Classifications

• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
  • A01N25/34—Shaped forms, e.g. sheets, not provided for in any other sub-group of this main group
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
  • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
  • A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
  • A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
  • A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
  • A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/716—Glucans
  • A61K31/722—Chitin, chitosan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
  • A61K31/727—Heparin; Heparan
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
  • A61K31/731—Carrageenans
• • 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
  • A61K47/56—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
  • A61K47/61—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 an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule the organic macromolecular compound being a polysaccharide or a derivative thereof
• • 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
  • A61K47/6949—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 inclusion complexes, e.g. clathrates, cavitates or fullerenes
  • A61K47/6951—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 inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
• • 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
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
  • A61K9/1605—Excipients; Inactive ingredients
  • A61K9/1629—Organic macromolecular compounds
  • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
• • 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
  • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
  • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
  • A61K9/51—Nanocapsules; Nanoparticles
  • A61K9/5107—Excipients; Inactive ingredients
  • A61K9/513—Organic macromolecular compounds; Dendrimers
  • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B82—NANOTECHNOLOGY
  • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
  • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
  • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
  • Y10T428/00—Stock material or miscellaneous articles
  • Y10T428/29—Coated or structually defined flake, particle, cell, strand, strand portion, rod, filament, macroscopic fiber or mass thereof
  • Y10T428/2982—Particulate matter [e.g., sphere, flake, etc.]

Definitions

• Microparticles and nanoparticles consisting of hydrophobized polysaccharides and alpha-cyclodextrin
• the invention relates to microparticles and nanoparticles consisting of hydrophobized polysaccharides and alpha-cyclodextrin.
• the invention also relates to their use as an encapsulation system.
• particles capable of containing and vectoring or encapsulating an active ingredient are the subject of active research. These particles must be capable of trapping a substance of interest, transporting it in the body to the target cell or tissue, and then releasing it without altering its structure. It is important that these particles are not toxic. But there is a big disadvantage related to their preparation. Indeed, it often requires, during a step at least, the use of an organic solvent and / or the use of surfactants, non-biocompatible, sometimes under very important acid conditions. The removal of the solvent and / or surfactant molecules is often long, incomplete and adds a cost to the production of the particle. The residual traces, toxic, can also contribute to degrade the active substances immobilized in the particles. This is why it is sought to produce these particles in an aqueous medium preferably, from biocompatible and biodegradable polymers.
• Polysaccharides form a class of polymers very interesting in the field of encapsulation.
• these polysaccharides are chitosan, chitin, hyaluronic acid and glycosaminoglycans (GAGs).
• Chitosan is a linear heteropolymer of N-acetyl-D-glucosamine and ⁇ -linked D-glucosamine (1-4) according to the formula:
• M represents the number of D-glucosamine units
• ⁇ n represents the number of N-acetyl-D-glucosamine units
• m in relation to the total number of units is greater than 50%. It has the advantage of being biocompatible and mucoadhesive. Nanoparticles of chitosan have been used as adjuvants for mucosal vaccination in animals (Annales de Médecine Vcierinaire, 2003, 147, 343-350).
• An adjuvant is a substance that, when administered together with an antigen, increases the immune response to that antigen.
• the advantage of mucosal vaccination is the introduction of an immune response to the entry gate of microbes. Vaccines administered alone by mucosal route have low bioavailability. They must be co-administered with substances that promote their penetration or with adjuvants.
• Chitin is a linear heteropolymer of N-acetyl-D-glucosamine and ⁇ -linked D-glucosamine (1-4) according to the formula:
• M represents the number of D-glucosamine units
• ⁇ n represents the number of N-acetyl-D-glucosamine units
• the percentage of m in relation to the total number of units is less than 50%.
• Chitin is a powerful moisturizing agent, an effective sensor of heavy metals, responsible for a large number of contact allergies. It is used in the treatment of burns because it has healing properties. Chitin is also used to filter wastewater: it forms ionizable chains that make it possible to fix the organic elements. It is also inserted in the industrial food (juice manufacturing), as a technological auxiliary.
• GAGs glycosaminoglycans
• HSV herpes simplex virus
• GAGs and sulfated polysaccharides showed antibacterial activity against: Staphylococcus aureus, Gardnerella vaginalis, Mycobacterium tuberculosis, Listeria monocytogenes, Neisseria gonorrhoeae, Helicobacter pylori, Yersinia enterocolitica, Mycoplasma pneumoniae, Streptococcus mutas, Chlamydia trachomatis, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa. (US 2005/0203055).
• the heparin coating of abiotic surfaces has reduced Candida albicans adhesion by approximately 50% (Infection and Immunity. 1993, 61 (11), 4560-4568).
• HIV human immunodeficiency virus
• HSV human immunodeficiency virus
• HPV bovine papillomavirus
• sulfated polysaccharides such as cellulose sulfate, carrageenan, dextran sulfate, dextrin sulfate have shown antimicrobial activity.
• Hyaluronic acid helps protect the joints by increasing the viscosity of the synovial fluid and making the cartilage more elastic.
• Hyaluronic acid is the only non-sulfated GAG.
• Hyaluronic acid is a natural constituent of the dermis that plays an important role in the hydration, tone and elasticity of the skin.
• Polysaccharides grafted with hydrophobic chains are amphiphilic systems capable of self-associating spontaneously in aqueous medium in the form of heart-crown type micelles capable of accommodating an active ingredient ⁇ Drug Discovery Today, 2012, 17, 623-629). But their solubility in aqueous medium decreases because of the presence of hydrophobic grafted chains.
• cyclic polysaccharides such as cyclodextrins
• cyclodextrins causes the formation of particles constituting inclusion systems, soluble in aqueous medium.
• inclusion systems soluble in aqueous medium.
• Variable size and structure, ranging from the nanoparticle to the hydrogel, are able to vectorize a substance of interest.
• Gref et al (US2005 / 004348A1) describe particles obtained by grafting one or more (for example two) cyclic polysaccharides, such as beta-cyclodextrin, onto a biodegradable polymer such as poly ( ⁇ -caprolaetone) and their use. as vectors of active substances.
• the cyclic polysaccharide is covalently bound to the biodegradable polymer.
• This same patent describes the formation of nanoparticles by mixing a dextran bearing lauryl chains and a beta-cyclodextrin polymer.
• Bochot et al (US2006 / 0188464A1, EP1590077B1, International Journal of Pharmaceuticals, 2007, 339, 121) describe, in their publication and their applications, beads whose size varies from 1 to 3 mm, obtained by mixing an oil and a solution of water. alpha-cyclodextrin. They do not use linear polysaccharides described above and, on the other hand, the size of the particles formed is millimetric.
• One of the aims of the invention is to provide inclusion complexes between a polysaccharide and a cyclodextrin.
• Another object of the invention is to provide microparticles or nanoparticles formed from the aforementioned inclusion complexes.
• Another aim is to provide a process for the simple preparation of said particles without necessarily using organic solvents or surfactants.
• the invention relates to the use of said particles as such, without necessarily adding substance (s) of interest.
• the invention relates to the use of said particles to encapsulate one or more substance (s) of interest.
• the invention relates to an inclusion complex formed between:
• polysaccharide and the cyclodextrin being non-covalently bound.
• the invention relates to an inclusion complex formed by the interaction between:
• a polysaccharide comprising hydrophobic groups covalently bonded to said polysaccharide
• inclusion complex refers to a system that results from the interaction of a "host” molecule that admits within its cavity one or more other "guest” molecules without any covalent linkage. established.
• polysaccharide refers to a carbohydrate macromolecule formed by the linking of a large number of elemental sugars.
• the majority of the polysaccharides are hydrophilic, they carry -OH and / or -NH 2 groups , and / or -COOH, and / or -CH 2 -OH, and / or -SO 3 " , or groups derived therefrom. The nature of these groups makes it possible to differentiate them from a structural point of view and from a point of view of the physicochemical and biological properties
• polysaccharide refers here to a linear or branched polysaccharide.
• hydrophobic group-containing polysaccharide means that the polysaccharide has been "hydrophobized” by grafting, on -OH and / or -NH 2 groups , and / or -COOH, and / or -CH 2 -OH groups, and / or or -S0 3 " of alkyl chains which, by their nature, are hydrophobic because of their apolar nature.
• The” polysaccharide having hydrophobic groups "is therefore an amphiphilic polysaccharide.In certain conditions, these polysaccharides are capable of self-association for form heart-crown type micelles in an aqueous medium ⁇ Drug Discovery Today, 2012, 623-629).
• Cyclodextrin (or cycloamylose) is a cyclic oligosaccharide of ⁇ -D-glucopyranose linked by ⁇ (1-4) linkages. It is a molecule-cage of natural origin that can encapsulate various molecules, including molecules of therapeutic interest. There are different sizes, each having the shape of a "lampshade”. It carries hydrophilic groups (-OH) located outside, the assembly delimiting a relatively hydrophobic cavity. This amphiphilic character allows the cyclodextrin to include in its cavity hydrophobic molecules to form water-soluble inclusion complexes. Its biodegradable nature predisposes it to important applications in the food, cosmetics and pharmaceutical fields. Encapsulation in cyclodextrins makes it possible to protect fragile molecules or to ensure their slow and controlled release.
• alpha-cyclodextrin instead of a cyclodextrin polymer is an obvious economic and regulatory advantage because this cyclodextrin is commercially available and is recognized as a pharmaceutical excipient accepted by the majority. pharmacopoeia.
• Polysaccharide and cyclodextrin being non-covalently bound means that the interactions between these two molecules are Van der Waals bonds and / or hydrogen bonds, and / or electrostatic bonds, and / or hydrophobic bonds, and not covalent bonds. These inclusion complexes are thus exclusively formed by non-covalent bonds by simple mixture of alpha-cyclodextrin and polysaccharide grafted with hydrophobic groups. Thus, using the same procedure and varying the type of these non-covalent interactions, one can form particles of varying size and structure.
• the invention thus relates to an inclusion complex formed by the interaction between at least:
• a polysaccharide chosen from chitosan, dextran, hyaluronic acid, amylose, amylopectin, pullulan, heparin, chitin, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin; sulfate, cellulose sulfate, dextran sulfate, dextrin sulfate, starch, pectin, alginates, carrageenans, fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinan, polymannuronic acid, and derivatives thereof, said polysaccharide having hydrophobic groups selected from alkyl groups, linear or branched, containing from 2 to 1000 carbon atoms, or linear or branched, especially linear alkenyl groups, which may contain at least 1 double bond C C, said hydrophobic groups being covalently bound to said polysaccharide,
• polysaccharide and the cyclodextrin being non-covalently bound.
• the polysaccharide is composed of at least 3 saccharide units, its molar mass being in particular between 100 Da to 1000000 kDa, and in particular equal to 20 kDa, 145 kDa or 250 kDa.
• the polysaccharide is composed of at least 3 saccharide units, its molar mass being in particular between 5 kDa and 100,000 kDa, and in particular equal to 20 kDa, kDa or 250 kDa.
• the ratio between the concentration of the cyclodextrin and the concentration of the polysaccharide is from 10 -6 to 90,000, in particular from 4 to 15, and in particular equal to 10.
• the ratio between the concentration of the cyclodextrin and the polysaccharide concentration is from 0.01 to 1500, in particular from 4 to 15, and especially equal to 10.
• This parameter is very important because it makes it possible to modulate the size of the particles obtained from the aforementioned inclusion complexes by modifying the concentration ratio between the cyclodextrin and the polysaccharide.
