WO2004112476A1 - Procedes de conservation de biomateriaux - Google Patents

Procedes de conservation de biomateriaux Download PDF

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Publication number
WO2004112476A1
WO2004112476A1 PCT/GB2004/002704 GB2004002704W WO2004112476A1 WO 2004112476 A1 WO2004112476 A1 WO 2004112476A1 GB 2004002704 W GB2004002704 W GB 2004002704W WO 2004112476 A1 WO2004112476 A1 WO 2004112476A1
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WO
WIPO (PCT)
Prior art keywords
biomaterial
plastics material
cells
dried
encapsulated
Prior art date
Application number
PCT/GB2004/002704
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English (en)
Inventor
Alan Garth Tunnacliffe
Maximino Manzanera
Susana Vilchez Tornero
Arcadio Garcia De Castro
Original Assignee
Cambridge University Technical Services Limited
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
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Application filed by Cambridge University Technical Services Limited filed Critical Cambridge University Technical Services Limited
Publication of WO2004112476A1 publication Critical patent/WO2004112476A1/fr

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    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
    • A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
    • 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
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A01N1/0205—Chemical aspects
    • A01N1/0231—Chemically defined matrices, e.g. alginate gels, for immobilising, holding or storing cells, tissue or organs for preservation purposes; Chemically altering or fixing cells, tissue or organs, e.g. by cross-linking, for preservation purposes
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/22—Bacillus
    • 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
    • A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
    • A01N63/20—Bacteria; Substances produced thereby or obtained therefrom
    • A01N63/27—Pseudomonas
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2013—Organic compounds, e.g. phospholipids, fats
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/2004—Excipients; Inactive ingredients
    • A61K9/2022—Organic macromolecular compounds
    • A61K9/205—Polysaccharides, e.g. alginate, gums; 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A61K9/2806—Coating materials
    • A61K9/2833—Organic macromolecular compounds
    • A61K9/284—Organic macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyvinyl pyrrolidone
    • 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
    • A61K9/20—Pills, tablets, discs, rods
    • A61K9/28—Dragees; Coated pills or tablets, e.g. with film or compression coating
    • A61K9/2806—Coating materials
    • A61K9/2833—Organic macromolecular compounds
    • A61K9/2853—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyethylene oxide, poloxamers, poly(lactide-co-glycolide)

Definitions

  • This invention relates to methods and means for the preservation of biomaterial in an active condition for extended periods.
  • Freeze-drying is often used for preservation and storage of biomaterial.
  • the subject to be preserved is placed in solution and the solution is then frozen.
  • the frozen solid is then exposed to a vacuum under conditions where it remains solid and the water and any other volatile components are removed by sublimation.
  • freeze-drying is widely used, freeze-dried bacteria are unstable at ambient temperatures and require refrigeration. Significant reductions in viability are observed and the approach is generally time- and energy-intensive and thus expensive.
  • biomaterial in a dried state is resistant to organic solvents and can be encapsulated in an active form in solid plastics materials, such as polystyrene. Furthermore, cells encapsulated in this way have been found to remain in an active and viable form for long periods at ambient temperature.
  • Biomaterial encapsulated as described herein is resistant to physical, chemical and environmental stress and can be moulded and subjected to manufacturing processes without significant reductions in activity and/or viability.
  • the present invention in various aspects, provides methods and means for the stabilisation and preservation of active biomaterials. These methods may be useful, for example, in protecting biomaterial from humidity, oxidation and other stresses such as oxidation that would otherwise compromise activity and/or viable. Biomaterial preserved as described herein is stable for extended periods and may be suitable for long-term storage.
  • One aspect of the present invention provides a method of preserving biomaterial in an active condition comprising; drying active biomaterial in the presence of a glass forming stabiliser, admixing the dried biomaterial with a plastics material liquid, and; causing said plastics material to solidify to encapsulate the biomaterial therein.
  • the biomaterial may be dried to below about 10% residual moisture. Under these conditions, the stabiliser coalesces to form a non-crystalline, vitreous, solid physical state (i.e. a glass) .
  • the particles of organic glass which are formed by drying the stabiliser encase the biomaterial and provide stability by dramatically slowing the rate of chemical reactions.
  • the dried biomaterial thus comprises active biomaterial encased in the organic glass which is formed by the stabiliser.
  • Glass-forming stabilisers suitable for use in methods of the invention are preferably insoluble in organic solvents and soluble in water.
