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WO1987004367A1 - Membranes covalentes - Google Patents

Membranes covalentes Download PDF

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Publication number
WO1987004367A1
WO1987004367A1 PCT/US1987/000098 US8700098W WO8704367A1 WO 1987004367 A1 WO1987004367 A1 WO 1987004367A1 US 8700098 W US8700098 W US 8700098W WO 8704367 A1 WO8704367 A1 WO 8704367A1
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WO
WIPO (PCT)
Prior art keywords
σompound
proσess
σells
σlaim
membrane
Prior art date
Application number
PCT/US1987/000098
Other languages
English (en)
Inventor
Franklin Lim
Lloyd Thomas Hall, Iii
Original Assignee
Ltl Associates
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
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Publication of WO1987004367A1 publication Critical patent/WO1987004367A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • 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
    • A61K9/5026—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone, poly(meth)acrylates
    • 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
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/06—Making microcapsules or microballoons by phase separation
    • B01J13/14—Polymerisation; cross-linking
    • B01J13/16—Interfacial polymerisation

Definitions

  • the present invention relates to semipermeable membranes, and more particularly to membranes which are formed covalently.
  • U.S. Patent No. 4,352,883 to Franklin Lim discloses a icroencapsulation technique which can be used to encapsulate solid or liquid material within semipermeable or substantially impermeable capsule membranes.
  • the process disclosed in the 'S83 patent begins by suspending the core material-in a solution of a water-soluble substance which can be reversibly gelled. The resulting solution is formed into droplets which are gelled to produce discrete shape-retaining temporary capsules. A permanent semipermeable membrane is then formed around the temporary capsules, followed by reliquifying the gel within the capsules.
  • Core materials discussed in the '883 patent include living cells and finely divided living tissue which are suspended in an aqueous medium which is physiologically compatible with the cells or tissue.
  • the preferred water-soluble substance therein for forming the temporary capsules is a gum, a preferred gum being sodium alginate.
  • the temporary capsules are formed by subjecting the droplets to a solution of multivalent cations, such as an aqueous solution of calcium chloride.
  • the ⁇ apsular membrane is stated to be formed by contacting the temporary capsules with a polymer of a molecular weight between 3000 and 100,000 daltons which has free a ino groups which displace the calcium ions of the calcium alginate bonds.
  • crosslinking polymers are polylysine and other cationic polyamino acids.
  • sodium alginate is the water-soluble substance
  • the gelled core material is reliquified by removal of the calcium ions contained in the capsules, thereby resolubilizing their interior.
  • the present invention is a process for forming a permanent, covalently cross-linked semipermeable membrane.
  • a first compound which possesses multiple reactive functional groups which may have previously been produced by reaction with a polyfunctional activating agent, is reacted with a se ⁇ ond ⁇ ompound with whi ⁇ h it forms covalent bonds, resulting in a permanent semipermeable membrane.
  • the present invention is a universal process which allows the fast, essentially instantaneous, formation of a semipermeable membranes with a designable pore size.
  • a permanent covalent membrane can be formed in a single step without the gelling and reliquification steps required by the prior art processes.
  • the process comprises bringing together two compounds, each containing multiple functional groups capable of covalent bond-forming reaction with that of the other. At least one of the two species must be in solution.
  • the other reacting specie can be either in solution, in the form of a biological or non-biological semi-solid or solid, or in an emulsion.