• the average particle size may increase as the ratio of cyclodextrin concentration to polysaccharide concentration decreases.
• the ratio is less than 0.01, no particles are formed. If the ratio is greater than 20, particles are formed. Beyond a concentration ratio of 1500, and especially for a concentration greater than 50 g / L, alpha-cyclodextrin is no longer soluble in water.
• the solubility of the polysaccharide is not a limiting factor in the formation of the particles, because they form even if the polysaccharide is used as a suspension in water.
• a suspension polysaccharide can be used to formulate the nanoparticles and the microparticles has the advantage of obtaining more concentrated particles. Thanks to this new technology, polysaccharides that were difficult to formulate in the form of nanoparticles or microparticles because of their solubility problems in water (especially chitin) thus find a new use.
• this process is simple, reduces environmental pollution because solvents, surfactants or reagents are not necessarily used. It also decreases energy consumption. It does not necessarily use a heating step if a purification step after their preparation.
• the polysaccharides retain their antimicrobial properties even after their formulation in the form of nanoparticles and microparticles
• the invention particularly relates to an inclusion complex in which the polysaccharide is selected from chitosan, dextran, hyaluronic acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin sulfate, cellulose sulfate, dextran sulfate, dextrin sulfate, starch, pectin, alginates, carrageenans, fucan, curdlane, xylan, polyguluronic acid, xanthan, arabinan, polymannuronic acid, and their derivatives, and is especially chitosan.
• the polysaccharide is selected from chitosan, dextran, hyaluronic acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose,
• They can be neutral (dextran for example), or globally positively charged (chitosan for example) or negatively (heparin, hyaluronic acid, pectin).
• the polysaccharides have intrinsic properties even without adding active molecules.
• Chitosan has the advantage of being biocompatible, mucoadhesive and can be used as an adjunct for mucosal vaccination.
• Chitin has moisturizing and healing properties and has the ability to capture heavy metals and purify wastewater.
• GAGs and sulfated polysaccharides have activity to prevent, inhibit and / or treat fungal infections, bacterial, viral and / or parasitic on biotic or abiotic surfaces.
• Hyaluronic acid is known to promote hydration, tone and elasticity of the skin and to protect the joints by increasing the viscosity of the synovial fluid and making the cartilage more elastic.
• Biofilms are defined as an organized community of cells attached to a biotic or abiotic surface, inserted into an extracellular material. Biofilms are the predominant form of life of microorganisms and constitute a mode of resistance of these to antimicrobial agents. They can colonize biotic surfaces such as the surface of cells, tissues or organs (such as the skin and mucous membranes of the mouth, nose, eye, ear, vagina, rectum and / or the digestive tract) or abiotic surfaces such as plastic, glass, metal or any other material on which microorganisms can develop.
• the degree of substitution of the polysaccharide by the hydrophobic groups is from 0.001 to 100%, in particular from 0.05 to 50%.
• the degree of substitution of the polysaccharide by the hydrophobic groups is between 0.1% and 70%, in particular equal to 2%, 13% or 17%. .
• the degree of substitution reflects the number of hydrophobic groups bound to 100 saccharide units of the polysaccharide chain. It is determined by the experimental conditions of the grafting and can be measured by nuclear magnetic resonance (RM) or by elemental analysis for example.
• RM nuclear magnetic resonance
• the hydrophobic groups are not aromatic groups.
• fatty acids used for the grafting of the hydrophobic chains on the polysaccharide are in particular lauric acid, palmitic acid, oleic acid, stearic acid, linoleic acid, this list being by no means exhaustive and limiting.
• the hydrophobic groups are covalently attached to the polysaccharide by a nitrogen atom of said polysaccharide.
• This relates to polysaccharides having amino groups, especially chitosan and heparin.
• amino groups may undergo an N-acylation reaction by reaction with a fatty acid or a fatty acid derivative such as a fatty acid chloride or a fatty acid anhydride.
• the hydrophobic groups are covalently attached to the polysaccharide by one or more oxygen atoms of said polysaccharide.
• the hydrophobic groups are covalently attached to the polysaccharide by one or more oxygen atoms of said polysaccharide, and in particular at the level of the carboxylic function COOH and / or a CH 2 -OH group and / or -SO 3 "of said polysaccharide.
• -OH groups are numerous on polysaccharides.They react with fatty acids or fatty acid derivatives such as chlorides. acid, the acid anhydrides, to give esters.
• the hydrophobic group of the fatty acid or its derivative is thus grafted to the polysaccharide by one of its oxygen atoms, in the form of an acyl group: it is an "O-acylation" reaction.
• the O-acylation reaction leads to the formation of easily ester-degradable ester bonds after in vivo administration.
• the hydrophobic groups are covalently attached to the polysaccharide by a nitrogen, phosphorus or sulfur atom and by atoms of oxygen of said polysaccharide in the proportions of 0.001 to 100%.
• the hydrophobic groups are covalently attached to the polysaccharide by a nitrogen, phosphorus or sulfur atom and by atoms of oxygen of said polysaccharide in the proportions of 0.5 to 20%.
• the polysaccharide contains amino groups and hydroxyl groups, by reaction with fatty acids or fatty acid derivatives such as acid chlorides, acid anhydrides, it can undergo both N-acylation and O-acylation.
• the amino groups are more reactive than the hydroxyl groups.
• the cyclodextrin CD has the formula:
• R, R and R identical or different, in particular identical, are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, selected from methyl, ethyl, propyl, isopropyl, amino groups, groups; ammonium -NH 3 , or -S0 4 " -sulphate groups, and are in particular hydrogen atoms or methyl groups, said CD being talpha-cyclodextrin ( ⁇ -CD) in monomer form.”
• Cyclodextrins "(CD ) are cyclic oligomers of ⁇ -D-glucopyranoses linked by ⁇ (1-4) linkages
• Three families are mainly used: ⁇ -, ⁇ - and ⁇ -cyclodextrins respectively 6, 7 or 8 glucopyranose subunits.
• the cyclodextrins are therefore caged molecules capable of accommodating molecules by inclusion, in particular species of hydrophobic nature.
• the size of the cavity depends on the nature of the cyclodextrin.
• the inner part of the cavity is hydrophobic, the outer part is hydrophilic.
• the -OH groups may be substituted, in particular with methyl, hydroxypropyl or sulphobutyl groups. Substitutions can increase the solubility of the cyclodextrin.
• the cyclodextrin only used in the invention is ⁇ ' ⁇ -cyclodextrin.
• the advantage of this cyclodextrin is related to its small size which allows it to interact with the hydrophobic chains linked to the polysaccharide.
• the cyclodextrin is functionalized with a ligand chosen from antibodies, antibody fragments, receptors, lectins, biotin or its derivatives.
• ligand refers to a molecule capable of covalently binding to CD.
• the ligand is chosen from protein compounds involved in particular in the recognition and / or neutralization of pathogens (anti-bodies or fragments, receptors, lectins), involved in the metabolism of fatty acids (biotin and derivatives).
• the cyclodextrin in the inclusion complex of the invention, is charged or uncharged. In yet another aspect, in the inclusion complex of the invention, the cyclodextrin is substituted or unsubstituted.
• substituted cyclodextrin is meant, for example, an alkyl-substituted cyclodextrin, for example a methylated cyclodextrin, a hydroxyalkyl group, a maltosyl group, a galactosyl group, or any other molecule,
• the polysaccharide is a chitosan bearing hydrophobic groups
• ⁇ the cyclodextrin is ⁇ a-CD.
• Chitosan a linear polysaccharide of natural origin, randomly composed of ⁇ - (1-4) linked D-glucosamine deacylated units to acetylated N-acetyl-D-glucosamine units, is produced by chemical or enzymatic deacetylation of chitin. .
• Chitosan is biocompatible, biodegradable. It is non-toxic, bio-adhesive because of the interaction between the positive charges in acidic medium (pH ⁇ 6.0-6.5) carried by the amine functions and the negative charges carried by the biological membranes. It also has antibacterial and anti viral activity.
• Chitosan is soluble in an aqueous solution of acetic acid in low concentration by protonation of the amino groups present.
• the glucoside chain of chitosan is essentially hydrophilic. But it is possible to graft groups, in particular hydrophobic groups, on the amino and hydroxyl groups.
• the polysaccharide then becomes amphiphilic and can form micelles, nanoparticles but also hydrogels at higher concentration by self-association in an aqueous medium. It is therefore possible to encapsulate active ingredients in the objects it forms. It can be used to encapsulate active ingredients (paclitaxel, ibuprofen, ).
• the chitosan carries hydrophobic groups grafted at certain nitrogen atoms and has the formula:
• M represents the number of units of desacetylated units
• N represents the number of units of acetylated units
• DDA degree of deacetylation
• R 4 represents the hydrophobic group and is chosen from:
• alkyl group linear or branched, containing from 1 to 20 carbon atoms, in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -C] 3 ⁇ 4,
• the chitosan carries hydrophobic groups grafted at certain nitrogen atoms and has the formula
• M represents the number of units of D-glucosamine units
• N represents the number of units of N-acetyl-D-glucosamine units
• DDA degree of deacetylation
• R 4 represents the hydrophobic group and is chosen from:
• a linear or branched alkyl group containing from 1 to 1000 carbon atoms in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• the 50% DDA is the limit between chitin and chitosan: when the DDA is less than 50%, it is called chitin, otherwise it is called chitosan.
• the - (CH 2 ) 14 -CH 3 group comes from palmitic acid
• - (CH) 16 -CH 3 comes from stearic acid
• - (CH 2 ) 7 -CH CH-CH 2 - (CH 2 7 -CH 3 is derived from linoleic acid
• - (CH 2 ) 7 -CH CH- (CH 2 ) 7 -CH 3 is derived from oleic acid.
• the N-acylated chitosan is obtained from an N-acylation reaction in the presence of a coupling agent, EDCI (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide), which reacts with the groups carboxylic acids of the fatty acid (oleic or palmitic for example) to form an intermediate ester capable of binding to the free amino functions of chitosan (Lee, KY, et al., Structural Determination and Interior Polarity of Self-Aggregate Prepared from Deoxycholic Acid-Modified Chitosan in Water., Macromolecules, 1998, 31, 2, 378-383).
• the following diagram shows the sequence of reactions carried out to obtain an N-acylated chitosan, grafted with chains derived from oleic acid. We proceed in the same way with other fatty acids such as palmitic acid for example.
• the IR and NMR spectra of the grafted chitosan make it possible to demonstrate the secondary amino function obtained after N-acylation and the presence of the grafted alkyl chains.
• the chitosan in the inclusion complex, carries hydrophobic groups grafted at oxygen atoms from -OH groups and / or -CH 2 OH groups attached to the ring.
• chitosan and having the formula:
• n, m and R have the meanings given above.
• the chitosan in the inclusion complex, carries hydrophobic groups grafted at oxygen atoms from -OH groups and / or -CH 2 OH groups attached to the ring.
• chitosan and having the formula:
• ⁇ R represents
• n, m and R have the meanings given above, and provided that R
• the infrared (IR) spectra of the products obtained show a characteristic band of an ester bond at 1700 cm -1 , while the proton MR spectra compared to that of the native chitosan make it possible to establish the presence of the alkyl chains.
• the chitosan carries hydrophobic groups grafted at certain oxygen atoms and certain nitrogen atoms, and has the formula:
• n, m and R 4 have the meanings given above.