  • glass-forming stabilisers examples include non-reducing carbohydrates, such as trehalose, hydroxyectoine, maltitol (4-0- ss-D-glucopyranosyl-D-glucitol) , lactitol (4-O-ss-D- galactopyranosyl-D- glucitol) , palatinit [a mixture of GPS (oe- D-glucopyranosyl-1-6-sorbitol) and
  • GPM ⁇ -D-glucopyranosyl-l-O-6-mannitol
  • its individual sugar alcohol components GPS and GPM and hydrogenated maltooligosaccharides and maltooligosaccharides .
  • Non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other straight chain polyalcohols such as neotrehalose, lactoneotrehalose, galactosyl-trehalose, sucrose, lactosucrose, raffinose, stachyose and melezitose may also be used.
  • suitable glass-forming stabilisers include amino acids such as hydroxyectoine .
  • the amount of glass-forming stabiliser used in the present methods will depend on several variables, most particularly, the nature of the stabiliser and the biomaterial that is being stabilized and the method of drying.
  • hydroxyectoine may be used at between 0.5M and 2M and trehalose at between 0. IM and 1.5M.
  • Biomaterial dried with the glass forming stabiliser is resistant to the plastics material liquid (i.e. it retains activity) .
  • Biomaterial which is not dried with the glass-forming stabiliser is not resistant to the plastics material liquid.
  • the dried biomaterial may be admixed with the plastics material liquid in a particulate form i.e. particles of biomaterial may be mixed with and encapsulated by the plastics material liquid.
  • the dried biomaterial may be in a non- particulate form and may, for example, be shaped, moulded, or formed into a solid 3 dimensional object such as a block, tablet, ingot, nugget, patch, sheet or ball of dried biomaterial.
  • Such objects may be generated, for example, by compressing the dried biomaterial using standard techniques.
  • Objects comprising or consisting of dried biomaterial may be admixed with the plastics material liquid by immersing, suspending and/or mixing the objects in the plastics material liquid or by applying the plastics material liquid to the object, for example as a coating, in order to encapsulate the dried biomaterial.
  • a coating may be applied by any convenient technique, including spraying or brushing.
  • Plastics materials suitable for encapsulating biomaterial include synthetic, semi-synthetic and natural organic polymers.
  • the plastics material may be non- biodegradable, for example polystyrene, polyvinylchloride polycarbonate, polyethylene, Mylar, cellophane, polyacrylates, ethylene-vinyl acetate polymers and other acyl substituted cellulose acetates and derivatives thereof, non-erodible polyurethanes, polyvinyl fluoride, poly (vinyl imidazole) , chlorosulphonated polyolifms, polyethyleneoxide, polyvinyl alcohol, or nylon.
  • the plastics material may be biodegradable or bioerodible, for example poly (lactide) (PLA) , poly (glycolic acid) (PGA) , poly ⁇ lactide-co-glycolide) (PLGA) , and other poly (alpha-hydroxy acids), poly (caprolactone) , polycarbonates, polyamides, polyanhydrides, polyamino acids, polyortho esters, polyacetals, polyhydroxyalkanoates, polycyanoacrylates, degradable polyurethanes and other organic matrices, such as gums, latex/rubber and resins.
  • Suitable plastics may, for example, be isolated from natural sources, including bacteria.
  • the plastics material is preferably soluble in an organic solvent.
  • the plastics material liquid preferably comprises a plastics material dissolved in an organic (i.e. non-aqueous) solvent.
  • the biomaterial, in the form of organic glass particles, is then admixed with this solution.
  • the organic solvent is preferably a non-polar organic solvent, such as hexane, acetone, chloroform, toluene or xylene. Polar solvents such as ethanol may also be useful for some applications.
  • the organic solvent may be progressively removed from the plastics material solution. This may be achieved by any convenient means, for example air-, freeze-, spray/freeze- or vacuum-drying. As the organic solvent is progressively removed, the plastics material solidifies and encapsulates the cells that are contained therein. In other embodiments, the plastics material liquid may be solidified by other means such as cooling, photo- polymerisation, heat-induced polymerisation, or chemical-induced polymerisation. After preservation as described above and, optionally storage for an extended period, a method may comprise disrupting the plastics material to expose the encapsulated biomaterial.
  • the plastics material may be physically disrupted, for example by piercing or tearing the material or by eroding or abrading its surface, or chemically or biochemically disrupted, for example by reaction with one or more external agents.
  • External agents may, for example, include organic solvents which dissolve the plastics material.
