WO2000001373A1 - Matieres et procedes d'encapsulation - Google Patents

Matieres et procedes d'encapsulation Download PDF

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Publication number
WO2000001373A1
WO2000001373A1 PCT/GB1999/002159 GB9902159W WO0001373A1 WO 2000001373 A1 WO2000001373 A1 WO 2000001373A1 GB 9902159 W GB9902159 W GB 9902159W WO 0001373 A1 WO0001373 A1 WO 0001373A1
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Prior art keywords
capsules
chitosan
membrane
reaction
molecular weight
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PCT/GB1999/002159
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English (en)
Inventor
Artur Bartkowiak
David Hunkeler
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Ecole Polytechnique Federale De Lausanne
Kiddle, Simon
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Application filed by Ecole Polytechnique Federale De Lausanne, Kiddle, Simon filed Critical Ecole Polytechnique Federale De Lausanne
Priority to EP99929581A priority Critical patent/EP1094792A1/fr
Priority to AU46366/99A priority patent/AU4636699A/en
Publication of WO2000001373A1 publication Critical patent/WO2000001373A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—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
    • A61K9/5005—Wall or coating material
    • A61K9/5021—Organic macromolecular compounds
    • A61K9/5036—Polysaccharides, e.g. gums, alginate; 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
    • A61K9/5089—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
    • A61K2035/128—Immunoprotecting barriers, e.g. jackets, diffusion chambers capsules, e.g. microcapsules

Definitions

  • the present invention relates to materials and methods relating to encapsulation, and in particular to encapsulating biological materials in capsules by complex formation between oppositely charged polymers.
  • Encapsulation is currently employed in the food, agriculture and biotechnology and biomedical industries. Examples of these and potential applications are the encapsulation of islets for the treatment of diabetes mellitus, the use of encapsulated bioartificial organs targeted at treating neurodegenerative disorders such as Parkinson's disease, Alzheimer's disease and Huntington' s chorea, and in the control of chronic pain and the administration of human growth factors .
  • One approach for forming capsules is based on complex formation between oppositely charged, high molecular weight polymers.
  • a solution of a first polymer including a material to be encapsulated is preformed in droplets between about 0.2 -5.0mm in diameter and contacted with the second oppositely charged polymer.
  • droplets of the first polymer and the material to be encapsulated are generated using a capillary or spraying device and contacted with the second polymer, e.g. by falling into a precipitation bath.
  • the reaction between the polymers at the surface of the droplet forms a membrane around a liquid core including the encapsulated material .
  • PECs polyelectrolyte complexes
  • GB 2 135 954 A (Dautzenberg et al) describes a method of forming capsules from a pair of oppositely charged polyelectrolytes or a single polyelectrolyte and low molecular weight organic counter ions.
  • preformed particles of a polyelectrolyte are contacted with a second polyelectrolyte or organic counter ions so that a membrane forms around the droplet thereby producing a capsule.
  • the examples in this reference employ high molecular weight polyelectrolytes such as cellulose sulphate, carboxymethylcellulose or alginate.
  • the lowest molecular weight polyelectrolyte disclosed is polydiallylmethylammonium chloride (40kD) , from which capsules were formed with sodium cellulose sulphate at pH 7.0.
  • the reaction described in this application requires high levels of the organic counter ionic material to provide the necessary the mass transfer driving force to form a dense membrane skin. Further, the use of the organic counter ions suffers from the disadvantage that these materials can be toxic, the method does not provide a satisfactory trade-off between the permeability and the mechanical strength of the capsules, and the reaction times are exceedingly long (tens of minutes) .
  • EP 0 152 898 A describes the use of oppositely charged polymers solutions to encapsulate biological materials, exemplifying the use of chitosan and alginate. This method uses high molecular weight polymers and consequently is carried out below pH 6.6 to ensure that these materials are soluble. Further, it relies on polyvalent metal cations (e.g. Ca 2+ ions) present in the cationic polymer solution to gel or harden the anionic polymer during capsule formation.
  • polyvalent metal cations e.g. Ca 2+ ions
  • US Patent No:4,808,707 discloses a method of forming capsules using chitosan and alginate, employing a pH of 6.1 or less and esterifying a proportion of the alginate in an attempt control the permeability of the membrane around the capsules.
  • the reaction was also carried out in the presence of polyvalent metal ions (Ca 2+ ) , as with EP 0 152 898 A to promote gelling of beads during capsule formation.
  • US Patent No: 5, 462, 866 (Vanderbilt University) describes a method for producing uniform capsules by forming capsules by individually enveloping droplets of polyanion solution with a collapsing annular sheet of polycation solution while the sheet is travelling downward at the same velocity as the droplets.