• the chitosan carries hydrophobic groups grafted at certain oxygen atoms and certain nitrogen atoms, and has the formula:
• ⁇ R represents
• n, m and R 4 have the meanings defined above, and subject to
• the polysaccharide is a dextran bearing hydrophobic groups, attached by oxygen atoms of said dextran and containing groups of formula
• p R 4 has the meanings indicated above,
• ⁇ * represents dextran
• ⁇ the cyclodextrin is ⁇ a-CD.
• Dextran is a dextrose polymer.
• the IR spectrum of O-palmitoyl-dextran shows an absorption band corresponding to the carbonyl ester group.
• the polysaccharide is hyaluronic acid bearing hydrophobic groups, attached by oxygen atoms of said hyaluronic acid and representing groups of formula
• Cyclodextrin is a-CD.
• the polysaccharide is an amylopectin bearing hydrophobic groups attached by oxygen atoms of said amylopectin and representing groups of formula
• ⁇ the cyclodextrin is ⁇ a-CD.
• the polysaccharide is a pullulan bearing hydrophobic groups attached to the oxygen atoms of said pullulan and resenting groups of formula
• n R 4 has the meanings given above,
• Cyclodextrin is a-CD.
• Pullulan consists of maltotriose units.
• the three units of glucose that make up maltotriose are linked by a saccharide bond of the ⁇ -1,4 type, while the maltotrioses are connected to each other by osidic bonds of the ⁇ -1,6 type. It is grafted, in particular by palmitic acid.
• the polysaccharide is a heparin carrying hydrophobic groups fixed by nitrogen atoms of said heparin and containing groups of formula
• 0 * represents heparin
• the polysaccharide is a heparin bearing hydrophobic groups attached by oxygen atoms of said heparin, these oxygens possibly originating from the hydroxyl or carboxyl groups of heparin, and representing groups of formula
• ⁇ and the cyclodextrin is the ct-CD.
• the polysaccharide is a carrageenan carrying hydrophobic groups fixed by nitrogen atoms of said sulfate carrageenan and containing groups of formula
• polysaccharide is a carrageenan bearing hydrophobic groups attached by oxygen atoms of said carrageenan, these oxygens possibly originating from hydroxyl or carboxyl groups of carrageenan, and representing groups of formula
• ⁇ and the cyclodextrin is ⁇ a-CD.
• the polysaccharide is a hyaluronic acid bearing hydrophobic groups attached by nitrogen atoms of said hyaluronic acid and representing groups of formula in which
• the polysaccharide is a hyaluronic acid bearing hydrophobic groups attached by oxygen atoms of said hyaluronic acid, these oxygens possibly originating from the hydroxyl or carboxyl groups of hyaluronic acid, and representing groups of formula
• ⁇ * represents hyaluronic acid
• ⁇ and the cyclodextrin is ⁇ a-CD.
• the polysaccharide is a glycosaminoglycan bearing hydrophobic groups attached by nitrogen atoms of said glycosaminoglycan and representing groups of formula
• polysaccharide is a glycosaminoglycan bearing hydrophobic groups attached by oxygen atoms of said glycosaminoglycan, these oxygens possibly originating from hydroxyl or carboxyl groups of glycosaminoglycan, and representing groups of formula
• n R 4 has the meanings given above,
• ⁇ and the cyclodextrin is ⁇ a-CD.
• the polysaccharide is a carrageenan sulfate bearing hydrophobic groups attached by nitrogen atoms of said carrageenan sulfate and representing groups of formula
• a * represents carrageenan sulfate
• polysaccharide is a carrageenan sulfate bearing hydrophobic groups attached by oxygen atoms of said carrageenan sulfate, these oxygens possibly originating from hydroxyl or carboxyl groups of carrageenan sulfate, and representing groups of formula
• ⁇ and the cyclodextrin is ⁇ a-CD.
• the polysaccharide is a dextran sulphate carrying hydrophobic groups attached by nitrogen atoms of said dextran sulphate and containing groups of formula
• ⁇ R 4 has the meanings indicated above,
• n * represents the. dextran sulfate
• polysaccharide is a dextran sulfate bearing hydrophobic groups attached by oxygen atoms of said dextran sulfate, these oxygens possibly coming from hydroxyl or carboxyl groups of dextran sulfate, and representing groups of formula in which
• ⁇ and the cyclodextrin is ⁇ a-CD.
• the polysaccharide is a sulfate cellulose carrying hydrophobic groups attached by nitrogen atoms of said cellulose sulphate and representing groups of formula
• the polysaccharide is a cellulose sulfate carrying hydrophobic groups attached by oxygen atoms of said cellulose sulfate, these oxygens possibly originating from hydroxyl or carboxyl groups of cellulose sulfate, and representing groups of formula
• ⁇ and the cyclodextrin is ⁇ a-CD.
• the polysaccharide is a heparan sulphate carrying hydrophobic groups attached by nitrogens of nitrate representing groups of formula
• ⁇ R 4 has the meanings indicated above,
• the polysaccharide is a heparan sulfate bearing hydrophobic groups attached by oxygen atoms of said heparan sulfate, these oxygens being able to from the hydroxyl or carboxyl groups of heparan sulfate, and representing groups of formula
• ⁇ and the cyclodextrin is ⁇ a-CD.
• the polysaccharide is a keratan sulfate bearing hydrophobic groups fixed by nitrogen atoms of said keratan ulfate and representing groups of formula
• the polysaccharide is a keratan sulfate bearing hydrophobic groups attached by oxygen atoms of said keratan sulfate, these oxygens possibly coming from hydroxyl or carboxyl groups of keratan sulfate, and representing groups of formula
• n * represents keratan sulfate
• ⁇ and the cyclodextrin is the a-CD.
• the polysaccharide is a chondroitin sulfate bearing hydrophobic groups attached by nitrogen atoms of said chondroitin sulfate and representing groups of formula in which
• the polysaccharide is a chondroitin sulfate bearing hydrophobic groups attached by oxygen atoms of said chondroitin sulfate, these oxygens possibly originating from hydroxyl or carboxyl groups of chondroitin sulfate, and representing groups of formula
• n R 4 has the meanings given above,
• n * represents chondroitin sulfate
• ⁇ and the cyclodextrin is ⁇ -CD.
• the polysaccharide is a sulfate dextrin carrying hydrophobic groups attached by nitrogen atoms of said sulfate dextrin and containing groups of the formula
• polysaccharide is a sulfate dextrin carrying hydrophobic groups attached by oxygen atoms of said sulfate dextrin, these oxygens possibly originating from hydroxyl or carboxyl groups of dextrin sulfate, and representing groups of formula
• the invention also relates to a particle of size ranging from 10 nm to 100,000 nm containing inclusion complexes formed by the interaction between at least
• a polysaccharide comprising hydrophobic groups covalently bonded to said polysaccharide, optionally functionalized with a ligand chosen from antibodies, antibody fragments, receptors, lectins or biotin or its derivatives,
• a cyclodextrin in the form of a monomer optionally functionalized with a ligand chosen from antibodies, antibody fragments, receptors, lectins or biotin or its derivatives.
• the invention also relates to a particle of size ranging from 50 nm to 10,000 nm containing inclusion complexes between:
• a polysaccharide comprising hydrophobic groups covalently bonded to said polysaccharide, optionally functionalized with a ligand chosen from antibodies, antibody fragments, receptors, lectins or biotin or its derivatives,
• a cyclodextrin in the form of a monomer optionally functionalized with a ligand chosen from antibodies, antibody fragments, receptors, lectins or biotin or its derivatives.
• the invention relates to nanometric particles, ranging in size from 10 nm to 1000 nm and micrometric particles of size ranging from
• the invention relates to nanoscale particles having a size of from 50 nm to 1000 nm and micrometric particles ranging in size from 1000 nm to 10000 nm.
• the invention relates to a particle of
• a polysaccharide chosen from chitosan, dextran, hyaluronic acid, amylose, amylopectin, puUulane, heparin, chitin, cellulose derivatives, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin sulfate, cellulose sulfate, dextran sulfate, dextrin sulfate, starch, pectin, alginates, carrageenans, fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinane, polymannuronic acid, and derivatives thereof, comprising hydrophobic groups selected from linear or branched alkyl groups or linear or branched alkenyl groups and carrying from 1 to 4 double bonds C C, conjugated or otherwise, said groups hydrophobic agents being covalently bonded to said polysaccharide, optionally functionalized with a ligand chosen
• a monomeric ⁇ -cyclodextrin optionally functionalized with a ligand selected from antibodies, antibody fragments, receptors, lectins or biotin or its derivatives.
• the sizes of the particles formed were evaluated by quasi-elastic light scattering (DQEL) on the one hand and by transmission electron microscopy (TEM) on the other hand.
• DQEL quasi-elastic light scattering
• TEM transmission electron microscopy
• Size plays a very important role regarding the amount of encapsulated active ingredient, the applications envisaged, and the route of administration of the active ingredient.
• the invention also relates to a particle of size ranging from 10 nm to 100000 nm containing inclusion complexes formed by the interaction between at least:
• a polysaccharide chosen from chitosan, dextran, hyaluronic acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose derivatives, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin sulfate, cellulose sulfate, dextran sulfate, dextrin sulfate, starch, pectin, alginates, carrageenans, fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinane, polymannuronic acid, and derivatives thereof, comprising hydrophobic groups selected from alkyl groups, linear or branched, containing from 2 to 1000 carbon atoms, or linear or branched, especially linear alkenyl groups, which may contain at least least 1 C C double bond, said hydrophobic groups being covalently bonded to said polysaccharide,
• an ⁇ -cyclodextrin in the form of a monomer optionally functionalized with a ligand chosen from antibodies, antibody fragments, receptors, lectins or biotin or its derivatives.
• the invention also relates to particles of size ranging from 10 nm to 100000 nm containing inclusion complexes formed by the interaction between:
• a mixture of at least two polysaccharides chosen from chitosan, dextran, hyaluronic acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose derivatives, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin sulfate, cellulose sulfate, dextran sulfate, dextrin sulfate, starch, pectin, alginates, carrageenans, fucan, curdlane, xylan, acid polyguluronic acid, xanthan, arabinan, polymannuronic acid, and derivatives thereof, comprising hydrophobic groups chosen from linear or branched alkyl groups containing from 2 to 1000 carbon atoms, or linear or branched alkenyl groups, in particular linear, which may contain at least one C C double bond, said hydrophobic groups being covalently bonded to said polysacc
• At least one ⁇ -cyclodextrin in the form of a monomer optionally functionalized with a ligand chosen from antibodies, antibody fragments, receptors, lectins or biotin or its derivatives.
• the invention also relates to an encapsulation system containing one or more previously defined particles, and a substance used for its properties in the pharmaceutical, paramedical, medical device, animal feed, agrochemical, medical, cosmetic and veterinary fields. agri-foodstuffs, pesticides, cosmetic textiles, perfumery, the environment (eg water pollution control) or in the paint, building and / or automobile industry.
• a medical device is an instrument, apparatus, equipment or software for use in humans or animals for the purposes of: diagnosis, prevention, control, treatment or mitigation of disease , diagnosis, control, treatment, mitigation or compensation of injury or disability, study or replacement or modification of anatomy or physiological process, mastery of conception .
• the substance has pharmaceutical properties and is chosen from inorganic compounds and organic, synthetic or natural compounds.
• the encapsulation system previously defined according to the invention can be used to prepare appropriate compositions in the pharmaceutical, medical, paramedical, medical device, animal feed, cosmetic, veterinary, food, pesticide, cosmeto-textile, perfumery, environmental (eg water pollution control) or in the paint, packaging, building and / or automobile industry.