  • Biodegradable plastics materials such as PLGA may be gradually dissolved or "dismantled" by biological agents, e.g. enzymes, phagocytic cells or micro-organisms.
  • Exposed biomaterial may then be placed in an appropriate medium or carrier and optionally combined with other constituents, for example an adjuvant, prior to use.
  • the invention includes methods of reconstituting biomaterial preserved by the methods described herein.
  • the nature and amount of medium or carrier used for reconstitution will depend upon the biomaterial as well as its intended use.
  • biochemical degradation of the plastics material may occur in situ and no further steps are required.
  • the plastics material may degrade within the body to expose active biomaterial with therapeutic properties.
  • Active biomaterial may comprise or consist of viable cells, for example viable unicellular organisms or multi-cellular lower eukaryotes, such as nematodes; and/or other membrane structures, including lipid or phospholipid membrane structures such as liposomes.
  • active biomaterial may comprise active viruses and viral particles.
  • a viable cell is a cell that is capable of performing normal cell functions, including replication and cell division.
  • a cell may be from a naturally occurring or wild type organism. Alternatively, the cell may have been treated, engineered or mutated prior to preservation as described herein. For example, the cell may have been rendered transfection-competent or may contain recombinant nucleic acid.
  • Cells encapsulated as described herein may be homogeneous, e.g. identical cells of a particular species, strain or isolate, or heterogeneous, e.g. a library of cells each containing a different recombinant nucleic acid.
  • Cells suitable for preserving in accordance with the present invention include prokaryotic cells, such as bacterial cells and eukaryotic cells including yeasts, in particular desiccation tolerant yeasts (e.g. brewers' yeast and bakers' yeast) and mammalian cells, such as tissue culture cells, organ cultures, sperm and egg cells.
  • prokaryotic cells such as bacterial cells and eukaryotic cells including yeasts, in particular desiccation tolerant yeasts (e.g. brewers' yeast and bakers' yeast) and mammalian cells, such as tissue culture cells, organ cultures, sperm and egg cells.
  • the cell is a non-anhydrobiotic cell (i.e. a cell which is sensitive to desiccation) .
  • Non-anhydrobiotic prokaryotic organisms are generally non-sporulating (i.e. do not form spores) and include, for example, gram-negative bacteria such as E. coli , S . typhimur ⁇ um and P. putida .
  • suitable organisms include Salmonella spp for live vaccines, Rhizobiu spp. for nitrogen fixation,; Pseudomonas spp or Rhodococcus spp for biodegradation/bioremediation, and Lactobacillus spp. or Bifidobacterium spp for probiotics and dairy use.
  • Multi-cellular lower eukaryotes may include nematode worms, which may, for example, be useful as biopesticides .
  • stability of a microorganism may be improved by culturing under conditions that increase the intracellular concentration of trehalose or other stabiliser prior to drying in the presence of glass forming stabiliser.
  • conditions of high osmolarity i.e. high salt concentration
  • a method may comprise determining the viability of a cell before or after encapsulation in a plastics material.
  • Cell viability may be determined using any of a number of techniques known in the art, including for example, a plate assay for colony forming units (CFU) . Viability may for example, be determined after disruption of the plastics material but before application and/or administration of the preserved cells.
  • CFU colony forming units
  • Biomolecules may also be preserved using the techniques described herein.
  • a method of preserving an active biomolecule may comprise; drying the biomolecule in the presence of a glass-forming stabiliser, admixing the biomolecule with a liquid plastics material, and; solidifying the plastics material to encapsulate the biomolecule therein.
  • a biomolecule may include nucleic acids, polysaccharides, lipids, vitamins, cofactors and small organic molecules which have a biological activity, for example a pharmacologic, immunogenic, and/or physiologic effect by local and/or systemic action.
  • Biomolecules may include DNA or RNA containing genes or parts of genes or genetically active sequences, and small bioactive molecules including drugs.
  • Biomaterial and biomolecules according to some embodiments may specifically exclude polypeptides .
  • Active biomolecules may be therapeutic agents and may include anti-infectives such as antibiotics and antiviral agents; analgesics and analgesic combinations; local and general anesthetics; anorexics; antiarthritics; antiasthmatic agents; anticonvulsants; antidepressants; antihistamines; anti- inflammatory agents; antinauseants; antineoplastics; antipruritics; antipsychotics; antipyretics; antispasmodics; cardiovascular preparations (including calcium channel blockers, beta-blockers, beta-agonists and antiarrythmics) ; antihypertensives; diuretics; vasodilators; central nervous system stimulants; cough and cold preparations; decongestants; diagnostics; hormones; bone growth stimulants and bone resorption inhibitors; immunosuppressives; muscle relaxants; psychostimulants; sedatives; tranquilizers; proteins, peptides, and fragments thereof (whether naturally occurring, chemically synthesized or
  • An active biomolecule may be comprised within a lipid membrane.