  • This approach was employed as high molecular weight polyelectrolytes form viscous solutions leading to polyanion droplets distorting on impact with the surface of the polycation solution, and form irregular and highly distorted capsules.
  • the patent discloses that the problem arises as a 0.2% solution of high molecular weight chitosan (>100kD) is about 10 times as viscous as a calcium chloride solution.
  • Example 4 in this patent mentions "a 0.2% citosan [sic] solution with an approximate molecular weight of 20,000 daltons" .
  • the chitosan used in this example is a high molecular weight polymer, probably having a molecular weight of 200kD.
  • GB 2 145 992 A discloses a method for encapsulating cells by cross-linking droplets of chitosan by exposing the droplets to anionic materials such as polyaspartic acid and polyglutamic acid.
  • anionic materials such as polyaspartic acid and polyglutamic acid.
  • the chitosan has a high molecular weight and requires acidic pH to form solutions in water.
  • US Patent No: 5, 089,272 (Shioya et al) relates to a method of producing capsules using ionic strength to adjust the permeability of the capsule.
  • capsules are formed from a polyanionic polysaccharide and chitosan obtained from natural source materials having a molecular weight of 2800kD and 1600kD.
  • the present invention relates to methods of producing capsules from oppositely charged ionic polymer solutions, wherein one of the polymers is oligomeric.
  • one of the polymers is oligomeric.
  • the use of an oligomeric polymer provides microcapsules having good mechanical properties and allows capsules to be formed at neutral or alkaline pH.
  • the use of oligomers in conjunction with higher molecular weight polyelectrolytes can help to ameliorate some of the problem associated with the prior art methods discussed above.
  • the present invention provides a method for encapsulating a core material within a capsule having a permeable or semipermeable membrane using a complex formation reaction between oppositely charged polymers, the method comprising: forming a droplet from a solution of an inner polymer comprising at least one positively or negatively charged polymer and the core material; and, contacting the droplet with a solution of an outer polymer comprising at least one polymer having an opposite charge to the polymer or polymers forming the droplet so that the polymers react to form a membrane around the droplet, wherein one of the polymers is an oligosaccharide with a molecular weight (MW) of less than 10. OkD and the reaction is carried out at a pH between about 6.6 and 7.5.
  • MW molecular weight
  • the present inventors surprisingly found that there is a significant correlation between the solubility of polysaccharides such as chitosan and their molecular weight, and that the use of an oligomeric polymer can both improve the solubility of the polymer at physiologically relevant pH and provide capsules having good mechanical properties.
  • the present invention provides capsules having useful strength and permeability at physiologically appropriate pH by employing chitosan having a molecular weight of less than 20. OkD and more preferably, less than 10. OkD.
  • the oligosaccharide is produced from a commercially available natural source materials such as chitosan (modified chitin) . While these materials are readily available, in general they have relatively high molecular weights and may require pretreatment to reduce their molecular weight so that they can then be used in the above method. Conveniently, the pretreatment is carried out using radical degradation to break down the polysaccharide so that it has a molecular weight below 10. OkD and is soluble at the pH of the encapsulation reaction, e.g. using methods such as radical degradation with peroxide, hydrolytic-enzymatic degradation, ⁇ -ray irradiation or acidic hydrolysis.
  • the oligosaccharide has the further advantage that it is degraded in a controlled way, providing a consistent material and a degree of quality control required in many biomedical applications.
  • the oligosaccharide has a degree of deacetylation >90%, and more preferably >95% and/or a polydispersity in MM less than 2.0, more preferably less than 1.8, and still more preferably less than about 1.5.
  • the method does not require the use of high temperatures or polyvalent metal ions to promote gelling of the polymer solution and/or the use of organic counter ions in a precipitation bath.
  • multivalent ions such as calcium, barium or iron are used to gel the polyelectrolyte.
  • These beads are then coated with a solution of an oppositely charged polyelectrolyte.
  • the solid bead is then converted into a permeable capsule by liquefying the gel, generally via the addition of a chelating agent such as EDTA or sodium citrate.
  • a chelating agent such as EDTA or sodium citrate.
  • these materials can be toxic and have a detrimental effect when present in methods for encapsulating biological materials.
  • the method is preferably a single step reaction, in contrast to many of the prior art multi-step processes.
  • the polymers are naturally occurring polymers to promote biocompatibility with encapsulated biological materials.
  • the use of oligosaccharides or polysaccharides is especially preferred.
  • the polymers can be negatively charged polymers such as alginate, i - carrageenan or ⁇ -carrageenan or carboxymethylcellulose. Chitosan is a preferred positively charged polymer.