• the encapsulated substance may possess pharmaceutical properties and be an active ingredient for therapeutic use. It may belong to the following list, the latter being in no way limiting, to the group of compounds with vitamin properties, in particular vitamin A, vitamin E, vitamin C, vitamin K, vitamin B, vitamin D , antitumor agents, in particular paclitaxel, docetaxel, tamoxifen, doxorubicin, painkillers, in particular paracetamol, anti-inflammatories, in particular diclofenac, ibuprofen, ketoprofen, antibiotics, in particular penicillins, tetracyclines and antifungals, in particular ketoconazole, clotrimazole, nystatin, chlorhexidine and its derivatives, antiparasitic agents, in particular albendazole, metronidazole, enzymatic agents, in particular alkaline phosphatase, acetylcholinesterase, alcohol dehydrogenase, hormonal agents, in particular testosterone, levonorgestrel
• This substance for therapeutic use can be used in humans and animals.
• the encapsulated substance may also have cosmetic properties and belong to the group of compounds with anti-inflammatory, anti-aging, anti-ultraviolet (anti-UV), depigmenting, healing, moisturizing, fragrancing, deodorizing, antibacterial, antiperspirant, cleansing, coloring, conservative.
• anti-UV anti-ultraviolet
• the substance has food properties and belongs to the group of compounds with vitamin, enzymatic and sweetening properties. It can also be an essential oil, a dye, a preservative, an antioxidant, a probiotic.
• Some molecules or families of molecules that can be encapsulated are: molsidomine, ketoconazole, gliclazide, diclofenac, levonorgestrel, paclitaxel, docetaxel, tamoxifen, hydrocortisone, pancratistatin, ketoprofen, diazepam, ibuprofen, nifedipine, testosterone, tamoxifen, furosemide, tolbutamide, chloramphenicol, benzodiazepines, naproxen, dexamethasone, diflunisal, anadamide, pilocarpine, daunorubicin, doxorubicin, essential oils, terpenes, terpenoids.
• a solvent or a mixture of solvents especially: alkyl acetate (ethyl acetate, butyl acetate, methyl acetate), acetone, acetonitrile, acetic acid, methanoic acid, ammonia, acetic anhydride, aniline, anisole, benzene, butanol, butanone, chlorobenzene, chloroform, cyclohexane, cyclopentane, dichloroethane, dichloromethane, diisopropyl ether, dimethylformamide, dimethylsulfoxide, dioxane, water, ethanol, glycol ether, diethyl ether, ethylene glycol, heptane, hexamethylphosphoramide, hexane, methanol, methyl ethyl ketone, nitrobenzene, pentane, percgloroethylene, propano
• alkyl acetate ethyl acetate,
• compositions containing the encapsulation system with the active ingredient are therefore the medical, veterinary, cosmetic, cosmeto-textile, the environmental field particularly related to the depollution of water, the field of paints, building or the automobile, perfumery.
• the invention also relates to the use of previously defined particles.
• the invention thus relates to the use of said particles as medicaments, especially as adjuvants for vaccination, treatment of burns, and / or for cicatrization.
• the invention also relates to the use of said particles in the preparation of medicaments having at least one activity for preventing, inhibiting and / or treating fungal, bacterial, viral and / or parasitic infections on biotic or abiotic surfaces.
• the invention also relates to the use of said particles as veterinary medicaments, especially as adjuvants for vaccination.
• the invention also relates to the use of particles as a cosmetic agent, especially as an anti-aging agent, depigmenting, healing, moisturizing, perfuming, deodorant, antiperspirant, cleanser, dye, preservative.
• the invention also relates to the use of particles for the implementation of a method for preparing devices, in particular healing dressings, said devices comprising said particles, and being capable of releasing said particles or one or more substance (s) active (s) of interest contained in said particles.
• the invention also relates to a pharmaceutical composition containing as active substance the substance encapsulated in the particles, in solid form, or in the form of solution or suspension in a physiological medium, optionally enriched with an excipient such as glucose, sucrose or any other pharmaceutically acceptable excipient, usable for the routes:
• parenteral oral, cutaneous, subcutaneous, nasal, pulmonary or ocular and, for any mucosal administration, or at a specific site (tumor, light of certain blood vessels),
• pills tablets
• soft capsules hard capsules
• capsules powders, granules, soluble or dispersible tablets, patch, implant, suppositories, solutions, suspension, syrup, pastes, creams, gels, emulsions, sprays, lotions, ointments, shampoos.
• the invention also relates to a pharmaceutical composition containing as active substance a substance encapsulated in inclusion complexes or in particles defined above, in association with a pharmaceutically acceptable vehicle, in solid form, or in the form of a solution or suspension. in a physiological medium, usable by the ways:
• compositions in the pharmaceutical field are tablets, soft capsules, hard capsules (capsules), powders, granules, patches, implants, suppositories, solutions, suspensions, syrups, pastes, creams, gels, emulsions, sprays, lotions and ointments.
• Advantageous forms in the paramedical field are dressings, catheters, compress, gauze, hydrophilic cotton, physiological saline, spray, etc.
• Implants in the field of medical devices are implants, prostheses, instrument washer-disinfectors, compresses, dressings (especially healing), sprays, gauzes, hydrophilic cotton, etc.
• oral forms tablettes, powders, soft capsules, hard capsules (capsules), granules, pastes, solutions, suspensions), injectables (solutions, suspensions) and topical agents, the action of which may be local or systemic (spray, necklaces, ear loops, powders, lotions, ointments, shampoos, patch, emulsions, milk, gel, cream).
• Advantageous forms in the food field are solutions, emulsions, pastes, gels, powders used alone or included in food preparations; in the field of food supplements: mainly oral forms (powders, tablets, capsules, granules, soft capsules or hard capsules (capsules), pastes, solutions, suspensions, infusions).
• the invention also relates to a cosmetic composition containing as active substance the substance encapsulated in the particles, and containing cosmetically acceptable excipients, usable in the form of gels, pastes, ointments, lotions, creams, milks, sticks, shampoos, powders, aerosols, patches.
• the invention also relates to a process for preparing an inclusion complex comprising a mixing step:
• a polysaccharide comprising hydrophobic groups covalently bound by a nitrogen atom or by one or more oxygen atoms to said polysaccharide
• the process for preparing the invention of an inclusion complex comprises a mixing step:
• a polysaccharide comprising hydrophobic groups covalently bound to the polysaccharide by a nitrogen atom of said polysaccharide
• the method for preparing the invention of an inclusion complex comprises a step of mixing at least:
• a polysaccharide comprising hydrophobic groups covalently bound to the polysaccharide by a nitrogen atom of said polysaccharide
• the invention also relates to a method for preparing an inclusion complex as defined above, comprising a mixing step of at least
• a polysaccharide in the form of a suspension in a solvent especially water comprising hydrophobic groups covalently bound to the polysaccharide by oxygen atoms of said polysaccharide, and
• polysaccharide having hydrophobic groups of formula:
• R 4 represents
• a linear or branched alkyl group containing from 1 to 1000 carbon atoms in particular the groups - (CH 2 ) 4 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• R, R and R are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, selected from methyl, ethyl, propyl, isopropyl, amino -NH 2 groups, ammonium groups - NH 3 , or sulfate groups --SO4 and are especially hydrogen atoms or methyl groups,
• said CD being alpha-cyclodextrin ( ⁇ -CD) in monomeric form, to obtain an inclusion complex in which said polysaccharide and cyclodextrin are non-covalently bound.
• the invention also relates to a process for preparing an inclusion complex as defined above, comprising a step of mixing at least:
• a polysaccharide comprising hydrophobic groups covalently bound to the polysaccharide by oxygen atoms of said polysaccharide
• ⁇ -cyclodextrin in the form of a monomer suspended in a solvent, in particular water,
• polysaccharide having hydrophobic groups of formula:
• a linear or branched alkyl group containing from 1 to 1000 carbon atoms in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• R, R and R are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, selected from methyl, ethyl, propyl, isopropyl, amino -NH 2 groups, ammonium groups - NH 3 + , or sulphate -SO 2 - groups, and are in particular hydrogen atoms or methyl groups,
• said CD being alpha-cyclodextrin ( ⁇ -CD) in monomeric form
• the invention also relates to a process for preparing an inclusion complex as defined above, comprising a step of mixing at least:
• polysaccharide having hydrophobic roups of formula:
• R 4 represents A linear or branched alkyl group containing from 1 to 1000 carbon atoms, especially the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• R, R and R are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, chosen from methyl, ethyl, propyl, isopropyl, amino -NH 2 groups, ammonium groups - NH 3 + , or sulfate groups -SO 4 2- , and are in particular hydrogen atoms or methyl groups,
• said CD being alpha-cyclodextrin ( ⁇ -CD) in monomeric form
• the process for preparing an inclusion complex comprises a mixing step
• a polysaccharide comprising hydrophobic groups covalently bound to the polysaccharide by oxygen atoms of said polysaccharide
• the process for preparing an inclusion complex comprises a step of mixing at least:
• a polysaccharide comprising hydrophobic groups covalently bound to the polysaccharide by oxygen atoms of said polysaccharide
• the invention also relates to a method for preparing an inclusion complex comprising a mixing step:
• a chitosan comprising hydrophobic groups covalently attached to chitosan by one or more oxygen atoms of said chitosan,
• M represents the number of units of D-glucosamine units
• N represents the number of units of N-acetyl-D-glucosamine units
• the degree of deacetylation representing the percentage of m in relation to the total number of units is greater than 50%
• R 4 represents a hydrophobic group and is chosen from:
• R 1 , R 2 and R 3 are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, selected from methyl, ethyl, propyl, isopropyl, amino groups -NH 2 , ammonium groups - NH 3 + , or sulfate groups -SO 4 " , and are in particular hydrogen atoms or methyl groups,
• said CD being alpha-cyclodextrin ( ⁇ -CD) in monomeric form
• the process for preparing an inclusion complex comprises a mixing step:
• a plurality of polysaccharides comprising hydrophobic groups covalently attached to said polysaccharides by a nitrogen atom and / or by one or more oxygen atoms of said polysaccharides, the polysaccharides being chitosan, dextran, hyaluronic acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose derivatives, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin sulfate, cellulose sulfate, dextran sulfate, dextrin sulfate starch, pectin, alginates, carrageenans, fucan, curdlan, xylan, polyguluronic acid, xanthan, arabinan, polymannuronic acid, and their derivatives,
• polysaccharides and the cyclodextrin are non-covalently bound.
• the use of several polysaccharides may have the advantage of modulating the properties of the particles by modifying the ratio between the polysaccharides. Thus, it is expected that the size, overall charge and target applications of these particles may be modulated.