  • Active biomolecules are commonly labile and preservation in accordance with the present methods allows the activity of the biomolecule to be maintained for extended periods without the need for expensive storage conditions.
  • the plastics material liquid Before solidifying, the plastics material liquid may be placed on a support, for example a glass slide. In some preferred embodiments, the liquid is placed in a mould or applied as a coating. Moulded plastics materials and plastics material coatings comprising active biomaterial (i.e. active biomolecules or cells or both) have a range of applications. For example, biomaterial encapsulated in a plastics material may be applied as a coating to a biosensor component or seed.
  • active biomaterial i.e. active biomolecules or cells or both
  • the invention encompasses a seed coated with a plastics material comprising encapsulated biomaterial, in particular viable cells such as rhizobacteria, as described herein, and a method of coating a seed with a plastics material comprising producing a plastics material solution which comprises such biomaterial as described herein, contacting a seed with the solution, and solidifying or fixing the plastics material on the seed to form a coating of encapsulated biomaterial.
  • a seed may be immersed in a plastics material solution which comprises dried cells of a beneficial prokaryotic organism, for example a root colonising organism such as P. putida or a nitrogen fixing organism such as Rhizobium spp.
  • a beneficial prokaryotic organism for example a root colonising organism such as P. putida or a nitrogen fixing organism such as Rhizobium spp.
  • the seed is then removed from the solution and dried to remove the organic solvent and fix the plastics material on the seed to produce a coating comprising the encapsulated cells of the organism.
  • the present methods also allow the use of biomaterial in manufacturing processes for which the biomaterial would otherwise be too unstable.
  • the invention encompasses extrusions and mouldings comprising preserved biomaterial and methods of producing such products .
  • a method of producing a moulding containing active biomaterial may comprise providing a plastics material solution which comprises biomaterial as described above, adding the solution to a mould and solidifying the plastics material, for example by drying to remove the solvent .
  • the mould may be removed.
  • the use of moulds to shape a range of plastics materials is well known in the art.
  • standard injection moulding techniques may be used.
  • the mould may be of any shape, for example a particle, tablet, pill, block, sheet or strip, depending on the application to which the moulding is to be put.
  • a plastics material moulding comprising encapsulated active biomaterial may be useful as a component of a biosensor.
  • a biosensor may comprise bacteria which are capable of detecting specific toxic compounds, or pathogens, or general toxicity, for example by modulating their respiration and/or proliferation e.g. the baroxymeter (Tzoris et al (2002) Anal. Chim. Acta 460 257-272) .
  • Moulded plastics materials comprising encapsulated active biomaterial may be useful as foodstuffs or food supplements (e.g. neutraceuticals) .
  • probiotic bacteria such as Lactobacillus spp. or Bifidobacterium spp. may be encapsulated within a plastics material such as PLGA (biodegradable) and ingested directly by an individual. Degradation of the plastics material within the body exposes the bacteria to produce the beneficial effect.
  • the plastics material comprising the probiotic cells may be moulded into a pill or tablet to facilitate ingestion. In other embodiments, the plastics material may be disrupted and the exposed cells used to supplement other foodstuffs prior to ingestion.
  • the encapsulation of biomaterial in moulded plastics materials may be useful in the formulation of biopharmaceuticals and vaccines.
  • immunogenic bacterial cells such as M. bovis BCG, Salmonella spp. or Lactococcus spp. (vaccine vectors) may be encapsulated within a plastics material such as described above allowing the storage of the immunogenic biomaterial for extended periods without the need for specialised or expensive storage conditions.
  • the plastics material may be broken or pierced to expose the immunogenic biomaterial prior to or during administration or the plastics material may degrade within the body to expose the immunogen.
  • Biomaterial encapsulated as described herein in a biodegradable plastics material such as PLGA may be conveniently administered transdermally to an individual using needle-less pneumatic delivery devices which are well known in the art (see for example US6475181, US6063053, US5899880, US5630796, US6004286)
  • an adjuvant may be added in an amount sufficient to enhance the immune response to the immunogen.