  • the multiple functional groups may require activation or derivitization to produce reactive chemi ⁇ al groups capable of covalent bond formation with the functional groups of the other reacting compound.
  • Typical examples of covalent bonds formed in this manner are ester bonds, peptide bonds, disulfide bonds, and bonds formed by free radical reactions.
  • Activation of functional groups may be accomplished by initiators or activators such as light or
  • E SHEET temperature or by specific cross-linking reagents which, possess at least two types of reactive groups, hereafter referred to as "polyfunctional a ⁇ tivating agents".
  • Membrane pore size ⁇ an be ⁇ ontrolled by ⁇ hoosing or adjusting the density (number) and distribution of the rea ⁇ tive groups in the reactants, adjusting the concentration of the reactants, or adjusting the reaction ⁇ onditions, such as time or temperature.
  • Nonbiologi ⁇ al solids ⁇ an include surfa ⁇ es as diverse as plasti ⁇ , metal, or ceramic.
  • a membrane ⁇ ould be formed on the surfa ⁇ e of ⁇ a sphere of sintered inorganic parti ⁇ les such as that des ⁇ ribed in International Appli ⁇ ation PCT/US85/01893, in ⁇ orporation herein by referen ⁇ e.
  • Certain solids or semi-solids will ⁇ ontain a suffi ⁇ ient number of fun ⁇ tional groups for use in the process without any modification.
  • pro ⁇ ess of the present invention is also ⁇ apable of produ ⁇ ing mi ⁇ ro ⁇ apsules substantially instantaneously with a true ⁇ ovalently linked polymeri ⁇ membrane, as opposed to the reversible polyele ⁇ trolyte ⁇ omplex type of membrane of the prior art.
  • substantially instantaneously is meant, in fewer than about five se ⁇ onds.
  • production of the covalently linked polymeric membrane is substantially complete in one second or less, particularly when living material is involved. Otherwise, the ⁇ hemi ⁇ als and ⁇ onditions used may adversely a fe ⁇ t its viability.
  • biologically labile materials including living cells
  • the extrusion of the suspension solution into a solution containing a compound with the appropriate complementary reactive or activated functional groups results in the production of permanent micro ⁇ apsules ⁇ ontaining the biologi ⁇ ally labile material.
  • the reacting compounds will have a molecular weight between about 2,000 and about 100,000 daltons, preferably in excess of about 10,000 daltons.
  • the tissues, organelles, or cells to be encapsulated in ac ⁇ ordan ⁇ e with the pro ⁇ ess of the present invention are prepared in a ⁇ cordan ⁇ e with well-known prior art te ⁇ hniques.
  • the material to be en ⁇ apsulated is suspended in a suitable aqueous medium whi ⁇ h does not interfere with the en ⁇ apsulation pro ⁇ ess.
  • a physiologi ⁇ al saline solution is suitable for this purpose.
  • a medium is utilized whi ⁇ h is suitable for aintenan ⁇ e and for supporting the ongoing metaboli ⁇ pro ⁇ esses of the parti ⁇ ular material involved.
  • the average diameter of the material being encapsulated can vary widely between less than a mi ⁇ ron to several millimeters.
  • samples of materials as divergent in size as mammalian Islets of Langerhans (50-200 mi ⁇ rometers in diameter) , Herpes simplex virus (30-40 nanometers in diameter) , or immunoglobulins ⁇ an all be en ⁇ apsulated by the pro ⁇ ess in this appli ⁇ ation.
  • the ⁇ ore material to be en ⁇ apsulated ⁇ an be dis ⁇ rete living ⁇ ells, viable tissue of plant or animal origin, or Protista su ⁇ h as viruses.
  • Islet of Langerhans, individual hepato ⁇ ytes, organelles, or other tissue units may be en ⁇ apsulated as desired.
  • ⁇ ells whi ⁇ h ⁇ an be en ⁇ apsulated in ⁇ lude thymi ⁇ ⁇ ells, thyroid ⁇ ells, liver ⁇ ells, (e.g., hepato ⁇ ytes) , adrenal ⁇ ells, blood ⁇ ells, lymphoid ⁇ ells, and pituitary ⁇ ells.
  • i ⁇ roorganisms may be en ⁇ apsulated as well as non-living materials of biologi ⁇ al or non-biologi ⁇ al origin.
  • the pro ⁇ ess of the present invention allows the in situ formation of a membrane on a desired surfa ⁇ e.
  • This surfa ⁇ e if ne ⁇ essary, may be modified before use in the pro ⁇ ess, for example, by a ⁇ tivation or by the addition of rea ⁇ tive sites.