• the process for preparing an inclusion complex comprises a mixing step: A polysaccharide chosen from chitosan, dextran, hyaluronic acid, amylose, amylopectin, puUulane, heparin, chitin, cellulose derivatives, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin sulfate, cellulose sulfate, dextran sulfate, dextrin sulfate, starch, pectin, alginates, carrageenans, fucan, curdlane, xylan, polyguluronic acid, xanthan, arabinan, polymannuronic acid, and their derivatives, and is especially chitosan, said polysaccharide comprising hydrophobic groups of formula:
• ⁇ * represents the polysaccharide
• ⁇ R 4 represents:
• alkyl group linear or branched, containing from 1 to 20 carbon atoms, in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 6 -CH 3 ,
• ⁇ P is an integer equal to 6
• R, R and R are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, selected from methyl, ethyl, propyl, isopropyl, amino -NH 2 groups, ammonium groups - NH 3 , or sulfate groups -SO 4 and are in particular hydrogen atoms or methyl groups,
• said CD being alpha-cyclodextrin ( ⁇ -CD) in monomeric form
• the process for preparing an inclusion complex comprises a mixing step of at least
• ⁇ * represents the polysaccharide
• ⁇ R 4 represents:
• a linear or branched alkyl group containing from 1 to 20 carbon atoms in particular the groups - (CH 2 ) 4 -CH 3 or - (CH 2 ) 16 -CH 3,
• R, R and R are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, selected from methyl, ethyl, propyl, isopropyl, amino -NH 2 groups, ammonium groups - N3 ⁇ 4, or sulfate groups -SO 4 ' , and are in particular hydrogen atoms or methyl groups,
• said CD being alpha-cyclodextrin ( ⁇ -CD) in monomeric form, to obtain an inclusion complex in which said polysaccharide and cyclodextrin are non-covalently bound.
• the process for preparing an inclusion complex comprises a step of mixing the polysaccharide:
• aqueous solution at a concentration of from 0.01 to 9000 g / l, in particular of from 1 to 600 g / l, and in particular approximately equal to 10 g / l, the aqueous solvent being chosen from pure water, an aqueous solution of pH ranging from 1 to 7 or 7 to 14, in particular from 5 to 7, or a physiological saline solution optionally enriched with glucose or containing any other excipient for pharmaceutical, medical, paramedical, medical devices, food animal, cosmetic, veterinary, agri-food, pesticides, cosmeto-textiles, perfumery, environmental (eg water pollution control) or in the paint, packaging, building and / or automotive industry .
• environmental eg water pollution control
• an aqueous medium chosen from pure water, an aqueous solution of pH of from 1 to 7 or 7 to 14, in particular of from 5 to 7, or a saline solution optionally enriched with glucose or containing any other excipient for pharmaceutical, medical, paramedical, medical devices, animal nutrition, cosmetics, veterinary, agri-food, pesticides, cosmeto-textiles, perfumery, environmental (eg water depollution) or in the industry paintings, packaging, building and / or automobile,
• the process for preparing an inclusion complex comprises a step of mixing the polysaccharide
• aqueous solution at a concentration of from 1 to 150 g / l, in particular of from 5 to 50 g / l, and in particular approximately equal to 10 g / l, the aqueous solvent being chosen from pure water, a solution of aqueous pH ranging from 1 to 7 or 7 to 12, in particular from 5 to 7, or a physiological saline solution optionally enriched with glucose or containing any other excipient for pharmaceutical, cosmetic, agri-food or veterinary use,
• an aqueous medium chosen from pure water, an aqueous solution of pH ranging from 1 to 7 or from 7 to 12, in particular from 5 to 7, or a saline solution optionally enriched with glucose or containing any other excipient for pharmaceutical, cosmetic, agri-food or veterinary use,
• the step of mixing the polysaccharide in aqueous solution or dispersion is carried out at a concentration of from 0.01 to 9000 g / l, in particular from 1 to 600 g / l. L, and in particular approximately equal to 10 g / l, with a cyclodextrin, especially ⁇ ' ⁇ -CD, at a concentration of from 0.01 to 9000 g / l, in particular of from 1 to 300 g / l, and in particular about 200 g / L.
• the step of mixing the polysaccharide in aqueous solution or dispersion is carried out at a concentration of from 1 to 150 g / l, in particular from 5 to 50 g / l, and in particular approximately equal to 10 g / l, with a cyclodextrin, ⁇ ' ⁇ -CD, at the concentration of 1 to 150 g / l, in particular of 5 to 50 g / l, and in particular approximately equal to 10 g / L.
• the process for preparing an inclusion complex comprises a step of mixing the polysaccharide in dispersion in an aqueous medium
• the concentration of said polysaccharide being from 0.01 to 9000 g / l of aqueous medium, in particular ranging from 1 to 600 g / l of aqueous medium, and in particular being approximately equal to 1 g / l or approximately equal to 10 g / L of aqueous medium,
• a cyclodextrin, ⁇ ' ⁇ -CD at a concentration of from 0.01 to 9000 g / l of aqueous medium, in particular of from 0.01 to 300 g / l of aqueous medium, and in particular of approximately 10 g / L aqueous medium.
• the process for preparing an inclusion complex comprises a step of mixing the polysaccharide in dispersion in an aqueous medium
• the mass of said polysaccharide being from 1 to 150 g / l of aqueous medium, in particular being from 1 to 10 g / l of aqueous medium, and in particular being approximately equal to 1 g / l or approximately equal to 10 g / l of aqueous medium,
• a cyclodextrin, ⁇ -CD at a concentration of from 1 to 150 g / l of aqueous medium, in particular of from 1 to 50 g / l of aqueous medium, and in particular of approximately 10 g / l of aqueous medium.
• the complexes are also formed when the polysaccharide is used in the form of a suspension.
• the process for preparing an inclusion complex comprises a step of mixing the polysaccharide with a cyclodextrin, ⁇ -CD,
• the mass percentage of said polysaccharide being from 0.01% to 90%, and especially from 0.5% to 50%
• the weight percentage of the cyclodextrin being from 0.01% to 90%, and especially from 0.5% to 50%
• the mass percentage of the water or of the aqueous medium being comprised from 10% to 99.99%, and especially from 50% to 99.5%.
• the process for preparing an inclusion complex comprises a step of mixing the polysaccharide with a cyclodextrin, ⁇ -CD,
• the mass percentage of said polysaccharide being from 0.1% to 15%, and in particular from 0.5% to 5%,
• the weight percentage of the cyclodextrin being from 0.1% to 15%, and especially from 0.5% to 5%,
• the mass percentage of water or of the aqueous medium being comprised from 70% to 99.9%, and in particular from 90% to 99%.
• stirring in particular magnetic
• stirring speed of between 10 and 1000 rpm, in particular at a stirring speed of from 100 to 500 rpm, and in particular at a stirring speed of approximately equal at 200 rpm, at a temperature of 1 to 100 ° C, in particular at a temperature of 10 to 35 ° C, and in particular at a temperature of about 20 ° C,
• the duration of the stirring being from 6 hours to 15 days, in particular from 24 hours to 96 hours, and in particular approximately equal to 72 hours.
• stirring in particular magnetic stirring at a speed of between 50 and 1000 revolutions / minute, in particular at a stirring speed of 100 to 500 revolutions / minute, and in particular at a stirring speed of about equal to at 200 rpm, at a temperature of 4 to 60 ° C, in particular at a temperature of 10 to 35 ° C, and especially at a temperature of about 20 ° C,
• the duration of stirring being from 24 hours to 15 days, in particular from 24 hours to 48 hours, and in particular approximately equal to 36 hours.
• the process for preparing an inclusion complex also comprises a step for preparing a polysaccharide bearing hydrophobic groups, by reaction of N-acylation
• R 4 represents: A linear or branched alkyl group containing from 1 to 1000 carbon atoms, in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• the process for preparing an inclusion complex also comprises a step for preparing a polysaccharide bearing hydrophobic groups, by reaction of N-acylation
• R 4 represents:
• a linear or branched alkyl group containing from 1 to 20 carbon atoms in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• the N-acylation reaction relates in particular to chitosan. It is carried out by the action of an acid chloride (Li, Y.-Y. et al., 2006, J Appl Polym Sci 102, 1968-1973), a carboxylic acid, less reactive than the chloride of acid, in the presence of the coupling agent EDCI, or by the action of acid anhydrides (Lee, KY et al., 1995, Biomaterials 16, 1211-1216, Lee, MY et al, 2005, Int J Biol Macromol, 36, 152-158; Mourya, VK and Inamdar, NN (2008) Fact Sheet Polymer 68 (6), 1013-1051).
• Another embodiment of the process for preparing an inclusion complex comprises a step for preparing a polysaccharide bearing hydrophobic groups, by reacting O-acylation between said polysaccharide dissolved in methanesulphonate and 1 to 20 equivalents per polysaccharide unit. of fatty acid chloride, the reaction being carried out at ambient temperature,
• a chitosan comprising hydrophobic groups covalently attached to chitosan by a nitrogen atom and / or by one or more oxygen atoms of said chitosan,
• a chitosan comprising hydrophobic groups covalently attached to chitosan by a nitrogen atom and / or by one or more oxygen atoms of said chitosan,
• the process for preparing an inclusion complex comprises a mixing step between
• M represents the number of units of desacetylated units
• N represents the number of units of acetylated units
• the degree of deacetylation representing the percentage of m in relation to the total number of units is greater than 50%
• R 4 represents a hydrophobic group and is chosen from:
• alkyl group linear or branched, containing from 1 to 20 carbon atoms, in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 6 -CH 3j
• R, R and R are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, selected from methyl, ethyl, propyl, isopropyl, amino -NH 2 groups, ammonium groups - N3 ⁇ 4, or sulfate groups -SO 4 " , and are in particular hydrogen atoms or methyl groups,
• said CD being alpha-cyclodextrin ( ⁇ -CD) in monomeric form, at a concentration of from 1 to 150 g / l of aqueous medium, in particular of from 1 to 50 g of aqueous medium, and in particular of approximately 10 g / l of aqueous medium,
• aqueous solution at the concentration of from 1 to 150 g / l, in particular being from 1 to 10 g / l, and in particular being approximately equal to 1 or approximately equal to 10 g / l,
• the aqueous solvent being chosen from pure water, an aqueous solution of pH ranging from 1 to 7 or from 7 to 12, in particular from 5 to 7, or a physiological saline solution optionally enriched with glucose or containing any other excipient to pharmaceutical, cosmetic, agri-food or veterinary use,
• an aqueous medium chosen from pure water, an aqueous solution of pH ranging from 1 to 7 or 7 to 12, in particular from 5 to 7, or a saline solution optionally enriched with glucose or containing any other excipient for pharmaceutical, cosmetic, agri-food or veterinary use
• the mass of said polysaccharide being from 1 to 150 g / l of aqueous medium, in particular being from 1 to 10 g / l of aqueous medium, and in particular being equal to 1 g / L or 10 g / L of aqueous medium
• the mass percentage of said polysaccharide being from 0.1% to 15%, and in particular from 0.5% to 5%,
• the weight percentage of the cyclodextrin being from 0.1% to 15%, and especially from 0.5% to 5%,
• the mass percentage of the water or of the aqueous medium being from 70% to 99.9%, and especially from 90% to 99%,
• said mixture being carried out with stirring, in particular magnetic stirring at a speed of between 50 and 1000 revolutions / minute, in particular at a stirring speed of 100 to 500 revolutions / minute, and in particular at a stirring speed approximately equal to 200 rpm, at a temperature of 4 to 60 ° C, in particular at a temperature of 10 to 35 ° C, and especially at a temperature of about 20 ° C,
• the duration of the stirring being between 24 hours and 15 days, in particular between 24 hours and 48 hours, and in particular approximately equal to 36 hours,
• the process for preparing an inclusion complex comprises a mixing step between
• ⁇ R represents
• R 4 represents a hydrophobic group and is chosen from:
• ⁇ is a linear or branched alkyl group containing from 1 to 1000 carbon atoms, in particular the groups - (CH 2 ) 4 -CH 3 or - (CH 2 ) 16 -CH 3;
• R represents at least one group of formula
• M represents the number of units of D-glucosamine units
• N represents the number of units of N-acetyl-D-glucosamine units
• R, R and R are hydrogen atoms, alkyl groups having 1 to 3 carbon atoms, chosen from methyl, ethyl, propyl, isopropyl, amino -NH 2 groups, ammonium groups - NH 3 + , or sulphate groups -S0 4 2 ⁇ , and are in particular hydrogen atoms or methyl groups,
• said CD being alpha-cyclodextrin ( ⁇ -CD) in monomeric form
• aqueous solvent chosen from water pure, an aqueous solution of pH of 1 to 7 or 7 to 14, in particular of 5 to 7, or a saline solution optionally enriched with glucose or containing any other excipient for pharmaceutical, medical, paramedical, medical devices animal feed, cosmetics, veterinary, agri-food, pesticides, cosmeto-textiles, perfumery, environmental (eg water pollution control) or in the paint, packaging, building and / or building industry automobile,
• an aqueous medium chosen from pure water, an aqueous solution of pH of from 0 to 7 or 7 to 14, in particular of from 5 to 7, or a physiological saline solution optionally enriched with glucose or containing any other excipient for pharmaceutical, medical, paramedical, medical devices, animal nutrition, cosmetics, veterinary, food, pesticides, cosmetic textiles, perfumery, environmental (eg water depollution) or in the industry paintings, packaging, building and / or automobile.