  • the adjuvant may be added to the biomaterial before desiccation and preservation or may be separately reconstituted along with the biomaterial prior to use.
  • Suitable adjuvants are well known in the art. The precise choice of adjuvant depends in part on the stability of the vaccine in the presence of the adjuvant, the route of administration, and the regulatory acceptability of the adjuvant, particularly when intended for human use. For instance, alum is approved by the
  • FDA Food and Drug Administration
  • a pharmaceutical composition comprising encapsulated biomaterial may comprise one or more added materials such as carriers, vehicles, and/or excipients.
  • carriers such as carriers, vehicles, and/or excipients.
  • Carriers substantially inert materials which are non- toxic and do not interact with other components of the composition in a deleterious manner. These materials can be used to increase the amount of solids in particulate pharmaceutical compositions.
  • suitable carriers include water, silicone, gelatin, waxes, and like materials.
  • excipients include pharmaceutical grades of dextrose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran, starch, cellulose, sodium or calcium phosphates, calcium sulfate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycols (PEG) , and combinations thereof.
  • a charged lipid and/or detergent in the pharmaceutical compositions.
  • Such materials can be used as stabilizers, anti- oxidants, or used to reduce the possibility of local irritation at the site of administration.
  • Suitable charged lipids include phosphatidylcholines (lecithin) .
  • Detergents will typically be a nonionic, anionic, cationic or amphoteric surfactant.
  • suitable surfactants include, for example, TergitolTM and TritonTM, surfactants (Union Carbide Chemicals and Plastics, Danbury, Conn.), polyoxyethylenesorbitans, e.g., T EENTM. surfactants (Atlas Chemical Industries, Wilmington, Del.), polyoxyethylene ethers, e.g., Brij , pharmaceutically acceptable fatty acid esters, e.g. lauryl sulfate and salts thereof (SDS) , and like materials.
  • preserved fluorophores may be used in organic light-emitting diodes in which synthetic fluorophores based on the active centres of molecules such as GFP or phycoerythrin (You et al . , Adv. Mater. 12, 1678-1681, 2000) , are embedded in a polymer layer, possibly together with a dye to act as an emissive centre.
  • Encapsulated fluorescent biomaterials may also be used in products such as hair gel or skin paint, lettering or graphics, decorations for clothes, toys etc.
  • Moulded plastics materials comprising encapsulated biomaterial may also be useful in the development of medical devices, for example contact lenses containing pharmaceuticals; implants or patches containing drugs or vaccines; stents for repairing or maintaining blood vessel function, made from PLGA containing anticoagulants or molecules to discourage blood cell attachment; cervical rings containing, for example, antibiotics; and biodegradable plastic thread for surgical stitching containing growth factors for tissue repair.
  • medical devices for example contact lenses containing pharmaceuticals; implants or patches containing drugs or vaccines; stents for repairing or maintaining blood vessel function, made from PLGA containing anticoagulants or molecules to discourage blood cell attachment; cervical rings containing, for example, antibiotics; and biodegradable plastic thread for surgical stitching containing growth factors for tissue repair.
  • Techniques for the production of medical devices from moulded plastics materials are well known in the art .
  • Anti-rejection drugs such as rapamycin and cyclosporin may also be incorporated into medical devices by the encapsulation techniques described herein. Conveniently, anti-rejection drugs are encapsulated in biodegradable plastics materials, such as PLGA to provide an active effect as the plastics moulding breaks down.
  • Release of biomaterial from the plastics material may be instantaneous or controlled to provide delayed, gradient or staged release profiles. Multiple reagents may be laminated together for simultaneous or staged release. Release might be controlled in various ways, for example through erosion of biodegradable plastics (sustained or gradient release) ; through multiple layers of degradable or semi-porous plastics (staged release) ; through the above, plus mechanical disruption or other method of disruption.
  • a pharmaceutical cell, particle or molecule may be released from a subcutaneous implant as the biodegradable plastics material casing breaks down.
  • Initial and booster doses of biopharmaceutical may be provided by means of a composition comprising two or more layers of biodegradable plastics material. Examples of suitable materials are described above.
  • a "dummy" layer between two “bioactive layers” comprising encapsulated biomaterial may be used to cause delayed release of the second booster dose of biomaterial.
  • Small drug molecules may be encapsulated as described herein in a plastics material moulded into a therapeutic patch. Biodegradation of the patch where it contacts the skin allows the transdermal administration of the small drug molecule.