  • An example of su ⁇ h a surfa ⁇ e would be the exterior surfa ⁇ e of an inta ⁇ t organ or ⁇ ell.
  • a membrane ⁇ an be formed on any surfa ⁇ e, in ⁇ luding plasti ⁇ , ⁇ ellulose, metal or cerami ⁇ , provided suitable rea ⁇ tive sites ⁇ an be formed.
  • the present invention also in ⁇ ludes embodiments where the ⁇ ore material is en ⁇ losed by the semipermeable ⁇ ovalent membrane.
  • en ⁇ losed is meant that the ⁇ ore material is surrounded by a ⁇ ontinuous wall of whi ⁇ h at least a portion is semipermeable ⁇ ovalent membrane.
  • a preferred set of rea ⁇ tants are ⁇ hitosan a ⁇ etate and polya ⁇ ryli ⁇ a ⁇ id.
  • the ⁇ arboxyli ⁇ a ⁇ id moieties of the polya ⁇ ryli ⁇ a ⁇ id ⁇ an be a ⁇ tivated, for example, by rea ⁇ tion with Woodward's Reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) . While the a ⁇ tivating group does not form a part of the final produ ⁇ t when Woodward's Reagent K is used for a ⁇ tivation, it is also a ⁇ eptable for the a ⁇ tivating group to be in ⁇ orporated into the membrane.
  • Woodward's Reagent K is a well known material for enzyme immobilization by the formation of water-soluble enzyme-polymer ⁇ onjugates.
  • Typi ⁇ al of su ⁇ h prior art enzyme immobilization ⁇ onjugates are alpha- ⁇ hymotrypsin on polya ⁇ ryli ⁇ a ⁇ id, ⁇ arboxy methyl ⁇ ellulose, and poly-L-glutami ⁇ a ⁇ id.
  • a preferred polyfun ⁇ tional a ⁇ tivating agent is Woodward's Reagent K
  • numerous other polyfun ⁇ tional a ⁇ tivating agents are known whi ⁇ h will, for the purposes of the present invention, a ⁇ t in the same manner as Woodward's Reagent K, provided suitable rea ⁇ tants are ⁇ hosen.
  • su ⁇ h polyfun ⁇ tional a ⁇ tivating agents in ⁇ lude m-maleimidobenzoyl-N- hydroxysu ⁇ inimide ester; maleimidobenzoylsulfosu ⁇ inimide-ester; N-su ⁇ inimidyl-(4-iodoa ⁇ etyl)a inobenzoate; su ⁇ inimidyl 4-(N-maleimidomethyl)- ⁇ y ⁇ lohexane-1- ⁇ arboxylate; sulf ⁇ succinimidyl(4-i ⁇ doa ⁇ etyl) aminobenzoate; and sulfosu ⁇ inimidyl- 4-(N-maleimidomethyl) ⁇ y ⁇ lohexane-1-carboxylate.
  • a ⁇ arboxyli ⁇ a ⁇ id group- ⁇ ontaining polymer is a ⁇ tivated by rea ⁇ tion with Woodward's Reagent K, and then the amino groups of the se ⁇ ond ⁇ ompound rea ⁇ t with the a ⁇ tivated ⁇ arboxylate groups.
  • the amino groups of the chitosan salt react with the a ⁇ tivated carboxylate groups of the polya ⁇ ryli ⁇ a ⁇ id to form a permanent ⁇ ovalent membrane.
  • This embodiment illustrates the rea ⁇ tion between a polyamino ⁇ ompound and a poly ⁇ arboxylate ⁇ ompound.
  • the ⁇ hitosan salt and polyacryli ⁇ acid are only_-being used as examples of suitable polyamino ⁇ a d poly ⁇ arboxylate rea ⁇ tants.
  • the present invention is appli ⁇ able generally to rea ⁇ tions between a wide variety of ⁇ ovalently rea ⁇ tive spe ⁇ ies, in ⁇ luding, but not limited to, polyamino and polysulfhydryl ⁇ ompounds, poly ⁇ arboxyli ⁇ acids, polymeric aryl azides, and polymeric n-hydroxysuc ⁇ inimide esters.
  • a hydroxyl group- ⁇ ontaining polymer su ⁇ h as dextran is a ⁇ tivated by rea ⁇ tion with the photoa ⁇ tivated ⁇ ross-linking agent sulfo-SANPAH (sulfosu ⁇ inimidyl-6-(4'-azido-2'-nitrophenylamino) hexanoate) , and then rea ⁇ ted with the amino groups of ⁇ hitosan a ⁇ etate.
  • Su ⁇ h photoa ⁇ tivation is typi ⁇ ally a ⁇ omplished over a period of 5 - 45 minutes.
  • the solution ⁇ ontaining the ⁇ ore material is formed into droplets of a desired size.
  • the drop formation may be condu ⁇ ted by known methods.
  • An exemplary pro ⁇ edure follows.
  • the apparatus ⁇ onsists of a housing having an upper air intake nozzle and an elongate hollow body fri ⁇ tion fitted into the stopper.
  • a 10 ⁇ syringe equipped with a syringe pump is mounted atop the housing with, e.g., a 20G by 1-1/2" needle squared off at the end whi ⁇ h passes through the length of the housing.
  • the interior of the housing is designed su ⁇ h that the tip of the needle is subje ⁇ ted to a ⁇ onstant air flow whi ⁇ h a ⁇ ts as an air knife.
  • the syringe pump is actuated to incrementally force droplets of solution or suspension from the tip of the needle. Each drop is "cut off" by the air stream and falls approximately 2 to 10 cm into the vessel containing the other rea ⁇ tant where the ⁇ ovalent membrane substantially immediately forms by rea ⁇ tion between the two rea ⁇ tants.