• the mass of said polysaccharide being from 0.01 to 9000 g / L of aqueous medium, in particular ranging from 1 to 60 g / L of aqueous medium, and in particular being equal to 1 g / L or 10 g / L of medium aqueous,
• the mass percentage of said polysaccharide being from 0.01% to 90%, and especially from 0.5% to 5%,
• the weight percentage of the cyclodextrin being from 0.01% to 90%, and especially from 0.5% to 5%,
• the mass percentage of water or of the aqueous medium being from 10% to 99.99%, and especially from 50% to 99.5%,
• said mixture being carried out with stirring, in particular magnetic stirring at a stirring speed of between 10 and 1000 rpm, in particular at a stirring speed of 100 to 500 rpm, and especially at a stirring speed about 200 rpm, at a temperature of 1 to 100 ° C, in particular at a temperature of 10 to 35 ° C, and especially at a temperature of about 20 ° C,
• the duration of the stirring being between 12 hours and 15 days, in particular between 24 hours and 96 hours, and in particular approximately equal to 72 hours,
• the invention also relates to particles obtained according to the methods described above.
• the invention also relates to a chitosan bearing hydrophobic groups having for formula II:
• a linear or branched alkyl group containing from 1 to 20 carbon atoms in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• M represents the number of units of desacetylated units
• N represents the number of units of acetylated units
• the degree of deacetylation representing the percentage of m relative to the total number of units is greater than 50%.
• the invention also relates to a chitosan bearing hydrophobic groups having for formula II:
• ⁇ R represents
• a linear or branched alkyl group containing from 1 to 1000 carbon atoms in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• R represents at least one group of formula
• M represents the number of units of D-glucosamine units
• N represents the number of units of N-acetyl-D-glucosamine units, provided that the degree of deacetylation (DDA) representing the percentage of m relative to the total number of units is greater than 50%.
• DDA degree of deacetylation
• the invention also relates to a carrageenan bearing hydrophobic groups having the formula I and / u II:
• R represents
• a linear or branched alkyl group containing from 1 to 1000 carbon atoms in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 16 -CH 3 ,
• R represents at least one group of formula
• the invention also relates to a heparin carrying hydrophobic groups having the formula:
• ⁇ R represents
• R represents
• a linear or branched alkyl group containing from 1 to 1000 carbon atoms in particular the groups - (CH 2) 4 --CH 3 or - (CH 2 ) 6 --CH 3 ,
• R represents at least one group of formula
• the invention also relates to an amylopectin bearing hydrophobic groups having the formula:
• R represents a hydrogen atom
• alkyl group linear or branched, containing from 1 to 1000 carbon atoms, in particular the - (CH 2) 14 -CH 3 or - (CH 2 ) 6 -CH 3 groups ,
• R represents at least one group of formula
• the invention also relates to a pullulan carrying hydrophobic groups having the formula:
• R represents
• R represents at least one group of formula
• the invention also relates to a dextran bearing hydrophobic groups having the formula:
• ⁇ R represents
• alkyl group linear or branched, containing from 1 to 20 carbon atoms, in particular the groups - (CH 2) 14 -CH 3 or - (CH 2 ) 1 -CH 3 ,
• R represents at least one group of formula
• the invention also relates to a pectin bearing hydrophobic groups having the formula:
• ⁇ R represents
• R represents
• alkyl group linear or branched, containing from 1 to 20 carbon atoms, in particular the groups - (CH 2 ) 14 -CH 3 or - (CH 2 ) 6 -CH 3 ,
• R represents at least one group of formula
• the invention also relates to hyaluronic acid bearing hydrophobic groups having the formula:
• ⁇ R represents
• R represents
• a linear or branched alkyl group containing from 1 to 1000 carbon atoms in particular the groups - (CH 2 ) 14 -CH 3 or T (CH 2 ) 16 -CH 3 ,
• R represents at least one group of formula
• the invention also relates to a chitin bearing hydrophobic groups having the formula:
• R represents
• a linear or branched alkyl group containing from 1 to 1000 carbon atoms in particular the groups - (CH 2 ) 4 -CH 3 or - (CH 2) 16 -CH 3 ,
• M represents the number of N-glucosamine units
• ⁇ n represents the number of N-acetyl-glucosamine units
• the invention also relates to a method for preparing an encapsulation system comprising a step of mixing particles containing inclusion complexes according to the invention,
• said substance being previously dissolved in an aqueous medium such as water, physiological saline, said medium optionally being enriched with glucose or containing any other excipient for pharmaceutical, medical, paramedical, medical devices, animal feed, cosmetic, veterinary, agro food, pesticides, cosmeto-textiles, perfumery, environmental (water pollution for example) or in the industry of paints, packaging, building and / or automobile.
• an aqueous medium such as water, physiological saline
• said medium optionally being enriched with glucose or containing any other excipient for pharmaceutical, medical, paramedical, medical devices, animal feed, cosmetic, veterinary, agro food, pesticides, cosmeto-textiles, perfumery, environmental (water pollution for example) or in the industry of paints, packaging, building and / or automobile.
• said medium optionally containing a cosolvent selected from ethanol or acetone, or a surfactant selected from polysorbate derivatives, in particular Tween 80 or Tween 40, to obtain particles containing inclusion complexes containing said substance.
• a cosolvent selected from ethanol or acetone
• a surfactant selected from polysorbate derivatives, in particular Tween 80 or Tween 40
• the process for preparing an encapsulation system according to the invention comprises a mixing step:
• a polysaccharide comprising hydrophobic groups covalently attached to the polysaccharide by a nitrogen atom and by oxygen atoms of said polysaccharide,
• said polysaccharide being selected from chitosan, dextran, acid, hyaluronic acid, amylose, amylopectin, pullulan, heparin, chitin, cellulose derivatives, heparan sulfate, dermatan sulfate, keratan sulfate, chondroitin sulfate, cellulose sulfate, dextran sulfate, dextrin sulfate, starch, pectin, alginates, carrageenans, fucan, curdlane, xylan, polyguluronic acid, xanthan, arabinan, polymannuronic acid, and derivatives thereof, and is especially chitosan,
• the mass percentage of said solvent being from 0 to 50% and in particular approximately equal to 25%
• the mass percentage of said polysaccharide being from 0.1% to 15%, and in particular from 0.5% to 5%,
• the weight percentage of the cyclodextrin being from 0.1% to 15%, and especially from 0.5% to 5%,
• the percentage by weight of the water or of the aqueous medium being from 30% to 99.9%, and especially from 90% to 99%,
• the step of mixing the process for preparing an encapsulation system comprises a step of dissolving the active ingredient in an aqueous medium, followed by the addition in the medium of the amphiphilic polysaccharide and ⁇ -cyclodextrin.
• the mixture is placed under magnetic stirring for 3 days. But in a practical way, it is possible to mix all the components at the same time.
• the particles can then be isolated
• FIG. 1 represents the characteristic IR spectrum of N-acylated chitosan by oleic acid (1) in comparison with that of native chitosan (2).
• Figure 2 represents the characteristic IR spectrum of N-acylated chitosan by oleic acid (1) in comparison with that of native chitosan (2).
• Figure 2 shows the formula of unmodified chitosan and its 1 H-NMR spectrum (300 MHz) in DCl at 1% v / v in D 2 0.
• the hydrogen atoms are numbered on the formula of the compound, these numbers appearing on the peaks of the NMR spectrum.
• Figure 3 shows the 1H-NMR spectrum (300 MHz) of chitosan after N-acylation with oleic acid solubilized in DCl at 1% v / v in D 2 0.
• Figure 4 shows the IR spectrum of O-palmitoyl-cbitosan (1) in comparison with native chitosan (2).
• Figure 5 shows the proton NMR spectrum in DMSO-d 6 , O-palmitoyl-chitosan.
• Figure 6 shows the IR spectrum of O-palmitoyl-pullulan (1) compared to that of native pullulan (2).
• Figure 7 shows the IR spectrum of O-palmitoyl amylopectin (1) compared to that of native amylopectin (2).
• Figure 8 shows the IR spectrum of O-palmitoyl dextran (1) compared to that of native dextran (2).
• Figure 8bis shows the IR spectrum of O-palmitoyl-dextran
• Figure 9 shows the IR spectrum of chitin esterified with palmitic acid (1) in comparison with that of native chitin (2).
• Figure 9-bis shows the IR spectrum of O-palmitoyl-chitin
• Figure 10 shows the solid 13 C-NMR spectrum of O-oleoyl-chitin.
• Figure 11 shows the characteristic IR spectrum of O-palmitoyl heparin (1) compared to native heparin (2).
• Figure 12 shows the characteristic IR spectrum of O-palmitoyl heparin (1) compared to native heparin (2).
• Figure 12 shows the characteristic IR spectrum of ⁇ -striatoyl-carrageenan (1) in comparison with native carrageenan (2).
• Figure 13 shows the characteristic IR spectrum of O-palmitoyl hyaluronic acid (1) in comparison with native hyaluronic acid (2).
• Figure 14 shows the images obtained by transmission electron microscopy of different particle preparations.
• FIG. 15 represents the ⁇ , ⁇ and ⁇ cyclodextrins. In the figure, the dimensions are given in Angstroms.
• FIG. 16 represents the effect of the concentration of O-palmitoyl heparin DS 1% on the D h of the nanoparticles.
• FIG. 17 represents the effect of the concentration of ⁇ -CD on the D h of the microparticles composed of O-palmitoyl heparin DS2.
• FIG. 18 represents the observation in MET of particles composed of O-palmitoyl heparin DS2
• FIG. 19 represents the effect of the concentration of ⁇ -CD on the D h of the microparticles composed of O-palmitoyl-carrageenan DS I.
• Figure 20 shows the effect of the concentration of O-palmitoyl-carrageenan DSI on the D h of the microparticles.
• Figure 21 shows the effect of the ⁇ -CD concentration on the D h of O-pallitoyloyl-carrageenan DS2 particles.
• Figure 22 shows the effect of the concentration of ⁇ -palmitoyl-carrageenan DS3 on the D h of the microparticles formed.
• Figure 23 shows the effect of the concentration of ⁇ -palmitoyl-carrageenan DS3 on the D h of the microparticles formed.