  • Therapeutics such as drugs or probiotic bacteria may also be encapsulated in a gum matrix according to methods of the invention and delivered to the individual via chewing the gum.
  • Moulded plastics materials comprising encapsulated viable biomaterial or biomolecules may also be useful in other applications, for example in agriculture, ecology or bioremediation in which it is desirable to preserve and store active biomolecules or viable microorganisms for extended periods prior to use.
  • bioremediation bacteria such as Pseudomonas spp. or Rhodococcus spp. may be stored in an encapsulated form.
  • the encapsulated bacteria may be applied rapidly to the pollution, either by physically disrupting the encapsulating plastics material prior to application or by allowing the plastics material to degrade in si tu to release the bacteria.
  • beads of biodegradable plastics material containing bioremediation microorganisms may be ploughed into affected soils to provide steady release of organisms over time as the lamination breaks down.
  • Microorganisms which accumulate heavy metals may be incorporated into the machine or device containing the pollutant, so that if it is disposed of incorrectly, micro-organisms are already present at site of disposal .
  • biodegradable plastic bags containing encapsulated micro-organisms may be used to store compostable waste materials to accelerate the composting process.
  • nutrients, fertilizer or biopesticides may be encapsulated in a plastics material which is extruded as a plastic sheet using conventional techniques.
  • Suitable biopesticides may include, for example, Bacillus spp, Pseudomonas spp, or entomopathogenic eukaryotes such as nematodes .
  • the sheet containing the embedded biomaterial may be useful in cultivating or mulching plants.
  • Preserved bacterial cells for example Lactobacillus spp., Bifidobacterium spp., Streptomyces spp. may also be useful for example in the dairy industry, for example, for the production of cheese and other diary products .
  • Encapsulated cells as described herein may also be useful in the biotechnology industry, for example for seeding bioreactors and fermentation vessels.
  • Vessels may, for example, be seeded with tablets or wafers of encapsulated cells derived from a single original stock, providing uniformity and consistency.
  • the surface of the tablet may be abraded by mechanical stirring or shaking to release the encapsulated cells and seed the medium.
  • Table 1 shows the percentage survival of wet and dried cells in organic solvents.
  • Table 2 shows survival values (total viable cell numbers) of plastic encapsulated P. putida or spores of B. subtilis after treatment with different disinfectants.
  • P. putida KT2440 and E. coli MC4100 bacteria were grown in Luria Broth (LB) or in M9 minimal medium with glucose (20 ⁇ iM) as the sole carbon source at 30 °C for P. putida and at 37 °C for E. coli as previously described (Garcia de Lau et al (2000) supra) .
  • LB Luria Broth
  • M9 minimal medium with glucose (20 ⁇ iM) as the sole carbon source
  • glucose (20 ⁇ iM) as the sole carbon source
  • To generate HMM NaCl was added to M9 at a final concentration of 0.4 M and 0.6 M for P. putida and E. coli , respectively (Manzanera et al . 2002 supra).
  • B . subtilis was grown in M9 with glycerol 0.5% (v/v) as sole C- source. To obtain spores from B . subtilis, 10 7 -10 9 cells were collected by centrifugation. The pellets were washed with sterile distilled water and incubated at 72°C for 30 minutes. Around 30% of spores per vegetative cell were obtained. Viability of spores did not changed despite of being dried or not .
  • Drying was performed in serum vials under vacuum without freezing in a modified freeze dryer (Dura-Stop ⁇ P; FTS Systems, Stone Ridge, N.Y.) at 30 °C shelf temperature and 15 mTorr (2 Pa; 2 x 10 "5 atm) for 20 h followed by temperature ramping of 25°C/min with a 15- min pause after every increase of 2 °C up to a maximum temperature of 40°C. Samples were sealed under vacuum and stored for variable periods at 30°C after which they were resuspended in LB to a total volume of 1 ml .
  • a modified freeze dryer Dura-Stop ⁇ P; FTS Systems, Stone Ridge, N.Y.
  • serial dilutions in LB were plated on LB agar plates, incubated at 37°C for 24 hr, and counted to determine CFU/ml .
  • the tolerance to different pure organic solvents of dried samples of E. coli and P. putida in presence of trehalose and hydroxyectoine was compared with that of spores of B. subtilis .
  • Osmotically induced cells from the gram-negative microorganisms were vacuum-dried on trehalose (IM) plus PVP 1.5% (w/v) , or hydroxyectoine (IM) plus PVP 1.5% (w/v) .