  • the distan ⁇ e between the tip of the needle and the surfa ⁇ e of the solution in the vessel is great enough, in this instan ⁇ e, to allow the solution or suspension to assume the most physi ⁇ ally favorable shape; i.e., a sphere (maximum volume for minimum surfa ⁇ e area) .
  • Air within the vessel bleeds through an opening in the stopper. This results in the formation of a shape-retaining permanent covalent capsule containing the suspended tissue and its medium.
  • the capsules colle ⁇ t in the solution as a separate phase and may be separated by de ⁇ antation.
  • the produ ⁇ tion of mi ⁇ ro ⁇ apsules by the pro ⁇ ess of the ⁇ urrent invention may in some ⁇ ir ⁇ umstan ⁇ es require the use of rea ⁇ tive polymers or se ⁇ ondary membrane treatments whi ⁇ h may adversely effe ⁇ t the ⁇ ore material to be en ⁇ apsulated.
  • the pro ⁇ ess of the ⁇ urrent invention in ⁇ ludes treatments of this sort, whi ⁇ h may be a ⁇ omplished as follows: i) The nas ⁇ ent mi ⁇ ro ⁇ apsules are allowed to _.._-pass through a fine mist of the harsh,
  • .-rea ⁇ tive reagent for a defined period of time.
  • This time period may be sele ⁇ ted by ⁇ ontrolling the mist-filled distan ⁇ e and/or the mist density through whi ⁇ h the ⁇ apsule falls, or by ⁇ ontrolling the amount of time that the ⁇ apsule is suspended in su ⁇ h a mist by a ⁇ ounter ⁇ urrent air flow; ii)
  • the nas ⁇ ent ⁇ apsules may be ⁇ aused to pass more or less perpendi ⁇ ularly through a 'liquid ⁇ urtain' of the harsh reagent.
  • This liquid ⁇ urtain is produ ⁇ ed, for example, by for ⁇ ing the harsh reagent through an orifi ⁇ e the geometry of whi ⁇ h is ⁇ hosen to ⁇ ause a flattened, laminar sheet of liquid to be formed.
  • the thi ⁇ kness of this liquid sheet is adjusted so that the mi ⁇ ro ⁇ apsule ⁇ an pass through the liquid without signifi ⁇ ant deformation of the mi ⁇ ro ⁇ apsule; or iii) the par i ⁇ les or droplets may be en ⁇ apsulated using a preformed oil-in-water emulsion in whi ⁇ h the polyfun ⁇ tional cross- linking reagent has been dissolved in the organic phase.
  • the particles or droplets are added, such that they are contacted essentially only by the aqueous environment.
  • Contact of the emulsion with the particles to be encapsulated allows cross-linking to oc ⁇ ur in a ⁇ ontrolled manner as the ⁇ ross-linking reagent diffuses into the aqueous phase.
  • the mi ⁇ ro ⁇ apsules may be treated repetitively with one or more reagents by the use of su ⁇ h limited ⁇ onta ⁇ t treatment systems if this is desirable for a parti ⁇ ular appli ⁇ at.ion.
  • Su ⁇ h repetitive treatments may, for example, be produ ⁇ ed by multiple discrete passes through a minimal conta ⁇ t system, or by the use of a ⁇ as ⁇ ade of su ⁇ h dis ⁇ rete systems, as required.
  • the ⁇ ontrolled release of low mole ⁇ ular weight spe ⁇ ies from mi ⁇ ro ⁇ apsules ⁇ an be used in a wide variety of appli ⁇ ations. These in ⁇ lude (but are not limited to) drug delivery systems, ⁇ osmeti ⁇ preparations, water treatment systems for aquariums, ⁇ ontrolled release of reagents into tissue ⁇ ulture systems, and other appli ⁇ ations involving the measured release of hydrophii ⁇ spe ⁇ ies into an aqueous environment.
  • pro ⁇ ess of the present invention it is possible to produ ⁇ e hydrophili ⁇ mi ⁇ ro ⁇ apsules of extremely limited permeability.
  • Su ⁇ h ⁇ apsules ⁇ an be used to produ ⁇ e a ⁇ ontrolled release system for suitably ⁇ hosen ⁇ hemi ⁇ al spe ⁇ ies having mole ⁇ ular weights in the range of about one hundred to five thousand daltons. Release of the ⁇ hosen species from the micro ⁇ apsules ⁇ an be ⁇ aused to o ⁇ ur over a ⁇ hosen period ranging from several minutes to more than twenty four hours.
  • the permeability of the ⁇ apsules is determined by varying the ⁇ on ⁇ entrations of the ⁇ onstituent polymers in the membrane, by varying the pH of the solutions ⁇ ontaining the ⁇ apsules, or by treating preformed ⁇ apsules with suitably ⁇ hosen polymeri ⁇ or non-polymeri ⁇ spe ⁇ ies.