  • the oligosaccharide has a molecular weight less than 20. OkD, more preferably less than 10. OkD, and more preferably between about 0.5 and about 9. OkD, more preferably between about 0.6 and about 6. OkD and most preferably between about 1.0 and about 3. OkD.
  • the other polymer has a molecular weight in the range 100-lOOOkD, more preferably in the range 100-500kD.
  • the oligomeric polymer is positively charged, e.g. using the combination of oligochitosan and alginate.
  • the polymer solutions may include low molecular weight monovalent metal salts (e.g. Na + or K + ) , or agents to promote isotonicity (e.g. mannitol) or buffer solutions.
  • low molecular weight monovalent metal salts e.g. Na + or K +
  • agents to promote isotonicity e.g. mannitol
  • buffer solutions e.g. mannitol
  • these and other additives can be included to help to maintain the osmotic pressure between the cells and polymer solutions in equilibrium.
  • suitable anionic polymers for capsule formation include alginate, carboxymethylcellulose, xanthan, hyaluronic acid, gellan gum, cellulose sulphate, carrageenans (kappa- or iota-) and polyacrylic acid.
  • Preferred cationic polymers include chitosan, glycol chitosan, chitosan derivatives, polyallylamines, quaternised polyamines, polydiallyldimethyl ammonium chloride (polyDADMAC) , polyDADMAC-acrylamide, polytrimethylammoethylacrylate-co-acrylamide, polymethylene-co-guanidine, polyvinylamine .
  • the capsules can be produced by dropping one of the polymer solutions into a bath of the other.
  • the concentration of the polymers is in the range 0.1 to 5.0%, more preferably in the range 0.5 to 2.0%.
  • the droplets can be formed using passing one of the polymers through a capillary, by extrusion with gas or a liquid, using a spinning disk or by electrostatic generation.
  • the droplets are formed from the higher molecular weight polymer, with the oligomeric polymer being the outer polymer.
  • the capsules produced using the method have a diameter between 50 ⁇ m and 5.0mm.
  • the diameter of the capsules can be controlled by varying the flow rate of the inner polymer solution or by controlling physical parameters such as the applied voltage, if electrostatic droplet generation is used, or the fluid stripping velocity, if the capsules are made by air/fluid stripping.
  • the membrane around the capsules is typically 5 to 400/xm thick, and more preferably 50 to 200 ⁇ m thick. Preferably, the membrane is permeable or semi-permeable .
  • the encapsulation method can also allow the molecular weight cut off of the membrane to be controlled between about 1 to about 300kD by selecting the polyelectrolyte, oligomer or monovalent salt concentration, pH, temperature or reaction time.
  • the core material can be encapsulated by mixing it with the polymer solution which is formed into droplets.
  • the method is particularly useful for encapsulating biological or biologically active materials, including cells, bacteria, enzymes, antibodies, drugs, cytokines or hormones, as it can be carried out biologically compatible pH, preferably at a pH greater than about 6.5, more preferably at a pH between about 6.6 and 7.5, more preferably at a pH between about 6.8 and 7.4 and more preferably at a pH between about 7.0 and 7.4, and more preferably at a pH between about 7.2 and 7.4.
  • the present invention provides a method which additionally comprises the steps of: selecting a physical property required of a capsule; selecting the molecular weight of an oligosaccharide between 0.5 and 20. OkD that will provide capsules having the physical property when reacted with a core material and an oppositely charged polymer; reacting the polymers and core material as described above to produce the capsules; wherein the physical property is deformability and/or mechanical strength.
  • the capsules have one or more of : (a) a high deformability of >70%, more preferably
  • the present invention provides a method which comprises following the encapsulation of a core material as described above, the further step of formulating the capsules in a composition or other product .
  • the present invention provides a polymeric capsule comprising a semipermeable membrane encapsulating a core material produced by the above method. In a further aspect, the present invention provides a polymeric capsule comprising a semipermeable membrane encapsulating a core material produced by the above method for use in a method of medical treatment .
  • Figure 1 Schematic representation of capsule formation via polyelectrolyte complexation.
  • A droplet generation apparatus
  • B contacting of oppositely charged polymers
  • C capsule with membrane.
  • Figure 2 Variation in the mechanical properties of the chitosan-alginate capsules with changes in the molecular weight of chitosan.
  • Figure 4 Permeability of capsules synthesized in various solutions (for conditions see Table 1) . Solute diffusion of dextran of different molar masses.
  • Figure 5 Relative cross linking density of capsule membranes in function of membrane/capsule volume ratio for conditions see Table 1.
  • the arrows indicate the direction of the pH increase (from 6.0 to 7.0) .