  • Cells presented a standard tolerance to desiccation in the short term at values between 60% (for P. putida when dried in hydroxyectoine and 80% (for E. coli when dried in trehalose) .
  • Fresh dried samples containing approximately 10 8 cells of E. coli and P. putida were mixed with 200 ⁇ l pure acetone, chloroform, or ethanol, and incubated with these solvents for at least 5 min.
  • a vacuum chamber at low pressure was used to remove the solvents.
  • the time of solvent removal varied from 25 min for samples with acetone, or 30 min for samples with chloroform, to 135 min for samples with ethanol.
  • the survival of the organic solvent treated bacteria was compared with that of non-treated cells.
  • Plastic layers were shredded with a sterile blade razor and incubated at the same conditions previously mentioned. Growth was detected in less than 24 hours; the culture was pure and consisted of the same strains included in each plastic layer.
  • Serial dilutions of bacteria in the dried state Dried bacteria in trehalose were reduced to powder in a mortar. "Dilutions" in the dried state was made by mixing 100 mg of powdered dried bacteria with 900 mg of powdered trehalose. This mixture was called dilution 10 "1 and serial 10-fold “dilutions” were then made from this in trehalose.
  • Tablets were prepared from a powdered mixture of dried cells and trehalose. 500 mg of a mixture of trehalose and 10 4 dried cells were compressed in a KBR press (Spectralab) at 10 atms pressure for 1-5 min. The resulting tablets were 3 mm in height and 14 mm in diameter. To check any possible deleterious effect of the pressure on the cells, 500 mg of same mixture (trehalose and dried-cells) were dissolved in 25 ml of sterile phosphate buffer before and after moulding. After plating 100 ⁇ l of each suspension, a similar number of cfu was found. This result indicated that the pressure applied has no detrimental effect on viability of stable-dried cells.
  • a layer of polystyrene was applied (by paint-brushing) to tablets containing dry cells, prepared as described above.
  • the plastic solution was prepared from 200 mg of polystyrene dissolved in 1 ml of a 1:1 (v/v) mixture of chloroform and acetone. Laminated tablets were allowed to dry for 3 hours
  • Laminated or non-laminated tablets were incubated in 25 ml of the following solutions: phosphate buffer pH 7, 70% ethanol, 4% formaldehyde, 0.1 M K 2 Mn0 4 , 1% bleach, 1/80 solution of seldine (commercial iodine solution) , 10 mM NaOH (pH 12) , 10 mM HC1 (pH 2) .
  • seldine commercial iodine solution
  • 10 mM NaOH pH 12
  • 10 mM HC1 pH 2
  • P. putida MAX10 a bioluminescent P. putida was constructed by insertion of the lux operon from Photorhabdus luminescens into P. putida chromosomal DNA.
  • the resulting strain, named P. putida MAX10 was used in the assay below described.
  • Corn seeds were sterilised by incubation in ethanol 70% (v/v) for 5 min. Followinged by a second incubation in sodium hypochlorite 20% (v/v) . A final set of 5 washes in sterile distilled water was performed to remove traces of ethanol or bleach. Finally, the seeds were air dried in a sterile flow cabinet
  • Sterile sweet-corn seeds were coated with cells by immersion in a mixture containing dried P. putida MAX10, chloroform and polystyrene (as previously described) . After soaking for 2 minutes, the seeds were withdrawn from the mixture and allowed to air-dry for 10 min. Seeds were sowed onto sterile agar plates 1, 30 and 90 days after coating and were incubated at 30°C in the dark. Root colonization was followed over time.
  • Root size was observed to reach several cm two days after germination.
  • Plastic lamination of phospholipid structures Phosphatidyl ethanolamine (PE) labelled with the fluorescent compound nitrobenzoxadiazole (NBD) was used to test the applicability of plastic lamination to membrane systems.
  • PE Phosphatidyl ethanolamine
  • NBD nitrobenzoxadiazole
  • NBD-PE was dissolved at 0.25 mg/ml in 1 M trehalose and a small volume dropped onto a microscope slide which was then placed on an aluminium heating block at 70 °C overnight. The trehalose solution was observed to form a clear glass which incorporated many vesicular structures similar to that formed in aqueous solution.
  • N.D non detected.

Abstract

L'invention concerne des procédés et un organe permettant de conserver des cellules ou d'autres matériaux dans un état viable pendant de longues périodes. Le biomatériau est séché en présence d'un stabilisateur vitrifiant, puis il est encapsulé dans un matériau en plastique liquide qui est ensuite solidifié. Les biomatériaux conservés de cette façon présentent une grande gamme d'applications.