  • the kineti ⁇ s of release from the ⁇ apsules are also influen ⁇ ed by the ⁇ harge, molecular weight, and solubility of the species released.
  • permeability modifying agents su ⁇ h as polylysine, polyargenine, polyornithin , polyethylenimine, glu ⁇ osamine, potassium, ⁇ hloride and sodium a ⁇ etate.
  • a typi ⁇ al treatment would be for from about one minute to an hour, preferably about two to about ten minutes, of residen ⁇ e in a 0.1M solution of the agent in a saline solution.
  • the mi ⁇ ro ⁇ apsules should be gently kept in suspension during this treatment.
  • a substantial ex ⁇ ess of liquid e.g., at least ten ⁇ apsule volumes, should be employed to ensure adequate ⁇ onta ⁇ t between the surfa ⁇ e of the ⁇ apsules and the treating solution.
  • Capsules formed by the pro ⁇ ess of the present invention have essentially the same utilities as those of the prior art, and thus also the same ⁇ advantages, and are in fa ⁇ t useful in appli ⁇ ations where the polyele ⁇ trolyte ⁇ omplex type mi ⁇ ro ⁇ apsules of the prior art would not be useful be ⁇ ause of potential adverse ioni ⁇ intera ⁇ tions, or la ⁇ k of permanen ⁇ y of the polyele ⁇ trolyte ⁇ omplex membrane.
  • the pro ⁇ ess of the present invention is useful for the en ⁇ apsulation of biologi ⁇ ally active material such as enzymes, hormones, and antibodies.
  • the process of the present invention is also useful for the encapsulation of viable Islets of Langerhans.
  • the resulting capsules when placed in a medium containing the nutrients and other materials necessary to maintain viability and support in vitro metabolism of the tissue will maintain their complete physiological functional integrity.
  • Other types of ⁇ ells similarly ⁇ an be en ⁇ apsulated in a physiologi ⁇ ally a ⁇ tive state.
  • the ⁇ apsules of the present -invention would also be useful for tissue implantation into a mammalian body.
  • Cell ⁇ ultures en ⁇ apsulated as des ⁇ ribed above may be suspended in ⁇ ulture media designed spe ⁇ ifi ⁇ ally to satisfy all of the requirements of the particular ⁇ ell type involved and will ⁇ ontinue to ⁇ arry out normal metaboli ⁇ pro ⁇ esses in the ⁇ apsules. Nutrients required by the ⁇ ells will normally be of suffi ⁇ iently low mole ⁇ ular weight so that their diffusion into the mi ⁇ ro ⁇ apsule will be substantially unimpeded by the membrane.
  • the membrane may be ⁇ onstru ⁇ ted in su ⁇ h a way as to limit the es ⁇ ape of the ⁇ ells* high mole ⁇ ular weight etaboli ⁇ produ ⁇ ts, thereby allowing the isolation of these produ ⁇ ts from the relatively smaller volume of the intra ⁇ apsular medium rather than the larger volume of the extra ⁇ apsular medium.
  • This ⁇ on ⁇ entrating effe ⁇ t of mi ⁇ ro ⁇ apsular membranes is an advantage that the present invention shares with the mi ⁇ ro ⁇ apsules of the prior art.
  • the en ⁇ apsulated ⁇ ells may be ⁇ ultured under ⁇ onditions of, e.g., temperature, pH, and ioni ⁇ environment, identi ⁇ al to ⁇ onventional ⁇ ultures.
  • ⁇ ell-produ ⁇ ed produ ⁇ ts may be harvested from the extra ⁇ apsular medium or from within the ⁇ apsules by ⁇ onventional techniques. Examples of cell produced products which may be harvested include insulin, glu ⁇ agon, prola ⁇ tin, somatostatin, thyroxin, steroid hormones, pituitary—hormones, interferons, FSH, and PTH.
  • EXAMPLE 1 To illustrate the ⁇ ontinuing viability of mammalian ⁇ ells after en ⁇ apsulation by the pro ⁇ ess of the present invention, myeloma ⁇ ells (ATCC CRL1580; P3x63 Ag 8.653 [non-se ⁇ reting mouse myeloma]) were en ⁇ apsulated into ⁇ apsules formed by rea ⁇ tion of ⁇ hitosan a ⁇ etate with polya ⁇ ryli ⁇ a ⁇ id whi ⁇ h had previously been a ⁇ tivated by rea ⁇ tion with Woodward's Reagent K.
  • the myeloma ⁇ ell/ ⁇ hitosan acetate suspension was added dropwise to the activated polya ⁇ ryli ⁇ a ⁇ id by the pro ⁇ edure des ⁇ ribed above.
  • the ⁇ apsules were washed four times with physiologi ⁇ al saline, and then pla ⁇ ed in a suitable medium (Dulbe ⁇ o's Minimal Essential Medium (Gib ⁇ o) supplemented with 20% fetal ⁇ alf serum) . After one hour, viability of the ⁇ ells was verified by trypan blue ex ⁇ lusion.
  • a suitable medium Dulbe ⁇ o's Minimal Essential Medium (Gib ⁇ o) supplemented with 20% fetal ⁇ alf serum