  • Capsules with a permeable, or semipermeable wall, and a liquid core can be formed by introducing the liquid droplets from aqueous solution of an anionic polyelectrolyte into an aqueous solution of a countercharged cationic polyelectrolyte (Figure 1A) .
  • the capsule wall consists of polymer complex which is a product of the reaction of oppositely charged polyelectrolytes ( Figure IB) .
  • the anionic polymer is generally interior to the cationic polymer, the reverse can also be applied.
  • Figure 1C shows a schematic of a membrane generated by the interaction of the two polymers .
  • Section A and examples 1 to 23 describe microcapsules production with different polymers and reaction conditions. Following on from this, a series of thermodynamic and kinetic experiments using the preferred chitosan/alginate system are described in section B. Finally, in vivo implantation experiments are described in section C.
  • Keltone HV - Sodium Alginate (lot. 54650A) was obtained from Kelco/NutraSweet (San Diego, CA, USA) .
  • An intrinsic viscosity [ ⁇ ] of 780mL/g was measured in 0. IM NaCl at 20°C in a capillary viscosimeter (Viscologic TI 1, SEMA Tech, France) . This reflects to MM of 390kD.
  • Chitosan Samples with varying molar masses (l-10kD) were obtained in controlled radical degradation via continuous addition of hydrogen peroxide (0.8-6.4mMol/g of polysaccharide) to 2.5% chitosan solution of pH 3.5 to 4.0 at 80°C.
  • Molar masses of chitosan samples were estimated by GPC at a flow-rate of 0.5 mL/min.
  • a Shodex OHpak SB-803 HQ column (Showa-Denko Company, Tokyo, Japan) was employed as the stationary phase, using 0.5M acetic acid/0.5M sodium acetate as an eluent, as recommended.
  • Polyethylene glycol standards (PSS, Mainz, Germany) were used for column calibration and as a relative reference for MM calculation. All other reagents were of analytical grade.
  • the degree of chitosan amino group ionization ( ⁇ ) at different pHs were determined from potentiometric titration curves, where 20 ml of 0.1% polymer solutions (pH 2.5) were titrated with 0.02M NaOH.
  • Capsules were produced from a pair of oppositely charged polysaccharides. A 0.5-2% aqueous sodium alginate was prepared in deionized water or 0.9% NaCl. Approximately 4mL of this solution was introduced into a 5mL disposable syringe with a 0.4mm flat-cut needle (Becton Dickinson AG, Basel, Switzerland) .
  • the droplets were sheared off for 60 sec at a flow rate lmL/min (kdScientific syringe pump - Bioblock Scientific, Frenkendorf, Switzerland) into 20mL of solution of 1% chitosan (M n varied between 2-3kD) at pH 6, 6.5 and 7.0 previously adjusted with IM NaOH respectively.
  • the resulting microcapsules (2.5-3mm in diameter) were allowed to harden for 20 minutes under gentle stirring (200rpm) with small magnetic bar, filtered and rinsed with the solvent used for preparation of the polysaccharide solutions. Collected microcapsules (approx. 2mL in volume) were stored at 4°C in 0.9%
  • chitosan molar mass on the relative mechanical strength of the prepared chitosan/alginate capsules was determined as follows.
  • the oligochitosan generally had a "lower critical oligomer chain length" (M n >l-2kD) for stable capsule formation, with a maximum in mechanical capsule strength observed at a MM of 2-3kD. All oligochitosan samples used in the mechanism and kinetics studies were prepared within this range (2.2-2.8kD) .
  • Chitosan Conversion Determination Samples of 400mL of chitosan solutions were withdrawn from the reaction bath every 5 minutes during capsule formation and characterized by using the aforementioned chromatographic method. By assuming that the total concentration of the solute is proportional to the GPC elution curve area (Beers' Law) , the conversion and MM of chitosan were calculated from the respective chromatogram derivatives.
  • the dextran concentrations were proportional to the maximum heights of the detected chromatographic peaks (Beers' Law) and calculated with respect to the initial polymer standard concentration, which is the concentration of the dextran standard with the defined MM at time 0 (immediately following the addition of 2ml of standard solution into 1ml of capsules) .
  • the membrane permeability calculated from decrease in dextran concentration, was maximal at 33.3% of polymer diffusion.
  • the cut-off of the microcapsules was defined as the lowest MM of dextran for which diffusion was smaller than 2% after 3 hours.
  • capsules were produced according to the following protocol.
  • Solvents used included water, as well as aqueous solution of low molecular weight monovalent metal salts, and isotonic or buffer solutions.
  • reaction time varied between 1 minute and three hours.