PCT/GB2004/002704 2003-06-23 2004-06-23 Procedes de conservation de biomateriaux WO2004112476A1 (fr)

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WO2009038853A2 (fr) * 2007-06-29 2009-03-26 Biomatrica, Inc. Intégration du stockage d'échantillons et de la gestion des échantillons pour les sciences du vivant
WO2010132508A3 (fr) * 2009-05-11 2011-03-10 Biomatrica, Inc. Compositions et procédés utilisables à des fins de stockage d'échantillons biologiques
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US9376709B2 (en) 2010-07-26 2016-06-28 Biomatrica, Inc. Compositions for stabilizing DNA and RNA in blood and other biological samples during shipping and storage at ambient temperatures
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US9725703B2 (en) 2012-12-20 2017-08-08 Biomatrica, Inc. Formulations and methods for stabilizing PCR reagents
US9845489B2 (en) 2010-07-26 2017-12-19 Biomatrica, Inc. Compositions for stabilizing DNA, RNA and proteins in saliva and other biological samples during shipping and storage at ambient temperatures
US10064404B2 (en) 2014-06-10 2018-09-04 Biomatrica, Inc. Stabilization of thrombocytes at ambient temperatures
US10568317B2 (en) 2015-12-08 2020-02-25 Biomatrica, Inc. Reduction of erythrocyte sedimentation rate

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US8900856B2 (en) 2004-04-08 2014-12-02 Biomatrica, Inc. Integration of sample storage and sample management for life science
US9078426B2 (en) 2004-04-08 2015-07-14 Biomatrica, Inc. Integration of sample storage and sample management for life science
WO2007075253A2 (fr) * 2005-12-01 2007-07-05 Biomatrica, Inc. Integration de stockage d'echantillons et de gestion d'echantillons pour les sciences biologiques
WO2007075253A3 (fr) * 2005-12-01 2008-01-03 Biomatrica Inc Integration de stockage d'echantillons et de gestion d'echantillons pour les sciences biologiques
WO2009038853A2 (fr) * 2007-06-29 2009-03-26 Biomatrica, Inc. Intégration du stockage d'échantillons et de la gestion des échantillons pour les sciences du vivant
WO2009038853A3 (fr) * 2007-06-29 2009-10-15 Biomatrica, Inc. Intégration du stockage d'échantillons et de la gestion des échantillons pour les sciences du vivant
WO2010132508A3 (fr) * 2009-05-11 2011-03-10 Biomatrica, Inc. Compositions et procédés utilisables à des fins de stockage d'échantillons biologiques
US8519125B2 (en) 2009-05-11 2013-08-27 Biomatrica, Inc. Compositions and methods for biological sample storage
US9376709B2 (en) 2010-07-26 2016-06-28 Biomatrica, Inc. Compositions for stabilizing DNA and RNA in blood and other biological samples during shipping and storage at ambient temperatures
US9845489B2 (en) 2010-07-26 2017-12-19 Biomatrica, Inc. Compositions for stabilizing DNA, RNA and proteins in saliva and other biological samples during shipping and storage at ambient temperatures
US9999217B2 (en) 2010-07-26 2018-06-19 Biomatrica, Inc. Compositions for stabilizing DNA, RNA, and proteins in blood and other biological samples during shipping and storage at ambient temperatures
US9725703B2 (en) 2012-12-20 2017-08-08 Biomatrica, Inc. Formulations and methods for stabilizing PCR reagents
EP3007556A4 (fr) * 2013-06-13 2017-03-08 Biomatrica, INC. Stabilisation de cellules
US10064404B2 (en) 2014-06-10 2018-09-04 Biomatrica, Inc. Stabilization of thrombocytes at ambient temperatures
US10772319B2 (en) 2014-06-10 2020-09-15 Biomatrica, Inc. Stabilization of thrombocytes at ambient temperatures
US11672247B2 (en) 2014-06-10 2023-06-13 Biomatrica, Inc. Stabilization of thrombocytes at ambient temperatures
US10568317B2 (en) 2015-12-08 2020-02-25 Biomatrica, Inc. Reduction of erythrocyte sedimentation rate
US11116205B2 (en) 2015-12-08 2021-09-14 Biomatrica, Inc. Reduction of erythrocyte sedimentation rate

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