  • the ⁇ apsules were treated with a 0.65% by weight solution of ⁇ alcium chloride (pH 6.2).
  • the capsules remained inta ⁇ t, thereby demonstrating that a ⁇ ovalently bonded membrane had been formed, sin ⁇ e an ioni ⁇ ally bonded membrane made of these materials would have disintegrated under these ⁇ onditions.
  • saline In 20 ml of saline is dissolved 0.2 g of T40 dextran (m.w. 40,000). To this is added 5 mg of sulfoSANPAH (sulfosu ⁇ inimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate) (a produ ⁇ t sold by Pier ⁇ e Chemi ⁇ al Company, P. 0. Box 117, Ro ⁇ kford, Illinois 61105) . The resulting solution is pla ⁇ ed in a ⁇ losed ⁇ ontainer with a high pressure mer ⁇ ury light sour ⁇ e. The solution is irradiated for about 30 minutes while maintaining a temperature in the ⁇ ontainer-of about 80.C. After ⁇ ooling, the resulting a ⁇ tivated dextran is rehydrated to 1% by volume of dextran by the addition of saline.
  • sulfoSANPAH sulfosu ⁇
  • chitosan acetate is readily prepared by suspending 2 grams of chitosam in 200 ml of deionized water, adding 0.62 ml of a ⁇ eti ⁇ a ⁇ id, mixing until the ⁇ hitosan dissolves, and filtering to remove undissolved material.
  • the ⁇ ell- ⁇ ontaining ⁇ hitosan a ⁇ etate solution is added dropwise to the a ⁇ tivated dextran solution to form ⁇ ovalently bonded mi ⁇ ro ⁇ apsules.
  • Capsules were then formed by droplets of one weight per ⁇ ent ⁇ hitosan a ⁇ etate dissolved in physiologi ⁇ al saline. In ea ⁇ h ⁇ ase, ⁇ apsules readily formed which remained inta ⁇ t after treatment with 1.3% (w/v) aqueous ⁇ al ⁇ ium ⁇ hloride. This treatment led to crenation of the capsules, thus demonstrating their permeability to water.
  • the ⁇ apsules prepared using 0.08g and 0.0008g of Woodward's Reagent K for a ⁇ tivation were removed from the ⁇ al ⁇ ium ⁇ hloride solution and pla ⁇ ed in distilled water where they re-inflated in fewer than fifteen se ⁇ onds.
  • the same ⁇ apsules were pla ⁇ ed in an 11.9% by volume solution of hemoglobin in physiologi ⁇ al saline.
  • the ⁇ apsules loaded in approximately one minute with visible ⁇ renation. This indi ⁇ ated that mole ⁇ ules of approximately 65,000 daltons ⁇ ould easily pass through the membrane.
  • Capsules were washed twi ⁇ e in isotoni ⁇ saline and then treated twi ⁇ e with 0.25% w/v glu ⁇ osamine. Ea ⁇ h treatment lasted for four minutes. The ⁇ apsules were then washed in TRIS buffer (pH 7.0). The ⁇ apsules were suspended in a variety of solutes and allowed to equilibrate.
  • EXAMPLE 6 The following example illustrates the formation of sheets of enzyme-bearing, ⁇ ovalently ⁇ rossleaked membranes.
  • An a ⁇ tivated polya ⁇ ryli ⁇ a ⁇ id solution was prepared as des ⁇ ribed above in Example 1.
  • To five mL of the solution was added 16 g of ⁇ atalase and the solution stirred gently for about two minutes.
  • a mi ⁇ ros ⁇ ope ⁇ over slip was ⁇ oated with a 1% by weight solution of ⁇ hitosan a ⁇ etate prepared as des ⁇ ribed above in Example 1.
  • the ⁇ oated slip was slid into a ⁇ ontainer of the a ⁇ tivated polya ⁇ ryli ⁇ a ⁇ id solution and allowed to rea ⁇ t to form a membrane whi ⁇ h ⁇ ould be peeled off the slip.
  • the ⁇ atalase was also determined to retain its a ⁇ tivity as demonstrated by the evolution of oxygen gas when, exposed to hydrogen peroxide. Moreover, .the a ⁇ tivity.was seen even after the membrane had been washed with distilled water.
  • E SHEET EXAMPLE 7 The following example demonstrates the formation of ⁇ ovalently ⁇ rosslinked mi ⁇ ro ⁇ apsules whi ⁇ h bear immobilized antibodies. Magnetite parti ⁇ les were suspended in a 1% solutionof chitosan a ⁇ etate prepared in a ⁇ ordan ⁇ e with the pro ⁇ edure of Example 1 above. The resulting solution was sprayed into 200_uL of a solution ⁇ ontaining 2g of polya ⁇ ryli ⁇ a ⁇ id (mole ⁇ ular weight 5100 daltons) and O.lg of Woodward's Reagent K, the latter solution being at a pH of 5.6.
  • the resulting mi ⁇ ro ⁇ apsules were washed three times with isotoni ⁇ saline, and then 0.1 mL of the mi ⁇ ro ⁇ apsules were suspended in 0.5 mL of PBS (pH 8; 0.01M) .
  • PBS pH 8; 0.01M
  • the mi ⁇ ro ⁇ apsules were then washed three times with distilled water.