  • the capsules are stable after a few seconds, but 1 minute is a preferred minimum reaction time .
  • Alginate can be replaced by either iota-or kappa-carrageenan or carboxymethylcellulose (CMC) (Table I) •
  • Capsule size 50 ⁇ m to 5.0mm in diameter, exemplifying the ranges 3 -4mm, 0.4 -0.6mm and 0.8 -1.0mm.
  • FIG. 2 shows a schematic graph correlating the mechanical strength of the capsules with the molecular weight (MW) of the outer polymer (chitosan) . This shows that below 600 daltons, no capsules were precipitated under the conditions used, while above 10. OkD, the membrane formed had low mechanical strength. Between these extremes, stable capsule were generated with a maximum at a molecular weight of 2,000 daltons.
  • MW molecular weight
  • Example 1 The following examples are provided by way of illustration to demonstrate how the capsules can be produced under a range of different conditions.
  • Example 1
  • the droplets Immediately after entry into the precipitation bath, the droplets become covered with a membrane of the alginate/chitosan complex. After 20 minutes, the capsules were separated by decanting and washed with water.
  • the spherical transparent microcapsules with good mechanical properties have a diameter of 3 -4mm.
  • Example 2 Following the procedure of Example 1, 0.75-1% w/v of sodium alginate solution in 0.9% NaCl was added dropwise to precipitation bath of 1-2% w/v oligochitosan solution also in 0.9% NaCl.
  • the chitosan had molecular weight of 3-6 KD and a solution pH of 6.7.
  • the droplets become covered with membrane of the alginate/chitosan complex.
  • the capsules were separated by decanting and washed with 0.9% NaCl.
  • the spherical semi-transparent microcapsules with good mechanical properties have a diameter of 3 -4mm.
  • Example 2 Following the procedure of Example 1, 0.75-1% w/v iota- carrageenan solution in water was added dropwise to precipitation bath of 0.5-2 %w/v oligochitosan solution in water.
  • the chitosan had molecular weight 1-4 KD and its solution pH 6.9.
  • the droplets became covered with a membrane of the iota-carrageenan/chitosan complex.
  • the capsules were separated by decanting and washed with 0.9% NaCl.
  • the spherical semi-transparent microcapsules with good mechanical properties have a diameter of 3 -4mm.
  • Example 2 This example was carried out under the conditions used in Example 1 with a pH of 6.6.
  • Example 6 This example was carried out under the conditions used in Example 1 with a reaction time of 40 minutes.
  • Example 2 This example was carried out under the conditions used in Example 1 with a diameter of 0.8 -lmm.
  • Example 2 This example was carried out under the conditions used in Example 1 with a diameter of 0.4 -0.6mm.
  • Example 2 This example was carried out under the conditions used in Example 2 with a pH of 7.2 and the chitosan with a molecular weight of l-3kD.
  • Example 2 with a diameter of 0.4 -0.6mm.
  • Example 14 This example was carried out under the conditions used in Example 3 with a reaction time of 30 minutes.
  • Example 3 This example was carried out under the conditions used in Example 3 with a diameter of 0.8-lmm.
  • Example 3 This example was carried out under the conditions used in Example 3 with a diameter of 0.4-0.6mm.
  • Example 4 with a diameter of 0.8 -lmm.
  • Example 4 This example was carried out under the conditions used in Example 4 with a diameter of 0.4 -0.6mm.
  • Example 21 This example was carried out under conditions used in Examples 1,6,7 and 8 where alginate and chitosan solutions were prepared in MOPS or HEPES buffer solutions .
  • Membrane Formation Capsule permeability is controlled by the crosslinking density of the polymer network. As the membrane formation is an electrostatic process, pH and ionic strength influence complexation, with the protonation degree of chitosan being the key variable. Effect of Chitosan Protonation:
  • Chitosan is weak base and has therefore a limited solubility in the higher pH region, i.e. precipitation occurs when the pH exceeds 6.0-6.5.
  • precipitation occurs when the pH exceeds 6.0-6.5.
  • Figure 3 shows the ionization degrees of 50kD chitosan and of two oligochitsan samples as a function of pH. At a pH 4.0 all samples are in their fully protonated form. However, with increasing pH protonation becomes highly dependent on the MM.
  • the structure of the chitosan/alginate microcapsule membrane may be represented as alternative sequences of ionic interchain bonds and loop-like regions incorporating the uncoupled units of both chains. More compact membranes, with smaller "loops" and lower cutoffs, are formed at lower pHs, though only in the simple aqueous system (capsule prepared in water) .
  • the introduction of low MM ions changes the polyelectrolyte solution conformation.
  • longer chains, as in alginate transform from elongated into more compact coil structures.