• FIG. 23 (1) represents an image obtained by transmission electron microscopy of the preparation ⁇ -cyclodextrin / native chitosan / water (10/1/89)% indicating the absence of formation of nanoparticles or microparticles.
• Figure 23 (2) shows nanoparticles composed of ⁇ -cyclodextrin / CM6 / water (10/1/89)%.
• FIG. 24 (1) represents an image obtained by transmission electron microscopy of the nanoparticles at the laboratory scale.
• FIG. 24 (2) shows an image obtained by transmission electron microscopy of the nanoparticles at the pilot scale.
• CD cyclodextrin
• CM modified chitosan
• Da unit of molar mass in dalton, which corresponds to g L
• HSV the human herpes simplex virus
• HPV the human papillomavirus
• RSV Respiratory syncytial virus
• IR spectra ATR-FTIR, FT spectrometer TR-4100, JASCO: the principle consists in putting a crystal (diamond) in contact with the sample to be analyzed, before being crossed by the infrared beam.
• Particle size measurements The particle sizes were evaluated by the hydrodynamic diameter. Measurements of the average hydrodynamic diameters of the nanoparticles and microparticles were carried out with a Nano-ZS90 nanoseries Zetasizer from Malvern Instruments SA (Orsay, France) by quasi-elastic light scattering. The samples were previously diluted by taking 30 ⁇ of suspension of nanoparticles or microparticles and diluting them in 1 ml of MilliQ ® water. Diameters The hydrodynamics of the microparticles were remeasured by a laser granulometer (MasterSizer 2000) from Malvern Instruments SA (Orsay, France).
• TEM Transmission electron microscopy
• Castor oil comes from the company Croda, France.
• the molar mass of the depolymerized chitosan is determined by capillary viscometry.
• DDA degree of deacetylation
• the IR spectra of the grafted chitosan obtained were recorded.
• the two curves of FIG. 1 show a very wide band between 3430 cm -1 and 3440 cm -1 corresponding to the OH groups (FIG. 1).
• the native chitosan has a low absorption at the level of the vibration of the NH groups around 1578 cm- 1, after N-acylation, it is necessary to note the weak absorption of its derivatives in the bands 3000 cm -1 and 3600 cm -1 and the appearance of two bands 1636 cm -1 and 1657 cm -1 , characteristics of the carbonyl groups and secondary amides respectively and which confirm well the formation of the amide bond .
• For strips to 2922 cm “, 2853 cm” ⁇ 1457 cm "1 and 1198 cm they correspond to alkyl chains of the fatty acid.
• the characteristics of the modified chitosan CM1-CM9 are summarized in Table 1 below.
• the degree of substitution was calculated from the results of the elemental analysis of ⁇ -acylated chitosan and native chitosan.
• N-acyl chitosan For the characterization of N-acyl chitosan by proton NMR, a solution of polymer at a concentration of 5 g / L is prepared in deuterated water (D 2 0) in the presence of deuterated hydrochloric acid (DCl). This step allows the exchange of labile protons of the hydroxyl groups by deuterium atoms. The labile protons of the hydroxyl groups all resonant at the same frequency, their exchange by deuterium atoms makes it possible to eliminate the residual signal of the light water. In order to reduce the viscosity, the experiments were recorded at a temperature of 85 ° C with an acquisition number and a relaxation time of 5 and 1 seconds respectively.
• the chitosan (250 kDa, 2 g) is dissolved in 20 mL of methanesulfonic acid at room temperature with continuous magnetic stirring for one hour.
• the acid chloride (oleic or palmitic) is then introduced into the reaction medium. After 5 hours, the mixture is cooled in an ice bath to stop the reaction, a precipitate is formed. The precipitate is dialyzed for 12 hours and then neutralized with sodium bicarbonate. It is then dialyzed for 48 hours and lyophilized.
• the IR spectrum (FIG. 4) of O-palmitoyl-chitosan shows a characteristic band of carbonyls of an ester group at 1700 cm -1 and bands at 2916 cm -1 , 2849 cm -1 correspond to the alkyl chains of the acid.
• Example 5 Table 2, characteristics of modified chitosan CM10-CM12
• the characteristics of the modified chitosan CM10-CM12 are summarized in Table 2 below.
• the degree of substitution was calculated from the results of the elemental analysis of O-acylated chitosan and native chitosan.
• O-palmitoyl-pullulan was prepared according to the reference of Sunamoto et al (Sunamoto J, Sato T, Taguchi T, Hamazaki H, Naturally occuring polysaccharid derivatives which is an artificial cell wall on an artificial liposome, Macromolecules, 1992, 25, 5665-5670).
• Pullulan (5 g) is dissolved in anhydrous dimethylformamide (55 mL) at 60 ° C.
• 5 ml of anhydrous pyridine and palmitoyl chloride (5 equivalents per unit of glucose triholoside) are added to the resulting solution.
• the reaction mixture is stirred at 60 ° C for 2 h and then for 1 h at room temperature.
• the mixture is poured into ethanol (350 mL).
• the precipitate obtained is extracted and washed with ethanol and then with diethyl ether.
• the white solid obtained is dried under vacuum.
• Amylopectin (5 g) is mixed with anhydrous dimethylformamide. The mixture is stirred at 70 ° C. until the polysaccharide is completely dissolved. Then, anhydrous triethylamine and palmitoyl chloride are added and then heated under stirring for 2 hours. The solution is then diluted in methanol which is responsible for precipitation of O-palmitoyl amylopectin. The white solid obtained is filtered and then dried under vacuum.
• IR spectrum of O-palmitoyl amylopectin 1739 cm -1 band corresponding to the carbonyl of the ester group (FIG. 7), and in particular 2916 cm- 1 , 2849 cm -1 and 1462 cm- 1 bands corresponding to the alkyl chains fatty acid.
• Example 7bis Synthesis and Characterization of Amylopectin Derivatives Grafted with Palmitic Acid
• the (-palnitoyl amylopectin was prepared according to the reference of Sunamoto et al (Sunamoto J, Sato T, Taguchi T, Hamazaki H, Naturally occuring polysaccharid derivatives which is an artificial cell wall on an artificial liposome, Macromolecules, 1992, 25, 5665-5670)
• the amylopectin (5 g) is mixed with 55 ml of anhydrous dimethylformamide at 60 ° C. with continuous magnetic stirring
• To the solution obtained are added 5 ml of anhydrous pyridine, 1.2 ml of anhydrous DMF.
• the amount of palmitoyl chloride was varied depending on the degree of substitution desired.
• Dextran is suspended in anhydrous dimethylformamide. The mixture is stirred at 70 ° C until complete dissolution of the polysaccharide. Then, the anhydrous triethylamine and palmitoyl chloride are added and then heated with stirring for 2 hours. The solution is then diluted in methanol which causes precipitation of O-palmitoyl dextran. The white solid obtained is filtered and then dried under vacuum.
• IR spectrum of O-palmitoyl-dextran 1739 cm -1 band corresponding to the carbonyl of the ester group (FIG. 8), and in particular bands at 2914 cm -1 , 2853 cm -1, correspond to the alkyl chains of the fatty acid.
• Example 8bis Synthesis and Characterization of Dextran Derivatives Grafted with Palmitic Acid
• O-palmitoyl-dextran was prepared according to the protocol described by Sunamoto et al (Sunamoto J, Sato T, Taguchi T, Hamazaki H, Naturally occuring polysaccharid derivatives which is an artificial cell wall on an artificial liposome, Macromolecules, 1992 , 25, 5665-5670).
• Dextran (5 g) is mixed with 55 mL of anhydrous dimethylformamide at 60 ° C. 5 ml of anhydrous pyridine and palmitoyl chloride are added to the solution obtained. The reaction mixture is stirred at 60 ° C for 2 hours and then for 1 hour at room temperature. The mixture is poured into 350 mL of ethanol. The precipitate obtained is extracted and washed with ethanol and then with diethyl ether. The white solid obtained is dried under vacuum.
• Chitin was esterified with oleic acid according to the method described in reference (Yang B.Y., Ding Q, Montgomery R., Preparation and physical properties of chitin fatty acid esters, Carbohydrate Research, 2009, 344 (3) , 336-342).
• Chitin (7.5 g) previously dried under vacuum at 60 ° C. for 16 hours, is introduced into a mixture of trifluoroacetic acid (75 ml) and palmitic acid or oleic acid (28 g). The mixture is then heated to 70 ° C with continuous stirring for 30 min, then cooled to room temperature.
• trifluoroacetic acid causes the formation of the acid anhydride which is much more reactive than the carboxylic acid itself. Being strongly electronegative and having a strong acid power, trifluoroacetic acid causes the protonation of the amine functions, thus allowing a preferential esterification on the alcohol functions.
• O-oleoyl-chitin and O-palmitoyl-chitin Characterization of O-oleoyl-chitin and O-palmitoyl-chitin: Infrared spectroscopy analysis is a fast and efficient method for identifying the chemical groups of esterified chitin compared to the native chitin spectrum.
• the spectrum shown in FIG. 9 is typical of chitin, the frequency of the carbonyl (CO) regions of the amides between 1600 cm -1 and 1500 cm -1 is of great intensity.
• the band corresponding to amide I is divided into two peaks at 1654 cm "1 and 1619 cm".
• the band corresponding to amide II is unique and is 1556 cm -1 .
• FIG. 9bis With regard to the spectrum obtained from O-palmitoyl-chitin (FIG. 9bis), we observe a greater absorption for the bands at 1649 cm -1 and 1554 cm -1 , which explains the presence of the CO and amide II carbonyl groups. thus demonstrating N-acylation of the free amino functions of chitin.
• Figure 9bis (1) also shows bands at 2916 cm- 1 and 2849 cm- 1 corresponding to the alkyl chains of the fatty acid.
• Characterization by I3 C-NMR of the oleic acid-esterified chitin solid is particularly suitable for the characterization of this derivative which is insoluble in water and in the majority of organic solvents.
• the spectrum shown in FIG. 10 shows the presence of 9 resonance peaks whose values are shown in Table 3 below.
• Example 10a Synthesis and Characterization of Heparin Grafted with Palmitic Acid DS2
• the heparin esterified with palmitic acid is purified by dissolving in 10 ml of distilled water and adding NaCl until a NaCl concentration of 10% w / v is reached. Afterwards, 20 ml of methanol are added and the precipitate formed is recovered on a sintered glass filter and then washed with 100 ml of ethanol and then 100 ml of acetone. The solid is dried under vacuum at room temperature.
• Carrageenan (2 g) is suspended in 10 ml of dichloromethane and 1, 2 or 3 ml of palmitic acid chloride contained in a flask.
• O-palmitoyl-carrageenans obtained DSI, DS2 and DS3 in relation to the amount of acid chloride reacted.
• the flask is refluxed under continuous magnetic stirring for 72 h at 30 ° C.
• the solid is recovered on a sintered glass filter and then washed twice with 100 mL of ethanol.
• the solid is dried under vacuum at room temperature for 12 hours.
• Hyaluronic acid (2 g) is mixed with 10 ml of dichloromethane and 2 ml of palmitic acid chloride contained in a flask. The flask is refluxed under continuous magnetic stirring for 5 days at 30 ° C. Afterwards, the solid is recovered on a sintered glass filter and then washed twice with 100 ml of acetone. The solid is dried under vacuum at room temperature for 12 hours.
• Infrared characterization Figure 13: Characterization by infrared spectroscopy of ⁇ -palmitoyl-hyaluronic acid showed the presence of bands corresponding to the carbonyl groups of the ester function.