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Abstract

Selon un procédé de formation d'une membrane semi-perméable permanente réticulée par covalence, on fait réagir un premier composé ayant des groupes fonctionnels réactifs multiples, qui peuvent avoir été produits au préalable par réaction avec un activant polyfonctionnel, avec un deuxième composé. Les deux composés forment des liaisons covalentes entre eux et donnent une membrane semi-perméable permanente.
PCT/US1987/000098 1986-01-23 1987-01-23 Membranes covalentes WO1987004367A1 (fr)

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US82190386A 1986-01-23 1986-01-23
US821,903 1986-01-23
US88978786A 1986-07-28 1986-07-28
US889,787 1986-07-28

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988000237A1 (fr) * 1986-06-27 1988-01-14 Damon Biotech, Inc. Membranes covalentes
WO1991010425A1 (fr) * 1990-01-08 1991-07-25 Brown University Research Foundation Systemes d'extrusion de capsules de cellules
US5232712A (en) * 1991-06-28 1993-08-03 Brown University Research Foundation Extrusion apparatus and systems
US5283187A (en) * 1987-11-17 1994-02-01 Brown University Research Foundation Cell culture-containing tubular capsule produced by co-extrusion
US5418154A (en) * 1987-11-17 1995-05-23 Brown University Research Foundation Method of preparing elongated seamless capsules containing biological material
US5800829A (en) * 1991-04-25 1998-09-01 Brown University Research Foundation Methods for coextruding immunoisolatory implantable vehicles with a biocompatible jacket and a biocompatible matrix core
US5800828A (en) * 1991-04-25 1998-09-01 Brown University Research Foundation Implantable biocompatible immunoisolatory vehicle for delivery of selected therapeutic products