  • the short oligochitosan chains can more easily penetrate the alginate chain network and form thicker and less dense membranes.
  • Table 2 summarizes the chitosan MM bound within the capsule membrane after 5 and 20 min of reaction.
  • a very selective binding with respect to the MM of chitosan was observed during capsule formation in solutions which differ in pH and ionic strength. This effect is most prominent between capsules prepared in solutions with varying ionic strengths, such as water and 0.9% NaCl.
  • the lower MM portion of chitosan is involved in membrane formation in salt free system. This specific complexation is caused by the so called polyelectrolyte effect. It has been shown for a number of interpolymer reactions between mixtures of oligomers that preferential binding to the longest chains takes place. However, this conclusion was primarily based on hydrogen bond complexation systems.
  • the properties of the capsules vary remarkably as a function of the reaction conditions with the reaction time having the most profound influence on the process of binary capsule formation.
  • the mechanical strength of the capsules increases with reaction time due to the increase of the capsule wall thickness (Table 4) .
  • no significant differences in capsule permeability and cut-off were measured. This indicates that the process of "skin" formation during the first minutes of reaction controls the cut-off of the membrane.
  • the subsequent diffusion controlled building-up of the inner membrane is responsible for the capsule's mechanical resistance.
  • Table 5 illustrates that lower MM chitosans (2.6kD) form slightly denser membrane skins which limit oligocation diffusion and membrane build-up and, as a consequence, lead to mechanically less resistant capsules.
  • decreasing the oligochitosan concentration changes the kinetics of membrane formation, engendering a significant reduction in mechanical stability.
  • no significant difference in membrane permeability was observed.
  • the capsule membrane grows more slowly at higher alginate concentrations (Table 6) . Oligocation diffusion seems to be retarded by the higher concentration of the polyanion chain network, which leads to thinner but due to the higher charge density, denser membrane with lower cut-off and higher mechanical resistance.
  • the ionic strength and the pH of the solution utilized during the capsule formation process influence the structure of the alginate/oligochitosan membrane.
  • the presence of a low MM salt (0.9% NaCl) accelerates the diffusion of the oligocations and leads to thicker capsule walls, although with lower relative crosslinking density.
  • sodium chloride diminishes the effect of chitosan charge density on the permeability and the mechanical properties of capsules and shifts the cut-off of prepared membranes towards higher MM values.
  • the permeability of the new alginate-oligochitosan microcapsules therefore, depends mainly of two factors: (1) the density of the membrane which forms the outer shell influences the cut-off, which is particularly important for capsules prepared in water, and (2) the final membrane thickness controls the kinetics of solute diffusion and is more important for capsules synthesized in 0 . 9% NaCl .
  • the mechanical properties of capsules prepared at physiological conditions are primarily influenced by membrane thickness and can be controlled by parameters such as reaction time, oligochitosan MM and concentration.
  • the alginate concentration significantly effects both mechanical and porosity characteristic of the capsule membrane.
  • Keltone HV Keltone Chemical Company, UK
  • 500mL in 0.9% NaCl was filtered over 5mm and 0.22mm filters (high pressure sterile filtration; 200mL) .
  • the polyanion was extruded at 5mL/h using air stripping apparatus (22 G needle-Becton Dickinson, Ireland) into the chitosan receiving bath (30mL of 1% oligochitosan solution) and allowed to react in the bath for 5 minutes. After a further 5 minutes, the reaction solution was discarded over sterile plastic tube with nylon net. The capsules were then washed several times with PBS solution in a plastic tube (transferred 2x into the fresh PBS solution) .
  • the method described herein can be applied to the encapsulation of a wide variety of materials.
  • the method is particularly relevant to the encapsulation of biological or biologically active materials.
  • the encapsulation can be used to physically protect the encapsulated material, e.g. from mechanical or environmental, or where the capsules are for implantation into a human or animal, to help to prevent or reduce an immune rejection to the material.
  • Particularly preferred applications include:
  • Capsules (2.5 mm in diameter) were obtained through a reaction between a 1% alginate (Keltone HV) solution and a chitosan solutions (pH 7.0, 0.9% NaCl, 20 min. reaction time, ambient temperature) .

Abstract

L'invention concerne un procédé permettant d'encapsuler une matière de noyau dans une capsule pourvue d'une membrane perméable ou semi-perméable au moyen d'une réaction de formation complexe entre des polymères à charges opposées, un des polymères étant un oligosaccharide tel qu'un chitosane. La réaction est réalisée à un pH compris entre 6,6 et 7,5. Ce procédé permet de surmonter la faible solubilité de ces polysaccharides à masse moléculaire élevée à des pH neutres et alcalins, grâce à des capsules présentant une force et une perméabilité avantageuses.