• Example 13 Formation of microparticles and nanoparticles from ⁇ -cyclodextrin and acylated polysaccharides: N-palmitoyl-chitosan and O-oleoyl-chitosan
• microparticles and nanoparticles were formed from ⁇ -cyclodextrin and polysaccharide grafted with fatty acids.
• the examples of the polysaccharides used are grouped in the table below.
• the protocol consists of weighing ⁇ -cyclodextrin and the O- or N-acyl polysaccharide in a small flask. Subsequently, the distilled water is added to the mixture of ⁇ -cyclodextrin and the polysaccharide O- or N-acylated. The mixture is kept under magnetic stirring for 3 days.
• Table 4 Sizes of particles obtained from N-acylated or O-acylated chitosan An example of an image of particles observed by electron transmission microscopy is shown in FIG. 14.
• the protocol adopted for encapsulating the hydrophilic active principles is to dissolve the active principle in water at an initial concentration as indicated in Table 5 and then to add the amphiphilic polysaccharide and ⁇ -cyclodextrin.
• the mixture is kept under magnetic stirring for 3 days. After the 3 days of mixing, the concentration of the active ingredient that has not been encapsulated is determined in the supernatant of the preparation.
• the supernatant is separated either by simple sedimentation or by centrifugation of the microparticles, or after ultracentrifugation in the case of nanoparticles.
• Example of encapsulated active molecules Example 15 Preparation and Characterization of Nanoparticles Comprising O-Palmitoyl Heparin DSI
• the protocol consists of weighing O-palmitoyl heparin DSI and ⁇ -cyclodextrin in a vial. Subsequently, the water is added to the mixture. The whole is mixed for 72 hours at room temperature thanks to a magnetic bar.
• the O-palmitoyl heparin DSI concentrations of ⁇ -cyclodextrin are shown in Table 6.
• the results of hydrodynamic diameter measurements are shown in Table 6 and Figure 16.
• O-palmitoyl heparin DS2 and ⁇ -cyclodextrin were weighed into a vial. Subsequently, the water is added to the mixture. The whole is mixed for 72 hours at room temperature thanks to a magnetic bar.
• concentrations of ⁇ -cyclodextrin ⁇ -palmitoyl heparin DS 2 are shown in Table 7.
• the results of hydrodynamic diameter measurements are shown in Table 7 and Figure 17.
• Table 7 Effect of the variation of the concentration of ⁇ -CD on the hydrodynamic diameter of nanoparticles composed of heparin DS2
• Figure 18 shows an example of images obtained by electron transmission electron microscopy observations of nanoparticles composed of heparin DS2. These images obtained without contrast agents, show that these nanoparticles self-organized in a well structured manner in the form of a hexagon.
• the 0-palmitoyl-carrageenan DSI and ⁇ -cyclodextrin are weighed in a bottle. Subsequently, the water is added to the mixture. The whole is mixed for 72 hours at room temperature thanks to a magnetic bar.
• the O-palmitoyl-carrageenan de ⁇ -cyclodextrin concentrations and the hydrodynamic diameter measurement results are shown in Table 8.
• LO-palmitoyl-carrageenan DS3 as well as ⁇ -cyclodextrin are weighed into a vial. Subsequently, the water is added to the mixture. The whole is mixed for 72 hours at room temperature.
• the ⁇ -palmitoyl-carrageenan concentrations of ⁇ -cyclodextrin and the results of hydrodynamic diameter measurements are shown in Table 11. • Variation of the amount of fl-palmitoyl-carrageenan DS3.
• Table 11 Effect of variation of O-palmitoyl-carrageenan concentration on the hydrodynamic diameter of microparticles composed of carrageenan DS3
• the microparticles and nanoparticles were formed from ⁇ -cyclodextrin and N-palmitoyl-chitosan CM9.
• concentrations used are summarized in the table below.
• the protocol consists of weighing N-palmitoyl-chitosan, ⁇ -cyclodextrin and castor oil previously marked by Sudan III. The addition of Sudan III makes it possible to detect the instability phenomena of the preparations. Subsequently, the water is added to the mixture. The whole is mixed for 72 hours at room temperature.
• Table 12 Effect of variation of castor oil concentration on the hydrodynamic diameter of microparticles and nanoparticles composed of chitosan CM9
• the microparticles were formed from ⁇ -cyclodextrin and O-oleoyl-chitin DS 0.68%.
• concentrations used are grouped in the table below.
• the protocol consists of weighing O-oleoyl-cliitin, ⁇ -cyclodextrin and castor oil previously marked by Sudan ⁇ .
• Sudan III makes it possible to detect the instability phenomena of the preparations.
• the water is added to the mixture. Everything is mixed for 72 hours at room temperature.
• the preparations observed with the eye did not show phenomena of instability.
• the results of the size measurements are summarized in the table below.
• FIG. 23 (1) is an image obtained after observation by electron transmission microscopy which shows the absence of formation of nanoparticles or microparticles in comparison with the images of nanoparticles obtained with O-palmitoyl-chitosan CM6 / ⁇ -cyclodextrin / water (1/10/89)%.
• Lyophilization is a vacuum drying process at low temperature.
• the product containing water is previously frozen. Even today freeze-drying remains the method of choice for drying heat-sensitive products.
• microparticles were prepared from ⁇ -cyclodextrin and N-oleoyl-chitosan, CM4, DS 13.47%.
• the protocol consists of weighing N-oleoyl-chitosan and ⁇ -cyclodextrin. The concentrations used are shown in Table 14. Thereafter, water is added to the mixture. The whole is mixed for 72 hours at room temperature.
• Freeze-drying is carried out on 1 ml of preparation containing the microparticles previously frozen at (-20 ° C.) for 12 h, then placed in the freeze-dryer for 24 h. After freeze-drying, the preparations are resuspended in 1 mL of MilliQ ® water, we note that the macroscopic appearance of the samples has not changed. In order to ensure that the lyophilization step did not modify the physicochemical characteristics of the particles, measurements of the hydrodynamic diameter were made after lyophilization and compared with those obtained before lyophilization (Table 14).
• Scaling up is a necessity to develop this process on an industrial scale.
• a pilot consisting of a reactor stirred by a mechanical propeller type agitator.
• the stirring speed is set at 350 rpm.
• the regulation of the temperature at 25 ° C is ensured by a circulation of fluid (water / ethylene glycol) connected to a thermostat.
• the batches of 100 ml of microparticles are obtained by successively adding to the reactor (1 g) chitosan of molecular weight 250 kDa N-acylated with 10 equivalents of palmitic acid (CM9, DS 17.01%), 10 g of a-cyclodextrin and MilliQ ® water qs 100 mL.
• Table 16 Hydrodynamic diameters of microparticles composed of a mixture of O-.
• Table 17 Hydrodynamic Diameters of the Microparticles Comprising a Mixture of O-Palmitoyl-Chitosan and O-Palmitoyl-Pullulan
• Table 20 Hydrodynamic Diameters of the Microparticles Comprising a Mixture of O-Palmitoyl-Chitin and O-Palmitoyl-Pullulan
• Example 26 Antiviral activity of nanoparticles composed of O-palmitoyl heparin Cells. Kidney epithelial cells extracted from an African green monkey (cells
• Vero (ATCC CCL-81), Hep-2 and (ATCC CCL-23) human epithelial cells and kidney epithelial cells extracted from an African green monkey (MA-104, ATCC CRL-2378.1) are grown in monolayers. in Eagle's Minimum Essential Environment (MEAT) (Gibco BRL, Gaithersburg, MD) supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic-antifungal solution (Zell Shield, Minerva Biolabs GmbH, Berlin, Germany).
• MEAT Eagle's Minimum Essential Environment
• the 293TT cell line derived from human kidney embryonic cells transformed with simian virus 40 large T antigen (SV40), is cultured in Dulbecco's Modified Minimal Eagle Medium (DMEM) (Gibco-BRL, Gaithersburg, MD). ) supplemented with 10% fetal bovine serum (FCS; Gibco-BRL), 1% Glutamax-I (Invitrogen, Carlsbad, CA) and 1% non-essential amino acids (Sigma Aldrich, Steinheim, Germany). 293TT cells allow the expression of a high level of proteins from vectors containing the SV40 origin of replication due to over-replication of the expression vectors (Buck et al, 2004).
• DMEM Dulbecco's Modified Minimal Eagle Medium
• FCS fetal bovine serum
• Glutamax-I Invitrogen, Carlsbad, CA
• non-essential amino acids Sigma Aldrich, Steinheim, Germany
• HSV-1 and HSV-2 Clinical isolates of HSV-1 and HSV-2 have been provided by Professor M. Pistello (University of Pisa, Italy). The HSV-1 and HSV-2 strains were propagated and titrated by the Vero cell plate technique.
• the RSV strain A2 (ATCC VR-1540) was propagated and titrated by the Reed-Muench method on Hep-2 cells described previously (Donalisio et al, 2012).
• the human rotavirus strain Wa (ATCC VR-2018) was active with 5 ⁇ g / ml with porcine type IX pork trypsin (Sigma, St. Louis, Mo.) for 30 min at 37 ° C and propagated in patients.
• HPV PsV production Plasmids and 293TT cells used for pseudovirus (PsV) production were provided by John Schiller (National Cancer Institute, Bethesda, MD). The protocol details and plasmid maps of this study can be found at the following link: http://home.ccr.cancer.gov/lco/default.asp. HPV-16 pseudoviruses were produced according to previously described methods (Buck et al, 2005). Briefly, 293TT cells are transfected with plasmids expressing the major and minor capsid proteins of papillomavirus (respectively L1 and L2) and a reporter plasmid expressing secreted alkaline phosphatase (SEAP), pYSEAP.
• SEAP secreted alkaline phosphatase
• the capsids were matured overnight in a cell lysate, the clarified supernatant was then loaded over a density gradient of 27 to 33 to 39%, Optiprep (Sigma-Aldrich, St. Louis, MO), at room temperature for 4 hours. The material was then centrifuged at 340,000 g for 3.5 hours at 16 ° C in an SW50.1 rotor (Beckman Coulter, Inc., FuUerton, CA) and then collected by puncture at the bottom of the tubes. Fractions are assayed for purity in glycine-10% Tris-Sodium Dodecyl Sulfate (SDS) gels, titrated on 293TT cells for SEAP detection, and then pooled and frozen at -80 °. C for the desired duration. The L1 protein content of PsV stocks was determined by comparison with standard bovine serum albumin on SDS-polyacrylamide gels stained with Coomassie Blue.
• SDS Tris-Sodium Dodecyl Sulfate
• O-palmitoyl-hyaluronic acid and ⁇ -cyclodextrin are weighed in a bottle. Subsequently, the water is added to the mixture. The whole is mixed for 72 hours at room temperature. The concentration of ⁇ -cyclodextrin and the results of measurements of the hydrodynamic diameters are shown in Table 25.
• the protocol adopted for encapsulating the hydrophobic active principles is to dissolve the active ingredient in an ethanol / water mixture at an initial concentration as indicated in Table 26 and then to add the amphiphilic polysaccharide and ⁇ -cyclodextrin.
• the mixture is kept under magnetic stirring for 3 days. After the 3 days of mixing, the concentration of the active ingredient that has not been encapsulated is determined in the supernatant of the preparation. The supernatant is separated either by simple sedimentation or by centrifugation of the microparticles, or after ultracentrifugation in the case of nanoparticles.

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