la concentration micellaire critique,- Introducing amphiphilic compounds capable of forming a functional membrane (33) either in the aqueous solution of the detergent, or in the aqueous suspension before or after addition of the composition of polyionic polymers, and so that the concentration of the detergent remains higher than the critical micellar concentration,
- on élimine ensuite le détergent de la suspension.- then detergent is removed from the suspension.
38/ - Procédé selon la revendication 35, caractérisé en ce que : on introduit des composés amphiphiles aptes à former une membrane fonctionnelle (63) soit dans la solution aqueuse du détergent, soit dans la suspension aqueuse, puis on diminue la concentration du détergent dans la suspension aqueuse jusqu'à une concentration inférieure à sa concentration micellaire critique,38 / - Process according to claim 35, characterized in that: amphiphilic compounds capable of forming a functional membrane (63) are introduced either into the aqueous solution of the detergent or into the aqueous suspension, then the concentration of the detergent is reduced in the aqueous suspension up to a concentration lower than its critical micelle concentration,
- puis on place ensuite cette suspension aqueuse au contact d'un substrat (61) en phase solide,- then this aqueous suspension is then placed in contact with a substrate (61) in solid phase,
- puis on élimine le détergent non ionique. - then the non-ionic detergent is removed.
EP97911315A 1996-10-23 1997-10-22 Artificial polymeric membrane structure, method for preparing same, method for preparing this polymer, particle and film containing this structure Withdrawn EP0938580A1 (en)

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FR9613101 1996-10-23
FR9613101A FR2754828B1 (en) 1996-10-23 1996-10-23 ARTIFICIAL MEMBRANE STRUCTURE, METHOD AND POLYMER FOR PREPARING IT, METHOD FOR PREPARING THIS POLYMER, PARTICLE AND FILM COMPRISING THIS STRUCTURE
PCT/FR1997/001891 WO1998017818A1 (en) 1996-10-23 1997-10-22 Artificial polymeric membrane structure, method for preparing same, method for preparing this polymer, particle and film containing this structure

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