PCT/GB1999/002159 1998-07-06 1999-07-06 Matieres et procedes d'encapsulation WO2000001373A1 (fr)

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EP99929581A EP1094792A1 (fr) 1998-07-06 1999-07-06 Matieres et procedes d'encapsulation
AU46366/99A AU4636699A (en) 1998-07-06 1999-07-06 Materials and methods relating to encapsulation

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GB9814619.4 1998-07-06
GBGB9814619.4A GB9814619D0 (en) 1998-07-06 1998-07-06 Materials and methods relating to encapsulation

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Cited By (24)

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EP1129771A1 (fr) * 2000-03-04 2001-09-05 Primacare S.A. Microcapsules
WO2001087475A1 (fr) * 2000-05-19 2001-11-22 Ecole Polytechnique Federale De Lausanne Materiaux et procedes d'encapsulation
EP1184027A1 (fr) * 2000-09-01 2002-03-06 Primacare S.A. Composition pour baton applicateur cosmetique
US6733790B1 (en) 1999-07-02 2004-05-11 Cognis Iberia S. L. Microcapsules and processes for making the same using various polymers and chitosans
EP1502646A1 (fr) 2003-08-01 2005-02-02 The Procter & Gamble Company Microcapsules
WO2006064331A1 (fr) * 2004-12-17 2006-06-22 Medipol Sa Particules hydrophiles a base de derives de chitosanes cationiques
EP1702674A1 (fr) * 2005-03-16 2006-09-20 Cognis IP Management GmbH Perles visibles
EP1727525A1 (fr) * 2004-03-23 2006-12-06 Lytone Enterprise, Inc. Capsule contenant du chitosane noye ou encapsule
WO2007031812A1 (fr) * 2005-09-16 2007-03-22 Medipol Sa Particules a base de chitosane
CN1313157C (zh) * 2005-04-18 2007-05-02 天津大学 促进药物透皮传递的壳低聚糖组合物及其使用方法
US7641917B2 (en) 2001-05-30 2010-01-05 Csir Method of encapsulating an active substance
US7897555B2 (en) 2003-08-01 2011-03-01 The Procter & Gamble Company Microcapsules
WO2012099482A3 (fr) * 2011-01-18 2012-11-01 Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies (A4Tec) Dispositif, procédé et système pour la préparation de microcapsules
CN102824332A (zh) * 2012-09-14 2012-12-19 东北农业大学 一种抗菌肽缓释微胶囊的制备方法
RU2536225C1 (ru) * 2014-01-28 2014-12-20 Федеральное государственное бюджетное учреждение науки Тихоокеанский институт биоорганической химии им. Г.Б. Елякова Дальневосточного отделения Российской академии наук (ТИБОХ ДВО РАН) Средство, обладающее гастропротекторной активностью
WO2017089974A1 (fr) 2015-11-23 2017-06-01 Association For The Advancement Of Tissue Engineering Cell Based Technologies & Therapies Associação Composition comprenant des complexes de polyélectrolyte, procédés et utilisations de cette dernière
CN109172536A (zh) * 2018-09-28 2019-01-11 青岛大学 具有多层三明治结构的中空胶囊的制备及得到的中空胶囊
CN109172535A (zh) * 2018-09-26 2019-01-11 青岛大学 聚电解质氧化石墨烯中空胶囊的制备及得到的中空胶囊
CN109223726A (zh) * 2018-09-28 2019-01-18 青岛大学 一种新型中空胶囊的制备方法及得到的新型中空胶囊
CN109316460A (zh) * 2018-09-26 2019-02-12 青岛大学 一种多糖类聚电解质中空胶囊的制备方法及得到的中空胶囊
CN109338490A (zh) * 2018-09-30 2019-02-15 青岛大学 一种中空多层结构纤维的制备方法及得到的纤维与其应用
CN109350606A (zh) * 2018-09-26 2019-02-19 青岛大学 一种聚电解质中空胶囊的制备方法及得到的中空胶囊
CN109433122A (zh) * 2018-09-28 2019-03-08 青岛大学 一种无机/有机核壳结构胶囊的制备方法及得到的胶囊
WO2020221361A1 (fr) * 2019-04-30 2020-11-05 青岛大学 Procédé de préparation de fibre à l'oxyde de graphène, et fibre ainsi obtenue

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US6733790B1 (en) 1999-07-02 2004-05-11 Cognis Iberia S. L. Microcapsules and processes for making the same using various polymers and chitosans