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US4208309A (en) * 1977-05-20 1980-06-17 Rohm Gmbh Pearl polymer containing hollow pearls
US4219411A (en) * 1978-09-18 1980-08-26 California Institute Of Technology Cell sorting apparatus
GB2041517A (en) * 1979-01-09 1980-09-10 Fuji Photo Film Co Ltd Material and process for immunological assay
GB2094833A (en) * 1981-03-13 1982-09-22 Damon Corp Process and system for producing biological materials from encapsulated cells

Patent Citations (4)

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Publication number Priority date Publication date Assignee Title
US4208309A (en) * 1977-05-20 1980-06-17 Rohm Gmbh Pearl polymer containing hollow pearls
US4219411A (en) * 1978-09-18 1980-08-26 California Institute Of Technology Cell sorting apparatus
GB2041517A (en) * 1979-01-09 1980-09-10 Fuji Photo Film Co Ltd Material and process for immunological assay
GB2094833A (en) * 1981-03-13 1982-09-22 Damon Corp Process and system for producing biological materials from encapsulated cells

Non-Patent Citations (2)

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Title
Chemtech, January 1974 (American Chemical society, Washington, USA), W.R. VIETH et al., "Enzyme Engineering. Part II. Materials for Immobilized Enzyme Reactors" pages 47-54, see page 48 *
Journal of Pharmaceutical Sciences, Vol. 71, No. 8, August 1982 (American Pharmaceutical Association, Washington, USA), K. BALA et al.: "pH-Sensitive Microcapsules for Drug Release", see pages 960-962 *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988000237A1 (fr) * 1986-06-27 1988-01-14 Damon Biotech, Inc. Membranes covalentes
US5643773A (en) * 1987-11-17 1997-07-01 Brown University Research Foundation Preparation of elongated seamless capsules containing a coaxial rod and biological material
US5283187A (en) * 1987-11-17 1994-02-01 Brown University Research Foundation Cell culture-containing tubular capsule produced by co-extrusion
US5284761A (en) * 1987-11-17 1994-02-08 Brown University Research Foundation Method of encapsulating cells in a tubular extrudate
US5389535A (en) * 1987-11-17 1995-02-14 Brown University Research Foundation Method of encapsulating cells in a tubular extrudate
US5418154A (en) * 1987-11-17 1995-05-23 Brown University Research Foundation Method of preparing elongated seamless capsules containing biological material
WO1991010425A1 (fr) * 1990-01-08 1991-07-25 Brown University Research Foundation Systemes d'extrusion de capsules de cellules
US5800829A (en) * 1991-04-25 1998-09-01 Brown University Research Foundation Methods for coextruding immunoisolatory implantable vehicles with a biocompatible jacket and a biocompatible matrix core
US5800828A (en) * 1991-04-25 1998-09-01 Brown University Research Foundation Implantable biocompatible immunoisolatory vehicle for delivery of selected therapeutic products
US5834001A (en) * 1991-04-25 1998-11-10 Brown University Research Foundation Methods for making immunoisolatory implantable vehicles with a biocompatiable jacket and a biocompatible matrix core
US5869077A (en) * 1991-04-25 1999-02-09 Brown University Research Foundation Methods for treating diabetes by delivering insulin from biocompatible cell-containing devices
US5871767A (en) * 1991-04-25 1999-02-16 Brown University Research Foundation Methods for treatment or prevention of neurodegenerative conditions using immunoisolatory implantable vehicles with a biocompatible jacket and a biocompatible matrix core
US5874099A (en) * 1991-04-25 1999-02-23 Brown University Research Foundation Methods for making immunoisolatary implantable vehicles with a biocompatible jacket and a biocompatible matrix core
US6083523A (en) * 1991-04-25 2000-07-04 Brown University Research Foundation Implantable biocompatable immunoisolatory vehicle for delivery of selected therapeutic products
US6322804B1 (en) 1991-04-25 2001-11-27 Neurotech S.A. Implantable biocompatible immunoisolatory vehicle for the delivery of selected therapeutic products
US6960351B2 (en) 1991-04-25 2005-11-01 Brown University Research Foundation Implantable biocompatible immunoisolatory vehicle for delivery of selected therapeutic products
US5232712A (en) * 1991-06-28 1993-08-03 Brown University Research Foundation Extrusion apparatus and systems

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AU6940087A (en) 1987-08-14

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