EP1129771A1 (fr) * 2000-03-04 2001-09-05 Primacare S.A. Microcapsules
WO2001066240A1 (fr) * 2000-03-04 2001-09-13 Cognis Iberia, S.L. Produits de soin pour la peau
WO2001066241A1 (fr) * 2000-03-04 2001-09-13 Cognis Iberia, S.L. Microcapsules - vi
WO2001087475A1 (fr) * 2000-05-19 2001-11-22 Ecole Polytechnique Federale De Lausanne Materiaux et procedes d'encapsulation
EP1184027A1 (fr) * 2000-09-01 2002-03-06 Primacare S.A. Composition pour baton applicateur cosmetique
WO2002017859A1 (fr) * 2000-09-01 2002-03-07 Cognis Iberia, S.L. Preparations cosmetiques
US7641917B2 (en) 2001-05-30 2010-01-05 Csir Method of encapsulating an active substance
WO2005011855A1 (fr) * 2003-08-01 2005-02-10 The Procter & Gamble Company Microcapsules
US7435715B2 (en) 2003-08-01 2008-10-14 The Procter & Gamble Company Microcapsules
EP1502646A1 (fr) 2003-08-01 2005-02-02 The Procter & Gamble Company Microcapsules
US7897555B2 (en) 2003-08-01 2011-03-01 The Procter & Gamble Company Microcapsules
EP1727525A1 (fr) * 2004-03-23 2006-12-06 Lytone Enterprise, Inc. Capsule contenant du chitosane noye ou encapsule
EP1727525A4 (fr) * 2004-03-23 2009-12-09 Lytone Enterprise Inc Capsule contenant du chitosane noye ou encapsule
WO2006064331A1 (fr) * 2004-12-17 2006-06-22 Medipol Sa Particules hydrophiles a base de derives de chitosanes cationiques
EP1702674A1 (fr) * 2005-03-16 2006-09-20 Cognis IP Management GmbH Perles visibles
CN1313157C (zh) * 2005-04-18 2007-05-02 天津大学 促进药物透皮传递的壳低聚糖组合物及其使用方法
WO2007031812A1 (fr) * 2005-09-16 2007-03-22 Medipol Sa Particules a base de chitosane
WO2012099482A3 (fr) * 2011-01-18 2012-11-01 Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies (A4Tec) Dispositif, procédé et système pour la préparation de microcapsules
CN102824332A (zh) * 2012-09-14 2012-12-19 东北农业大学 一种抗菌肽缓释微胶囊的制备方法
RU2536225C1 (ru) * 2014-01-28 2014-12-20 Федеральное государственное бюджетное учреждение науки Тихоокеанский институт биоорганической химии им. Г.Б. Елякова Дальневосточного отделения Российской академии наук (ТИБОХ ДВО РАН) Средство, обладающее гастропротекторной активностью
WO2017089974A1 (fr) 2015-11-23 2017-06-01 Association For The Advancement Of Tissue Engineering Cell Based Technologies & Therapies Associação Composition comprenant des complexes de polyélectrolyte, procédés et utilisations de cette dernière
US10576042B2 (en) 2015-11-23 2020-03-03 Association For The Advancement Of Tissue Engineering Cell Based Technologies & Therapies Associaçao Composition comprising polyelectrolyte complexes, methods and uses thereof
CN109172535A (zh) * 2018-09-26 2019-01-11 青岛大学 聚电解质氧化石墨烯中空胶囊的制备及得到的中空胶囊
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CN109316460A (zh) * 2018-09-26 2019-02-12 青岛大学 一种多糖类聚电解质中空胶囊的制备方法及得到的中空胶囊
CN109350606B (zh) * 2018-09-26 2021-08-13 青岛大学 一种聚电解质中空胶囊的制备方法及得到的中空胶囊
CN109350606A (zh) * 2018-09-26 2019-02-19 青岛大学 一种聚电解质中空胶囊的制备方法及得到的中空胶囊
CN109433122A (zh) * 2018-09-28 2019-03-08 青岛大学 一种无机/有机核壳结构胶囊的制备方法及得到的胶囊
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CN109433122B (zh) * 2018-09-28 2021-11-26 青岛大学 一种无机/有机核壳结构胶囊的制备方法及得到的胶囊
CN109223726A (zh) * 2018-09-28 2019-01-18 青岛大学 一种新型中空胶囊的制备方法及得到的新型中空胶囊
CN109338490A (zh) * 2018-09-30 2019-02-15 青岛大学 一种中空多层结构纤维的制备方法及得到的纤维与其应用
WO2020221361A1 (fr) * 2019-04-30 2020-11-05 青岛大学 Procédé de préparation de fibre à l'oxyde de graphène, et fibre ainsi obtenue

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